Mycology and microbiology : a textbook for UG and PG courses 9788172339890, 8172339895, 9788172339913, 8172339917

Table of contents :
Title
The Authors
Foreword
Preface
Contents
A. Mycology
1.The Fungi
2.History of Mycology
3.General Characters of Fungi
4. International Code of Nomenclature for Algae,Fungi and Plants (Source: Wikipedia); Taxonomy of Fungi
5.Zoosporic Fungi
6.Zygomycota
7.Ascomycota
8.Basidiomycota
9.Anamorphic Fungi
10.Glomeromycota
11. Myxomycota and Plasmidiophoromycota(General Account)
B. Current Topics of Importance
12.Phylogeny, Evolution and Origin of Fungi
13.Biodiversity and Biotechnology of Fungi
14.Aeromycology
15. General Account of Plant Diseases Caused byFungi and their Control
16.Endophytic Fungi – Some Glimpses
17.Fungal Ecology
18.Fungal Genetics - General Account
19.Diversity and Conservation of Fungi
20. Lichens - Structure, Reproduction, Ecologicaland Economic Importance
21.Mushroom – Cultivation and Application
22.Mycorrhiza
23.Sexual Reproduction of Fungi - Recent Trends
24.Fungi in Miscellaneous Substrates
25.Entomogenous Fungi
26.Mycotoxigenic Fungi – Mycotoxins
27. Interaction of Fungi with Higher Plants -Some Paleobotanical Glimpses
28.Keratinophilic Fungi – General Account
29. Mycological Methods - Collection,Observation and Isolation
C. Microbiology
30.The Living Kingdom
31.Historical Developments in Microbiology
32.Microscopy
33.Bacteria
34.Viruses
35.Other Microorganisms
36. Diseases Caused by Bacteria and otherProkaryotes
37. Diversity and Conservation ofMicroorganisms
38. Molecular Methods for the Analysis ofMicrobial Communities
39.Some aspects of Applied Microbiology
40.Life Cycles in Microbes and Fungi
Glossary

Citation preview

Mycology and Microbiology

The Authors

Prof. C. Manoharachary Ph.D., D.Sc. was Head, Dept. of Botany, Osmania University, Hyderabad, did post doctoral work at UK, USA, and Germany, is NASI Senior Scientist - Platinum Jubilee Fellow. Guided 46 Ph.D. students, published over 435 research papers and authored/edited 24 books. He bagged several National awards and is Fellow of eight national academies. Prof. K.V.B.R. Tilak Ph.D. was Head, Division of Microbiology, IARI, New Delhi and NASI Senior Scientist and Platinum Jubilee Fellow. He was DAAD Fellow and Humboldt Fellow, Germany. Guided 30 Ph.D. students and published over 250 research papers. He is Fellow of six Academies and honoured with several national awards. Prof. K.V. Mallaiah, Ph.D. is founder of Department of Microbiology and served as Head, Depts. of Microbiology and Botany, Nagarjuna University, Guntur, A.P. He has guided 20 Ph.D. students and published over 150 research papers. He has authored ten books. He is Fellow of four academic societies. Dr (Mrs) I.K. Kunwar D.Phil. was Visiting Research Associate in USA and Research Scientist in the Department of Botany, Osmania University, Hyderabad. She has published more than 125 research papers and authored/edited 5 books.

Mycology and Microbiology (A Textbook for UG and PG Courses)

C. Manoharachary Department of Botany, Osmania University, Hyderabad-500007, Telangana

K.V.B.R. Tilak Department of Botany, Osmania University, Hyderabad-500007, Telangana

K.V. Mallaiah Department of Botany, Nagarjuna University, Guntur, A.P.

I.K. Kunwar Department of Botany, Osmania University, Hyderabad-500007, Telangana

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© 2016, Authors

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ISBN: 978-81-7233-991-3 (PB) 978-81-7233-989-0 (HB) eISBN: 978-93-86102-13-3

Printed in India

This book is dedicated to the Mycologists and Microbiologists of India

Preface

Fungal biology is an interesting branch of science as it plays an important role in the development of the biomedical and biotechnology sectors. Fungi are nonchlorophyllous living eukaryotes which are variable in form, behavior, function and life cycle. Fungi have created themselves specialized status of a kingdom by virtue of possessing well defined, typically chitinized cell wall and absorptive nutrition besides being ubiquitous as saprobes, symbionts, parasites or hyperparasites and cosmopolitan in distribution. Many natural processes are dependent on the interaction between fungi and their environment. Fungal kingdom is diverse and around 1.5 million is the estimated number. Around one lakh fungi are described from the world and 29000 fungal species are reported from India. India is the cradle for fungi, microbes, plants and other living organisms and this hidden wealth needs to be explored for human welfare in order to provide food security, nutritional security, health security and environmental security for the growing population. The variety and galaxy of fungi and their natural beauty occupy prime place in the biological world. Fungi are not only beautiful but play a significant role in the daily life of human beings besides their utilization in industry, agriculture, medicine, food industry, textiles, bioremediation, biocontrol, natural cycling of elements, as biofertilizers, in biotransformation, genetic manipulation and in many other ways. Fungal biotechnology has become an integral part of human welfare. Fungi are well recognized to produce a wide variety of chemical structures, several of which are most valuable pharmaceuticals, agrochemicals and industrial products. Saccharomyces cerevisiae was the first eukaryote available with a genome sequence. The recent developments in molecular biology have allowed mycologists to discover the unexplored or hidden wealth of fungi for the benefit of humanity. Microorganisms are microscopic organisms and include bacteria viruses, microalgae, yeasts, actinomycetes, protozoans and others. Microbes have the ability to breakdown complex chemical substances including hydrocarbons, pesticides, wastes and convert them into simpler substance. These are known to help in recycling of chemical elements and incorporate them into the soil, water and air. Autotrophic bacteria and algae play an important role in photosynthesis which is a food and oxygen generating process. Microbes have applied value and play important role in human welfare. Microbes are used in the synthesis of acetone, glycerin, organic acids, vitamins, amino acids, enzymes, alcohols, vaccines, antibiotics, drugs, in agriculture, bioremediation and others.

x

Biology and Biotechnology of Fungi and Microbes

There are several good text books available in Mycology and Microbiology and all such text books have worthy material. In recent times there have been changes in the nomenclature, classification, life cycle studies, relationship between asexual and sexual stages, origin and phylogeny, diversity, biotechnology and molecular approaches. This book includes a number of chapters dealing with fungal biology, diversity, taxonomy, biotechnology and topics of interest in mycology along with microbial diversity, their structure, reproduction, function and importance. The subject content suffices the needs of U.G. and P.G. syllabus of UGC, ICAR institutions and others. It is an outcome of our committed sincere and humble attempt of our 35 to 40 years of teaching and research experience and expertise. We sincerely hope that this text book will be highly useful to the student community, teaching fraternity, young and budding researchers, candidates preparing for competitive examinations and others. We hope that this humble academic venture will be worthy addition in understanding fungi and microbes. We express our thanks to Mr. Y.S.N. Murthy for his valuable technical help. Our grateful thanks are to Scientific Publishers (India), Jodhpur, India for their keen interest in bringing out this academic contribution and their concerted efforts are laudable. Our thanks are to the teaching community, research scholars, students, eminent mycologists and all others of India for their encouragement. We welcome your suggestions and critical opinion to for further improvement in adding new knowledge. We assure you that next edition will take care all such opinions. C. Manoharachary K.V.B.R. Tilak K.V. Mallaiah I.K. Kunwar

Contents

A. MYCOLOGY 1.

THE FUNGI

2.

HISTORY OF MYCOLOGY

3.

GENERAL CHARACTERS OF FUNGI

4.

5.

6.

1-3 4-10 11-37

Nutrition of Fungi

26

Growth of fungi

27

Reproduction in Fungi

29

INTERNATIONAL CODE OF NOMENCLATURE FOR ALGAE, FUNGI AND PLANTS; TAXONOMY OF FUNGI

38-46

Principles

38

History

39

Taxonomy of Fungi

40

ZOOSPORIC FUNGI (CHYTRIDIOMYCOTA: KINGDOM-STRAMENOPILA)

47-84

Phylum - Chytridiomycota

48

Class – Chytridiomycetes

50

Kingdom – Stramenopila

51

Class – Hyphochytridiomycetes

58

Phylum – Oomycota

59

Class – Oomycetes

59

Important Genera

62

ZYGOMYCOTA

85-109

Class- Zygomycetes

85

Class - Trichomycetes

94

Important Genera

96

xii 7.

8.

9.

10.

11.

Biology and Biotechnology of Fungi and Microbes ASCOMYCOTA

110-145

Classification

117

Class - Hemiascomycetes

118

Class – Plectomycetes

119

Class – Pyrenomycetes

119

Class – Discomycetes

123

Class – Loculoascomycetes

126

Class- Laboulbeniomycetes

128

Important Genera

129

BASIDIOMYCOTA

146-189

Classification

152

Class – Teliomycetes

154

Class – Hymenomycetes

158

Class- Gasteromycetes

166

Important Genera

171

ANAMORPHIC FUNGI

190-215

Class - Blastomycetes

196

Class - Hyphomycetes

197

Class - Coelomycetes

197

Important Genera

199

GLOMEROMYCOTA

216-220

Arbuscular Mycorrhiza

216

Benefits derived from Mycorrhiza by Host Plants

218

Morphological Diversity in Arbuscular Mycorrhizal Fungi

218

MYXOMYCOTA

221-224

Class - Myxomycetes

221

Plasmodiophoromycota

224

B. CURRENT TOPICS OF IMPORTANCE 12.

PHYLOGENY, EVOLUTION AND ORIGIN OF FUNGI Phylogenetic Hypotheses, EvolutionaryRrelationships and Circumscription of the Fungi

13.

BIODIVERSITY AND BIOTECHNOLOGY OF FUNGI What is Biodiversity?

239-242 240 243-254 243

Contents

xiii

Biodiversity of Fungi

243

Fungal Biotechnology

247

14.

AEROMYCOLOGY

255-258

15.

GENERAL ACCOUNT OF PLANT DISEASES CAUSED BY FUNGI AND THEIR CONTROL

259-265

Some Important Fungal Diseases of Crop Plants

260

Control of Plant Diseases Caused by Fungi

263

16.

ENDOPHYTIC FUNGI – SOME GLIMPSES

266-268

17.

FUNGAL ECOLOGY

269-278

18.

FUNGAL GENETICS - GENERAL ACCOUNT

279-283

19.

DIVERSITY AND CONSERVATION OF FUNGI

284-292

20.

21.

22.

23.

Diversity of Fungi

285

Conservation of Fungi

289

LICHENS - STRUCTURE, REPRODUCTION, ECOLOGICAL AND ECONOMIC IMPORTANCE

293-304

Occurrence

294

Thallus Structure

295

Nutrition and Growth

297

Reproduction

297

Classification of Lichens

299

Some Important Genera of Lichens

300

Importance of Lichens

301

MUSHROOM – CULTIVATION AND APPLICATION

305-317

Button Mushroom

306

Oyster Mushroom

310

Paddy Straw Mushroom

313

MYCORRHIZA

318-326

Mycorrhiza Types

318

Benefits Derived from Mycorrhiza by Host Plants

322

Morphological Diversity in AM Fungi

322

Ecological Diversity in AM Fungi

323

SEXUAL REPRODUCTION OF FUNGI – RECENT TRENDS Sexual Reproduction

327-341 328

xiv

24.

25.

26.

Biology and Biotechnology of Fungi and Microbes Taxonomic Implications of Sexual Reproduction

330

Fungal Mating-Type Locus

341

Genetic Diversity in Microsporidia

341

FUNGI IN MISCELLANEOUS SUBSTRATES

342-346

Thermophillic Fungi

342

Psychrophillic Fungi

344

Coprophilous Fungi

345

Phyllosphere Fungi

345

Fungi in the Atmosphere

345

Soil Fungi

346

Marine Fungi

346

ENTOMOGENOUS FUNGI

347-354

Effect of Environmental Factors

349

Development of Entomogenous Fungi for Pest Control

350

Entomophthorales

353

MYCOTOXIGENIC FUNGI – MYCOTOXINS

355-364

Mycotoxins

356

Mycotoxicoses

362

27.

INTERACTION OF FUNGI WITH HIGHER PLANTS – SOME PALEOBOTANICAL GLIMPSES

365-366

28.

KERATINOPHILIC FUNGI – GENERAL ACCOUNT

367-369

Dermatophytes 29.

368

MYCOLOGICAL METHODS - COLLECTION, OBSERVATION AND ISOLATION

370-378

Collection

370

Observation

372

Isolation Techniques

375 C. MICROBIOLOGY

30.

THE LIVING KINGDOM

397-400

31.

HISTORICAL DEVELOPMENTS IN MICROBIOLOGY

401-414

Beginnings of Microbiology

401

Golden Age of Microbiology

403

Pioneers of Microbiology

405

Contents

32.

33.

34.

35.

xv

Era of Molecular Biology

409

Developments of Soil Microbiology during 20th Century

410

Prospects and Challenges

413

MICROSCOPY

415-424

Bright Field Microscope

415

The Phase Microscope

417

The Electron Microscope

418

The Dark-Field Microscope

418

Microscopic Examination of Bacteria

419

Differential Stains

421

BACTERIA

425-494

Components of Bacterial Cell

426

Plasmids

433

Cultivation and Nutrition of Bacteria

438

Utilization of Nutritional and Energy Sources by Bacteria

444

Growth of Bacteria

445

Classification of Bacteria

452

Reproduction in Bacteria

470

Biotechnology of Bacteria

476

VIRUSES

495-506

Viruses

495

Bacteriophage

497

Virus Multiplication

498

Classification

502

Cultivation of Viruses

505

OTHER MICROORGANISMS

507-529

Actinobacteria (Actinomycetes)

507

Archaebacteria

509

Bioprospecting of Archaea

514

Mollicutes

515

Rickettsiae

518

Chlamydiae

518

Prions

519

Cyanobacteria (Blue-Green Algae)

520

xvi

36.

37.

38.

Biology and Biotechnology of Fungi and Microbes Selected Members of Cyanophyceae

525

Protozoa

526

DISEASES CAUSED BY BACTERIA AND OTHER PROKARYOTES Bacterial Diseases

531

Viral Diseases

536

Plant Diseases Caused by Spiroplasmas

540

DIVERSITY AND CONSERVATION OF MICROORGANISMS

40.

545-552

Biodiversity

545

Conservation

545

Utilization of Microbial Diversity for Sustainable Agriculture

550

MOLECULAR METHODS FOR THE ANALYSIS OF MICROBIAL COMMUNITIES Molecular Techniques to Study Natural Microbial Communities

39.

530-544

SOME ASPECTS OF APPLIED MICROBIOLOGY

553-556 553 557-581

Metabolism

557

Antibiotics

561

Environmental issues

563

Soil remediation

564

Immunology

564

LIFE CYCLES IN MICROBES AND FUNGI

570-582

Life cycles in fungi

570

Asexual life cycle in fungi

573

GLOSSARY

583-607

A. MYCOLOGY

Chapter - 1

The Fungi

Mycology has been defined as ‘the scientific study of fungi’ by Kirk et al (2008). Hippocrates first mentioned mushrooms when he wrote about their medicinal value in 400 B.C. Robert Hooke’s microscopic observation had been related to the moulds in his epoch-making work ‘Micrographia’ (1665). Pier Antonio Micheli (1679-1737), described and classified several hundred species of fungi in ‘Nova Plantarum Genera Iuxta Tournefortii Methodum Disposita’. Earlier to Hooke’s Micrographia, Carolus Clusius (1526-1609) had published ‘Rariorum Plantarum Historia’ comprising the part of ‘Fungorum in Pannoniis observatum brevis historia’. In ‘Fungi from West Pannonia’ he was the first to describe 86 mushrooms with water colors. German mycologist Anton de Bary (1831-1888) initiated the studies of fungi in the 19th century. de Bary (1866) in German brought out ‘Morphology and physiology of the fungi, lichens and myxomycetes’. Willam Delisle Hay in his ‘An elementary textbook of British fungi’ (1887), decribed Mycology as the study of Fungi. The Oxford English Dictionary, defined that mycology is the adaptation of the modern Latin ‘Mycologia’, which consists of ‘myco’ and ‘logia’, adapted from the Greek words literally meaning fungus science. McNicoll (1863), used the term ‘Mycetology’ instead of Mycology in his book ‘Dictionary of Natural History Terms with their Derivations’. What are Fungi? 1.

Fungi are non-photosynthetic living organisms, hence nutrition is heterotrophic and absorptive. The absence of chlorophyll has enforced them to be saprophytic, parasitic and symbiotic.

2.

Thallus is holocarpic (single celled) or eucarpic (filamentous mycelia). In case of mycelial fungi, being coenocytic (non-septate) and septate.

3.

Well defined cell wall is present. Mostly it is made of chitin, glucans, mannans and glycoproteins. In Oomycota it is made of β- glucans and cellulose.

4.

Fungal nuclei are eukaryotic, small, nuclear membrane does not get separated during cell division, nuclear status may be multinucleate (coenocytie mycelium), homokaryotic or heterokaryotic (haploid, diploid or dikaryotic mostly in septate mycelia).

2

Biology and Biotechnology of Fungi and Microbes

5.

Simple or complex life cycles are present.

6.

Asexual reproduction is by zoospores, sporangiospores and mitospores (conidia). Sexual reproduction is by plano-gemetic copulation, gametangial contact, gametangial copulation, somatogamy and spermatization. Sexual thalli may be homothallic or heterothallic.

7.

Asexual fruit bodies like Synnemata, Acervuli, Pycnidia, Sporodochia are found in fungi. Sexual fruit bodies may be open asci, loose fruit body, Cleistothecium, Perithecium, Apotheciun and Basidiocarps (Epigeal and Hypogeal).

8.

The resultant product of sexual reproduction being Oospore, Zygospore, Ascospore and Basidiospore.

9.

Fungi are ubiquitous, cosmopolitan is distribution and are known to colonize diversified habitats: soil, litter, water, animal excreta, seed, living plant parts, marine habitat, senescent leaves, leaf surface, root zone, root surface, dung, bark, air, extremophilic substrates etc.

Fungal diversity is the variety of life on earth from the genetic through the origanismal to the ecological levels (Kirk et al 2008). Number of fungi around the world has been estimated to be 1.5 million (Hawksworth 1991) and around one lakh fungal species so far have been described and documented (Kirk et al 2008). Around 29,000 fungal species are reported from India indicating that about one third of the fungal diversity of the globe exists in India (Manoharachary et al 2005). Threats of fungi throughout the world are of much concern since they are not only beautiful but also play a significant role in human welfare. Moore et al (2001) have suggested the following steps for fungal conservation (i) conservation of habitats, (ii) in situ conservation of non-mycological reserves/ecological niches, (iii) ex-situ conservation especially for saprotrophic species growing in culture. Many of the described species are known only as dead herbarium material and around 5-7% of species are isolated as pure cultures. Geographic location, climatic conditions, macro- and micro-ecological niches, substrate nutrients, distributed vegetation and fauna, and meteorological conditions are such factors that contribute for fungal distribution. Morphotaxonomic criteria have been in use since long time in identifying fungi. Methodologies in the field of systematics have undergone rapid and phenomenal changes during the past two decades. DNA based molecular methods are rapidly replacing and supplementing the conventional methods, such as microscopy, cultural and biochemical characterization. Nucleic acid probes are used for rapid identification of fungi. Polymerase chain reaction (PCR) offers several benefits over the other methods and has become a powerful tool in recent times along with metagenomics. Taxonomy is the mother of all sciences and naming of fungi, their characterization and identification are important. Though molecular tools are available and found useful, still morphological criteria form key factor in the identification of fungi. In the present situation there are only a small number of taxonomists available both in India and other parts of the world who can help in the identification. Identification of fungi and fungal pathogens are important. There is a

The Fungi

3

need to impart training to youngsters in the taxonomy of fungi to strengthen the process of identification of fungi through capacity building programmes. Nature represents a formidable part of bioactive compounds and is a strategic source for new and successful commercial products. Microbial sources such as fungi are well recognized to produce a wide variety of chemical compounds, several of which are most valuable pharmaceutical, agrochemical and industrial products. The world of fungi provides a fascinating and almost endless source of biological diversity which is a rich source for exploitation.

■■■

Chapter - 2

History of Mycology

Fungi and their utilization are known to humans since times immemorial. The utility of fungi began when men lived as hunter gatherers. Fungi remained as mystery though some information on plant and animals was known in Greek and Roman civilizations. The fundamental work of P.A. Micheli has established scientific approach on study of fungi. The improvements in microscopy lead to the understanding of mycelium and fruit bodies. Federico Cesi illustrated around 1620 species of fungi observed in Italy. P.A. Saccardo’s monumemtal work ‘Sylloge fungorum omnium hucusque cognitorum’ was published in 25 volumes. Bresadola completed ‘Iconographia Mycologica’, a complete atlas of mushrooms of the 20th century. The naming of fungi as per binomial nomenclature of Linnaeus was introduced for fungi by C.H. Persoon and E.M Fries in the early 19th century. Sculptures and etchings outlining the mushrooms date back to 1300 B.C. found in Mexico and Guatemala. Some prehistoric etchings were also found in ancient Egyptian, Indian and Sumarian civilizations. In medieval Europe, it was believed that fungi the work of devil who, when wandering on earth took on the guise of a fat Toad and ordered mushrooms to sprout from the earth for use as stools, thus generated false beliefs and superstitions. In the Palaeolitic Period or Old Stone Age (2,50,000 BC to 10,000 BC), Mesolithic Period or Middle Stone Age (10,000 BC to 8,000 BC) and Neolithic Period or New Stone Age (8,000 BC to 3000 BC) primitive people were familiar with edible and poisonous mushrooms with reference to their domestication, nutritive values, poisonous nature, religious and artistic influences. The Cavemen discovered Amanita muscaria var. muscaria as medicinal/ poisonous mushroom. This hallucinogenic mushroom was shown as ‘Round Heads’ in pictorial stages by archaeologists during 7000 BC to 5000 BC. In Denmark Fomes fomentarius of Polyporaceae was found associated with fragments of pyrites and silica in 6000 BC and also in Yorkshire (U.K). A megalithic monument common to Kerala is the ‘Kuda Kallu’ (umbrella stone) the most important concentrations of which are found in the inner regions of Trichur. A huge copper cauldron with umbrella type of mushrooms on its top was found in Hungary. The use of fungi in ancient times was regarded as something like evil and it can be assured with reason that they were used by wizards and professional poisoners.

History of Mycology

5

The symptoms leading to death as documented in some ancient documents actually lead to thinking that these deaths were caused by poisoning with Amanita phalloides. Documentation by Wasson and Wasson (1957) indicated that Roman Emperor Claudius Caesar (AD 54) was murdered by offering poisonous mushroom Amanita phalloides as mix with food in order to occupy the throne. Greek physician by the name Dioscorides has listed out the differences between edible and poisonous mushrooms. The warning that comes out of these studies is that edibility of a mushroom has to be testified by scientists (mycologist) and a certificate needs to be obtained by mushroom growers to this effect. Most of the mushrooms are death traps but for few like Button mushroom, Oyster mushroom, Paddy straw mushroom, Morel or Gucchi (Morchella esculenta) and few others are edible and nutritious. There are few records available about mushrooms as Soma and about some plant diseases caused by fungi in the Vedas and Hindu scriptures. The discovery of microscope in 17th century by Leeuwenhoek laid the foundation for the discovery of microbes and fungi. The Greek Theophrastus disciple of Aristotle defined fungi as ‘Imperfect plant, without roots, leaves, flowers and fruits’. Samorin (2002) studied the ancient mushroom sculptures and paintings and noted the drawings of Amanita muscaria, Psilocybe and others. One wall painting in central France showed the entry of Christ into Jerusalem greeted with a painting of a tree having mushroom like formations. Mycological traditions have been preserved for centuries and millennia following cross cultural pathway, while necessary faith was constantly enlivened by experiences made with some mushrooms. Andrea–Mattioli (1500-1577), made an interesting passage on fungi now identified as Laetiporus sulphureus which is sulphur yellow to orange and grows on woods in the mountains and is so large that sometimes it may weigh up to 100 pounds. Andrea Caesalpino (1519-1603) described fungi as plants without fruits and seeds. Robert Hook (1635-1703), was the first botanist to draw the illustrations of sporangia of Mucor and teleutospores of Phragmidium mucronatum. A huge amount of literature has accumulated on fungi from time to time. The following Table 2.1 enlists the glimpses of some important mycological contributions. Table 2.1. Some important mycological contributions. Year

Name

Contribution

1588

Porta G della

Mushroom spores as seeds

1590-1608

Jensen Z

Microscopic drawing of microbes

1601

Clusius CE

‘Rariorium Plantarum Historia’

1665

Hooke R

Micrographia

1673

Leeuwenhoek Anton van Microscope, yeast observation

1675

Sterbeeck FV

‘Theatrum Fungorum oft het tooneel der campernoelien’

1729

Micheli PA

‘Nova Genera Plantarum’

6

Biology and Biotechnology of Fungi and Microbes

Year

Name

Contribution

1801

Persoon CH

‘Synopsis Methodica Fungorum’

1821-1832

Fries FM

‘Systema Mycologicum’

1837-1854

Corda ACJ

‘Icônes Fungorum’

1860

Pasteur Louis

Fermentation using yeast

1861-1865

Tulsane LR, Tulsane C

‘Selecta Fungorum Carpologia’

1869

Fuckel L

Fungi Imperfecti

1872-1912

Brefeld O

Fungi in culture, slime molds

1874-1943

Butler EJ

Pythium, Allomyces, Fungi of India, HCIO, Plant diseases

1882

Saccardo PA

‘Sylloge Fungorum’

1883

Barclay A

Mushrooms

1883-1884

Koch Robert

Pour-Plate method, Koch’s postulates

1885

Lt. Col. Kirtikar

Fleshy fungi

1887

de Bary

Heteroecism, Comparative morphology and Biology of the Fungi, Phytophthora infestans

1895-1942

Mitra M

Cereal Rusts, Karnal Burnt of Wheat

1896-1952

Mundkur BB

Smuts, Fungal Diseases of Crops, Quarantine Regulations

1897-1971

Saksena SB

Soil fungi, Saksenaea vasiformis

1898-1973

Bagchee KD

Soil borne diseases

1903-1999

Tandon RN

Physiology of fungi, Mucorales

1904

Blakslee AF

Heterothallism

1911

Butler EJ

Allomyces

1912

Ajrekar SL

Smuts, Mucorales

1913-2001

Sadasivan TS

Root- and Soil-borne Diseases, Fungal enzymes

1914-1999

Thirumalachar MJ

Rusts, Smults, Antibiotics

1918-1947

Bose SR

Monograph on Polyporaceae

1921

Waksman SA

Soil - Dilution Plate Method, Streptomycin

1924

Burgeff H

Sex-hormones

1925-1955

Dodge BO

Neurospora

1927-1967

Craigie JH

Sex hormones in Rust Fungi

1928

Alexander Flemming

Penicillin

1929

Linder DH

Helicosporous fungi

1929-1957

Das Gupta SN

Aquatic fungi, Medical Mycology

1931-1960

Vasudeva RS

Fungi of India

History of Mycology

7

Year

Name

Contribution

1932-1934

Lindegren CC

Tetrad analysis in Neurospora

1933-1996

Bilgrami KS

Physiology of Fungi, Mycotoxigenic fungi

1934

Buller AHR

Fungal spore dispersal

1941

Beadle G and Tatum E

One gene one enzyme theory

1941

Emerson R

Life cycle of Allomyces

1948-1978

Ramakrishnan K

Coelomycetes

1948-1980

Thind KS

Aphyllophorales

1948-1980

Bhargava KS

Saprolegniaceae, Cytology of fungi

1948-1990

Mukerji KG

Mycorrhizae, Fungal taxonomy Chaetomium, Myxomycetes

1948-1990

Subramanian CV

Hyphomycetes , Taxonomy, Biology

1949

Agarwal GP

Entomogenous fungi

1949

Miller JH

Pyrenomycetes

1950-1995

Pavgi MS

Smuts, Fungi Imperfecti, Synchytrium, Chytrids

1952

Pontecorvo and Roper

Parasexuality

1952

Ainsworth GC

Classification of fungi, Medical Mycology, Dictionary of Fungi

1952

Middleton JT

Genus Pythium

1952-1982

Mehrotra BS

Mucorales

1953

Hughes SJ

Conidial ontogeny

1955

Moore RT

Helicosporae

1956

Johnson TW

Achlya, Aquatic fungi

1957

Hesseltine CW

Mucorales

1959-1961

Kamat MN

Cytology of fungi

1960-1990

Agnihotrudu V

Fungi Imperfecti

1961

Martin GW

Fungal Taxonomy

1962

Dwivedi RS

Rhizosphere fungi

1962

Moore RT and MC Alear JR

Dolipore Septum

1962-2002

Dayal Ram

Aquatic fungi, Nematode trapping fungi

1963

Ames LM

Chaetomium

1963

Gams W

Fungal Taxonomy

1963-1980

Sutton BC

Coelomycetes

1965

Ahmadjian V

Lichens

1965

Ingold CT

Fungal spore discharge

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Biology and Biotechnology of Fungi and Microbes

Year

Name

Contribution

1965

Muller G

Ascomycetes

1965

Raper KB and Fennell DI The genus Aspergillus

1965

Rifai MA

Trichoderma

1965

Scott KJ

Culturing of Rusts

1966

Meredith DS

Helminthosporium

1966-2001

Mehrotra RS

Phytophthora

1967

Hale ME

Lichens

1967

Martin P

Xylariaceac

1967

Pegler DN

Polyporaceae

1968

Barron G L

‘Hyphomycetes from Soil’

1968

Karling JS

Chytrids, Plasmdiophoralcs

1969

Alexopoulos CJ

Myxomycetes

1969

Harley JL

Ectomycorrhiza

1970

Garrett SD

Soil and root infecting fungi, Inoculum potential

1970

Bartnicki Garcia S

Fungal cell wall

1970

Lodder J

Yeasts

1970

Olive LS

Mycetozoa

1970-2008

Natarajan K

Agaricales

1971

Ellis MB

‘Dematiaceous Hyphomycetes’

1971

Fincham JRS

Fungal Genetics

1971

Kendrick WB

Taxonomy of Fungi Imperfecti

1971

Cunningham DD

Aeromycology, Mucorales, Rusts, Smuts,

1972

Bandoni RJ

Terrestrial, Aquatic Hyphomycetes

1972

Punithalingam E

Pycnidial Fungi

1973

Benjamin RK

Mucorales

1973

Dick MW

Zoosporic fungi

1973

Duddington CL

Nematode trapping fungi

1973

Gregory PH

Aeromycology

1973

Hiratsuka Y

Rust Fungi

1973

Korf RP

Discomycetes

1973

Luttrell ES

Loculoascomycetes

1973

Poelt J

Lichens

1973

Sparrow FK

Zoosporic fungi

1974

Gerdemann JW

Endogone

History of Mycology

9

Year

Name

Contribution

1975

Burentt JH

Fungal Genetics

1975

Cole GJ

Ultra Structure of Conidia

1975

Singer R

Agaricales

1975

Tubaki K

Hyphomycetes

1975

Waterhouse GM

Pythium, Peronospora

1975

Christensen CM

Mycotoxins

1975-2005

Lakhanpal TN

Myxomycetes, Agaricales

1975-2015

Manoharachary C

Soil fungi, Hyphomycetes, Mycorrhizal fungi, Agaricales Lichens, Aquatic fungi.

1977

Nawawi A

Aquatic fungi

1977

Neergaard P

Seedborne fungi

1978

Booth C

Fusarium, Ascomycetes

1978

Yarwood CE

Powdery mildews

1980

Webster J

Aquatic Fungi, General Fungi

1982-1950

Mehta KC

Wheat Rust

1991

Hawksworth DL

Biodiversity of Fungi

2001

Hibbett DS

Basidiomycota

2008

Kirk PM

Dictionary of fungi

Besides the above a number of mycologists have contributed their best from different parts of India, these include: Agarwal DK - Rusts, Smuts and Mucorales; Agarwal SC - Medical Mycology; Aneja KR- Fungi on weeds, diversity of fungi; Arora DK - Molecular taxonomy of fungi; Atri NS- Edible fungi; Awasthi BB – Lichens; Bagyanarayana G – Rusts; Banerjee SN- Wood rotting fungi; Baruha N- Phylloplane fungi; Bakshi BK– Ectomycorrhizae; Bhat DJ– Hyphomycetes; Chauhan RKS and Chauhan Shashi Seed fungi, Mycotoxigenic fungi; Chowdhry PN - Pythiaceous fungi, General fungi; Dargan JS – Xylaria; Dhingra GS - Corticioid fungi; Dubey NK - Plant microbe interactions; Gandhe RV - Smut fungi; Gangawane LV - Soil and Rhizosphere fungi; Govindu HC - Diseases of cereals; Hasija SK, Rajak RC, Pandey A, Jamaluddin Diversity and Taxonomy of fungi, Chytrids, Coelomycetes, Weed fungi and Forest fungi; Hosagoudar VB - Sooty molds and Meliolales; Jalali BL - Mycorrhizal fungi; Jha DK - Diversity of fungi, Mycorrhizae; Johri BN - Thermophilic fungi; Kamal Taxonomy of Cercosporaceous and allied fungi; Kapoor JN - Hyphomycetes, Mushrooms; Kaviarasan V - Higher Fungi; Kharwar RN - Endophytic fungi, Hyphomycetes; Kushwaha RKS - Keratinophilic fungi; Lodha BC - Coprophilous Ascomycetes; Mall Sudha and Mall OP - Seed-borne fungi, Root diseases; Mishra RR - Soil fungi; Mohanan C and Sankaran KV - Forest fungi; Mukadam DS - Seedborne fungi; Muthu Mary – Coelomycetes; Niranjana SR - Biocontrol agents; Padmanabhan SY - Rice diseases; Pande Alka – Ascomycetes; Prof. Patil MS - Rust fungi; Patil SD - Aquatic fungi; Patwardan PG – Lichens; Prakash HS - Seed-borne

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Biology and Biotechnology of Fungi and Microbes

pathogens; Prasad GS – Yeasts; Prashar IB - Higher fungi, Anamorphic fungi; Purkayastha RP – Phytoalexins; Raghuveer Rao P – Hyphomycetes; Raghukumar S and Chandrakala Raghumukar - Marine and deep sea fungi; Rai Bharat - Ecology of Fungi; Rama Rao P - Soil fungi; Raman N - Mycorrhizal fungi; Reddy SM Mycotoxigenic fungi; Rodrigues BF - Mycorrhizal fungi; Saikia UN – Hyphomycetes; Samajpati N - Taxonomy of fungi; Sarbhoy AK – Mucorales; Sathe V - Rust fungi; Satyanarayana T - Thermophilic fungi; Sharama MP - Ascomycota and Basidiomycota; Sharma - Higher fungi; Sharma J - Wood rotting fungi; Shekhar Shetty H - Seedborne fungi; Singh KP - Lichen taxonomy; Singh SK - Fungal Taxonomy; Singh S – Ectomycorrhizae; Singh D - Seed fungi; Sohi HS and Upadhyaya RC – Mushrooms; Sridhar KR - Aquatic hyphomycetes; Srinivasan MC – Entomophthora; Srivastava AK - Biocontrol fungi, culturing of fungi, endophytes; Sundaram Indira Kalyana – Myxomycetes; Suryanarayanan T - Endophytic fungi; Kumar S - Taxonomy and culturing of fungi; Tilak ST– Aeromycology; Trivedi PC Nematode trapping fungi; Upadhyay RS - Soil-borne diseases, Biocontrol; Upreti DK- Lichen metabolites and Vaidya JG - Higher Fungi. There are many eminent mycologists and young mycologists who have contributed for the growth of Mycology in India. India is a vast country and there might have been some left out names of mycologists. Further it is impossible to cover all such contributions. In the last ten years mycological research has been slowed down and number of senior mycologists either might have left this world or retired. The available number of such eminent mycologists in the taxonomy of fungi is reduced to 10 to 15, thus not only taxonomy of fungi has become forgotten science but the subject mycology has been getting raw real and step motherly treatment at different levels. However the biodiversity of fungi is the resource material for biotechnology and biotechnological products are worth millions of dollars hence requires attention.

■■■

Chapter - 3

General Characters of Fungi

Fungi are highly specialized; nonchlorophyllous, heterotrophic, eukaryotic organisms with hyphae possessing cell wall generally made up of fungal chitin, and reproduce both asexually and sexually by producing spores characteristic of each group. The fruit body forming macroscopic organisms like mushrooms, puff balls, bracket fungi, etc. Filamentous forms like Aspergillus, Penicillium, Mucor, etc., and unicellular forms like yeasts are important examples of fungi. The term fungus is derived from Greek word ‘sphongos’ meaning sponge and it refers to the soft fleshy sponge like fruit bodies of mushrooms. The study of fungi is called Mycology and it is derived from Greek words ‘mykes’ meaning mushroom and ‘logos’ meaning study or discourse. Etymologically, Mycology means study of mushrooms, because they are the prominent group known to man before the invention of microscope. Since the time of Anton von Leeuwen Hoek of Holland and Robert Hooke of England who first used the microscope to study the biological specimens during 1660’s a large number of microscopic fungi are discovered and the studies are still going on. The first major contribution to the study of modern mycology was made by Pier’ Antonio Micheli, an Italian Botanist who published the book ‘Nova Plantarum Genera’ in 1729, meaning new plant genera. These are nothing but fungi. Later, in the early part of 19th century, Christian Hendrick Persoon (1801), the famous Dutch mycologist wrote a monograph on fungi entitled ‘Synopsis Methodica Fungorum’. Elias Magnus Fries (1831) of Sweden published ‘Systema Mycolgicum’. The contributions of various mycologists like Tulsane brothers of France, M.J. Berkley of England, Anton de Bary of Germany, P.A. Saccardo of Italy, M.S. Woronin of Russia firmly established the science of Mycology. Distribution of fungi Fungi are ubiquitous and widely distributed in nature. They are found growing on all substrates with some organic matter and moisture. They are found in water, soil, on decaying plant and animal parts. Since they lack chlorophyll, they are heterotrophic in nutrition. Most of the fungi are saprophytic and grow on dead organic matter. Some of the fungi are parasitic on plants, causing various types of plant diseases. Some of the fungi enter into symbiotic relationship with other organisms. For example, with algae they form lichens, and with roots of higher plants they form mycorrhiza.

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Biology and Biotechnology of Fungi and Microbes

Status of fungi The fungi were traditionally treated as plants and studied by botanists. Theophrastus, the ‘Father of Botany’, in the first book on plants, ‘Historia Plantarum’, described fungi or agarics as rootless and leafless plants that arose from the excess moisture of soil or putrefying organic matter during rainy season. Carl Von Linnaeus (1753) who is considered as ‘Father of plant taxonomy’ treated fungi as a group in his 24th class of the plants, Cryptogamia. Anton van Leeuwenhoek of Holland and Robert Hooke of England started using microscopes to study the biological specimens in 1660s, and over a period of time a great deal of knowledge accumulated on microscopic forms of life. With increasing knowledge on microorganisms, the basic concept of treating all living organisms either as plants or animals was questioned. Ernst Haeckel (1866) a famous German Zoologist proposed 4 kingdoms. Apart from Metaphyta (green plants) and Metazoa (animals) he recognized two more kingdoms viz. Protophyta and Protozoa. He included fungi in Protophyta. However, proposal of 4 kingdom system was considered as revolutionary by the biologists of that time, and they began to treat microscopic forms of life as Protista. In the early 20th century, the importance of nucleus was recognized and basing on the presence or absence of membrane bound nucleus the microorganisms of Protista are recognized as two groups viz. prokaryotes and eukaryotes. Whittaker (1969) proposed 5 Kingdom systems and for the first time elevated the status of fungi and treated them as a separate kingdom on par with plants and animals. The 5 Kingdoms proposed by Whittaker are as follows 1.

Monera : Prokaryotic blue green algae, bacteria and related forms

2.

Protista : Eukaryotic unicellular organism mainly algae and protozoa

3.

Fungi : Organisms showing absorptive type of nutrition

4.

Plantae : Photosynthetic organisms

5.

Animalia : Organisms showing phagotrophic type of nutrition

Since the proposals of Whittaker many scientists began treating fungi as a separate kingdom ‘Mycetae’, In recent years, Cavalier Smith (1998) recognized two empires and 6 kingdoms in the living world. According to this system, the empires are Procaryota and Eukaryota. In the empire Procaryota has one kingdom Bacteria. In the empire Eukaryota, 5 kingdoms are recognized. They are Protozoa, Fungi, Chromista, Plantae and Animalia. Among these the first kingdom is at a lower level with reference to cell structure while the latter 4 kingdoms are at a higher level of cell structure. According to these proposals also the fungi are considered as a separate kingdom, the status of fungi as a separate kingdom is well established. The characters of fungi are very distinct and there is justification for treating fungi as a separate kingdom based on nutrition, storage material, biochemistry of cell wall, pattern of growth, protoplasmic continuity, hyphal anastomosis and genetic plasticity. 1. Absorptive type of nutrition: Fungi are heterotrophic organisms like animals, but there is a fundamental difference in nutrition of fungi and animals.

General Characters of Fungi

13

Animals show phagotrophic or ingestive type of nutrition i.e. taking in of solid food material and digesting them inside their body. Fungi show absorptive type of nutrition i.e. they secrete extracellular enzymes into the surroundings and degrade them into smaller units which can be absorbed by the cells directly. Such absorptive type of nutrition is seen in bacteria also but they are fundamentally different from fungi in nuclear organization. Bacteria are prokaryotes and fungi are eukaryotes. 2. Biochemistry of cell wall: Fungi are having true cell wall made of mostly fungal chitin. Cellulose is present in the cell wall only in Oomycetes, while the cell wall of plants is mainly composed of cellulose. The animal cells lack cell walls. Bacteria also possess a rigid cell wall but it is mainly made up of peptidoglycan layer but not cellulose or chitin. 3. Storage material: In plants the reserve food material is mainly starch, while in fungi reserve food material is mainly glycogen (animal starch). It is typical of animal cells but not of higher plants. 4. Growth: The primary growth of filamentous fungi is apical i.e. hyphae grow only at apices. Their walls may thicken considerably behind this point but they do not extend. This form of growth is in contrast with intercalary growth which is seen in other filamentous organisms in which almost any cell of the filament can enlarge and divide. 5. Protoplasmic continuity: The mycelium of filamentous forms of zoosporic fungi and Zygomycetes is made up of aseptate, coenocytic hyphae, hence protoplasm is continuous throughout the fungal body. The mycelia of fungi belonging to Ascomycotina, Basidiomycotina and most Deuteromycotina comprises of branched hyphae which are septate at frequent intervals. However, the septa have central perforations or pores which help in easy communication between the cells, thus maintaining the protoplasmic continuity throughout the mycelium. 6. Hyphal anastomosis: The most remarkable feature of fungal hyphae is the process of hyphal fusion or anastomosis. When compatible hyphae are growing nearby, they come in contact with one another and protoplasm from one hypha is transferred to the other. 7. Genetic plasticity: The fungal cells contain membrane bound nuclei. The cells are coenocytic. Mostly they are haploid and are of same genotype. However, because of fungal anastomoses and nuclear migration throughout, two or more nuclei in the same cell may be of different genotype, and such a condition is described as Heterokaryosis. The nuclear fusion may take place between homozygous nuclei or nuclei of different genotypes and hence some cells may contain nuclei of different genotypes. Such genotypic variability is not found in other groups of organisms. From the foregoing, it is considered by many mycologists that the fungi deserve to be treated as a separate kingdom. Ultra structure of a typical fungal cell The vegetative cells of fungi are typically eukaryotic with all the major structural components of other eukaryotic organism’s viz. plants and animals. The fungal cell differs fundamentally from a plant cell by the lack of chloroplasts, and differs from the animal cells in possessing a rigid cell wall.

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Biology and Biotechnology of Fungi and Microbes

Fungal species are morphologically variable, and range from unicellular forms to hyphal forms which may be either coenocytic tubular forms or septate hyphal forms. Further, the structure and components of fungal cells differ greatly at different growth stages in different parts, and also influenced by nutritional and environmental factors. Hence, to explain the structure of a fungal cell, the ultra structural features of a typical fungal cell are shown in the Fig. 3.1.

Fig. 3.1. Ultra structure of a typical fungal cell.

The structure of a typical fungal cell include outer cell wall, cell membrane that surrounds cytoplasm, and various cell inclusions in cytoplasm like nucleus, mitochondria, endoplasmic reticulum, ribosomes, vacuoles, reserve food materials and lomasomes. Golgi bodies or dictyosomes are present only in a relatively few species. Further, all the structures shown in the typical cell may or may not be present in a single cell and distribution also changes in different species of fungi. Various components of a fungal cell are given below. Cell wall: Fungal cell wall is rigid and formed with lamellae made up of a number of fibrils. Carbohydrates are major chemical constituent of cell wall along with proteins, lipids and other substances. The chemical composition of cell wall is different in different groups of fungi. In most of the groups chitin is the major constituent while in Oomycetes cellulose is the major constituent. Even though there are differences in chemical composition of cell wall, there are some general characters also. Chemical constituents of the cell wall occur as fibrillar structures in an amorphous matrix. Proteins are common constituent in all types of cell wall. Chitin and cellulose occur as microfibrils while proteins, mannans and glucans occur in amorphous form. A mature cell wall is made up of 4 tubular structures (Fig. 3.2).

General Characters of Fungi

15

Fig. 3.2. Different layers in fungal cell wall. 1. Plasma lemma. 2. Lower layer with chitin microfibrils. 3. Protein layer. 4. Sub outer layer. 5. Outer layer.

Each layer in the cell wall is made up of a specific chemical constituent. The outer layer is made up of amorphous glucose. Its thickness is 80-90 nm. The layer just below the outer layer is made up of glycoproteins and occurs in amorphous glucan matrix. Its thickness is 40-50 nm. The third layer from outside is made up of protein and its thickness is 8-10 nm. The inner layer is formed by chitin in microfibrillar structures in amorphous protein matrix. Its thickness is 20 nm. In the cell wall of some vegetative cells and spores, melanins and lipids are also present. Chemical constituents of the cell wall occur as fibrillar structures in an amorphous matrix. Proteins are common constituent in all types of cell wall. Chitin and cellulose occur as microfibrils while proteins, mannans and glucans occur in amorphous form. Plasma membrane: The fungal cell protoplast is surrounded by a unit membrane called plasma membrane or plasmalemma. Fungal cell membranes are rich in sterols and phospholipids. Other chemical constituents include proteins and carbohydrates. Glucosamine and glucose residues account for most of the carbohydrates. Mannose is also present in cell membrane of some fungi. Electron microscopic studies showed that the structure of fungal cell membrane is fundamentally similar to cell membranes of all other cellular organisms. Characteristically it is a triple layer with two electron dense layers separated by an electron-lucent-zone with a total width of approximately 8 nm. The lipids in the cell membrane are amphoteric, with hydrophobic portions inside and hydrophilic portions outside. It is called lipid bilayer (Fig. 3.3). Other constituents usually are embedded in the lipid bilayer. Plasma membrane usually closely adheres to the cell wall, but in some species it may become undulating or invaginated. It is a semipermeable membrane and helps in maintaining osmotic and electrolyte balance between the cell constituents and outside environment. Nucleus: The fungal cells are eukaryotic and possess one or more membrane bound well organized nuclei. The nuclei in the vegetative cells of most fungi are extremely small, and their study with light microscope is very difficult. In fact, there

16

Biology and Biotechnology of Fungi and Microbes

was much controversy over the presence of nuclei in the fungal cells until the facility of electron microscope came into use for studying biological specimens.

Fig. 3.3. Schematic representation of lipid bilayer of cell membrane.

The fungal nucleus is surrounded by a double layered membrane and the outer layer is continuous with endoplasmic reticulum (ER). When there is more than one nucleus in the cells, all the nuclei within the cell are interconnected by ER. There are numerous pores in the nuclear envelope and these allow the continuity of nucleoplasm and cytoplasm. The pores in the nuclear envelope may be randomly scattered all over the membrane or may be confined to specific regions as in the zoospores of Blastocladiella. The pores in the envelope may be of different shapes in different fungi. They may be circular (yeasts), hexagonal (Thraustotheca clavata) or octagonal (Boletus rubinellus). The behaviour of the nuclear envelope in fungi during mitotic cell division is quite distinct from that in other eukaryotic organisms. During mitosis, the nuclear membrane do not disappear but persist, and after the division of nuclear material, it stretches to form a dumb bell shaped structure which eventually constrict in the middle to separate the two nuclei. Hence mitotic divisions are considered as atypical and described as karyokinesis. Within the nuclear envelope, in the nucleoplasm, the fungal nucleus possesses chromatin strands and nucleolus. The chromatin strands mainly contain DNA and become organized into chromosomes during cell division. The fungal chromosomes are very small, poorly stained, and usually do not form distinct metaphase plate. The nucleolus, mainly consisting of RNA, appears like a dense, deeply stained area in the nucleoplasm of some fungi, while in others, nucleolus is indistinct and nucleoplasm appears homogenous. The behaviour of nucleolus during cell division varies in different genera. It is generally retained by most fungi during mitosis. Like nuclear membrane, it is also stretched out and divides between the daughter nuclei. Presence of a nuclear cap that surrounds the nucleus, mainly at the upper part, appears to be a peculiar feature of fungal nucleus in the zoospores of Chytridiomycetes. Nuclear cap is an electron dense, membrane bound structure (Fig. 3.4), and its significance is not clear. It is present only in the posteriorly uniflagellate zoospores but not others

General Characters of Fungi

17

Fig. 3.4. Structure of a zoospore of Blastocladiella emersonii, distinct nuclear cap (NC) surrounds the nucleus (N).

Mitochondria: Mitochondria in fungal cells are highly pleomorphic and range from small, spherical structures of 1µm to very long structures of 30 µm in length, and variable in thickness. Growth stage and environmental factors influence the number and size of mitochondria. In general they are sufficiently large in many fungi and can be seen under light microscope. They vary in number in different parts and at different growth stages, but usually abundant in the subapical regions of vegetative hyphae. Like in other eukaryotes, the mitochondria of fungal cells are also surrounded by a two layered membrane. The inner layer folds inwards to form cristae, which may be rather poorly defined. The outer layer is continuous with endoplasmic reticulum. The mitochondrial stroma contains DNA and ribosomes. The mitochondrian ribosomes are smaller than those present in the cytoplasm. Functionally they are centres of respiration, as in other eukaryotes. Endoplasmic reticulum (ER): The fungal cells, especially hyphal tips, contain an extensive system of tubular network of membranes that traverse the cytoplasm from nucleus to cell membrane. It is called endoplasmic reticulum. It is believed to originate from nuclear membrane and forms a network with all membrane bound organelles in the cell. The ER is described as two types viz. smooth and rough. In rough type, the surface of ER is coated with ribosomes, and in smooth type, the surface of ER do not contain ribosomes. In fungi, ER is mostly rough type. Ribosomes: In fungal cells, ribosomes are present in cytoplasm and mitochondria. They contain high proportion of proteins and RNA. In cytoplasm, the ribosomes are present in groups and are described as polyribosomes or polysomes. The ribosomes in cytoplasm are found in association with endoplasmic reticulum.

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Biology and Biotechnology of Fungi and Microbes

Golgi bodies: These are also called dictyosomes. They are not present in cells of all fungal species. They are recognized only in a few fungal genera such as Pythium, Rhizopus, Neurospora and Aspergillus. The golgi bodies are poorly defined in fungi and are composed of 4 or 5 flattened cisternae in Pythium, and in Rhizopus sexualis they form a ring like structure. Dictyosomes play an important role in hyphal growth. Small secretory vesicles are formed and released from ER and they fuse with dictyosomes, which in turn gives rise to larger secretory vesicles. Thus, dictyosomes are in a state of dynamic equilibrium of receiving, processing and releasing secretory vesicles. The secretory vesicles increase in size by themselves or by fusing with other vesicles. The vesicles contain polysaccharides needed in cell wall synthesis and possibly the material for plasma membrane also. The diagrammatic representation of the role of dictyosomes in cell wall formation is shown in the Fig. 3.5.

Fig. 3.5. Role of dictyosomes in hyphal growth. 1. Endoplasmic reticulum. 2. Formation of vesicles from ER. 3. Cisternae of dictyosomes to which vesicles fuse and reemerge. 4. Growth and movement of vesicles towards cell membrane. 5. Cell membrane. 6. Fusion of vesicles with cell membrane. 7. Cell wall.

Lysosomes: These are formed from golgi bodies. They contain digestive enzymes. These are not found in all fungal species. They were discovered in the vegetative cells of only some fungi like Phycomyces and Botrytis cinerea. They are bound by a single membrane and thought to be budded-off from golgi cisternae. In higher eukaryotic organisms lysosomes are rich in digestive enzymes and help in digestion of food material taken in. Since the fungi show absorptive type of nutrition, the function of lysosomes in a fungal cell is not clear. Lomasomes and Plasmalemmasomes: These specialized structures are found only in certain fungal cells. They are formed between cell wall and cell membrane. The surface of plasma membrane, at which cytoplasmic secretory vesicles fuse, invaginate creating a space between plasma membrane and cell wall. In these spaces, lomasomes and plasmalemmasomes are formed. Greenwood (1970) suggested that plasmalemmasomes are produced when balance between wall plasticity and turgor pressure is disturbed in such a way that more plasmalemma is produced than is needed to line the cell wall. Heath and Greenwood (1970) defined a

General Characters of Fungi

19

plasmalemmasomes as the ‘various membrane configurations which are external to the plasmalemma, often in a pocket projecting into cytoplasm and less obviously embedded in wall material. They defined a lomasome as ‘a membrane vesicular material embedded in the wall external to the plasmalemma’. Hence, plasmalemmasomes and lomasomes are similar in origin and nature, and when project into cytoplasm in the plasma membrane loop they are called plasmalemmasomes and when they become embedded in developing cell wall they are called lomasomes. Plasmalemmasomes are extremely pleomorphic, and these structures found in Coprinus sp. and Pythium sp. are shown in Fig. 3.6.

Fig. 3.6. Plasma membranes as visualized in (A) Coprinus and (B) Pythium.

Chitosomes: They are recognized in the hyphae of Mucor rouxii. They are either globose or pleomorphic, measure 40–70 µm in size. They mainly contain enzyme chitinsynthetase. This enzyme helps in synthesis of chitin microfibrils. Vacuoles: Fungal cells contain a large number of vacuoles. Yeast cells contain a single large vacuole. Since the vacuoles are surrounded by a membrane, they are also considered as cell organelles. The vacuoles stabilize the density and structure of the fungal cell. They contain food particles, pigments, waste materials etc. Cell inclusions: In addition to membrane bound organelles, fungal cells also contain non-membrane bound structures or bodies which are called inclusions. Among the fungal cell inclusions, reserve food materials are important. Glycogen, a polysaccharide is the main reserve food material in most of the fungal cells. Lipids or oil globules are also found in many fungi (e.g. Colletotrichum). The reserve food materials are more abundant in mature cells and reproductive structures than in young vegetative cells. Apart from the reserve food materials, calcium oxalate

20

Biology and Biotechnology of Fungi and Microbes

crystals and carotenoid pigments are also reported as inclusion bodies in various fungi. Cytoplasmic microtubules: They occur throughout the cytoplasm. They are very long and fine network of hollow fibres of about 25 nm diameter, parallel to the long axis of the hyphae. They are considered as cytoeskeletal elements and help in cytoplasmic streaming and stabilization of the cell shape. Microbodies: Frederick et al (1975) first reported the presence of microbodies in fungal cells. These organelles are round to oval structures of 1.5 to 2.0 µm in diameter. They are surrounded by a single unit membrane and are electron rich. Woronin bodies, which are associated with septal pores of some ascomycotina fungi, are formed from the microbodies. Unicellular and multicellular organization Based on the vegetative structure the fungi are mainly of two types. These are hyphal forms and unicellular forms. Fungal body made of hyphae is described as mycelium. Majority of fungi are mycelial forms. These are also called moulds. Among the unicellular forms yeasts are important. Apart from these two major types of fungal structures, mycelium with rhizoids and dimorphic fungi are also recognized. Among the hyphal forms some have regular septal formation while others are tubular without any septa. The fungi that occur both in unicellular form and hyphal form in different conditions are described as dimorphic fungi. In some yeasts which grow by budding, the daughter buds may not separate from the mother cell, and give rise to another bud, and the process may repeat forming a chain of cells. The yeast cell with a chain of buds formed from it appears like a mycelium and it is described as pseudo mycelium. Different forms of vegetative forms of fungi are given in the Fig. 3.7.

Fig. 3.7. Different types of mycelia in fungi. A. Unicellular form (yeast). B. Pseudomycelium. in yeasts. C. Coenocytic tubular mycelium. D. Septate mycelium.

General Characters of Fungi

21

Structure of unicellular fungi: Yeast cells may appear in different forms like round, oval, elongated or other structures. The cells are up to 1.5 µm in width and 5.3 µm in length. They do not possess flagella or other motile structures. Yeast cells possess a clear cell wall and a membrane bound nucleus. Along with the nucleus, there may be one or two prominent vacuoles in the cytoplasm. Sometimes the nucleus may not be clearly seen, being covered by a prominent vacuole. The reserve food material occurs as lipids, proteins and carbohydrates in the cell (Fig. 3.8).

Fig. 3.8. Structure of a yeast cell. 1. Cell wall. 2. Cell membrane. 3. Nucleus. 4. Mitochondria. 5. Vacuole. 6. Vacuolar membrane. 7. Vacuolar particles. 8. Bud scar. 9. Food particles.

The growth of yeasts occurs either by cell division or formation of buds. In some yeast species reproducing by budding, the buds do not immediately separate from the mother cell, and may become active to form another bud on it. Because of such active budding a chain of cells are formed, and it appears like a hyphal fragment. Such hyphae are described as pseudohyphae. Apart from yeasts, unicellular species are present in zoosporic fungi also, e.g. Olpidium, Anisolpidium, Olpidiopsis etc. The genus Olpidium is considered as simplest form of fungus among Eumycota. It mainly occurs in the roots of host plants. It is a unicellular, intracellular, parasitic form. Fungal body with rhizoids: The fungal body in the members of Chytridiomycetes possesses rhizoids. In the fungal genera belonging to the order Chytridiales like Chytridium, Cladochytrium etc there is no true mycelium. The fungal body is unicellular, and from that cell a large number of rhizoids develop and enter into the substratum. When this rhizoidal system is developed extensively it is called rhizomycelium. In rhizoids or rhizomycelium nuclei are not usually present, but the nuclei may pass through them. If the rhizomycelium is formed from a single cell it is described as monocentric form. If a number of cells develop on rhizomycelium it is described as polycentric form. In the order Blastocladiales, along with the rhizomycelium, erect mycelium is also formed. In the order

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Monoblepharidales, the erect system is well developed and rhizoidal system is reduced to a basal cell, called holdfast. The vegetative structures in the members of Chytridiomycetes are given in Fig. 3.9.

Fig. 3.9. Mycelial types in Chytridiomycetes. A. Unicentric mycelium. B. Polycentric mycelium. C. Allomyces mycelium. D. Monoblepharis mycelium.

In the genus Rhizopus of Zygomycotina, rhizoids are formed in groups at regular intervals from the lower side of the coenocytic mycelium and enter into the substratum. The hyphal region between two groups of rhizoids is described as stolon. Usually erect sporangiophores are formed either singly or in groups on the hyphae at the place where the rhizoids are formed on the lower side (Fig. 3.10).

Fig. 3.10. Mycelium of Rhizopus. 1. Rhizoids. 2. Stolon. 3. Sporangiophore.

Mycelial forms: Highly developed mycelium with hyphae is found in the members of Oomycetes, Zygomycotina, Ascomycotina, Basidiomycotina and Deuteromycotina. In the fungi belonging to Oomycetes and Zygomycotina, even though the mycelium is well developed with highly branched hyphae, septa are not found in the hyphae, and they appear as tubular filaments. The nuclei occur in the cytoplasm which is continuous throughout the mycelium, and hence the hyphae are described as coenocytic.

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23

In the members of Ascomycotina and Basidiomycotina, fungal hyphae possess septa giving cellular appearance. But, there are pores in the septa of these fungi, and hence the protoplasm shows continuity throughout the mycelium. The pores in the septa of ascomycetes are simple while those in basidiomycetes are more complex and are called dolipore septa (Fig. 3.11)

Fig. 3.11. Septal pores. a. Simple pore in Ascomycotina. b. Dolipore septal pore in Basidiomycotina.

Hyphal structures: In free living fungi, mycelium is usually highly developed and appears as white cottony growth on the substrata. In some species, some of the hyphae grow very closely, wound round one another and form special structures called rhizomorphs. The fungal tissues formed by the close association of the hyphae are described as plectenchyma. Two types of fungal tissues are commonly seen. 1. The structures formed by loose association of the hyphae where hyphal structures may be clearly seen. They are called prosenchyma or elongated parenchyma. 2. The fungal hyphae are closely woven without intercellular spaces and hyphal nature cannot be seen. Such tissue is described as pseudoparenchyma, and is commonly seen in structures like sclerotium, stroma etc. In asexual or sexual reproductive structures, the fungal tissue associated is commonly described as stroma. The hard fungal structures formed during unfavourable conditions are called sclerotia. Different types of hyphal structures are shown in Fig. 3.12.

Fig. 3.12. Hyphal arrangement in fungal tissues. A. Prosenchyma. B. Pseudoparenchymatous. tissue, C. Sclerotium. D. Cross section of sclerotium. E. Perithecial stroma.

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In parasitic fungi, the mycelium is usually not extensive. The hyphae, growing in the intercellular spaces of host plants, send in haustoria into the host cells to draw nourishment. The haustoria may be globose, elongated or branched (Fig. 3.13).

Fig. 3.13. Haustoria in fungi. 1. Fungal hypha. 2. Haustorium. 3. Host cell.

Dimorphic fungi: These are fungi which grow in different forms depending upon environmental or physiological conditions and show dimorphism. When these fungi are growing under favourable conditions on their natural substrata they show mycelial growth. When growing in adverse habitats or on nutrient media under unfavourable conditions, they show yeast like unicellular growth. Such a condition is commonly seen in Mucor rouxii, a free living fungus, and in smuts causing plant diseases. The fungi causing respiratory infections such as Histoplasma capsulatum, Blastomyces dermatidis, Coccidioides immitis, etc. occur as yeast like cells in the lungs or when growing on media at 37°C. They show mycelial growth in soil, which is their natural habitat or when growing on media at room temperature. These are some important examples of dimorphic fungi. Cell wall composition Like plant cells and bacteria, fungal cells and hyphae also possess a rigid cell wall giving a definite shape. The chemical composition of the fungal cell wall shows much variation and the variations follow taxonomic lines. Hence, the cell wall composition is an important criterion in fungal taxonomy and in establishing the relationships between various groups. The major chemical constituents of cell wall are polysaccharides which constitute 80-90% cell wall by weight and remainder consists of proteins, lipids and inorganic ions. Among the polysaccharides that occur in fungal cell walls, chitin and cellulose are important. Chitin, a strong but flexible nitrogen containing polysaccharide is a polymer of N-acetyl glucosamine. The OH group of carbon atom 2 of D-glucose units is replaced by N-acetyl aminogroup (NHCOCH3) to form Nacetyl glucosamine, and chitin is formed by polymerization of these units by β- 1,4 linkages. Cellulose is a polymer of D-glucose units by beta-1,4 glycosidic linkages. Chitin is the major constituent in the cell wall of majority of fungi belonging to zygomycetes, ascomycetes, basidiomycetes and deuteromycetes, while cellulose is the major constituent of fungi belonging to oomycetes. The structural formulae for the repeating units that make up cellulose and chitin are shown in the Fig. 3.14.

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Fig. 3.14. The structural formulae for the repeating units making up cellulose and chitin.

The other polysaccharides present in the cell wall of fungi include glucan (a homopolymer of glucose), mannan (homopolymer of mannose), glycogen (a homopolymer of glucose), chitosan (a polymer of D-glucosamine), polyuronides and some heteropolysaccharides. An elaborate review of the cell wall composition of fungal groups was made by Bartnicki-Garcia (1970) and he identified eight categories in cell wall of fungi basing on chemical composition mainly of polysaccharides. The categories are given in the Table 3.1. Table 3.1. Chemical composition of cell wall in different fungal groups. Major chemicals in Fungal cell wall Class

Genus

Cellulose - glycogen

Acrasiomycetes

Dictyostelium

Cellulose - β glucans

Oomycetes

Phytophthora

Cellulose - chitin

Hyphochytridiomycetes

Rhizidiomyces

Chitin - Chitosan

Zygomycetes

Mucor, Rhizopus

Chitin - β glucans

Chytridiomycetes Ascomycetes Basidiomycetes Deuteromycetes

Allomyces Neurospora Schizophyllum, Polyporus Aspergillus

Mannan - β glucans

Ascomycetes

Saccharomyces, Candida

Chitin – mannan

Basdiomycetes

Sporobolomyces

Glucosamine – galactose polymers

Trichomycetes

Amoebidium

From the table it is clear that cellulose is present only in limited number of lower groups of fungi. Cellulose is the major constituent along with glycogen in the spore walls of Acrasiomycetes, along with β glucan in Oomycetes and along with chitin in Hyphochytridiomyctetes. Glycogen, the major reserve food material of fungi, is present in the cell walls of only Acrasiomycetes. The cell wall composition of Zygomycetes is distinct from others with chitin and chitosan and without any glucans. Chitin along with β glucan is the major chemical constituent in cell walls of most groups of fungi including Chytridiomyctes (among zoosporic fungi), Ascomycotina, Basidiomycotina and Deuteromycotina. The cell wall composition of yeasts appears to be different as they contain mannan as the major constituent (60 – 70%) along with glucans in ascomycetous yeasts, and along with chitin in basidio-

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mycetous yeasts. The cell wall of fungi in Trichomycetes appears to be unique in having neither chitin nor cellulose. Trichomycetes is a group of uncertain affinity. Basing on the chemical composition of cell wall, some mycologists suggested separation of Oomycetes from other fungi, because they do not have chitin in their cell wall and contain mainly cellulose. However, there appears to be no mutual exclusion of chitin and cellulose in fungal walls because both are reported in the cell walls of some fungi like Rhizidiomyces of Hypochytriodiomycetes and Ceratocystis of Ascomycotina. Further, Lin et al. (1976) reported chitin also in the cell wall of an Oomycete, Apodachlya. The cell walls of fungi are not chemically inert. Some of the proteins associated with cell wall possess melanin pigments and lipids may also be present. The presence of melanin gives coloration. Lipids are present in large quantities in the cell wall of fungal spores belonging to Erysiphaceae. Factors affecting chemical composition: The chemical composition of fungal cells is not constant but changes with age and cultural conditions. Substances present in the cell walls of young hyphae may be replaced completely as it ages, or in older hyphae because of deposition of new substances. Those present at younger stage may be completely masked. Another factor that influences the chemical composition of cell wall is the chemical composition of media or substrata on which the fungus is growing. Environmental factors such as pH and temperature also show profound influence on the chemical composition of fungal cell wall. NUTRITION OF FUNGI Fungi are heterotrophic and gather nutrients from other sources. Like other organisms the fungi also require carbon, hydrogen, oxygen, nitrogen, potassium, phosphorus and sulphur in relatively large quantities, and hence they are called macronutrients. Along with the major nutrients, they also require magnesium, zinc, copper, boron, magnesium, molybdenum, iron, calcium and others in small quantities for their nutrition. These are called minor nutrients or micronutrients. The macronutrients like carbon, hydrogen and oxygen (CHO) are taken in the form of carbohydrates. Fungi can utilize different types of carbohydrates for their nutrition. Glucose is the most easily utilized carbohydrate. Among the other organic compounds, aliphatic hydrocarbons are also utilized easily but aromatic hydrocarbons are utilized only under special conditions. Among the nitrogen compounds fungi mostly utilize organic compounds like amino acids and proteins. Among the inorganic compounds ammonia and nitrates are easily taken up and used. Fungi take up phosphorus in the form of phosphates, and sulphur in the form of sulphates. Some fungi require vitamins and other growth factors also for their growth. Most of the fungi can synthesize them, but a few take them from the external sources. Fungi take up nutrients by the method of absorption. As the fungal cells have rigid cell wall, nutrients cannot pass into the cell directly. Hence, fungi produce and release various digestive enzymes into surroundings, hydrolyse the nutrients and

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dissolved nutrients are absorbed. Depending on the nature of nutrition, fungi can be divided into 5 nutritional types. They are 1. Obligate saprophytes. 2. Facultative saprophytes. 3. Facultative parasites. 4. Obligate parasites and 5. Symbionts. 1. Obligate saprophytes: Fungi growing on dead remains of animals and plants or on organic matter in soil are called obligate saprophytes. Majority of fungi belongs to this group. They do not have parasitic ability or ability to enter into symbiotic association with other organisms. They are described as free living fungi. It is because of the action of these fungi dead remains of plants and animals disintegrate and nutrients bound in them are released into the soil, making them available for use by other organisms. 2. Facultative saprophytes: Fungi growing as parasites on host plants, and in the absence of hosts growing in the soil as saprophytes are described as facultative saprophytes. Important examples for these are smut fungi. The saprophytic life of those fungi is relatively short. 3. Facultative parasites: The fungi commonly living as saprophytes and occur as parasites on hosts under favourable conditions are called facultative parasites. For example, soil fungi like Rhizoctonia, Pythium, Sclerotinia, Macrophomina etc. mainly grow as saprophytes, and become parasitic when susceptible host plants are growing nearby. They grow very rapidly in the host tissue, and kill the plants in a very short time, and re-enter the soil. In the host they first kill the cells and then derive nutrients from the dead cells. Hence they are described as necrotrophs. 4. Obligate parasites: These can grow only on living cells, and cannot grow outside the host. Important examples are rust fungi, powdery mildews and downy mildews. These fungi send in haustoria into the host cells and draw nourishment from them. They cannot take food from dead cells. Hence, they are called biotrophs. 5. Symbiotic nutrition: Some fungi form symbiotic association with other organisms usually autotrophs, and derive nutrients from them. For example, they form symbiotic association with algae to form lichens, with roots of higher plants to form mycorrhiza. In lichens, the fungi derive food from algal partner, and give protection to it and also provide minerals, water to algae to carryout photosynthesis. In mycorrhiza, the fungal partner gets nourishment from the host plant, and helps the host nutrition by extending the area of absorption through its hyphae extending from the root surface all around the root and absorbing phosphorus and minerals from large area and supplying them to the host plants. GROWTH OF FUNGI Fungi are worldwide in distribution. They grow extensively in some areas. Generally, the temperature range at which fungi can grow is 10-35oC and optimum range is 20-30oC. Some fungi are thermophilic and can grow even at 50oC or more, while some are psychrophilic and can grow at 0oC or less. However, majority of fungi are mesophilic. pH is very important in growth of fungi. Generally fungi are slightly acidophilic and grow luxuriantly at pH 5-6. The fungi do not need light for its growth, but some light is essential in spore formation.

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Most of the fungi are aerobes and oxygen is most important for the growth of fungi. Some yeast species can grow at low oxygen conditions, and they utilize the nutrients through fermentation process. Emerson and Heald (1909) discovered that the genus Aqualinderella fermentans belonging to Oomycetes is an obligate anaerobe. This can be considered as a unique organism. Under favourable conditions, the fungi can grow continuously without any limit. The growth occurs mainly at the apical region. The branches arise from main hyphae only from some distance below the fungal tip. Dichotomous or trichotomous growth, characteristic of some filamentous organisms, is not present in fungi. However, in the genus Allomyces, the erect branches show dichotomous growth. This may be considered as an exception rather than general condition.

Fig. 3.15. Structure of the apical part of a hyphal tip.

The tip of the fungal hyphae usually does not possess any cell organelles. There are a large number of vesicles in cytoplasm. Small vesicles are released from endoplasmic reticulum and contain material for cell membrane. These vesicles coalesce with cisternae of golgi apparatus, accumulate more amount of cell membrane material. They are released from the cisternae, move towards cell membrane and grow in size as they move towards the cell membrane. These vesicles

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coalesce with cell membrane and release their contents, and the membrane grows. The vesicles also contain an enzyme called chitin synthetase, and this enzyme helps in synthesis of chitin which helps in expansion of cell wall (Fig. 3.15). REPRODUCTION IN FUNGI Fungi reproduce in a number of ways. The methods of reproduction in fungi can be broadly divided into three type’s viz. vegetative reproduction, asexual reproduction and sexual reproduction. A. Vegetative reproduction: The fungi reproduce vegetatively by different methods such as 1. Fragmentation. 2. Chlamydospore formation. 3. Cell division. 4. Budding. 1. Fragmentation: When long hyphae break into small bits of hyphae of variable length and separate from the parent mycelium they can initiate new colonies of the same fungus independently. This type of reproduction is called fragmentation. 2. Chlamydospore formation: Some fungi like Fusarium, Colletotrichum etc. form thick walled vegetative cells during unfavourable conditions for growth. When the vegetative cell transform into thick walled resting structures, they are called chlamydospores (Fig. 3.16). They germinate and form new colonies under favourable conditions.

Fig. 3.16. Chlamydospores in vegetative hypha. 1. Vegetative hypha. 2. Chlamydospore

3. Cell division: Unicellular yeasts like Schizosaccharomyces always divide into two daughter cells by cell division. It is the simplest process of reproduction in unicellular forms. 4. Budding: Budding is the most common type of reproduction in yeasts. Small buds develop virtually at any place, on the cell surface of yeasts, gradually increase in size by passing of protoplast from mother cell to the growing bud. When nucleus divides and one enters the bud, it is separated from the mother cell by wall formation (Fig. 3.17). Such cells formed by budding are called blastospores.

Fig. 3.17. Vegetative reproduction in yeast. A. Cell division. B. Development of buds.

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B. Asexual reproduction The formation of distinct spores either in a specialized sac like structure called sporangium, or on the specialized stalked called conidiophores is referred to as asexual reproduction. 1. Sporangiospores: These spores are formed within a sac like structure called sporangium by cleavage of protoplasm. The sporangiospores can be divided into two types based on the presence or absence of flagella. a). Zoospores – spores with flagella, and b). Spores without flagella. a). Zoospores: All fungi belonging to Mastigomycotina produce motile flagellate zoospores during the asexual reproduction. The zoospores are formed by the division of protoplast in a sporangium. They are always single celled without a rigid cell wall but possess a plasmamembrane. They are motile by means of flagella. Basing on the nature and arrangement of flagella, zoospores are 4 of types (Fig. 3.18). They are:

Fig. 3.18. Types of zoospores. A. Posteriorly arranged single whiplash type flagellum. B. Anteriorly arranged single tinsel type flagellum. C. Anteriorly arranged two flagella of which one is whiplash type and the other tinsel type. D. Laterally arranged two flagella of which one projecting downward is of whiplash type and the other upward projecting is of tinsel type. E. Two whip lash type flagella.

i.

Posteriorly uniflagellate zoospores. The flagella are of whiplash type. These types of zoospores are characteristic of Chytridiomycetes.

ii.

Anteriorly uniflagellate zoospores. Flagella are of tinsel type. These types of zoospores are characteristic of Hyphochytridiomycetes.

iii.

Anteriorly biflagellate zoospores. One flagellum is of whiplash type and the other of tinsel type. They are characteristic of Lagenidiales.

iv.

Laterally biflagellate zoospores. The flagellum directed upwards is tinsel type and that projecting downwards is whiplash type. Such types

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of biflagellate zoospores are found in the members of the class Oomycetes. In Plasmodiophoromycetes both anterior and posterior flagella are of whiplash type. b). Spores without flagella: They could be aplanospores, arthrospores or conidia. i). Aplanospores: These are formed in members of Zygomycetes, especially those belonging to the order Mucorales e.g. Mucor, Rhizopus etc. The sporangium is a sac like structure formed at the tip of a specialized hyphal branch called sporangiophore. Sometimes the tip of the sporangiophore projects into sporangium and it is called columella. The protoplast cleaves to form uninucleate single celled spores or sporangiospores which are released on disintegration of sporangial wall (Fig. 3.19). These spores do not possess flagella, and are aerially dispersed. These are also called sporangiospores.

Fig. 3.19. Asexual reproductive structures in Mucor. A. Young sporangium. B. Undehisced mature sporangium. C. Aplanospores on exposed columella after dehiscence.

ii). Arthrospores: When thin walled vegetative hyphae fragment into individual cells which disperse, they are called arthrospores. They are mainly formed in powdery mildew fungi. The mycelium in these fungi is external on the host surface. Some hyphae grow vertically, and on these hyphal branches the cells from the tip downwards gradually transform into round, oval or barrel shaped thin walled structures. At maturity they detach, disperse and produce new colonies (Fig. 3.20).

Fig. 3.20 A-F. Stages in the development of conidia in Erysiphe

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iii). Conidia: They are mainly formed in the members of Ascomycotina and Deuteromycotina. They are formed on special fertile hyphae called conidiophores, instead of in the sac like sporangia. The fertile hyphae bear conidiogenous cells and conidia are produced either singly or in chains from these conidiogenous cells, e.g. Alternaria, Curvularia, Helminthosporium, Drechslera etc. (Fig. 3.21).

Fig. 3.21. Conidia in some genera of Deuteromycotina fungi. A. Alternaria. B. Curvularia. C. Helminthosporium. D. Drechslera.

The conidiophores may occur as individual structures or arranged into special asexual reproductive structures like pycnidia, acervulus, synnemata or sporodochium. The asexual fruiting bodies are shown in the Fig. 3.22.

Fig. 3.22. Asexual fruiting bodies in fungi. A. Pycnidium. B. Acervulus. C. Sporodochium. D. Synnema.

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C. Sexual reproduction Except in case of Deuteromycotina, the fungi in other groups reproduce sexually by producing specialized spores characteristic of the group. Oospores are the sexual spores in zoosporic fungi (mainly in Oomycetes), zygospores in Zygomycotina, ascospores in Ascomytotina and basidiospores in Basidiomycotina. These sexual spores are formed when two compatible strains of the same fungus species come together. Plasmogamy, karyogamy and meiosis occur at specified points or stages in the sexual reproduction. In Mastigomycotina and Zygomycotina, plasmogamy is immediately followed by karyogamy. The diploid zygotic nucleus may undergo meiosis either in the developing sexual spores or at the time of germination. However, in some Oomycetes, it is reported that vegetative mycelium is diploid and meiosis occurs in gametangia. In Ascomycotina, plasmogamy is not immediately followed by karyogamy but a brief period of dikaryotic stage occurs, but karyogamy is immediately followed by meiosis. In Basidiomycotina, plasmogamy occurs between two compatible primary mycelia and it results in dikaryotization. The dikaryotic mycelium is the major vegetative phase of the fungus. When fruit body is formed, in the tip cell of generative hypha (basidium), the two nuclei fuse and meiosis occurs immediately to produce four haploid basidiospores. Thus, the three important steps of sexual reproduction viz. plasmogamy, karyogamy and meiosis occur in all fungi showing sexual reproduction, but at particular stages characteristic of each group. The initial stage of sexual reproduction in fungi occurs by different methods and they can be broadly recognized into five types. 1. Planogametic copulation: This method is mainly found in class Chytridiomycetes of Mastigomycotina fungi. These fungi produce gametangia by transformation of the vegetative cells and motile gametes are formed in the gametangia. The gametes possess a whip lash type flagellum in the posterior region. Hence these gametes are called motile gametes. When the motile gametes are released from gametangia into surrounding water, they actively swim for some time, and when another compatible gamete comes in contact, the two motile gametes completely fuse with one another. Hence the method is described as planogametic copulation. Plasmogamy is followed by karyogamy and zygote is formed. When the two copulating gametes are identical, it is called isoplanogamy. It is mainly seen in members of Chytridiales, e.g. Chytridium. When the two copulating planogametes are different in their size, it is called anisogamy. It is mainly seen in members of the order Blastocladiales, e.g. Allomyces. In the fungi belonging to the order Monoblepharidales, male gametes are motile while female gamete or egg is nonmotile. The egg is not usually released from the gametangium, and the motile gamete enters the oogonium and fertilizes the egg. Such a situation in which motile male gamete fertilizes nonmotile egg is described as heterogamy, e.g. Monoblepharis. The zygote formed as a result of plasmogamy and karyogamy develops into a resting spore by forming a thick wall. They germinate

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under favourable conditions. The methods of sexual reproduction found in Chytridiomycetes are shown diagrammatically in Fig. 3.23.

Fig. 3.23. Types of sexual reproduction in Chytridiomycetes. A. Isogamy. B. Anisogamy. C. Heterogamy.

2. Gametangial contact: This type of reproduction is seen in members of Oomycetes and some members of Ascomycotina (Fig. 3.24). In this method, gametangia do not release any gametes. When two compatible gametangia come in contact with one another, usually a fertilization tube develops and nuclei from one gametangium are transferred to the other. The gametangia may differ in their size and/or shape, and they can be recognized as male and female gametangia. The gametangium that receives the nuclei is female gametangium and which gives the nuclei is male gametangium. Usually the female gametangia are bigger and male gametangia are smaller in size. In Oomycetes, the female gametangia are called oogonia and male gametangia are called antheridia. In some genera of oomycetes, oogonia produce one or more eggs or oospheres for e.g. Saprolegnia. In some genera, the protoplasm of oogonium is clearly differentiated into central ooplasm and peripheral periplasm. The male nuclei are liberated into ooplasm, e.g. Phytophthora, Albugo etc. After karyogamy, the developing zygote, called oospore develops a thick wall and undergoes a resting period.

Fig. 3.24. Gametangial contact type of sexual reproduction. A. In Oomycetes. 1. Oogonium. 2. Antheridium. 3. Fertilization tube. B. In Ascomycetes. 1. Asogonium. 2. Trichogyne. 3. Antheridium. C. In Ascomycetes. 1. Asogonium. 2. Antheridium.

General Characters of Fungi

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In the members of Ascomycotina, some fungi show gametangial contact method. In these fungi, the female gametangium is called ascogonium and male gametangium is called antheridium. In some genera (Talaromyces), the ascogonium is vertical, cylindrical and relatively bigger in size, while the antheridium is relatively small and narrow, and it wounds round the ascogonium. At the places of contact, the cell walls dissolves and male nuclei are transferred to ascogonium. In some genera, ascogonia are globose at the base and at the upper part it possesses a long, narrow, curved structure called trichogyne. The antheridium is relatively small, and when the trichogyne touches the antheridium, the male nuclei are transferred into ascogonium. The two compatible nuclei pair together forming dikayons. Ascigerous hyphae then develop from the ascogonium and asci, ascospores and ascocarps. Karyogamy occurs in crozier cell and meiosis follows immediately. 3. Gametangial copulation: This method is mainly found in the members of Zygomycetes. The two gametangia are usually identical in size and shape. When two compatible hyphae grow side by side, zygophores develop and grow towards one another. The tip part of each zygophore swells and is separated from the lower part by formation of a septum. The globose tip part is the gametangium and the basal part is suspensor. When the two compatible gametangia come in contact with one another, at the point of contact, the wall of gametangia dissolves and the protoplasts of the two gametangia fuse completely forming zygospores (Fig. 3.25). By developing a thick wall around, it transforms into a resting structure, which germinates under favourable conditions.

Fig. 3.25. Sexual reproduction in Mucor. A. Zygophores (+, -). B. Gametangia with suspensors. C. Fusion of gametangia. D. Formation of zygospore.

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4. Spermatization: This method of reproduction is found in some members of Ascomycotina and rust fungi of Basidiomycotina (Fig. 3.26). In some genera of ascomycetes, ascogonium is formed with a basal globose structure and upper trichogyne, but, antheridium is not formed. In such genera usually the male gametangium is a specialized structure called spermagonium. It appears like pycnidium. In spermagonia large numbers of spermatia are formed. These are unicellular spores without flagella. They disperse through air, and when come in contact with trichogyne, the male nucleus is transferred to ascogonium. Since fertilization occurs through spermatia, it is called spermatization.

Fig. 3.26. Spermatization. A. In Ascomycetes. 1. Spermagonium. 2. Spermatia. 3. Ascogonium. 4. Trichogyne. B. In Rust fungi. 1. Spermagonium. 2. Receptive hyphae. 3. Spermatia.

In rust fungi of basidiomycetes, spermagonia are formed on the upper surface of the host leaf. Spermatia are formed from the spermatiophores that line the inner surface of spermagonium. A large number of erect hyphae called receptive hyphae develop at the ostiolar region. Usually there is no compatibility between the spermatia and receptive hyphae formed in the same spermagonium because rust fungi are heterothallic. When compatible spermatia come into contact with receptive hyphae, spermatial nucleus transfers to receptive hyphae, and the two nuclei form dikaryon. 5. Somatogamy: In some ascomycetes (Peziza, Morchella) and majority of basidiomycetes (Agaricus) no sex organs are formed, and when two compatible hyphae come together, nuclear migration occurs from one hyphae to the other and the two nuclei pair together starting dikaryotic stage. Different methods of sexual reproduction in fungi are shown diagramatically in Fig. 3.27.

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37

Fig. 3.27. Different methods of sexual reproduction in fungi.

■■■

Chapter - 4

International Code of Nomenclature for Algae, Fungi and Plants (Source: Wikipedia); Taxonomy of Fungi

The International Code of Nomenclature for algae, fungi, and plants (ICN) is the set of rules and recommendations dealing with the formal botanical names that are given to plants, fungi and a few other groups of organisms, all those ‘traditionally treated as plants’. It was previously called the International Code of Botanical Nomenclature (ICBN); the name was changed at the International Botanical Congress in Melbourne in July 2011 as part of the Melbourne Code which replaces the Vienna Code of 2005. As with earlier codes, it takes effect as soon as ratified by the congress (on Saturday 23 July 2011), but the documentation of the code in its final form takes some time to prepare after the congress. Preliminary wording of some of the articles with the most significant changes has been published in September 2011. The name of the Code is partly capitalized and partly not. The lower-case for ‘algae, fungi, and plants’ indicates that these terms are not formal names of clades, but indicate groups of organisms that were historically known by these names and traditionally studied by botanists, mycologists, and phycologists. This includes bluegreen algae (Cyanobacteria); fungi, including chytrids, oomycetes, and slime moulds; photosynthetic protists and taxonomicallv related non-photosynthetic groups. There are special provisions in the ICN for some of these groups, as there are for fossils. The ICN can only be changed by an International Botanical Congress (IBC), with the International Association for Plant Taxonomy providing the supporting infrastructure. Each new edition supersedes the earlier editions and is retroactive back to 1753, except where different starting dates are specified. For the naming of cultivated plants there is a separate code, the international Code of Nomenclature for Cultivated Plants, which gives rules and recommendations that supplement the ICN. PRINCIPLES 1.

Botanical nomenclature is independent of zoological, bacteriological, and viruses nomenclature.

2.

A botanical name is fixed to a taxon by a type. This is almost invariably dried plant material and is usually deposited and preserved in a

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herbarium, although it may also be an image or a preserved culture. Some type collections can be viewed online at the websites of the herbaria in question. 3.

A guiding principle in botanical nomenclature is priority, the first publication of a name for a taxon. The formal starting date for purposes of priority is 1 May 1753, the publication of Species Plantarum by Linnaeus. However, to avoid undesirable (destabilizing) effects of strict enforcement of priority, conservation of family, genus, and species names is possible.

4.

The intent of the Code is that each taxonomic group (‘taxon’, plural ‘taxa’) of plants has only one correct name that is accepted worldwide, provided that it has the same circumscription, position and rank. The value of a scientific name is that it is an identifier. It is not necessarily of descriptive value.

5.

Names of taxa are treated as Latin.

6.

The rules of nomenclature are retroactive, unless there is an explicit statement that this does not apply. HISTORY

The rules governing botanical nomenclature have a long and tumultuous history, dating back to dissatisfaction with rules that were established in 1843 to govern zoological nomenclature. The first set of international rules was the Lois de la nomenclature botanique (‘Laws of botanical nomenclature’) that was adopted as the ‘best guide to follow for botanical nomenclature’ at an ‘International Botanical Congress’ convened in Paris in 1867. Unlike modern codes, it was not enforced. It was organized as six sections with 68 articles in total. Multiple attempts to bring more ‘expedient’ or more equitable practice to botanical nomenclature resulted in several competing codes, which finally reached a compromise with the 1930 congress. In the meantime, the second edition of the international rules followed the Vienna congress in 1905. These rules were published as the Règles internationales de la Nomenclature botanique adoptées par le Congrès International de Botanique de Vienne 1905 (or in English, International rules of Botanical Nomenclature adopted by the International Botanical Conference of Vienna 1905). Informally they are referred to as the Vienna Rules (not to be confused with the Vienna Code of 2006). Some but not all subsequent meetings of the International Botanical Congress have produced revised versions of these Rules, later called the International Code of Botanical Nomenclature. Some important versions are listed below (Table 4.1). The Nomenclature Section held just before the 18th International Botanical Congress in Melbourne, Australia in July 2011 saw sweeping changes to the way scientists name new plants, algae, and fungi. 1.

For the first time in history the Code now permits electronic-only publication of names of new taxa; no longer will it be a requirement to deposit some paper copies in libraries.

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2.

The requirement for a Latin validating diagnosis or description was changed to allow either English or Latin for these essential components of the publication of a new name.

3.

‘One fungus one name’ and ‘one fossil, one name’ are important changes for fungi and for fossils; the concepts of anamorph and teleomorph (for fungi) as well as morphotaxa (for fossils) have been eliminated.

4.

As an experiment with ‘registration of names’, new fungal descriptions will require the use of an identifier from ‘a recognized repository’; there are two recognized repositories so far, Index Fungorum and MycoBank.

5.

The title of the Code was broadened to make explicit that it applies not only to plants, but also to algae and fungi.

Table 4.1. International Code of Botanical Nomenclature - Some important versions. Year of adoption

Informal name

1905

Vienna Rules

1935

Cambridge Rules

1952

Stockholm Code

1969

Seattle Code

1975

Leningrad Code

1981

Sydney Code

1987

Berlin Code

1993

Tokyo Code

1999

St Louis Code, The Black Code

2005

Vienna Code

2011

Melbourne Code

TAXONOMY OF FUNGI Nature is bountiful of living organisms and 1.5 million of fungi are estimated to be present. Diversity of fungi and because of its large number and biotechnological importance taxonomy of this group has been the central point since long time. Many attempts are made to identify and classify the fungi based on morphological, anatomical, ultrastructure, cell wall composition, physiological, biochemical and reproductive features. Earlier fungi were treated as achlorophyllous plants and most of the taxonomic aspects were on par with cryptogams. However the peculiar features like chitin cell wall, absorptive nutrition, thallus organization like holocarpic and eucarpic, biochemistry and reproduction in fungi paved the way for a separate kingdom Mycota or Mycetae. The initial classification of fungi proposed were mostly based on sexual reproduction and for those where sexual stages were not available, were tentatively classified based on asexual reproductive units / structures.

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In modern times attempts were made to classify and establish the phylogenetic relationship based on biochemical and molecular aspects. Earlier Classifications Naturalists of 15th and 16th centuries studied the fungi based on their habitat association and classified them into: 1.

Terrestrial fungi – Growing in soil

2.

Hypogean fungi – Growing under the ground

3.

Epiphytic fungi – Growing on plants.

A number of mycologists like Micheli, Persoon, Fries, Tulsane and others have proposed different classifications, based on morphology and reproduction. Pier Andres Saccardo (1866) classified fungi into Phycomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes treating them as classes. Gwynne–Vaughan and Barnes (1926) divided the fungi into Phycomycetes, Ascomycetes, Basidiomycetes and Deuteromycetes. Gaumann and Dodge (1928), Bessey (1950), Martin (1961) and Alexopoulous (1962) proposed varied systems of classification of fungi. Ainsworth (1973) treated fungi as a separate kingdom and recognized Myxomycota and Eumycota as two divisions. Eumycota included Mastigomycotina, Zygomycotina, Ascomycotina, Basidiomycotina and Deuteromycotina as sub-divisions which were further divided into different classes. Earlier in 1866, Ernst Hackel proposed Protista, a third kingdom which does not belong to neither Plant Kingdom nor Animal Kingdom. R.H. Whittaker (1969) proposed a five kingdom system and fungi were accepted as one of the kingdom along with Monera, Plantae, Animalia, and Protista. Today the system of classification includes six kingdoms; Plants, Animals, Protista, Fungi, Archaebacteria and Eubacteria. This is based on cell type, complex or simple, their ability to make their food and number of cells in their body. Von Arx (1981) divided fungi into four classes namely, Myxomycota, Oomycota, Chytridimycota and Eumycota. Hawksworth and others (1995) classified fungi into four divisions namely Chytridiomycota, Zygomycota, Ascomycota and Basidiomycota. Fungi belonging to Deuteromycotina were treated as asexual states of Ascomycotina and Basidimoycotina. Further the classes like Oomycetes and Hyphochytridiomycetes were clubbed with diatoms, brown algae and other algae and treated as Kingdom Chromista. Hawksworth et al (1995) in their 8th edition of dictionary of fungi placed fungi in Kingdom Fungi in four Phyla viz. Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota. Myxomycetes and Plasmodiophoromycetes were classified as fungi like organisms under the Kindgom Protozoa with four Phyla viz. Acrasiomycota, Dictyosteliomycota, Myxomycota and Plamodiophoromycota. Classification of fungi by Alexopoules and Mims (1979) is as follows (Fig. 4.1)

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Fig. 4.1. Alexopoulous and Mims (1979) classification of Fungi

Ainsworth’s (1973) classification is given below KINGDOM FUNGI Key to divisions of fungi Plasmodium or pseudoplasmodium present ...................................... Myxomycota I Plasmodium or pseudoplamodium absent, assimilative phase typically filamentous ... ................................................................................. Eumycota II I. Myxomycota Key to classes of Myxmycota 1. Assimilative phase free- living amoebae which unite as a pseudoplasmodium before reprouction .................................... Acrasiomycetes Assimilative phase a plasmodium ....................................................................2

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2. Plasmodium forming a net work (“net plasmodium”) ........ Hydromyxomycetes Plasmodium not forming a net work ................................................................3 3. Plasmodium saprobic, free-living ................................................. Myxomycetes Plasmodium parasitic within cells of the host plant .......................................................................Plasmodiophoromycetes II. Eumycota Key to subdivisitions of Eumycota 1. Motile cell is (zoospores) present, perfect state spores typically oospores . ........................................................................... Mastigomycotina III Motile cells absent .............................................................................................2 2. Perfect state absent ............................................................ Deuteromycotina VII Perfect state present ..........................................................................................3 3. Perfect state spores zygospores ............................................... Zygomycotina IV Zygospores absent ............................................................................................4 4. Perfect-state spores ascospores ................................................. Ascomycotina V Perfect-state spores basidiospores ..................................... Basidiomycotina VI III. Mastigomycotina Key to classes of Mastigomycotina 1. Zoospores posteriorly uniflageflate (flagella whiplash-type) .......................................................... Chytridiomycetes Zoospores not posteriorly uniflagellate ............................................................2 2. Zoospores anteriorly uniflagellate (flagella tinsel-type) ..................................................... Hyphochytridiomycetes Zoospores biflagellate (posterior flagellum whiplash-type; anterior tinsel type); cell wall cellulosic ................................... Oomycetes IV. Zygomycotina Key to classes of Zygomycotina Saprobic or, if parasitic or predacious, having mycelium immersed in host tissue ..................................................................................... Zygomycetes Associated with arthropods and attached to the cuticle or digestive tract by a holdfast and not immersed in the host tissue .............. Trichomycetes V. Ascomycotina Key to classes of Ascomycotina 1. Ascocarps and ascogenous hyphae absent; thallus mycelial or yeastlike ................................................................................. Hemiascomycetes Ascocarps and ascogenous hyphae present; thallus mycelial ............................2

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2. Asci bitunicate; ascocarp an ascostroma ............................ Loculoascomycetes Asci typically unitunicate; if bitunicate, ascocarp an apothecium ................... 3 3. Asci evanescent, scattered within the ascomatous ascocarp which is typically a cleistothecium; ascospores aseptate .......................Plectomycetes Asci regularly arranged within the ascocarps as a basal or peripheral layer ............................................................................................................4 4. Exoparasites of arthropods; thallus reduced; ascocarp a perithecium; asci inoperculate .......................................... Laboulbeniomycetes Not exoparasites of arthropods .........................................................................5 5. Ascocarp typically a peritheciurn which is usually ostiolate (if astomous.asci not evanescent); asci inoperculaic with an apical pore or slit ....................................................................... Pyrenomycetes Ascocarp an apothecium or a modified apothecium, frequently macrocarpic, epigean or hypogean; asci inoperculate or operculate ............................................................................... Discomycetes VI. Basidiomycotina Key to classes of Basidiomycotina 1. Basidiocarp lacking and replaced by teliospores or chlamydospores (encysted probasidia) grouped in sori or scattered within the host tissue; parasitic on vascular plants ....................................... Teliomycetes Basidiocarp usually well-developed; basidia typically organized as a hymenium; saprobic or rarely parasitic ......................................................2 2. Basidiocarp typically gymnocarpous or semiangiocarpous; basidia phragmobasidia (Phragmobasidiomycetidae) or holobasidia (Holobasidio-mycetidae); basidiospores ballistospores........... Hymenomycetes Basidiocarp typically angiocarpous; basidia holobasidia; basidiospores not ballistospores .......................................... Gasteromycetes VII. Deuteromycotina Key to classes of Deuteromycotina 1. Budding (yeast or yeast-like) cells with or without pseudomycelium; characteristic true mycelium lacking or not well developed . … Blastomycetes Mycelium well developed, assimilative budding cells absent ............. ……… 2 2. Mycelium sterile or bearing spores directly or on special branches (sporophores) which may be variously aggregated but not in pycnidia or acervuli .................................................................... Hyphomycetes Spores in pycnidia or acervuli ............................................... ……Coelomycetes

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Though monophyletic Fungi have been classified in four phyla: Chytridiomycota, Zygomycota, Ascomycota and Basidiomycota (Barr 1992, Hawksworth et al 1995, Alexopoulos et al 1996); however current classification under Kingdom Fungi includes Glomales, Zygomycota, Blastocladiales (Chytridiomycota), Microsporidia, Ascomycota and Basidiomycota besides the Choanoflagellates and Haptophytes as out groups (James et al 2000) Schüßler et al in 2001 created a fifth phylum, the Glomeromycota for accommodating Arbuscular Mycorrhizal (AM) fungi thus invalidating their inclusion in Zygomycota. Schüßler et al (200l) has included five phyla in the kingdom Fungi viz; Chytridiomycota, Zygomycota, Glomeromycota, Ascomycota and Basidiomycota. The major groups of fungi and fungus-like organisms (after Alexopoulos et al 1996) are presented below. KINGDOM – FUNGI Phylum - Chytridiomycota Phylum - Zygomycota Phylum - Glomeromycota Phylum - Ascomycota Phylum - Basidiomycota KINGDOM - STRAMEMOPILA Phylum - Oomycota Phylum - Hyphochytriomycota Phylum - Labyrinthulomycota KINGDOM – PROTISTA Phylum - Plasmodiophoromycota Phylum - Dictyosteliomycota Phylum - Acrasiomycota Phylum - Myxomycota The classification scheme proposed by Blackwell and Spatafora (2004) is shown below. Fungi

Straminopila

Slime Molds

Chytridiomycota Zygomycota Ascomycota Basidiomycota

Oomycota Hyphochytriomycota Labyrinthulales Thraustochytriales

Plasmodiophorales Myxomycota Dictyosteliomycota Acrasiomycota

According to the universal phylogenetic tree, fungi are placed in the domain Eukarya. Using molecular systematics, organisms are grouped together-based on the

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molecular phylogeny of their nuclear SSU rRNA genes and the type of mitochondrial cristae present. According to this tentative phylogeny, the true fungi, slime molds and water molds form three distinct monophyletic branches. Accordingly, based on molecular systematics true fungi are placed in domain Eukarya. In this classification kingdom Fungi and Straminipila as true fungi are also considered. However slime molds are not taken into consideration but for the benefit of students a brief account of each kingdom of Eukaryota are given (Table. 4.2). Table 4.2. Brief account of some characters of Eukaryota. Character

Animalia

Chromista

Fungi

Nutrition

Heterotrophic Autotrophic Heterotrophic Autotrophic (phagotrophic (photosynthetic (absorptive/ (photosyor osmotrophic) or absorptive) osmotrophic) nthetic)

Cell wall

Absent, cellulosic material not produced

Often cellulose, Chitin and chitin and β – glucans β- glucans absent

Plantae

Protozoa Heterotrophic (phagotrophic or autotrophic)

Cellulose and Absent when other polysac- trophic, varies charides when present

Mitochon- Flattened Tubular drial cristae (rarely tubular)

Flattened

Flattened

Tubular

Flagellar mastigonemes

Absent

Absent

Not tubular

Absent

Tubular

Taxonomy of fungi has become a complicated issue and many mycologists have proposed different classifications based on morphology, ultrastructure, reproductive structures, and cell wall composition and often in recent times molecular biology has made inroads in making major contributions in understanding the biology and phylogeny of fungi. Kirk et al (2008) in the 10th coln of Dictionary of Fungi have recognized six phyla in Kingdom Fungi viz. Chytridiomyceta, Glomeromycota, Microsporidia, Zygomycota, Ascomycota and Basidiomycota. In Kingdom Protista three phyla were recognized viz. Hyphochytriomycota, Labyrinthulomycota and Oomycota.

■■■

Chapter - 5

Zoosporic Fungi (Phylum – Chytridiomycota: Kingdom-Stramenopila)

The fungi that produce motile zoospores in their life cycle are included in the subdivision Mastigomycotina. These are also called zoosporic fungi (Plate 5.1). They mainly occur in water and moist soils.

Plate 5.1. Zoosporic fungi. 1. Zoosporangium containing zoospores of Saprolegnia sp. 2. Released resting spore of Chytrid. 3. Released zoosporesof Pythium sp. at the mouth of zoosporangium

The zoospores may be uniflagellate or biflagellate (Fig. 3.18). The flagella are of two types: 1. Whiplash type, 2. Tinsel type. In whiplash type flagella the axis is smooth while in tinsel type flagella have small hair like mastigonemes on their axis.

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Uniflagellate zoospores are of two types: 1. Zoospores with posteriorly arranged whip lash type flagellum, and 2. Zoospores with anteriorly situated tinsel type flagellum. Biflagellate zoospores are also of two types: 1. Anteriorly situated two flagella and 2. Laterally situated two flagella. Usually whiplash type flagellum is pointed downwards while tinsel type flagellum is pointed upwards. The type of sexual reproduction also varies very much in this group. In morphologically simpler forms it may occur between planoisogametes, planoanisogametes or between a planogamete and an immotile egg. In relatively higher forms it may be oogamous type which occurs between the male gametangium described as antheridium, and the female gametangium described as oognium.The oogonium may have single or many oospheres (eggs) (Fig. 5.1).

Fig. 5.1. Different types of sexual reproduction in zoosporic fungi. A. Planoisogamy. B. Planoanisogamy. C. Heterogamy. D. Ferilization between oogonium with many oospheres and antheridium. E. Fertilization between oogonium with single oosphere and antheridium.

PHYLUM-CHYTRIDIOMYCOTA Chytridiomycota is a phylum of lower fungi in the kingdom Fungi. The Chytrids except the recently established order Spizellomycetales, were placed in the class Phycomycetes under the subphylum Myxomycophyta of the kingdom Fungi. Earlier they were placed in Mastigomycotina as the class Chytridiomycetes. Those fungi were included in the order Chytridiales. The term ‘chytrid’ refers only to members of Chytridiomycota. The chytrids were also included in Protoctista and are now classified in fungi. Chytrids the early diverging fungal lineages are saprobic, with degrading refractory materials such as chitin and keratin and sometimes are parasites on algae and sea faunae. The chitin cell walls, a posterior whiplash

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flagellum, absorptive nutrition, use of glycogen as an energy storage compound, and synthesis of lysine by the α-amino adipic acid (AAA) pathway of chytrids indicate that they are true fungi. There are over 750 chytrid species distributed among 7 orders. There has been a significant increase in the research of chytrids since the discovery of Batrachochytrium dendrobatidis, the causal agent of chytridiomycosis. Classification Phylum

: Chytridiomycota

Class

: Chytridiomycetes

Orders

: Chytridiales, Cladochytriales, Rhizophydiales, Polychytriales, Spizellomycetales, Rhizophlyctidales, Lobulomycetales, Gromochytriales, Mesochytriales, Monoblepharidomycetes

Life cycle The thallus is either holocarpic or eucarpic. In some rhizomycelium is alos present. Chytridiomycota reproduce by zoospores. In many members of Chytridiomycetes, sexual reproduction is not known. Asexual reproduction is through the zoospores derived through mitosis. Sexual reproduction of Chytridomycetes is by variety of methods. As a result of sexual reproduction zygote is formed that survives under adverse conditions. Sexual reproduction is by the fusion of isogametes (gametes of the same size and shape). This group includes plant pathogens (Synchytrium), some algal parasites which reproduce by oogamy: a motile male gamete that attaches itself to a non-motile structure containing the female gamete. In other fungi, two thalli produce tubes that fuse and allow the gametes to unite. In some rhizoids of compatible strains meet and fuse. Both nuclei migrate out of the zoosporangium and enter into the conjoined rhizoids where they fuse. The resulting zygote germinates and turn into a resting spore. Sexual reproduction is common and well known among members of the Monblephar-idomycetes. These chytrids reproduce through oogamy: the male is motile and the female is stationary. In Monoblephris oogonia, and antheridia are formed which give rise to gametes. On fertilization the zygote either becomes an encysted or motile oospore, which becomes a resting spore that later germinate and give rise to new zoosporangia. The germinated resting spore release zoospores that fall on suitable substrate for growth using chemotaxis or phototaxis. Some species encyst and germinate directly upon the substrate, others encyst and germinate a short distance away. Enzymes released from the zoospore help to break down the substrate and utilize it to produce a new thallus. Thalli are coenocytic and usually form no true mycelium (having rhizoids instead). Some chytrids are holocarpic, in which they only produce a zoosporangium and zoospores. Others are eucarpic, meaning they produce rhizoids, in addition to the zoosporangium and zoospores. Some chytrids are monocentric in which, single zoospore gives rise to a single zoosporangium. Others are polycentric, in which one zoospore gives rise to many zoosporangia connected by a rhizomycelium. Rhizoids do not have nuclei while a rhizomycelium has got nuclei. Growth continues until a new batch of zoospores is ready for release. Chytrids have a diverse zoospore release

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mechanisms that helps to group into the broad categories of operculate or inoperculate. Operculate discharge involves the complete or incomplete detachment of a lid-like structure, called an operculum, allowing the zoospores out of the sporangium. Inoperculate chytrids release their zoospores through pores, slits, or papillae. Fungi belonging to Chytridiomycota are traditionally delineated and classified based on development, morphology, substrate, and method of zoospore discharge. Single spore isolates display a great amount of variation in many of these features; thus, these features cannot be used as reliable criteria to identify a species. Currently, taxonomy in Chytridiomycota is based on molecular data, zoospore ultrastructure and some aspects of thallus morphology and development. CLASS - CHYTRIDIOMYCETES The one characteristic feature which distinguishes the members of Chytridiomycetes from other fungi is the production of motile cells (zoospores or gemetes) each with a single posterior whiplash flagellum. Other important features are 1. Coenocytic thallus - which may be unicellular or simple hypha or a well developed mycelium. 2. Conversion of zygote into a resting spore or resting sporangium except in Blastocladiales where it develops into a diploid mycelium. 3. Chitin is the chief constituent of the cell wall in Chytridiomycetes. Cellulose is also reported but usually it is indistinguishable. The members of this class are usually aquatic, but some however, are found in moist soil. Most of them occur on organic matter growing saprophytically, and some are parasitic on aquatic algae and plants growing in moist soils. The species of Olpidium and Physoderma are parasitic on economically important crop plants. Vegetative mycelium ranges from unicellular holocarpic forms (Olpidium) to eucarpic forms with rhizoids, and rhizomycelium (which comprises a system of hyphae like filaments which usually do not contain nuclei) to forms with true mycelium which is coenocytic. The mycelium of higher Chytridiomycetes may form pseudosepta. These are septum like partitions or plugs of chemical composition different from that of hyphal walls, which are deposited at intervals in the hyphae. Asexual reproductive structure is a sporangium, which produces uninucleate characteristic zoospores. Sexual reproduction may be a). Isogamy (Chytridiales), b). Anisogamy (Blastocladiales) or c). Heterogamy (Monoblepharidales). Chytridiomycetes is divided into orders namely Chytridiales, Spizellomycetales, Neocallimastigales, Blastocladiales and Monoblepharidales based on thallus construction, Zoospore ultrastructure, mode of plasmogamy and oospore / zygote formation. Order

: Chytridiales

Family

: Chytridiaceae (genera - Rhizophydium, Polyphagus)

Family

: Synchytriaceae (genus – Synchytrium)

Order

: Spizellomycetales

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Family

: Olpidiaceae (genus – Olpidium)

Family

: Urophlyctidiaceae (genus – Urophlyctis)

Order

: Neocallimastigales

Family

: Neocallimastigaceae (genus – Neocallimasti)

Order

: Blastocladiales

Family

: Coelomycetaceae. (genus – Coelomomyces)

Family

: Blastocladiaceae (genus – Blastocladiella, Blastocladia, Allomyces)

Order – Monoblepharidales Family

: Monoblepharidaceae (genus – Monoblepharis)

Family

: Gonapodyaceae (genus – Monoblepharella, Gonapodya) Order – CHYTRIDIALES

Members of this group are mostly aquatic, growing saprophytically on plant and animal remains in water, or parasitically in the cells of algae. Some species are terrestrial growing saprophytically on various plant and animal substrata in soil. Some species are parasitic on roots of higher plants. Synchytrium endobioticum is the economically most important pathogenic fungus, and it causes ‘black wart’ disease of potato’. The thallus is highly variable. In morphologically simpler forms such as Olpidium and Synchytrium it is holocarpic i.e. entire cytoplasmic contents or whole structure is transformed into a reproductive structure producing either zoospores or gametes. In many chytrids, the thallus is clearly defined into a vegetative part concerned with the nutrition of the organism and a reproductive part concerned with the formation of asexual or sexual structures. In such organisms the thallus is described as eucarpic. In eucarpic forms, thallus may be entirely within the host cell, and such a condition is described as endobiotic. In some forms, only rhizoidal system enters the host, and reproductive part remains outside the host. Such a situation is described as epibiotic. The zoospore on germination gives rise to a rhizoidal system which produces a reproductive structure. If there is a single sporangium produced per thallus, it is described as monocentric. If there is more than one sporangium per thallus, it is described as polycentric. The thallus variation in Chytridiales is shown in Fig. 5.2. Asexual reproduction In chytrids it is by means of zoospores. The zoosporangium is usually a spherical or pear shaped sac bearing one or more discharge tubes or exit papillae. The method by which zoospore release is achieved is used in classification and hence is important. The sporangia may be inoperculate, exooperculate or endooperculate.

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Fig. 5.2. Thallus variation in chytrids.

Inoperculate type: No operculum is formed. The discharge tube or exit papilla penetrates to the exterior of the host cell and its tip becomes gelatinous and dissolves releasing zoospores, e.g. Olpidium, Cladochytrium. Exooperulate type: The tip of the discharge tube breaks open and a circular dome shaped structure called operculum is formed. At the time of spore discharge, operculum is thrown out as lid, which remains attached to the exit tube at a point, e.g. Chytridium, Nowakowskiella. Enodooperculate type: The apex of the papilla dissolves; the pore is plugged with a clear gelatinous substance. Beneath this plug, cytoplasm deposits a convex solid operculum. Since it is formed within the papilla, it is called endooperculum. Although the size of the zoospore is roughly constant for a given species, the size of the zoosporangium varies and the number of zoospores produced per sporangium depends on its size. The zoospore is a spherical body with a long trailing whiplash flagellum. The period of zoospore movement varies from few minutes to several hours. Then zoospores come to rest and encyst. The flagellum may be withdrawn or it may be cast off. Germination is monopolar. In holocarpic forms entire protoplast leaving the cyst, enters the host. In monocentric forms, rhizoids develop from the cyst and enter the host and cyst develops into sporangium. In

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polycentric forms, germination results in rhizomycelium on which a swollen cell arises giving rise to further branches of rhizomycelium. Sexual reproduction It is isogamous type. Isoplanogametes unite to form zygote. It transforms into a thick walled resting sporangium. However, in many forms sexual reproduction has not been observed. The order is divided into 9 families: Order – BLASTOCLADIALES The blastocladiales are chiefly water moulds or soil inhabitants characterized by the production of thick walled, resistant resting sporangia, usually with pitted walls. The order is divided into 3 families 1. Coelomomycetaceae, 2. Catenariaceae, and 3. Blastocladiaceae. The members of Coelomomycetaceae are obligate parasites in the body cavities of mosquito larvae. The hyphae are naked i.e. without cell walls, and somewhat resemble the plasmodium of myxomycetes. The entire mycelium is converted into thick walled resting sporangia, which germinate, releasing a mass of zoospores. Sexual reproduction is completed in another host, Cyclops venalis a copepod. The best example for the family is Coelomomyces pleophoriae. In Catenariaceae, thallus is tubular and aseptate. It bears numerous rhizoids. Asexual reproduction is by formation of zoospores, and sexual reproduction is by copulation between isogamous planogametes. The members of the family Blastocladiaceae are morphologically well developed, and show well defined alternation of generations. The genus Allomyces, first discovered in India by E.J. Butler in 1911, is the best studied among the family. Vegetative structures The mycelium of Allomyces is well developed and consists of 1. Well branched rhizoidal system by means of which the fungus attaches itself to the substratum. 2. A stout or slender trunk like body. 3. Numerous side branches, which are dichotomously branched. The hyphae are non-septate, but pseudosepta in the form of thickened rims are present in some species. 4. The reproductive structures are formed on branches. In Allomyces macrogynus and A. arbuscula, the life cycle consists of isomorphic alternation of generation between diploid sporothallus and haploid gametothallus. The two types of thalli are indistinguishable morphologically until they form reproductive structures. Diploid sporothallus forms two types of sporangia. 1. Thin walled, elongated, colourless zoosporangia, and hence called mitosporangia, and 2. Thick walled, oval, pitted, resting sporangia, which contain melanin pigments, appear reddish brown. In the zoosporangia (also called mitosporangia), the protoplast divides mitotically during germination, giving rise to characteristic zoospores, which after a

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period of motility round up and germinate to give rise to sporothalli, thus repeating the diploid generation. The resting sporangia undergo a period of rest, and before germination meiosis take place and they produce smaller zoospores called meiospores. They germinate to produce gametothalli. The life cycle of Allomyces is shown in Fig. 5.3.

Fig. 5.3. Life cycle of Allomyces macrogynus.

Haploid gametothalli are monoecious and produce colourless female gametangia, and orange coloured male gametangia, usually on the same branch. The male gametangia are smaller than the female and may be borne on the latter (A. macrogynous) i.e. epigynous or below them (A. arbuscula) i.e. hypogynous. Both types of gametangia release motile gametes into the surrounding waters. The gametes are of general structure as zoospores.

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Attracted by the sexual hormone sirenin produced by female gametes, the male gametes copulate with the latter in pairs and karyogamy follows plasmogamy. The motile zygote comes to rest, loses it flagella, rounds up and soon germinates. First a germ tube is produced which develops into rhizoids. Then the main body of the zygote enlarges and gives rise to hyphae, which soon dichotomously branch and develop into diploid sporothallus. The presence of such isomorphic alternation of generations in Allomyces is unique in the zoosporic fungi. Order – MONOBLEPHARIDALES These are highly developed among Chytridiomycetes, and resemble closely the members of Blastocladiales. This group is characterised by oogamous type of sexual reproduction which is unique in that nonmotile oosphere is fertilized by motile male gametes.

Fig. 5.4. Life cycle of Monoblepharis polymorpha.

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This is a small order with three well recognized genera distributed in two families 1. Monoblepharidaceae, with a single genus Monoblepharis, and 2. Gonapodyaceae, with two genera, viz. Monoblepharella and Gonapodya. The lifecycle of Monoblepharis is shown in Fig. 5.4. Thallus is well developed and mycelial type. Hyphae are highly vacuolated and well developed with many branches. The basal cell representing rhizoidal system is not well defined. Asexual reproduction is by forming of zoosporangia. The sporangia are elongated and borne singly at the hyphal tips, and are not well distinguished from vegetative hyphae. The sporangia are separated by a septum. The zoospores are released through the tip of the sporangium. The same thallus produces gametangia, when subjected to higher temperatures. The gametangia are easily distinguishable into male and female. Male gametangia are known as antheridia. They are narrow, elongated and borne on the rounded, larger oogonium, the female gametangium. The protoplast of the oogonium becomes rounded and forms a uninucleate oosphere. An oosphere or egg is defined as ‘a single large spherical, naked nonciliate and practically nonmotile gamete’. In Monoblepharis it is uninucleate. After the antherozoids (male gametes) are released from the antheridia, they swim or creep over to the oogonia. A single sperm enters the oogonium through a papilla present in the oogonial wall, penetrates the oosphere and fuses with it (plasmogamy). The fertilized egg soon emerges from the oogonium and while still attached to the gametangium oogonial wall by a hyaline collar, secretes a thick wall around itself and develops into an oospore. Karyogamy is delayed until the oospore wall is partially formed. Oospore germinates under favourable conditions to produce vegetative thallus. Meiosis occurs during germination of the oospores. KINGDOM – STRAMENOPILA (STRAMINIPILIA) This includes diatoms, chrysophytes, brown algae and protozoa. This also includes Phyla - Hyphochytridiomycota, Labyrinthulomycota and Oomycota. Term stramenopiles has been introduced by D.J. Peterson in 1989 and criteria used are: 1.

Presence of tinsel type of flagellum with two rows of tubular tripartite hairs.

2.

Zoospores get a pull through water.

However in some fungi filamentous thallus is also present indicating their convergent evolution. Phylum – Hyphochytridiomycota This group of fungi is distributed in soil, fresh water and also in saline water. These fungi live as saprophytes or parasites on algae and also on some fungi as hyperparasites. These fungi evolutionarily are related to Oomycota. The cell wall is made up of chitin and cellulose. Zoospores bear one anteriorly inserted tinsel flagellum and sexual reproduction is known very poorly. It has got 2 families, 6 genera and 23 species eg: Hyphochytridium catenoides.

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Brief life cycle is:

Phylum – Labyrinthulomycota These fungi are commonly known as marine slime molds. These fungi live as parasites or saprophytes on molluscs, aquatic plants, organic detritus etc., Zostera marina, sea grass is parasitized by Labyrinthula zostereae. Thallus is covered with thin, golgi derived scales and ectoplasmic net is produced by sagenoges. Asexual reproduction is by biflagellate zoospores having long tinsel and shorter whiplash flagella. This phylum has two familes. 1.

Labyrinthulaceae

2.

Thraustochytriaceae.

The zoospores produced by Labyrinthula are biflagellate and contain a dark eye spot but lack surface scales. Thraustochytrium thalli are well composed of layered scales formed by golgi organelle. Zoospores are surrounded by a single layer of scales without an eye spot. Phylum – Oomycota These organisms belong to fungi and obtain nutrients through absorption. Many fungi are filamentous, mostly coenocytic, septa are formed in older hyphae rarely and nuclear status is diploid. Asexual reproduction is by biflagellate zoospores having one whiplash flagellum directed posteriorly and other being tinsel type with mastigonemes directed anteriorly. Zoospores get released one by one to the orfice (Saprolegnia) or in groups (Achlya) and in fungi like Pythium zoospores get released into vesicle. Zoosporangial proliferation is found in Saprolegnia and also rarely in Achlya, Protoachlya and Pythium. Diplanetism (one motile stage interspaced by encystment followed by another motile stage) is found in Saprolegnia. Cell wall is made up of  glucans and cellulose. Mitochondria may be with tubular cristae. Members of Oomycota undergo sexual reproduction by Oogamous / Gametangial contact and result in diploid oospore formation. These oospores may be single as in Phytophthora or many in Achlya, Saprolegnia etc. The mycelium is most commonly coenocytic and contains diploid nuclei. Oospores may be centric, eccentric and subcentric types. Oospores on germination give rise to zoosporangia which in turn release zoospores. Molecular analysis of Oomycota indicates their relationship with heterokont algae / chromista. However most of the mycologists still study oomycota alongwith true fungi because of their similarities in nutrition, parasitic nature, absence of chlorophyll and other characters. Oomycetes has two subclasses. Class

: Oomycetes

Sub class

: Saprolegniomycetidae

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Orders

: Rhipidiales, Leptomitales, Saprolegniales, Olpidiopsidales, Lagenismales, Myzocytiopsidales, Salilagenidales

Sub class

: Peronosporomycetidae

Orders

: Pythiales, Peronosporales, Sclerosporales CLASS – HYPHOCHYTRIDIOMYCETES

The Hyphochytridiomycetes are aquatic, fresh water or marine chytrid like fungi, whose motile cells are anteriorly uniflagellate, possessing a flagellum of tinsel type. They are parasitic on algae and fungi or saprobic on plant and insect debris in water. Thallus closely resembles that of Chytriales. It may be holocarpic or eucarpic. Holocarpic fungi are endobiotic. Eucarpic forms may be monocentric or polycentric with rhizoidal system.

Fig. 5.5. Life cycle of Rhizidiomyces apophysatus.

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The zoosporangia are inoperculate, and zoospores are released by dissolution of exit tubes. Zoospores are characteristic with anterior tinsel flagellum. Sexual reproduction is known only in a few species and incompletely known. The life cycle of Rhizidiomyces is shown in Fig. 5.5. This is a small class comprising a single order Hyphochytriales with three families. Family- Anisolpidiaceae: Thallus holocarpic, endobiotic, zoospores formed outside or inside sporangium, e.g. Anisolpidium, Canteriomyces. Family– Rhizidiomycetaceae: Thallus eucarpic, monocentric, epibiotic, zoospores formed outside or inside sporangium, e.g. Rhizidiomyces, Rhizidiomycopsis.

Family– Hyphochytriaceae: Thallus polycentric, zoosores formed inside sporangium or at the mouth of a discharge tube. Only one genus Hyphochytrium is recognized. PHYLUM – OOMYCOTA Class – OOMYCETES Fungi producing biflagellate zoospores are included in this single class. The zoospores possess two flagella either anteriorly or laterally. Of the two flagella one is tinsel type directed forward and the other whiplash type directed backward. Even though a few members are unicellular, majority members are mycelial forms with tubular hyphae without septa. The hyphae contain a large number of nuclei and are described as coenocytic. The cell wall is mainly made up of cellulose, but not chitin. Hence, these fungi are considered as distinct from other fungi. Sexual reproduction is by gametangial contact between oogonium and antheridium. The zygote develops into a thick walled structure called oospore. The oospores are resting structures and under favourable conditions they germinate to form vegetative mycelium. It was discovered that meiosis does not occur at the time of oospore germination and hence, the vegetative mycelium is diploid. Meiosis occurs in gametangia. This character is also distinctly different from other fungi, in which vegetative mycelium is essentially haploid. Oomycetes is subdivided into two sub-classes viz. Saprolegniomycetidae and Peronosporomycetidae. Some important orders are described below. Subclass - Peronosporomycetidae Order- Legenidiales This is a small group of aquatic fungi parasitic on algae, small animals and other forms of aquatic or semiaquatic life. Fungal mycelium may be unicellular or multicellular, and they are endobiotic and holocarpic forms. The order comprises three families viz. 1. Olpidiopsidaceae 2. Sirolpidiaceae and 3. Lagenidiaceae. In Olpidiopsidaceae (e.g. Olpidiopsis) thallus is unicellular and holocarpic. In other two families thallus is filamentous, though not well developed. There is no extensive mycelium but only a small filament, which may or may not be branched

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growing within a cell of an alga or other hosts. After the thallus has reached a certain stage of maturity, septa are formed which divide the tubular thallus into a few cells. Each of these cells now changes into a reproductive organ- a sporangium or a gametangium. The protoplast of a sporangium divides itself into a number of zoospores, probably as many as there are nuclei in the sporangium. The zoospores escape through one or more exit tubes formed in the sporangial wall and penetrating through the host cell wall. In the genus Lagenidium, a thin bubble like vesicle develops at the tip of exit tube. The sporangial protoplast moves through the exit tube into the vesicle, and the zoospores become differentiated there in. The zoospores are liberated into the surrounding water when the vesicle bursts. The zoospores swim around in the water for some time and eventually they come to rest on a susceptible host and penetrate it. Sexual reproduction is by gametangial copulation. The mating gametangia may be formed from adjacent cells of same thallus or two thalli lying side by side. During copulation, protoplast of one gametangium passes into another through a pore or tube. Only one nucleus from each protoplast is functional and the rest disintegrate. The zygote is eventually transformed into a resting spore by the formation of a thick wall. Meiosis presumably takes place when oospore germinates. Sub class - Saprolegniomycetidae Order- Saprolegniales These are generally called water moulds. Most of them are free living saprophytic forms, but a few are parasitic on fish and fish eggs. Asexual reproduction is by formation of biflagellate zoospores, and sexual reproduction by gametangial contact between oogonium and antheridium. The oogonium usually consists of 3 or more eggs and no periplasm is observed. The order includes 5 families. Family - Saprolegniaceae: This is typical of the order consisting of more advanced mycelial forms with filamentous thallus. In this family two genera viz. Saprolegnia and Aphanomyces consist of some species which cause diseases that are of considerable economic importance. Some species of Saprolegnia such as S. ferax and S. parasitica have been implicated in disease of fish and their eggs. Species of Aphanomyces are the causal agents of root rots of higher plants such as sugar beet, peas, and other crops. The vegetative thallus is eucarpic and filamentous. Zoosporangia are delimited by basal septa. The gametangia are always morphologically distinct. In each oogonium more than oosphere or egg is present, e.g. Saprolegnia, Leptolegnia, Achlya. Order – Leptomitales This is a small order of about 7 genera and 20 species, which are saprobic on other aquatic fungi. Leptomitus is common in sewage waters. In these fungi cytoplasm in the oogonium is clearly differentiated into central ooplasm and surrounding periplasm.

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In this order, two families viz. 1. Leptomitaceae and 2. Rhipidiaceae, are recognized. In general, the members of this group resemble the Saprolegniales. The somatic hyphae are truly aseptate, but constricted at regular intervals. The constrictions are sometimes plugged with granules of cellulin, giving the appearance of septa, and this distinguishes these fungi from Saprolegniales. Further, the Saprolegniales are unable to utilize sulphates, the Leptomitales reduce suphates and can utilize sulphur in their nutrition. Asexual reproduction is by means of terminal zoosporangia from which biflagellate zoospores are released. The sporangia of some species are elongated and resemble vegetative hyphae, while in many species they are pyriform. The species may be monoplanetic or diplanetic. Sexual reproduction is by gametangial contact. Each oogonium contains a single oosphere, and periplasm is present around the oosphere. Order – Pythiales This order has a single family Pythiaceae and 11 genera including Lagenidium, Pythium, Myzocytium, Perenophytophora and Phytopthora. These are aquatic amphibious or terrestrial, many of which are inportant parasites. The sporangiophores are indistinguishable from hyphae and of indeterminate growth. Family - Pythiaceae: The sporangiophores are similar to somatic hyphae (Pythium) or if different, they are of indeterminate growth (Phytophthora). The sporangiophores continue to grow indefinitely producing sporangia as they grow, resulting in sporangia of different ages from immature to mature on the same sporangiophore. The species are facultative parasites. The details of the genera Pythium and Phytopthora are given. Order – Peronosporales The members of this family are all obligate parasites of higher plants causing the diseases termed downy mildews. Sporangiophores are strikingly different from somatic hyphae and are of determinate growth. Sporangia are produced singly or in clusters at the tips of mature sporangiophores. No sporangia are produced until the sporangiophore completes its development and matures. Then a single crop of sporangia are produced and all the sporangia are of approximately same age. After the sporangia fall off, the sporangiophore withers and dies. The important genera are Peronospora, Psuedoperonospora, Plasmopara, Sclerospora, Basidiophora etc. The details of Peronospora, Plasmopara and Sclerospora are given. Family - Albuginaceae: Sporangiophores are distinct and sporangia formed in chains at the tips of short, stout, club shaped sporangiophores. The sporangiophores are of indefinite growth. A single genus Albugo is recognized in this family. All the species of Albugo cause white blisters on their host plants and are often called white rusts. The species are obligate parasites. The details of the genus are discussed. Family - Peronosporaceae: Periplasm inconspicuous, facultative parasites of plants, sporangia borne on sporangiophores of determinate growth.

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IMPORTANT GENERA GENUS: OLPIDIUM Class : Chytridiomycetes Order : Chytridiales Family : Olpidiaceae The family Olpidiaceae includes simplest of fungi, all of which are unicellular, holocarpic and endobiotic forms. There are several genera in the family and the genus Olpidium is the best known among them. There are about 25 species in the genus Olpidium and of these O. brassicae which causes root rot of cabbage, cauliflower and other crops is the most wide spread and agriculturally important. Olpidium viciae, parasitic on aquatic plant Vicia unijuga is the most thoroughly investigated and its life history was studied by Japanese mycologist Shunsuke Kusano (1912). The other important species in the genus include Olpidium endogonum which infects members of conjugales in algae, O. gregarium infects rotifer eggs, O. maritinum infects marine plants and O. uredinis infects urediniospores of rust fungi.

Fig. 5.6. Important stages in the life cycle of Olpidium viciae

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The life history of Olpidium viciae is given in the Fig. 5.6. It is parasitic on the leaves and stems of Vicia unijuga. When the infected parts are wet, the zoospores escape from the sporangium through the exit tube. After a brief period of swimming, they encyst on the surface of the host. Infection occurs through a minute pore digested in the host cell wall. Through the pore the protoplast of the parasite enters the host cell leaving the cyst on the cuticle. In the host cell, the protoplast of the fungus is situated near the host nucleus, and then secretes a membrane around itself. Then it transforms into a zoosporangium. The nucleus in the developing zoosporangium divides repeatedly. Each nucleus is surrounded by a bit of cytoplasm and becomes a zoospore. The zoospores, thus formed, escape and repeat the asexual life cycle. The sexual reproduction is by formation of resting spores which are believed to be formed by the fusion of motile gametes. The swarm cells released from a zoosporangium may behave either as zoospores or planogametes. The planogametes most frequently originate from different sporangia. The copulation of two gametes results in a motile zygote. The zygote infects a host cell in the same manner as does a zoospore, but infection by the zygote results in formation of thick walled resting sporangium. The resting spores are capable of over wintering. The resting spore is at first binucleate but before germination karyogamy takes place probably followed by meiosis. Several nuclear divisions result in a multinucleate structure, the protoplast of which eventually undergoes cleavage to form presumably uninucleate zoospores. These escape and may reinfect host plants. GENUS: CHYTRIDIUM Class: Chytridiomycetes Order : Chytridiales Family : Chytridiaceae The genus Chytridium is a large one with more than 25 species, and widely distributed in temperate climates. The important and well studied species in the genus is C. olla which is parasitic on oogonia and oospores of Oedogonium. Other species include C. sexuale which is parasitic on Vaucheria filaments, C. lecythii on rhizopod Lecythium, and C. cocconeidis on diatom Cocconeis. The members of the genus are eucarpic, epibiotic with extensive rhizoidal system. The sporangia develop from rhizomycelium and are globose with granular pale pink contents with a series of main axes branching into very fine extremities. When the sporangia are mature they dehisce by lifting off a large operculum and numerous posteriorly uniflagellate zoospores escape and swim for a few hours. On coming to rest, they encyst. The sexual reproduction has been described for C. sexuale by Koch (1951). At the time of sexual reproduction, the motile spores from female thallus attach to the host cell wall and germinate to form limited thallus. Then male motile gametes attach to the female thallus and encyst. The encysted motile cell empties its contents into the female thallus and combined protoplast move into the host cell and forms a swelling endobiotically. This swelling increases in size and become surrounded by a

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thick warty wall, thus forming a resting spore. The resting spore germinates under favourable conditions to form rhizomycelium (Fig. 5.7).

Fig. 5.7. Important stages in the Life cycle of Chytridium olla (A-D) and C. sexuale (E-K).

GENUS: SAPROLEGNIA Class : Oomycetes Order : Saprolegniales Family : Saprolegniaceae The genus Saprolegnia is the most common and widely studied genus in the family Saprolegniaceae. Saprolegnia ferax is the type species. The species of Saprolegnia are very common in soil and in fresh water. They are saprophytic on plant and animal remains but a few species such as S. ferax and S. parasitica have been implicated in diseases of fish and their eggs. Mycelium: Saprolegnia species form extensive much branched, coenocytic mycelium and it is easily visible to the naked eye as it forms a colony around some bits of decaying plant or animal tissue in water. The hyphal walls contain cellulose.

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Septa are formed in the somatic mycelium just below the reproductive organs separating them from the somatic hyphae, which generally remains aseptate. The hyphae vary considerably in diameter. In some species they are very wide while in others they are fine or narrow. Nutrition of the fungus is also interesting. It is unable to utilize nitrates but use organic nitrogen in the form of peptone or amino acids. So also sulphates cannot be utilized but organic sulphur in amino acids like cysteine, cystine and methionine is utilized. Glucose is the best source of carbon. pH range from 4.0 to 6.0 has been found to be optimum for growth. Asexual Reproduction: The zoosporangia are long cylindrical, terminal or slightly greater in diameter than the hyphae on which they are produced. The sporangia are delimited by a septum and young sporangia are full of dense granular protoplasm which is somewhat brownish under transmitted light. Sporangial proliferation occurs in Saprolegnia (Fig. 5.8). When a sporangium liberates all the zoospores produced in it, another or secondary sporangium is initiated at the basal septum and grows through the first sporangium, maturing within it or beyond it. Several sporangia may be formed one within the other, each maturing and shedding its spores before the next one is formed. The zoospores produced in the sporangia are pear shaped and bear two flagella at the apex. After a period of swimming, they encyst. Instead of germinating by a germ tube, the cyst gives rise to a secondary zoospore which is kidney shaped bearing two oppositely directed flagella at the concave side of the zoospores. The fungus is described as dimorphic and diplanetic. Polyplanetism may also occur in some species of Saprolegnia.

Fig. 5.8. A. Mature sporangium in Saprolegnia. B. Internal proliferation. 1, 2. Empty sporangial cases. 3. Developing sporangium.

In addition to the production of zoosporangia, the fungus also produces chlamydospores, sometimes called gemmae.

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Sexual Reproduction: When conditions are favourable, sexual reproduction occurs in which copulation is by gametangial contact between oogonium and antheridium. The sex organs are produced in close proximity on same hyphal branch (monoclinous) or on different branches (diclinous). The oogonium is usually a globose or elongate structure formed terminally or in intercalary position, and its contents differentiate into one or more uninucleate oospheres or eggs. The antheridium is an elongate and multinucleate structure. The sex organs in Saprolegnia are shown in the Fig. 5.9.

Fig. 5.9. Oogonium and antheridium in Saprolegnia sp. A. Terminal oogonium with attached antheridium. B. Intercalary oogonium.

On coming in contact, antheridium produces fertilization tube which penetrates the wall of oogonium and branch out to reach each oosphere present in the oogonium. The antheridial nuclei now migrate from antheridium to oosphere through fertilization tube, and one nucleus enters each oosphere, and approaches its nucleus. Then the two nuclei fuse and form diploid zygotic nucleus. After fertilization, a thick wall develops around each oosphere converting it into an oospore. The wall of the oospore is smooth. After a prolonged period of rest, the oospores are released from the disintegrating oogonial wall. The oospore germinates by giving rise to a germ tube. At the tip of the short germ tube a globose vesicle is differentiated and the contents of the germ tube are transferred into it, and the vesicle is transformed into a sporangium. Meiosis occurs during germination and the sporangial nuclei are haploid. The life cycle of Saprolegnia is shown in Fig. 5.10.

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Fig. 5.10. Life cycle of Saprolegnia.

GENUS : ACHLYA Class : Oomycetes Order : Saprolegniales Family : Saprolegniaceae The genus Achlya is a member of the family Saprolegniaceae, and some species like A. bisexualis, A. ambisexualis and others are well known for the studies on the discovery of a series of hormones involved in sexual reproduction in

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zoosporic fungi. The species of Achlya are common in soil and in water logged plant debris such as twigs and some are reported naturally occurring as pathogens of fish. Mycelium: Achlya species form extensive much branched, coenocytic mycelium and it is easily visible to the naked eye as it forms a colony around some bits of decaying plant or animal tissue in water. The hyphal walls contain cellulose. Septa are formed in the somatic mycelium just below the reproductive organs separating them from the somatic hyphae, which generally remains aseptate.

Fig. 5.11. Asexual reproductive structures in Achlya colorata. A. Zoosporangium showing a clump of primary cysts at the mouth. B. Full and empty primary cysts. C. Stages in the release of secondary zoospores from a primary cyst. D. Secondary zoospores. E. Secondary cyst. F. Secondary cyst germinating by means of a germ tube.

Asexual Reproduction: It is also similar to that described for Saprolegnia. The zoosporangium is a long cylindrical structure. The contents of the sporangium cleave to form zoospores. The flagellation of the spores is not clear. At the time of

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liberation, they do not swim away but aggregate at the mouth of the hollow sporangium, and encyst. The bunch of encysted zoospores appears like a loose spore ball at the mouth of the sporangium. Thus the first motile phase is a very brief one. The encysted zoospore releases a secondary zoospore. After a brief period of swimming it encysts and germinates by germ tube or releases another secondary zoospore. Repeated emergence of secondary zoospores may occur (Fig. 5.11). Sexual Reproduction: The method of sexual reproduction is basically oogamous type which is similar to that in Saprolegnia. The type of sexual reproduction and hormonal interaction in the process are described by John Raper (1951, 57) in Achlya bisexualis and A. ambisexualis. These are heterothallic and hermaproditic species which show four types of thalli viz. pure male thallus, pure female thallus, potentially male thallus and potentially female thallus. The potentially male thallus behaves like a male thallus in presence of a pure female thallus or potentially female thallus, but in the presence of a pure male thallus it behaves as a female thallus. Likewise potentially female thallus behaves as a male thallus in presence of a pure female thallus. When compatible thalli grow in close proximity, a system of four distinct hormones becomes operative sequentially in the sexual process. The sexual process is initiated by female vegetative hyphae which releases hormone A. It induces the formation of antheridia by male thallus. This substance was later isolated, characterized, and is called antheridiole. The antheridial branches produce hormone B and it induces the formation of oogonial initials by the female thallus. Then the oogonial initials produce hormone C which attracts antheridial branches of male thallus to oogonial initials of female thallus. On contact the delimitation of antheridium is induced thigmotropically. Then the antheridial branches produce hormone D which induces the formation of a wall at the base of each oogonium, thus delimiting the female sex organs. Sexual process in heterothallic species of Achlya relating the sequence of morphological developments to the origins and specific activities of sexual hormone is shown in Fig. 5.12. GENUS : PYTHIUM Class : Oomycetes Order : Pythiales Family : Pythiaceae The genus Pythium (Pythien = to cause rot) was established by Pringsheim in 1858 the types species being P. monosporum. Some important other species are P. aphanidermatum, P. debayanum, P. butleri, P. ultimum, P. graminicoloum etc. The genus is world side in distribution and is represented by about 90 species. Several species occur as saprophytes in water, soil or decaying organic matter. Many species are parasitic on plants with wide host range, causing diseases like root rots, stem rot, fruit rot and most importantly damping-off disease of germinating seeds and developing seedlings. Damping-off of tobacco, tomato, chillies, brassicas, legumes etc. are very serious especially in seed beds causing heavy yield losses.

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Fig. 5.12. Sexual process in heterothallic species of Achlya involving sex hormones.

Vegetative structure: Mycelium is composed of coenocytic, thin, slender hyhpae, which may develop septa at the time of formation of reproductive structures and in old hyphae. In parasitic species the hyphae in the host tissue are both intercellular as well as intracellular. Intercellular hyphae may form haustoria but intracellular mycelium lacks haustoria. The genus is easily culturable and forms white cottony mycelium on agar medium. Asexual Reproduction: This occurs by formation of zoospores formed within sporangia. The zoosporangia in different species vary in form. Three principal types of zoosporangia are recognized – lobate, apical and intercalary (Fig. 5.13).

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Fig. 5.13. Different types of sporangia in Pythium spp.

1.

Filamentous sporangia: These types of sporangia are scarcely distinguished from vegetative hyphae. They are slender, simple or branched filaments of the same size as the vegetative hyphae, e.g. P. monosporum, P. gracile, P. papillatum etc.

2.

Lobed sporangia: In some species the sporangia are inflated and lobed. e.g. P. aphanidermatum, P. graminicola.

3.

Globose sporangia: In many species (e.g. P. debaryanum) the sporangia are globose, terminal or intercalary, cut off by a septum from vegetative hyphae. The sporangia are multinucleate. The cleavage of cytoplasm to form zoospores begins in the sporangium but completed in a thin walled vesicle that is extruded from the sporangium. The vesicle enlarges and about 8 to 20 zoospores are released by breakdown of the vesicle wall.

The zoospores are broadly bean shaped with two lateral flagella. After swimming for some time, they encyst and germinate by germ tubes to form new mycelium. Repeated emergence of zoospore has also been reported. Sporangial proliferation occurs in some species such as P. undulatum and P. proliferum. In some species like P. ultimum, the sporangia are separated from mycelium and germinate by germ tube, thus showing the tendency of conidia. Sexual Reproduction: It is oogamous type. Most species are homothallic, while some species such as P. sylvaticum, P. splendens and P. heterothallicum are heterothallic. The female sex organ is oogonium and the male sex organ is antheridium. Oogonia develop as terminal or intercalary spherical swellings which become cut off by cross wall from the rest of mycelium. The young oogonium is multinucleate and cytoplasm within it differentiates into a multinucleate central mass called ooplasm and peripheral multinucleate mass called periplasm. Many earlier workers assumed that the vegetative hyphae are haploid and meiosis occurs during the germination of oospore. However, Sansome (1961, 1963) claimed that meiosis occurred in oogonia and antheridia of P. debaryanum. In this and other species, out of nearly 32 nuclei formed by meiosis only one egg nucleus survives in the centre of ooplasm while the remaining nuclei degenerate. Nuclei of periplasm also degenerate. Antheridia arise as club shaped swollen hyphal tips either as branches of stalk of oogonia (monoclinous) or on separate hyphae (diclinous). One to several antheridia can be seen around each oogonium. Young antheridia are multinucleate but all nuclei except one degenerate at maturity. The surviving nucleus undergoes meiosis to have

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four haploid nuclei. The antheridia contact the oogonium and penetrate the oogonial wall by a fertilization tube. Only one male nucleus is transferred from antheridium to the oogonium. Karyogamy between male nucleus and the egg nucleus occurs and the remaining nuclei of antheridia and oogonial periplasm degenerate. Fertilized egg secretes a double wall and has plenty of reserve food material. The oospores may undergo a period of rest of several weeks before they germinate. Germination may be by means of a germ tube or by the formation of a vesicle in which zoospores are differentiated. The details of different stages in the life cycle of P. debaryanum are shown in Fig. 5.14.

Fig. 5.14. Life cycle of Pythium debaryanum.

GENUS : PHYTOPHTHORA Class : Oomycetes Order : Pythialaes Family : Pythiaceae

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The name of the genus indicates that it is a plant destroyer (phyto = plant; phthora = destroyer), and indeed some species are certainly destructive parasites. About 40 species are known in the genus, and of them Phytophthora infestans is the first described and the best known. While some species may live saprophytically as water moulds, the majority are pathogenic forms probably have no prolonged free living saprophytic existence. It is the causal organism of late blight disease of potato and caused Irish famine of 1845-46. The species of the genus are having a wide host range. P. infestans is confined to the members of Solanaceae, but others have much wider host range. For example, P. cactorum has been recorded from over 40 families of flowering plants causing a variety of diseases such as damping-off, root rot and fruit rot. Other important species in the genus and the diseases they cause are given below. Pathogen

Disease

Phytophthora antiquorum

Blight of colocasia

P. arecae

Koleroga of areca palms

P. cryptogea

Foot-rot of tomato seedlings

P. erythroseptica

Pink rot of potato

P. fragariae

Red core of strawberries

P. palmivora

Bud rot of palms

P. nicotianae

Seedling blight of castor

Mycelium: It is aseptate, coenocytic, producing branches at right angles, often constricted at their point of origin. Within the host, mycelium is intercellular but finger like haustoria are formed which penetrate the host cells. Asexual Reproduction: It is by sporangial formation. The sporangiophores emerge through the stomata. The first formed sporangium is terminal, and the hyphae bearing it may push it to one side and proceed to form another sporangium and the process is repeated continuously. Thus the first formed sporangium is at the base and youngest at the top. The sporangiophores show unlimited growth. The mature sporangium has a terminal papilla and it is separated from the sporangiophore by a thickening of wall material which forms a basal plug. In terrestrial forms, the sporangia are detached, possibly aided by hygroscopic twisting of the sporangiophore by drying and disperse by wind before germinating, but in aquatic forms zoospore release commonly occurs while the sporangia are still attached. In these aquatic species, internal proliferation of sporangia may occur. The germination of sporangia may be either direct by germ tube or by forming zoospores. Below 15oC uninucleate zoospores are produced while above 20oC multinucleate germ tubes arise. With increasing age, sporangia lose their capacity to produce zoospores. When zoospores are formed, they are typically differentiated within the sporangium, and not in vesicle as in Pythium. The uninucleate, laterally biflagellate zoospores swim for a time, often attracted chemotactically to host tissue, then encyst and germinate by germ tube, but repeated emergence (polyplanetism) has occasionally been reported.

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Sexual Reproduction: Some species of Phytophthora are homothallic and form oospores readily in culture derived from single uninucleate zoospores. e.g. P. cactorum, P. erythroseptica. Majority of species, e.g. P. infestans, P. palmivora etc., are heterothallic, and normally form oospores only when different isolates are paired together i.e. they are self incompatible. When conditions are favourable for sexual reproduction, two types of sex organs viz. antheridia and oogonia are formed, and the reproduction is oogamous type. Oogonium is a bigger and spherical structure while antheridium is a smaller and club shaped structure. Two distinct types of antheridial arrangement are found. In P. cactorum and others, antheridia are laterally attached to oogonium, and it is called paragynous type. In P. infestans, P. erythroseptica and others, the oogonia during development penetrate the antheridium, grow through it, inflate and develop into a spherical oogonium, with antheridium persisting as a collar around the base of the mature oogonium. This type of arrangement is called amphigynous type.

Fig. 5.15. Life cycle of Phytophthora infestans.

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Both the oogonia and the antheridia are initially multinucleate, but as the oospore matures only a single nucleus remains at the centre, while the remaining nuclei are included in the periplasm. Fertilization tubes have been observed and a single nucleus is introduced from the antheridium into the oogonium. The fusion between the egg nucleus and the antheridial nucleus is delayed until the oospore wall is mature. This wall has a thin exospore derived from the periplasm composed of pectic substances, and a thicker endospore of cellulose, protein and possibly other reserve substances. Such oospores do not germinate immediately, but undergo a period of maturation lasting several weeks or months. Before germination, nuclear fusion occurs, and the fusion nucleus divides several times mitotically to become multinucleate. During germination a short germ tube is formed from the oospore and at the tip of the germ tube a sporangium is formed and it forms zoospores. The details of lifecycle of Phytophthora infestans are shown in Fig. 5.15. GENUS: PERONOSPORA Class : Oomycetes Order : Peronosporales Family : Peronosporaceae The genus Peronospora is relatively large and widely distributed, and all the species in the genus are obligate pathogens causing economically important disease called downy mildew disease on higher plants. Peronospora parasitica occurs on a number of cruciferous plants like cabbage, cauliflower, turnip, radish, mustard etc. Peronospora tabacina causes blue mould of tobacco. It is a downy mildew disease of the crop, and the name refers to the bluish-purple colour of the sporangia which is a feature of many species of Peronospora. P. destructor causes downy mildew disease of onion crop while P. farinosa causes downy mildew of sugar beet. Among the others, species which attack legume crops like P. viciae on peas and beans, P. trifoliarum which causes downy mildew of clover are important. Vegetative structures: The species of the genus are obligate parasites (biotrophs) and have no free living vegetative phase outside the host they attack. The fungus attacks all aerial parts, but is conspicuous on the leaves. Mycelium comprises of intercellular coenocytic hyphae with large finger shaped or clavate or branched haustoria which occupy major part of the host cell. The haustorium does not actually invade the host cell but is ensheathed by a layer of callose material which is often visible as a thickened collar around the base of haustorium. At the point of penetration the haustorium is surrounded by a sheath of host wall material. It is enclosed for the whole of its length by a further sheath. The plasma membrane of the haustorium invaginates at certain points to form lomasomes. Mycelium shows limited growth, and aggregate below the substomatal cavity. The upper side of the infected portion of the leaf becomes yellowish brown and appears like a leaf spot. Asexual Reproduction: It is by the formation of sporangia on distinct sporangiophores which come out of the infected leaf in groups from stomata. At first the mass of sporangiophores appear like a loose cottony growth on the lower surface of the leaf. Soon they turn pink or bluish in color as the sporangia are formed. Each sporangiophore comprises of a stout long main axis of about 100 to 300 µm in

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length. It remains unbranched in the lower part, but shows dichotomous branching 6– 8 times in the upper part. The branches become progressively thin and the tips of finer branches are incurved. Sporangia are formed singly at tip of each incurved fine branch. The tips of the sporangiophore branches are long, slender and pointed. These tips are often described as sterigmata of the sporangiophores. The sporangia are hyaline, broadly oval or ellipsoidal in shape, measure around 25-30 x 15-20 µm and bear a papilla at the tip. The contents of the sporangium are light pink or bluish in colour. They are easily detached and become airborne. Detachment of the sporangia is possibly by hygroscopic twisting of the sporangiophores related to changes in humidity in the atmosphere. In P. tabacina, it has been suggested that changes in turgor of sporangiophores occur with parallel changes in water content of the tobacco leaf. It has also been claimed for this fungus that the sporangia are discharged actively by energy applied at the point of attachment of sporangia. The sporangia when deposited on a suitable host surface, germinate under favourable conditions. The sporangial germination in the species of Peronospora is always by means of a germ tube and not by formation of zoospores. Hence, some mycologists prefer to describe them as conidia and the sporangiophores are called conidiophores. The germ tube enters the host by penetrating the epidermal cells.

Fig. 5.16. a. Symptoms of host leaf infected by Peronospora sp. b. Conidiophores and conidia. c. Oospore

Sexual Reproduction: It is by gametangial contact. The species of Peronospora may be homothallic or heterothallic, and there is evidence that it is a strain character. Towards the end of cropping season, the sex organs are formed throughout the infected tissue. Both antheridia and oogonia are formed either on the same hyphal branch or on different closely formed hyphae. Oogonium is a relatively bigger globose structure and antheridium is a smaller elongated structure. Meiosis occurs in the gametangia followed by repeated meiotic divisions making the gametangia multinucleate. In the oogonium only a single functional nucleus remains in the central region and the rest of the nuclei move to the peripheral region. The male nucleus from antheridium is transferred to oogonium by the formation of a

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fertilization tube. The male and female nuclei do not fuse immediately but fusion is delayed until the oospore wall is partially formed. The oospore wall is very tough, and remains viable for long periods. The oospores over-winter in soil and give rise to new infection in the next crop season. In P. destructor and some other species, the oospores germinate directly by a germ tube and infect the host, while in P. tabacina the formation of zoospores has been reported during germination (Fig. 5.16). GENUS: PLASMOPARA Class : Oomycetes Order : Peronosporales Family : Peronosporaceae The genus Plasmopara is one of the common genera of downy mildew fungi. Plasmopara viticola is a widely distributed species which causes downy mildew disease on grapes (Vitis vinifera) wherever they are grown. The other species include P. nivea which causes downy mildew on members of umbelliferae, P. australis which causes downy mildew of cucurbits, P. halstedii which attacks sunflower and other members of compositae, P. pusilla attacks Geranium, and P. pygmaea causes downy mildew of Anemone nemorosa. Of all the species of Plasmopara, P. viticola, which causes downy mildew of grapes, is of great historical importance. The disease on grapes became very serious and wide spread during later part of 19th century in France, known for its wine industry. Because of the disease yield of grapes is drastically reduced and the French wine industry was in doldrums. Prof. Alexis Millardt of Bordeaux University visiting a farm house found that plants nearer to the path were having white coating and were relatively free from the disease than the interior plants without white coating. He learnt that the plants near the path are sprayed with lime and copper sulphate. He extensively experimented on fungicidal activity of copper sulphate and lime, and introduced in 1886 a copper based fungicide by the name Bordeaux mixture to control the downy mildew disease on grapes. It was the first commercially introduced fungicide. Later it was found to be effective against a number of foliar pathogenic fungi, and ruled the fungicide markets of the world for about half a century. Mycelium: The species of Plasmopara are obligate (biotrophic) pathogens and there is no vegetative growth outside the host tissue. The mycelium is mainly found in the leaves of the infected plants, and it may infect other parts also. Mycelium is strictly inter cellular and consists of coenocytic, thin walled, hyaline hyphae with granular protoplasm. These hyphae are 12 to 60 µm in width. The organic connection with the host is established by producing knob like spherical haustoria into the host. Asexual Reproduction: Sporangiophores arise from hyphae congregated in the substomatal space. One to 20 sporangiophores emerge from a single stoma. Sometimes, they may emerge directly through the cuticle. On young berries they come out through lenticles. These spore bearing structures are mostly produced during night under conditions of high humidity. They are 300 – 500 x 7-9 µm in size. Branching of the sporangiophore is almost at right angle to the main axis and at regular intervals. The final branches arise from the apex. From the lower branches

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secondary branches are also produced. From the apex of each branch 2-3 sterigmata arise and bear sporangia. The sporangia are thick walled, oval or lemon-shaped, and measure 15-30 x 11-18 µm. Two types of sporangial germination have been observed. In P. pygmaea there were no zoospores but the entire sporangial contents escape and later produce a germ tube. In other species the sporangia germinate by means of zoospores. The production of zoospores may be inside or outside in a vesicle. The zoospores are pear shaped and 7-9 µm in size. They have two apical flagella which may be 30 µm long. Sexual Reproduction: It is typically by the gametangial contact between oogonium and ascogonium. The oospores are produced mostly in tissues adjacent to the midrib. They are thick walled and 25-26 µm in diameter. They germinate by producing a germ tube that bears an apical sporangium. The details of different stages in the life cycle of Plasmopara viticola are shown in Fig. 5.17.

Fig. 5.17. Life cycle of Plasmopara viticola.

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GENUS : SCLEROSPORA Class : Oomycetes Order : Peronosporales Family : Peronosporaceae The genus Sclerospora is a relatively large genus and about 10 species are reported from India. All the species in the genus are obligate parasites on higher plants causing downy mildew symptoms on leaves. It is distinguished from other species in the family by the production of long stout sporangiophores that are heavily branched at the apex, each branch bearing a sporangium at the tip, and by the presence of persisting oogonial wall around the smooth thick walled oospore giving it a rough appearance. Most important species in the genus is S. graminicola which causes downy mildew and green ear disease of pearl millet (bajra). Others include S. sorghi, which causes leaf shredding disease of great millet (sorghum), S. maydis which causes downy mildew of maize, S. sacchari, which causes downy mildew of sugarcane and others. S. graminicola is the thoroughly studied species Vegetative structures: The fungus is an obligate parasite (biotroph). The mycelium is found in all parts of the systemically affected plants. Hyphae are strictly intercellular, and provided with knob like unbranched haustoria. The haustoria are formed mainly in the mesophyll cells in leaves and young tissues in the stem. In the leaves it is restricted to mesophyll tissue, rarely penetrating the epidermal layer. The hyphae may penetrate fibrovascular bundles in the leaf, but xylem and phloem are not attacked. The hyphae branch freely in the intercellular spaces. They have gelatinous cell wall and clear protoplasm. Asexual Reproduction: The sporangiophores arise from the internal mycelium which develops in the air space below the stomata. They emerge through the stomata either singly or in groups. Each sporangiophore is a broad, hyaline, nonseptate hypha measuring about 100 µm in length and up to 15 µm in width. It is unbranched in the lower part, but a few, about 2-6, short thick braches are formed di- or trichotomously at the tip. The tips of the branches are slightly swollen and sporangia are borne on the swellings. Sporangia are hyaline, thin walled, broadly elliptical with a papilla at the free end. The sporangia vary in size with host, but measure in the range of 15-35 x 10-25 µm. They germinate by liberating numerous zoospores which are irregularly reniform and biflagellate. They swim for a while, come to rest, encyst and germinate by germ tubes. Different stages in the development of Sclerospora graminicola sporangia are shown in the Fig. 5.18. Sexual Reproduction: It is oogamous type, and by gametangial contact between oogonium and antheridium. The sex organs are formed mainly in the leaves. The oogonia are usually terminal, occasionally intercalary. A mass of protoplasm with about 50 nuclei enters the oogonium from the mycelium. After the oogonium has expanded it is cut-off by a septum. The nuclei are at first shrivelled in appearance but may enlarge and, as they approach the metaphase become oriented in the region which is to become the boundary between ooplasm and periplasm. The nuclei pass through a meiotic division. A coenocentrum develops near which a single nucleus is found. The nucleus divides once and one daughter nucleus serves as the female functional gamete. The antheridium contains 3-4 nuclei, and also undergoes meiosis

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slightly ahead of the division in the oogonium. The fertilization tube penetrates the oogonial wall and passes rapidly to the oosphere where it discharges one nucleus and quickly disintegrates. The coenocentrum also disappears. Differentiation of the oospore wall follows by secretions from the ooplasm. A thin outer layer makes up the exospore and a thicker layer forms the endospore. The original oogonial wall shrinks to touch the new oosporial wall at many points giving the entire oospore an elliptical to angular or irregular shape although the oospore proper is usually spherical. Under the high power of microscope the mature oospore shows three walls, the exosporium, mesosporium and endosporium. The endosporium is smooth, yellow in color and of even thickness. Exospsorium is tawny in appearance and deeper colored. After a period of rest, they germinate by germ tubes. The strains of S. graminicola in India mainly produce a large number of oospores which are very abundant in the infected tissues, while in some species like S. philippinensis oospores are not yet observed.

Fig. 5.18. Stages in the development of sporangiophore in Sclerospora graminicola.

GENUS: ALBUGO Class : Oomycetes Order : Peronosporales Family : Albuginaceae There are about 30 species in the genus Albugo. This genus is also called Cystopus. The genus was first described by C.H. Persoon in 1801 as Albugo. Later, in 1840s Levellie described the genus as Cystopus. Since the genus name was first published validly as Albugo, it should be treated as Albugo, and Cystopus is a later synonym.

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All the species in the genus are obligate parasites on higher plants. On the host plants they produce white rust disease. Due to the fungal infection white blisters are formed on host leaves and stem. The blisters appear as pustules formed by rust fungi but white in colour. Hence the disease is called white rust disease. In the blisters a large number of sporangia are formed in a mass. Due to the incidence of large number of pustules on leaves, the photosynthetic area is reduced, and the formation of blisters on stem weakens it. The fungus may also infect the inflorescence during severe infections. Because of infection the peduncle becomes thick, and floral parts become green leaf like structures. The species of Albugo infecting the members of the families like Amaranthaceae, Cruciferae, Convolvulaceae etc. are given below. Species

Host(s)

Albugo bliti

Amaranthaceae

A. candida

Cruciferae

A. ipomoeae- panduranae

Evolvulus alisinoides

A. evolvuli

sweet potato

A. oxidentalis

Spinach species

A. platensis

Boerhaavia species

A. portulacae

Portulaca species

Mycelium: All the species of Albugo are obligate parasites. Hence, vegetative growth occurs only in host tissue. The hyphae are septate, hyaline, branched filaments. They grow in the intercellular spaces in the host tissue. The hyphae send in small, globose structures into the host tissue to draw nourishment. They are called haustoria. Asexual Reproduction: Albugo species reproduce asexually by production of sporangia and sexual reproduction by gametangial contact. The details of reproduction are presented in Fig. 5.19. Asexual reproduction: The mycelium grows in the intercellular spaces of the host tissue. After a period of growth for 4 or 5 days after infection, mycelium aggregates below the epidermis. The sporangiophores are produced in a layer below the epidermis. The sporangiophores are short, erect, unbranched, cylindrical or club shaped, and produce sporangia at the tip in chains. The tip of the sporangium bulges into a globose structure with accumulation of protoplast with 5-8 nuclei. Then it is cut off from the sporangiophore with formation of a septum. The process repeats a number of times to produce a chain of sporangia, with first formed sporangium at the apex and youngest at the base. Such an arrangement of sporangia is described as in basipetal succession. The sporangiophore, though shows continuous growth producing sporangia, they remain almost constant in size and shape. As the sporangia are formed in large numbers and accumulate below the epidermis, the epidermis, at the region of the sporangial mass, bursts open because of the internal pressure exposing the sporangia. This appears like a pustule. Since the pustules formed by the sporangia are white in colour, and resemble the pustule formation in rust disease, the

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infection caused by Albugo is described as white rust. The sporangia formed in a chain are attached to one another and usually held together by slime masses. After the exposure of the sporangia, the slime evaporates, and the sporangia become dry and separated. Then they are dispersed through the air like conidia. When the sporangia get are deposited on a susceptible host surface, they germinate under favourable conditions. Two types of sporangial germination have been observed.

Fig. 5.19. Life cycle of Albugo candida. A. Vegetative hyphae on host cell. B. Sorus with sporangia. C. Sporangium. D. Liberation of zoospores. E. Zoospores. F. Encystment of zoospores. G. Germination of cysts. H. Gametangial contact. I. Fertilization tube releasing male nuclei into oogonium. J. Nuclear fusion. K. Oospore formation in oogonium. L. Germination of oospore. M. Motile zoospores. N. Germination of zoospores.

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1. Zoospore production: When the temperature is 15oC or below and there is abundant moisture or a thin film of water on the host surface, the protoplast of the sporangium cleaves to produce biflagellate zoospores, and they are released by splitting of the sporangial wall. The zoospores are kidney shaped and laterally biflagellate. After a period of motility, the zoospores encyst. The cyst germinates giving rise to an infection peg which enters the host leaf through stomata. 2. Direct germination: when the temperature is 20oC or more and there is no moisture on the host surface, the sporangia do not produce zoospores, but germinate directly to produce a germ tube which enters the host leaf through stomata. Sexual reproduction: Albugo species are homothallic. The male gametangium (antheridium) and female gametangium (oogonium) form on the same thallus and carry out sexual reproduction. The gametangia are formed on the tips of the vegetative hyphae growing in the host tissue. Oogonium is globose, multinucleate at first with 6–12 nuclei. These nuclei undergo repeated divisions to form about 200 nuclei. The cytoplasm in the oogonium is then differentiated into a clear, dense central part called centroplasm or ooplasm, and surrounding less dense cytoplasm called periplasm. The antheridia are relatively small structures formed on separate hyphae. They are club shaped. At maturity the oogonium and antheridium occur side by side. This condition is described as paragynous. Depending on the activity of the nuclei in oogonium, two types of conjugation occur. In some species (e.g. Albugo candida), both oogonium and antheridium contain only one active nucleus each. At the place of gametangial contact, a fertilization tube develops from the antheridium and enters the oogonium and releases the active nucleus into centroplasm. Then the two active nuclei fuse producing diploid zygotic nucleus. In some species (e.g. Albugo bliti) both antheridium and oogonium are multinucleate. In the oogonium, at first, there may be about 300 nuclei, and among them 40 to 50 remain in centroplasm and rest move into the periplasm during the differentiation. Then the nuclei in the centroplasm undergo further division to form about 80 to 100 nuclei. In the antheridium there are about 30-35 nuclei in the beginning and they repeatedly undergo division to form about 100 nuclei. All the male nuclei are passed into centroplasm of oogonium through fertilization tube. The male and female nuclei pair together and karyogamy occurs producing a large number of zygotic diploid nuclei. Since all the zygotic nuclei occur in the single oospore that develops after karyogamy, it is called compound oospore. In both the methods of fertilization, after the formation of diploid nuclei a thick wall is formed around the centroplasm which transforms into oospore. The oospore wall is very thick, and two layers can be distinguished, the outer exospore and inner endospore. The exospore shows various types of thickenings, which may be tuberculate, porous, reticulate etc. The type of thickening is useful in identification of different species in the genus. As in other oomycetes, in the genus Albugo also meiosis occurs in gametangia. Hence the vegetative mycelium is diploid. Germination of oospores: The oospores are resting structures and after a long period of rest they germinate under favourable conditions. The germination of

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oospores occurs by two methods. Both the types may be found in the same species (e.g. Albugo candida). The outer layer of the oospore (exospore) cracks open and inner endospore comes out as a vesicle. The protoplast of the oospore is transferred into the vesicle, and zoospores are formed in the vesicle. The zoospores are released after the disintegration of the vesicle membrane. During germination oospore directly gives rise to a short germ tube, and a vesicle is formed at the tip of the vesicle. The protoplast of the oospore is transferred into the vesicle, and zoospores are formed in the vesicle. Life cycle: In Albugo species vegetative mycelium is diploid. Meiosis occurs in gametangia to produce haploid nuclei. The fusion of the haploid nuclei results in diploid stage. The life cycle of Albugo is shown in the Fig. 5.20.

Fig. 5.20. Life cycle of Albugo.

■■■

Chapter - 6

Zygomycota

The fungi that produce coenocytic mycelium and reproduce asexually by sporangiospores or aplanospores and sexually by zygospores are included in this subdivision. In the subdivision two classes are recognized 1. Zygomycetes, that typically produce zygospores, and 2. Trichomycetes, a group of uncertain affinity. CLASS- ZYGOMYCETES The fungi with typical characters of the group are placed in this class. Three orders are recognized in this class. Order- Mucorales: These fungi are mostly free living saprophytes. Order- Entomophthorales: These are mostly insect parasites. Order- Zoopagales: It is a relatively small group of soil fungi that occur as parasites or predators on soil protozoa and soil nematodes. Order – MUCORALES The fungi included in this group occur in different habitats. Most of them are free living saprophytes (Mucor, Rhizopus, etc.), some are coprophilous growing on dung (e.g. Pilobolus), and some are mycoparasites growing on other fungi (e.g. Piptocephalis). Saksenea vasiformis is a weak wound parasite and is important in medical mycology. The most important characters of these fungi are: 1.

Filamentous hyphae without septa.

2.

Asexual reproduction by aplanospores produced in sporangia.

3.

Sexual reproduction by gametangial copulation producing thick walled zygospore.

Mycelium: The mycelium of the fungi included in this group is made up of branched filamentous hyphae without septa. Cytoplasm is freely distributed throughout the mycelium. A large number of nuclei are present in cytoplasm and hence they are described as coenocytic. Protoplasm shows rapid streaming throughout the mycelium. The cell wall is made up of chitin and chitosans, cellulose is absent. In some genera like Rhizopus, a group of rhizoids are formed from hyphae at regular intervals. Rhizoids help mycelium adhere to the substratum and to draw nourishment from the substratum.

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In some fungi like Mucor rouxii, thallus shows dimorphism. Under normal conditions, the fungus produces normal coenocytic, filamentous branched hyphae, but under anaerobic conditions or in environment with high CO2 concentration, the fungus shows unicellular yeast like growth. The concentration of glucose in the growth medium also appears to be an important factor in determining whether a particular species will produce mycelium or yeast cells. Concentration of glucose in excess of 5 percent induces yeast like growth in some species even under aerobic conditions. Asexual reproduction: In this group of fungi, asexual reproduction occurs by production of nonmotile aplanospores or sporangiospores in sporangia. The sporangia are formed at the tip of sporangiophores. The sporangium may be globose or oval, and the tip of the sporangiophore penetrates the sporangium as dome shaped structure called columella. The cytoplasm above the columella in the sporangium divides into uninucleate portions and each bit is transformed into a non motile spore. When the sporangium wall disintegrates the spores are liberated and disperse through air. In addition to the typical columellate sporangia, two other types of small noncolumellate spore bearing sacs are also formed. These are: 1.

Small, globose sac like structures with limited number of spores called sporangiola.

2.

Narrow, cylindrical or finger shaped structures called merosporangia in which the spores are formed in uniseriate fashion.

There is much variation in the asexual reproductive structures in this group, and these are of taxonomic significance also. The asexual reproductive structures in some important genera are described below. 1.

In Mucor, Rhizopus and other genera of Mucoraceae, typical columellate sporangia are formed.

2.

In the genus Pilobolus, the sporangiophore is a distinctly long structure which is bulbous at the base, narrow in the middle and bulged like a vesicle below the sporangium. The basal part is called trophocyst, and the vesicle is described as subsporangial vesicle. The sporangium formed at the tip is thick walled with limited number of thick walled spores, and the columella is flat disc like structure at the base of the sporangium. The protoplast in the subsporangial vesicle is under great pressure, and when it bursts the sporangium is thrown out with force up to a distance of two meters.

3.

In the genus Mortierella, the tip of the sporangiophore is pointed and a thin walled sporangium is formed at the tip. The sporangial wall is evanescent, and dissolves during spore formation, leaving a group of spore mass at the tip of the sporangiophore.

4.

In the genus Choanephora, apart from the typical columellate sporangia, a large number of sporangiola are formed on separate sporangiophores.

5.

In the genus Thamnidium, columellate sporangia are formed at the tip and sporangiola are formed on the branches on the same sporangiophore.

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6.

In the genus Cunninghamella, on the upper part of the club shaped sporangiophore, single celled sporangiola are formed. The spore wall and sporangial wall are closely attached to one another, appearing like a single structure. They are separated from the sporangiophore and disperse through air like conidia.

7.

In the genera Syncephalastrum and Piptocephalis, the sporangiophore forms a vesicle at the tip, and a large number of merosporangia develop from the vesicle. Each merosporangium bears about 10 spores in uniseriate fashion.

8.

In the genus Dimargaris, merosporangia are formed with only two spores.

9.

In the genus Kickxella, single spored merosporangia are formed on special structures called sporocladia. The merosporangia are formed in a layer on the underside of each sporocladium, and entire structure appears like a comb.

The asexual reproductive structures are shown diagrammatically in Fig. 6.1.

Fig. 6.1. A-J. Asexual reproductive structures in various genera of Zygomycotina. A. Mucor. B. Pilobolus. C. Mortierella. D. Choanephora. 1. Columellate sporangium. 2. Sporangiola. E. Thamnidium. 1. Columellate sporangium at the tip of sporangiophore. 2. Sporangiola on the branches of sporangiophore. F. Cunninghamella. G. Syncephalastrum. H. Dimargaris. I. Kickxella 1. Sporocladium. 2. Single celled merosporangia. J. Entomophthora. 1. Sporangiophore. 2. Sporangium.

Sexual reproduction: Sexual reproduction takes place by the conjugation between two gametangia which are usually coenocytic, globose and of same size and shape. The gametangial copulation results in the formation of a thick walled zygospore. In some species, gametangia formed on the hyphal branches of same mycelium can conjugate, and such species are called homothallic species, e.g. Mucor genevensis, Zygorynchus sp., etc. In some species, gametangia do not form unless

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two compatible mycelia of the same species are growing side by side. Such a phenomenon of presence of two morphologically similar but sexually different mycelia in the same species is described as heterothallism. American geneticist, A.F. Blakeslee (1904) first discovered the phenomenon in Mucor mucedo, and it was noticed in a number of other species also. Since the two strains participating in sexual reproduction are morphologically similar in all respects, they were designated as (+) and (-) strains. In heterothallic species, when two compatible strains are growing side by side zygophore initials are formed on hyphae from the two strains facing one another (Fig. 6.2). They gradually grow towards each other and when they come in contact at the tips, the gametangia are delimited from the basal part called suspensor. The wall between the gametangia disintegrates at the point of contact and the two protoplasts fuse. Each gametangium consists of a large number of nuclei, and after protoplast fusion, the compatible nuclei pair together and karyogamy occurs between them to form zygotic nuclei. A thick wall develops around the zygospore and the wall of gametangium remains adhered to the zygospore wall. Hence the entire structure is often described as zygosporangium. After a long period of rest the zygospore germinates by giving rise to a germ tube which forms a vesicle at the tip. Meiosis occurs during germination. The protoplast cleaves in the vesicle to form non motile spores

Fig. 6.2. Sexual reproduction (gametangial copulation) in Mucorales. A. In Mucor with equal suspensors. B. In Zygorhynchus with unequal suspensors. C. In Phycomyces with tong shaped suspensors.

Burgeff (1924) explained that the phenomenon of heterothallism is controlled by hormones in Mucorales. He explained that the compatible hyphae possess preformed prohormones and when they are growing side by side, the prohormones diffuse through the medium and induce the production of hormones in the opposite mating type. The hormones diffuse through the medium and induce the formation of zygophore initials. The growth of zygophores towards one another is called zygotropism. It is reported that the zygotropic movement is influenced by prohormones rather than hormones. When the zygophores come in contact with one another, the delimitation of gametangia occurs due to thigmotropic reaction. In zygomycetes, trisporic acid A, B, C are recognized as prohormones. Methyl -4-OH

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trisporate, dimethyl compounds and β-carotene are identified as hormones. The role of hormones in sexual reproduction is shown in Fig. 6.3.

Fig. 6.3. Effect of hormones on sexual reproduction in heterothallic species.

In the order Mucorales, 14 families are recognised. Separation is based on the nature of asexual means of reproduction including the nature of the sporangium and the sporangiophores. This is because the sexual state often is not known or is hard to induce in many species. Family - Mucoraceae: The largest family in the order with about 20 genera. Mostly occurring in soil as free living saprophytes. Some species are dung inhabitants. Some are storage fungi growing on stored grains. Some are parasites on mushrooms (Syzygites, Dicranophora and Spinellus). Species of Mucor, Rhizopus and Actinomucor are of economic importance because of their use in fermented foods (tempeh, sufu, lao-chao) and in steroid transformations. The hyphae are typically aseptate, tubular and coenocytic. In Rhizopus and Actinomucor rhizoidal system is present. All species of the family have large multispored sporangia which contain well defined columellae. The zygospore in Mucor is formed as a result of the fusion of two gametangia delimited from the suspensor by a septum. The suspensors are typically enlarged, equal and opposite to one another. The two gametangia fuse and this result in a zygospore which is always brown to black, heavy walled and covered with rough blunt conical projections or warts. Important genera in the family include Mucor, Actinomucor, Rhizopus, Phycomyces, Zygorynchus, Syzygites, etc. Family - Pilobolaceae: They are dung fungi. All species possess sporangia with dark coloured, persistent walls containing many spores, and sporangium is with well defined columella. Sporangiophores are usually large and elongate and often phototropic. In Pilobolus and Utharomyces special swollen areas separated from the vegetative mycelium by a septum are found at the base of the sporangiophores. The swellings are on, or more often in, the substratum and are called trophocysts The sporangia are thick walled, produced terminally on long sporagiophore with trophocyst and subsporangial vesicle. They are liberated with force by the burst of subsporangial vesicle. The zygospores are formed between the tong shaped suspensors. Only three genera are recognised. The best known is Pilobolus and the other two are Pilaria and Utharomyces. Family - Choanephoraceae: Fungi included in this family are saprophytes or weakly parasitic forms occurring on many crop plants. Asexual reproduction is by sporangia and sporangiola. All members are characterized by possessing large columellate sporangium with persistent sporangial walls which break open as two

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halves. The spores are characteristically striate with bristle like appendages at their ends. In addition to the sporangium, they possess distinct fruiting stalks which bear single celled sporangiola. Three genera viz. Choanephora, Blakeslea and Gilbertiella are recognized in the family. Of these the first two are widely distributed and well studied. Family - Thamnidiaceae: This family consists of 7 genera (Thamnidium, Thamnostylum, Cokeromyces, Chaetocladium, Dicranophora and Helicostylum). They are worldwide in distribution and commonly found on dung, mushrooms, soil and decaying vegetation. In the genus Thamnidium the asexual reproduction is by production of both collumellate sporangia as well as non columellate small sporangiola on the same sporangiophore. The columellate sporangia are formed at the tip and sporangiola on the side branches. In the genus Helicostylum and other genera only sporangiola are produced. Sexual reproduction is by production of zygospores formed between enlarged nearly equal suspensors. Family - Cunninghamellaceae: The fungi in the family produce single spored sporangiola on club shaped sporangiophores. Sexual reproduction is typically as in Mucor. Four genera are known viz. Cunninghamella, Mycotypha, Phascolomyces and Thamnocephala. Of these only Cunninghamella is ubiquitous and is commonly encountered in soil all over the world. Family - Mortierellaceae: The family is composed of species which produce from one to many spored sporangia with columellae lacking or only vestigial. In the species with only one spore the sporangial wall can readily be seen as a separate structure from the wall of the sporangiospore. The fungi in the family produce noncolumellate sporangia on tapering sporangiophores. The single celled spores are termed stylospores. Sexual reproduction is by formation of zygospores between tong shaped suspensors and surrounded by sterile hyphae. The zygospores with a tuft of sterile mycelial covering appear as primitive sporocarp. The genera recognized are Mortierella, Dissophora, Haplosporangium, and Aquamortierella. Family - Syncephalastraceae: A single genus Syncephalastrum with a single species racemosum is recognized in the family. It is a common saprophytic species worldwide in distribution. The fungus typically produces merosporangia on globular vesicle. It is a heterothallic species. Zygospore is readily formed between two equal gametangia in compatible aerial hyphae. Family - Piptocephalidaceae: The members are parasitic on mycelium of other Mucorales, and on ascomycetes. The vegetative mycelium is delicate and fine. Asexual reproduction is by many spored merosporangia resembling those of Syncephalastrum. Two genera are recognized in the family. They are Piptocephalis and Syncephalis. Family - Dimargaritaceae: A small family in which mycelium is septate and produce two spored sporangiola. The sporangiospores are arranged in a linear fashion and hence, are considered as reduced merosporangia. Four genera are recognized viz. Dimargaris, Dispira, Spinalia and Tieghmiomyces. They are either parasites or facultative parasites on other Mucorales. Family - Kickxellaceae: In the fungi included in this family vegetative mycelium is septate from the begining, asexual reproduction is by one celled

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sporangiola produced on special structures called sporocladia. Zygospores are produced from progametangia that are undifferentiated from the hyphae. The members of the family are found in soil, dung, dead insects etc. In the family eight genera are recognized. Important are Kickxella, Spiromyces, Linderina, Coemansia, etc. Family - Endogonaceae: The family is of great interest because it contains mycorrhizal associates of roots of wide range of herbaceous and woody hosts from all groups of vascular plants. More than 85% of land plants have mycorrhizal association with these fungi. The important genera are Endogone, Glaziella, Modicella etc. In these fungi described as arbuscular mycorrhizae (AM), no sporangia are produced. Zygospores are produced in clusters and surrounded by web of sterile hyphae and the entire structure is called sporocarp. Family - Helicocephalidaceae: The fungi occur mainly in actively decomposing organic matter where soil fauna, mainly nematodes are present. They are often included in the order Zoopagales since they are associated with nematodes and other soil fauna. In these fungi the asexual spores are produced in helical chains. Family - Saksenaceae: Two genera viz. Saksenaea, named after Indian mycologist Ram Kumar Saksena, and Echinosporangium are included in this family. Both are soil borne. In the genus Saksenaea, columellate sporangia are produced on long sporangiophore like in Mucor but the upper part becomes elongated giving the sporangia flask like appearance. The spores are released through the long neck of the sporangium. Zygospore formation has not been observed in any member of the family. Family - Radiomycetaceae: Two genera viz. Radiomyces and Hesseltinella are recognized in this family. Hesseltinella was isolated from paddy soil, and two species of Radiomyces are isolated from mouse and lizard dung. The vegetative mycelium of both genera possesses stolons and rhizoids like in Rhizopus. Asexual reproduction is by production of sporangiola which are formed on secondary vesicles formed on enlarged primary vesicle. Some sporangia bear long spinose processes. The zygospores are formed as in Mucor. Order - Entomophthorales: The order Entomophthorales is an interesting group of fungi parasitic on both plants and animals or saprobic. There are six genera all of which are placed in a single family Entomophthoraceae. The best known genus is Entomophthora, the species of which are insect parasites. Vegetative structures: The mycelium is limited and is divided by septa with uninucleate or multinucleate segments. There is a tendency for the mycelium to fragment into forms called hyphal bodies. Asexual Reproduction: From the hyphal bodies, a sporangiophore develops which may be undivided (E. muscae) or divided at the upper part to form a number of upright branches (E. sepulchralis). At the tip of the sporangiophore or its branches a single smooth sporangium is formed. It contains a single spore, and the spore wall and sporangial wall are closely appressed. Hence it is often described as conidium. It is forcibly discharged. In addition to the smooth conidia, more pointed ciliate conidia are also formed in some species (E. coronata). If the conidium falls on a suitable host substratum, it germinates and enters the host. If the conidium falls on a unsuitable

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substratum, it germinates and produce a secondary conidium terminally at the tip of germ tube. Secondary conidia are also forcibly discharged. Tertiary conidia may also be formed if it falls on a non-host surface. The process of conidial formation continues for 3 or 4 generations or up to the exhaustion of the conidial protoplast. Sexual Reproduction: The Hyphal bodies act as gametangia and copulate. No special gametangia are formed. The zygospore develops as a lateral outgrowth or bud arising between the two fusing hyphal bodies. The asexual and sexual reproductive structures of Entomophthora are shown in Fig. 6.4.

Fig. 6.4. Conidiophore with conidia, hyphal bodies of Entomophthora.

Order - Zoopagales: The zoopagales consist of microscopic fungi occurring in soil, in rotting vegetation of many kinds and in water. The great majority are either predacious on rhizopod protozoa or on free living nematodes or are endoparasitic in them. Two genera Bdellospora and Amoebophilus are ectoparsitic on amoebae. The predaceous species of zoopagales possess a mycelium of branched, nonseptate hyphae to which the prey adheres owing to the secretion of sticky substance by the hyphae. After capture, the protozoa are invaded by fine, branched haustoria arising from the hyphae at the point of contact. Species that capture

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nematodes intrude a system of trophic hyphae into the body of the victim. In either case the body contents of the prey are consumed by the fungus. In endoparasitic forms, the mycelium is a thallus of variable shape, the commonest form being a broad, filamentous structure coiled in a helix of about one and half turns. Asexual reproduction is by conidial formation, the mode of production is variable. Most of the members produce thread like, spindle shaped or globose spores that are borne singly, in chains or in loose beads at the tips or sides of hyphae. The spores are not forcibly discharged. At maturity, they simply break off and germinate on a suitable substrate. Sexual reproduction, where known, is by zygospore formation, by fusion of a pair of gametangia, which usually consists of undifferentiated hyphal branches in mycelial species. The order consists of about 10 genera, and they are recognised in two families. The predacious fungi are placed in the family Zoopagaceae, and the parasitic forms are placed in the family Cochlonemaceae. The representative genera of the order are shown in Fig. 6.5.

Fig. 6.5. Representative forms of Cochlonema verrucosa, Zoopagales. A. Amoeba enveloping a single thallus of the parasitic fungus. B. Dying amoeba with three internal thalli of the parasite. C. Chains of spores shown in the section with a, b, and c representing corresponding points. D. Mature spores. E. Zygosporangia, one containing a nearly mature zygospore.

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CLASS TRICHOMYCETES The fungi included in this class are distinct from others and are obligately associated with animals of the class Arthropoda, such as insects, millipedes and crustaceans. The relationship between the fungus and the host is not clear whether it is parasitism or commensalism or symbiosis. Hence, their association is described as obligate association. The fungi grow within the guts of their host, most of which are aquatic. Most trichomycetes are formed primarily in the hind gut where they attach to the chitinous gut lining by means of a special structure called hold fast. The fungal body is made up of an erect, branched or unbranched hypha with or without septal formation. They appear like trichomes or leaf hairs, and hence the name trichomycetes. The mycelium never penetrates tissues of the host and obtain their nourishment from the contents of the gut lumen and hence not parasitic in the usual sense. Trichomycetes have been found more or less all over in the world, probably limited only by the distribution of their hosts. They are not host specific and some times more than one species occurs within the same host. They must normally be teased out of their hosts in order to be studied. Sexual reproduction occurs in only a few species. Their taxonomic position is controversial and their placement in Zygomycotina may be considered tentative. Mycelium: It is very limited in extent and may be branched or unbranched. Some have regularly occurring septa, while others lack septa except at the base of reproductive structures. Asexual Reproduction: It occurs by different types of cells like trichospores, sporangiospores, arthrospores or amoeboid cells. In one order (Harpellales) special structures known as trichospores are produced. A trichospore is an exogenous indehiscent, usually elongate sporangium containing a single uninucleate sporangiospore and having one to several basally attached filamentous appendages contiguous with the sporangial wall. The long appendages are thought to function in the passive transmission of the spores by becoming entangled in material on which the host organisms feed. Sexual reproduction: Although sexual reproduction has not been confirmed in trichomycetes, structures referred to as zygospores have been reported in 12 out of 16 genera belonging to the single order Harpellales. They are thought to be sexual spores since they typically form after conjugation between two thalli. The representative genera of the order are shown in Fig. 6.6. Classification: The class Trichomycetes is divided into four orders. Order - Harpellales: Mycelium comprises septate, branched or unbranched hyphae, give rise to trichospores. They are restricted to aquatic larvae. Two families viz. Harpellaceae and Genistellaceae are recognized. Order - Amoebidiales: It is a small order, containing unbranched aseptate mycelium. Asexual reproduction is by sporangiospores or amoeboid cells. A single family Amoebidiaceae is recognized with two genera. Order - Asellariales: It is a small order with 3 genera placed in a single family Asellariaceae. Mycelium is made up of branched septate hyphae, and asexual reproduction is by arthrospores.

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Fig. 6.6. Representative forms of Trichomycetes.

Order - Eccrinales: It is the largest of the four orders. Mycelium is unbranched and aseptate. Asexual reproduction is by sporangiospores usually produced singly in series of sporangia that release the spores in sequence. Three families are recognized. They are 1. Palavasciaceae, 2. Parataeniellaceae, and 3. Eccrinaceae The broad outline of the classification of Zygomycotina is given in Table 6.1. Table 6.1. Outline classification of Zygomycotina. Class

Order

Family

Zygomycetes

Mucorales

Mucoraceae, Pilobolaceae, Choanephoraceae, Thamnidiaceae, Cunninghamellaceae, Syncephalastraceae, Piptocephalidaceae, Dimargaritaceae, Kickxellaceae, Mortierellaceae, Endogonaceae, Helicocephalidaceae, Radiomycetaceae, Saksenaceae

Entomophthorales

Entomophthoraceae

Zoopagales

Zoopagaceae, Cochlonemaceae

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Class

Order

Family

Trichomycetes

Harpellales

Harpellaceae, Genistellaceae

Amoebidiales

Amoebidiaceae

Asellariales

Asellariaceae

Eccrinales

Eccrinaceae, Palavasiaceae, Paratienellaceae

IMPORTANT GENERA GENUS : MUCOR Class: Zygomycetes Order : Mucorales Family : Mucoraceae The genus Mucor is a relatively large genus with about 80 species, and is worldwide in distribution. Common species are Mucor hiemalis, M. mucedo, M. racemosus, M. strictus, M. rouxii etc. Most of the species are soil borne, free living saprophytes. They readily grow on a number of organic substrates with some moisture. The growth of the fungus on exposed bread, vegetables, etc. is very common, and hence it is called common bread mould. The growth of the fungus first appears as white cottony mass which soon turns black with formation of numerous sporangia. Hence, it is also called black mould. Some species like M. javanicus is employed in fermentative production of alcohol. Some species of Mucor such as M. circinelloides, M. racemosus, M. spinosus, etc. are opportunistic pathogens of humans causing infections referred to as mucoromycosis.

Fig. 6.7. Mycelium of Mucor with erect sporangiophores. 1. Mycelium. 2. Sporangiophore. 3. Sporangiospores. 4. Sporangium. 5. Columella. 6. Exposed columella after spore release.

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Mycelium: In Mucor species the mycelium comprises of highly branched aseptate, coenocytic, coarse, tubular hyphae, hyaline, white or light brown in colour. Generally when the spores are deposited on a suitable substratum, they germinate and grow rapidly to form white cottony growth on the substratum. The hyphae easily penetrate the substratum, secrete extracellular enzymes, and digest the material. The mycelium is mainly made up of chitin and chitosan, but not cellulose. Even though the hyphae are aseptate, the cross walls may be produced to separate reproductive structures. The mycelium of Mucor with sporangiophores, sporangia and spores is shown in Fig. 6.7. Dimorphism is observed in a few species of the fungus. For example, Mucor rouxii grows as aseptate mycelium under normal conditions, but shows yeast-like unicellular growth under anaerobic conditions when carbon dioxide content is more, and medium is very rich in glucose. Asexual reproduction: Asexual reproduction is by nonmotile sporangiospores formed in globose or pear shaped sporangia formed at the tip of a branched or unbranched sporangiophore. The sporangiophores develop as coarser, blunt-tipped, aerial hyphae which grow away from the substratum. The tip expands to form the sporangial initial containing numerous nuclei which continue to divide. A dome shaped septum is laid down cutting off a distal portion, which contains the spores, from a central cylindrical or subglobose spore free core, the columella. The protoplasm in the sporiferous region cleaves to form uninucleate bits that eventually round-off and become the spores. The cleavage of the protoplast is accomplished by the fusion of cleavage vesicles associated with endoplasmic reticulum which is continuous with the nuclear envelop. The sporangial wall often darkens and become rough. At maturity the sporangial wall dissolves or breaks into pieces to liberate the aplanospores (Fig. 6.8). In many common species such as Mucor hiemalis, the sporangium becomes converted at maturity into a ‘Sporangial drop’. The sporangial wall dissolves and the spores absorb water so that the tip of the sporangiophore bears a drop of liquid containing spores adhering to columella. In M. mucedo spores are embedded in mucilage. The spore masses are not readily detached by wind, but possibly disperse by rain-splash or insects or after drying. In some species such as M. plumbeus sporangial wall breaks into pieces and spores are dispersed by air currents or mechanical agitation. Different stages in the development of sporangium are shown in Fig. 6.8. Sexual reproduction: In the genus Mucor, sexual reproduction takes place by copulation of two multinucleate gametangia that are mainly similar in structure. The first step in the process is formation of special hyphae called zygophores. The compatible zygophores are attracted to one other and fuse in pairs at their tips to form a fusion septum. The two zygophore tips swell to form progametangia. A septum termed gametangial septum then forms near the tip of each progametangium separating it into two cells, a terminal gametangium and a subtending stalk called suspensor. The fusion septum then dissolves, the protoplasts of the two gametangia mix, karyogamy eventually takes place. The cell formed by the fusion of the two gametangia is initially referred to as prozygosporangium. It enlarges, develops a thick multilayered wall, and becomes the zygosporangium in which a single zygospore develops.

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Fig. 6.8. Asexual reproduction in Mucor. A. Coenocytic mycelium with sporangiophores. B. Young sporangium. C-E. Sporangial development. F. Ruptured sporangium. G. Spores.

The zygospores of Mucor need a period of dormancy before they germinate. The period of dormancy may vary from a few to several months. Even after the dormancy period, the percentage of zygospore germination is very low. At the time of germination, the zygosporangium cracks open and a sporangiophore typically emerges and develops a sporangium called germsporangium, at its tip. Meiosis takes place either some time before or more usually during the process of zygospore germination. In homothallic species, all the sporangiospores in the germsporangium are genetically same, and give rise to homothallic mycelia. In the hetero- thallic species two types of spores i.e. + and – strains are formed. In Mucor hiemalis 90 percent spores found are of + type, and the rest of – type. In Mucor

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mucedo, all spores in a germ sporangium are of same type either + or – type. In this species resting sporangium is multinucleate containing several diploid nuclei. Before the germination, meiosis takes place and apparently all but one of the nuclei formed after meiosis disintegrates. The surviving nucleus multiplies and become incorporated in the spores. Important stages in the life cycle of Mucor are shown in Fig. 6.9.

Fig. 6.9. Life cycle of Mucor. A. Mycelium (+ strain). B. Sporangiophores and sporangia. C. spores. D. Spore germination and repetition of asexual cycle. E-H. Asexual cycle of (-) strain. I-L. Isogametangial copulation between (+) and (-) strains resulting in zygospore formation. M. Germination of zygospore. N. Formation of germ sporangium.

GENUS : RHIZOPUS Class : Zygomycetes Order : Mucorales Family : Mucoraceae The genus is represented by nearly 50 species, and is worldwide in distribution. It occurs mainly in soil and its spores spread aerially and on deposition grow on fruits, food stuffs, all kinds of decaying matter and as a laboratory contaminant. One very common species is R. stolonifer (= R. nigricans), the common bread mould.

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Some species are economically useful and used in industrial fermentations. R. oryzae is used in alcoholic fermentation, R. nodosus and R. sinensis are employed in lactic acid fermentation, R. oligosporus is used in food industry for fermentation of soybean to prepare tempeh. Vegetative Structures: The vegetative structure is mycelial, hyphae are branched and coenocytic. The characteristic feature is the presence of rhizoids at the base of sporangiophores and the stoloniferous habit of mycelium. Aerial hyphae when touch the substratum bears rhizoids and sporangiophores. The mycelium is differentiated into rhizoids, stolons and sporangiophores. The rhizoids are repeatedly branched, penetrate the substratum and anchor the mycelium. The stolons are aerial and arching, running horizontally and parallel to the substratum. At some points of contact with the substrate, the stolons develop tufts of rhizoids and enter into the substratum. The sporangiophores grow upwards from the stolons exactly opposite the rhizoids, singly or in clusters. Hyphae with rhizoids and sporangiophores are shown in Fig. 6.10. Asexual reproduction: It occurs by means of aplanospores produced in the sporangia. The sporangiophores develop opposite to the rhizoids, and are relatively shorter, stout and stiff than those produced in Mucor. Sporangia develop terminally on sporangiophores which may be simple or branched. The apex of aerial hypha swells and cytoplasmic mass along with nuclei move into this part. The swollen part enlarges and develops into a large, globose structure, and it is the young sporangium. On maturity, the contents of sporangium become differentiated into a thick dense layer of cytoplasm with many nuclei towards the peripheral region beneath sporangial wall, and a vacuolated portion towards the centre. A dome shaped septum is then laid down cutting off a distal peripheral portion known as sporiferous region, and a central cylindrical or subglobose spore free core, the columella. The contents of the distal portion become cleaved into 2 to 10 nucleate spores. When the sporangia are mature, the sporangiophores may be seen as coarser, blunt tipped, aerial hyphae growing away from the substratum. At the time of spore release, the sporangial wall cracks into a number of small fragments, the columella collapses and the spores are quickly blown away by wind.

Fig. 6.10. Vegetative structures of Rhizopus. 1. Rhizoids. 2. Stolon. 3. Sporangiophore.

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Sexual reproduction: Most species of Rhizopus are heterothallic, a few like R. sexualis are homothallic. Sexual reproduction is isogamous by conjugation of two identical coeno-gametangia to give rise to the zygospores.

Fig. 6.11. Life cycle of Rhizopus stolonifer.

In heterothallic species, zygospores are formed only when mycelia of compatible strains contact each other (Fig. 6.11). Each branch swells at the tip to develop a progametangium. Dense cytoplasm and numerous nuclei flow to the contacting tips which enlarge further. A septum then separates the terminal portion of the gametangium from the remaining part of the progametangium, which is now called the suspensor. Since gametangia are undifferentiated mass of multinucleate protoplast, they are called coeno-gametangia. As the gametangia mature, the

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separating wall dissolves and the protoplasts of both mix with each other. Nuclear fusion occurs between nuclei of (+) and (–) strains to give numerous diploid nuclei in each zygospore. The young zygospore, containing many diploid nuclei, lies within the parent gametangial wall. It later enlarges and secretes several layered thick wall around it. As the zygospore matures, it breaks up the original gametangial wall into fragments which fall apart exposing the outer, thick, spiny, dark exospore. The wall of mature zygospore is probably 5 layered, two layers in outer exospore and 3 in the inner endospore. The zyspore germinates after a long period of rest. During germination the outer wall cracks and the inner comes out in the form of a germsporangiophore which bear a single terminal germ sporangium containing many spores. Karyogamy in zygospore is delayed which generally occurs during germination. Meiosis probably occurs during formation of germ sporangium. Of the 4 haploid nuclei formed after meiosis (from each diploid nucleus) three degenerate. Hence, the germ sporangia in some species (R. stolonifer) contain either all (+) or all (–) spores or a mixture of two types. The spores in the sporangium are initially uninucleate but this nucleus divides later making them multinucleate. GENUS: PHYCOMYCES Class: Zygomycetes Order : Mucorales Family : Mucoraceae The genus Phycomyces is a common member of the family Mucoraceae. The likely substrates are fatty products, oilcakes, bread, dung and other substances rich in fatty materials. Three species are recognized in the genus, two of which have been used extensively in experimental studies. Of these two species, Phycomyces blakesleeanus found on the dung of animals has been used most widely. Phycomyces has attracted much attention because the species are used extensively for the investigation on phototropism. The photo receptor in the sporangiophore is a flavoprotein located in the plasma membrane. P. blakesleeanus is used for the bioassay of thiamine, a vitamin required as a growth factor. Mycelium: It is typically made up of coenocytic, aeptate, tubular hyphae as in other members of mucoralean fungi. Asexual reproduction: It is by the production of non-motile, sporangiospores. The genus is characterized by the production of very long sporangiophores with a metallic luster that may attain a height of over 80 mm. The sporangia produced are very large and columellate. Sexual reproduction: It is typically by gametangial copulation, but the size and shape of the gametangia as well as the nature of zygophoric hyphae differ from that of Mucor. The zygophores consist of intricately branched lobes that interdigitate with one another. These closely appressed structures then grow up from the substratum and give rise to a gametagial apparatus resembling calipers holding a zygosporangium between their tips. The appendages then develop from the suspensors, branch irregularly, and darken forming a loose cover around the zygosporangium (Fig. 6.12).

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Fig. 6.12. Phycomyces blakesleeanus. E. Sporangiophore. F, G. Gametangia. H. Development of zygospore.

In the zygosporangium of Phycomyces blakesleeanus both karyogamy and meiosis are delayed until germination occurs. Some nuclei fuse at this time and some remain unfused. Some of the diploid nuclei then undergo meiosis, but others do not. This condition results in a mixture of haploid + and – and diploid nuclei. When these become incorporated in the spores, the germ sporangium obviously contains at least three kinds of spores. The diploid spores give rise to homothallic mycelia, or if the nuclei are reduced either in the spores or in the hyphae during division, the spores may give rise to heterokaryotic mycelia that behave as homothallic. The proportion of diploid nuclei diminishes with each asexual generation until no more diploid spores are formed. GENUS: PILOBOLUS Class: Zygomycetes Order : Mucorales Family : Pilobolaceae

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The genus name Pilobolus literally means hat thrower, referring to the sporangial discharge in the fungus. It is a common coprophilous fungus occurring on the dung of herbivorous animals such as horse, buffalo, camel, cow, rat, donkey, goat, rabbit, deer, etc. Common species are Pilobolus kleinii, P. longipes and P. crystallinus. Vegetative structure: Mycelium is typically aseptate, tubular and grows on the dung of herbivorous animals as white cottony growth. It readily grows on common media. All species require a special growth factor called coprogen for their better growth. It is identified as ferrichrome. Asexual reproduction: The genus Pilobolus reproduces asexually by sporangiospores and the sporangial apparatus is characteristic with specialized structures (Fig. 6.13). The sporangiophore is having a basal trophocyst, middle stalk and a subsporangial vesicle below the sporangium. The development of sporangiophore begins with the formation of swollen bulbous structure from surface mycelium. It is called trophocyst. Sporangiophore develops from the trophocyst towards light and its tip enlarges to become the sporangium. As the sporangium develops a bulbous vesicle called subsporangial vesicle is formed below the sporangium. The trophocyst and young sporangiophores are yellow due to carotene content. The sporangium is flattened; with dark black wall, shiny, tough and unwettable. At the base of the sporangium is a conical columella which is separated from the spores by a pad of mucilage. At maturity the sporangium cracks open by a suture running around the base, just above the columella. The spores are, however, not released at this time because they are prevented from escaping by the mucilaginous pad which protrudes through the crack in the sporangium wall as a ring of mucilage. The subsporangial vesicle is turgid and it contains liquid under pressure. It has been estimated that the osmotic pressure of liquid is of the order of 5.5 bars. Drops of excreted liquid commonly adhere to the sporangiophore. Eventually, the subsporangial vesicle explodes at a line of weakness just beneath the columella. The sporangia can be projected vertically upwards for as much as 2 meters and horizontally for up to 2.5 meters. The velocity of projection varies between 4.7 to 27.5 m/sec. with a mean of 10.8 m/sec. The violently discharged sporangium may deposit on herbage surrounding the substratum. The sporangium deposits on the herbage in such away that the mucilaginous ring adheres to it. As the drop of liquid dries the mucilage becomes more firmly attached, and dried sporangia are extremely difficult to detach from vegetation. The spores are not released at the time of sporangial discharge but only after the sporangia have been eaten by an animal then they are released into the gut. Passage through the alimentary canal stimulates their growth in the dung of the animal. Sexual reproduction: All the species of Pilobolus are heterothallic, and sexual reproduction is typically by the copulation of compatible isogametantgia, as in Mucor. Zygospores are known for a few species only. These are located on the substrate and are formed between tong shaped suspensors. The zygospore wall is thick and smooth or nearly so and light brown to black.

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Fig. 6.13. Sporangiophore, sporangium, subsporangial vesicle and sporangiospores of Pilobolus.

GENUS : SYNCEPHALASTRUM Class: Zygomycetes Order : Mucorales Family : Syncephalastraceae The family Syncephalastraceae comprises of a single genus Syncephalastrum and a single species racemosum. The organism is worldwide in distribution, and a common inhabitant of soil and dung. It is also often isolated from grains in storage. Mycelium: The mycelium grows rapidly and the branched sporangiophores may be modified into stolon like structures which produce rhizoids. They are produced in cultures only when the culture becomes old. Asexual Reproduction: It is typically by the production of multispored, cylindrical merosporangia in large numbers on the surface of vesicular structures at the tips of simple or branched sporophores or sporangiophores. The merosporangia arise synchronously from the vesicles of the sporangiophores. The aerial hyphae begin to produce sporophores and it starts with swelling of the tips of aerial hyphal branches into vesicular structures. The vesicles contain large number of nuclei and give rise to merosporangia. Several finger like out growths develop from the vesicles and enlarge to form merosporangia. Spore formation in merosporangia occurs by protoplasmic cleavage and wall differentiation. The contents of each merosporangium differentiate into a single row of 5-10 sporangiospores, each containing one to three nuclei. The sporangial wall shrinks at maturity so that the spores appear in a chain similar to that of Aspergillus. The spore heads remain dry and entire rows of spores are detached by wind leaving abstriction scars on surface of vesicles. Recession of sporangiospores may occur basipetally, leaving segments of

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merosporangia attached to vesicle. The receded spores are smooth walled but remnants of merosporangial wall may remain attached to their surface. Asexual reproductive structures are shown in Fig. 6.14.

Fig. 6.14. Asexual reproductive structures in Syncephalastrum. A. Sporangiophore with merosporangia at the apex. A1. Merosporangium with spores.

Sexual Reproduction: Syncephalastrum racemosum is a heterothallic species. The gametangia are readily produced when compatible hyphae grow together. Zygospores are formed as a result of copulation between equal gametangia in the aerial hyphae. The zygospores are dark and roughened. They are similar to those of Mucor in formation and appearance. GENUS : CUNNINGHAMELLA Class: Zygomycetes Order : Mucorales Family : Cunninghamellaceae The species of Cunninghamella are worldwide in distribution. In India, about seven species are recognised. These occur as saprobes in soil and decaying vegetation. The species are quite commonly encountered on nuts in the warmer regions of the world Mycelium: It is typically mucoraceous, consisting of hyaline, aseptate, coenycytic, much branched hyphae. The fungus can grow luxuriantly on a simple medium with inorganic nitrogen. Asexual reproduction: is characterized by the formation of monosporous sporangiola which are often described as conidia of the fungus. They are hyaline and

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are borne on swollen, globose vesicles on branched or unbranched conidiophores. The conidia are also interpreted as one spored sporangiolum. However, the original wall of the conidium is two layered, and there is no evidence of synthesis of a new layer at or before germination, and the germ tube is continuous with a wall layer whose appearance is consistent with it being a chemically changed part of the original wall rather than an entirely new wall formed de novo. In some species like C. echinulata and C. elegans, the conidia are spiny but in others they are smooth. Asexual reproductive structures are shown in Fig. 6.15.

Fig. 6.15. Asexual reproductive structures in Cunninghamella.

Investigations on the differentiation of wall layers of ungerminated spores of C. elegans have demonstrated the presence of a thin outer electron-dense inner layer corresponding to the sporangiospore wall. Hence, the functionally similar conidia are ontogenetically equivalent to monosporous sporangiola. Sexual reproduction: It is typically by isogametangial copulation as in Mucor. Most of the species are heterothallic in nature. GENUS : CHOANEPHORA Class: Zygomycetes Order : Mucorales Family : Choanephoraceae Species of Choanephora are found on flowers and fruits of higher plants as well as in soil. C. cucurbitarum is one of the commonest species and it causes blossom-blight and soft rot of cucurbits and other plants. The crops infected are red pepper, colocasia, okra, cowpea, common bean etc. Mycelium: The mycelium typically consists of well developed, tubular coenocytic hyphae. In parasitic forms, mycelium is internal in the host tissue. It rapidly devastates the aerial infected parts and soon appears on the surface of the rotting tissue as white cottony growth.

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Asexual Reproduction: In general Choanephora species produce two types of spore bearing structures: 1. Multispored, columellate sporangia and 2. Monosporus, non-columellate sporangiola. The two types of asexual reproductive structures are shown in Fig. 6.16.

Fig. 6.16. Sporangia and sporangiola and spores of Choanephora. A. Sporangiophore with drooping sporangium. B, C. Conidiophores and conidia. D. Dehisced sporangium showing striate spores with appendages. E. Sporangiolum. F. Conidium.

The sporangiophores are usually ovoid or fusiform. They are irregularly or racemosely branched. The sporangial wall breaks open as two halves to release the spores. Sporangiospores are often striate, brown to purple, and possess long, stiff hair like spines at their ends. Monosporus sporangiola develop from the swollen vesicles. The sporangiolar initials of C. cucurbitarum develop synchronously from fertile vesicle as conical projections which later swell at their apices and become ellipsoidal. The monosporus sporangiola remain attached to the vesicle by short pedicels. The wall of the sporangiolum is continuous with that of the fertile vesicle and it is fused to the spore wall. The monosporous sporangiola resemble the sporangiospores in shape but are

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nonciliate and have longitudinal striations with a short hyaline appendage at the base. In addition to these, intercalary chlamydospores with thickened walls are also formed. Sexual Reproduction: Choanephora is heterothallic. Gametangial copulation is typically as in Mucor. The zygospores are borne between tong shaped suspensors, and are slightly roughened.

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Chapter - 7

Ascomycota

The fungi in this group are generally called Ascomycetes. This is the largest group of fungi with more than 2700 genera and 28,000 species. The major characteristic feature is production of sexual spores called ascospores in specialized sac like structure called ascus (Plate 7.1). They are widely distributed and found throughout the world in all hospitable environments. They are mostly free living saprophytes observed in soil, dung, decaying plant parts and on various substrata in water. Some are parasitic on plants and animals causing various types of diseases. Aflatoxin producing species of Aspergillus are asexual stages of ascomycete genera such as Eurotium, Sartorya and Emericella. There are some beneficial organisms also present in this group, and unicellular yeasts are most important fungi in various industries like bakeries, breweries, in the production of vitamins and enzymes.

Plate 7.1. Ascomycete members. 1. Perithecia of Chaetomium sp. 2. Ascocarp of Ceratocystis sp. 3. Apothecia of Peziza sp. 4. Ascospores of Neurospora sp.

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Mycelium: The vegetative thallus of Ascomycetes is mycelium composed of much branched hyphae except in unicellular yeasts. The hyphae are septate, and the septa are perforated by simple pores in the middle, and the nuclei, mitochondria and other cell organelles can pass through the pores (Fig. 7.1). Hence, the protoplast of the mycelium is continuous. In some species the pores appear to be blocked on either side by dense particles (Fig. 7.1). The cell wall is made up of chitin and glucans, cellulose is absent. In the cell wall of yeasts, glucans and mannans are the main constituents.

Fig. 7.1. Septal pore in the hyphae of Ascomycetous fungi. A. 1. Nuclear migration through septal pore. B. 1. Septal pore plugged with membrane bound particles. 2. Nucleus.

Asexual reproduction: It occurs essentially by means of producing conidia, which are formed externally on conidiogenous cells. The conidiophores may be single and free, or may be organized into specialized structures like synnemata, sporodochium, acervulus or pycnidium. The asexual fruiting structures are described in Chapter 9. The conidia also vary from small, single celled structures to very large multicellular forms. Asexual reproduction is also brought out by vegetative means like fragmentation of hyphae and chlamydospore formation. In unicellular yeasts, reproduction occurs through cell division and budding. Sexual reproduction: Sexual reproduction in unicellular yeasts occurs by gametangial copulation. The yeast cells that act as gametangia and their protoplasts fuse completely. In mycelial forms, sexual reproduction may occur by gametangial contact, spermatization or somatogamy (Fig. 7.2). The gametangial contact occurs in fungi belonging to plectomycetes like Eurotium, Talaromyces, etc., and in the members of Erysiphaceae. In the members of Pyrenomycetes like Neurospora, spermatization occurs. Somatogamy occurs in Discomycetous fungi like Peziza, Morchella, etc. In ascomycetes, the female gametangium is ascogonium and male gametangium is antheridium. The ascogonium may be of two types. 1. Elongate or club shaped or cylindrical structure, and 2. Globose base with upper thin, long, slightly curved trichogyne which acts as a receptive hypha receiving male nucleus either from antheridium or spermatium. Antheridium is usually small, cylindrical or club shaped structure.

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Fig. 7.2. Types of sexual reproduction in Ascomycetous fungi. A. Somatogamy in Yeasts. B. Gametangial contact – antheridium wound round cylindrical ascogonium. C. Gametangial contact – ascogonium with trichogyne contacting antheridium. D. Spermatization – ascogonium with trichogyne receiving spermatium from spermagonium. E. Somatogamy between vegetative hyphae.

Development of asci: In unicellular yeasts, karyogamy follows somatogamy and zygote is formed. The cell is then called ascus mother cell. The nucleus undergoes meiosis to form four haploid nuclei, and four ascospores are formed by free cell formation.

Fig. 7.3. A-G. Development of ascogenous hyphae from ascogonium and ascus formation. 1. ascogenous hypha. 2. Crozier. 3. Completely bent crozier. 4. Nuclear fusion in subapical cell. 5. New ascogenous hypha. 6. Ascus mother cell. 7. Meiotic division in developing ascus. 8. Mature ascus.

In mycelial forms, karyogamy does not follow somatogamy and the two nuclei pair together to form dikaryotic stage. A number of papillae are produced on the ascogonial wall, and they enlarge into hyphae like structures called ascogenous hyphae. The pairs of nuclei enter into these hyphae. Septa are formed in the ascogenous hypha in such a way that each cell receives two compatible nuclei. The ascogenous hypha elongates and bends at the tip to form a hook cell called crozier.

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The two nuclei in the crozier divide in such a way that of the four nuclei formed, two compatible nuclei occur at the tip of the crozier and one above it and one below it. Then the septa are formed to separate single celled tip cell and the third cell and two nuclei are in the hook or crozier cell. Then the crozier cell transforms into ascus mother cell. Karyogamy occurs in it followed by meiosis producing four haploid nuclei. Each of these nuclei undergoes one mitotic division resulting in formation of 8 haploid nuclei. Each nucleus is surrounded by a bit of cytoplasm and transforms into an ascospore by a process called free cell formation (Fig.7. 3). Ascocarps: As the asci are developing, sterile mycelium around the developing asci is stimulated to form a thick cover around the asci, and the entire structure is called ascocarp. Usually the ascocarp possesses a clear well defined wall called peridium. Except the members of Hemiascomycetes, all other fungi produce distinct ascocarps. According to the structure, the ascocarps are mainly of four types (Fig. 7.4).

Fig. 7.4. Types of ascocarps. A. Cleistothecium. 1. Peridium. 2. Ascus. B. Perithecium. 1. Ostiole. 2. Periphyses. 3. Hymenium. C. Apothecium. 1. Asci. D. Psedothecium. 1. Ostiole. 2. Asci.

a)

Cleistothecium: The ascocarp in which globose, evanescent asci are arranged randomly without forming a hymenium is described as cleistothecium. Usually peridium completely envelops the asci without an ostiole. The ascospores are unicellular. This type of ascocarp is characteristic of the class Plectomycetes.

b)

Perithecium: The ascocarps which are globose, oval or flask shaped structures with an ostiole at the tip for the release of ascospores, and asci

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are arranged in a clear layer called hymenium, are described as perithecia. The perithecial fungi are placed in the class Pyrenomycetes. The presence or absence of hymenium is an important character rather than the presence or absence of ostiole. Hence, the ascocarps of powdery fungi, which were earlier regarded as cleistothecia, because of the absence of ostiole, and placed in the class plectomycetes, are now considered as perithecia because they have hymenium, and placed in the class pyrenomycetes. The internal structure of the ascocarp is called centrum. Based on the centrum types, three groups are recognized (Fig. 7.5).

Fig. 7.5. Perithecial centrum types in Pyrenomycetes. A. Xylaria type centrum. 1. Ascus 2. Paraphyses. B. Diaporthe type centrum. 1. Ascus. C. Nectria type centrum. 1. Ascus. 2. Apical paraphyses.

Xylaria type centrum: The typical perithecium with basal hymenium interspersed with sterile structures called paraphyses. Diaporthe type centrum: In mature ascocarp the hymenium is composed of only asci, and paraphyses are absent, because they are evanescent and dissolve during development. Nectria type centrum: Sterile hyphae grow downwards from the upper part of the ascocarp. These are called apical paraphyses. Basal paraphyses are usually absent. c)

Apothecium: This is a cup shaped structure with open hymenium composed of cylindrical asci interspersed with long paraphyses. This type of ascocarp is characteristic of the class Discomycetes.

d)

Pseudothecium or ascostroma: They appear like perithecia but do not possess a clear peridium. Hence they are called pseudothecia. Since the asci are formed in locules formed in a stroma, they are also called ascostroma, and the fungi are placed in the class Loculoascomycetes. Another important character is the presence of bitunicate asci. In the

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asci the outer layer is rigid, and inner layer is elastic. At the time of maturity, the inner layer pierces through outer layer and comes out. Structure of the asci: With the formation of ascospores, the ascus development is complete. The asci may be globose, oval, clavate or cylindrical. Some are stalked while some are sessile. Usually the ascus is a sac like structure without septa. However, septate asci are reported in some but they are rare. Different types of asci are shown in Fig. 7.6.

Fig. 7.6. Different types of asci. A. Globose ascus. B. Stalked ascus. C. Septate ascus (rare type). D. Club shaped ascus. E. Cylindrical ascus.

Each ascus wall is made of two layers. The inner layer is called endotunica and the outer layer exotunica. Basing on the bebaviour of the wall layers three types of asci are recognized. They are 1.

Prototunicate asci: In this type the wall layers become evanescent quite early in the developmental stages, and mature ascospores lie free in the ascocarp cavity.

2.

Unitunicate asci: In this type the two wall layers are persistent and behave as a single unit throughout.

3.

Bitunicate asci: In this type, the wall layers are persistent but behave differently. The outer exotunica is rigid while the inner endotunica is elastic. At the time of spore release, the outer layer ruptures and inner layer balloons out.

Release of ascospores: The asci in Plectomycetes are prototunicate and dissolve usually at the maturity, and the ascospores are released into the ascocarp centrum. In the unitunicate asci the release of ascospores occurs through the apical region either through a small or broad pore or through the narrow elongate pore in the thickened ascal apex. When the ascal apex is thickened having a long narrow channel like pore, the ascospores are released one by one, as the bullets come out of the pistol, and hence the mechanism is described as ballistospore method. Such method of ascospore release is found in the members of Pyrenomycetes.

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In the members of the class Discomycetes, especially those belonging to Pezizales, the broad pore at the ascal apex is covered by a shield shaped structure called operculum. At maturity, the operculum is thrown out and all the ascospores in the ascus are released simultaneously. A number of asci mature at a time and release the spores at a time and the released spore mass appear like puff of smoke. Hence this method of spore release is described as puffing. The ascal tips helping in release of ascospores are shown in the Fig. 7.7.

Fig. 7.7. Different types of ascal tips. A. Ascus without any pore. B. Ascus tip with a small pore. C. Thick ascal tip with a narrow long pore. D. Ascus with operculum. 1. Ascal pore. 2. Operculum. E. Bitunicate ascus with projecting endotunica. 1. Endotunica. 2. Exotunica.

Fig.7.8. Different types of ascospores.

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Ascospores: The ascospores vary greatly in size, shape, colour, wall ornamentation and other characters. In size they range from 1-1000 µm, but most of the spores are in the range of 10-30 µm. They may be single celled or multicellular. The unicellular spores may be globose, oval, pulley shaped or hat shaped. The multicellular forms may have only transverse septa or have both transverse as well as longitudinal septa. Some are long thread like. Various types of ascospores are shown in the Fig. 7.8. CLASSIFICATION In recent classification of Phylum Ascomycota based on the occurrence and structure of ascocarps, now three subphyla are recognized. Subphylum Taphrinomycotina Class Taphrinomycetes Order Taphrinales Class Schizosaccharomycetes Class Pneumocystidiomycetes Class Neolectomycetes Subphylum Saccharomycotina Class Saccharomycetes Order Saccharomycetales Subphylum Pezizomycotina Class Pezizomycetes (operculate discomycetes) Order Pezizales Class Dothideomycetes Order Dothideales Order Capnodiales Order Myriangiales Order Pleosporales Order Botryosphaeriales Order Hysteriales Order Patellariales Class Eurotiomycetes Order Eurotiales Order Onygenales Order Coryneliales Order Mycocaliciales Order Chaetothyriales Order Pyrenulales

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Order Verrucariales Class Laboulbeniomycetes Order Laboulbeniales Order Pyxidiophorales Class Lecanoromycetes Many orders of Lichens Class Arthoniomycetes Order Arthoniales Class Leotiomycetes Order Helotiales Order Erysiphales Order Rhytismatales Order Cyttariales Order Thelebolales Class Sordariomycetes Order Sordariales Order Ophiostomatales Order Diaporthales Order Coniochaetales Order Chaetosphaeriales Order Hypocreales (includes Clavicipitales) Order Microascales Order Melanosporales Order Coronophorales Order Xylariales Order Trichosphaeriales Order Phyllachorales Order Meliolales Order Lulworthiales Order Calosphaeriales The classification followed here is of Ainsworth (1973). He classified Ascomycotina into six classes: Hemiascomycetes, Plectomycetes, Pyrenomycetes, Discomycetes, Laboulbeniomycetes, and Loculoascomycetes. CLASS - HEMIASCOMYCETES These are considered primitive Ascomycetes and are characterized by the absence of ascocarp. The asci are formed singly usually following karyogamy, but

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ascigerous hyphae are not formed. The yeasts of this class are highly important economically. Three orders are recognized in this class. They are Protomycetales, Endomycetales and Taphrinales. Order - Protomycetales: This is a poorly known group of fungi parasitic on higher plants producing galls and discolorations. The chief characteristic feature is a spore sac believed by some mycologists (Gaumann, 1952; Martin, 1961) to represent a compound ascus i.e. synascus, and others (Bessy, 1950) consider it as a sporangium. A single family Protomycetaceae is recognized in the order. Order – Endomycetales: Members of this group are sometimes known as Saccharomycetales also. It includes yeasts which produce ascospores and other related fungi in which the zygote develops directly into an ascus. They may be unicellular, or have pseudomycelium or well developed septate or coenocytic mycelium. Most of the members are saprobic, rarely parasitic. Some members are important human and animal pathogens. Yeasts are very important in alcoholic fermentation and bread making. The order has four families. Order - Taphrinales: They are parasitic on higher plants and ferns, cause malformations of the tissues resulting in leaf curl, witches brooms, plum pockets and blister like lesions. Members form subcuticular or subepidermal layer of ascogenous cells from the mycelium and asci are arranged in a palisade layer. It has single family Taphrinaceae and a single genus Taphrina. CLASS – PLECTOMYCETES This class includes the fungi that produce simple cleistothecium type ascocarp. The ascocarp peridium varies from none to thin wefts of hyphae forming a covering over asci, to a definite pesudoparenchymatous peridium. Ostiole is absent. Paraphyses are completely absent. The asci do not form a hymenium and are randomly scattered in the ascocarp centrum. They are typically thin walled, globose to pyriform, and evanescent i.e. prototunicate. The ascospores are unicellular. A single order Eurotiales is recognized in this class. Majority of Eurotiales are saprophytes in soil or on substrata such as dead insects, wood, dung, hair, bones, feathers etc. Skin diseases of humans and other animals may be caused by parasitic species of form genera Trichophyton and Microsporum, which are conidial stages with teleomorphs in Arthroderma and Nannizzia respectively. The causal agent of serious and often fatal disease of humans Histoplamosis is caused by Histoplasma capsulatum, the anamorph of Emmonsiella capsulata. The two most common genera of fungi viz. Aspergillus and Penicillium, have their perfect stages in Plectomycetes. Some Aspergillus species such as A. flavus and A. fumigatus cause animal and human diseases known as aspergillosis. Aspergillus flavus produces aflatoxins. The most important antibiotc penicillin is produced by Penicillium notatum & P. chrysogenum. CLASS – PYRENOMYCETES The pyrenomycetes are defined as ascomycetes with ascocarps entirely surrounded by a peridial wall, and containing unitunicate asci which are primarily

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arranged in a hymenial layer. In general, the ascocarp is provided with an apical opening (ostiole) which is lined by periphyses. The ascocarp is usually a flask shaped structure with globose base and a short or long beak, giving a flask shaped appearance. The class is divided into four orders viz. 1. Erysiphales. 2. Meliolales. 3. Coronophorales and 4. Sphaeriales. The first three orders are small, specialized groups each with a single family. The ascocarps in these three orders are atypical perithecia. All the rest of the fungi with typical perithecia are placed in the order Sphaeriales comprising 15 families. Hence, the classification of perithecial fungi is considered as fluid.

Fig. 7.9A-F. Appendages and asci in the ascocarps of Erysiphales. A-D. Type of appendages on ascocarps. E-F. Variation in number of asci within the ascocarp.

Order - Erysiphales: A single family Erysiphaceae is recognised in this order. All the members of the group are ecologically obligate ectoparasites causing conspicuous diseases called powdery mildews. Ectoparasitic mycelium produces enormous number of phototropic erect conidiophores and conidia. Mycelium, conidiophores and conidia are hyaline which in mass appear as white powder on the

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surface of the infected plant parts which are mainly leaves. Hence, the diseases are called powdery mildews. The important genera in the group are Erysiphe, Spaerotheca, Podosphaera, Microsphaera, Uncinula and Phyllactinia. These are recognised based on the type of appendages on the ascocarps which may be mycelioid or dichotomously branched, circinate or bulbous (Fig. 7.9). The ascocarp centrum in this group is described as Phyllactinia-type. It is characterized by the presence of unitunicate asci in a basal hymenium and absence of paraphyses. The ascocarp generally lacks an ostiole. Order - Meliolales: A single family Meliolaceae is recognized. The members are called black mildews. More than 1800 species are described basing on the host range. They are ectoparasitic fungi mainly found in tropics. In these fungi, mycelium consists of superficial, brown, septate hyphae which are firmly attached to the host surface by two types of hyphopodia viz. 1. Capitate hyphopodia and 2. Mucronate hyphopodia. The fungi produce no conidial stages. Ascocarps with peridium completely enclosing the asci often with rudimentary ostioles are formed. Ascocarps show Phyllactinia type centrum. The ascospores are dark brown 2-3 septate phragmospores. Representative members of the order are shown in the Fig. 7.10.

Fig. 7.10. A-C. Characters of meliolales. Meliola caesariae. a- ascospore; b- capitate hyphopodium, c- mucronatehyphopodium, d- germinating ascospore. B. Asterdiella homalii-angustifolii. a - ascocarps, b- capitate hyphopodium, c- mucronate hyphopodium, d- ascospore. C. Meliola homaliicola. a- ascospore, b- seta, c- capitate hyphopodium, d- mucronate hyphopodium.

Order – Coronophorales: A single family Coronophoraceae is recognized. These are wood inhabiting fungi. Mycelium is scanty. Ascocarps develop within the substrata, and they lack a true peridium. True ostioles are absent, but openings develop at the apex by disintegration of the cells. Asci are unitunicate. Thus, these fungi are considered as loculate ascomycetes with unitunicate asci. Order – Sordariales: Majority of perithecial fungi are placed in this order. Ascocarps are spherical or flask shaped. They are mostly ostiolate and have a bright fleshy or dark carbonaceous peridium. Asci are unitunicate, and arranged in a basal hymenium. The asci may be spherical, clavate or cylindrical in shape. Paraphyses are mostly present. Ostiole is lined by periphyses. The order, as conceived by Muller and Von Arx (1973) in ‘The Fungi – An advanced treatise’ Volume 4A, comprises 15 families.

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Family - Ophiostomataceae: Asci are formed at various heights in the ascocarp cavity and no hymenium is formed. The ascus walls are evanescent and the ascospores are released into the ascocarp cavity. The peritheciea are provided with an ostiole and a long beak of neck. The ascospore mass oozes out through long neck. The best known example is Ceratocystis ulmi which cause Dutch elm disease. Order - Xylariales: Typical perithecia with cylindrical asci arranged into a basal hymenium interspersed with persistent paraphyses are formed. The centrum type is described as Xylaria – type. The members of the family are free living macrofungi. Important examples are Xylaria, Hypoxylon, Nummularia, etc. Family - Diatrypaceae: These fungi have Xylaria type centrum. Ascocarps are formed in stroma composted of both host tissue and fungal hyphae. Paraphyses are formed but gelatinize when mature. Important examples are Diatrype, Diatrypella, Eutypa, etc. Family - Clavicipitaceae: Most important genus is Claviceps, the species of which are pathogenic on crop plants causing ergot disease. Sclerotia are the resting structures which germinate to give rise to a stipe which at the bulbous tip bears ascocarps arranged in semicircular fashion; the ascocarp centrum is Xylaria-type. Ascospores are very long and thread like. The large macroscopic sclerotia contain a large number of alkaloids and are the source for producing LSD, one of the first narcotic drugs to be manufactured. Family - Polystigmataceae: Pathogenic fungi producing foliar disease called tar- spots on grasses and other host plants. The ascocarps are formed in the host tissue. The best example is Phyllachora. Family - Amphispheariaceae: Ascocarps may be immersed in the host tissue, arranged around a collective ostiolum or may have individual ostiole. The ascocarp centrum is Xylaria-type. Ascospores are one or two celled, often fusiform. Important examples are Physalospora, Amphisphaeria, Hyponectria, etc. Family - Sphaeriaceae: The fungi produce typical ascocarps with Xylaria-type centrum, and they may or may not form in stroma. Stromata bright crust like or dark. Ascomata are small, brown or black, developing superficially, ascospores one or two celled brown to black. Important genera are Trichosphaeria, Calosphaeria, Eriosphaeria, etc. Order – Diaporthales: The fungi included in this family are plant pathogens or saprophytes, perithecia produced in a stroma, and Diaporthe type centrum characterized by evanescent paraphyses, asci formed in a hymenium with short stalks. The stalks are evanescent at maturity releasing the asci into the ascoma. Two important genera are Diaporthe and Endothia, the later causes severe blight disease of chest nut. Family - Melanosporaceae: The fungi are nonstromatic and have Diaporthetype centrum. The ascospores are characteristically single celled and dark colored. The ascospores are released into the ascocarp cavity and ooze out through ostiole in a cirrhus. The best known examples are Melanospora, Chaetomium, etc. Family - Halosphaeriaceae: Marine perithecial fungi are placed in this family. The centrum is Diaporthe-type. They are parasitic on red algae or saprobes. The

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ascocarps are immersed in the substratum, ascospores occur in gelatinous mass. Important examples are Halosphaeria, Nautosphaeria, Corollospora, etc. Family - Hypocreaceae: The family includes fungi in which the ascocarps are fleshy, bright, often white, yellow, red, green or blue, and are produced either superficially on a stroma or immersed in stroma. Ascocarp centrum is Nectria-type, and is characterized by formation of apical paraphyses which grow down as palisade layer and finally disintegrating as asci grow up among them. Important genera are Hypoxylon, Nectria, Gibberella, etc. Order - Hypocreales: These are mostly mycoparasites on fruit bodies of basidiomycetes causing symptoms of hypertrophy. The perithecia are immersed in a loose prosenchyma, and show Nectria type-centrum. Important example is Hypomyces. Family - Coryneliaceae: This is a family of uncertain affinity. The fungi produce unitunicate asci in ascostroma, impotant examples are Corynelia, Coyneliopsis, Triposora, etc. Family - Verrucariaceae: All lichenised fungi that produce perithecia are placed in this family. CLASS – DISCOMYCETES The fungi that produce apothecia are placed in the class Discomycetes. Apothecium is defined as an open ascocarp. Discomycetes include the cup fungi, the earth tongues, morels and the truffles. Fruiting bodies of some species are brilliantly coloured red, yellow or orange. Some others are brown to black. Besides the typical cups or discs, there are sponges and bells, saddles and tongues, and brain like fruiting bodies and winged ascocarps, and some resemble small leather bags filled with jelly. All these different kinds of apothecia have one characteristic in common – they are open, they bear their asci on the surface or in large open cavities, and they puff their spores out in clouds in some cases with a hiss. The apothecium consists of three parts viz. hymenium, hypothecium and excipulum (Fig. 7.11). The hymenium is the layer of asci that lines the surface or hollow part of the disc, cup, saddle or other variously shaped structures. It is made up of club shaped or cylindrical asci, usually with many or few paraphyses among them. The paraphyses may be as long as the asci or somewhat shorter. In some species the tips of the paraphyses may be branched. The tips of these branches may unite above the asci and form a layer called epithecium. Hypothecium is a thin layer of interwoven hyphae immediately below the hymenium. The apothecium proper i.e. the fleshy part of the ascocarp that supports the hypothecium is called excipulum. It consists of two parts, the ectal excipulum, which is the outer layer of the apothecium, and medullary excipulum which is the inner portion. In classifying these fungi, the type of ascus and its dehiscence are more important than the shape and structure of ascocarps. The Discomycetes occur both on the ground (epigeal) and below the ground (hypogeal). Majorities are epigeal and only a small group is hypogeal. Among the epigeal forms, two major types are recognized, 1. Operculate forms, and 2. Inoperculate forms. Operculum is a cap like or lid like structure at the tip of the asci. At the time of spore discharge, the

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operculum is thrown out and the spores are released at a time like a puff or cloud of spores.

Fig. 7.11. Details of typical apothecium in Discomycetes.

The class is divided into 7 orders. Of these, only the members of the order Tuberales are hypogeal, while the rest are epigeal. Among the epigeal forms the members of Pezizales and Cyttariales are operculate while the members of Helotiales, Ostropales and Phacidiales are nonoperculate. Order – Mediolariales: This is a very small order in which a single family Medeolariaceae with a single genus Medeolaria was recognised. The species of fungus are pathogenic on North Indian cucumber Medeola and cause rosette symptoms with shortened internodes. The nature of ascus tip has not been studied. Order – Cyttariales: This is also a very small order with single family Cyttariaceae and single genus Cyttaria. About 10 species are recognised in the genus and all are pathogenic on species of Nothofagus producing galls on branches. The genus is characterized by presence of long cylindrical asci which open by a broad pore. The order is considered as operculate since the asci have a broad pore even though the operculum as such has not been observed and considered as lost during development. Order – Pezizales: This is a large order of operculate and epigean cup fungi. They are mostly free living saprophytes growing on soil and other substrata. They produce apothecia of various sizes and shapes. The fruit bodies are composed of

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tissues which are usually fleshy, somewhat brittle to leathery and may rarely be gelatinous. The asci are arranged in a distinct hymenium interspersed with paraphyses. The asci are cylindrical, and the ascus apex has apical or sub-apical lid, the operculum, which is thrown back at maturity to release ascospores. These fungi are of little importance except for edible species of morels. In this order seven families are recognized. 1. Pezizaceae, 2. Morchellaceae, 3. Helvellaceae, 4. Pyronemataceae, 5. Ascobolaceae, 6. Sarcoscyphaceae and 7. Sarcosomataceae. Order – Helotiales: This is a relatively large and diverse order of inoperculate Discomycetes comprising about 150 genera and 2000 species. The members are mostly free living saprophytes occurring on decaying plant parts. Some are plant pathogens causing serious diseases of economically important crop plants. Among the pathogenic species, Sclerotinia fructigena and S. fruiticola cause brown rot of apples and other fruits. Sclerotinia sclerotiorum attacks a diversity of host plants such as carrots, potatoes etc. Ascocarps are clavate, pileate, cupulate or discoid in shape, stipitate or sessile, small, often inconspicuous, superficial or immersed in host tissue. The asci are clavate or cylindrical with an apical pore through which the ascospores are violently discharged. The ascospores are round, ellipsoidal or elongated, septate or nonseptate. Even though the fruit bodies are often small and inconspicuous, the fruit bodies of Geoglossum and related genera such as Trichoglossum, Spathularia etc. are described as earth tongues, because the fruit bodies are club shaped or fan shaped or tongue shaped. They commonly occur on rotting wood or decaying leaves or on organic matter rich soil. In the order 8 families are recognized. 1. Ascocorticiaceae, 2. Dermataceae, 3. Geoglossaceae, 4. Hemiphacidiaceae, 5. Hyaloscyphaceae, 6. Leotiaceae, 7. Orbiliaceae and 8. Sclerotiniaceae. Order – Ostropales: This is a small order of inoperculate fungi, with a single family Stictidaceae. The members are saprobes or parasites of herbaceous stems and bark, and some form crustose lichens. The asci are very long and cylindrical. The young ascal apex is greatly thickened and traversed by a pore through which filiform ascospores are forcibly discharged in most of the genera, a character similar to the members of clavicipitaceae of Pyrenomycetes. The common genus in the group is Cordyceps. Order – Phacidiales: All the members of the order are pathogenic fungi infecting higher plants producing tar-spot symptoms on leaves and other infected parts. The ascocarps are formed in the host tissue and covered by black membrane. The asci are inoperculate. Three families are recognized in the order. They are Rhytismataceae, Cryptomycetaceae and Phacidiaceae. Order – Tuberales: The members of Tuberales, commonly called truffles, are highly prized edible Ascomycetes. They are most common in France and Italy. The fruit bodies are hypogeal and remain closed in most species, and are fleshy to leathery in texture and globose in shape. The hymenium lines a single or complex series of locules. The asci may be globose or oval with 8 or less number of ascospores, and the number varying among the asci in the same species. The

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ascospores are unicellular, hyaline or brown, often spherical, smooth or spiny. Many species of tuberales form sheathing mycorrhiza with tree like Pinus, beech, oak, etc. The mycelium of truffles can be grown in artificial media in the laboratory, but no fruit bodies are formed in culture. However, scientists in France and Italy have succeeded in artificially synthesizing truffle mycorrhizae by inoculating susceptible tree seedlings with spores or mycelium of edible fungus. The most common species are Tuber melanosporum (black or perigord truffle), T. magnatum (white truffle) and T. aestivum (summer truffle). Four families are recognized in the order. They are 1. Elaphomycetaceae, 2. Terfeziaceae, 3. Geneaceae, and 4. Tuberaceae. CLASS – LOCULOASCOMYCETES The two most important characters of the fungi included in this class are 1. Asci are bitunicate and 2. The ascocarp is a pseudothecium. The most dependable character of this subclass is presence of bitunicate asci. The inner wall (endotunica) expands two or three times the length of the outer wall (ectotunica) at the spore discharge (Fig. 7.12).

Fig. 7.12. Rapid expansion of the endotunica (EN) of the bitunicate ascus. A. The mature obpyriform ascus has an ectotunica (EC) which surrounds the ascus and an endotunica deposited in the upper part of the ascus and within ectotunica. B. Ectotunica ruptured and endotunica elongated. C. Extended endotunica.

The bitunicate asci are produced in the locules in the fungal tissue called stroma. The locules are formed in the stroma either lysogenously or schizogenously. An opening is formed above the locules for the release of ascospores, and the entire structure gives the appearance of a perithecium, but there is no clear peridium around the locules. Since the asci are formed in locules, they are described as loculoascomycetes. Based on the development and nature of the components of the ascocarps, three types of centrum are recognized in this order.

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Elsinoë type centrum: In this type of centrum, ascogenous hyphae develop from the ascogonium, spread out into the stroma and develop asci individually. A locule is formed around each ascus. The monoascus locules are often scattered, but a fertile region may be differentiated in the stroma, and locules may be developed in a single irregular layer. Dothidea type centrum: In this type, the locules are delimited around the ascogonia and the asci, originating at a single point, push up into the pseudoparencyma of the locule and spread out like a fan. Either a pore is dissolved in the stoma over the mature asci or an ostiole with periphyses develops. No sterile threads of any sort develop among the asci. Pleospora type centrum: In this type, vertical sterile hyphae, the pseudoparaphyses, originate in the stroma where the ascogonium is located. A locule then develops in the region around the ascogonium and pseudoparaphyses. Asci now develop among the pseudoparaphyses and grow upward between them. A pore is dissolved in the stroma above the mature asci. Five orders are recognized in this class. Order – Myriangiales: The order comprises fungi with typically globose, thick walled asci that develop singly in the uniascal locules separated by the stromatic tissue. The locules may be distributed at various levels or form a single layer in a fertile portion of the stroma. The important genera of the order include Piedraia hortai, the causal organism of blak piedra of humans; the species of Myriangium are parasitic on scale insects, and the species of Elsinoë and Bitancourtia are parasitic on leaves and stems of angiosperms. Four families are recognized in the order. They are 1. Myriangeaceae. 2. Atichiaceae. 3. Saccardiaceae and 4. Sccardinulaceae. Order – Dothideales: The members of the order have Dothidea type centrum. The pseudothecium have a group of basal asci without any paraphyses. The most important genera include Mycosphaerella which is a large genus with about 1000 species and its conidial stages like Cercospora, Septoria, etc. cause important leaf spot diseases. The genus Mycosphaerella is placed in the family Dothideaceae, which is sometimes called Mycosphaerellaceae. The sooty moulds included in this order are epiphytic fungi growing on honey dew exudates of insects on leaves. The species of Capnodium are widespread epiphytes in tropics. The order includes 8 families 1. Dothideaceae, 2. Dotheoraceae, 3. Capnodiaceae, 4. Englerulaceae, 5. Trichothyriaceae, 6. Chetothyriaceae 7. Pseudospheariaceae and 8. Parodiopsidaceae. Order – Pleosporales: The members of this order are characterized by the Pleospora – type centrum in which the asci develop among psuedoparaphyses. These fungi chiefly occur on wood and on dead herbaceous stems. Some are important plant pathogens. The conidial stages of Cochliobolus and Pyrenophora are parasitic on grasses and cereals. Species of Pleospora are pathogenic on wide range of cultivated leguminous crops. Venturia inaequalis causes apple scab, one of the devastating plant diseases. Some are common coprophilous fungi. For example, Sporormia is very common on dung of herbivorous animals.

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The order includes 8 families. They are 1. Pleosporaceae, 2. Sporormiaceae, 3. Venturiaceae, 4. Botryospheriaceae 5. Mesneriaceae, 6. Mycoporaceae, 7. Dimeriaceae and 8. Lophiostromataceae; Order – Hysteriales: A small group of fungi producing ascocarp described as hysterothecium. It is a boat shaped pseudothecium opening by a longitudinal slit and becoming apothecioid when moistened. Asci are long cylindrical structures formed in a basal hymenium. The asci grow among persistent pseudoparaphyses as in Pleospora type centrum. Six families are recognized in this order. 1. Hysteriaceae, 2. Arthoniaceae, 3. Opegraphaceae, 4. Phillipsellaceae, 5. Patellariaceae and 6. Leconactidiaceae. Order – Hemisphaeriales: A large group of loculoascomycetes with flattened ascocarps. They occur primarily on living leaves, young stems etc. as hyperparasites on superficial fungi or as ectocommensals apparently growing on plant exudates. Ascocarp is a flat plate of cells with a central pore and is appressed to the host surface, and develops beneath the superficial mycelium. Ascocarp centrum resembles Pleospora-type. 11 families are recognized. 1. Microthyriaceae. 2. Trichopeltinaceae. 3. Munkiellaceae. 4. Micropeltidaceae. 5. Asterinaceae. 6. Briefeldiellaceae. 7. Aulographaceae. 8. Parmulariaceae. 9. Stephanothecaceae. 10. Schizothyriaceae and 11. Leptopeltidaceae. CLASS - LABOULBENIOMYCETES The organisms now classified in this group were first noticed by French entomologists Alex Laboulbene and August Rouget in 1840, and the fungus was first described in 1853 as Laboulbenia rougetii, in honour of their discoverers. These fungi are parasitic mostly on insects, and a few are parasitic on marine red algae. The mycelium is very much reduced, asexual reproduction is absent and sexual reproduction is by producing asci in perithecia. Each ascus produces only four ascospores which are either one or two celled (Fig. 7.13). Two orders are recognized in this class.

Fig. 7.13. Thallus and ascocarp of Laboulbenia.

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Order – Laboulbeniales: The fungi parasitic on insects are placed in this order. Ascospores are two celled. Three families were recognized in the order. 1. Laboulbeniaceae, 2. Ceratomycetaceae, and 3. Peyritshiellaceae. Order – Spathulosporales: The fungi parasitic on red algae are placed in this order. The ascospores are one celled. A single family Spathulosporaceae is recognized. A single genus Spathulospora was recognised with 5 species. IMPORTANT GENERA GENUS : TAPHRINA Class : Hemiascomycetes Order : Taphrinales Family : Taphrinaceae The genus Taphrina is the only one genus recognized in the family Taphrinaceae and the order Taphrinales. All the species in the genus are parasitic on vascular plants causing malformations of the tissues they attack and produce symptoms such as leaf curl, blisters, witch’s broom etc. They are all ecologically obligate pathogens. Some species cause severe damage to crop plants. Species causing major diseases are given in Table 7.1. Table 7.1. Taphrina species causing diseases. Pathogen

Disease

Taphrina deformans

Peach leaf curl

T. maculans

Leaf blotch of turmeric

T. pruni

Plumpockets on Prunus

T. cerasi

Witch’s broom of cherries

T. caerulescens

Leaf spot of Quercus

T. ulmi

Leaf spot of Ulmus

T. laurencia

Pteris quadriaurita (Pteridophyte)

The mycelium infects only superficial cells. It may be subcuticular or intra epidermal or sometimes mycelium may become intracellular in the leaf tissue. Asexual Reproduction: There is no special asexual reproduction process as such. However, ascospores may show the process of budding producing a large number of cells called blastospores. Sexual reproduction: Taphrina produces no sex organs. Ascospores or blastospores are haploid and uninucleate. They are aerially dispersed, and when they are deposited on a suitable host surface they germinate. During germination the nucleus divides into two and pairing occurs and they divide synchronously. Alternatively the two germ tubes from compatible cells may fuse and dikaryotization occurs. The mycelium grows in subcuticular space or in the epidermal cells. When mycelium accumulates, and form a layer, the outer layer breaks into components of

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binucleate cells and it becomes ascogenous cell in which karyogamy occurs. The diploid nucleus divides mitotically and a transverse septum forms to produce a basal cell and apical ascus mother cell. Meiosis occurs in the ascus mother cell and 8 nuclei are formed. Ascospores are formed by free cell formation. The ascospores are violently discharged, and are aerially dispersed. The aerial ascosspores are deposited in large numbers on the leaves of nearby plants and get severely infected. The leaf spots on turmeric caused by Taphrina maculans, and mycelium and asci of the pathogen are shown in the Fig. 7.14.

Fig. 7.14a-c. Taphrina symptoms on turmeric leaf, and mycelium and asci in epidermal cells

GENUS : SACCHAROMYCES Class : Hemiascomycetes Order : Endomycetales Family : Saccharomycetaceae Yeasts are economically very important, and among the yeasts the species of Saccharomyces are most useful. About 40 species are recognized in the genus Saccharomyces, and of these the best known is S. cerevisiae, the strains of which are

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used in the fermentation of certain beverages like beers and wines, and also in baking industry. Saccharomyces and other yeasts carry out fermentation of carbohydrates to produce alcohol and carbon dioxide (CO2). Alcohol is the main product in breweries while CO2 is useful in bakeries. Because of their extensive use in industry, they are used in preparation of yeast cakes for use in the industry. S. cerevisiae is found in nature on the surface of ripe fruits. Grape wines are often made by spontaneous or natural fermentation by yeasts growing on the surface of grapes. The species of Saccharomyces used in preparation of beverages are given in Table 7.2. Table 7.2. Yeast species and their usage. Yeast species

Beverage/ type

Saccharomyces cerevisiae

Cultivated yeast

S. ellipsoides

Wine yeast

S. pombe

Preparation of African beer from pearl millet

S. mellaci

Preparation of Jamaican rum

S. piriformis

Ginger beer yeast

Fig. 7.15. Structure of a typical yeast cell.

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All yeasts, including the species of Saccharomyces, are unicellular. The vegetative cells of Saccharomyces cerevisiae are generally diploid. Even though the cells appear colorless individually, on artificial solid media, colonies appear as white, cream coloured or tinged with brownish pigment. The cells are elliptical and about 6 – 8 x 5-6 µm in size. The cells contain a large membrane bound vacuole and a small nucleus. It is often found between the vacuole and a developing bud. Other inclusions are mitochondria, storage granules, endoplasmic reticulum (Fig. 7.15).

Fig. 7.16. Life cycles of Saccharomyces.

Asexual reproduction: In yeasts asexual reproduction is by budding. A mature yeast cell giving rise to another cell by budding is shown in the Fig. 3.15. The cells of Saccharomyces can give rise to buds from all over the surface and, the scars where buds were formed previously appear crater like raised scars. The point of origin of buds is at the poles of the yeast cell. Possibly it is because the internal fluid pressure in an ellipsoidal cell is exerted maximally against the cell wall at the point of maximum curvature. The cell wall then bulges and stretches out to form the bud. Various organelles accumulate in the newly formed bud whose wall appears as an extension of the parent cell but is actually newly synthesized. When the bud is fully formed centripetal formation of septa occurs at the isthmus joining the bud with the

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mother cell. A primary septum composed of chitin is first formed and is followed by a secondary septum composed of glucan. Then separation of the bud from the mother cell takes place. Bud car is evident on the mother cell opposite a birth scar on the daughter cell. A bud scar resembles a crater on the surface of the mother cell with a raised circular rim. By counting the bud scars on a cell, one can calculate the number of buds that have been produced from it. Calculations based on the average surface area of a yeast cell shows that the maximum number of scars which could be accommodated would be about 100. But, actual counting of scars present reveals a maximum of 23. At the optimum temperature of 30oC, the time taken for completing bud formation and separation is about 100 minutes. Sexual reproduction: Many strains are heterothallic and ascospores are of two mating types. In Saccharomyces, the vegetative cells are both haploid and diploid and are of equal importance. When two compatible haploid cells copulate plasmogamy is followed by karyogamy and the diploid cells are formed. They asexually reproduce by budding. When diploid cells undergo meiosis 4 ascospores are formed. These also reproduce by budding. Since both haploid and diploid stages are important in the life cycle of the fungus, it is described as haplo-diplontic life cycle (Fig. 7.16). GENUS : EUROTIUM Class: Plectomycetes Order : Eurotiales Family : Eurotiaceae The genus Eurotium is one of most important fungus in the class Plectomycetes. It is the perfect stage of some species of widely known form genus Aspergillus. About 20 species of Eurotium are known. They are widely distributed in soil. The spores are aerially dispersed and cause spoilage of food stuffs. Mycelium comprises of well developed, hyaline, septate hyphae, which are profusely branched. The individual cells of the hyphae are multinucleate. Asexual Reproduction: Asexual stage of the genus Eurotium mostly belongs to the Glaucus group of Aspergillus. Mycelium produces abundant conidiophores (Fig. 7.17). They arise singly from vegetative hyphae. The hyphal cell that gives rise to conidiophore is called foot cell. Conidiophores are long, erect and terminate in a bulbous head, the vesicle. As the multinucleate vesicle develops, a large number of conidiogenous cells are produced over its entire surface. One or two layers of conidiogenous cells (sterigmata) may be produced according to the species. When two layers of sterigmata are present, the secondary ones are conidiogenous. The conidia bearing cells, whether they are primary or secondary, are typically flask shaped and termed phialides. In the asexual stages of Eurotium, the phialides are borne directly on vesicles. The conidia are formed inside the tip of the phialide, which is actually a tube. A portion of the mycelium with a nucleus at the tip of the sterigmata is delimited by a septum. The protoplast rounds off, secretes a wall of its own within the tubular phialide and develops into a conidium. A second protoplast below the first develops into a spore and pushes the first spore outward, so that a chain of spores is formed as the phialide protoplasm continues to grow and cut off

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more conidia. Because the conidiophores and conidia are produced in great abundance, the colony colour is predominantly that of conidial mass.

Fig. 7.17. Conidiophores and hypha of Aspergillus state of Eurotium sp. A. Conidiophore with one row of sterigmata. B. Conidiophore with two rows of sterigmata. C. Hypha showing multinucleate condition.

Sexual reproduction: In Eurotium, sex organs antheridia and ascogonia, are produced close to each other on somatic hyphae. Both of them are multinucleate and elongate structures. They coil around each other. Whether or not the antheridium is functional, pairing of nuclei takes place in the ascogonium. If nuclei from the antheridium have entered, they pair with the ascogonial nuclei, otherwise the ascogonial nuclei themselves pair. Then the ascogonium produces a number of ascogenous hyphae that branch within the developing ascocarp. Asci are formed at different levels. At an early stage, the cleistothecium begins to develop as a single layer of cells around the sex organs. It matures into a small globose ascocarp with smooth wall that is generally yellow. Asci are globose, ovoid or pear shaped. They are evanescent, dissolving soon after ascospore formation, leaving the ascospores free within the cleistothecium (Fig. 7.18).

Fig. 7.18. Cleistothecia of Eurotium sp. A. Ascocarp showing asci. B. Ascocarp centrum with ascospores after disintegration of asci.

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The ascospores are fundamentally shaped like pulley wheels, although furrow may be absent in some species. The outside wall of the two halves is variously modified and sculptured, so the side view gives the appearance of a pulley wheel, in the face view the ascospores may appear round, star shaped, and so on. Eight ascospores are usually produced in each ascus. The ascospores germinate to give rise to germ tubes that develop into mycelia. GENUS : TALAROMYCES Class : Plectomycetes Order : Eurotiales Family : Eurotiaceae The genus Talaromyces consists of 15 species. The genus is well known for its conidial stage which is present in the form genus Penicillium. The genus Penicillium comprises of a large number of species. Among the Penicillium species, mostly those having biverticillate conidiophores belong to the genus Talaromyces. Mycelium is composed of well developed, highly branched, septate hyphae. The presence of septa gives cellular appearance to the hyphae. But the septa possess a simple pore in the middle through which cytoplasm and nucleus can pass. Hence the protoplasm is continuous throughout the mycelium. Each cell may have more than one nucleus. When mycelium is growing on a substratum, it sends in hyphae into the substratum to draw the nourishment. As the mycelium grows, at first, it appears like a white cottony growth on the substratum, and as conidiophores and conidia develop, it gradually turns to characteristic colour. The colonies appear in various shades of green, blue, brown etc. The colour is due to the colour of conidia. The mycelium and conidiophores are hyaline.

Fig. 7.19. Monoverticillate conidiophore of Talaromyces sp.

Asexual reproduction: When the mycelium is growing under favourable conditions, some hyphae grow vertically. After some growth, these vertical hyphae gives rise to two or three branches called rami. On the rami, the conidiogenous cells

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called phialides develop. When only a single series of phialides are directly formed on the conidiophores, the condition is described as monoverticillate. When two series of cells are formed on rami, the upper one is the phialide and the lower supporting cell is described as metula (pl: metulae). Such a condition is described as biverticillate. In some species, a number of branches are formed on a conidiophore and phialides develop on them. Such a situation is described as polyverticillate. In all these conditions, conidia develop only on phialides. In the asexual stages of Talaromyces conidiophores are mostly biverticillate. Phialide is a small flask shaped structure with globose or oval basal part and tubular upper part called neck. The conidia are formed in the neck region. After one conidium is formed it is pushed up and another conidium is formed at the same place. The process repeats. Thus a number of conidia are formed in a chain with first formed oldest conidium at the top and youngest at the base. Such a chain formation is described as in basipetal succession. The conidia are single celled and uninucleate. The conidiophores and phialides with conidial chain appear like a brush. In Latin penicillus means a small brush. Since the conidia are formed on erect conidiophores, and are small, globose, light and dry, they can come into air very easily due to wind turbulence or any mechanical disturbance, and are aerially dispersed. Sexual reproduction The sexual reproduction was thoroughly studied in Talaromyces vermiculatus by Dangeard in 1907. The important stages in the life cycle of this species are shown in Fig. 7.20. Talaromyces vermiculatus is a homothallic species. Both male (antheridium) and female (ascogonium) gametangia are formed on the hyphae of same mycelium. Ascogonium is a long cylindrical structure. It is uninucleate at first but with repeated mitotic division it becomes multinucleate with about 64 nuclei. Antheridium is relatively thin, small, cylindrical structure which grows helically coiled around the ascogonium. The antheridium is also uninucleate at first but becomes multinucleate as it matures. The fully formed antheridium wound round only up to half the height of ascogonium. The area where the tip of antheridium comes in contact with ascogonium, the wall between the two gametangia dissolves, and protoplasts of the two becomes continuous. However, according to Dangeard, there is no evidence that the male nuclei enter ascogonium. The nuclei in the ascogonium itself pair together. Then the transverse septa are formed in the ascogonium to make it a multicellular structure. Each cell contains two nuclei in a pair. This is called dikaryotic stage. Small papillae develop on the side wall of each dikaryotic cell and grow into ascogenous hyphae. The two nuclei in the dikaryotic cell undergo mitotic divisions simultaneously and the two nuclei enter the ascogenous hyphae, and the other two nuclei remain in the ascogonial cell. The growing ascogenous hyphae become transversely septate and each cell of the ascogenous hyphae also contains a pair of nuclei. The top cell of the ascogenous hyphae becomes curved and this curved cell is described as ‘crozier’. In the crozier cell the two nuclei undergo mitotic division. During the nuclear divisions the spindles are arranged in an inclined position in such a way that the two daughter nuclei, one from each parent nucleus, is at the top of the crozier cell, one at the lower region. Then the septa are formed in such a way that the

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tip cell contains one nucleus, the cell below the hook cell contains one nucleus, and the curved cell contains two compatible nuclei. The top cell of the ascogenous hypha is thus actually the penultimate cell. It is described as the ascus mother cell because the two compatible nuclei fuse in this cell. The diploid nucleus immediately undergoes meiotic division forming four haploid nuclei. Then each nucleus undergoes one mitotic division producing eight haploid nuclei. As this is occurring in the ascus mother cell, the uninucleate tip cell bends and touches the third cell which is also uninucleate. At the point of contact the wall dissolves, and the nucleus from the tip cell enters the third cell making it dikaryotic. From this cell another ascogenous hypha develops. The ascus mother cell with its eight nuclei enlarges. A bit of cytoplasm surrounds each of the eight nuclei and it is transformed into an ascospore by forming a wall around each nucleus with its cytoplasm. Such a pattern of cell development is described as free cell formation. With the completion of ascospore formation, ascus development is complete, and the shape of the ascus is typically globose and it is prototunicate. As the ascospores are being formed in the ascus, the vegetative hyphae from the base of the ascogonium are stimulated and grow and surround the developing asci, thus forming an ascocarp. The peridium completely encloses the ascocarp without an ostiole. As the asci are prototunicate, the ascus wall becomes evanescent by maturity, and the ascospores are released into the ascocarp centrum. The ascospores may differ in their size but all of them are single celled and usually pulley shaped. The ascospores are released only on disintegration of the peridium, they become easily airborne and spread to long distances.

Fig. 7.20. Important stages in the life cycle of Talaromyces vermiculatus. A. Vegetative hyphae. B. Conidiophore. C. Conidia. D. Germinating conidium. E. Gametangia. 1. Ascogonium. 2. Antheridium. F. Formation of septa in ascogonium. G & H. Formation of cleistothecium . 1. Ascus. 2. Sterile hyphae. I. Ascospores.

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Life Cycle: The life cycle of Talaromyces vermiculatus is shown in the Fig. 7.21.

Fig. 7.21. Life cycle of Talaromyces.

GENUS : CHAETOMIUM Class : Pyrenomycetes Order : Sordariales Family : Chaetomiaceae Chaetomium is a large genus with more than 200 species. Many of them cause decay of cellulose rich substrata such as textiles in contact with soil, straw, dung and wood which may undergo a superficial decay known as soft rot. The breakdown of cellulose by Chaetomium is brought about by a powerful cellulase. When fed with sucrose C. globosum breaks down the disaccharide into its two hexose moieties, glucose and fructose, but the glucose moiety is absorbed preferentially. Little sucrose is directly used. C. thermophile is a thermophilic species. Some species of Chaetomium e.g. C. cochliodes, are antagonistic to soil-borne and seed borne fungal pathogens, and the possibility of using them to control plant diseases has been investigated. An antibiotic chaetomin, has been isolated form C. cochliodes. Mycelium is composed of well developed, highly branched, septate hyphae. Each cell may have more than one nucleus. When mycelium is growing on a substratum, it sends hyphae into the substratum to draw the nourishment. Asexual reproduction: Conidial states are rare in Chaetomium, but simple phialides and phialospores occur in C. elatum and C. globosum while C. piluliferum forms both phialospores and globose thalloconidia of the Botryotrichum type. Chaetomium trigonosporum has conidia belonging to the form genus Scopulariopsis.

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Sexual reproduction: Most species of Chaetomium are homothallic but some, e.g. C. cochliodes, are heterothallic. Ascogonium is a coiled structure. Antheridium may or may not be formed, and even if formed is non functional. Ascocarp develops from ascogonium. The perithecia are produced superficially without a stroma. The perithecia are barrel shaped. Investing hyphae arise from ascogonial stalk or from adjacent vegetative cells surrounding the ascogonium. The centrum is at first filled with hyaline pseudoparenchyma. At the apex of the perithecium, certain of these cells become meristematic and give rise to the elongated periphyses which line the ostiole. Ascogenous hyphae develop from the ascogonium at the base of the centrum and, at about this time, the surrounding pseudoparencyma cells of centrum deliquesce to form a cavity. In some species, croziers have been reported, but in others they may be absent. The asci are produced in basal tufts, are club shaped or broadly oval, in some they may be cylindrical. The asci of all species except two species viz. Chaetomium hispidum and C. tetrasporum, have eight spores. The ascal wall deliquesces before the spores are mature.

Fig. 7.22. Ascocarp, asci and ascospores of Chaetomium. A. Ascocarp with terminal and lateral hairs and emerging ascospores in a cirrhus. B. Asci in different stages of growth. C. Ascospores.

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Paraphyses are formed in the initial stages but never survive until the spores are mature. Paraphyses of two types have been described in some species. In C. brasiliense, lateral paraphyses i.e. paraphyses arising from the pseudoparenchyma cells outside the hymenium, have been reported, while hymenial paraphyses have been reported in C. globosum. The paraphyses are evanescent and disappear before the ascospsores mature. This type of perithecium development is described as Xylaria-type because asci develop among paraphyses even if they deliquesce before ascospore maturity. Characteristic feature of the ascocarp is the presence of conspicuous hairs at the apex (terminal) and sides (lateral) of the perithecia. Perithecial hairs develop early from the external cell layer. In C. elatum, one of the commonest species growing on damp rotting straw, the terminal hairs are dichotomously branched. In others e.g. C. cochliodes, the body of the perithecium bears straight or slightly wavy, unbranched hairs, while the apex bears a group of spirally coiled hairs. The hairs may be smooth, roughened or ornamented and the type of ornamentation is an aid in identification. When perithecia are ripe, a column like mass of black ascospores arises from the apex. The spore column (cirrhus) results from the breakdown of the asci within the body of the perithecium i.e. the asci do not discharge their spores violently. In most species, the spores are lemon shaped with a single germ pore. The perithecium, asci and ascospores of Chaetomium are shown in Fig. 7.22. GENUS : NEUROSPORA Class : Pyrenomycetes Order : Sordariales Family : Sordariaceae The genus Neurospora comprises of 12 species. The ascospores are dark brown or black with nerve-like ribs on the outer wall that characterize the genus Neurospora and give it its name. Neurospora sitophila is the most important species, and the others include N. crassa, N. tetrasperma, N. terricola, N. dodgei, etc. Neurospora sitophila is commonly referred to as red bread mould or bakery mould, because it frequently infests bakeries and causes considerable damage. When it invades the mycological or bacteriological laboratory as a contaminant, it plays havoc with cultures and is difficult to exterminate because of its rapidly creeping aerial mycelium, and the production of enormous quantities of easily dispersible conidia. The mycelium of N. sitophila consists of numerous branched hyphae. The hyphal cells are multinucleate. The mycelium is pigmented with the amount of pigment varying with substratum. Its aerial hyphae form a mass of mycelium easily recognized by the pink masses of oval conidia borne in chains on branched conidiophores. Asexual Reproduction: Neurospora produces multinucleate macroconidia and uninucleate microconidia. Both are able to germinate and form mycelium. The fungus can propagate itself indefinitely by asexual means alone. Conidial stage is known as Monilia sitophila.

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Sexual Reproduction: A young ascogonium, which is a curved, septate hypha with several nuclei in each cell, is surrounded with several layers of interwoven hyphae. This structure is termed protoperithecium. Certain cells of the ascogonium produce long trichogyne like branches, which penetrate the sterile hyphal layers surrounding the ascogonium. The male elements are represented by microconidia produced in chains on microconidiophores. A conidium or germ tube can also supply nuclei to the receptive trichogyne. Spermatization can be brought about not only by the microconidia but also by macroconidia or conidial germ tube.

Fig.7.23. Life cycle of Neurospora sitophila.

Fusion between trichogyne and the fertilizing cell (i.e. conidium, microconidium or conidial germ tube) is followed by migration of one or more nuclei from the fertilizing cell down the trichogyne into the ascogonial cell. The development of the perithecium follows a pattern characteristic of ascomycetes. The sterile layer of protoperithecium increases in size and organizes itself into a perithecium whose apex develops into an ostiole. The young asci of Neurospora are binucleate descendants of the nuclei of two mating types. Nuclear division takes place in each young ascus followed by three successive nuclear divisions during which meiosis is completed. At maturity the asci contain 8 linearly

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arranged nuclei. These with a portion of the surrounding cytoplasm finally segregate from residual cytoplasm by free cell formation. Mature perithecia are dark colored, pyriform, beaked and contain numerous octosporous asci but not paraphyses at maturity. Of the 8 ascospores in an ascus, four are of one mating type and the other four of another mating type. Neurospora tetrasperma contains only four ascospores in an ascus, and each ascospore contains two of the original 8 ascal nuclei. Normally spores of this species germinate and produce self fertile mycelium. N. sitophila and N. crassa are heterothallic. Incompatibility is controlled by a pair of alleles A and a. The important stages in the life cycle of N. sitophila are shown in Fig. 7.23. GENUS : SORDARIA Class : Pyrenomycetes Order : Sordariales Family : Sordariaceae The genus Sordaria comprises about 10 species, and include S. fimicola, S. brevicollis, S. heterothallis, S. sclerogenia, S. macrospora, A. humana, etc. Perithecia of Sordaria are common on the dung of herbivores and occasionally on other substrata. Sordaria fimicola is the most common and widely studied species. It is used in experiments on nutritional requirements, physiology of fruiting, spore liberation and genetics. The genus is characterized by the dark brown ascospores surrounded by a gelatinous sheath. Mycelium is composed of well developed, highly branched, septate hyphae. When mycelium is growing on a substratum, it sends hyphae into the substratum to draw the nourishment. Asexual Reproduction: Sordaria fimicola produces neither microconidia nor macroconida. It solely reproduces by ascospores explosively discharged from ostiolate perithecia. Sexual reproduction: Sordaria fimicola is homothallic. S. brevicollis, S. heterothallis and S. sclerogenia are heterothallic. Gametangial contact occurs by ascogonia and antheridia. Some workers have reported hyphal fusion. The ascogonia are formed within a stroma or are free in the mycelium. Branches from the stalk cell of the ascogonium from neighbouring vegetative hyphae envelope the ascogonium to form a spherical mass of tissue, the peithecial initial. The outer layers of this mass become differentiated into a perithecial wall. The central portion develops into a centrum composed of pseudoparenchyma cells later producing the perithecial cavity. The asci expand as a group into the disintegrating centrum pseudoparenchyma and ultimately form a layer lining the base of the perithecial cavity. The apical region of perithecia is more or less an elongated perithecial neck. The neck has an opening the ostiole and is lined on the inside by hairs called periphyses. The development of perithecium is Diaporthe type. Biotin plays an important role in perithecial formation and

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maturation. There is a direct correlation between available supply of biotin and percentage of mature ascospores produced. The perithecial neck in Sordaria is positively phototropic. As the asci mature, they swell and fill the upper part of the perithecium. One of the asci stretches, and pushes through the ostiolar opening. Its base remains attached to the perithecial wall. As its tip protrudes, the ascus discharges all its spores explosively, collapses and disintegrates, to be followed by other asci in succession. The spores are flung out for a distance of up to 8 cm. Each ascus is about 13 µm wide, and the apical apparatus of ascus is only about 4 µm wide, the spores are gripped as they leave the ascus. The ascospores of S. fumicola have a distinct mucilage envelope. After the ascospores are discharged, they germinate and produce mycelium when conditions are favourable. The details of ascocarp and release of ascospores are shown in Fig. 7.24.

Fig. 7.24. Ascocarp of Sordaria fimicola. A. V.S. of a perithecium, B. An ascospore with mucilaginous epispore. C-F. Stages in the release of ascospores.

GENUS : PEZIZA Class : Discomycetes Oomycetes Order : Pezizales Family : Pezizaceae Peziza is represented by about 100 species. It lives as saprophyte on dung, rotten wood, heaps of manure and soils rich in organic matter. The genus is cosmopolitan and forms apothecia crowded together in green humus, heavily manured soil and dung. Because of their cup shaped apothecia they are commonly known as cup fungi. Some important species in the genus are P. vesiculosa, P. aurantia, P. rapanda, P. fuckeliana, P. ostracoderma etc. The ascocarps vary in size

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from minute to large conspicuous structures. Peziza vesiculosa is one of the largest cup fungi. The ascocarps of P. aurantia are small and brilliantly orange in colour. The mycelium is well developed and profusely branched, ramifying inside the substratum or soil. The hyphae are septate and the hyphal cells are uninucleate. As the mycelium is not visible the presence of Peziza can be noticed where cup shaped apothecia appear above the substratum. Asexual reproduction: It is not common. P. vesiculosa and some other species produce conidia. The conidial states are described in form genera Oedocephalum and Chromelosporium. Microconidia are also reported in some species. Sexual reproduction: In Peziza sex organs are completely absent. The mycelium grows in all directions forming a tangled mass. Fusion of hyphal cells takes place resulting in dikaryotic cells. This is called somatogamy. These dikaryotic cells give rise to ascogenous hyphae. The cells of ascogenous hyphae function as ascus mother cell (P. vesiculosa). In some species the penultimate cell produced after crozier formation functions as ascus mother cell (P. catinum). The two nuclei in such cells fuse to form synkaryon and the diploid nucleus thus formed divides by meiosis and later by mitosis giving rise to 8 ascospores. The asci grow erect and lay side by side parallel to each other. As these changes are taking place the vegetative hyphae start growing around the asci resulting in the formation of fruiting body called apothecium. It is cup shaped and sessile, about 2-6 cm in diameter. The inner lining of the cup is smooth and brown in colour. It is the fertile layer called hymenium. It contains numerous erect and long asci. They lie parallel to each other. They are intermingled with sterile hypae called paraphyses.

Fig. 7.25. Ascocarps, asci and ascospores of Peziza. A. A group of Peziza cups. B. Vertical section of ascocarp. C. Hymenium. 1. Ascus. 2. Paraphysis. 3. Subhymenium.

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In the ascus 8 ascospores are present in an oblique row. The region below the hymenium is called subhymenium. The rest of the apothecium is made up of compactly interwoven hyphae called excipulum. When the asci mature, they stretch so that the terminal part of the ascus opens as a lid releasing a cloud of ascospores. The method of spore release in Peziza has been studied in detail by Buller (1934). The ascospores are dispersed by air currents. Each ascospore is smooth, elliptical. On reaching the substratum, it germinates giving rise to germ tube which develops mycelium in the substratum. Apothecia, hymenium, asci and ascospores of Peziza sp. are shown in Fig. 7.25.

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Chapter - 8

Basidiomycota

The phylum Basidiomycota includes such well known macrofungi like mushrooms, polypores, puff balls, earth stars, bird’s nest fungi and others (Plates 8.1, 8.2). The group also includes important plant pathogenic fungi which cause rust and smut diseases of higher plants. About 1100 genera and 16,000 species are described in this group. The most important character of the group is production of sexual spores called basidiospores on specialized structure described as basidium (pl. basidia). The basidia in macrofungi are formed in fruit bodies called basidiocarps, but basidiocarps are not formed in pathogenic fungi. Over a period of time, some of the common macrofungi came to be known by their common names (Table 8.1). Some of the macrofungi and their common names are:

Plate 8.1. Basidiomycota. 1. Polyporus sp. 2. Dictyospora sp. 3. Urediniospores of Puccinia. 4. Coprinus sp.

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Plate 8.2. Basidiomycota. 1. Russula sp. 2. Boletus sp. 3. Phallus sp. 4. Laccaria sp. 5. Scleroderma sp. 6. Psilocybe sp.

Table 8.1. Common name and technical name of some macrofungi Technical name of Macrofungi

Common name

Family

Agaricus campestris

Field mushroom

Agaricaceae

Agaricus bisporus

Button mushroom

Agaricaceae

Amanita muscaria

Fly agaric

Amanitaceae

Amanita caesaria

Caesar’s mushroom

Amanitaceae

Amanita phalloides

Death cup

Amanitaceae

Amanita virosa

Destroying angel

Amanitaceae

Coprinus atramentarius

Inky cap mushroom

Coprinaceae

Termitomyces heimii

Termite nest fungus

Coprinaceae

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Technical name of Macrofungi

Common name

Family

Strobilomyces

Old man of the woods

Boletaceae

Psilocybe mexicana

Sacred mushroom

Stophariaceae

Lactarius deliciosus

Saffron milk-cap

Russulaceae

Pholiota samino

Viscid mushroom

Cortinariaceae

Volvariella volvacea

Paddy straw mushroom

Pluteaceae

Pluteus cervinus

Saw dust mushroom

Pluteaceae

Pleurotus spp.

Oyster mushroom

Tricholomataceae

Lentinus edodes

Shiitake mushroom

Tricholomataceae

Marasmius oreades

Fairy ring fungus

Tricholomataceae

Omphalotus olearius

Jack-o-lantern

Tricholomataceae

Armillariella mellea

Honey mushroom

Tricholomataceae

Flammulina velutipes

Enokitake mushroom

Tricholomataceae

Cyathus spp.

Bird’s nest fungus

Cyathaceae

Phallus spp.

Stink horn

Phallaceae

Lycoperdon spp.

Common puff ball

Lycoperdaceae

Clavatia gigantea

Giant puff ball

Lycoperdaceae

Tulostoma spp.

Stalked puff ball

Tulostomataceae

Geastrum spp.

Earth star

Geastraceae

Clavarius spp.

Coral fungi

Clavariaceae

Polyporus, Ganoderma, Fomes, etc.

Bracket fungi

Polyporaceae

Hydnum sp.

Tooth fungi

Hydnaceae

Auricularia polytricha

Wood-ear fungus

Auriculariaceae

Mycelium: The mycelium of basidiomycetous fungi consists of well developed, septate hyphae. The cell wall is made of chitin and glucans, cellulose is absent. In free living, saprophytic macrofungi, extensive vegetative mycelium develops in soil or in other substrata while in plant pathogenic forms (rusts and smuts) mycelium is limited in the host tissue, and the hyphae sends in haustoria into the host cells to draw nourishment. The hyphae of basidiomycetous fungi are septate and the septa possess a pore. Because of the septal pores the protoplasm is continuous throughout the mycelium. The septal pore in basidiomycetes is barrel shaped and is described as dolipore septum (Fig. 8.1). Endoplasmic reticulum forms a layer of covering on either side of the pore and it is described as parenthosome. The parenthosome is not continuous but has a number of pores and hence there is no obstruction to cytoplasmic streaming. However, in rust fungi and smut fungi dolipore septum is absent. They contain a simple pore in their septal walls.

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Fig. 8.1. Structure of dolipore septum. 1. Layers of cell wall. 2. Endoplasmic reticulum. 3. Middle lamella. 4. Barrel shaped structure. 5. Septal pore. 6. Parenthosome

The mycelium of these fungi passes through three stages called primary mycelium, secondary mycelium and tertiary mycelium. Primary mycelium: This is formed when basidiospores germinate and produce fungal mycelium comprising of uninucleate hyphal cells. This uninucleate stage is called primary mycelium. It is usually very much limited. Secondary mycelium: When two compatible primary mycelia are growing side by side, somatogamy occurs between them forming dikaryotic stage. The nuclei divide mitotically and spread throughout the mycelium, thus the entire mycelium becomes dikaryotic. This dikaryotic stage of the mycelium is described as secondary mycelium. The dikaryotic mycelium may also form when germ tubes from two compatible basidiospores fuse, or by spermatization of primary mycelium. Tertiary mycelium: The mycelium that forms basidiocarps is called tertiary mycelium. Like in secondary mycelium, all the hyphal cells of tertiary mycelium are also dikaryotic.

Fig. 8.2. Development of clamp connection in secondary and tertiary mycelium. A. tip of a dikaryotic hypha. B-E. Method of formation of clamp connection.

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In secondary and tertiary mycelium, during cell divisions specialized structures called clamp connections are formed, and help to maintain dikaryotic condition of the daughter cells with two compatible nuclei (Fig. 8.2). Asexual reproduction: In Basidiomycotina fungi, asexual reproduction is not generally found. A few species produce conidia. In some species the vegetative cells separate as individual cells and disperse. Such individual cells which disperse are described as arthrospores. In rust fungi urediniospores are considered as conidia. In smut fungi basidiospores germinate to produce more cells which grow by budding as yeasts until the dikaryon is established. Sexual reproduction: In Basidiomycetes no specialized sexual reproductive structures are formed. Plasmogamy occurs either by somatogamy or spermatization to form dikaryotic mycelium, called as secondary mycelium which grows extensively. When conditions are favourable, the fruit bodies called basdiocarps are formed by mycelium termed as tertiary mycelium. Even though each dikaryotic cell is potentially capable of forming basidiospores, karyogamy occurs only in specialized cells called basidia. The basidia are formed at the tip of fertile hyphae. The fertile hyphae form a layer called hymenium. In agarics the gills are lined with hymenium, in polypores hymenium lines the pores. Hymenium formed on gills is shown in Fig. 8.3.

Fig. 8.3. Hymenium in basidiomycetous fungi. 1. Cystidium. 2. Basidiospores. 3. Basidium.

In the hymenium, some sterile structures are also present interspersing basidia. These are called cystidia which usually have a narrow stalk and upper globose structure which may be bigger than basidia. Basidium: Basidium may be defined as a structure bearing on its surface a definite number of basidiospores (usually four). The two nuclei in the basidium fuse and meiosis follows immediately after karyogamy producing four haploid nuclei. As meiosis is occurring, four sterigmata develop on the top of the basidium. One haploid nucleus enters each sterigma and one basidiospore is formed at the tip of the sterigmata. Based on the structure, the basidium is described as of two type viz. holobasidium and phragmobasidium. Holobasidium is a single celled, typically club shaped structure with four small sterigmata at the top. It is mainly found in Gasteromycetes and majority of Hymenomycetes. It is often described as a typical basidium. However, in some small groups like the members of Tulsanellales and Dacrymycetales, even though the basidium is single celled, it is not typical, and shows some structural variations. In

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the order Tulsanellales, the sterigmata are very big and prominent structures. In the order Dacrymycetales, the basidium is a curved structure with U-shaped appearance. Phragmobasidium is a multicellular structure usually with four cells. This type of basidium develops due to formation of transverse or longitudinal septa after meiosis. It is found in the members of the subclass Phragmobasidiomycetidae and members of the class Teliomycetes. Up to the stage of meiosis, the basidium is single celled, but as meiosis occurs and four nuclei are formed, basidium becomes septate to form a four celled structure. The septa are formed longitudinally in the order Tremellales, while the septa are formed transversly in Auriculariales, Septobasidiales and the members of Teliomycetes. Different types of basidia are shown in Fig. 8.4.

Fig. 8.4. Basidium types in different genera

Depending on the stage of development, the basidium is described as of two type’s viz. Probasidium and Metabasidium. The stage or part of the basidium in which karyogamy occurs is called probasidium, while the stage or part of the basidium in which meiosis occurs is described as metabasidium. In the members of Phragmobasidiomycetidae, the single celled stage in which karyogamy occurs is probasidium and septate structure with four cells formed after meiotic division is metabasidium. In rust fungi and smut fungi, the teliospores represent probasidium and promycelium formed due to germination of teliospore represent the metabasidium. Basidiospores: Basidiospores are typically unicellular, and may be globose, oval, elongated or variously shaped. They may be colourless or pigmented. In many cases, the pigments are very dilute and visible only in spore mass but not in individual spores. The spore prints may be of different colours like white, green, red,

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pink, different shades of brown etc. The basidiospore wall is usually thick and made up of two layers. The outer layer is called epispore and the inner layer is called endospore. In most of the species the spore wall is smooth, but in some the outer layer may be having different types of thickenings. In the genus Ganoderma, the outer layer is smooth, while conspicuous spiny thickenings are seen on the inner layer. The outer layer appears as a hyaline covering over the spines. Different types of basidiospores are shown in the Fig. 8.5.

Fig. 8.5. Different types of basidiospores

Basidiospore release: In majority of Hymenomycetes, basidiospore is formed on the tip of sterigma in an inclined position, and is actively discharged. Very close to the point of its attachment to the sterigma, the spore has a minute projection referred to as hilar appendix. A small bubble or drop forms at the hilar appendix as the spore matures. It is thought to be involved in active release of basidiospore. It was first described by A.H.R. Buller and hence is described as Buller’s drop. At the time of spore release, the drop bursts and the spore is thrown out with force (Fig. 8.6). Various other theories such as electrostatic repulsion, jet propulsion, surface tension, rounding off of turgid cells etc. have also been proposed to explain the active release of basidiospores.

Fig. 8.6. Liberation of basidiospore. 1. Sterigma. 2. Basidiospore. 3. Buller’s drop. 4. liberated basidiospores.

CLASSIFICATION The recent Classification of the Phylum Basidiomycota is as follows: Phylum Basidiomycota Class Urediniomycetes Order Uredinales

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Family Pucciniaceae (Genera – Puccinia, Gymnosporangium, Uromyces, Hemilea) Family Melampsoraceae (Genus – Melampsora) Order Septobasidiales (Genera – Septobasidium, Uredinella) Order Sporidiales (Genera – Rhodosporidium, Aessosporon) Class Ustilaginomycetes Order Exobasidiales (Genus – Exobasidium) Family Graphiolaceae Order Ustilaginales Family Ustilaginaceae (Genus – Ustilago) Family Tilletiaceae (Genera – Tilletia, Urocystis) Class Basidiomycetes Subclass Tremellomycetidae Order Tremellales Family Tremellaceae (Genus – Tremella) Family Exidiaceae (Genus – Exidia) Order Auriculariales Family Auriculariaceae (Genera – Auricularia, Helicobasidium) Order Filobasidiales Family Filobasidiaceae (Genus – Filobasidium) Order Dacrymycetales Order Tulasnellales Order Ceratobasidiales Family Ceratobasidiaceae (Genus – Thanatephorus) Subclass Agaricomycetidae Order Agaricales Family Agaricaceae (Genera – Agaricus, Lepiota, Podaxis) Family Pluteaceae (Genera – Amanita, Volvariella) Family Nidulariaceae (Genus – Cyathus) Family Strophariaceae (Genus –Psilocybe) Family Coprinaceae (Genus – Coprinus) Family Cortinariaceae (Genus – Cortinaritus) Family Tricholomataceae (Genera – Tricholoma, Termitomyces, Mycena, Crinipellis, Clitocybe) Family Marasmiaceae (Genera – Marasmium, Armillaria) Family Pleurotaceae (Genus – Pleurotus)

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Family Lycoperdaceae (“Puff-Ball” family) (Genera – Lycoperdon, Calvatia, Langermannia) Family Schizophyllaceae (Genus – Schizophyllum) Order Boletales Family Boletaceae (Genus – Boletus) Family Paxillaceae (Genus – Paxillus) Family Sclerodermataceae Order Phallales Family Phallaceae (Genus – Phallus) Family Geastraceae (Genus – Geastrum) Order Rssulales Family Russulaceae (Genus – Lactarius) Family Stereaceae (Genera – Stereum, Helicobasidium) Order Thelephorales Family Thelephoraceae Order Cantharellales Family Cantharellaceae (Genus – Cantharellus) Family Hydnaceae (Hedgehog fungi) (Genus – Hydnum) Order Hymenochaetales Family Hymenochaetaceae Order Polyporales Family Corticiaceae (Genus – Corticium) Family Fomitopsidaceae (Genera – Daedalea, Fomitopsis, Pitoporus) Family Ganodermataceae (Genera – Ganoderma, Trametes) Family Sparassidacea (genus – Sparassis) Family Stecchnariaceae (Genus – Irpex) Family Meruliaceae (Genus – Chondrostereum) Family Phanerochaetaceae (Genus – Phanerochaete) Family Polyporaceae (Genera –Poria, Fomes, Polyporus, Lenzites) The classification followed here is of Ainsworth (1973). He recognized three classes Teliomycetes, Hymenomycetes and Gasteromycetes. The classes are based on the presence or absence of basidiocarp and its development; septate or unseptate nature of basidia; and the nature of basidiospore discharge. CLASS – TELIOMYCETES This class includes the plant pathogenic fungi commonly known as rusts and smuts. They do not form basidiocarps. The septal pore of mycelium is simple, not

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dolipore type, and clamp connections are absent. They are characterized by the production of thick walled binucleate resting spores called teliospores or telutospores. During germination of teliospores, karyogamy occurs and the diploid nucleus enters the germ tube and undergoes meiosis producing four haploid nuclei. Then the germ tube becomes transversely septate producing four celled structure. From each cell one basidiospore is formed. In rust fungi the basidiospores are produced on small sterigmata while in most smut fungi the basidiospores are budded out directly from the cells without sterigmata. The basidiospores are not actively discharged. The class comprises two orders viz. Uredinales – the rust fungi, and Ustilaginales – the smut fungi. Order - Uredinales: There are about 100 genera and 4000 species, all causing rust disease on higher plants, gymnosperms and pteridophytes. They are ecologically obligate. In the life cycle of rust fungi, five types of spores are formed. They are 1. Pycniospores or spermatia, 2. Aeciospores, 3. Urediniospores, 4. Teliospores (also described as teleutospores) and 5. Basidiospores. These spores are formed sequentially in a cyclic fashion in the life cycle, and are designated as 0, I, II, III and IV spore types or stages. The pycnia or spermagonia were first recognised by Persoon in 1800. But their role or importance was not discovered at that time. Hence they were called zero stage. Their function in the life cycle was discovered by Craigie, a Canadian Mycologist in 1927. All these five types may or may not be formed in the same species of a rust fungus. One or two spore types may be missing in the life cycle. When all the five types of spores are formed, the life cycle is described as macrocylic. If one or two spore stages are missing in the life cycle, it is described as microcyclic. The rust species which completes the life cycle producing all the spore types on a single host are described as autoecious species. Among the autoecious species, Puccinia asparagi, P. helianthi, etc. are important. The species which require two different hosts to complete the life cycle are described as heteroecious. In rust fungi, teleutospore stage is considered as sexual stage since during the germination of teleutospores the two compatible nuclei fuse and karyogamy is followed by meiosis. The host on which perfect stage (sexual stage) occurs is considered as the primary host and the other as alternate or secondary host. The best example for the heteroecious rusts is Puccinia graminis tritici, which completes life cycle on wheat and barbery. There may be no connection between primary and alternate hosts as shown below (Table 8.2). Table 8.2. Primary and alternate host groups of some rust fungi. Rust fungus

Primary host (group)

Alternate host (group)

Puccinia graminis

Grasses (monocots)

Barberry (dicot)

Puccinia sorghi

Maize (monocot)

Oxalis stricta (dicot)

Puccinia penniseti

Bajra (monocot)

Brinjal (dicot)

Cronartium ribicola

Ribes (dicot)

Pinus strobus (gymnosperm)

Uredinopsis osmundae

Osmunda (Pteridophyte)

Abies balsamea (gymnosperm)

Uromyces pisi

Pisum sativa (dicot)

Euphorbia cyparissias (monochlamydae)

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Different types of teleutospores produced in rust fungi are shown in the Fig. 8.7.

Fig. 8.7. Different types of teleutospores in rust fungi. A. Uromyces. B. Pileolaria. C. Puccinia. D. Uropyxis. E. Xenodochus. F. Phragmidium. G. Nyssopsora. H. Ravenalia.

Traditionally two families viz. Pucciniaceae with stalked teliospores, and Melampsoraceae with sessile teliospores, are recognised in rust fungi. Now some authors recognise three families with sessile teliospores. Family - Pucciniaceae: Rusts which produce stalked teliospores in telial sorus, e.g. Puccinia, Uromyces, etc. Family - Melampsoraceae: Rusts which produce sessile teliospores arranged in a single layer are placed in this family. The best example is Melampsora Family - Coleosporiaceae: Rusts which produce sessile teliospores in one or two layers, and teliospores become septate and 4-celled structure during germination. Thus there is no promycelium, but the basidiospores are produced one from each cell of the four celled structure. Example: Coleosporium. Family - Cronartiaceae: The sessile teliospores are formed in chains. Example: Cronartium ribicola. Apart from the above four families, there are some rust fungi which produce only the uredinial stage or aecial stage. Since no teleutospore stage is present, they are treated as imperfect rust fungi. Since they can be identified as rust fungi, they are not placed in Deuteromycotina, but treated as ‘uredinale imperfecti’ group. In this group 5 form genera are recognized. 1.

Form genus Uredo: All imperfect rusts which produce only uredinial stage are placed in the form genus Uredo.

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The aeciospore producing fungi are recognised in 4 form genera. Different types of aecia are shown in the Fig. 8.8.

Fig. 8.8. Surface view and sections of aecial cups in Caeoma, Aecidium, Roestelia and Peridermium.

2.

Form genus Caeoma: fungi which produce diffuse aecia without peridium.

3.

Form genus Aecidium: fungi which produce typical aecial cups.

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4.

Form genus Roestelia: Peridium of the aecial cup over grows and the rim extends above the cup and rolls backwards.

5.

Form genus Peridermium: The peridermium completely covers the aecium.

Order – Ustilaginales: The members of this order are generally known as smut fungi. About 50 genera and 1100 species are recognized, and they attack host plants spread over 75 families of angiosperms. The members of graminae and cyperaceae are most frequently attacked. They produce smut sori manly in the place of grains, but other parts are also infected. Unlike the rusts, smuts are culturable, and in culture many fungi showed yeast like growth. In smut fungi the life cycle is simple. The basidiospores, often described as sporidia, are uninucleate. Dikaryotization takes place by fusion of any two compatible cells, two basidiospores, mycelial fragments or germ tubes etc. The dikaryotic mycelium infects the host and when a mass of mycelium is formed, the mycelium fragments into individual cells and each cell becomes a thick walled round spore called smut spore or chlamydospore or teliospore. During germination of the teliospores, karyogamy occurs in the spore and the diploid nucleus moves into germ tube and undergoes meiosis. The germ tube becomes septate, either three celled or four celled. Basidiospores are formed from the side of the cells of promycelium by budding without sterigmata. In three-celled promycelium the tip cell bears two spores one at the top and one laterally. In some fungi, generally called bunts, promycelium do not become septate but bear eight filiform basidiospores on the tip and two basidiospores fuse while still attached. Based on the method of spore germination, two families are recognized. Family - Ustilaginaceae: Teliospores germinate to form septate promycelium and, usually each cell produces one basidiospore. Example: Ustilago, Sphacelotheca, Tolyposporium, etc. Family - Tilletiaceae: Promycelium is aseptate and bears eight filiform basidiospores at the top. Examples: Tilletia, Neovossia, Entyloma, etc. CLASS – HYMENOMYCETES This is a very large group and is divided into two subclasses based on the structure of basidium. They are 1. Holobasidiomycetidae and 2. Phragmobasidiomycetidae. Subclass – Holobasidiomycetidae: It comprises the majority of hymenomycetes in which the basidium is a single celled holobasidium. It includes such well known macrofungi like mushrooms and polypores. The fruit bodies of agarics are soft, fleshy, evanescent, and the hymenium lines the gills or lamellae. Polypores include fungi which produce basidiocarps which are corky, leathery or woody and perennial, but not soft and evanescent. Usually the hymenium lines the pores in these fungi. In addition to these two groups (orders Agaricales and Aphyllophorales), the subclass includes four other small orders viz. Dacrymycetales, Exobasidiales, Brachybasidiales and Tulasnellales in which

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basidium is single celled but show much variation from typical holobasidium of Agaricales. In earlier literature these four groups weree called Heterobasidiomycetes. Order - Brachybasidiales: It is a small group of fungi parasitic on higher plants. Fruit bodies are not formed. The basidia are bisterigmate producing only two spores each. A single family Brachybasidiaceae is recognized in the order. Order - Dacrymycetales: The members of this order are characterized by formation of forked basidium producing two basidiospores. These fungi occur as saprophytes on decaying wood producing gelatinous or waxy fruit bodies. They are bright coloured, usually yellow or orange. A single family Dacrymycetaceae is recognized in the order. Order - Agaricales: The order Agaricales, commonly called ‘gill fungi’, comprises 270 genera and about 4000 species. The fruit bodies are called mushrooms. They are soft, fleshy and evanescent, mainly occurring during rainy season. They are terrestrial or lignicolous growing saprophytically. Some of the mushrooms are edible and people prefer them for delicacy and nutritional value. Some edible mushrooms are cultivated, and mushroom cultivation is a multimillion industry. Some common mushrooms are shown in Fig. 8.9.

Fig. 8.9. Some common mushrooms. A. Amanita (with volva and annulus). B. Agaricus (with only annulus). C. Volvariella (with only volva). D. Marasmius (in clusters). E. Pleurotus (funnel shaped pileus). F. Lactarius (funnel shaped convex pileus.

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The mycelium of Agaricales is typically basidiomycetous. The dikaryotic mycelium is the major vegetative stage which occurs in the substratum. In grass lands and open spaces, mycelium grows circularly, and continues to grow year after year, and produce a crop of basidiocarps at the periphery during rainy season. As the fruit bodies are formed in circular fashion, they are commonly called as ‘fairy rings’, in the belief that they represent the path of dancing fairies. Marasmius oreades and other mushrooms are generally called fairy ring fungi. The fruit bodies of Agaricales are well developed with stipe (stalk) and pileus (cap). The tissues comprising a mushroom consist of closely packed dikaryotic hyphae. Three basic types of mushroom development are recognized viz. gymnocarpic, pseudoangiocarpic and hemiangiocarpic. In gymnocarpic type of development, the hymenium remains always naked, and only stipe and pileus are prominent structures. In pseudoangiocarpic development, hymenium becomes enclosed by the incurving margin of the pileus, and at maturity it breaks exposing the hymenium. In hemiangiocarpic type of development, the fertile part (hymenium) is enclosed by tisssues of the basidiocarp. Typically the margin of the pileus is connected to the stipe by a membrane called inner veil. As the fruit body matures, veil is severed from the margin of the pileus and remains attached to the stipe in the form of a ring or annulus. In some species veil tears in such a way that broken membranes hang down from the cap like a thin cobweb curtain called cortina. In some members like Amanita species, entire primordium is also covered by a membranous structure called universal veil. As the fruit body expands, the universal veil tears and leaves a cup shaped body at the base of the stipe, and it is called volva. The remnants of universal veil that covers pileus are often seen in the form of scales on the cap. These vestigial structures resulting from the hemiangiocarpous mode of development are important in the classification of Agaricales. In Agaricales, hymenium is found lining the lamellae or gills, found beneath the pileus. Gills are generally thin strips of tissue radiating from the margin of the pileus towards the stalk. The gills may be free from the stalk and in them the pileus and stalk can be easily separated without disturbing gills. Gills may be directly attached to the stalk, and such gills are described as adnate gills. If the gills run down the stipe for a distance, such gills are described as decurrent gills. The nature of gill attachment to the stalk is an important taxonomic character. The inner tissue of the gills is called trama. Basically a trama consists of plectenchymatous tissue made up of elongated hyphal cells. Some specialized structures like sphaerocysts are present in fungi like Russula and Lactaria. The hymenium is formed on either side of the gills and each basidia bears four basidiospores which are actively discharged. An interesting phenomenon of the mycelium of a number of agarics is ‘bioluminescence’. Organic matter or pieces of decaying wood penetrated by the mycelium of bioluminecent fungi glow in the dark, like the wood pieces infected with Armillariella mellea. The basidiocarps of Omphalotus olearius, the jack-olantern mushroom has luminescent gills. The basidiocarps of Mycena lux-coeli are also bioluminescent. Traditionally all mushrooms are placed in a single family Agaricaceae, and genera are grouped into various sections based on the colour of the spore prints like

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melanosporae (spore print brown to black), ochrosporae (spore print rusty brown), rhodosporae (spore print pink or red), leucosporae (spore print white) etc. However, at present 16 families are recognised in the order, and they can be conveniently divided into various groups based on the color of spore print (Table 8.3). The groups have no taxonomic importance. Table 8.3. Division of mushrooms on the basis of colour of spore print Group

Family

Colour of the spore print

Melanosporae

Agaricaceae

Chocolate brown

Coprinaceae

Dark brown to black

Gomphidiaceae

Olive brown

Paxillaceae

Coffee brown

Strophariaceae

Yellowish to dark brown or purple

Bolbitaceae

Clay brown

Cortinariaceae

Rusty brown

Pluteace

Red spore print, free gills

Entolomataceae

Red spore print, attached gills

Amanitaceae

White spore print

Hygrophoraceae

White spore print (waxy gills)

Lepiotaceae

White spore print (annulus movable)

Tricholomataceae

White spore print

Russulaceae

White spore print (spaerocysts in trama)

Boletaceae

Fruit body fleshy, hymenium lines pores

Cantherellaceae

Basidiocarp leathery, lamellate

Ochrosporae Rhodosporae Leucosporae

Other families

Family - Agaricaceae: Previously all mushrooms were included in this family. Now it has three genera viz. Agaricus, Cystoagaricus and Melanophyllum. The family as represented by Agaricus, is characterized by producing a typical basidiocarp with a white to brown or gray pileus, gills are present on the underside of the pileus, gills are free, and the pileus can be easily separated from the pileus, the gills are light coloured when young, often pink or white, but eventually darken with maturity. The stipe is long, round with an annulus but no volva is formed at the base. The spores are dark brown in colour. Various species are found on well manured often grassy areas. A. campestris is the common field mushroom. A. rodmani is quite large. Both the species are eminently edible. Agaricus bisporus (= A. brunnescens) is the most popular cultivated mushroom. But, two other species viz. A. placomyces and A. silvaticus may cause gastrointestinal disturbance in some individuals. The other two genera are very small with limited distribution. Family - Amanitaceae: The family includes well known white spored genus Amanita. It produces typical fruit bodies with a stipe and pileus. Gills are free and the stipe is having an annulus, and possesses a clear cup shaped volva at the base. Fruit bodies are very attractive. Particularly striking are Amanita virosa with pure white

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fruit bodies, but it is very poisonous and called destroying angel. Amanita muscaria, commonly called fly agaric or divine mushroom, are beautiful with yellow, orange or brilliantly red cap speckled with scales or patches of white tissue. It is a hallucinogenic fungus. Amanita phalloides is a deadly poisonous mushroom, and is often responsible for deaths due to mushroom poisoning. It is described as ‘death cup’. Order - Boletales Boletales contain over 1300 species with a diverse among of fruit bodies like boletes earthballs, puffballs and false truffles most of the make are sapropic followed on the wood of fallen trees or in the soil at the base of trees. Family - Boletaceae: Boletes typically appear like other agarics with fleshy fruit body made up of stipe and pileus, but on the underside of the cap they bear pores instead of gills. The fruit bodies are often large and brightly coloured. Cap color range from almost black through various shades of brown, yellow and red. The caps may be glabrous or covered with long shaggy hairs. They are worldwide in distribution and important examples are Boletus, Strobilomyces, Suillus, Lecinium etc. Boletus mirabilis and B. edulis are excellent edible mushrooms, but some cause gastrointestinal problems. Family - Bolbitaceae: The stipe and pileus are separable to confluent. The pilei have a hymenium cuticle or cellular layer. Spore deposit is bright to dull rusty brown and the spores have a distinct apical pore, or in a few pores are very minute. The stipe is central. The members grow on humus and organic debris. The type is Bolbitus. Other genera are Agrocybe, Conocybe, Pholiotina etc. Family - Coprinaceae: Three genera viz. Coprinus, Panaeolus and Psathyrella are placed in this family. Black spores characterize the well known genus Coprinus whose members are commonly known as inky cap mushrooms since at maturity the gill of many species deliquesce into black inky liquid that drips from the disintegrating cap. The fruit bodies of Panaeolus are strikingly similar to Coprinus, but most species of Panaeolus are poisonous and hellucinogenic. Family - Cortinariaceae: The members of the family are characterized by rusty brown to cinnamon brown spores. In the genus Cortinarius, a delicate spider web like veil called cortina is present hanging down the pileus. Other genera included are Galerina, Inocybe etc. Of these, species of Galerina are deadly poisonous. Family - Entolomataceae: The stipe and pileus are confluent, a veil typically is absent. The spore deposit is reddish, cinnamon to brownish vinaceous. Spores are non-amyloid, typically angular or grooved. The lamellar trama is regular. The pilear cuticle is chiefly of appressed hyphae or hyphae with cystidiform end cells more or less ascendent. The type genus of the family is Entoloma. Other genera are Clitopilus and Rhodocybe. Family - Gomphidaceae: The spore deposit is olive brown. The lamellae are typically decurrent, close to distinct and thickish. The spores are large and boletoid in shape. The pleurocystidia are long (up to 100 µm or more) and subcylindric to

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narrowly clavate in shape. The species form mycorrhizae with conifers. The type is Gomphidium and other genera are Cystogomphus and Chroogomphus. Family - Hygrophoraceae: This family includes a single genus Hygrophora. The gills are thick, adnate to decurrent. The gills are covered with waxy material that can be detected by crushing the gills between the thumb and forefinger. The spores are white. The fruit bodies have very colourful caps, which are either yellow orange or red. Family - Lepiotaceae: The family includes three genera viz. Lepiota, Leucoagaricus and Chlorophyllum. They are commonly found in lawns and pastures. The fruit bodies of Lepiota resemble those of Agaricus, but produce white spores. In the genus Chlorophyllum the spores are green coloured. Fruit bodies have conspicuous annulus on the stipe, and volva is absent. The annulus is movable like a ring on the stipe. Family - Paxillaceae: The pileus and stipe when present are confluent. The spore deposit is ochraceous to coffee brown to olivaceous. The lamellae are well formed and thick to thin and close; at times they are intervenose. The hymenophore is typically readily separable from the pileus. The type is Paxilla. Other genera are Phylloporus, Linderomyces, Neopaxilla etc. Family - Pluteaceae: This family is characterized by pink colour of spore mass. Two genera viz. Volvariella and Pluteus are included in this family. Volvariella (Paddy straw mushroom) is the cultivated mushroom, and Pluteus is widely distributed in nature Family - Russulaceae: Only two genera viz. Russula and Lactarius are recognized in the family. The members are distinguished from others by the presence of sphaerocysts, the large globose or ovoid cells, in the body of pileus and trama of the gills. The gills are attached and spores are white. The members of the genus are large, often brilliantly coloured and the gills are brittle. In the genus Lactarius watery or milky juice exudes from the cut ends of the gills. Lactarius deliciosus is a very good edible species. Russula emetica is a beautiful but probably poisonous species. Family - Strophariaceae: The family includes the genera Stropharia, Nematoloma and Psilocybe. All have attached gills and produce dark purple brown spores. Among the members, the species of Psilocybe are hallucinogenic fungi. Psilocybe mexicana is used in the religious festivals of Mexican region and is called ‘sacred mushroom’. Family - Tricholomataceae: It is a very large family composed of whitespored species with attached gills. Members of the family are found on many different substrates and in many different habitats. The species of Pleurotus (Oyster mushroom) and Lentinus edodes (shiitake mushroom) are important cultivated mushrooms. Armillariella mellea (honey agaric) produces honey coloured fruit bodies in clusters and commonly grow on dead tree stumps. The fruit bodies of Marasmius oreades are small but tough mushrooms with thin stalks. They grow in a circular fashion in grass land and are described as ‘Fairy-ring fungus’. The fruit bodies of Omphalotus olearis (Jack-o lantern) are relatively large with orange caps and decurrent gills that are bioluminescent. Other common genera are Tricholoma, Panus, Clitocybe, etc.

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Family - Cantherellaceae: The members included in this family produce fruit bodies which are leathery and possess lamellae. The type is Cantherellus. Most authors place this family in the order Aphyllophorales. Order - Aphyllophorales: This is a very large order comprising about 400 genera and 1200 species in 23 families. All of them produce open hymenium formed on various types of structures but not on gills. The hymenium may line pores, coral like branches, teeth like projections etc. The basidiocarps of the fungi are corky, leathery, woody and perennial, but not soft and evanescent. Most of the fungi are free living saprophytes occurring on ground or decaying plant parts. However, some of the members of the group are wood inhabiting causing serious wood rot. Some form fruit bodies on forest trees which partially encircle the stem, and are called shelf fungi or bracket fungi.

Fig. 8.10. Various types of fruiting bodies produced by Aphyllophorales

Traditionally, the order (previously called Polyporales) is divided into four families viz. Polyporaceae, Clavariaceae, Hydnaceae and Thelephoraceae, based on gross morphology of basidiocarp and orientation of hymenium. At present, basing on

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more stable microscopic characters such as hyphal systems, formation of hymenium and nature of basidiocarp development, 23 families are recognized. They can be divided into five major groups viz. 1. Poroid families, 2. Clavarioid families, 3. Hydnaceous families, 4. Thelephoroid families and 5. Lamellate families. These groups have no taxonomic significance, but convenient groups for the study of this large order. Various types of fruit bodies produced by members of Aphyllophorales are shown in Fig. 8.10. Poroid families: In five families of Aphyllophorales, the hymenium is typically organized into circular structure leaving a pore at the centre. The basidiocarps are pileate, corky or woody and the lower surface shows numerous pores. The families are 1. Polyporaceae, 2. Ganodermataceae, 3. Fistulinaceae, 4. Bondarzewiaceae and 5. Podoscyphaceae. Clavarioid families: The fruit bodies form erect clavate or coralloid branches and hymenium on the external surface of the branches. Such organization is found in four families viz. Clavariaceae, Clavulinaceae, Lachnocladiaceae and Sparassidaceae. Hydnaceous families: In this group the basidiocarps are mushroom like with stalk and pileus. The construction of the fruit body is monomitic with generative hyphae only and, hence, the fruit bodies are smooth like those of agarics. On the lower side of the pileus, spines or teeth like projections are present and hymenium is formed on these teeth like structures. Such structures are found in five families viz. Hydnaceae, Auriscalpiaceae, Bankereaceae, Echinodontiaceae and Hericaceae. Thelephoroid families: The basidiocarps are simple, sessile and resupinate (crust like or flat on the substrate). They are membranous, leathery or hard, with monomitic construction. Hymenium is formed variously. Seven families can be recognized in this group. They are Thelephoraceae, Corticiaceae, Coniophoraceae, Stereaceae, Punctulariaceae, Hymenochaetaceae and Gomphaceae. Lamellate families: The hymenium is formed on gill like structures. The fruit body is leathery and perennial, but not fleshy and evanescent. Two families viz. Cantherellaceae and Schizophyllaceae can be included in this group. In Cantharellaceae, the basidiocarps are half funnel shaped and on the outer surface thin folds resembling gills are formed and hymenium is formed on these structures. In Schizophyllaceae, small, leathery fruit bodies are formed on groups on fallen logs and decaying wood. The gills on the lower surface of the pileus are typically split in the middle, and they are described as ‘split gills’. Such gills are formed in the only Genus Schizophyllum. Subclass - Phragmobasidiomycetidae It is a small group which comprises fungi whose metabasidium is typically septate and divided into four cells either by transverse or longitudinal septa. It includes three orders viz. Tremellales, Auriculariales and Septobasidiales. Order - Tremellales: They are commonly called ‘jelly fungi’ because of the gelatinous jelly like nature of basidiocarps. They are saprophytic, and on substrata

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form a thin layer of gelatinous hyphae and the basidiocarps appear as cushion shaped thin layer, and they produce oval to globose basidia. They are probasidia in which karyogamy occurs, and then it is divided into 4 cells by longitudinal septa. Each cell bears an elongated sterigma and one basidiospore is formed at its tip. The order comprises of three families 1.

Family - Tremellaceae: Basidia asymmetrically on sterigmata.

noncatenulate,

spores

borne

2.

Family - Sirobasidiaceae: Basidia catenulate, maturing basipetally, protosterigmata sporoid, deciduous. Single genus Sirobasidium is recognized.

3.

Family - Hyaloriaceae: Basidia not catenulate, prosterigmata not deciduous, Spores nonapiculate, borne symmetrically on cylindrical, filiform, nonspiculate stermata.

Order - Auriculariales: Members of this group are free living saprophytes that occur on wood. They form ear shaped, gelatinous basidiocarps, and hence are described as ‘wood-ears’. They produce single celled binucleate basidia. After the karyogamy, meiosis occurs to produce four haploid nuclei, and then it is divided by transverse septa into four cells. Each of the four cells bear a long sterigma forming one basidiospore at the tip. It is a small group with a single family Auriculariaceae. The members of this group are saprobic on decaying plant parts. Order - Septobasidiales: This is a small group of insect parasites. Mycelium is limited and produce thick walled spores which function as probasidia and gives rise to a transversely septate four celled metabasidium. From each cell of the metabasidium one basidiospsore is formed on a small sterigma, as in rust fungi. A single family Septobasidiaceae is recognized in the order. CLASS - GASTEROMYCETES This class includes such commonly known macrofungi like puff balls, earth stars, stink horns, bird’s nest fungi etc. They are quite common in a variety of habitats. All of them are free living occurring on soil, below the soil (hypogean), on decaying plant parts, on dung and other substrates. Some gasteromycetes are mycorrhizal, forming symbiotic association with roots of higher plants. It is an unnatural assemblage of basidiomycetes that have the common negative character that the basidiospores are not discharged violently. Instead of asymmetrically poised basidiospores as found in hymenomycetes, in gasteromycets badisiopores are usually symmetrically poised on their sterigmata or are sessile. They are termed statismospores. Fruit bodies remain closed or open only after the spores are mature. Basidium is a holobasidium. Clamp connections may or may not be present. Dolipore septa are common. The fruit bodies have a distinct outer wall called peridium. Within the peridium, the spore bearing part is described as gleba. Presence or absence of hymenium is considered as a fundamental character. The class includes nine orders. In some of them hymenium is formed during spore production while in others hymenium is not formed at any stage of development. Five different types of hymenia are recognised based on their structure and

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development. They are lacunar type, forate type, auleate type, pileate type and multipileate type. In this class, nine orders are recognised. Hymenium is formed at some stage of development in the members of five orders, and hymenium is not formed at any stage in the members of other orders. Orders in which hymenium is present are 1. Podaxales, 2. Phallales, 3. Hymenogastrales, 4. Lycoperdales and 5. Gauteriales. Orders in which hymenium is not formed at any stage are 6. Tulostomatales 7. Sclerodermatales, 8. Melanogastrales and 9. Nidulariales. Order - Podaxales: They are very common in grass lands, and the fruit bodies appear like unopened Coprinus fruit bodies. They are relatively large measuring up to 25 cm tall and 5 cm wide. The important characters are peridium single, membranous fragile, dehisce around the base where stipe joins the columella. Hymenium is pileate, spore mass appears black, but the individual spores are reddish brown. A single family Podaxaceae is recognized in the order. Podaxis pisstilaris is the well known fungus of this group. Order - Phallales: The members of the order are commonly called ‘stink horns’ because of the fetid odour that accompanies the exposure of gleba. The fruit bodies are simple, hollow, column like receptacle which bears gleba on its outer surface near the top. Some are hypogeal and others, mainly the members of Phallaceae and Clathraceae are epigeal. The young fruit body is whitish egg shaped, of hen’s egg size. The development of basidia and basidiospores occur within the egg shell. At maturity the eggs hatch. The pressure caused by enlargement of internal structures breaks the peridium and a long spongy receptacle emerges carrying the gleba on the surface of the tip. The egg shell (peridium) remains as a volva. After exposure, gleba undergoes autodigestion. The spore mass is enmeshed in a foul smelling gelatinous greenish matrix. Flies attracted by the odor visit the fruit bodies, and spores cling to the body and mouth parts, and are disseminated. Six families are recognized in the order. Of them two families viz. Phallaceae and Clathraceae are epigeal and conspicuous. The remaining four families viz. Protophallaceae, Clastulaceae, Hysterangiaceae and Gelopellidaceae are hypogeal or inconspicuous. The genus Phallus is the best known stink horn. Family - Phallaceae: Receptacle simple, hollow column bearing the mucid, usually stinking gleba near the top on the outside. Development is pileate. Family - Clathraceae: Peridium dehiscent, receptacle present, peridial sutures present, epigeal, gleba glutinous, receptacle sessile or stipitate consisting of a spherical network or of several columns united at the top, or of spreading arms. Gleba usually borne on the inside of the receptacle. Development of fruit body multipileate. Family - Claustulaceae: Peridium dehiscent, receptacle present, sessile, globose with a nonmucid sweet smelling gleba borne on the inside. A single monotypic genus is recognized viz. Claustula fischeri.

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Family - Protophallaceae: Peridium indehiscent, receptacle absent, usually hypogeal, gleba soft but not glutinous, gelatinous layer well developed but interrupted by peridial sutures, gleba divided into many lobes, development multipileate. Family - Hysterangiaceae: Peridium indehiscent, recepticle absent, usually hypogeal, gleba soft but not glutinous, gelatinous layer below the peridium, poorly developed, often cartilaginous. Family - Gelopellidaceae: Peridium indehiscent, receptacle absent, usually hypogeal, gleba soft but not glutinous, gelatinous layer continuous. Gleba more or less globose mass with central columella, development pileate. Order – Hymenogastrales: The members of the order are intermediate in structure between hymenomycetes and Gasteromycetes. Most of them are hypogeal and are often called ‘false truffles’. They are also described as secotioid fungi- the agarics in which cap failed to expand. Some members form mycorrhizal association with roots of higher plants. Three families viz. Hymenogastraceae, Gastroporaceae and Protogastraceae are recognised. Family - Hymenogastraceae: Fruit bodies usually more than 1cm in diameter, peridium of one or two indistinct layers; gleba of a labyrinthine cavity; basidia fusiform one to four spored; spores ellipsoidal, coloured, often longitudinally ribbed, irregularly warted or occasionally smooth. A single genus Hymenogaster with about 70 species is recognized. Family - Protogastraceae: Fruit bodies minute to 500 µm in diameter; globose with a single globose glebal chamber; hymenium not convoluted; basidia subfusiform, one to four spored; spores shortly ellipsoidal, smooth, brown, filling the glebal cavity at maturity. Includes a single monotypic genus Protogaster (P. rhizophilus) on living roots of Viola. Family - Gastroporiaceae: Fruit-bodies globose up to 1.5 cm in diameter. Peridium double; outer peridium chalk white, flocculent, of collapsed hyphae mixed with oxalate crystals, innner peridium membranous, cracking irregularly at maturity; gleba homogeneous, olivaceous, powdery, basidia usually eight spored, spores ochraceous, globose to subglobose, ornamented with a few small irregular warts; cystidia and capillitium absent. Contains a single genus Gastroporium with species G. simplex, associated with grasses, etc. in xerothermin situations, Mediterranean and Central Europe. Order - Lycoperdales: Puff balls and earth stars belong to this group. The fruit bodies are epigeal, globose, a few cm in diameter. The giant puff ball (Clavatia gigantea) may be up to 70–80 cm in diameter. The fruit bodies are sessile and liberate spores from an apical pore when pressure is applied. The peridium consists of two layers, outer exoperidium and inner endoperidium. The exoperidium which may be warty, spiny or granular withers away leaving thin membranous endoperidium which has a small central opening (ostiole) from which the spores are puffed when an object strikes the surface of the basidiocarp. Four families are recognized in the order.

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Family - Lycoperdaceae: The members are called puff balls. They grow on tree stumps, decaying logs or on ground in woods and lawns. Gleba is powdery mixed with capillitium at maturity. The genus Lycoperdon is the common puff ball. Family - Geastraceae: The members are called ‘earth stars’. In these fungi, the exoperidium of the fruit body splits along the radial lines when wet and opens out in the form of a star. The inner peridium remains intact with a central apical pore. Family - Arachniaceae: The fungi of the family form hypogeal fruit bodies. They are globose and 1–2 cm in diameter. Family - Mesopellidaceae: The members of this family are either epigeal or hypogeal. The peridium of fruit body is very thick, and it may be three to four layered. Order – Gautieriales: It is a small group of forest, mycorrhizal fungi. Fruit bodies are usually hypogeal, peridium thin, gleba cartilaginous. In this order, a single family Gautieraceae with a single genus Gautieraia is recognized. Order - Melanogastrales: Resemble Hymenogastrales but without hymenium; fruit bodies hypogeal, and mycorrhizal. These are also called false truffles. Two families are recognized, 1. Melanogastraceae, and 2. Torrendiaceae. Family - Melanogastraceae: Fruit bodies hypogeal, irregularly globose, islands of primary basidia gelatinize to give way to secondary basidia. Important examples are Melanogaster, Leucogaster, etc. Family - Torrendiaceae: Pseudostem present or absent, primary basidia scattered through maturing gleba, partially collapsing but persisting, forming foci towards which secondary fertile basidia grow, terrestrial or marine. Important examples are Torrendia and Nia. Order - Tulostomatales: These are called stalked puff balls. They resemble puff balls but with a stalk. Hymenium is not formed at any stage. Two families recognized, 1. Tulostomataceae, and 2. Calostomataceae. Family - Tulostomataceae: Peridium relatively simple, often not divisible into well defined layers, usually apically dehiscent, stipe well developed. Gleba powdery with well developed capillitium, basidia pleurosporus. Important examples, Tulostoma, Schizostoma, Phelloria. Family - Calostomataceae: Peridium complex consisting of four clearly defined layers, the outer most layer gelatinous spiny, the second pigmented, the third very horny, the innermost membranous and remaining attached to the outer layers only at the top around the star shaped apical pore and so hanging loose inside the horny layer. Stipe consisting of a continuation of the outer and the horny layers. Gleba pale of clay like texture with annularly thickened capillitium, initially surrounded by secondary fertile basidia. Spores large and often extravagantly ornamented, with a reticulum or long spines. A single genus Calostoma with about 10 species is recognized in the family. Order - Sclerodermatales: They are called stony puff balls. In these fungi, peridium is very hard. Hymenium is not found. Among these fungi, Pisolithus tinctorius is a very important ectomycorrhizal fungus, and it is used extensively for inoculation of seedling of trees used in afforestation programmes. Four families are

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recognized 1. Sclerodermataceae, 2. Glischrodermataceae, 3. Broomeiaceae, 4. Astraceae. Family - Sclerodermataceae: Fruit body simple, often strikingly yellow or orange, peridium not divided into separable layers, dehiscent by attribution from the apex, ocassionally irregularly stellate; glebal islands consisting of groups of basidia which discharge their spores early, development being continued through the agency of placental hyphae; spores globose, brown with well developed spiny or reticulate ornamentation, capillitium absent. Important genera are Scleroderma and Pisolithus. Family - Glischrodermataceae: Fruit bodies pulvinate to subglobose, frequently gregarious, often on a common subiculum, peridium thin, usually tough dehiscing apically, basidia very large, branched and lobed and bearing numerous spores on short sterigmata over the entire surface; spores globose reticulately wrinkled or warted. Important genera are Glischrodrema and Lycoperdella. Family - Broomeiaceae: Fruit body compound, consisting of numerous spore sacs seated on a more or less massive stroma; outer peridium forming a thin universal veil breaking away at maturity, inner peridia dehiscing by apical stroma, capillitium present. Important genera are Broomeia and Diplocystis. Family - Astraeaceae: Fruit body sessile, initially subglobose but with thick and complex outer peridium which usually opens stellately from the apex to form an earth star, occasionally indehiscent; inner peridium of one or two layers; gleba with abundant capillitium; Spores irregularly warted or reticulated, globose or subglobose. Important genera are Astraeus and Endogonopsis. Order - Nidulariales: The members of this order are called ‘Bird’s nest fungi’. The fruit bodies are inverted bell shaped, and contain peridioles which are glebal structures containing spore mass. They are attached to the inner surface of the fruit body by a sheath and purse. When rain drops fall on the peridiole, they are shot out. According to Ingold (1972) the throw occurs to a maximum height of 569 cm. Two families are recognized, 1. Nidulariaceae, and 2. Sphaerobolaceae. Family - Sphaerobolaceae: Fruit bodies up to three mm in diameter, initially globose, growing on wood or dung; peridium of six layers, dehiscing by stellate apical splitting leaving the single globose peridiole free in the peridial cup, inner peridial layers becoming detached from the outer except tips of the rays; differential absorption of water by the layers of the inner cup causing it to revert, shooting the peridiole away; spores ellipsoid, smooth, hyaline, 10 um in diameter. A single genus Sphaerobolus with two species recognized in the family. Family - Nidulariaceae: Fruit bodies one mm to one cm in diameter, initially globose, usually becoming pulvinate or obconical. Peridium of one to three layers, dehiscing either irregularly or by a circumscissile epiphragm, in which case the remains of the peridium form a cup. Peridioles occasionally one, usually several to many per fruit body. Basidia acrosporous, four spored with mucilaginous coat. Five genera are recognized in the family. They are Nidularia, Nidula, Crucibulum, Mycocalla and Cyathus. Of these Cyathus is the well known or familiar bird’s nest fungus.

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IMPORTANT GENERA GENUS : PUCCINIA Class : Uredinomycetes Order : Uredinales Family : Pucciniaceae The species of Puccinia are obligate parasites on higher plants causing rust disease. About 700 species were described in the genus so far, and of them about 125 species are recorded in India. They attack a number of crop plants, weeds and trees. Important crop plants attacked by rust fungi include wheat, barley, sorghum, maize, bajra, groundnut, sunflower and others. Of all these, the black stem rust of wheat caused by Puccinia graminis tritici is the most serious disease causing severe yield losses. The rust is known since a long time. In the first century B.C., Romans believed that the rust disease of wheat is caused by the anger of ‘rust God’ Robigo and used to perform a function called ‘Rubigalia’ on a day which corresponds to 25th April in modern calendar, and sacrificed the red blood of a black dog to please the rust God. Whatever may be the reason for the belief of Romans, one thing that becomes clear from this is that the wheat rust was a very serious disease even more than 2000 years back, and the symptoms of the rust disease remained almost unchanged during this period. Some important rust diseases of crop plants caused by Puccinia species are given below (Table 8.4). Table 8.4. Some important diseases caused by Puccinia species. Crop

Common name of the disease

Puccinia species

Wheat

Black stem rust

Puccinia graminis tritici

Wheat

Yellow stripe rust

P. striiformis

Wheat

Orange leaf rust

P. recondita

Maize

Maize rust

P. maydis, P. sorghi

Sorghum

Sorghum rust

P. purpurea

Bajra

Bajra leaf rust

P. penniseti

Groundnut

Peanut rust

P. arachidis

Sunflower

Sunflower rust

P. helianthi

In the life cycle of Puccinia species five types of spores are formed. They are 1. Pycniospores or spermatia, 2. Aeciospores, 3. Urediniospores, 4. Teliospores (also described as teleutospores) and 5. Basidiospores. These spores are formed sequentially in a cyclic fashion in the life cycle, and are designated as 0, I, II, III and IV spore types or stages. All these five types may or may not be formed in same species of Puccinia. One or two spore types may be missing in the life cycle. When all the five types of spores are formed, the life cycle is described as macrocylic. If one or two spore stages are missing in the life cycle, it is described as microcyclic. The species of Puccinia which complete the life cycle producing all the spore types on a single host are described as autoecious species. The species which requires two

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different hosts to complete the life cycle are described as heteroecious. The host on which perfect stage (sexual stage) occurs is considered as the primary host and the other as alternate or secondary host. Among the autoecious species, Puccinia asparagi, P. helianthi etc. are important, while among the heteroecious species, Puccinia graminis is very important. This species produces 0 and I stages on barberry, and II and III stages on wheat. In rust fungi, teleutospore stage is considered as sexual stage since during the germination of teleutospores the two compatible nuclei fuse and karyogamy is followed by meiosis. Hence wheat is considered as the primary host and barberry as the secondary host. Among the other heteroecious species, Puccinia sorghi produces II and III stages on maize, while O, I stages occur on Oxalis stricta. In the life cycle of Puccinia penniseti, II and III stages occur on bajra, and O and I stages occur on brinjal. Life cycle of Puccinia: Among all the species of Puccinia, Puccinia graminis tritici causing black stem rust of wheat was most thoroughly studied, and hence, it is taken as a good example for the life cycle of Puccinia species. Puccinia graminis tritici is a macrocylic, heteroecious rust completing its life cycle on two hosts viz. wheat and barberry. Stages on wheat: Due to infection by Puccinia graminis tritici two stages are formed on wheat. They are uredinial stage and telial stage. Uredinial stage: When aeciospores released from infected barberry bushes are deposited on wheat leaves, they germinate under favourable conditions and germ tubes enter the leaves through stomata. The stems may also be infected. After an incubation period of about one week, the mycelium aggregates below the epidermis and produces large number of urediniospores. As the mass of urediniospores increase, the epidermis ruptures due to internal pressure, and the urediniospores are exposed. The spots formed by the rupture of epidermis are called pustules. They are also called uredinia or urediniosori. The pustules on wheat leaves and structure of uredinial sorus are shown in the Fig. 8.11.

Fig. 8.11. Uredinia and uredinial sorus in Puccinia. A. Uredinial pustules on wheat leaf. B. Structure of uredinial sorus.

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The urediniospores become easily airborne and when they are deposited on wheat leaves or other plants they germinate and readily infect the leaf, and after an incubation period of about one week, new pustules are formed. Since the uredinial pustules are formed repeatedly in the same crop season, it is called ‘repeating stage’, and the urediniospores are described as ‘conidia of rust fungi’. When the urediniospores are deposited on wheat leaves, they germinate under favourable conditions. High relative humidity, near saturation point, or free water is essential for germination of urediniospores, and optimum range of temperature is 20 – 25oC. The germ tubes grow and form a round thin globose structure called appressorium. The appressorium helps to keep the germ tube attached to the leaf surface. From the underside of the appressorium, an infection peg develops and enters the leaf through the stomata. The tip of the infection peg expands in the substomatal cavity, and it is described as substomatal vesicle. Hyphae develop from substomatal vesicle and spread in the intercellular spaces. These hyphae send in small, globose haustoria into the host cells to draw nourishment. Up to this stage the reserve food material in the urediniospore itself provides energy for the growth. Once the organic connection is established between the pathogen and the host, the mycelium develops rapidly and aggregates below the epidermis in about four or five days after infection. From this aggregated mycelium urediniospores are produced on small, evanescent stalks. As the spore mass increases, pressure builds up on the epidermis covering the developing pustule, and it eventually ruptures exposing the spore mass. This spot formed by the rupture of epidermis is called pustule. In each pustule thousands of spores are produced. These urediniospores are round to oval in shape, thick walled and measure 20–25 µm in size. Small spines or thickenings may be present on the spore wall. These are called echinulations. Each spore has two nuclei, and there are four or more germ pores in the wall. The urediniospore germinates and germ tubes come out through these germ pores. More than one germ tube may come from a single spore but only one shows further growth. The urediniospores are small, dry, round, thick walled, coloured spores produced in large numbers on infected leaves, and hence, easily come into the air by mechanical disturbances or turbulence of air, and can spread to long distances on wind currents. The long distance dispersal of urediniospores of wheat rust fungi was first reported by Stakman and his coworkers in USA during 1920s and by Prof. K.C. Mehta and his coworkers in India during 1930s and 1940s.

Fig. 8.12. Telia and telial sorus in Puccinia. A. Black telial pustule (1) on stem and leaf of wheat. B. Structure of telial sorus.

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Telial stage: By the time the wheat crop reaches harvesting stage, teliospores begin to form in the same pustule in which urediniospores formed earlier. Later formed pustules produce only teliospores. They are formed both on leaves as well as stems. They appear flat dark brown or black. By the time the telial sori or pustules are formed, the crop matures and the leaves dry up, and drop off. Hence they are seen prominently on stems. Therefore, the disease is described as black stem rust. By the time of the telial stage, the crop is in seed filling stage called ‘dough stage’. If the telial stage is very severe, seed filling is very poor. The teliospores are smooth, thick walled, two celled and the tip of the upper cell is pointed. Each cell contains two nuclei. They measure 30 – 40 µm in length and 15 – 20 µm in width. They have long persistent stalks to which the spores are firmly attached. Hence, they do not become airborne easily. The telial sorus is shown in Fig. 8.12. Teliospore germination and formation of basidiospores: After harvesting of wheat, the teliospores remain dormant in dried leaves for a long time. When favourable conditions return the teliospores germinate giving rise to two germ tubes, one from each of the two cells of the spore. The upper cell of the spore possesses a germ pore at the tip and the lower cell has germpore at one side just below the septum. Hence, the germ tube comes from the tip of the upper cell, and laterally from lower cell. As the cells begin to germinate, the two nuclei in the cell fuse and meiosis follows karyogamy producing four haploid nuclei. These haploid nuclei enter into the germ tube. Then transverse septa develop in the germ tube resulting in formation of four uninucleate cells. This structure is described as promycelium. From each cell of the promycelium a small sterigma develops and one basidiospore is formed at the tip of each sterigma. Thus four basidiospores are formed. Of the four basidiospores thus formed, two are of (+) strain and two are of (-) strain. They are released passively and aerially dispersed. They cannot infect wheat plant, but can infect alternate host, the barberry bush. The germination of the teliospore and formation of basidiospores is shown in the Fig. 8.13.

Fig. 8.13. Germination of teliospore and formation of basidiospores. A. Formation of promycelium through germination of upper cell of teliospore. B. Formation of promycelium through germination of lower cell of teliospore. 1. Teliospore. 2. Promycelium. 3. Basidiospores.

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In basidiomycetes, the cell in which karyogamy occurs is described as probasidium and the structure producing basidiospores is described as metabasidium. Hence, the teliospore may be considered as probasidium and promycelium as metabasidium. Stages on barberry: In the life cycle of Puccinia graminis tritici 0 and I stages are formed on barberry plants. Stage ‘0’ is called spermagonium stage, and stage ‘I’ is called aecial stage. Spermagonium stage: When the basidiospores are deposited on barberry leaves, they germinate under favourable conditions by giving rise to germ tubes which enters the leaf either through stomata or by penetrating the epidermal cells directly. In the leaf the hyphae grow in the intercellular spaces and send in small globose haustoria into the host cells. Some of the basidiospores are of (+) strain and others are of (-) strain. When mycelia of these strains are growing both the strains produce spermagonia on the upper epidermis. The spermagonium, also called pycnium, is a flask shaped structures with round basal part and narrow beak shaped upper part, also called neck. The neck part projects to the outside by piercing through epidermis. The neck part is lined by large number of periphyses. When the spermagonia are fully formed, they appear like yellow or orange coloured pustules on the upper surface of barberry leaves. From the inner wall of the spermagonium long, erect, pointed cells develop and these are called spermatiophores. At the tip of the spermatiophores small, globose, smooth, hyaline unicellular spermatia are formed in chains with basipetal succession. They are also called pycniospores. These spores mixed with honey coloured fluid formed in the spermagonium ooze out through the ostiole. From the entrance of spermagonium, a number of erect hyphae, which are longer than periphyses, develop and project out prominently. These are called receptive hyphae. Spermogonia on barberry leaf and their structure is shown in Fig. 8.14.

Fig. 8.14. Spermagonia on barberry leaf and structure of spermagonia. A. Groups of spermagonia on barberry leaf. B. Structure of spermagonium. 1. Spermatia. 2. Receptive hyphae.

The rust fungi are heterothallic, and the spermatia and receptive hyphae of the spermagonium are not compatible with each other because they are of the same

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strain. Hence, the receptive hyphae can receive the nucleus only from a compatible strain formed either on the same leaf or on other leaves or plants. When a compatible spermatium or pycniospore is deposited on receptive hypha, the nucleus from the spermatium enters into receptive hypha. The compatible nucleus that entered the receptive hypha repeatedly undergoes mitotic divisions, and the nuclei spread throughout the mycelium. The two compatible nuclei in each cell of the mycelium pair together. Thus, the dikaryotization occurs. In the life cycle of Puccinia, the occurrence of spermagonium was reported in early 19th century by Persoon (1801), Unger (1833) and others, but its role in dikaryotization was explained for the first time by Craige in 1927. Till then the role of this stage was not known. Hence it is described as ‘0’ stage. Hiratsuka and Cummins (1963) reported that about 11 types of spermagonia are formed by Puccinia species. Of these, flask shaped spermagonium is the most common type. Aecial stage: The mycelium formed in barberry leaves due to infection by basidiospores form spermagonia on the upper surface, and the same mycelium form aecial primordia below the lower epidermis. However, until dikaryotization takes place the aecial primordia are inactive. When the entire mycelium becomes dikaryotic, the aecial primordial are stimulated to form aecial cups on the lower surface of the leaf. The aecial cups are surrounded by a clear peridium, and hence they have a clear shape. When the aecial cups rupture the epidermis to expose the aeciospores, the aecial pustules are formed. The aecial pustules always form on the lower surface of the leaf, and occur in concentric rings (Fig. 8.15).

Fig. 8.15. Aecial sori on barberry leaf. A. Aecial cups on barberry leaf. B. Structure of aecial sorus. 1. Peridium. 2. Aeciospores.

The aeciospores are formed on generative cells arranged in a layer at the base of the aecial cup. The spores are formed in chains with basipetal succession. They are single celled, globose and measure 20-25 µm in diameter. The aecial chains are closely packed in the aecial cups, and due to this the spores may appear angular or polygonal. They disperse through the air. They can infect only wheat leaves, but not barberry leaves. On wheat leaves, the infection due to aeciospores results in the formation of uredinial pustules.

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Life cycle: In the genus Puccinia, basidiospores are uninucleate and haploid. The cells of mycelium formed in barberry leaves are also at first uninucleate. When the receptive hyphae receive compatible nucleus from spermatium, dikaryotization occurs. The aeciospores formed on dikaryotic mycelium are also dikaryotic. The infection of wheat leaves by aeciospores result in uredinial stage producing dikaryotic urediniospores. The teliospores produced on wheat leaves are also dikaryotic. When the teliospores germinate, karyogamy occurs immediately followed by meiosis. The basidiospores formed on the promycelium are uninucleate and haploid. Thus in the life cycle of Puccinia the diploid stage is confined to the zygotic nucleus only, and dikaryotic stage is the predominant one. The life cycle of Puccinia graminis tritici is shown in the Figs. 8.16 and 8.17.

Fig. 8.16. Important Stages in the life cycle of Puccinia graminis tritici. A. + and – strains of basidiospores. B. Spermagonia on barberry leaf. C. Plasmogamy between receptive hypha and pycniospore. D. Aecial cups on barberry leaves. E. Uredinia on wheat leaf. F. Telia on wheat leaf. G. Teliospore. H. Karyogamy and meiosis forming + and – strains of basidiospore.

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Fig. 8.17. Life cycle of Puccinia.

Other species of Puccinia on wheat: Apart from Puccinia graminis tritici, two other species of Puccinia viz. P. striiformis and P. recondita also infect wheat. Puccinia striiformis: Due to infection of this species yellow pustules are formed on wheat leaves, in between the veins. The pustules are small and form one below the other giving a striped appearance. Hence it is called stripe rust or striiformis rust. Since the pustules are yellow in colour, the rust is also described as yellow rust or yellow stripe rust. This rust occurs mainly in temperate countries and is relatively rare in tropics. It is mostly confined to leaves, and other aerial parts are infected only in severe conditions. The fungus produces mainly uredinial sori. The formation of telial sori is occasional. When formed, the stalks of the teliospores are short and do not rupture the epidermis exposing the sori. The part of the leaf where telial sori are formed appears slightly bulged. The teliospores have two or three cells and the tip of the spore is flat rather than pointed. The teliospores germinate after a period of rest producing basidiospores, but the basidiospores cannot infect wheat plant. Hence, this species is considered as heteroecious species, but the alternate host on which spermagonial and aecial stages are formed, has not yet been discovered. Puccinia recondita: This species infects mainly the leaves and incidence on other aerial parts is rare. Hence this rust is described as ‘rust’. The uredinial pustules are orange in colour. Therefore, this rust is also described as ‘orange rust’ or ‘orange leaf rust’. This rust mainly produces uredinial pustules on wheat plant, and the

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incidence of telial pustules is occasional. When telial sori are formed they are relatively large with groups of paraphyses between the teliospores. The paraphyses divide the telial sori into two or three compartments or chambers. Teliospores are two or three celled, and the tip of the top cell is flat. The fungus produces uredinial and telial stages on wheat, and the spermagonial and aecial stages are formed on alternate host which may be a species of Thalictrum or Isopyrum fumariodes. Hence, this rust is a macrocyclic, heteroecious rust. A comparative account of the three rusts on wheat is given in the Table 8.5. Table 8.5. Comparison of three rust diseases on wheat Character

Black stem rust

Yellow stripe rust

Orange leaf rust

Causal organism

Puccinia graminis tritici

Puccinia striiformis

Puccinia recondita

Affected parts

Leaves and stem

Mainly leaves, in severe Mainly leaves conditions other parts

pattern of development

Randomly

Mainly between veins one below the other

Randomly

Colour

Reddish brown

Yellow

Orange

Size

Big, fully opened

Small, not fully opened Intermediate

Affected parts

Mainly on stems, also on leaves

Leaves

Leaves

Pustules

Black

Pustules not formed

Pustules not formed

Stalk of teliospore

long

Short

Short

Number of cells in a Usually 2 teliospore

2 or 3

2 or 3

Shape

Upper cell tip pointed

Upper cell tip flattened

Upper cell tip flattened

Paraphyses

Not present

Present

Present in groups dividing the sorus into chambers

Stages on wheat

II, III

II, III

II, III

Alternate host

Berberis vulgaris

Not known

Species of Isopyrum, Thalictrum

Uredinial sori

Telial sori

The problem of wheat rust disease in India Wheat is the most important cereal crop of North India. It is cultivated in Central India to some extent, but confined to cool hilly regions in South India. Indogangetic plains are the major areas of wheat cultivation. It is attacked by a

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number of diseases, and the rust disease is the most important one occurring regularly causing severe yield losses. In North India wheat is sown in October – November period, and is harvested in March – April period. October-November period is favourable for the incidence of rust disease on wheat in North India, but the crop is not attacked by the pathogen during this period. The orange leaf rust caused by Puccinia recondita appears in December, yellow stripe rust caused by Puccinia striiformis appears in January, and the rust caused by Puccinia graminis tritici appears in February. In North India barberry bushes are absent and the summer temperatures are very high. Hence urediniospores on wheat leaves cannot survive the hot summer temperatures. For black stem rust disease or other rusts to occur, the urediniospores have to come from far off places. The epidemiology of wheat rusts in North India was extensively studied by Prof. K.C. Mehta and his associates of Agra University during 1920-40 periods. For their study, they established more than 60 research stations in hilly regions as well as plains throughout India. To trace the movement of the airborne urediniospores they used adhesive coated slides tied to balloons, kites etc. They studied the incidence and concentration of airborne urediniospores all over India. Based on their studies K.C. Mehta proposed that urediniospores of Puccinia graminis tritici and other rusts come to North India from Nilgiri and Pulney hills of South India, where the wheat is cultivated during summer, and rust is severe on this crop. He also considered Central Nepal in Himalayan region as another source contributing to the primary source of inoculum to the wheat crop in Indo-Gangetic plains to start wheat rust epidemics. During 1960-70 period, Dr. L.M. Joshi, Dr. Nagarajan and their associates at Indian Agricultural Research Institute (IARI), New Delhi, reinvestigated the epidemiology of wheat rusts using latest techniques. Throughout India, they established wheat research centres and also mobile units, to study the onset and progress of wheat rust in different parts of India. They analyzed meteorological data on wind movement and velocities, and satellite pictures to trace the path of wind currents at upper strata of the atmosphere over India. Basing on their studies they confirmed that the wheat crop grown in Nilgiri and Pulney hills of South India release clouds of urediniospores into air that spread to central India and North India. Even though Prof. Mehta considered Central Nepal as another important source of primary inoculum, IARI scientists reported that there is no possibility of urediniospores spreading from central Nepal because due to very low temperatures in Himalayan region, the uredinial pustules formed are very small and urediniospores formed in the sori are very few. Further, the incidence of rust in Himalayan region is not severe because of very long incubation period. Disturbances in Bay of Bengal resulting in severe cyclones are very common during November. During these cyclones the urediniospores are released in massive numbers from wheat crop on Nilgiri and Pulney Hills due to heavy turbulence. They spread to at least 1000 km northwards on high wind speeds associated with cyclonic storms, deposit in Central India within three or four days. Thus, the primary inoculum is brought about 1000 km nearer to North Indian wheat crop, due to November cyclones in Bay of Bengal. Meteorological records during 1890–1973

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revealed that in 28 years November cyclones occurred in Bay of Bengal, and wheat rust was severe in North India during these cyclone years. Before 1960, wheat in Central India was cultivated at the same time when it was cultivated in North India. Because of increased irrigational facilities wheat crop is sown in Central India during August or early September. Hence, the urediniospores coming from South India first infect wheat crop in Central India and by the time wheat crop comes up in North India, the primary source for rust is very near. Hence, the wheat crop is infected quite early in North India resulting in severe yield losses. At present, rice crop is being cultivated in North India as alternate crop during rainy season. Because of this practice wheat sowings are delayed. Hence, it is also resulting in exposure of wheat crop to rust infection for longer periods resulting in rust severity. Control of wheat rust: Rust is an aerially dispersed disease, and it is very difficult to control the disease once established in any region. Wheat is the most important cereal crop in the temperate countries, and is extensively cultivated in thousands of hectares occurring continuously. It makes the control of the disease very difficult. A large number of methods were employed to control the disease. Some important methods used to control the wheat rusts are as follows. 1.

Eradication of alternate host: The wheat stem rust is a heteroecious macrocylic rust completing life cycle on wheat and barberry plants. The alternate host is the primary source of inoculum for the main crop. Even before the life cycle of the rust was completely studied, the empirical observations led to the realization that wheat rust is always severe when the barberry bushes are present in the area. It led to the eradication of barberry bushes for the control of the wheat rust. It is one of the earliest measures taken to control the rust disease. Many European countries legally banned the cultivation of barberry bushes in the 17th Century.

2.

After harvesting of the crop, the infected leaves and stems should be burned to reduce the source of uredinial pustules, which act as primary inoculum, in the absence of barberry bushes.

3.

In the areas where there are no alternate hosts or local uredinial source, the disease recurs through the airborne urediniospores coming from long distances. Hence, cultivation of short duration cultivars reduces the risk of exposure of the crop for longer periods to the rust disease, reducing the severity of the disease.

4.

Early sowing is another practice, especially in places where irrigation facilities are available. It reduces the severity of the disease because by the time the primary inoculum from a distant source arrives, the crop will be in an advanced stage of growth.

5.

Mixed cropping i.e. growing wheat crop along with another crop which is not susceptible to the rust, in the same field reduces the disease severity, because the second crop acts as a barrier for free dispersal of the rust spores. Growing tall plants around the field also reduces severity by interfering with the spread of infection from other fields.

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6.

It is observed that the use of high nitrogenous fertilizers increase disease severity. Hence NPK fertilizers with relatively low nitrogen percentage are useful to reduce disease severity.

7.

Chemical control is practiced since long time to control the rust disease. Sulphur dusting is the earliest chemical control measure used. Bordeaux mixture was used for a long time in the early 20th century. Dithane compounds, tin compounds, nickel compounds etc. were also tried for the control of the rust. But all these are contact fungicides which protect the crop as long as they are on the surface of the leaf, and new growth cannot be controlled. At present a number of systemic fungicides are available and they are used for control of the rust. However, chemical control is effective only in varieties that show some field resistance to the rust.

8.

Use of resistant varieties is the best method for control of the rust disease of wheat. However, there are a number of physiological races of rust fungi; cultivated varieties of wheat resistant to one race of the pathogen may be susceptible to other races. Further, new races are being produced continuously by the pathogen in nature. Hence, breeding for disease resistance is a continuous process. GENUS : USTILAGO

Class : Ustilaginomycetes Order : Ustilaginales Family : Ustilaginaceae The genus Ustilago comprises of about 300 species, and all of them cause smut disease on higher plants, especially, graminaceous hosts. The term smut refers to the mass of dark powdery spore mass formed in the sori formed by the pathogen on the infected host. Various parts of the plant are infected, but most commonly infected part is inflorescence forming smut sori instead of grains. Generally no symptoms appear on the infected host until the smut balls are formed. However, on maize, the infection results in formation of galls on various infected parts including stalks, leaves and ears. In sugarcane, the infected inflorescence is transformed into a long, tapering, whip like structure. Some important species of Ustilago and the diseases they cause are given below (Table 8.6) The life cycle of the smut fungus starts with the germination of the resting spores. The smut spores are functionally equivalent to the teliospores of rust fungi. These teliospores are binucleate, globose in shape, smooth, light brown in colour, measure 6-10 µm in diameter. During the process of maturation or just before the germination the two nuclei fuse forming diploid nucleus. Germination of resting spores: The teliospore germinates to produce a germ tube or promycelium, into which the diploid nucleus passes and undergoes meiosis. Then the promycelium develops three transverse septa to cut off four cells, each containing a single haploid nucleus. Within each cell the nucleus divides and a

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daughter nucleus passes into a sporidium budded off from the cell. The four celled promycelium is metabasidium and the sporidia are considered as basidiospores. Table 8.6. Some important diseases caused by Ustilago spp. Pathogen

Disease

Ustilago tritici

Loose smut of wheat

U. avenae

loose smut of oats

U. nuda

Loose smut of barley

U. hordei

Covered smut of barley

U. kolleri

Covered smut of oats

U. maydis

Smut of maize

U. scitaminea

Whip smut of sugarcane

In Ustilago longissima, the cause of leaf stripe of Glyceria spp. the teliospores on germination do not produce obvious promycelium but merely a short tube from which sporidia are budded off successively. In Ustilago violacea, causing smut of Caryophyllaceae members, the teliospores germinate by producing three celled promycelium and from each cell a sporidium is formed by budding. They detach from the teliospore and may continue to develop sporidia even after separation. The bipolar nature of incompatibility in smuts was first demonstrated in this fungus. In Ustilago nuda, the cause of loose smut of wheat and barley, there are no sporidia. A septate promycelium develops on germination and becomes primary mycelium. Dikaryotization: Most species of Ustilago are heterothallic and show bipolar heterothallism controlled by a single gene and two alleles. Of the four cells of promycelium two contain one mating type and other two the opposite mating type. The sporidia produced are thus of two mating types. In some species two sporidia (basidiospores) of opposite mating type may copulate, in other species a basidiospore of one mating type may unite with a hypha issuing from a basidiospore of the opposite type. In Ustilago nuda, the dikaryotic phase is established by fusion of germ tubes derived from the individual uninucleate cells of the promycelium. Regardless of how dikaryotization takes place, it is the binucleate mycelium that carries on the life history of the fungus. Uninucleate mycelium seems to be of short duration and seldom grows very much. Normally it bears no clamp connections and forms no teliospores. Sorus development: During sorus development, dikaryotic hyphae proliferate and mass together in the intercellular spaces, often destroying the softer internal host tissues, but remain enclosed in the host epidermis. Eventually all hyphae are composed of binucleate cells. After the nuclear fusion, the walls thicken and gelatinize. Meanwhile the cells enlarge to become globose. The spore initials are at first enclosed in a gelatinous matrix, which disappear at maturity. The ripe,

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uninucleate, diploid teliospsores have thick, usually dark walls, which may be almost smooth or ornamented by spines or reticulations. Dissemination of smut spores: The teliospores are commonly dispersed by wind and germinate by a variety of methods. The secondary mycelium in the host may produce branches that reach the surface of the host and give rise to several crops of binucleate conidia that are disseminated by wind and initiate new infections. Later binucleate mycelium forms smut ball (sorus) in which teliospores develop. Over wintering: The smut fungi over winter in the teliospore stage or as mycelium in the host. Ustilago maydis over winters as teliospores that may survive in the soil or in the corn debris. Life cycle: The important stages in the life cycle of Ustilago maydis are shown in Fig. 8.18.

Fig. 8.18. Life cycle of Ustilago sp.

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GENUS : TILLETIA Class : Ustilaginomycetes Order : Ustilaginales Family : Tilletiaceae The genus Tilletia is a large genus with more than 75 species, and worldwide in distribution, causing bunt disease in higher plants. The genus was established by Tulasne brothers in 1847 and it was named after Mathaieu Tillet who established in 1755 the causal relationship between the common bunt disease and the bunt ball dust. The two most important hosts for the fungus are wheat and rice, the two major cereal crops of the world. Tilletia caries and T. foetida cause of bunt of wheat which is often described as common bunt or stinking smut or hill bunt of wheat. Tilletia barclayana causes bunt disease of rice. Tilletia caries is the best studied species.

Fig. 8.19. Life cycle of Tilletia caries.

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The stinking smut is a systemic disease resulting from seedling infection. The symptoms of the disease are not usually evident until the heading stage. After the ears have emerged the presence of the disease can be easily detected. The smutted ears are darker green then normal and remain green longer. The smutted grains yield a soft black pasty mass. In mature grains the black pasty interior changes to an oily powder, the characteristic feature of the spore mass. The presence of the smut in the field can be detected by a foul smell caused by the presence of a volatile compound, trimethylamine. The black mass consists of dark brown spores which are globose, thick walled with reticulate thickening. They measure about 15-25 µm in diameter. The spores readily germinate by giving rise to a stout tubular structure called promycelium. The two haploid nuclei in the spore fuse to form a diploid nucleus. It enters the promycelium and undergoes meiosis to produce four haploid nuclei. They mitotically divide once to form eight haploid nuclei. As the nuclear divisions are occurring, the promycelium produces at its tip, eight filiform, hyaline structures called primary sporidia. The nuclei from the promycelium pass into these sporidia. The uninucleate primary sporidia fuse in pairs, while still attached to the promycelium to form the characteristic H-shaped structures. After fusion the primary sporidia become binucleate, they germinate by giving out a hypha which bears secondary sporidia. These are sickle-shaped, hyaline spores, and they germinate to produce the infection thread. Infection of the host takes place at the seedling stage, when teliospores present on the surface of the seed or in the soil germinate. The life cycle of Tilletia caries is shown in the Fig. 8.19. GENUS : AGARICUS Class : Hymenomycetes Order : Agaricales Family : Agaricaceae Agaricus is a well known genus with about 200 species which are cosmopolitan. These are commonly found growing on ground in pastures. A. campestris (L. campester = relating to plain or flat field) is the common field mushroom. It grows amongst grass in pastures, on old lawns etc. A. brunnescens (syn. A. bisporus) is the cultivated mushroom of commerce and is characterized by two spored basidia. The fruit body of cultivated mushroom is usually more robust, and the flesh is thicker and firmer than in the wild field mushroom i.e. A. campestris. There is some question whether the cultivated species has an exact counterpart in nature. The strains presently used have been selected for desirable characteristcs over a long period of time. Apart from the cultivated mushroom, the genus contains some other edible mushrooms including A. Campestris and A. rodmani, but other species such as A. placomyces, A. silvaticus and A. xanthodermus may cause gastrointestinal disturbances in some individuals. The generic name Psalliota has earlier been used for these species.

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Mycelium normally inhabits the ground and has a tendency to grow in all directions from a central point forming a large, invisible circular colony. When the time for sporulation arrives, the fruit bodies are produced in the periphery of the colony and thus form a ring. This ring is called a fairy ring because of an old superstition that mushrooms growing in a circle represent the path of dancing fairies. The mycelium of Agaricus undergoes three stages viz. 1. primary mycelium which is homokaryoic, 2. secondary mycelium which is dikaryotic and heterokaryotic formed from somatogamy between two compatible mycelia, and 3. tertiary mycelium which forms the fruit bodies. Basidiocarp: The basidiocarp of Agaricus is a well developed mushroom, the most conspicuous parts of which are the stipe (stalk) and pileus (cap). The tissues comprising a fruit body consist of closely packed dikaryotic hyphae that arise from the somatic hyphae growing within the particular substratum supporting the growth of the fungus (Fig. 8.20).

Fig. 8.20. Agaricus campestris. A. Button stage. B. Mature bruit body.

To the casual observer the fruit bodies seem to appear overnight. This is not a totally accurate assessment, however, since the sudden appearance of basidiocarp usually results primarily from the rapid enlargement of cells already contained in a fruit body primordium or button stage as called in cultivated mushroom. The type of development is described as hemiangiocarpic. This type of development is characterized by the fact that even during the earliest stages of basidiocarp development the hymenium or fertile layer is enclosed by tissues of the basidiocarp. Typically the margin of the pileus is connected to the stipe by a membrane technically known as inner veil. The hymenium is not exposed until the cap expands, shortly before the spores mature and are discharged from the basidia. The veil often becomes severed from the margin of the pileus and remains attached to the stipe in the form of a ring or annulus (Fig. 8.21).

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Fig. 8.21. Hemiangiocarpic type of development of the fruit body.

Fig. 8.22. Life cycle of Agaricus campestris.

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Hymenium is found beneath the pileus. It lines the lamellae (gills). Gills are generally thin strips of tissue radiating from the margin of the pileus toward the stalk. The gills are not attached to stipe and are described as free gills. Because of free gills the stipe can be easily separated from the pileus. In the early stages the gills are light pink or rose coloured. The pink coloration of the young gills is due to cytoplasmic pigment in the spores. Later, the gills turn a purplish brown, due to the deposition of dark pigments in the spore wall. Gill structure is described as aequihymeniferous (equal hymenial) type. The term aequihymeniferous refers to the fact that the hymenium develops in an equal manner all over the surface of the gill i.e. basidial development is not localized at any one point of the gill. Each basidium is usually having four sterigmata and a basidiospore at the tip of each sterigma. The colour of the spore print is chocolate brown. The basidiospores are forcibly discharged from the basidia and are aerially dispersed. When the spores deposit on a suitable surface they germinate under favourable conditions and the life cycle is repeated. Life cycle: The life cycle of the fungus starts with germination of basidiospores and producing primary mycelium. Two primary mycelia developing from compatible strains of the fungus fuse to form dikaryotic secondary mycelium and continue the lifecycle (Fig. 8.22).

■■■

Chapter - 9

Anamorphic Fungi

The fungi in which sexual stage is absent or not observed so far are included in this group known as anamorphic fungi (Deuteromycotina or Fungi Imperfecti or Mitosporic fungi) (Plates 9.1, 9.2). These are also called imperfect fungi. It is a large group with more than 1680 genera and 17000 species. Among these fungi 95% were considered as conidial stages belonging to Ascomycetes group. The mycelium of these fungi is similar to that of Ascomycotina fungi. Perfect stages of some fungi are found in Basidiomycetes. Hence, we may consider the Deuteromycetes as ‘conidial stages of Ascomycetes and rarely of Basidiomycetes, whose sexual stages have not been discovered or do not exist.’

Plate 9.1. Anamorphic fungi. 1. Conidia of Alternaria alternata. 2. Synnemata of Graphium sp. 3. Aspergillus sp. 4. Soil dilution plate with fungal colonies.

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Plate 9.2. Anamorphic fungi. 1. Curvularia sp. 2. Nigrospora sp. 3. Drechslera sp. 4. Penicillium sp.

However, there is no one to one relation between perfect and imperfect stages. Different conidial fungi may have perfect stages in same genus of Ascomycetes, and species of same form genus may have perfect stages in different genera. Some common examples are shown in Table 9.1. It is clear from the above that the identification and classification of fungi included in Deuteromycetes is based on convenience rather than phylogenetic relationships. Mycelium: The mycelium in most of the imperfect fungi consists of septate, branched hyphae, and is similar to that in ascomycetous fungi. Asexual reproduction mainly occurs through formation of conidia. The hyphae having conidiogenous cells are present on conidiophores. The conidia and conidiophores, their morphology, structure and other characters are considered for identification and classification of these fungi.

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Table 9.1. Imperfect and perfect stages of some anamorphic fungi. Imperfect stage

Perfect stage

Aspergillus fumigatus

Sartorya fumigata

A. nidulans

Emericella nidulans

Septoria avenae

Leptosphaeria avenae

Septoria rubi

Mycosphaerella rubi

Cercospora arachidicola

Mycosphaerella arachidis

Ramularia fragariae

Mycospaerella fragariae

Monilia fructigena

Sclerotinia fructigena

Monilia sitophila

Neurospora sitophila

Alternaria species

Pleospsora species

Stemphylium species

Pleospora species

Drechslera sativa

Cochliobolus sativa

Monilia sitophila

Neurospora sitophila

Alternaria species

Pleospsora species

Stemphylium species

Pleospora species

Drechslera sativa

Cochliobolus sativa

Drechslera teres

Pyrenophora teres

Drechslera turcica

Trichometasphaeria turcica

Conidia: The conidia formed in this group are highly variable in their morphology structure, size, shape and other characters. Basing on the characters of conidia, Saccardo (1809) divided the conidial fungi into seven sections. The sections of Saccardo have no taxonomic status, but are useful for description of conidia. The sections proposed by Saccardo are as follows. 1.

Amerosporae: The single celled conidia which are round, oval or elongated. These spores are called amerospores. Three subsections are recognized. The hyaline conidia are described as Hyalosporae, the coloured spores are called Phaeosporae, and slightly curved spores are called Allantosporae.

2.

Didymosporae: Two celled spores are included in this section. These conidia are called didymospores. They are mostly oval in shape. Two subsections are recognized in this section as Hyalodidymae - the hyaline conidia and Phaeodidymae – the coloured conidia.

3.

Phragmosporae: Long conidia with transverse septa are included in this section. The conidia in this section are called phragmospores. Two subsections are recognized in this section. They are Hyalophragmiae – with hyaline conidia, and Phaeophragmiae – with coloured conidia.

4.

Dictyosporae: The conidia with both transverse and longitudinal septa are included in this section. The two subsections recognized in this

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section are hyalodictyae – with hyaline conidia, and phaeodictyae – with coloured conidia. The conidia of this group are called dictyospores. 5.

Scolecosporae: The conidia which are thin, long and thread like are included in this section. The conidia may be hyaline or coloured. The conidia of this group are called scolecospores.

6.

Helicosporae: Helically coiled spores either hyaline or coloured are included in this section. They are described as helicospores.

7.

Staurosporae: The conidia with centrally radiating branches, irrespective of the colour are included in this section. They are called staurospores.

Different types of conidia observed in Anamorphic fungi are shown in Fig. 9.1.

Fig. 9.1. Different types of conidia found in Deuteromycetes. A–D. Amerospores. E-F. Didymospores. G-H. Phragmospores. I-J. Dictyospores. K-L. Scolecospores. M-N. Helicospores. O-P. Staurospores.

Conidiophores: Conidiophores are highly variable in their structure. They may occur individually or organized into specialized asexual fruit bodies. The various types are: Micronematous: Conidiophores are similar to vegetative hyphae and not distinct. Macronematous: Conidiophores are distinct from vegetative hyphae. Mononematous: Conidiophores are free, may occur singly or in fascicles

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Synnemata: A large number of conidiophores are aggregated along their longitudinal axis, and the structure appears like a broom stick. Sporodochium: It is a cushion shaped structure in which short conidiophores arise from central region. Acervulus: It is typically a flat or saucer shaped bed of generally short conidiophores growing side by side and arise from stromatic mass of hyphae. Long, Sterile, thick walled structures called setae may be present in the stroma interspersing the conidiophores. Pycnidium: They are globose or flask shaped bodies that are lined on the inside with conidiophores. They usually possess an ostiole through which conidia formed on conidiophores ooze out. Different type of asexual fruit bodies found in Anamorphic fungi are shown in Fig. 9.2.

Fig. 9.2. Asexual fruit bodies in Deuteromycetes. A. Pycnidium. B. Acervulus. C. Sporodochium. D. Synnema.

Conidiogenesis: The pattern of conidial development is called conidiogenesis. The conidia may be formed singly or in groups. The conidia produced in chains are described as catenulate. The conidia are formed from a conidiogenous cell which may be distinct or integrated with conidiophore. There are different types of specialized conidiogenous cells, and among them phialide and annelid are important. The process of conidiogenesis is mainly of two types, thallic mode, and blastic mode. Of these two types, blastic mode of conidiogenesis is very common and occurs in majority of fungi. Out of 285 dematiaceous hyphomycete genera 275 fungi

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show blastic mode of conidiogenesis and only 12 show thallic mode of conidiogenesis. Thallic mode of conidiogenesis: In this method, an entire preexisting hyphal cell is transformed into a conidium. The cell may be either terminal or intercalary. The thallic conidia are often described as arthroconidia. Thallic mode is of two types viz. holothallic and enterothallic. 1.

Holothallic mode: If all the layers of the wall of conidiogenous cell are involved in the formation of the conidium wall, the conidium is said to be holothallic.

2.

Enterothallic mode: If the outer layer of the wall of conidiogenous cell does not become part of the conidium wall, the conidium is described as enterothallic mode.

Of the two types, holothallic conidia are more common than enterothallic conidia. Blastic mode of conidiogenesis: Marked enlargement of the recognizable conidial initial takes place before it is delimited from the conidiogenous cell by a septum. Blastic mode is also mainly of two types viz. holoblastic and enteroblastic. 1.

Holoblastic method: Both outer and inner walls of blastic conidiogenous cell contribute towards the formation of conidia. In this mode two types are recognised viz. monoblastic and polyblastic.

2.

Monoblastic mode: If the conidiogenous cell blows out at a single point it is called monoblastic mode.

3.

Polyblastic mode: If the conidiogenous cell blows out at several points it is called polyblastic mode.

4.

Enteroblastic method: Only inner wall of the conidiogenous cell or no wall layer of conidiogenous cell contributes to the formation of new conidium it is called enteroblastic method of conidiogenesis. In this method three types are recognized viz. tretic, phialidic and annelidic. i.

Tretic mode: When conidium develops by protrusion of inner wall through a channel or pore in the outer wall, it is described as tretic mode of conidiogenesis. This type of conidia are also called porospores. e.g. Helminthosporium.

ii.

Annelidic mode: Conidiogenous cell produces blastic conidia in a basipetal succession, and typically elongates with the production of each conidium. As the first conidium detaches from the conidiogenous cell it leaves a ring like scar on its surface near conidiogenous locus. The numbers of scars indicate the number of conidia produced. Example Scopulariopsis.

iii.

Phialidic mode: The conidiogenous cell is a flask shaped structure called a phialide. The conidia formed are described as phialospores. Even if a large number of conidia are produced, the phialidic cell retains its shape and size, and does not enlarge. The best examples for this mode are Aspergillus and Penicillium.

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The types of conidiogenesis methods are shown in Fig. 9.3.

Fig. 9.3. Methods of conidiogenesis. A-B. Holoblastic method. C-D. Enteroblastic methods.

Classification: The classification of these fungi is based on conidiophore structures at macrolevel. Three classes are recognized in this subdivision. Unicellular fungi that reproduce mainly by budding ....................... Blastomycetes Mycelial fungi that produce conidia on distinct conidiophores ....... Hyphomycetes Mycelial fungi that produce conidia in pycnidia or acervulus .......... Coelomycetes CLASS : BLASTOMYCETES Vegetative structures comprise of yeast like organisms with or without pseudomycelium. Mycelium, if present, not well developed. They reproduce by budding producing blastospores. The yeasts that do not show sexual reproduction are included in this class. They are often called imperfect yeasts. The class comprises two orders viz. Sporobolomycetales and Cryptococcales. Order - Sporobolomycetales: The fungi in which blastospores are actively discharged are placed in this order. Since, the buds are discharged forcibly, the spores are also called ballistospores. The fungi included are imperfect stages of basidiomycetes. A single family Sporobolomycetaceae is recognized in the order. Family - Sporobolomycetaceae: The family includes four genera of which Sporobolomyces is the important one. It is considered as the most common fungus colonising the leaves and releases the spores abundantly into air in the early hours of the morning. It is also called ‘mirror yeast’. Order - Cryptococcales: The imperfect stages of ascomycetous or basidiomycetous yeasts that do not actively discharge the spores are placed in this order. None of them produce ballistospores. A single family Cryptococcaceae is recognized in the order. Family - Cryptococcaceae: The family is a relatively large one with about 15 genera. They include some important human pathogens like Candida, Cryptococcus, Trichosporon, Malassizia etc. The species of Candida are important fungi associated with human body, and it is an opportunistic pathogen causing a variety of symptoms

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in different parts of the body collectively called Candidiasis. Cryptococcus neoformans is an important pathogen causing deep mycosis. Malassizia furfur is a skin pathogen. Trichosporon colonizes the skin of the scalp causing white piedra. CLASS : HYPHOMYCETES Majority of the fungi included in this class produce conidia on distinct conidiophores which occur individually or form sporodochium or synnemata. Some fungi which do not produce any conidia are also included in this class. Two orders viz. Moniliales and Agonomycetales are recognized in this class. Order - Moniliales: This is a very large order with more than 1000 genera and 9000 species. All of them produce conidia. Four families are recognized in the order. Family - Moniliaceae: The fungi having hyaline mycelium and unorganized hyaline conidiophores are placed in this family. They include some very common saprophytic fungi such as Aspergillus and Penicillium, human pathogens like dermatophytes (Trichophyton, Microsporum, Epidermophyton), dimorphic fungi (Histoplasma, Coccidioides), and also predacious fungi. Family - Dematiaceae: The fungi with dark coloured mycelium and conidiophores are placed in this family. They include some very common saprophytic and plant pathogenic fungi like Alternaria, Curvularia, Helminthosporium, Drechslera, Cercospora, Nigrospora, etc. Family - Tuberculariaceae: The fungi in which the conidiophores are produced from stromatic mass of tissue described as sporodochium are placed in this family. The common examples are Fusarium, Epicoccum, etc. Family - Stilbellaceae: The fungi in which conidiophores are aggregated in the lower part to produce an erect columnar structure called synnemata (singular synnema) are placed in this family. Some common examples are Isariopsis, Phaeoisariopsis, Graphium, etc. Order - Agonomycetales: The fungi that do not produce even conidia were previously called mycelia sterilia. These are now placed in this order with a single family. Family - Agonomycetaceae: This is an artificial assemblage of fungi in which asexual reproduction has not been observed, or not present. About 40 genera are recognised. There are some important plant pathogens like Rhizoctonia, Sclerotium, etc. CLASS : COELOMYCETES The fungi which produce conidia in fruit bodies that have a coelome known as pycnidia and acervulus, are called coelomycetes. Two orders viz. Sphaeropsidales and Melanconiales are recognized in this class. Order - Sphaeropsidales: The fungi producing conidia in pycnidia are placed in this order. These are commonly called pycnidial fungi. Four families are recognized in this order.

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Family - Sphaeropsidaceae: The fungi produce typical pycnidia which are hard, leathery, dark coloured and ostiolate. Examples: Septoria, Phoma, Phyllosticta, etc. Family - Nectrioidaceae: The fungi produce pycnidia which are light coloured, soft and waxy. Important examples are Zythia, Aschersonia, etc. The genus Aschersonia comprises about 50 species which are pathogenic to scale insects which infest crop plants. Some species of the genus are used as biocontrol agents against the insects. Family - Leptostromataceae: The family includes leaf colonizing saprophytes. These fungi produce pycnidia in which the upper part is well developed but the lower part does not show perceptible development. Examples: Leptostroma, Discosia, Melasmia, etc. Family - Excipulaceae: The fungi included in the family produce pycnidia which open out early in the development to produce a cup or saucer shaped structure. Examples: Excipula, Sporonema, etc. Order - Melanconiales: The fungi producing conidia in acervulus are placed in this order. A single family is recognized in the order. Family - Melanconiaceae: It includes all the acervulus producing fungi. Important examples are Colletotrichum, Pestalotia, Pestalotiopsis, Monochaetia etc. Broad outline classification of Anamorphic fungi (Deuteromycotina) is given in the Table 9.2. Table 9.2. Outline classification of Anamorphic fungi (Deuteromycotina). Class

Order

Family

Blastomycetes

Sporobolomycetales

Sporobolomycetaceae

Cryptococcales

Cryptococcaceae

Moniliales

Moniliaceae Dematiaceae Stilbellaceae Tuberculariaceae

Agonomycetales

Agonomycetaceae

Spaeropsidales

Sphaeropsidaceae Nectrioidaceae Leptostomataceae Excipulaceae

Melanconiales

Melanconiaceae

Hyphomycetes

Coelomycetes

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IMPORTANT GENERA GENUS: ALTERNARIA Class : Hyphomycetes Order : Moniliales Family : Dematiaceae Alternaria is one of the important genera of Anamorphic fungi (Deuteromycotina). About 150 species are described in the genus. They are worldwide in distribution. They disperse through air, and produce colonies on various substrata. Most of the species are free living saprophytes, and a few cause important plant diseases. The conidia deposited in house dust cause allergy. Some species cause rotting of stored fruits, vegetables etc. The most important character of the fungus useful for identification is the structure of the conidia which are light to dark coloured, dictyospores with a short or long prominent beak. Alternaria tenuis is the type species of the genus. Various species like A. tenuis, A. tenuissima, A. humicola, etc. are saprophytes. They mainly grow on fallen plant parts. A number of species cause plant diseases mainly leaf spots and blight. They are physiologically weak parasites, causing serious infection on senescent leaves and other plant parts. Some important plant disease causing species of Alternaria, their hosts and disease they cause are given below in the Table 9.3. Table 9.3. Alternaria species pathogenic to crop plants Alternaria species Alternaria triticina A. solani A. brassicae, A. brassicicola A. ricini A. sesame A. macrospora A. longipes A. alternata

Host crop Wheat Potato Brinjal Cabbage, Cauliflower, and other cruciferous crops Castor Sesame Cotton Tobacco Onion

Disease Leaf spot, leaf blight Leaf spot and blight Leaf spot and blight, fruit rot Leaf spot Leaf spot Leaf spot Leaf spot Leaf spot Leaf blight

The mycelium of Alternaria species is composed of light brown coloured septate much branched hyphae. In saprophytic species it grows extensively on the substratum. In plant pathogens the conidia germinate on host leaf surface and the germ tube enters the leaf either through stomata or by direct penetration. In the host tissue the mycelium grows in the intercellular spaces and sends in haustoria into the host tissue to draw nourishment. Some species, especially those causing blight diseases, produce pathotoxins, kill the host cells and feed on the dead cells. Hence, the pathogenic species are described as nectrotrophs. The toxin produced by Alternaria species is identified as alternaric acid. Alternaria species can be cultured easily on common laboratory media. Potato dextrose agar (PDA) and, Czapek-dox agar are most widely used for culturing of

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Alternaria species. The colonies develop very quickly on the medium. At first the colonies are white but gradually change into various shades of dark colors such as dark brown, black, grey etc. Colonies of some species are characteristically black with greenish tinge. The colonies are round and slightly raised. Colonies of some species show characteristic zonations also. Numerous conidia are formed in the colonies. Asexual reproduction: After the vegetative mycelium is well developed, branches from some hyphae transform into specialized conidiophores and produce conidia. In pathogenic species the conidiophores penetrate through stomata or epidermis and produce conidia on the leaf surface. The conidiophores are distinct from vegetative hyphae, and they remain as individual structures but not organized into any special asexual fruit bodies. The conidia develop from the tip of the conidiophore by blastic method in acropetal chains. Conidia: The conidia of Alternaria species are multicellular with two clear parts, conidial body and beak. The conidial body is usually club shaped, with relatively big and thick cells. The septa are formed both horizontally and vertically. Hence, these are called dictyospores. At the anterior part there is a beak with uniseriate smaller cells. The beak may be very short or very long (Fig. 9.4).

Fig. 9.4. Alternaria symptoms on leaves and conidia. A. Alternaria leaf spot on potato. B. Alternaria blight on wheat leaf. C. Chain of conidia on conidiophore.

From the apical cell of the beak, another conidium is formed by budding. The process repeats forming a chain of cells. The length of the beak varies. The arrangement of conidia in chains is described as catenulate. In the conidial chain of Alternaria, the first formed mature conidium is at the base and youngest developing conidium is at the tip. Such type of arrangement of the conidia is described as acropetal. Depending on the length of the conidial chain, the species of Alternaria are divided into three groups. 1.

Non catenate species: The species with very long conidia measuring up to 100 to 200 µm, usually do not form conidial chains, and hence are described as non-catenate species. In these species the beak is usually very long compared to the body of the conidium, e.g. A. solani, A. brassicae, A. porri, A. crassa, etc.

2.

Brevi catenate species: The length of the conidium in this group is medium, measuring 50-100 µm, and beak is also medium in length. The conidia are formed in short chains having less than 10 conidia in a chain, e.g. A. longipes, A. sesami, A. amaranthi, etc.

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3.

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Longi catenate species: These species produce short conidia with a short beak of a few cells. They form very long chains with 10 to 15 conidia or more, e.g. A. tenuis, A. brassicicola, etc.

Dispersal of Alternaria conidia: The conidia of Alternaria species are dry without any slime and hence, they can easily come into the air and disperse. They are not released actively but come into air by the turbulence of the air or any other mechanical disturbances. In the parasitic species, the conidia are formed on the leaves, and in saprophytic forms they are formed on the substratum in large numbers. The wind speed and turbulence are usually more at noon or early evening hours, and hence the conidia also usually disperse more during day time. Aerobiological surveys conducted in different parts of the world also revealed that the conidia of Alternaria are important constituents of air spora during day time. Germination of conidia: The airborne conidia when deposited on a substratum with some organic matter and moisture, readily germinate under favourable conditions. Temperatures in the range of 25-30°C and 90-100% relative humidity or moisture are favourable for conidial germination. The germ tubes may arise from any cell of the conidia. Usually 5–6 cells of a conidium germinate at a time. In free living species, the germ tubes gradually elongate to form mycelium. In pathogenic species the germ tubes enter the host tissue and mycelium develops. Sexual stage: Most of the species do not show sexual reproduction. However, a few species may show sexual stage occasionally. The sexual stage of the Alternaria species has been identified as belonging to the genus Pleospora of Ascomycetes. GENUS : DRECHSLERA Class : Hyphomycetes Order : Moniliales Family : Dematiaceae The genus Drechslera is a common member of dematiaceae. It was first established by Ito in 1930 to include some species of Helminthosporium that differ from the type species in the mode of germination. Later it was redefined by Shoemaker (1959, 1962) based on the mode of conidiogenesis. The genus Drechslera is characterized by percurrent or lateral proliferation of conidiophore to produce conidia always at the growing tip, while in Helminthosporium the conidiogenesis is tretic resulting in production of porospores. The species commonly found on graminaceous hosts include rice, wheat, oats, corn, sorghum, sugarcane etc. A number of species are important pathogens on the cultivated plants. Brown spot of rice caused by Drechslera oryzae, and D. maydis which causes Southern leaf blight of corn are the most important pathogens which caused severe yield losses. The failure of rice crop in 1940s was very widespread and it resulted in great Bengal famine in 1945-46. Southern leaf blight decimated corn crop in Southern United states in the summer of 1970, and it has been estimated that financial losses in the year alone are over one billion dollars. Apart from these two species, other important species of Drechslera that cause plant diseases are given in Table 9.4.

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Some species grow as weak pathogens or as saprophytes on different plant parts. The symptoms caused by D. oryzae on rice, the conidiophores and conidia of the fungus are shown in the Fig. 9.5. The mycelium in pathogenic species consists of both intercellular and intracellular hyphae. The hyphae are subhyaline to light brown in colour. In cultures, mycelium varies from grey to olive brown to dark brown in colour. Table 9.4. Plant diseases caused by Drechslera species Pathogen

Disease

Drechslera sacchari

Eye spot and seedling blight of sugarcane

D. halodes

Leaf spot of coconut and other hosts

D. heveae

Bird’s eye spot of rubber

D. teres

Seedling blight, foot rot and early blight of barley

D. graminea

Leaf stripe of barley

D. turcica

Northern leaf blight of maize

D. stenophila

Brown stripe of sugarcane

D. solani

Silver scruf of potato

D. rostrata

Leaf spot of various cereals, millets and other grasses

Fig. 9.5. Drechslera oryzae – symptoms and conidia. A. Symptoms of brown spot on rice leaf. B. Conidia and conidiophores.

Asexual reproduction: The conidiophores arise as lateral branches from the hyphae. They emerge in tufts through the stomata when developing from the internal mycelium. They are stout, erect, unbranched except a0t the base where they sometimes form branches. The colour of the conidiophore is dark at the base resembling that of hyphae, and gradually becomes lighter towards the tip. They are geniculate possessing knee-like bends the points where the conidia are attached. The conidiophores vary in size and are usually 100 to 175 µm long and 5-7 µm wide.

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The conidia are characteristically large phragmospores i.e multicellular conidia possessing only transverse septa. The conidia vary in size according to the species and environmental conditions They measure 50-100 µm in length and 10-20 µm in width. The number of septa varies from 4-10 or more and it depends on the length of the conidium. Based on septation two types of conidia are recognized. 1.

Euseptate conidia: These types of conidia are surrounded by a single wall and have true septa formed as inward extensions of the lateral walls. The septum develops as a closing diaphragm and usually a pore remains in the centre connecting the two cells.

2.

Distoseptate conidia: They have a common outer wall enclosing more or less spherical cells each of which is surrounded by an individual wall. Apparently the wall of young conidium is double. Invaginations of the inner wall divide the protoplast into a series of cells with pores in the inner walls connecting the adjacent cells.

The conidia are thick walled, clavate, obclavate, curved, cylindrical and the shape is often characteristic of the isolate. The conidia of Drechslera are commonly airborne and disperse through wind. Active discharge of conidia has been reported in some species like D. turcica. The discharge was associated with drying or wetting which cause tension in the protoplast and a gaseous phase develops. The tension is released with the formation of a suture in the wall which causes conidium to be projected with force. The conidia may also be released through the force of electrostatic repulsion. However, active discharge of conidia does not occur in all the species. In D. maydis conidia do not detach easily and considerable external force may be required to separate the conidia from conidiophore. It is estimated that a wind velocity above 5 m/sec is required for the release of conidia in this fungus. Perfect stages: The perfect stages of some species of Drechslera are found in the genera belonging to ascomycetes. The perfect stages have been found in more than one genus (Table 9.5). Table 9.5. Conidial stage of Drechslera species and their perfect stage Conidial stage

Perfect stage

Dreschslera oryzae

Cochliobolus miyabeanus

D. sativa

Cochliobolus sativus

D. graminea

Pyerenophora graminea

D. teres

Pyrenophora teres

D. turcica

Trichometasphaeria turcica

GENUS : CURVULARIA Class : Hyphomycetes Order : Moniliales Family : Dematiaceae

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The form genus Curvularia includes over 35 species, which are saprophytes and at the most weak pathogens on various plants in the tropics. Sometimes they attack plants, particularly when young or senescent. Many are probably secondary invaders. Curvularia lunata is cosmopolitan and common on crop plants in tropical parts of the world. A range of different disease symptoms are associated with Curvularia species. On rice and other crops they are reported to cause leaf spots, leaf blights, kernel rots, roots rots, seedling blights, grain discolorations, grain lesions and grain deformations. The characteristic features of the form genus are formation of macronematous, mononematous, erect conidiophores, bearing spores spirally or in whorls. The spores are usually curved, the third cell from the base of the spore is larger than the rest, and the end cells are paler. In some species (C. cymbopogonis) the base of the conidium bears a protruberant hilum. In C. inaequalis, the first formed conidium develops tretically i.e. as poroconidium. At the apex of the elongating conidiophore a tiny apical pore forms at the tip by dissolution of the outer wall, and a spherical cytoplasmic bubble is blown out through the pore. The first conidium assumes an obovoid shape, and after it has matured, the conidiophore develops a new subterminal growing point, from which a second conidium initial arises. The process is repeated so that a succession of new apices are formed, each terminating in a conidium (Fig. 9.6). The term sympodula has been applied to this type of conidiophore apex.

Fig. 9.6. Conidiophores and conidia of Curvularia lunata.

The perfect state of Cuvularia species where known are species of Cochliobolus. GENUS : CERCOSPORA Class : Hyphomycetes Order : Moniliales Family : Dematiaceae

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The genus Cercospora (Cercos = tail; spora = spore) was first established by George Fresenius in 1863. Cercospora apii, which causes blight of celery (Apium graveolens) is the type species. More than 3000 species are described in the genus. Some important species are given in Table 9.6. Table 9.6. Some important species of Cercospora causing disease. Cercospora species

Disease

Cercospora apii

Blight of celery

C. arachidicola

Tikka disease of groundnut

C. beticola

Blight on sugar beet

C. musae

Sigatoka disease of banana

C. nicotianae

Frog eye leaf spot of tobacco

C. canescens

Leaf spot of many legumes

C. capsici

Leaf spot of chillies

C. solanacearum

Leaf spot of brinjal

C. rodmanii

Leaf spot of water hyacinth

The epidemic outbreak of cercosporosis is reported in various crops and it causes severe yield losses. Due to infection by Cercospora species defoliation of the host leaves is heavy and plant dries up. This devastating nature of the disease has been reported to be useful at least in one case to eradicate the concerned host plant. For example Cercospora rodmanii is used to control water hyacinth, which clogs the water ways to the crop plats. Even though human infection with plant pathogens is rare, it is reported that Cercospora apii is a human pathogen. It was isolated from severely infected patient in Indonesia. It causes severe lesions on the face making it look awful. Mycelium: The mycelium consists of very fine to coarse, hyaline to colored, septate hyphae, which are mostly internal but in some they are both internal and external (e.g. C. arachidicola). In some species which cause sooty patches on leaves of Hibiscus species, and others, mycelium is mainly external. When young, the mycelium is invariably hyaline or with slight yellowish tinge, but as it grows older, it usually takes a shade of olive or brown colour. In host tissue, the hyphae are usually intercellular sending in round or lobed haustoria into the host cells. In some species which cause rapid blight, hyphae become intracellular and do not produce haustoria. Asexual Reproduction: Some of the hyphal tips round off and aggregate below the stomatal cavity to produce a stroma from which the conidiophores arise. Extent of stroma varies greatly from a few cells to large sclerotia like compact, tuberculate, pseudoparenchymatous structures which may extend to the surface of the host tissue. The conidiophores emerge from the stromata either through the stoma or by rupturing the epidermis. Sometimes both types of emergence may be found in a single species. The number of conidiophores formed on a stroma varies considerably and they may be few to many, and loose to dense.

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The conidiophores are hyaline to various shades of brown to dark brown, straight or curved, simple or branched, sometimes septate and geniculate. At the point of geniculations scars may be very prominent to insignificant. The geniculations are due to prolific growth of conidiophore which bears conidium at the tip. While young conidium is being formed, the conidiophore pushes it aside laterally and grows a little further and bears another conidium, and so a number of conidia are formed on a single conidiophore. The conidia are of different shapes but have a breadth to length ratio of 1:10 to 1:150. They are mostly acicular but may be cylindrical, obclavate or even fusiform. The conidia are usually hyaline, 1-12 or more septate in the size range of 2-8 x 20 – 200 µm or more. Cercospora species do not grow readily in artificial media. The conidiophore and conidia of a typical species of Cercospora are shown in Fig. 9.7.

Fig. 9.7. Conidiophores and conidia of Cercospora sp.

The perfect stage is reported only for a few species, and where it is reported it is an ascomycetous genus Mycosphaerella. For example the perfect state of Cercospora arachidicola has been reported as Mycosphaerella arachidis. But it is not of common occurrence. Mycosphaerella belongs to the family Dothideaceae, of

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the order Dothideales in the class of Loculoascomycetes of subdivision Ascomycotina Sections in the genus: As the number of species described in the genus became very large, attempts have been made to split them into various sections based on the characters of stroma, conidiophores and conidia. Penzes (1927) proposed three sections in the genus based on the conidial characters like colour, septation and size: 1. Brachycercosporae … Conidia brown, 3–5 septate, and size 20-60 x 5-7 µm. 2. Mediocercosporae ..... Conidia subhyaline, 5-7 septate, 40-80 x 3.5-5.5 µm. 3. Macrocercosporae ..... Conidia hyaline, multiseptate, 60-150 x 3.5–4.5 µm. Solheim (1930) proposed 21 sections mainly based on four characters. 1.

Mycelium internal or both internal and external.

2.

Conidiophores simple or branched, and type of branching.

3.

Stroma tuberculate or not.

4.

Shape of the conidia.

The recognition of sections in the genus is considered as artificial, and later authors separated some species into other already described genera or erected new genera. Splitting of the genus: The species originally described in the genus Cercospora are now separated and placed in a number of genera like Pseudocercospora, Cerosporidium, Cercosporella, Passallora, Ramularia, Mycovellosiella, etc., all of which together are described as ‘Cercospora-complex’. Von Arx (1983) distinguished 4 groups of genera in ‘Cercospora-complex’ 1.

Conidiogenous structures hyaline or subhyaline, scars unthickened, flat and broad.

2.

Conidiogenous structures hyaline or subhyaline, scars slightly thickened and protruberant.

3.

Conidiogenous structures pigmented, scars unthickened and broad.

4.

Conidiogenous structures pigmented, scars thickened, dark and bulging.

The genus Cercospora is placed in the 4th group GENUS : FUSARIUM Class : Hyphomycetes Order : Moniliales Family : Tuberculariaceae The genus Fusarium was established by Link in 1809, and Fusarium roseum is the type species. The genus comprises of a large number of species and many forms within each species. The identification of the genus is easy because of the characteristic hyaline fusoid or fusiform conidia they produce, but there is no general agreement on species determination within the genus.

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There are two main systems of classification of the genus. 1.

Wollenweber and Reinking (1935) proposed that the genus can be divided into 16 sections basing on cultural characteristics and spore measurements. They recognized 65 species, 55 varieties and 22 forma speciales.

2.

Snyder and Hansen (1940, 1945) pointed out that variability within the species and single spore isolates was great enough to invalidate many species identified in Wollenweber system. They proposed that the number of species be reduced to those that can be reliably described on the basis of morphological differences. They proposed that all species that cause wilt disease be placed in one species F. oxysporum and earlier species recognized by host species they attack be recognized as forma speciales. In the same way, all species in the section Martiella were placed in a single species F. solani. They recognized only 9 species and 34 forms. Both the systems have their adherents. Booth (1971, 1977) of Commonwealth Mycological Institute recognized about 50 species in the genus.

All species of Fusarium have a saprophytic stage. The species are widely distributed in soil and on organic substrates, and have been isolated from frost of Arctic glaciers and from sand of Sahara desert. They are abundant in cultivated soils both in temperate and tropical regions. Many species are only mild facultative parasites. Some are primarily decay causing organisms on dormant vegetables and fruits. Some are mild root parasites. Some are primarily cortical invaders causing pre-emergence damping-off, foot rots and stem cankers. The most important pathogens are those that cause serious vascular wilt disease. Some important pathogenic species of Fusarium are given below (Table 9.7). Table 9.7. Pathogenic species of Fusarium causing disease Species

Disease

Fusarium oxysporum

Wilt disease

F.o. f.sp. pisi

Wilt of peas

F.o. f.sp. udum

Wilt of pigeon pea

F.o f.sp. vasinfectum

Wilt of cotton

F.o. f.sp. cubense

Wilt of banana (Panama disease)

F.o. f.sp. lycopersicae

Wilt of tomato

F.o. f.sp. lini

Wilt of flax

F. solani

Root rot of solanaceous vegetables

F.s. f.sp. phaseoli

Dry root rot of beans

F.s f.sp. coeruleum

Dry root rot of potatoes.

F. moniliforme

Stalk rot of sugarcane

F. moniliforme

Foot rot of rice (bakanae disease)

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Species

Disease

F. graminearum

Damping-off of wheat and other cereals

F. culmorum

Damping off of wheat, oats and barley

F. nivale

Damping off of oats

F. equiseti

Root rots and fruit rots of many crops

F. semitectum

Fruit rots of many crops

Mycelium: It is extensive. Hyphae septate with multinucleate cells, much branched. In the host it is both intercellular and intracellular and sometimes occurs in xylem vessels. It grows easily on ordinary culture media. The white colonies develop with some tinge of pink, purple or yellow. Wilt fusaria are known to produce a toxin called fusaric acid, a non specific wilt toxin. Gibberellin, an important growth promoting substance was first discovered during the bakane or foolish seeding disease of rice. Sawada (1912) was the first to suggest that bakane effect might be due to a substance produced by fungus causing the disease. Kurosawa (1924) demonstrated that the culture filtrate of Gibberella fujikuroi can stimulate the growth of rice seedlings, and the principle came to be known as Gibberellin. Reproduction: The members of the genus Fusarium typically produce two types of conidia that are termed macroconidia and microconidia based on the size. Both types are produced from phialides. The conidiophores are variable, slender or stout, simple or branched irregularly. A whorl of phialides is formed at the tips of the branches. They are either single or grouped with small sporodochia. Some members of the genus never produce sporodochia, a fact that makes inclusion of Fusarium in the family Tuberculariaceae somewhat arbitrary. Macroconidia are long, multiseptate, crescent or fusiform in shape, and are generally borne in sporodochia but may also occur separately. The spores are hyaline, 3–4 or more septate. Microconidia are very small conidia produced from the tips of simple or branched conidiophores, which are indistinguishable from hyphae. Conidia vary in form from round to oval, occurring in chains or often formed in small masses. Both types of conidia are dispersed either by rain splash or dry wind currents. They germinate to form new mycelium. In addition to the two types of conidia, many species often produce chlamydospores under unfavourable conditions, and some species produce sclerotia, both of which help in perennation. Chlamydospores are round to oval thick walled cells formed in the hyphae. They may be formed singly or in pairs or in chains. They become separated from the parent hyphae after maturation and function as resting spores. Under unfavourable conditions, the cells of the conidia also often become thick walled chlamydospores. Sclerotia are hard masses of closely packed thick walled hyphae that are formed in some species. They function as storage organs and also serve as a means of perennation and vegetative propagation. Asexual reproductive structures are shown in the Fig. 9.8.

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Fig. 9.8. Conidiophores and conidia of Fusarium solani. A. A portion of sporodochium showing conidiogenous cells. B. Macroconidia. C. Microconidia. D. Terminal and intercalary chlamydospores.

Perfect stages: Some species produce ascigerous stages in culture. All the species of Fusarium do not have a single type of ascigerous stage. The perfect stages belonging to different genera of the class pyrenomycetes of ascomycotina are formed (Table 9.8). Table 9.8. Fusarium species with their perfect stages. Conidial stage name

Perfect stage name

Fusarium moniliforme

Gibberella fujikuroi

F. solani

Nectria haematococca

F. tabacinum

Micronectriella cucumeris

F. nivale

Calonectria nivalis

F. decemcellulare

Calonectria rigidiuscula

GENUS : PHOMA Class : Coelomycetes Order : Sphaeropsidales Family : Sphaeropsidaceae The genus Phoma includes about 40 species. Actually, about 2000 species were described in the genus; most of them were reduced to synonyms because they are based on hosts rather than the characters of the fungus. The fungus is a facultative parasite and many species are pathogenic to crop plants and others. Many species have a wide host range and occur on a number of hosts. They are soil borne and exist saprophytically on plant debris. Some important plant pathogens are given in (Table 9.9).

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Table 9.9. Diseases caused by some Phoma species. Pathogen

Disease

Phoma glomerata

Blight of vines, flowers and grapes

P. glomerata

Leaf and fruit spot of apple

P. glomerata

Damping off of conifers

P. glomerata

Secondary invaders causing rot of vegetables and fruits

P. prunicola

Leaf spot of apple and pear

P. medicaginis

Foot and collar rot of peas

P. destructiva

Leaf spot and fruit rot of tomato

P. betae

Black leg and damping off of beet

P. lingam

Black leg of crucifers

Fig. 9.9. Pycnidium of Phoma. A. L.S. Pycnidium. B. Portion of pycnidial wall showing conidiogenous cells and conidia (pycniospores)

Mycelium is hyaline to brownish in colour, septate, branched, and produce characteristic asexual fruiting strutures called pycnidia. The pycnidia are dark coloured, flask shaped structures with an apical circular opening the ostiole. The inner surface of the pycnidium is lined by a layer of hymenium. The conidiophores are indistinct, and conidiogenous cells are single celled, phialidic, integrated or discrete, ampulliform or doliiform in structure. The conidiogenesis is enteroblastic

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and the tips of the conidiogenous cells open by a minute aperture. Spore development is monopolar by repetitive budding. The conidia are hyaline, single celled or occasionally become single septate. They are thin walled and of various shapes like ellipsoidal, cylindrical, fusiform, pyriform or globose. Numerous hyaline conidia are formed and ooze out from the ostiole as a tendril-like mass described as cirrhus. Asexual reproductive structures are shown in the Fig. 9.9. In addition to pycnidium, some species produce dictyochlamydospores which are dark, irregularly ovate, with a number of transverse and longitudinal septa. Perfect stages: The Perfect stages of Phoma species are found in different ascomycetous genera belonging to pyrenomycetes such as Pyrenochaeta, Didymella and Leptosphaeria. Allied genera: There is a great deal of confusion with the identity of Phoma species. The genera like Phyllosticta and Macrophoma are also having the same characters as Phoma. If they grow on leaves, they are called Phyllosticta, while similar fungi growing on stems is called Phoma or Macrophoma. If the conidia measure up to 15 µm they are placed in the genus Phoma and if the conidia measure over 15 µm they are placed in Macrophoma. Hence, this group of pycnidial fungi needs further taxonomic clarification. GENUS : COLLETOTRICHUM Class : Coelomycetes Order : Melanconiales Family : Melanconiaceae One of the most commonly encountered form genera in Melanconiales is Colletotrichum. Over 1000 species were described in the genus Colletotrichum based on host species, but based on cultural studies Von Arx (1957) recognized only 20 species and made rest of the species as synonyms. For example about 600 synonyms exist for Colletotrichum gloeosporioides. Some authors recognize form genus Gloeosporium, and distuinguish it from Colletotrichum on the basis that the acervuli of Gloeosporium lack setae. The current tendency is to combine these two genera under Colletotrichum. Sutton (1980) of Commonwealth Mycological Institute recognized 22 species in the genus Colletotrichum based on cultural characters. Several species of Colletotrichum are parasitic on plants causing diseases of leaves, young twigs, fruits and vegetables. The diseases caused by Colletotrichum are commonly called anthracnoses, because of soot like black colour of the lesions due to the presence of numerous dark setae. Some important species are given in Table 9.10. Mycelium is at first hyaline turning dark brown at maturity. It is septate, highly branched. The contents also become denser with age. In culture, the thick walled mycelium aggregate to form a structure resembling sporodochium from which conidiophores develop. In the host, mycelium occurs in the intercellular spaces and aggregates below the epidermis to form a saucer shaped structure called acervulus. In fairly old mycelium, some thick walled dark brown, irregular or angular cells develop with one

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or more large oil drops within them. These are formed in either terminal or intercalary cells of hyphae. These get detached and are capable of germination after a long period of rest. Thus these behave as chlamydospores. Table 9.10. Some Colletotrichum species causing disease. Pathogen

Disease

Colletotrichum falcatum

Red rot of sugarcane

C. lagenarium

Anthracnose of cucurbits

C. lindemuthianum

Anthracnose of beans

C. gloeosporioides

Anthracnose of mango, and various hosts

C. gossypii

Boll rot of cotton

C. graminicola

Red rot of sorghum and other grasses

C. circinans

Onion smudge

C. capsici

Fruit rot and die back of chillies

C. musae

Anthracnose of banana

C. coffeanum

Coffee berry disease

C. lini

Seedling blight of flax

Reproduction: It occurs by means of conidia. The fruiting structures are acervuli. In the host, the conidia are formed in acervuli which develop from the stromatic mass of hyphae formed just beneath the epidermis. In grains, this mass is formed just below the pericarp. Asexual reproductive structures are shown in the Fig. 9.10.

Fig. 9.10. Acervulus and conidia of Colletotrichum graminicola. A. Portion of host leaf showing acervuli. B. An acervulus. C. Conidiophores and conidia.

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The acervulus is a saucer shaped structure, and is surrounded by stiff dark brown, unbranched hairs called setae. The setae measure 100-200 µm in length and 5-10 µm in width. The conidiogenous cells form a closed packed palisade like layer of phialides. The phialides are very small producing phialoconidia at their apex. The conidia are aseptate, single celled, fusiform, slightly curved or sickle shaped, hyaline or slightly pink coloured with rounded ends. They vary in size but on an average they measure 15-30 µm in length and 3- 6 µm in width. They accumulate under moist conditions as glistening mass held in place by setae. The conidia germinate by two polar germ tubes. They enter the host through stomata or directly, and on culture media grow as mycelium. If the conditions are not favourable for infection, the germ tube shows limited growth and produces globose appressoria which become thickened and function like chlamydospores. Perfect stage: The perfect stage for some species is found in ascomycetous genus Glomerella, of family Sphaeriaceae class Pyrenomycetes. Some species showing perfect stages are as follows (Table 9.11). Table 9.11. Conidial and perfect stages of some Colletotrichum species. Conidial stage

Perfect stage

C. gloeosporioides

Glomeralla cingulata

C. lindemuthianum

G. cingulata

C. gossypii

G. gossypii

C. graminicola

G. graminicola

C. falcatum

G. tucumanensis

The genus Glomeralla produces typical flask shaped or globose, ostiolate perithecia. Asci are clavate and irregularly biseriate. The ascospores are hyaline and single celled. GENUS : PESTALOTIOPSIS Class : Coelomycetes Order : Melanconiales Family : Melanconiaceae Pestalotiopsis species are frequently isolated from decaying leaves and plant debris, but some are parasitic and are destructive pathogens of economically important plants. Pestalotiopsis theae causes grey blight of tea and cause enormous losses in the yield from tea plantations. It also attacks a number of other plants like mango, palm, cotton etc. Other species in the genus include P. palmarum, P. guepinii, P. papposa, etc. and attack various crop plants. Among the species of Pestalotiopsis, P. theae is the most extensively studied species. It occurs in epidemic form in various parts of the world including Asia, Africa, South America and Australia. Symptoms caused by the fungus begin as small brown leaf spots later enlarging to 1 cm diameter or more. On the upper surface it shows a greyish centre with light to dark brown margins. Lesions are usually circular

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or oval with concentric zonations due to the production of dark acervuli. Coalescence of the spots may also occur. The leaves of any age are attacked but old leaves about to fall are infected more. Stalk rot of tea cuttings has also been described from India.

Fig. 9.11. L.S. of Acervulus and conidia of Pestalotiopsis.

The characteristic feature of P. theae conidia on Camillia sinensis is the production of long, hyaline apical appendages usually three, rarely two or four with their slightly swollen tips. The conidia are fusiform, straight, five celled, with three median dark cells, and emerging as black masses from acervuli. The mycelium is intercellular, and sparsely septate. The acervuli are irregularly distributed on both surfaces of leaves, but most frequently these are epiphyllous and are globose to lenticular in shape. Acervuli rupture the epidermis by a pore which becomes wide and angular (Fig. 9.11). On PDA medium the acervuli develop black spore masses. Colonies usually show a clear diurnal zonation in the mycelial growth and acervuli formation.

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Chapter - 10

Glomeromycota

Several symbiotic groups, phosphorus solubilizers, plant growth promoters, and other such beneficial important micro-organisms are reported from different soils. Balanced microbial systems contribute to the sustainability in agriculture, forestry, and their management. In this regard, mycorrhiza has a substantial role. The term mycorrhiza (fungus root) was coined by Frank (1855). Harley and Smith (1983) defined mycorrhiza as an association between fungal hyphae and roots of higher plants concerned with absorption of mineral substances from the soil. Brundrett (2004) defined mycorrhiza as a symbiotic association for one or both partners between a fungus and a root of living plant that is primarily responsible for nutrient transfer. Mycorrhizas were earlier classified into arbuscular, ectomycorrhizal and orchid mycorrhizal categories based on the relative location of fungi in the roots. ARBUSCULAR MYCORRHIZA Arbuscular mycorrhiza is the most common mycorrhizal association. They have a widespread distribution throughout the plant kingdom and form mutualistic relationship with most of the vascular plants. Families that rarely form arbuscular mycorrhiza include Cruciferae, Chenopodiaceae, Polygonanceae and Cyperaceae. Families that do not form arbuscular mycorrhiza include Pinaceae, Betulaceae, Fumariaceae, Commelinaceae, Urticaceae and Ericaceae. The fungal partner belongs to Glomeromycota. The fungus forms vesicles within or between cortical cells that act as storage organs. Arbuscules are formed within the cortical cells and these provide a large surface area of contact between host root and fungus. The genera, which from AM (arbuscular mycorrhizal) fungal association are Acaulospora, Ambispora, Archeospora, Dentiscutata, Fuscutata, Diversispora, Entrophospora, Geosiphon, Gigaspora, Glomus, Intraspora, Kuklospora, Otospora, Pacispora, Paraglomus, Racocetra, Quatunica, Centrospora and Scutellospora (Schenck and Perez 1990; Schüßler et al 2010) etc. (Fig. 10.1). Besides morphological, anatomical, histochemical, and biochemical tools, molecular and genetical tools are also used for identification of mycorrhizal fungi, and to explore the structural and regulatory genes in both fungi and plants that permit mycorrhiza formation. Of the seven types of mycorrhizas, the two prevalent mycorrhizal types are the ectomycorrhizas (common with woody species related to forestry) and the arbuscular mycorrhizas (more often associated with the herbaceous plants with relevance to horticultural, ornamental, medicinal and crop plants).

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Fig. 10.1. Classification of arbuscular mycorrhizal fungi up to genus level

A large proportion of land area in India shows clear evidence of soil degradation due to salinity, alkalinity, soil erosion, water logging, and so on, which

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in turn affects the country’s productive resource base. The fundamental problem that the country is facing today is the rapidly increasing pressure of population on the limited resources of land. In order to meet the pressure of population, it is essential to efficiently manage the agricultural inputs for sustaining high crop productivity on long-term basis, with minimum damage to environment. Biofertilizers are used in order to reduce the cost and harm rendered by agrochemicals. Mycorrhiza helps the plants to acquire mineral nutrients from the soil, especially immobile elements such as phosphorous, zinc, copper and mobile elements such as sulphur, calcium, potassium, iron, magnesium, manganese, chlorine, bromine, and nitrogen. They also help in increasing the extent of soil particle aggregation. Further, most of the economically important plants have been found to be mycorrhizal, and the subject is currently gaining much attention in agriculture, horticulture, and forestry. It is also known that Glomalean fungi existed 400, 000, 000 years ago and helped in the colonization of land by primitive plants. Thus, primary land plant establishment was also due to mycorrhiza. The mycorrhizal symbiotic association appears to have evolved as a survival mechanism for fungi and higher plants, thus allowing each to survive in the existing environment of low temperature, soil fertility, drought, diseases, extreme environments, and other stress situations. Mycorrhizas offer primary biological defence to host plants against stress for crops and forest trees. BENEFITS DERIVED FROM MYCORRHIZA BY HOST PLANTS Mycorrhizal association helps in increased nutrient and water uptake by absorption through improved absorptive area, and translocation of elements to host tissues and their accumulation. Due to the unique ability of mycorrhiza to increase the uptake of phosphorous by plants, mycorrhizal fungi have the potential for utilization as a substitute for phosphatic fertilizers. Mycorrhizal fungi improve host nutrition by increasing the delivery of phosphorous and other minerals to roots and plants. Ectomycorrhizal fungi permeate the F and H horizon of forest floor and, thus, minerals get mobilized in these zones. This is followed by their absorption before they reach subsoil system. AM fungi are known to degrade complex minerals and organic substances in soil and, thus, make essential elements available to host plants. Mycorrhizal association is known to offer resistance in host plants to drought and plant pathogens. It also increases the tolerance of the plant to adverse conditions and helps in the production of growth hormones like auxins, gibberellins, and growth regulators such as Vitamin B. Mycorrhizal fungi contribute to organic matter turnover and nutrient cycling in forest and crop land ecosystems. They help in soil aggregation, soil-stabilization, and increase soil fertility. Mycorrhizas are symbiotic and not parasitic; therefore, they live hand-in-hand with other living organisms and are non-pollutants. MORPHOLOGICAL DIVERSITY IN ARBUSCULAR MYCORRHIZAL FUNGI Morphological characters that are stable and discrete are used in identification and classification of AM fungi. Some of the important morphological characters considered are described below.

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Hyphal characters Vegetative hyphae have been differentiated functionally into infective, absorptive, and runner hyphae. Fungal hyphae may be with H-shaped parallel connections as in Glomus; constricted hyphae near branch points as in Acaulospora and Entrophospora; and coiled, irregularly swollen hyphae with lateral projections or knobs as in Gigaspora and Scutellospora. The mycelia also form specialized structures like arbuscules, vesicles, and auxiliary cells. Abrupt narrowing-off of branch hyphae forms arbuscules in Gigaspora. In Glomus, arbuscules are formed by reduction in hyphal width. Vesicles in Glomaceae are subglobose to elliptical whereas in Acaulosporaceae they are pleiomorphic and knobby. Subtending hyphae The stalk of the spore known as subtending hypha or sporophore has importance in identification. Subtending hypha may be absent (Acaulospora), simple, straight or recurved (Glomus), swollen and straight but often appear sessile due to detachment from saccule and two scars present on either side of the spore (Entrophospora) or sporophore bulbous (Gigaspora and Scutellospora). Spores, sporocarps, and sub-cellular structures Spores may be formed singly or aggregated in loose/compact (with or without peridium) sporocarps. Spores in AM fungi may be azygospores or chlamydospores. Chlamydospores are seen in Glomus and Scutellospora. Azygospores are seen in Acaulospora, Entrophospora, Gigaspora, and Scutellospora. The colour of the spores varies from hyaline, yellow, reddish-brown, orange, brown, and black. Size of the spores is also variable. It ranges from 50-250 µm (diameter) in Glomus, Acaulospora, and Entrophospora, whereas the diameter of spore exceeds 300 µm in Gigaspora and Scutellospora. The shape of spores may be globose, subglobose, ovoid, pear-shaped, ellipsoid, oboviod, reniform or irregularly elongated. The cytoplasm within the spores may appear reticulate in a polygonal pattern or may be vacuolated because of the presence of many lipid droplets of variable sizes. Seven kinds of wall layers have been described in AM spores, namely evanescent, unit, laminated, membranous, coriaceous, amorphous, and expanding. Spores of different genera also differ in the number of wall layers. Spores of Gigaspora and Glomus have one to two wall layers, whereas Acaulospora, Scutellospora, and Entrophospora have more than three wall layers. Ecological diversity provides useful information regarding biogeographical distribution, dispersal patterns, and competitive interactions by member organisms in plant communities and soil microorganisms. The biogeographical distribution, dispersal patterns, and competitive interactions of AM fungi with plant communities and soil microorganisms are as follows.

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The available information indicates that there is more or less uniform distribution of AM fungi, though some may predominate in certain areas with broad ecological range. AM fungi are mostly present in the top 15-30 cm of soil and their numbers decrease significantly below the top 15 cm of soil. The distribution of species of AM fungi varies with climatic and edaphic conditions. For example, Gigaspora and Scutellospora are common in tropical soils, whereas Acaulospora species favour soils of pH below 5. AM fungi are indigenous to soils throughout the world. Many AM species are present in most of the continents of the world. Active dispersal of AM fungi is usually by spread from one living root to another through AM propagules, like mycelia and spores, which can be moved by biotic and abiotic agents. Dispersal of spores over greater distances is dependent upon passive dispersal by wind and water, especially in arid environment. Animal dispersal of AM spores is well documented and occurs through ingestion and egestion of spores. AM fungi are geographically ubiquitous and commonly associated with plants in agriculture, horticulture, pastures, and tropical forests. About 90% of vascular plants establish mutualistic relationship with AM fungi. The occurrence of AM fungi in roots has been reported from an exceptionally wide range of plants. Besides roots, colonization has been reported in other plant parts; for example, in leaves of Salvinia, in senescent leaves of Fumaria hygrometrica, and in decaying peanut leaves and rhizomatous tissues of Zingiber officinale. Colonization has also been reported from scales of Colocasia antiquorum, Elettaria cardamomum, Musa paradisica, Sansevieria trifasciata, garlic, and ginger. AM interactions bring about certain changes in the host physiology. These include increased production of cytokinins due to the inhibition of ctokinin degradation by compounds produced by, the fungus or plant as a result of the interaction. The presence of two gibberllin-like substances in culture extracts of Glomus mosseae and increased nitrate reductase activity has also been reported in mycorrhiza inoculated plants.

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Chapter - 11

Myxomycota and Plasmidiophoromycota (General Account)

Myxomycota are referred to as slime molds. These are presently classified in the Kingdom Protista. Slime molds were thought to be fungi (= Kingdom Mycetae) as they produce spores in sporangia, a characteristic feature of some fungi. The assimilative stage in this organism is similar to that of an amoeba, called myxamoeba. The myxamoeba is a uninucleate, haploid cell not enclosed by a rigid cell wall. It ingests its food by means of phagocytosis. In this process of ingestion, the food particles, usually bacteria become surrounded by the pseudopodia of the myxamoeba then get engulfed. They are surrounded by a membrane or food vacuole. The hydrolytic enzymes that are secreted digest the food. The assimilative stages in fungi are mycelium and single cell, both of which are surrounded by a rigid cell wall and obtain their food by means of absorption. For these reasons the mycologists recognized slime molds as separate group. However, this group has been studied in mycology as a matter of tradition and not because they are thought to be related to fungi. CLASS: MYXOMYCETES Approximately 500 species are reported and all are found on moist soil, decaying wood, and dung. Most of the species are found throughout the world. Life Cycle of Myxomycetes Physarum polycephalum and Didymium iridis are being used as examples to represent the myxomycete life cycle. These are selected since much literature exists on these species and their life cycles are well known. The spores of slime molds are globose, uninucelate, haploid and spore surface may be smooth, spiny to reticulate. Spores of P. polycephalum and D. iridis are spiny. The spore wall is made up of cellulose and is only one of two stages where a cell wall is formed. The other stage that forms a cell wall is the microcyst, on germination; the spore breaks open and releases a single, uninucelate myxamoeba which moves by amoeboid motion and ingest food, through phagocytosis. Later it feeds and grows, reproduces asexually by mitosis and cytokinesis.

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This stage may proliferate for any length of time provided nutrients are available and the environment is favourable. In the presence of free water myxamoeba get differentiated into flagellated swarm cells. Although two flagella are present, one of them is long, anteriorly directed and the second one is very short. During unfavorable conditions, the protoplast of the myxamoeba or swarm cell can roundup and form a thin, cellulose protective layer around itself, called the microcyst, which offers protection. 1.

Typical spore of Myxomycetes is a haploid, globose, uninucelate structure. In the case of P. polycephalum, the surface is spiny.

2.

Spore germination occurs by the cracking of the spore wall and releasing a single myxamoeba in P. polycephalum.

3.

Myxamoeba is usually the assimilative stage that ingests food by phagocytosis, but during sexual reproduction myxamoebae also function as gametes (isogametes).

4.

When free water is available, myxamoebae can become flagellated and swim through the water.

5.

During conditions unfavorable for myxamoebae growth, the cells may round up and form the resistant microcyst stage.

After some time, when a critical number of swarm cells or myxamoebae are formed, sexual reproduction occurs and these vegetative stages function as gametes. In P. polycephalum, the swarm cells act as gametes and are derived from a common myxamoeba, hence are self sterile. The gametes that are derived from a different population of myxamoebae (the different mating strains are designated as a1, a2, a3) undergo syngamy. Once the compatible strains come into contact with one another and fusion occurs to form the zygote and then undergo numerous mitotic divisions to form the large, multinucleate plasmodium. This is referred to as the cellular slime mold because the plasmodium state of the lifecycle is not composed of many cells. It is a single, multinucleate cell and there is also an assimilative state that consumes food by phagocytosis. However, the plasmodium is a diploid structure and is much larger. In P. polycephalum, it is a bright yellow, slimy structure while in D. iridis the plasmodium is colorless. Under unfavorable conditions, the plasmodium forms a protective, brittle layer and becomes dormant. This dormant stage is termed a sclerotium and is composed of smaller multinucleate cells called macrocysts under favorable conditions. Each macrocyst can give rise to a new plasmodium. 1.

The Physarum polycephalum has a bright yellow plasmodium. The plasmodium results from syngamy of two compatible myxamoebae, followed by numerous mitotic divisions.

2.

Plasmodium of Didymium iridis is colorless.

3.

When conditions become unfavorable, a plasmodium can become dormant; forming a resistant stage that is darker, yellowish-orange colored called sclerotium.

4.

Sclerotium is actually composed of smaller units called macrocysts. The number of nuclei in each macrocyst is variable.

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The plasmodium migrates and feeds for a period of time before being converted to numerous sporangia. In P. polycephalum, the exhaustion of food leads to formation of sporangia. The plasmodium stage persists for some time. Light appears to be another stimulus to fruiting in this species. The sporangium in D. iridis, is light blue, globose produced on a yellowish stipe while in P. polycephalum the sporangium is dark gray to almost black, lobed and is produced on a yellowish stipe. The fragile, outer layer of the sporangium is the peridium, which may be persistent or degenerate by the time the sporangium is ready to disperse its spores. In Didymium iridis, the light blue powdery appearance of sporangium is due to calcium carbonate crystal present on the peridium surface. 1.

Physarum polycephalum sporangium has numerous lobes. Calcium carbonate is also present on the peridium surface of this species, but is not obvious.

During formation of sporangium, the plasmodium becomes denser and forms a thick sheet called the hypothallus. The protoplasm of the plasmodium then becomes knotted into discrete nodules, representing sporangial primordia. The nodules elongate and as development continues the basal portion becomes the stalk, decreases in diameter while the upper portion becomes the sporangium proper and develops the finger-like projection characteristic of P. polycephalum. In D. iridis, the sporangium would become a single, globose sac. On the completion of movement of protoplasm into the sporangium, the stalk becomes more constricted and is without protoplasm. Spore formation along with the formation of cell walls takes place around the diploid nuclei. The nucleus in each spore undergoes meiosis to produce four haploid nuclei, and of which three degenerate. Only one myxamoebae results from each spore. The interior of the sporangium is of branched, thread-like capillitium. The capillitium arises from coalescene of vacuoles, which contain various materials from the protoplasm, especially calcium carbonate (CaCO3), in P. polycephalum and related species. However, there are variations in sporangial types and structures. Diachea leucopodia is another species with sporangia that have stipes. Trichia favoginea is a species that produces sessile sporangia. Lycogala epidendrum is the example of a species that produces aethalia. An aethalium resembles a sessile sporangium but is much larger. The larger size is thought to have evolved from many smaller sporangia that have fused. Fuligo septica also produces aethalia, they are reported to be the largest known aethalium. Hemitrichia serpula produces plasmodiocarps. The spores and capillitium of this sporangium type retains the shape of the plasmodial stage. Stemonitis sp. is the example of a stipitate sporangium in which the peridium disintegrates at maturity thereby leaving the capillitium and spores exposed. This species also differs in that stipe development continues within the sporangium. The extension of the stipe is the columella. Capillitium (pl. = capillitia) are filamentous structures that usually develop with spores within sporangia. They are thought to function in the retention of spores in the sporangia thus allowing gradual dispersal of spores over a long period of time.

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Some spores are always retained that are dispersed later, possibly during favorable period. Capillitia are often ornamented and have been used in defining some taxa in Myxomycetes. PLASMODIOPHOROMYCOTA These fungi are biotrophic or obligate parasites and are difficult to grow on artificial media under laboratory conditions. They attack members of Brassicaceae and cause club-root of crucifers. The pathogen of clubroot of crucifeos being Plasmodiophora brassicae and Spongospora subterranean causes powdery scab of potato. Other fungi are known to attack roots and shoot of wild plants, aquatic plants besides attacking algae and fungi. The segregation of genera is based on the arrangement of resting spores in the host tissue. Asexual reproduction is by zoospores which are biflagellate. The peculiar features being that the flagella are of unequal length and both are whiplash type, known as anisokont. Plasmodiophora brassicae infects crucifers and results in club root formation due to hypertrophy and

Fig. 11.1. Life cycle of Plasmodiophora brassicae

Myxomycota and Plasmidiophoromycota

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hyperplasia. Infected tissues show the presence of plasmodia, resting spores and zoosporangia also. The resting spore wall may contain chitin and presence of cellulose may be seen occasionally in P. brassicae. Such structures may be absent in Worononia and others. The resting spore releases zoospores which infect the host tissue. Later on zoosporangia are formed and these are thin walled. Some reports indicate formation of indistinguishable plasmodial stages. These may give rise to resting spores. Possibility of sexual fusion has been assumed before the formation of resting spores. In some genera plasmodium may give rise to resting spores. The life cycle of Plasmodiophora brassicae is given in Fig. 11.1.

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Schüßler A, Schwarzott D and Walker C 2001 A new fungal phylum the Glomeromycota; phylogeny and evolution. Mycological Research 105 1413-1421 Scott KJ and Chakravarthy AK (eds) 1982 The Rust Fungi, Academic Press, New York Seifert KA, Morgan Jones GA, Gams WA and Kendrick B (eds) 2011 The Genera of Hyphomycetes, CBS Biodiversity Series 10, CBS Fungal Diversity Centre, Utrecht, The Netherlands Shoemaker RA 1959 Nomenclature of Drechslera and Bipolaris, grass parasites segregated from 'Helminthosporium'. Canadian Journal of Botany 37 879-887 Shoemaker RA 1962 Drechslera Ito. Canadian Journal of Botany 40 809-836 Singer R 1986 The Agaricales in Modern Taxonomy, Koeltz Scientific Books, Germany Snyder WC and Hansen HN 1940 The species concept in Fusarium, American Journal of Botany 27 64-67 Snyder WC and Hansen HN 1945 The species concept in Fusarium with reference to Discolor and other Sections. American Journal of Botany 32 657-666 Solheim WG 1930 Morphological studies of the genus Cercospora, Illinois Biological Monographs 12 1-85 Sparrow FK Jr 1960 Aquatic Phycomycetes, Univ of Michigan Press, Ann Arbor Subramanian CV 1971 Hyphomycetes, ICAR Publ, New Delhi Subramanian CV 1983 Hyphomycetes: Taxonomy and Biology, Academic Press, London Sutton BC 1980 The Coelomycetes, CMI Kew, UK Sutton BC 1980 The Coelomycetes: Fungi Imperfecti with Pycnidia Acervuli and Stromata, Commonwealth Mycological Institute Tubaki K 1981 Hyphomycetes, their Perfect and Imperfect Connections, J. Cramer Udagawa S and Marinaga T 1984 Spore germination and successive growth of coprophilous fungi, in Progress in Microbial Ecology (eds) KG Mukerji, VP Agnihotri and RP Singh, Print House (India), Lucknow pp 325-339 Unger F 1833 Die Exantheme del' PRanzen und einige mit diesen verwandte Krankheiten del' Gewiichse, pathogenetisch und nosographisch dargestellt. Carl Gerold, Wien von Arx JA 1957 Die Arten der Gattung Colletotrichum Cda, Phytopatholgiche Zeitschrift 29 413-468 von Arx JA 1981 Genera of fungi sporulating in pure culture. 3rd edn J Cramer, pp 424 von Arx JA 1983 Mycosphaerella and its anamorphs, Proceedings K. Nederl. Akad. Wet. Ser. C 86 15-54 Walting R, Frankland JC, Ainsworth AM, Issac S and Robinson CH (eds) 2002 Tropical Mycology vol 2 Micromycetes, Cab International Wasson VP and Wasson RG 1957 Mushrooms, Russia and History, Pantheon Books NY Vol I and II Webster J 1970 Coprophilous fungi Transactions British Mycological Society 54 161-180 Webster J and Weber R WS 2007 Introduction to Fungi. 3rd edn Cambridge Press Whittaker RH 1969 New concepts of kingdoms of organisms. Science 163 150–160

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Questions

Give short answers 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.

What is Nomenclature of fungi? Differentiate between RAPD and RFLP. What is PCR? Define fungi. What is Saccardo’s contribution? List out five eminent mycologists of India. What is Whittaker’s classification? Describe fungal cell wall? What are lysosomes and what is their function? Describe fungal nucleus? Define citosomes. Describe dolipore septum. What is the difference between holcarpic and eucarpic thalli? What are haustoria? What is Bartniclci – Garcia’s contribution? What are obligate parasite?s What are chlamydospores? What is budding? How many types of flagella are there? Define zoospore and its’ function. What are arthrospores? What are sporangiospores? Define acervulus. Define synnema. What are the differences between Isogamy and Anisogamy? What is gametangial contact, cite examples. What is a zygospore?

Questions 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 51. 52. 53. 54. 55. 56. 57. 58. 59. 60. 61. 62. 63. 64. 65.

What is somatogamy and is it advanced or primitive? What is ICN and its’ role? In the classification of fungi what is von Arx’s contribution? What is Blackwell and Spatofora’s classification? Describe thallus variation in Chytrids. Briefly mention about Blastocladiales. What is the difference between oosphere and oospore? Describe the release of zoospores in Pythium. What is sporangial proliferation? What are antheridiol and oogoniol? Defineh heterothallism. What are amphigynous antheridia? What are rhizoids and stolons? What are sporangia, sporangiole and merosporangium? What is Burgeff’s contribution? Describe Entomophthora. What are Trichomycetes and their relevance? Differentiate between zygospore and zygophore. What is columella and it’s function? Describe zygospore in Phycomyces. How are the sporangia in Choanephora? Describe ascus formation. Differentiate between unitunicate and bitunicate asci. What are the different types of fruit bodies in Ascomycota? What are the various centrum types? Describe the various appendages on fruit bodies of Erysiphales. What are oidia? Differentiate between foot cell and hulle cells. Differentiate between Biverticillate and Multiverticillate. What are phialoconidia? What are setae or hairs, and their importance in Chaetomium? What is meant by one gene – one enzyme theory? What isw tetrad analysis? What is the difference between periphyses and paraphyses? What is clamp connection? What is the difference between phragmobasidium and holobasidium? What is Buller’s phenomenon? What is pleimorphisom?

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Biology and Biotechnology of Fungi and Microbes 66. 67. 68. 69. 70. 71. 72. 73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91.

What is basidiocarp? Differentiate between smut and rust. What are aecial and pycnial stages? Differentiate between micorcyclic and macrocyclic rusts. What are covered smuts and loose smuts? Describe parasexuality. Differentiate between basidiospores and sporidia. Distinguish between monophyletic and polyphyletic origin. What is Hughe’s contribution? What are thallic conidia? What are holoblastic and phialidic conidia? What is the difference between blastoconidia and tretic development? Distinguish between acroauxic and basauxic conidiophores. Describe synnema with examples. Distinguish between acervulus and sporodchium. What are euseptate and distoseptate? What is Von Arx’s Cercospora complex? What are anamorph and teleomorph? Distinguish between pycnidium and cirrhus. Describe conidial appendages with examples. Elaborate AM fungi. What are the benefits from mycorrhizal fungi? Distinguish between plasmodium and sorus. What is clubroot of crucifers? Give a brief account of Plasmodiophora life cycle. What is conidial ontogeny?

Give long answers 1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

Define fungi. Substantiate it with characters? List out major mycological contributions? Discuss in detail the thallus structure in fungi? Give an account of fungal ultra cell structure? Elaborate the chemistry of fungal cell wall? Write in detail about sexual reproduction in fungi? Discuss in detail the asexual reproduction by zoospores? Elaborate ICN of fungi with details? Outline the recent classifications of fungi? Give an account of zoosporic fungi?

Questions 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47.

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Give a general account of Straminopila? Discuss the life cycle of Olpidium? Disuss in detail the biology of Chytridium? Give an account of sexual reproduction in Achlya? Write about biology and importance of Phytophthora? Differentiate between Plasmopara and Peronospora asexual reproduction? Give a detailed account of life cycle of Sclerospora? Give general account of Mucorales? Discuss about sexual reproduction in Mucorales? Discuss in details about Pilobolus? Write about biology and importance of Phycomyces? Differentiate between Syncephalastrum and Cunninghamella? Give salient features of Ascomycota? Elaborate on fructifications of Ascomycota and their importance in classification? Give a general account of Loculoascomycetes? Discuss about life cycle of Taphrina? Differentiate between ascus types, ascus dispersal and types of ascospores? Differentiate between Eurotium and Talaromyces? Outline the life cycle of Chaetomium and its importance? Elaborate the life cycle of Neurospora? Differentiate between fruitbodies of Sordaria and Peziza? Give a detailed account of Basidiomycota? Give a general account of Teliomycetes? What are Hymenomycetes? Discuss the general characters? Outline the life cycle of wheat rust and control measures of the disease? What is pleiomorphism and discuss along with examples? Why smut diseases are important and give a general account? Discuss in detail the biology of Agaricus? Write in detail about asexual reproduction in Anamorphic fungi? Discuss in details about asexual fruit bodies? Gove an account of Alternaria? Differentiate between Drechslera and Curvularia? Discuss in details about Cercospora? How important is Fusarium? Give an account? Differentiate between Phoma and Colletotrichum? Briefly discuss about Pestalotia? Discuss in detail about Glomeromycota?

B. CURRENT TOPICS OF IMPORTANCE

Chapter - 12

Phylogeny, Evolution and Origin of Fungi

Fungal Kingdom embodies heterotrophic organisms which share some characters of animals in having chitinous cell wall, glycogen as reserved food, and few other characters. Further some similarities also exist in having spores or gametes with a single, smooth, posteriorly inserted flagellum. Fungi and other heterotrophic organisms such as choanoflagellates and mesomycetozoa are now grouped in opisthokonta (Cavalier Smith 1987). This argument has also been supported based on phylogenetic data including for nuclear small sub-unit ribosomal RNA gene. The earlier classification of fungi based on morphology needs updating as phylogenetic data has become essential when morphological criteria are convergent, reduced or missing among the taxa. This has been documented with reference to fungal species that reproduce sexually, due to the fact that sexual reproduction has been the basis for classification. However, the molecular characters analysis allows asexual fungi to be placed among their nearest relatives. Many phylogenetic studies suggest that two phyla Chytridiomycota and Zygomycota are not monophyletic. Chytrids seem to be paraphyletic, comprising Blastocladiomycota, Chytridomycota and Neocallimastigomycota, and genera like Olpidium and Rozella are of uncertain phylogenetic position. Both these genera are reduced endoparasites and seem to belong to isolated, phylogenetic lineage. Fungi with coenocytie hyphae or pseudoseptate hyphae and thick walled spores were placed in Zygomycota. Evidence for monophyletic origin is lacking and reconstruction of Zygomycota lead to the creation of fair unordered phyla namely; Entomophoromycotina, Kickxellomycotina, Mucoromycotina and Zoopagomycotina (Hibbett et al 2007). Separation of arbuscular mycorrhizal fungi that lack septa in hyphae and zygospores, have been accommodated in newly emerged phylum Glomeromycota (Schüßler et al 2001). The presence of septate hyphae has been considered as closely related character of Ascomycota and Basidiomycota. The SSU rDNA, RNA polymerase and mitochondrial genome sequence provided strong support for such relationship, a sub-Kingdon, Dikarya has been proposed for such fungi. Fungal classification and criteria have been changing from time to time, hence not static. The molecular data of Trichomycetes had demonstrated that they have some link with Mesomycetozoa, the Protista group. Hyphochytridiomycota and slime molds which were considered as fungi are now placed under Stramenopiles. Another revealation with phylogenetic evidence indicates that Protists and Micorsporidia are

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closely related to fungi. These must have been derived from Zygomycota. Steenkamp et al (2006) demonstrated that nucleariid amoebae are sister group of fungi. Regarding evolution of fungi, Wang et al (1999) reported that fungi diverged from other life forms around 1500 million years age. Glomalean fungi branched out from higher fungi 570 million years ago. Fungi might have colonized in Cambrian and such fossil evidence came during Devonian in Rhynie chert. Available fossil fungal evidence is meager. Typical fungal forms have been noticed in Proterozoic period. More recent studies estimate the arrival of fungi at about 760-1060 ma. Probably the fungi might have colonized during Cambrian (546-4883.3 ma) long before the land plants. Ascomycota and Basidiomycota diverged and all modern classes of fungi were present in the late Carboniferous era (316.1-2990 ma). Fungi became dominant in Permian – Triassic extinction event (251.4ma). Very little is known about evolutionary relationships among fungi. Recent data indicates that simple morphology, the lack of a useful fossil record and fungal diversity have been major impediments to achieve progress. Fungal evolution has been brought out based on comparative morphology, cell wall composition, cytological data, ultra structure and cellular metabolism. Fossil data and phylogenetic analysis have shown that the fungal kingdom is part of terminal radiation of eukaryotic organisms which occurred one billion years ago. The number of nucleotide substitutions in DNA sequences is directly proportional to the time passed and so the number of base changes can be used to estimate the data of evolutionary radiation using the data pertaining to the appearance of fossilized clamp connections from fossil record. Thus the absolute timing of the origin of fungal groups has been estimated. Zygomycota, Ascomycota and Basidiomycota, might have diverged from Chytridiomycota 550 million years ago. The split between Ascomycota and Basidiomycota occurred around 400 million years ago after the plants invaded the land. Ascomycetes might have evolved since the origin of angiosperms in the last 200 million years. PHYLOGENETIC HYPOTHESES, EVOLUTIONARY RELATIONSHIPS AND CIRCUMSCRIPTION OF THE FUNGI The monophyletic origin of ‘fungi’ is the dogma that made mycologists to explore the fungi and their relationships. In this theory, it is thought that all the fungi were derived from an algal ancestor that lost its ability to photosynthesize and gave rise to the flagellate fungi. This ancestor was thought to be a member of the Chytridiomycota, which was morphologically similar to their modern counterparts and produced posteriorly, uniflagellated zoospores. Like many of its modern counterparts, this ancestral chytrid was aquatic, but over time, became more adapted to the terrestrial environment. The loss of flagella and the evolution of the zygospore gave rise to the Zygomycota whose members were believed to be morphologically similar to Mucor or Rhizopus, which produce large, single, multispored, columellate sporangia. As this group evolved, the number of sporangiospores in a sporangium became reduced and the columella was lost, thereby the sporangiole was evolved, with the most advance

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members producing single-spored sporangioles, e.g. Cunninghamella. This led to the evolution of the conidium, an asexual spore typically produced by the Ascomycota and Basidiomycota where an anamorph stage is produced. Another line that arose within the Zygomycota was that which included the Endogonales. The zygospores produced in this line were uninucleate. This line was believed to have given rise to the Ascomycota. The link between the Zygomycota and Ascomycota was made with a fungus that resembled Dipodascopsis (Ascomycota). The Taphrina-like ancestor gave rise to other members of Ascomycota and ancestral Basidiomycota, which diverged into two lines. One line produced microcyclic rusts, which produced only teliospores and basidiospores and led to the present-day rusts. The other line produced the ancestor of the present day Auriculariales, which had a poorly differentiated basidiocarp. This line gave rise to the remaining basidial and basidiocarp types in the Basidiomycota. Bessey believed that the Zygomycota were derived from the Oomycota rather than Chytridiomycota, based on similarities in morphology of their sporangia and that both phyla produced coenocytic mycelium. Bessey, Sachs, Denison and Carrol believed that the Ascomycota were derived from the Floridean red algae (Rhodophyta). Although Kohlmeyer, Kohlmeyer and Kohlmeyer did not advocate the direct derivation of the Ascomycota from the Rhodophyta, they postulated a hypothetical, common ancestor that gave rise to both phyla. Demoulin had a similar hypothesis, but believed that both the Ascomycota and Basidiomycota were derived from a red algal ancestor. During this old era, characteristics not previously incorporated into determining fungal phylogeny and new tools that became available brought forth new theories on the interrelationship of the different fungal taxa. Some new criteria that were considered in fungal phylogeny included: 1.

Cell biological data on mitochondrial cristae, organelle distribution, flagellation, mitosis.

2.

Biochemical data on cell wall constituents, amino acid synthesis and enzymology.

This indicated that the Chytridiomycota should be classified in the kingdom Myceteae, with the fungi; the Oomycota should be in the kingdom Stramenopila with the Chrysophyta and Phaeophyta as proposed by Pringsheim and Kriesel. Although their theory did turned out to agree with this modern interpretation in phylogeny, they had based their classification of the Oomycota only on superficial morphological similarities. The Hyphochytriomycota were also first classified in this group this time. The different phyla of slime molds were also shown to be more distantly related than once believed, and also, it is noteworthy that the Rhodophyta is unrelated to the Ascomycota. Although phylogeny in the higher taxa of fungi seems to be more resolved, the lower taxa now seem to be more confused. DNA sequence analysis does not support the Dipodascopsis-like hypothetical ancestor that was once thought to link the Zycomycota to the Ascomycota. Thus, we no longer have a link between the two phyla. Another Ascomycota dogma that has been demonstrated to be incorrect is the monophyletic origin of the ascosporogenous yeasts. Members of Saccharomycetales

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no longer appear to be monophyletic and are more distantly related to other ascosporogenous yeasts. Phylogeny within the Basidiomycota has also become more confused. The relatedness of taxa within this phylum, based on the morphology of the basidium, which was the main character used to classify fungi in this phylum, is not in agreement with DNA sequence analysis. Instead, it appears that the septal pore apparatus is a better indicator of relationship between members of the Basidiomycota. Thus, with the exception of the Tremellales, all of the various basidial types, that are formed in basidiocarps now appear to be more closely related than once believed, i.e. Agaricales, Gasteromycetes, Auriculariales, Dacrymycetales, etc. are now believed to form a monophyletic group with the Tremellales as a sister group. The Ustilaginomyetes and Uredinomycetes are believed to be more distantly related.

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Chapter - 13

Biodiversity and Biotechnology of Fungi

WHAT IS BIODIVERSITY? Biological biodiversity means ‘the variability among living organisms from all sources including inter alia, terrestrial, marine and other aquatic ecosystems and the ecological complexes of which they are part. It is a basic resource that sustains the human race and it also includes diversity within species, between species and of ecosystem’ (United Nations environment programme 1992). Biodiversity is the most significant global and national asset and is the enduring resource for supporting the sustained existence of human beings. It includes diversity in form, from gene to the individual organism and then on to the population, community, ecosystem and biosphere level. The term biodiversity embraces genetic diversity, species diversity, ecosystem diversity and agro biodiversity. 5-50 millions species of living form exist on the globe and of which 1.5 million are fungi. The vast numbers of fungi that are recorded exceed 98,483 (Hawksworth, 1991, 1997, Kirk et al 2008). India is located in South Asia, between latitudes 6° and 30° and longitudes 69° and 97° E. It has total geographical area with a land mass of 329 million hectares with Himalayas in the north, the Bay of Bengal in the east, the Arabian Sea in the west and Indian Ocean in the south as boundaries. The richness of biodiversity is due to immense variety of climate, altitudinal conditions and soil types. BIODIVERSITY OF FUNGI The number of fungi recorded in India exceeds 29,000 species, the largest biotic community recorded after insects. These organisms are without plastids, with absorptive nutrition, never phagotrophic, without an amoeboid pseudopodial phase, cell walls mostly containing chitin and glucans, mitochondria with flattened cristae, peroxisomes nearly present. Individual cisternae present, thallus being unicellular or filamentous and consisting of septate or aseptate (coenocytic), haploid hyphae, mostly non flagellate, when present always lacking mastigonemes, reproducing sexually or asexually, the diploid phase generally short lived, saprobic, mutualistic or parasitic. Kirk et al (2008) in the 10th edn of Dictionary of the fungi have recognized six phyla viz. Chytridiomycota, Glomeromycota, Microsporidia, Zygomycota, Ascomycota and Basidiomycota. One third of global fungal diversity exists in India and thus India can claim its richness in fungi with mega-diversity identity (India with 2.4 % of the total

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biodiversity with species count of 0.130 million). The beauty and endless variety of fungi never cease to enchant biologists all over the globe. India has always been a strong centre and cradle for fungi. The fungal diversity unraveled by mycologists through conventional techniques is only a fraction of the amazing real diversity. In order to explore the unexplored and unexplorable, the fungal specialists have to look up for newer tools, innovation, creation of novel media, selective enrichment techniques besides taking into account various ecological parameters. The most important aspect being to adopt molecular techniques such as the use of DNA and RNA probes and immunological techniques. RFLP and RAPD can be used for species/strain delineation. Ecological parameters include the identification of unique ecological niche and search for fungi in environs experiencing extremes of salinity, atmospheric pressure, temperature, pH and pollution. In-depth study of fungal diversity is linked with its utilization and conservation. The fossil record of fungi dates back to the early phanerozoic and into the proterozoic geological (Pirozynski and Hawksworth 1988). The existence of fossil fungi indicates their evolutionary significance besides helping in solving certain phylogenetic complexities. In India several new fungal genera are recorded in spite of having a long fossil history. Zoosporic fungi Fungi belonging to Zoosporic fungi form a prevalent group of fungi in water. It comprises members of Chytridiomycetes, Hyphochytridiomycetes and Oomycetes. These fungi are known to colonize diversified habitats, which includes water, humid soils, insects, keratin, chitin, angiosperms, pollen grains and other by living either as saprophytes or parasites. All these fungi have been arbitrarily grouped together in this sub-division on the basis of zoospore and oospore. This division of fungi comprises 204 genera and 1160 species. Chytridiomycetous fungi occur as saprobes on plants and animals, remain in water while some members being parasites on algae and aquatic animals. Dick (1976), Fuller and Poyton (1964), Manoharachary (1981, 1991) and Sparrow (1960) have compiled lot of information on the morphotaxonomy, ecology, physiology, methodology and activity of flagellate fungi. The Hyphochytridiomycetes are aquatic whose thallus is holocarpic or eucarpic, monocentric or polycentric and their vegetative system is rhizoidal or hypha like with intercalary swellings. Sparrow (1960) has discussed various aspects of this group of fungi. The Oomycetes contain 74 genera and 580 species, which are mostly aquatic living either as parasites or saprophytes. Das Gupta (1982) and Manoharachary (1982) have made pioneer studies pertaining to the floristics, taxonomy and ecology of aquatic fungi from India. Chytridiales is the largest and least understood order of the Chytridiomycetes containing nearly 80 genera and many species, which are poorly described besides the families and genera are not phylogenetically based (Karling 1977, Longcore 1996). The Oomycetes are the largest group of heterotrophic Stramenopiles and most of them are the inhabitants of fresh water, humid soils while some are the indwellers of salt water habitats besides being parasitic. The cell wall is made up of β-1,3 - and β-1,6 - glucose with a small amount of cellulose. Some members have chitin deposits. Barr (1980) has recognized a new order Spizellomycetales under

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Chytridiomycetes. The fungi belonging to this order occur in soil and are seldom found in strictly aquatic habitats. Both saprophytic and parasitic forms are present and the thalli are monocentric. Sexual reproduction is unknown. Zoospores of this order have a nucleus that is usually spatially associated with or connected to the kinetosome. Zoospores lack rumposomes and microtubule rootlets, when present do not arise from the side of the kinetosome, they arise from a microtubule organizing center (MTOC) near the kinetosome. Zoospore ribosomes are generally dispersed and there are usually several lipid globules in the spores. A new nomenclature has been used for the fungi having flagella with mastigonemes that are hollow or straw like. All these fungi have mitochondria with tubular cristae and synthesize lysine via diaminopimelic acid pathway. These fungi are known as Stramenopiles and information available about this group of fungi is little. Many solid agar media such as mineral agar, corn meal, oatmeal, potato sucrose agar, glucose, peptone, Brassica agar media, hemp seed agar and other media have been used to culture the members of Mastigomycotina. Altogether 260 zoosporic fungi are reported from India which includes, predominantly occurring members of Oomycota and less frequently occurring chytrids. Manoharachary (1981) has classified the aquatic fungi into low temperature species, moderate temperature species, constant species and high temperature species. It is stated that warm weather retards the growth, multiplication of moulds. A pH range between 5.8 - 7.0, organic matter, nitrates, aerobic water and phosphates have been directly correlated with the abundance of Mastigomycotina under Indian conditions. Three different oospore types are found among Mastigomycotina members namely, centric, subcentric and eccentric. Eccentric oospore bearing zoosporic fungi are abundantly found in tropical and subtropical water bodies, centric / subcentric oospore bearing fungi are abundant in temperate climate. Zygomycotina It is an assemblage of fungi, which reproduce asexually by sporangiospores which are dispersed either violently or passively by wind, rain or animals. Sexual reproduction is by gametangial copulation and results in the formation of a zygospore. Fungi of this subdivision are ubiquitous in soil and dung, occurring mostly as saprophytes although few are parasitic on plants and animals. Trichomycetous fungi live in the guts of arthropods. Around 1000 fungal species are reported from India. These fungi are important in industry, food and in understanding the physiology, biochemistry and genetics. Saksenaea vasiformis has been the indigenous fungus, which has become important in medical mycology. An evolution of sporangiospore to single sporangiole or conidia, has been observed from Rhizopus to Cunninghamella. Ascomycotina Ascomyceteous fungi comprise of a wide variety of fungi differing in their morphology, ontogeny, ascocarp details, ascus organization, nature of ascospores, ultra structure and other parameters besides occurring on diversified habitats. Ascomycotina is the largest Phylum of the fungi encompassing 2,700 genera and

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28,500 species. Yeasts are common in moist, sugar rich environment like plant surfaces and fruits but are also prevalent in soil, fresh and marine water bodies. Yeasts are important in several industrial fermentations like brewing and baking. The mycelial members like Chaetomium, Xylaria, Neurospora, Sordaria and Ascobolus are the common saprophytes in soil, plant and animal remains. Some fungi such as Lulworthia and others are common in estuarine environment. All are important in decomposition processes, because of their abilities to degrade cellulose and other polymers. Fungi like Tuber and Truffels form ectomycorrhizas on forest trees. Fungi like Arthroderma and Nannizzia spp. parasitize man and cause diseases although they do not form sexual stages on the host. Many members of Ascomycotina viz: Taphrina, Ceratocystis, Erysiphe, Sphaerotheca, Claviceps, Phyllactinia etc., parasitize plants and cause huge losses. Lodder (1970) has classified the yeasts based on guanine and cytosine content of DNA, electron microscopic studies, morphological structures, life cycles and physiological tests. Luttrell (1973) has classified Ascomycotina based on uni- and bitunicate asci, ascospore structure, ornamentation, septation, colouration, ascocarp morphology, ontogeny of ascocarp, ascospore release, ultra structure of ascospore and others. Around 5,000 species of Ascomycotina have been reported from India. Fungi belonging to Laboulbeniomycetes which are exo-parasites on insects need to be studied in-depth. Basidiomycotina Largely fleshy fungi which include toadstools, bracket fungi, fairy clubs, puff balls, stinkhorns, earthstars, bird nest fungi and jelly fungi are the members of this group. These fungi live as saprophytes and some are serious agents of wood decay. Some toad stools which are associated with trees form mycorrhiza, a symbiotic association (Harley 1969) but some are severe parasites e.g: Armillaria mellea which destroys a wide range of woody and herbaceous plants. Some fleshy fungi being poisonous are notorious, while majority of these are harmless and some members of them are edible (Ramsbottom 1953). There is no full proof method to separate the edible from the poisonous mushrooms. The various sizes, colours and shapes of mushrooms have attracted the attention of both naturalists and artists who have depicted them in their drawings, paintings, sculptures, etc. In nature mushrooms grow wild in almost all types of soils on decaying organic matter, wooden stumps, etc. They appear in all seasons, but mainly during the rainy season whenever organic matter or its decomposed products are available. There are more than 2,000 species of edible mushrooms reported in literature from different parts of the world. Singer (1986) has reported 1320 species belonging to 129 genera under Agaricales. Deuteromycotina The Deuteromycotina (Fungi Imperfecti or Mitosporic fungi or Anamorphic fungi) constitute an artificial group and are the asexual phases of Ascomycotina and Basidiomycotina. The multiplication occurs by the production of mitotic spores or conidia from specialized hyphae called conidiophores. The Conidiophores are single in hyphal forms or grouped, or in various structures, the fruit bodies are called condioma or conidiomata. The production of conidia or conidiogenesis occurs by various means and on various structures. Some Fungi Imperfecti may produce

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several distinct types of conidial apparatus. Most of the asexual or imperfect forms are the anamorphs of Ascomycetes and Basidiomycetes. Conidial ontogeny formed the basis for the identification and segregation of fungi imperfecti. Hughes (1953) has visualized thallic and blastic, as the two basic developmental patterns of fungi. Louis Rene and Charles Tulsane wrote in 1811 at the end of their work ‘In order to study the hidden marvels of these fungi, one must devote a great deal of labour and patience, but in gazing upon them when one discovers them, how much greater is the Joy’. The Deuteromycetes comprise 1,700 genera of Hyphomycetes, and 700 genera of Coelomycetes that cover some 20,000 known species. These fungi colonize, survive and multiply in air, litter, soil or any other substrate. These fungi contribute to the bio-degradation, recycling of organic matter and enzyme production while some are important in nutrition, industrial production, antibiotic production, as immunoregulators, as bio-control agents besides causing profound mycoses, allergies and plant diseases. Around 8,000 Fungi Imperfecti are reported from India and such fungi are indexed by various authors in their volumes ‘Fungi of India’ (Butler and Bisby, 1960, Bilgrami et al 1981, 1991, Sarbhoy et al 1996, Jamaluddin et al 2004). There is a hidden wealth of fungi in soil, water, air, litter and other habitats besides being on many other microecological niches. FUNGAL BIOTECHNOLOGY Communities of saprophytic, parasitic and endophytic fungi living in the forest ecosystem contribute significantly to the species diversity. Studies have revealed that tropical fungi colonizing diversified habitats harbour diverse microfungi in abundance (Hyde 1997). It is becoming clear that both micro and macrofungi of the tropics are potential sources of biotechnology, industrially important and pharmaceutically valuable organic molecules. Realizing this, Rossman (1994) and Hawksworth (1997) have advocated complete and urgent recovery, inventory and investigation of mycobiota from forests and other localities of tropical belt since this region of the world is presently under stress. The microbiological conversion of carbohydrates to cellular material and other useful products has become increasingly important and interest in cell mass production is constantly growing. Saccharomyces cerevisiae plays significant role in the formation of biomass from carbohydrates. 20 amino acids were found to be the components of protein in yeast and on average yeast contain 10.8% nucleic acids. Lipid content of yeast is 8% and yeast lipids include sterols. The filamentous fungi contain many species capable of hydrocarbon oxidation. However, because of their average growth form they have seldom been extensively studied in the context of the production of microbial biomass with hydrocarbons as feed stock. One exception is the kerosene fungus Cladosporium (Amorphotheca) resinae, which is important in the field of petroleum product contamination. Filamentous fungi have also been used to produce different types of metabolites from alkane media. Cunninghamella, Mucor, Aspergillus, Botrytis, Fusarium, Paecilomyces, Sporotrichum, Verticillium and others are reported to contain

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hydrocarbon assimilating species or strains. Hansenula capsulata, Pichia lindnerii, Torulopsis glabrata and Candida boidinii are selected as methylotrophic yeasts. More than half of the total production of plant residues remain unused, mainly straw and leaves and often also wastes from agricultural, forest and industrial production processes. These different materials are partially burned or disposed of on land for soil amelioration after being shredded or composted. By suitable treatment they can be converted into substrates for the cultivation of higher fungi. Fruit bodies serve as delicious food, spent mushroom compost can be utilized as humus fertilizer and widely grown substrates e.g. straw, might be valuable as upgraded feed for animals. This process has an advantage where waste is profitably removed and reintegrated by way of natural processes into the ecosystem followed by the mobilization of cellulose or hemicellulose through mineralization. Hardly usable substrates are transformed into consumable protein rich biomass. Edible fungi represent a well characterized microbial biomass which is well accepted by the consumer. The consumption of edible fungi as food and drugs is closely related to the history of mankind. Lentinus edodes and Volvariella volvacea belong to those species which have been cultivated for 2000 years in Asiatic countries. Agaricus bisporus was first mentioned in the seventeenth century in France. More recently Agaricus bitorquis and Pleurotus species have also been produced commercially. Edible fungi are cultivated worldwide under various climatic conditions. In the last two decades Taiwan has become the third largest producer of mushrooms in the world. China is the important producer of Volvariella volvacea. Mushrooms are considered more nutritious than many other vegetables. The nutritive value of some quality mushrooms is nearly equals to that of milk. These mushrooms contain high level of essential amino acids, less carbohydrate, more fibre, vitamins, unsaturated fatty acids with a mineral content exceeding that in fish and meat and twice that in most vegetables. Fruiting bodies, in general, on dry weight basis, contain about 32% protein, 2% fat, less carbohydrates and rest in fibre and ash constituting the minerals. More than 2000 species of mushrooms are reported to be edible throughout the world. Around 150 species belonging to 30 genera are regarded as prime edible mushrooms. Over 60 are cultivated commercially and have reached an industrial scale in many countries. Mushrooms have great demand in food industry because of flavourants associated with mushrooms. Mushrooms have long been used for their medicinal and tonic properties in China, Japan and other countries. A number of mushrooms possess alkaloids or chemicals of pharmaceutical importance. Mushrooms contain antitumor glucans and an immunomodulatory protein FIP-fve, recently isolated from Flammulina velutipes. Mushrooms are also important in industries, bio-transformation of penicillin, as biopolymer in bioabsorption and other uses. Mushrooms like Ganoderma, Polyporus, Lentinus and Pleurotus have been used as ornamentals. Species of Ganoderma and others have been employed in cosmetic industry. Ganoderma lucidum has been used as a biosorbent of copper and chromium. Spent mushroom compost consisting of degraded cellulose, hemicelluloses and lignin serves as an effective soil fertilizer and conditioner. Spent substrate has also been used as a source of saccharifying enzymes and for production of single cell protein. Spent substrate contains more sugars which are produced by

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enzymatic hydrolysis of substrate by mushrooms. Spent substrate is a rich source for biogas production. Exploitation of many hidden activities of edible mushrooms is essential for mankind. Molecular tools like genetic engineering, protoplast fusion and others will be useful for the development of highly productive newer strains of edible Mushrooms. Soil fungi influence plant growth and health of the plant. In mycorrhiza, the plant root-fungus symbiotic association plays a key role in the effective uptake of phosphorus by mycorrhizal roots besides enhancing plant growth. Entomogenous fungi are found in all taxonomic groups, although the Deuteromycotina, Chytridiomycetes and Zygomycotina contain many of the useful species. Verticillium lecanii parasitic on aphids and whiteflies, Hirsutella thompsonii pathogen of eriophyid mites, Beauvaria bassiana and Metarhizium anisopliae have received utmost consideration and were also commercialized. The key disadvantages of fungi as microbial pesticides being rather restricted range of environmental conditions suitable for their use. Role of fungi is well established in fermentation technology and using these industrial processes, many metabolites are commercially exploited for their antibiotic properties. Cephalosporin, penicillin and griseofulvin are derived from fungi. Yeasts form an important agent in fermentation to produce ethanol from sugar or after hydrolysis from starch and cellulose. Fungi are involved in the production of food stuffs, bread, fermented milk, yoghurt, cheese and various yeast preparations. There are many foods used in Asian and oriental cuisines, for example sufu, tempeh and miso whose production is dependent on fungi. Many different industrial organic acids are produced by representatives of a wide range of fungal genera. These include citric acid (Aspergillus, Mucor and Penicillium), fumaric acid and lactic acid (Rhizopus) and gluconic and itaconic acids (Aspergillus). Rennin is produced by Rhizomucor and Rhizopus, riboflavin by Eremothecium and various fungi imperfecti are employed in steroid transformation. Production of mycelial paper, involves the addition of mycelium of Phytophthora and other members of the mastigomycotina to wood pulp. Today three species of fungi all ascomycetes namely Neurospora crassa, Aspergillus nidulans and Saccharomyces cerevisiae are employed as model system in genetics. Transformation systems are available for all three species and it is easy to isolate their DNA. Fungi are good candidates for employing them in degrading refractory substrates, cellulose, lignin, chitin, keratin and other substrates. Fungi like A. niger and A. oryzae are regarded as safe by the food and drug administration. Molds also secrete several hydrolytic enzymes that digest the substrate. Around 20 standard amino acids are produced commercially through fermentation using fungi. Hansenula anomala is used to produce tryptophan, Saccharomyces cerevisiae in the production of lysine, methionine is produced by Candida tropicalis and several marine fungi are found to accumulate alanine. Pullulan and Scleroglucan have greater significance as biopolymers. Several fungi are identified to produce lipids. Saccharomyces, Torulopsis and others have been found to be potential producers of glycerol. Several vitamins including vitamin B12, biotin, folic acid, pantothenic acid, pyridoxine, riboflavin and thiamine are the products of fungi. Eremothecium and Ashbya are important since these fungi can make up to 2.5 and 7g/liter of vitamin

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B12, respectively. Blackeslea trispora, a heterothallic fungus of Mucorales group is extremely rich in mycelial β-carotene, as a potential source of carotenoids. Industrial enzymes are used in food processing and in particular amylase, glucoamylase and glucose isomerase, are exploited for the production of glucose and fructose syrups. The solubilization of insoluble phosphorus by the fungi is one of the vital microbial processes in soil. Several fungi bring about solubilization by producing various types of organic acids. Besides solubilizing phosphorus, these organisms release sufficient amounts of soluble phosphorus into its surroundings. The most efficient phosphate solubilizing fungi belong to Aspergillus and Penicillium. Fungal toxins play a significant role in the health of humans and animals. Aflatoxin elaborated by Aspergillus flavus is commonly found associated with animal feed and in some food stuffs also. Aflatoxin B1, B2, B2a, G1, G2, G2a, M1 and M2 have been isolated and characterized. Other Aspergilli produce ochratoxin, sterigmatocystin which are commonly found associated with food stuffs and they seem to be carcinogenic. Penicillium urticae, P. expansum and others were found to produce patulin, a mycotoxin clinically found to be carcinogenic. Around 20 fungi have been found to cause mycotoxicoses in animals. Ergot fungus is known to elaborate atleast seven alkaloids including LSD. Ergot itself has a respectable place in pharmacopia. Several fungi have been found to cause mycotic diseases and in particular in tropics. Coccidioidomycosis, Blastomycosis, Histoplasmosis, Aspergillosis, Candidiasis have also been recognized with greatly increased frequency due to the loss of immunocompetance of individual host. Cryptococcosis has a worldwide distribution and is found to be prevalent in India and the organism Cryptococcus neoformans is being considered as a diagnostic tool for AIDS. Fungal biomass produced from Penicillium chrysogenum is used as fertilizer (Bisol) or as cattle feed. The same fungus is one component of biosorbent M, which enables the extraction of uranium and radium from atomic industrial waste water. Cyclosporine A is the most often used powerful immunosuppressant and is extracted from members of hyphales. Genera such as Arthrobotrys, Dactylella, Dactylaria and Monacrosporium are predators used against pathogenic nematodes. Trichoderma viride, T. harzianum and Verticillium biguttatum are the common fungal antagonists used as biocontrol agents to control pathogenic types of Fusarium, Rhizoctonia, Macrophomina and Sclerotium. Phellinus weirii which causes rot of telephone poles is controlled by Scytalidium. Fusarium oxysporum f.sp. cannabis is used as bioherbicide for suppressing marijuana plant. Colletotrichum gloeosporioides and C. truncatum are also used as bioherbicides. Pulmonary aspergillosis (Aspergillus), lymphatic sporotrichosis (Sporothrix schenckii), equine lymphatic histoplasmosis (Histoplasma), onchomycosis (Graphium), ringworm, chromomycosis (Phialophora), skin and respiratory allergies and such type of mycotic infections are of great importance in tropical health management. Such mycotic infections need to be tackled with all seriousness. Plant diseases such as blights, wilts, rust, smuts, cankers, leaf spots, blasts, etc. on a number of crop plants and forest plants have become important as they decrease country’s economy. Biodeterioration is a biological process that contributes to deterioration or destruction of material resulting in economic loss. Biodegradation refers to the biological breakdown of undesirable materials or compounds to

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harmless or tolerable products. A vast range of materials used by man, viz. food, feed, fabric and wood to numerous agricultural material and undesirable materials including sewage, coal mine, waste from paper pulp mills, sugar and oil refineries, industrial effluents, pesticides, detergents degraded by fungi are second to none in their armory of enzymes and ability to degrade substrates of all kinds. Application of mycoherbicides to control weeds has been an age old approach through classical methods in which plant pathogens are released to control weeds through natural spread. Control of skeleton weed by Puccinia condrillina in Australia, Puccinia abrupta var. parthenicola for the control of Parthenium hysterophorus, Alternaria alternata and Cercospora rodamanii for the control of water hyacinth and others clearly indicate the biopotential of fungal pathogens in the biological control of weeds. A mycoherbicide is a formulation of fungal pathogens that kills weed plants by causing disease at a lethal or threshold level. Thermophilic fungi have been playing key role in nature’s economy ever since they were recognized. Thermophiles have ability to degrade organic matter acting as biodeteriorators thus elaborating extracellular and intracellular enzymes, amino acids, antibiotics, phenolics, polysaccharides and sterols. They are also involved in the production of nutritionally enriched feeds and single cell protein besides their involvement in genetic manipulations. Fungi like Thermomyces, Sporotrichum and Torula are found to produce amylases. A number of thermophiles viz: Acremonium cellulophilum, Aspergillus fumigatus, Chaetomium thermophile, Thermoascus aurantiacus and others were reported to degrade cellulose and produce extracellular enzymes. Xylanases, lipases and proteases were also found to be elaborated by thermophilic fungi. Bioactive compounds such as antibiotics including myriocin are synthesized using fungi. Important cellulolytic and lignolytic enzyme producing thermophilic fungi like Chaetomium cellulolyticum and Sporotrichum pulverulentum are the most commonly used thermophiles for upgradation of animal feed and to produce single cell protein from lignocellulosic wastes. The disposal of municipal wastes, refuse and industrial effluents, has become a serious problem and bad odour emitted is of serious concern. Fungi like Phanerochaete, Mucor, Sporotrichum and others have showed their abilities in the treatment. Plant disease epidemics have become important as they cause huge losses thus dwindling country’s economy. In order to make agriculture sustainable, it is desirable that we should resort to integrated disease management (IDM) which would help not only in sustainability but also in preserving the ecological balance and protection of environment. Many unheated buildings of historic and artistic value with wall paintings such as old churches and caves, provide favourable conditions for the growth of fungi. Deterioration of wall paintings by fungi is largely promoted by the dust which covers the paintings. Species of Penicillium, Aspergillus, Cladosporium, Aureobasidium, Alternaria and Phoma are some of the fungal genera reported. Nearly 25 fungal species have been reported as responsible agents of deterioration of paintings in Ajanta caves. Therefore it is recommended using a low molecular polymer biocide at a lower concentration to prevent fungal attack. Study of degradation of buildings and

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archaeological timber has revealed the association of Serpula, Coniophora, Asterostroma and other fungi. Fungal Products Biotechnology is the application of living organisms and their components to industrial products and processes is not an industry in itself, but an important technology that will have large impact on many different industrial sectors in future. Traditional products include bread, beer, cheese and wine. In textile the enzymatic removal of starch sizes from woven fabrics has been in use for most of this century and the fermentation vat is probably the oldest known dyeing process. The new millennium biotechnology has seen very rapid development in genetic manipulation. It has the possibility of tailoring organisms in order to optimize the production of established or novel metabolites of commercial importance and of transferring genetic material from one organism to another. Biotechnology also offers the potential for new industrial processes that require less energy and are based on renewable raw materials. Today pure strains of yeast are normally employed and 1.5 million tons of baker’s yeast is produced worldwide every year. Cultivation of edible mushrooms in outdoor has been practiced for hundreds of years. Even today only a handful of species are grown commercially on a large scale although these represent a multibillion dollar industry. A recent innovation in food technology has been the development of Quorn, a mycoprotein from a filamentous fungus, Fusarium venenatum. The filamentous nature of the biomass is responsible for meat like texture and appearance of the final product, probably the most thoroughly tested food even to appear on supermarket shelves. Annual sales of Quorn are now in excess of £15 million in the U.K. Many important industrial products are now produced from fungi using fermentation technology. A wide range of enzymes are excreted by fungi and play an important role in the breakdown of organic materials and many of these enzymes are now produced commercially. Most of them are used in food processing. It was recently discovered that cellulase enzymes could replace the pumice stones used by industry to produce stone-washed denim garments. Another novel textile application for cellulase enzymes is in bio-polishing, the removal of cellulosic fibres which eliminates pilling making the fabrics smoother and cleaner looking. Cyclosporins were first isolated from Tolypocladium inflatum in 1976, as anti-fungal compounds and later shown to possess immunosuppressive activity. Cyclosporin A is currently the most widely used drug. Chitin and its derivative chiotsan have a wide range of industrial applications. These biopolymers are currently obtained commercially by treatment of prawn-crab shell wastes. Filamentous fungi grown under controlled conditions are an attractive source of chitin and chitiosan where a high quality product is required (cosmetic, medical, pharmaceutical application). Many fungi produce pigments during their growth which are substantive as indicated by the permanent staining and that is often associated with mildew growth on textiles and plastics. Some fungal pigments have been shown to be anthroquinone derivatives, resembling the important group of vat dyes. A recent report also indicates that lignin degrading fungi can even degrade synthetic textile polymers such as nylon previously thought to be non-biodegradable.

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It is a known fact that many modern drugs are the purified form of chemicals present in traditional medicines and only a few fungi have been used as traditional medicines. The caterpillar fungus (Cordyceps sinensis) is a traditional medicine that has been widely used as a tonic or medicine by the Chinese for hundreds of years. The use of this fungus was relatively unknown in this country until it was credited for the success of Chinese women athletes at the National games in Beijing, in 1993. The success was attributed to intensive training as well as a stress relieving tonic prepared from the caterpillar fungus. Scientists have already identified several compounds and some isolates that might be useful when purified as drugs to combat some types of cancer and lymphocytes. One commercial product of Trichoderma which is marketed is ‘BINA T — SEEPIC’, can be easily applied to the soil in the form of granules for biological control of soil-borne diseases. Biological control of weeds with plant pathogen has come to be recognized in recent years as safe and practical means of weed management. Presently there are many mycoherbicides registered, labeled and sold each year in the United States. The first ‘DeVine’, a formulation of Phytophthora palmivora was registered in 1981 for the control of milkweed and is marketed by Abbott Laboratories, Chicago, USA. ‘COLLEGO’, a formulation of Colletotirchum gloeosporioides used for the control of northern joint vetch in rice and soybean field. ‘CASST’, a formulation of Alternaria cassiae was developed for the control of Cassia obtusifolia. ‘BIOMA’, a formulation of Colletotrichum gloeosporioides f. sp. malvae for the control of Malva pusilla and Cercospora rodmanii formulation for the control of water hyacinth are in the way of evaluation for the control of above said weeds. Entomogenous fungi have become important in controlling several insect pests causing huge losses in crop and forest plants. Metarhizium anisopliae, which is a common pathogen of soil dwelling and pasture insects, has been produced on large scale as ‘Metaquinol’ in Brazil and is used against leaf hoppers. Hirsutella thompsonii infects only mites in tropical or subtropical regions and is marketed in USA as ‘Mycar’. Verticillium lecanii attacks aphids and scale insects in tropics and subtropics and was registered for use in UK as ‘Vertelac’ and ‘Mycota’ for the control of aphids and glass house whitefly, respectively. The above mycological documentation sufficiently convinces that the genetic diversity of fungi is vast by design and apparently crucial for life to continue. Saprophytic and parasitic fungi help in creating the organic components of the top soil, in alliance with myriad number of bacteria, insects and other organisms. A number of primary, secondary and tertiary saprobic fungi render wood into biodynamic soil components. These soils benefit plants that in turn use photosynthesis to manufacture their own food. Fungi can selectively raise or lower soil pH, increasing the usability of existing soil without the need for additional adjustment prior to use. Composting of organic matter through mushroom cultivation is a significant tool for the restoration, replenishment and remediation of earths over burdened ecosphere. Modern technologies for clearing and developing land for human use destroy mycorrhizal fungi, reducing ability of plants to thrive in manmade environments and forcing us to resort to fertilizer and other artificial means of promoting plant growth. It is now possible to reintroduce mycorrhizal fungi to the soil, replenishing and re-vitalizing it in an effective, safe and 100% natural way. ‘Mycogrow’ is the product of mycorrhizal fungi. It is designed for everyone from the

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home gardener / landscaper to the professional forestry manager, promoting faster growth, speeding transplant recovery and reducing the need for fertilizers and additives. A mixture of 12 species of ecto- and endomycorrhizal fungi has been developed into ‘Plant Revolution’ and ‘Plant Success’ tablets promoting fast plant and root growth, increased nutrient and water uptake besides reducing the fertilizer use. Many more fungi which are hidden underneath the ecological niches need to be surveyed, cultured, studied, classified, conserved and utilized for human welfare. Fungi are cosmopolitan, ubiquitous, beautiful, economically useful, used for human welfare, micro-, macroscopic and are the natural scavengers, hence deserve promotion. Our dedicated approach is essential in studying them, classifying them, growing and multiplying them.

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Chapter - 14

Aeromycology

Fungal spores form an important constituent of bioaerosol. Fungi are often well adapted to airborne dispersal of their spores having either tall conidiophores that penetrate into or through the laminar boundary layer or specialized liberation mechanisms that forcibly eject spores through this layer. In the course of evolution the fungi have probably exploited the wind for their dispersal more thoroughly than any other group of organisms and consequently dominate the airspora (80 - 90%). India has the unique distinction of being one of the earliest countries where aerobiological studies in general and aeromycological studies in particular were initiated (Cunningham 1873). Environmental mycology or aeromycology constitutes one of the major aspects mainly because of the dominance of fungal spores in the ambient air, which has naturally resulted in the accumulation of voluminous literature on aeromycology. However, in general aeromycological investigations take into account the identification of source, mode of release, dispersal, deposition, impaction and effects of impaction on the various living systems. The fungal spores and hyphal fragments are commonly recorded in the air and are important for the survival and subsequent continuation of generations. Many of the fungal spores are endowed with unique structures and capacity to survive under unfavourable environmental conditions and this probably accounts for their predominance in the air. Nearly all the spores are essentially dispersive units and their significance as gene dispersal units should not be lost sight of. The modern trend also takes into consideration the effect of pollutants by gases on airborne spores. Studies of such interactions constitute aeroecology. The spores of the fungi are major components responsible for allergic disorders since the spores are inhaled and deposited on sensitive mucosa. The spores of Mucor and related genera contain glucan, polyglucosamine (chitin) and melanin, 1.3% sporopollenin the same component is found in zygospores of Mucor mucedo and also in pollen walls. Cunningham’s (1873) air monitoring work over the Presidency jails of Calcutta forms the pioneer work in India. Harz deserves the credit for demonstration of viable microbial forms in the upper air at an altitude of 1500 - 2300 m during ballon ascent and identified spores of Pericona, Penicillium and Cladosporium. In the earlier period air monitoring was carried out by exposure of sticky coated slides on kites or ballons which clearly revealed the presence of variety of

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fungal spores. The use of volumetric sampler in recent years provides both qualitative and quantitative data which is of great value in correct interpretation of results and comparison with other data from other regions. The fungal propagules represent about 80 to 90% forms in the airspora mainly because of the wind dissemination and a variety of mechanisms developed by the fungi in efficiently liberating and dispersing the reproductive propagules. Such liberated spores get into air and subsequently transported. The flight route of fungal spores is mainly determined by wind speed and direction, and in the process turbulence gets funneled with distance. Wind transport involves the upward air currents, velocity and downward movement of wind. All are equally responsible for transport of fungal spores. In aeromycological studies it is usually observed that with the onset of rains there is a temporary increase in the dry spore fungi in airspora, however, later on a marked decrease of the spore content of air occurs due to ‘washing off’ by rains. In most of the dematiaceous fungi quite a large number of examples can be cited in which drying leads to deflations of the cells of conidium bearing structure e.g. Zygosporium and Deightoniella. Blowing off by wind with low humidity occurs in Trichothecium roseum. Among important contributors to airspora the spores of Fomes, Ganoderma, and Rhizopus are prominent during dry weather conditions. In the case of Botrytis, spores are abundant in air during day-time periods of low relative humidity; however, a heavy night time shower may also result in rapid built up of spore concentration. Rain splash is a significant mechanism for release of spores and some spores with such device do contribute to airspora. In the majority of Ascomycotina (with ascus bursting mechanism) and basidiomycotina (by drop - exertion mechanism) spores are violently discharged for subsequent transport through air currents. In general, for spore liberation in fungi, turgidity of cells is essential. Dew has a little effect on spore discharge but a light rainfall of even 0.2mm leads to abundant release of ascospores. The effect of rain fall on release of ascospores might be immediate or delayed. It is only in recent years aeromycologists have paid attention to the problem of deposition and here again it has revealed that spore size is highly significant. The principle mechanism is sedimentation under the influence of gravity impaction, including turbulent deposition and rain wash out. It must be remembered here that turbulent desposition would deposit as many spores on lower side as on the upper surface of horizontal slide exposed a foot or so above ground level. Experimental aeromycological work of Gregory (1959) using wind tunnel has already indicated that the efficiency of impaction directly increases with spore size and wind velocity. Gregory reported that very small spores of giant puffball, Calvatia gigantea, were not impacted at all on any of the cylinders with different wind velocities while large spores of club moss Lycopodium were freely deposited. Considering the efficiency of impaction, Gregory recognized certain spores as impactors. He pointed out that spores of many stem and leaf parasitic fungi, such as conidia of Helminthosporium being relatively large are good impactors. On the other hand relatively small sized spores of Penicillium, Aspergillus, Mucor and Trichoderma are poor impactors and these are normally brought to earth by rain wash/splash.

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It is assumed that, form of the spore is of great significance in relation to deposition but there is no experimental evidence of the deposition of elongated spores compared with the spherical ones as far as the airspora is concerned. It is a common observation that great many airborne fungi have more or less spherical spores with about 10 µm diameter. Gregory (1959) has developed a formula to describe dispersal in mathematical terms based on Sutton’s theory of eddy diffusion without consideration of spore size and rate of fall. Scrotter (1966) criticizes the neglect of rate of fall as a factor in aerial dispersion and considered it to be an important factor in determining the range of spore flight. Evidence on this matter has been presented by Sreeramulu and Ramlingam (1961) which indicate that spore size is an important factor in dispersal. It appears that natural selection has tended to limit the size of airborne spores. Aeromycological investigations have brought out some salient features of airborne spores. The predominant forms of airspora have significant relevance to numbers, shape, size and morphology and these are produced on large scale in fungi for example in Aspergillus, Cladosporium, Mucor, Penicillium, Phoma, Thielaviopsis and smut spores. These small sized spores have tendency to form groups or cluster which form a single dispersal unit which essentially is an adaptive and advantageous feature. Among the large size spores like, obclavate or muriform conidia of Alternaria, Helminthosporium, Curvularia and Cercospora have lower cellular contents as compared to their volume in addition to the presence of empty spaces in spores, which also helps to become airborne and float for long time. Bhati and Gaur (1979) have stressed the importance of structural aspects in airborne spores. Among the typical shapes of spores, obclavate or muriform Alternaria; cylindrical or polymorphous in Cladosporium; central cell being large in Curvularia, Spondylocladiella; fusiform or falcate in Fusarium; barrel shape in Geotrichum; elliptical or curved in Rhizoctonia and multiradiate with appendages in Tetraploa are some of the adaptive features which provide buoyancy to float in air for longer periods. Spore trapping in the immediate vicinity of the source has revealed a number of periodic patterns of the recurrence of fungus spores in the air and many of which are related to diurnal rhythm of alternating day and night while others are related to water relations of reproductive structures and some reflect changes in the climatic cycles. Sreeramulu (1967) recognized five distinct types of circardian periodicity patterns for fungal spores clearly signifying the peak concentration of spores in air and its relevance to time of day or night. Tilak, an eminent aerobiologist has worked extensively on outdoor and indoor mycoflora and on other aerobiological problems. The spores or fungal propagules are of a variety of forms. All these forms of spores and conidia range from 3-200 µm, most of these are about 10 µm in diameter. The spores are often liberated in the air in massive concentration and remain in the air for a long time. The major component of airspora is the fungal spores. Studies on aeromycology are of much importance in: 1.

Disease forecasting in crops and initiation of remedial measures.

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2.

Evaluating the damage and nature of infection or contamination of paintings, sculptures, idols, monuments and other related cultural heritage structures.

3.

Identification of aero-allergens and suggesting remedial measures.

4.

Understanding the air-borne bio deteriorating agents.

5.

Air-borne fungi serve as bio-indicators of pollution.

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Chapter - 15

General Account of Plant Diseases Caused by Fungi and their Control

Disease in plants may be defined as ‘a structural abnormality, or a physiological disorder that is sufficiently permanent and affects the entire plant or part of it, reducing the yield and causing economic loss’. Even though plant diseases are known since prehistoric time, the microbial cause of plant disease was discovered only during 1850’s. Among the microbial plant pathogens, fungi are the most important group. Fungal pathogens cause various types of plant diseases. Fungal diseases of crop plants often occur in epidemic proportions resulting in severe yield losses running into thousands of crores of rupees every year. Complete devastation of food crops by fungal pathogens resulted in wide spread famines. Among these, the great Irish famine of 1845-46 is very important one, and it was owing to the failure of potato crop due to ‘late blight’ disease caused by Phytophthora infestans. During this famine an estimated 1.5 million people died of starvation. In 1942, a wide spread famine occurred in Bengal province of India. The Bengal famine was due to failure of rice crop because of brown leaf spot leading to economic crisis. During 1870’s coffee plantations in Srilanka were completely devastated by rust disease caused by Hemileia vastatrix, resulting in abandoning the coffee plantations entirely. It lead to collapse of the economy of that small Island country, because it was the major commercial crop of the country at that time. Some very important fungal diseases of a few crop plants are given in the Table 15.1. Table 15.1. Important fungal diseases of some crop plants Crop

Disease

Pathogenic fungus

Wheat

Black stem rust Yellow stem rust Orange leaf rust Smut disease Powdery mildew Blast Brown leaf spot

Puccinia graminis-tritici P. striformis P. recondita Ustilago tritici Erysiphe graminis tritici Pyricularia oryzae Fusarium oxysporum Helminthosporium oryzae

Rice

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Crop

Disease

Pathogenic fungus

Jowar Pearl millet (Bajra) Cotton Groundnut

Grain smut Green ear Wilt Tikka leaf spot

Sugarcane

Red rot Whip smut Dieback and fruit rot Club root White rust Downy mildew Powdery mildew Early blight Late blight

Sphacelotheca sorghi Sclerospora graminicola Fusarium oxysporum Cercospora arachidicola Cercosporidium personatum Colletotrichum falcatum Ustilago scitaminea Colletotrichum capsici Plasmodiophora brassicae Albugo candida Plasmopara viticola Uncinula necator Alternaria solani Phytophthora infestans

Chillies Cabbage and Cauliflower Mustard Grapes Potato

Fungi attack all parts of the plant. Among the diseases of above ground parts of the plant are leaf spots, blights, rust, smuts, powdery mildews, downy mildews etc., and often causing epidemic outbreaks. Among the diseases that attack root system are damping off, root rots, wilts etc. A brief account of some important fungal diseases of crop plants is given below. SOME IMPORTANT FUNGAL DISEASES OF CROP PLANTS 1. Leaf spot diseases Leaf spots are the most conspicuous of all plant diseases. Spots on leaves develop due to limited necrosis (death) of host tissue because of infection by a pathogen. The spots vary from very small to large in size, round to irregular in outline, brown, black, red or of various colours. But the characters of spots are usually well defined for a host-pathogen combination. They appear prominently because the necrotic spots contrast with the lush green colour of the leaves. The leaf spots are caused by a number of fungi including species of Alternaria, Cercospora, Septoria, Colletotrichum, Helminthosporium, Drechslera, Phoma, Ascochyta, Mryrothecium etc. Leaf spot disease is very severe on groundnut, wherever the crop is grown. In India groundnut-leaf spot disease is called tikka disease. It is caused by two pathogens viz. Cercospora arachidicola and Cercosporidium personatum. The spots are dark, round to irregular and appear when the crop is about six weeks old. It gradually increases in severity causing severe leaf fall towards the end of the crop season. During epidemics the losses due to the disease maybe more than 50%. 2. Blight disease The diseases in which the infected region of the plant gives burnt-appearance are called blights. The pathogen once entered the host, spreads very rapidly and kills

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the entire plant in a very short period under favorable conditions. Late blight disease of potato is the best example for blight disease. It is caused by Phytophthora infestans. The disease appears in the field at the time of flowering, first on lower leaves and spreads very rapidly under favourable conditions. In severe conditions the yield loss is almost 100%. 3. Blast disease Sudden death of floral parts giving burnt appearance is described as blast. The blast disease is very common on many grasses and on rice crop and it is the most important fungal disease. Rice blast is caused by Pyricularia oryzae (Magnaporthe grisea). It attacks the crop at all stages of growth. In seed beds the disease appears as reddish or bluish flecks on leaves. In the rapidly growing crop the disease appears as characteristic acute spindle shaped leaf spots, internodes are attacked during rapid tillering stage, the neck of the panicle is attacked during inflorescence emerging stage. In severe cases, the yield losses may be upto 50-75%. 4. Rust disease Rust disease is caused by the members of the order Uredinales of Teliomycetes. There are about 4000 species of rust fungi distributed over 100 genera, and all of them are ecologically obligate pathogens showing high degree of host specificity. They attack both Angiosperms and Gymnosperms. Black stem rust of wheat caused by Puccinia graminis tritici is economically the most important disease, causing great yield loss in wheat, the most important cereal crop of western world. Hence, it is one of the most thoroughly and extensively studied plant diseases. Other important rust diseases include Yellow stripe rust of wheat caused by Puccinia striformis, orange leaf rust caused by Puccinia recondita, bean rust caused by Uromyces appendiculatus, coffee rust caused by Hemileia vastatrix, and linseed rust caused by Melampsora lint etc. The rust disease spreads by air borne urediniospores of the pathogen and the incubation period is relatively short. Hence, a number of disease cycles occur within a crop season, thus building up the epidemic very rapidly. The rust disease is described as ‘Compound interest’ disease. The yield losses are very high. 5. Smut diseases The fungi belonging to the order Ustilaginales of Teliomycetes are called smut fungi. They are are called smuts because infection results in formation of black, dusty, spore mass that resembles soot or smut. All smut organisms are plant pathogens and mostly attack floral parts, especially ovaries. There are about 1100 species of smut fungi that attack angiosperm hosts distributed in over 75 families of flowering plants. Important smut diseases of crop plants include stinking smut of wheat caused by Tilletia caries, whip smut of sugar cane caused by Ustilago scitaminea, loose smut of oats caused by Ustilago avenae. Sorghum vulgare, often described as great millet, is the major food crop of semi-arid tropics. It is attacked by a number of smut diseases like grain smut, loose

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smut, long smut, head smut etc. The grain smut is caused by Sphacelotheca sorghi and is the most important one. It infects the crop during seed germination and disease appears when the inflorescence comes out. Instead of seeds, the smut balls are formed in the panicle. From outside the seeds appear normal but inside are filled with smut spore mass. In the systemically infected plants, almost all grains are transformed into smut balls; hence, the yield loss is very high. 6. Powdery mildews Powdery mildew disease is caused by the members of Erysiphaceae, all of which are essentially, obligate ectoparasites on host plants. The pathogen produces enormous number of hyaline conidia on host surface giving the appearance of white powder on the infected surface, hence the name powdery mildew. Mildew means fungus. Powdery mildews are recorded on more than 8000 host species spread over 149 families of angiosperms. Powdery mildew of wheat caused by Erysiphe graminis var. tritici is a serious disease reducing yield. Powdery mildew is a serious disease on various crops including on peas, cucurbits etc. 7. Downy mildews Downy mildew disease is caused by the members of the family Peronosporaceae of Oomycetes. The disease is characterized by the appearance of a large number of sporangiophores and sporangia on the lower surface of the leaves in the infected region, hence the name downy mildew. The sporangiophores entangle together and appear as brownish or purplish patches of cottony growth. Downy mildew fungi include species of Peronospora, Pseudoperonospora, Plasmopara, Sclerospora and Peronosclerospora. Downy mildew is a serious disease on grapes, onions, cucurbits, sorghum, maize etc. The affected plants are generally weak and show stunted growth. 8. Anthracnoses The diseases caused by Colletotrichum species are generally called anthracnoses. Red rot of sugarcane caused by Colletotrichum falcatum is one of the most important plant diseases. The pathogen grows in the ground tissue of the stem and from disintegrating cells red gummy substance oozes out. It is absorbed by the cell wall and surrounding tissue appears red, hence the named red rot. The infected stems become hollow and sucrose is transformed into glucose. Hence, sugar cannot be prepared from the juice of infected stems. Diseases caused by Colletotrichum species are also important on chillies, turmeric, banana, cotton etc. 9. Damping off Disease It is essentially a disease of germinating seeds and seedlings. When the seeds are germinating, emerging radical is attacked by the pathogen, and seedling never comes above the soil surface. It mostly goes unnoticed in the field and farmers attribute it to the poor quality of seed. When the seedling comes out of the soil, the pathogen may attack the hypocotyl region of the young seedling and cause rotting of the tender tissue at the infected part. As a result the seedling collapses. This is called

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post emergence damping off. The disease is severe in the first week of seedling growth, and the seedling becomes gradually resistant, as the mechanical tissues develop. Damping off disease is mainly caused by species of Pythium, Rhizoctonia and other soil borne fungi. It is present in seed beds with poor drainage. 10. Wilt disease It is a disease of vascular tissue of the plant. The pathogen mainly grows in the xylem vessels and impairs the water relations of the plant. The infected plants fail to uptake and translocate water, resulting in wilting. The leaves are first affected. They lose their turgidity, become flaccid, droop and then drop off. Among the pathogens that cause wilt, the species of Fusarium, especially F. oxysporum is very important. Wilt is a serious disease on various crop plants like cotton, pigeon pea, linseed, banana etc. Before the introduction of exotic, long stapled cottons, cotton wilt was considered as a very serious disease of indigenous cottons in India. In India the pathogen is Fusarium oxysporum f.sp. vasinfectum. Once the pathogen is established in the field, it is very difficult to control it, because it can grow in soil saprophytically in the absence of host crop. 11. Root Rots Wilt symptoms may also appear in plants suffering from rotting of roots but the root rots essentially differ from the specific areas infected. In root rots the pathogens infect and cause rotting of cortex and medulla of root tissue, but never enter the vascular system. The severity of symptoms in root rots depends on the extent of rotting. Root rots are caused by a number of pathogens belonging to the genera such as Pythium, Phytophthora, Rhizoctonia, Sclerotium, Phymatotrichum etc. 12. Abnormal growths Fungal infections of plants may sometime cause abnormal growths due to hypertrophy and hyperplasia of the infected tissues. For example, Plasmodiophora brassicae causes club root disease of cruciferous vegetable crops such as cabbage, cauliflower etc. Infection leads to the increase of growth factors like IAA content and it causes hypertrophy of the tissue. The spindle shaped growth results as IAA content gradually decreases on either side of infected area. Multiple infections lead to irregular swelling and loss of function. Curling of leaves and other infected parts of peaches is a very serious disease and the pathogen is Taphrina deformans. CONTROL OF PLANT DISEASES CAUSED BY FUNGI For control of plant diseases caused by fungi, a number of methods are employed. They include exclusion (quarantine), eradication of primary inoculums, sanitation of the field, use of fungicides and disease resistant varieties. 1. Plant Quarantine: It is a legal restriction on the movement of diseased plants or plant parts, to prevent entry of the pathogens into an area where those pathogens are not known to be present. Such restrictions may operate between

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countries or even between states within the country to implement such restriction, governments establish plant quarantine stations at all international airports, seaports and land ports. Plant materials can be neither exported nor imported without a certificate from quarantine department. 2 Eradication: To prevent the pathogen entry into the field, various methods are employed depending upon the nature of primary inoculum for the disease. For control of wheat rust, eradication of Berberis vulgaris bushes was taken up in European countries, because Berberis is the alternate host for the pathogen and provides primary inoculum to the wheat crop infection. Collateral hosts and volunteer plants are eradicated before the main crop is sown, to prevent the inoculum coming from such sources. To prevent the seed-borne inoculum from reaching the field, seed treatment is employed. For removal of externally seed-borne inoculum, the seeds are treated with mercuric preparations like Ceresan, Agrosan etc. It is called seed disinfestation. The seeds are treated with systemic fungicides and the process is called seed disinfection. To protect the seeds and young seedlings from attack of soil borne pathogens, the seeds are coated with fungicides such as Thiram, Captan etc, and such a process is called seed protection. 3. Sanitation: Maintenance of healthy crop fields is called field sanitation. Various methods are employed for maintaining field sanitation area which include a) removal of diseased plant debris and its burning, b) ploughing to bury the fallen diseased leaves, twigs etc in the soil, c) hot weather, deep ploughing, d) use of chemicals to disinfect fallen plant debris, e) regular weeding in and around the fields. These measures reduce the inoculum density in the field. 4. Cultural practices: Various cultivational practices for reducing the disease severity include selection of the field, use of organic manure, correction of nutrient deficiencies in the soil, use of early maturing varieties, adjustment of sowing dates, mixed cropping, crop rotation etc. Crop rotation is the most suggested method for control of soil-borne diseases. In this method, main crop is replaced with an entirely different crop which is immune to the soil-borne pathogen attacking main crop. Cereal-legume rotation is considered as an ideal method. Mixed cropping is useful in reducing the severity of airborne diseases of the main crop. The crop used for mixed cropping should be taller than the main crop so that it reduces free dispersal of air-borne pathogens attacking the main crop. 5. Chemical control: Chemicals have been used as fungicides for centuries, but the first fungicide of known chemical composition was bordeaux mixture developed in 1886 against downy mildew disease of grapes. It was the major fungicide against all major plant diseases for over 50 years. From 1940s onwards a large number of chemicals were developed for use as fungicides. The fungicides are essentially of two types, basing on their mode of action viz., protective fungicides and systemic fungicides. i). Protective fungicides: Protective fungicides are those that protect the host surface as long as they are in contact with the surface. They protect the areas covered

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by them. They usually are not taken up by the plant. Their duration of activity is relatively short and needs frequent application for control of plant diseases in the field. Compounds of copper, sulphur and mercury were first developed as protectant fungicides, and now a number of other compounds are also used as fungicides. a) Copper fungicides: Copper compounds like copper sulphate, copper oxychloride, copper carbonate, cuprous oxide etc. are used in preparation of a number of fungicides. They are used against a number of foliar diseases. b) Sulphur compounds: Both inorganic and organic sulphur compounds are used as fungicides. Among inorganic sulphur preparations, sulphur dust, lime sulphur, wettable sulphur etc. is important. Among organic sulphur compounds, Dithio-carbamates are extensively used. They include thiram, ziram, nabam, ferbam, zineb, maneb, mancozeb etc. They are most extensively used protectant fungicides against a number of foliar fungal diseases. c) Mercury fungicides: Commercial preparations of mercury compounds include ceresan, agrosan, panogen etc, and they are mainly used for seed treatment. d) Other fungicides: Other protectant fungicides include quinone compounds (ehioranil, dichlone etc), benzene compounds (brassicol, kitazinatc), heterocyclic compounds (captan, captafol etc), Organic compounds (brestan, du-ter). ii). Systemic fungicide: These are chemicals which when applied are taken up by the plant and get translocated to various parts of the plant. They persist in the tissues for a relatively long period to ward off infection by a parasite or destroy the already established infection. Oxathins are the first systemic compounds developed in 1966, and a large number of sytemic fungicides are available. These include cathoxin, oxycarboxin, benomyl, carbendazim, tridemolph, kitazin, hinosan. Carbendazim is marketed as bavistin, and it is a broad spectrum systemic fungicide effective against a large number of plant diseases. Benomyl is very effective against diseases caused by Cercospora species. Hinosan is the fungicide used against blast of rice. 6. Use of resistant varieties: The cultivated varieties of crop plants are usually susceptible to pathogens prevalent in that area. For development of resistance, it is important to find cultivars or their wild relatives with some stable resistance. It is done by three methods viz, selection, introduction and breeding. Selection involves collection and propagation of seeds from individual plants that survived a severe epidemic outbreak of a disease. Introduction involves import of exotic varieties of the crop from other countries. For example, the problem of wilt disease of cotton was almost solved by the use of imported long stapled cottons in place of indigenous short stapled cottons. Breeding for disease resistance is the process of crossing between susceptible cultivated varieties with a resistant wild relative. Use of resistant varieties is the best method for control of plant diseases. But it is a prolonged and never ending process because as the scientists develop resistant varieties through breeding, the pathogen breeds for virulence in nature.

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Chapter - 16

Endophytic Fungi – Some Glimpses

Biodiversity is the variability among living organisms from all the sources including terrestrial, marine and other aquatic ecosystems and ecological complexes of which they are part and this includes diversity in species, between species, ecosystems and genes. Biodiversity is dynamic, the genetic composition of species change over the time in response to natural and human induced selection pressure, the occurrence and relative abundance of species in ecological communities change as a result of ecological and physical factors. Endophytic fungi are those fungi that colorize internal tissues of plants. Pathogenic fungi are not included in this group. Endophytic fungi colonize the internal tissues from that of bryophytes to angiosperms including lichens. Endophytic fungi are symbiotic/mutualistic, beneficial non-mycorrhizal that partner with many plants from grasses to trees. The mycelial threads spread between cell walls but do not enter them. These fungi enhance plant growth, have ability to absorb nutrients while starving off parasites, predation from insects, herbivores and other fungi. These are not true saprophytes or parasites but are in a class of their own. Some endophytic fungi sporulate but many do not sporulate. Clavicipitaceae endophytes and Acremonium colonize many grasses while conifers have many fungi belonging to Ascomycota and Basidiomycota as endophytes. Palms get colonized by Pestalotiopsis, Glomerella, Phomopsis, Colletotrichum and other fungi. The endophytic fungi of mangroves mostly belong to Hyphomycetes. Most of the Angiosperms get colonized by Ascomycetes, Basidiomycetes and Hyphomycetes. Some workers have also considered arbuscular mycorrhizal (AM) fungi as naturally occurring endophytes in 80% of plants. Arbuscular mycorrhizal fungi represent an obligate symbiotic group distinct from the rest of the soil microbial biomass. Mycorrhizal symbiosis is a dynamic mechanism and the process affects the physiological/ biochemical aspects of the host. AM fungi are unique as they are partly inside and partly outside the root. The vesicles, arbuscules and hyphae are formed inside the root and do not encounter competition and antagonism from soil microbes to the host rhizosphere. Class I endophytes may enhance the ecophysiology status of plants to counter abiotic stress such as drought and heavy metal contamination. This group represents

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a small number of phylogenetically related clavicepitaceous fungi that are fastidious in culture and limited to some warm and cool season grasses. The benefits conferred by this group depend on host genotype, and environmental variables. Transmission is vertical and material transfer to offspring is through seed. Class II members belong to Ascomycotina and Basidiomycotina and offer stress tolerance to host plants. Some endophytes avoid stress through plant symbiosis; tolerate sea water levels of water salinity and also detrimental effects of temperature. These classes of endophytes so far examined have shown an increase in host shoot and/or root biomass due to induction of plant hormones besides conferring habitat adaption. The fungi that colonize plants do not activate host defenses. Class III endophytic fungi are distinguished on the basis of occurrence and horizontal transmission. Hosts include non-vascular/ vascular/ woody/ herbaceous angiosperms. Single plant may harbor scores of different fungi. These groups are hyperdiverse endophytes and are associated with leaves of tropical trees. Biomass production on inculcation of these fungi are very low and are rarely isolated from seeds. These fungi are implicated in disease resistance, herbicide deterrence and changes in abiotic stress. Class IV group of endophytic fungi are restricted to plant roots having melanized septa including inter-, intracellular hyphae and microsclerotia confined to non-mycorrhizal plants with distribution from antarctic, arctic, alpine, sub-alpine, temperate to tropical ecosystems. It has been reported in the literature that brown, black pigment bearing fungi are found associated with plant roots and are mostly sterile. Dark septate endophytes either belong to Ascomycota or Basidiomycota. Endophytic fungi associated with host tissue (Leaf, root, stem, petiole, floral parts etc.) are collected, treated with 70% alcohol or 0.5-3.5% sodium hypochlorite for one to two minutes, rinsed in sterile water many times and plated in sterile nutrient agar media or water agar medium. In some other method, the epidermis is removed and tissues are treated as described above and plated on agar media. After an incubation of 5 days, the fungi are observed and identified up to species level wherever possible using different manuals and monographs. However majority of the endophytic fungi are sterile. Molecular techniques like ITS, RAPD, RFLP, AFLP, PCR and Metagenomics are employed including biochemical characterization. Naming of new fungi is done as per Melbourne code (1st Jan 2013) and new taxa are registred in Mycobank. Endophytic fungi have abundant secondary metabolites. Taxol, the world’s first billion dollar drugs highly functionalized diterpenoid was isolated from endophytic fungus Taxomyces andreanae in 1993 from the inner bark of Taxus brevifolia. Taxol has been confirmed as an anticancer compound. Pestacin and isopestacin, the antioxidant compounds were isolated from Pestalotiopsis microspora, the endophyte of Terminalia morobensis. The secondary metabolites from the endophytic fungi include amides, amines, indole derivatives, pyrrolizidines steroids, terpenoids, diterpenes, isocoumarin, quinines, flavoniods, phenols, aliphatic compounds, ergot alkaloids, lasidiplodin, cyclosporine and others.

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Tropical trees are rich in endophytic fungi and diversity. A non-peptidal fungal metabolite was isolated from Pseudomassaria, an endophytic fungus associated with African rain forest near Kinshasa in the Democratic Republic of the Congo. This compound has got insulin-mimetic agent and unlike insulin, is not destroyed in the digestive tract and may be given generally. This may lead to new therapy for diabetics if the chemical is commercialized.

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Chapter - 17

Fungal Ecology

Ecology is the study of organisms in relation to their environment. The population of organisms may interact in different ways e.g. commensalism, protocooperation, mutualism, neutralism, ammensalism, parasitism and predation etc. In fungi mostly autecological studies have been done. Fungi are ubiquitous and occur wherever decaying organic matter is present. Fungi could be terrestrial or aquatic. Terrestrial fungi could be terricolous, humicolous, ruperstral, phoenicoid, coprophilous and fumicolous. Those growing on animals and human beings include biophagus, dermatophytes and keratinophiles. Fungi inhabiting plants and their parts offer more diversity eg. fruticolous, graminicolous, culmicolous, remicolous, ovaricolous, lignicolous, pyroxylophicolous, fungicolous, agaricolous and mangilicolous besides phylloplane, rhizoplane and mycorrhizal fungi. Population of fungi in the soil is extremely diverse and is influenced by a number of biotic and abiotic factors. Fungi are an integral part of the living world, so their activities have a direct bearing on human welfare e.g. cause of allergies in humans, incidence of epiphytotics. Ecological studies of soil fungi have helped in understanding etiology of several crop diseases and their importance in decomposition of organic matter thus in cycling of nutrients. Ecology in modern times has been variously defined. Daubenmire (1947) defined it as ‘study of reciprocal relations between organisms and their environment’. Phillipson (1966) considers it as ‘branch of science which deals with the relationship between living things and their physical environment together with all the other living organisms within it’. According to Misra (1978), it is a study of dynamic interaction of biotic and abiotic components of an ecosystem and generates newer information not contained in the individual subsystems. Ecology, therefore, studies not only the structure and function of the components but also the information that they generate from interactions. Krebs (1985) defined ecology as ‘the scientific study of the interactions that determine the distribution and abundance of organisms’. According to Dix and Webster (1995) ‘ecology can be defined as the study of organisms in relation to their environment’. However, it is significant also to define what aspect of their relationship is to he studied viz., distribution, relative abundance, amount of living biomass, activity, viability, survival, form in which the fungus is present, reproductive capacity, and competitiveness. Ecosystem has been elaborated as ‘an open dynamic system with input, output and feedback energy and matter’. Misra (1977) is of the opinion that this concept of

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ecosystem enables us to comprehend the structure and function of community environment complexes in terms of energy and nutrient requirements and their transformations. There is no limit to the size of an ecosystem except the one put by researchers for the sake of study. The whole biosphere can be regarded as an ecosystem with subsystems of oceanic, terrestrial and freshwater habitats and community or we can take small ponds and pools or even, growth of a small patch of lichens on a tree bark, as examples of ecosystem. Population of two species may interact in different ways. According to Odum (1971), it may take the shape of commensalism in which one population is benefitted but the other is not affected, or protocooperation, in which both populations benefit by the association but the relations are not obligatory, or mutualism in which the growth and survival of both populations are benefitted and neither can survive under natural conditions without the other. Then there may be neutralism in which neither population is affected by association with each other, ammensalism in which one population is affected and the other is not affected, competition - direct inhibition type in which both populations actively inhibit each other, competition - resource use type in which each population adversely affects the other in the struggle for resources in short supply. Parasitism and predation is in which one population adversely affects the other by different mode of attack but is nevertheless dependent on the other. A review of literature on fungal ecology (Lynch 1983; Frarikiand 1984; Waiting et al 2002) reveals that the study has been largely on fungus autoecology i.e. relation of individual to its environment, though studies on synecology (composition of communities and relation to their environments) have also been done in some instances. One of the adequate tools for such studies is the comparative survey of fungus populations in various habitats. In general, fungal population varies considerably from one ecological area to the other and that plant cover and amount of organic matter associated with some other factors has a marked influence on the distribution of various species. The techniques and parameters used for such studies vary with different habitats or variations in a particular habitat. In the study of microorganisms and their populations, Cooke (1984) rightly observed that in order to compare the amount of growth or the activity of the various groups of organisms involved in any dynamic population, a technique should be chosen on which to have one’s concept of the ecosystem and its activities determining the nature of the population, the types of organisms involved and their relative importance within the group, as well as between the groups. Fungi are ubiquitous organisms that occur abundantly wherever decaying organic matter is found. Since they are unable to manufacture their own food they have to depend on preformed organic matter These are grouped into ‘decomposers’ and ‘reducers’ as they reduce the products formed by the activities of both producers and consumers to the original elements from which producers change these elements into organic matter. The number of fungi is enormous. It is estimated that there may be as many as 1.5 million species (Hawksworth 1991). In some form or the other, fungi are present wherever they are searched for. There are air-borne fungi, water fungi, marine fungi,

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terrestrial fungi, soil fungi, sand-dune fungi and those living in wet places and marshes, etc. (Manoharachary et al 2005). Tropical fungi are essential to the survival of other organisms, like termites culturing mushrooms, gardens of leaf cutter ants (Fisher and Stradling 2002), moths and spiders camouflaged against lichens (Gilbert 2000). Diversity of fungi becomes apparent by a cursory look at the designations of fungi on that basis. Among the terrestrial, we have terricolous (living on the ground), humicolous (living in or on soil), ruperstral (living on walls or rocks), phoenicoid (living on burnt areas), pyrophilous (fireloving), anthracophilous (coal-loving), carbonicolous (coal-inhabiting, fire place fungi), coprophilous (living on dung) and fumicolous (living on animal droppings) fungi. Those found on animals and human beings include biophagus (living on another living organism), dermatophytes (living as parasite on skin, hair, nails. feathers) and keratinophiles (having affinity with keratin). Plants and their parts offer more diversity. There are fructicolous (living on shrubs), graminicolous (living on grasses), culmicolous (living on stem/culms of grasses), remicolous (living on branches), fruticolous (1iving on fruits), ovaricolous (living on ovaries), lignicolous (living in or on wood), pyroxylophilous (living on burnt wood), fungicolous (living on fungi), agaricolous (living on agarics) and manglicolous (living on mangroves) fungi. Then there are phylloplane, rhizosphere, rhizoplane and mycorrhizal fungi. In an environment, which is conducive to good growth, there is a wide scope for survival and development. If a fungus is able to maintain itself in a particular environment, it is thought to adapt itself to the prevailing conditions. If the environment becomes hostile, natural population will not survive and any variant or mutant or other species might compete and take their place. Why a particular species is dominant in a particular situation and not so in others can only be understood if we know about the physiological behaviour of the fungus. The characteristic life style of a fungus, one finds that it has so evolved that it is able to deal effectively with wide fluctuations in their environment due to exhaustion of food supply, variations in temperature and moisture. It has a vegetative mycelial phase during which asexual spores are produced in large numbers and in a very short time. These spores, under favourable conditions, are capable of immediate germination and colonise suitable substrates. Under unfavourable conditions, it takes up sexual mode of reproduction in which dormant, long lived, resistant type of spores are formed. Other forms of resting spores include chlamydospores and sclerotia from the vegetative mycelium. These forms enable the fungus to perennate. In the absence of organic matter, no fungi are known to grow and reproduce in air. Many fungi, however, are well adapted to wind dissemination for they produce and liberate innumerable tiny and light spores into the air. The dispersal of aerial spores in time and space involves liberation, transport and deposition on host surface. During the process of germination, they are exposed to various factors of the environment like air currents, desiccation, temperature and other solar radiations. Air currents disseminate them to long distances, and their effects on spore dispersal are complex and varied. A steady wind blowing strongly carries spores in a horizontal direction but breezes with irregular gusts blowing in different directions cause

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multidirectional dispersal and deposition. The spore load of specific areas is usually considered a ‘population’. Spore concentration in an area fluctuates according to meterological conditions such as temperature, humidity or according to seasons in particular. Several techniques have been developed for trapping the fungal spores in the air. These methods rely mostly on the fall of spores by gravity. Most of the studies deal with the list of species found together with the data about their abundance and rarity. Spore surface is of paramount significance in the ecology of fungi having aerial spores (Tewari 1984). The functional parameters of the spore surfaces are poorly understood and they need further studies to elucidate the interaction between surfaces of fungal spores and their host plants. The retention of deposited spores varies with the ultrastructure of the spore wall and with electrostatic and other features of the phyllosphere. Studies of fungal populations of the air and their behaviour are important in epiphytotics and allergic ailments in human beings. Aquatic fungi or water moulds as the name indicates have water as their medium of life and are adapted to this environment. They are found not only in pools, ponds, lakes, streams, rivers and marshes but in marginal and strictly humid terrestrial habitats (Manoharachary et al 2005). Some fungi have also been reported from thermal springs, logs, acidotrophic lakes, inland salt pools and lakes. They thrive for the most part in water but are able to spend part of their life cycle in soil also. Apart from living as saprobes these may parasitise aquatic plants and animals, mostly fishes and their eggs. Though most of these belong to mastigomycotina but certain hyphomycetes also live as saprophytes on the decaying leaves and submerged wood (Tsui et al 2003) in water. Their spores are well adapted to live in an aquatic environment. Ecological conditions in various bodies of water are different in a stream or river. The upper reaches may have leaves and other parts of overhanging trees or other vegetation, dead bodies of animals, humans and their feces as the organic substrate. As we go down the stream addition of drainage from agricultural lands, urban household sewage and industrial waste will be there. Therefore, there will be different types of organic enrichment at different points during its course. The fungal population at the source when carried downstream will be faced by other organisms adapted to the downstream environment and will have to compete with them, those which are able to adapt to new environment will survive, others will perish. Ponds and lakes have different ecological areas; water mass having saprophytic water moulds and parasites of phytoplanktons; submerged structures like living plants, branches, twigs, fruits, culms and leaves colonized by various groups of fungi and bottom mud’s which support only a limited type of fungus flora. Ecological studies of water moulds have been mostly descriptive and qualitative. Methods of study include collection of water samples and then baiting, baiting in natural waters, collection and incubation of organic substrates and dilution and plating on solid substrates. By using these methods the activities of microorganisms in any environment are apt to be over emphasized or underemphasized depending upon the technique used. Gessner et al (2003) very systematically did qualitative and quantitative analyses of aquatic hyphomycetes in streams. Diversity of hyphomycete conidia was assessed by microscopy and DGGE (Raviraja et al 2005).

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Whenever estimates of total populations have been made these deals with only one kind of organism and, no attempt has been made in which members of a total population of microorganisms i.e. fungi, bacteria and protozoa have been considered. And this is no simple matter and changes in the total organisms of the ecosystem of a stream, pond or lake are difficult to determine directly. Not very long ago, increasing interest has been shown in fungi which adapt themselves to marine habitat. Nutritional requirements of marine fungi together with the studies of effect of temperature, salinity and pH on their growth and distribution have been done. Exposed wooden and cordage material and algae have been found to furnish nutrients to marine fungi. Not much information is available on microbiological and biochemical changes which occur in submerged plant materials immersed in salt or brackish water. Mangrove swamps serve as ecotones between land and sea. Mangrove mud is the result of plant debris accumulated by the growing vegetation and soil brought by rivers. Water logging caused by the regular sea tides makes these soils swampy. Leaf, stem, and wood debris provide litter to the substrate and due to biodegradation of cellulosic material forming humus and biorefuse (of plants and animals), a series of complex habitats are formed. In addition to decaying material, mangrove trees themselves are fascinating, because bases of their trunks and pneumatophores are permanently or intermittently submerged while upper parts of roots and trunks are always above water. Consequently two different types of habitats are found on the same plant at the same time. Marine fungi are found on the lower parts and terrestrial fungi on the upper. Several workers have studied the dominance and successional pattern of marine fungi attacking submerged wood, colonization of wood and pneumatophore pieces. Adaptations for salinity tolerance have also been studied. A study of ecological factors prevalent in the mangrove swamps like salinity, temperature, organic matter, aerobic conditions and very high amount of moisture content indicate that fungi native to such swamps do develop certain degree of adaptation to these extreme conditions due to their prolonged effect (Garg et al 1984). Soil has five major components mineral matter, organic matter, water, air and a living population. Soil habitat has been studied more intensively than any other habitat. The organic layers, which include litter, the fermentation layer, duff and humus, on the surface of mineral soil are generally considered as a part of soil complex. Population of microorganisms in the soil is extremely diverse and a number of biotic or abiotic factors influence the existence and growth of fungi either singly or in combination. In a forest cover fungal population differs from substrate to substrate. Fungi present in the litter are different from those found on fruits and fruit parts, twigs, branches, stumps, rotting logs and decaying wood. Other fungi grow in fermentation layer, duff or humus layer of the litter, and still others in deeper layers. In meadows and prairies, fungi have been observed mostly on surface areas and there is a considerable difference between populations associated with grasslands and forests. Substrate in a farm soil is entirely different because of mulches, fertilizers, crop remains, weed trash and so is the fungus population.

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Depending on the habitat soil fungi are classified (Garrett 1956) into: 1) those living saprobically in soil where they develop only on dead organic matter; 2) weak plant pathogens living in the soil, capable of using soil organic matter but eveloping as occasion arises on young roots or senescent roots; 3) ecologically obligate plant pathogens including those species which can parasitize the actively growing roots of plants, produce disease like vascular wilts, and following the death of the host, utilize its tissues as nutrients until their activity is arrested by more actively competitive saprobic fungi; 4) obligate plant parasites such as rusts, powdery- and downy mildews which apparently require living plant material as substrate; and 5) symbionts like mycorrhiza. On the basis of their nutritional requirements fungi have been grouped as saprophytic sugar fungi, cellulose decomposing fungi, pectin and cutin decomposing fungi and lignin attacking fungi. Ecology of soil fungi has been studied from many aspects and the studies have led to the control of several diseases of plants. Rhizosphere is a specialized ecological niche where soil is subjected to specific influence by plant roots due to the exudates from root cells and sloughing off of root tissues. Rhizosphere represents a poorly defined zone of soil with a microbiological gradient in which maximum changes in the population of soil microflora occur in soil adjacent to roots and declining with increasing distance (Manoharachary and Mukerji 2006). Region of the external surface of plant root together with any closely adhering soil particles of soil or debris is called rhizoplane. Root exudates stimulate microbial activity selectively in rhizosphere and rhizoplane regions. There is an intense competitive activity by the obligate saprobes, unspecialized root parasites and rootinhabiting fungi depending on their behaviour towards exudates while in case of root diseases the pathogen has to react with the rhizosphere and rhizoplane fungi before entering the root system. These may show antagonism and check its advancement. Various factors and the mechanism responsible for fungistasis i.e. failure of spores to germinate in mineral soil have not yet been fully determined. Many fungi, which function as oligotrophs can grow at very low carbon concentrations in low nutrient flux environments, and produce very fine mycelial networks to felicitate substrate collection (Wainwright 1993). Fungal hyphae get associated with the roots of higher plants in a symbiotic, nonpathogenic relationship and form a compound structure known as mycorrhiza. There may be ectomycorrhiza, or an endomycorrhiza or an intermediate type ectendomycorrhiza. In the mycorrhizal formation the higher plant, the symbiotic fungus and the environmental conditions are closely linked together to each other. Mycorrhizas are formed by about 90% of the plant species. In many of these associations 10-30% of the food produced by the plant moves through to the fungi. The fungal mycelia are adept at extracting minerals especially phosphorus and nitrogen from the soil and pass these through to the plants. Mycorrhizal fungi may also protect the plant against pathogenic fungi and microorganisms. The process of mycorrhiza formation depends on many factors on behalf of the host as well on behalf of the fungal partner. These factors, on the other hand are regulated by the environment, such as soil conditions. Symbiosis in mycorrhiza is still not fully elucidated. The controversial points may be partially reconciled if more information is available on the composition and activity of accompanying population of

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microorganisms, day length and seasons, soil type and soil properties and treatments of plants before beginning of experiments. Brundrett (2004) gave diversity and classification of mycorrhizal associations. In endotrophic forms the host response may involve far more complex physiological activities. Soil fertility due to amendments with mineral fertilizers or organic matter affects the activity and survival of these forms. Interaction of endotrophic fungi with other microorganisms is also being studied. Endotrophic fungi are regarded as highly obligate symbionts, which cannot be cultured. Mycorrhiza mainly helps in phosphorus nutrition provided the soils are deficient in ‘P’. Usar soils are different from other soils in having high pH, high osmotic concentration, high temperature during summer coupled with high salt concentration and relatively high degrees of solar radiations. These soils have very low and negligible level of nutrition. The question is often asked, whether the fungi with an active life in these soils are natural inhabitants or it is an indication of ecological specialization. High pH growth optima and osmotic tolerance of strains of fungi growing on usar soils as compared to their fertile counterparts indicate their adaptation to these extreme ecological conditions. There is a rich variety of life in soil or debris. Close physical associations among different group of microorganisms have been conducive to the development of many predatory and parasitic associations. These predacious fungi not only attack adult soil nematodes and protozoa but nematode eggs also. Information about the basic knowledge of these fungi is inadequate. Coprophilous fungi occur normally on animal excreta and prefer this substratum for their growth and reproduction. They form a unique ecological group. The composition of excreta varies from animal to animal; therefore little generalization can be made. The fungus in different stages of its life cycle has to pass through different forms of environments. The spores of a fungus are ingested by an animal and have to face high temperature and unfavourable digestive juices in their gut. After being excreted, they germinate and grow in the dung and produce their fruit bodies. Spores of some of the coprophilous fungi from these fruit bodies are liberated with force and land in such a way that they get attached to some herbage to be ingested by grazing animals. Phenomenon of fungal succession on dung is explained on nutritional hyphothesis where Mucorales (sugar fungi) appear at early stages of succession. When the soluble sugars are exhausted, Ascomycetes and Deutereomycetes grow dominantly due to their cellulose decomposing ability and finally the dung is occupied by lignolytic basidiomycetes. Fungal flora of various dung types, excreta or droppings differ considerably, difference being attributed to different feeding habits of animals. Some like cattle and rabbits feed on herbs, some are carnivorous and others like mouse feed on grains and other foodstuffs, lizards on insects, birds on fruits and grains. The condition within the gut is also varied. The soil is considered to be a reservoir into which spores and other propagules of coprophilous fungi fall and active life is observed on the dung above soil. These

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fungi may grow in soil and successfully or unsuccessfully compete with the terrestrial fungi. Estimate of ascomycetous coprophilous fungi in terrestrial environments is handicapped because of lack of experimental methods. More research work is needed for accurate ecological estimates of the coprophilous fungi in soil. Phoenicoid fungi are able to grow on burnt areas or areas sterilized by intense heat. Fires stimulate the spore germination as well as stimulate the sclerotia of some species to produce fruiting bodies. Many of the microorganisms are killed by heating which would often compete with these fungi, or phoenicoid fungi are antagonistic to them. The phoenicoid fungi take advantage of this reduced competition to colonize burnt areas. The soil chemistry is also changed by fires. The ash temporarily increasing the alkalinity and many fungi respond primarily to increased alkalinity, rather than the fire itself. There are certain unexplained changes in the available nitrogen also. The literature on plant pathology is replete with data and information concerning plant disease fungi from various aspects. There are not only specialized studies on the physiology of host-parasite relations, physiological specialization of parasitic fungi, environmental factors related to epiphytotics, relation of weather to fungal diseases, manipulation of the soil for the control of diseases, decay of forest trees, effect of air currents on the transportation of airborne spores of fungi but attempts have also been made to apply synecological techniques to the study of plant disease fungi. More work is, however needed on the ecological studies on fungi in harvesting, storage and transportation of both perishable and non-perishable plant products. Seed is exceedingly dynamic as an ecosystem. It changes its habitat owing to its migration from one place to another i.e. from plant in the field to distributing or disseminating agencies or to storage and ultimately to the field soil. Once the seed is drilled in the soil, a new environment is set up and is controlled by seed activity and soil composition. Fungi in the spermosphere and in or around the seed respond to various microclimatic pressures such as temperature, moisture and also the structure and composition of seed and its physiological activity. These fungi play an important role in the germination and development of the seedling and also in the deterioration of the seed in storage. Another aspect of association of fungi with plants is the study of leaf surface microflora of plants as phylloplane/ phyllosphere microflora. Leaves of plants act as spore traps of varying efficiency and certain fungi show diurnal periodicity of release. Various parameters taken into consideration in phylloplane studies include microclimate, anatomical features of leaf such as wax, pubescence, epidermal contours and physiological variations between leaves of different plants and between leaves on the same plant, which may cause further variation. Leaf environment is not very favourable for the fungal colonists. There is considerable fluctuation in the relative humidity, temperature and exposure to ultraviolet radiation in the daylight, though the spores derive benefit from the nutrients diffused by the leaf, algae, pollen grains, and honeydew formed by the aphids on the leaf surface.

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In addition to fungi, pollen grains, bacteria, algae and lichens are present on the leaf surface and interaction between these different organisms and between the organism and green plant requires further study. Similarly, the studies of interactions, between both saprophytic and parasitic fungi with members of the leaf fauna are awaiting development. The role of phylloplane fungi vis-a-vis leaf pathogenic fungi needs further elucidation. Their association with the members of the leaf microfauna has to be fully explored and the methods of survival and UV radiations need to be fully elucidated. Human pathogenic fungi have been isolated from soils indicating that it may be a reservoir of these disease agents. It has been ascertained in certain cases that the epidemiology of disease is intimately connected with the ecology of fungi. Many of the dermatophytes show geographic specialization. Since the substrates in various parts of the human body where pathogenic fungi attack, are so different from those present in the soil, their ecological association as saprophytic fungi is very interesting. How far these pathogenic fungi act as effective members of the soil fungal populations is yet to be determined. It will entail the use of different techniques. Keratinophilic fungi generally grow on tissues of human and animal skins, hair, nails, hooves and feathers. Some of these fungi are capable of attacking native keratin by penetration and by enzyme activity and are represented by dermatophytes. The other ecological group is of saprophytic species which attack keratin by simple hyphal penetration and by surface growth utilizing the more easily decomposable compounds of keratin. Ecological habitats of these fungi are the soils from densely populated areas, public gardens, cemeteries, playgrounds, poultry farms, cattle sheds and other places with decomposed organic matter. More studies are needed for understanding the role of these fungi in nature and their nutritional requirements. Over the last decade the incidence of fungal diseases in humans has increased markedly. Upto 20% of the population is affected due to the infections of skin, hair, nail and mucous membranes (Evans and Ashbee 2002). Some fungal infections occur mainly in tropical or sub-tropical parts of the world and in general, people from temperate zone especially Europe have little immunity to them. Increased leisure and business travel to the exotic tropical locations have resulted in higher prevalence of unusual fungal diseases (Evans and Ashbee 2002). Population studies of fungi along with other microorganisms in a habitat are vital to the understanding of ecology and to accomplish this we cannot do better than to quote Cooke (1984) ‘Studies of the populations of each of these types of organisms indicate the almost complete separation of bacteriology, mycology, algology and protozoology when, approaching populations in the polluted environment’. Fungi are an essential part of the living world and so their activities have a direct bearing on human welfare. Fungal populations of the air are important to know the causes of allergies in human beings, incidence of epiphytotics of crops and forecasting of crop diseases leading to control measures. Detailed knowledge of degrading microorganisms and the substances attacked and different ecological conditions would make it possible to either increase the efficiency of degradation of

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certain cellulosic and lignicolous materials which are more resistant to attack by common fungi or to prevent their activities. Ecological studies of soil fungi have helped in understanding the etiology of several crop diseases and their importance in decomposition of plant remains and as agents for beneficial C and N cycles in soil. Mycorrhizal ecophysiology will be helpful in maximizing the productivity and stabilization of mixed communities. Saprobic ecological association involved in human diseases and reports of fungi present in human environment are good basic findings suggestive of a potential danger posed by these fungi. Soils differ in their physico-chemical composition. Altitude, vegetation, crop debris, gaseous phases, water holding capacity, relative humidity, rainfall and plant residues have their own role and implication in the distribution, seasonal variation, quantitative composition and qualitative account of fungi. The cultivated soils which receive chemical fertilizers and green manure always harbour heavy fungal population represented by Mucorales, Aspergilli, Fungi-imperfecti and few Ascomycetes. It is observed that the soils supporting crops have more number of fungi when compared with virgin/forest soils. However, the forest soils are represented by a wide variety of fungi and many a times new fungi are found associated with them. In tropical soils pH plays a decisive role. Acidic soils (pH 4.5 – 6.8) harbour more fungal species than neutral to alkaline soils (pH 7.0-8.5). Saline/mangrove soils harbour salt tolerant fungi, the fungal number and species being very less. Among the mineral salts, nitrates of ammonium, Potassium and Sodium are positively correlated. It is also generally observed that lesser the phosphorus more the diversity of fungi including arbuscular mycorrhizal fungi. Fungi-imperfecti dominates many of the soils of this category. The organic carbon is another important parameter which is correlated positively with the fungal numbers and specific composition of fungi. Higher the altitude, greater the vegetation, and the fungal species composition varies characteristically. Many studies have shown that vegetation influences the quantity and quality of fungal biomass and this is very much true for temperate and dry deciduous forests. Further, the fungal species composition is different in red sandy loam soils when compared with clay soils. From the point of vegetation it is observed that Aspergilli are more dominant in tropical dry deciduous forests and soils supporting crops, while Penicillia dominate in the temperate forest soils and soils supporting Eucalyptus plantations. Therefore, the soil physico-chemical factors along with vegetation and other parameters have greater impact on soil fungi. Soil is a dynamic ecosystem and maintains equilibrium in spite of having diverse physicochemical factors. As a result, most of the soils harbour the same fungal species for some years until and unless they get disturbed/ damaged through pollution and by other means.

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Chapter - 18

Fungal Genetics - General Account

Genetics is the science of heredity dealing with the resemblances and differences of related organisms, resulting from the interaction of their genes and the environment and also of the structure, function and transmission of genes. Fungal nuclei are small. The nuclear membrane is double layered and has many large gaps. During mitosis the nuclear membrane does not get separated. In mitosis intra-nuclear chromosomes do not get arranged systematically on to equatorial plate but are randomly distributed. Meiotic division is also intra nuclear. During meiosis the nucleolus and nuclear envelop degenerate by the end of prophase and gets distributed irregularly on spindles in metaphase. There are a number of compatible and alternate genetic systems in fungi. Heterokaryosis It is a situation that occurs in some fungi in which nuclei of different genetic constitution occur in the hyphal cell as a result of non-sexual cell fusion of two genetically different types of hyphae. In heterkaryotic condition hyphae and mycelium carry two different types of nuclei. Incompatibility is the situation in which mating is prevented between strains having different factors so that out breeding is prevented and inbreeding is encouraged. Heterokaryosis arises in the following ways: 1.

By the inclusion of non-identical nuclei in a single spore, e.g. Neurospora tetrasperma, Podospora anserina.

2.

By the fusion of vegetative cells.

3.

Occurrence of mutation in a homokaryon which is many nucleated.

4.

Haploid homokaryon fuses to form diploid nuclei which survive, multiply and disperse among haploids.

Heterokaryosis is common in fungi belonging to Ascomycotina, Basidiomycotina and Anamorphic fungi. Heterokaryosis plays an important role in many pathogenic fungi such as rusts, smuts and others. Homothallism It is the condition exemplified by the homothallic species. It is the condition of being self fertile, able to reproduce sexually without partner. Hyphae which are self

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compatible, in them sexual reproduction occurs in the same organism by meiosis and genetic recombination. It is the fusion of hyphac that results in dikaryotic or diploid condition. In this process nuclei of both mating types occur in the same haploid thallus. Homothallism is most common and occurs in all groups except Basidiomycetes. The dioecious fungi cannot be homathallic and exhibit no mating types. Homothallism is also found in zoosporic and Ascomycetous fungi. Homothallic fungi form two types of gametangia on the same thallus. Heterothallism Heterothallism is the condition of being self sterile. Male and female sex organs are developed in Ascobolus magnificus requiring partner for sexual reproduction. Hyphae are incompatible with each other and each requiring contact with another hypha or compatible mating type, hyphal fusion forms dikaryon or diploid structure. The term homothallism and heterothallism were first applied by Blakeslee (1904) to the method of zygospore formation in Mucorales. He considered these terms equivalent to monoecious and dioecious of higher plants. Heterothallism has been used as the equivalent of haplodioecism in the case of Dictyuchus monosporus. Whitehouse distinguished the first type as morphological, and second type as physiological, for haploid, incompatibility. The term physiological heterothallism is further determined either by two allelomorphs at one locus or multiple allelomorphs at one or two loci. The last one i.e. multiple allelomorphic physiological heterothallism is characteristic of Hymenomycetes and Gasteromycetes in which 35 percent are heterothallic and bipolar with one locus and 55% are heterothallic tetrapolar with two loci. The term relative was used by Pontecorvo in the formation of crossed asci in excess of 50% by the combination of certain homothallic strains of Aspergillus nidulans. Dia-phoromixis. In this there exist more than two types of nuclei, amongst which two are compatible. Bipolar Heterothallism This consists of two groups or mating types of individuals that differ in their genetic make up for their compatibility factor. This is also called unifactorial system in which compatibility is controlled by alleles of a single factor (gene). After meiosis a single spore carries one allele. As a result two spores are produced and each spore upon germination gives rise to a clone of uninucleate cells or a thallus with one nuclear type. These are represented by + or –, A and a, or a and A, A1 and A2. This has been explained in Neurospora, Mucor, Phycomyces and Ascobolus. Multiple factor heterothallism is controlled by multiple factors at each genetic locus Bipolar multiple allele factor heterothallism is controlled by multiple allele that occur at single genetic locus, instead of a pair of allele. Tetrapolar Heterothallism This is also called multiple factor heterothallism. In this compatibility is controlled by two unlinked mating type loci, A and B, are polymorphic and contain

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a, b as sub loci. This mating system is called tetrapolar. This is observed in Coprinus cinereus. Parasexuality It is a process in which plasmogamy, karyogamy and haplodization takes place not at specified points in the life cycle of some fungi like Aspergillus nidulans, Verticillium etc. It is a process occurring in filamentous fungi by which partial genetic recombination of hereditary properties occurs not by true sexual reproduction but through mitotic crossing over. It was discovered by Pontecorvo and Roper in 1952 in filamentous fungi. The important steps are 1. The production of diploid nuclei in heterokaryotic haploid mycelium. 2. The multiplication of diploid nuclei along with haploid nuclei in a heterokaryotic mycelium 3. The sorting out of diploid homokaryon. 4. Segregation and recombination by crossing over of mitosis. 5. Haploidisation of the diploid nuclei. The results are similar to those achieved by meiosis but instead of a regular sequence of events in time as in the meiotic cycle, the various steps occur at different times. It may not be accompanied by a sexual cycle. Pontecorvo and his group obtained data from the parasexual cycle for mapping the eight chromosomes of Aspergillus nidulans. The research of Beadle and Tatum (1945) on auxotrophic mutants in Neurospora led to the ‘one gene – one enzyme’ discovery hypothesis. This relationship turned out to be between genes and polypeptide chains or, one step further back, between genes and RNA transcripts. The analysis of simple gene-protein relationships was the demonstration of relation between the amino acid sequences of polypeptide chains and the coding sequences in the genes. Sherman and Stewart (1971) have shown way in their study of the effects of mutations in the Saccharomyces cerevisiae cycl gene on the amino acid sequence of cytochrome-c. Most cytochrome-negative mutations had the effect of converting single amino acid codons to chain terminators, and reversion of these mutants was due either to the restoration of the original amino acid or its replacement by a new one consistent with a single base-pair change in the DNA. It is established that gene clusters are involved in galactose utilization in budding yeasts and quinate utilization in Neurospora were the most extensively worked upon and analysis of closely linked mutations affecting the same area of metabolism revealed no single genes but clusters of functionally related genes encoding different enzymes in different messenger RNAs, but with common transcriptional regulation. This multistep biosynthetic pathway is dependent on several enzyme activities, capable of individual elimination by mutation, were in several cases shown to be under the control of single gene, sometimes called ‘clustergenes’ encoding single multifunctional polypeptide chains. One of the excitements in the late 1950s was the discovery of occasional complementation between allelic mutations, resulting in the lack of the same enzyme, but producing the enzyme activity when put together in heterokaryons (Neurospora) or diploids (Saccharomyces). Such Allelic complementation’s, hardly

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disturbed the ‘central dogma’, but it did call attention to the fallibility of the complementation test as a test for allelism. This is due to the piecing together of fragments of a single polypeptide chain, which demonstrated mainly in bacteria, or, where the enzyme normally consists of identical and symmetrically related subunits, to the formation of hybrid dimmers of oligomers. The earlier observations on auxotrophic mutants with simple nutritional requirements encouraged a rather simple view of the relationships between genes, proteins and phenotypes. When attention shifted away from presence or absence of enzyme activities to regulatory functions, things became very much more complicated. All proteins can be made more thermolabile by sequence changes, all kinds of protein encoding genes can be detected in this way. An outstanding example of the use of temperature-conditional mutants is the study of the genetic control of cell division – a subject of obvious interest in connection with cancer research. The work was started in S. cerevisiae by L. Hartwell in Seattle with the isolation of a host of temperature-sensitive mutants unable to divide properly at the restrictive temperature. The fungal contribution, from the yeasts in particular, remains of great importance, but it has become integrated with biochemistry / molecular biology as a whole. Meiotic recombination The importance of fungi for formal genetics is that all four products of a single meiotic cell can be recovered and characterized. Analysis of fungal meiotic products in tetrads permitted precise genetic confirmation of the features of meiotic recombination, reciprocal crossing over involving only one chromatid of each chromosome, random involvement of chromatids, and the postponement of the splitting of centromeres until the second division. In the budding yeast Saccharomyces cerevisiae Carl and Gertrude Lindegren found that the Mendelian expectation of 2:2 segregation of allelic differences held in most tetrads, a proportion of several percent exhibited 3:1 or 1:3 ratios, as if one copy of one allele had been converted to the others. Later in the 1950s the same phenomenon, at variable and usually lower frequency, was demonstrated in the form of 6:2 and 2:6 asci in the eight spored Ascomycetes Sordaria fimicola, Ascobolus stercorarius and Neurospora crassa. The analysis in the first two of these species was greatly facilitated by the availability of mutant alleles affecting ascospore colour and therefore scorable in undissected asci. Occasional octads, sometimes as numerous as the 6:2s and 2:6s, segregated 5:3 or 3:5 indicating half-chromatid conversion (single-strand transfer without mismatch correction). Mating type The heredity of mating type in the heterothallic fungi was always an inescapable topic in fungal genetics, since the gene differences determining it were necessarily segregating in every meiotic cell. It was known in the 1940s that heterothallic ascomycetes had two mating types determined by what appeared to be two alleles at a single locus. On the other hand, basidiomycetes, including the smuts

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(Ustilaginales) and the mushroom-like fungi (Agaricales), had multiple mating types, often determined by multiple alleles at two different loci A and B. The early interest in mating types was largely concentrated on their effectiveness as devices for encouraging out breeding. It was pointed out (Mather 1942, Whitehouse 1949) that although all the mating-type system prevented self-fertilization, the multiple mating types had the additional hypothetical advantage of favouring mating of unrelated strains over sib mating, an advantage that was enhanced in the two-locus systems compared with single-locus systems. Mutation is a sudden change occurring in the fungi, plants, microbes etc., Mutation are found in some fungi like Neurospora crassa, Aspergillus nidulans, Ustilago maydis, Saccharomyces and in few other fungi. These mutations are mainly molecular mutations. The changes occur in the base pair sequence of DNA including small deletions. Point mutations occur within the protein coding region of a gene. Silent mutations code for the same amino acid and protein mutations which code for a different amino acid. Nonsense mutations are those in which a stop codon replaces an amino acid codon. Frameshift mutation causes a change in the reading frame leading to introduction of unrelated amino acids into the protein. Mutation also involves large scale changes in chromosome structure which can affect the functions of numerous genes. Mutations arise spontaneously at low frequency owing to the chemical instability of purine and pyrimidine bases and to errors during DNA replication. Induced mutations are possible due to application of chemicals such as colchicines etc.

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Chapter - 19

Diversity and Conservation of Fungi

The original biosphere of Earth was formed by microorganisms. They made conditions suitable for the evolution and existence of macroscopic life forms and also continue to drive and greatly influence many of the necessary biogeochemical cycles. Moreover, nearly all of the present-day biodiversity among the eukaryotes is microbial, being generated by the protists, algae and fungi. Various types of bacteria provide the biodiversity within the prokaryotes. As a result ‘the tree of life is largely a tree of microorganisms, much of the diversity on Earth is microbial with the plants and animals appearing as small, terminal branches’ (Staley 1997). Biodiversity was first defined as taxonomic diversity - the number of species. More recently, biodiversity has been broadened to include biological and geographic entities such as genetic diversity (genes, chromosomes), taxonomic diversity (species, genera, families, phyla, etc.) and biogeographic diversity (biogeographic regions, landscapes, ecosystems, habitats). Biodiversity always refers to the genetic or taxonomic variability, within a specific area or region. Table 19.1. Comparison of known and estimated species of some selected groups of organisms (Hawksworth 1991, Manoharachary et al 2005) Group

Expected Total Species

Known

(%)

Vascular plants

2, 70,000

220,000

81

Bryophytes

25,000

17,000

68

Algae

60,000

40,000

67

Fungi

15, 00,000

1, 00,000

6.6

Bacteria

30,000

3,000

10

Viruses

130,000

5,000

4

Insects

1, 50, 00,000

9, 50,000

6.4

The biodiversity of earth is amazing as it supports between 5-50 million species of plants, animals, fungi, protozoa, and bacteria. Despite two centuries of research, systematists have described only about 1.5 million species. The ecology, or role of these species in ecosystems, has been studied for less than one percent. We know more about large, economically important plants and animals than we do about fungi and bacteria, despite their important ecological roles. Only about one lakh

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species of fungi have been described of the estimated 1.5 million species (Table 19.1). Recent research suggests that fungal biodiversity is much higher than previously thought. More than 140 different species of microfungi have been found in very small samples of fresh and decayed leaves from the forest floor of tropical rainforests. The number of macrofungi alone in United States has been estimated to range from 5,000 to 10,000 species (http://www.herbarium.usu.edu/fungi/ funfacts/Biodiversity.html). BIODIVERSITY OF FUNGI Until the mid of the 21st century mycologists focused their work on particular crop plant pathogens, but in the latter part the focus was extensively spread to survey and systematics, leading to increase in information about some classes like Deuteromycetes, cup fungi etc. and some genera of fungi representing large number of species like Agaricus, Alternaria, Cercospora, Puccinia and Septoria, etc. These genera many not be having restricted distribution but may be observed in many habitats. Microfungi Microfungi are those fungi with relatively small to microscopic (in general, less than 1 mm) reproductive structures. These are represented by many taxonomic groups. Mostly Chytridiomycetes, Zygomycetes, some Ascomycetes and majority members of Deuteromycetes. Conidial stages of Basidiomycetes are also considered as microfungi. Microfungi in the tropics are rather uncommon yet they probably constitute the majority of the world’s fungi. The number of species of microfungi is estimated at 7,00,000 to 9,00,000 of which only 10-30% are known (Rossman 1994). The ubiquitous and widespread nature of microfungi presents a challenge to those with desire to understand and study their biodiversity. These fungi are more mysterious and unknown in the tropical region than in temperate regions. The minute size of the reproductive structures make it more complicated to locate their existence and identify them. For some groups of microfungi, it is possible to scrutinize the substrates (using specific techniques of isolation) on which they occur, such as soil, leaf litter, air, or interior of living plant or dead wood logs, etc. The techniques used to isolate these fungi vary considerably according to the substrates. Bills and Polishook (1994), isolated up to 145 different fungal species from 0.1 ml particle suspension from one gram of tropical leaf litter, using modified particle filtration isolation procedure. Macrofungi Macrofungi is an artificial group of fungi with sporophore/ sporocarps, which are clearly visible to naked eye, i.e. larger than 1mm. The sporocarp is an indicator for the presence of macrofungi and its mycelium in the substrate, but absence of sporocarps does not necessarily mean the absence of the fungus. The number of sporocarps does not necessarily reflect the abundance of mycelium. The analysis of communities of macrofungi is called as mycocoenology and the methodology of

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which is described by Arnolds (1992). Macrofungi are contributed by the members from Holobasidiomycetes, Heterobasidiomycetes and some orders of Ascomycetes. Most of the macrofungi are involved in the decomposition of complex organic molecules such as cellulose, hemicelluloses and lignin. There are different types of habitats or niches of the macrofungi, such as soil inhabiting, symbiotic association especially ectomycorrhizal, parasitic like heart rot, root rot of plants etc. The macrofungi are specialized and restricted in their distribution. This group of fungi shows strong temporal dynamics due to their short longevity, strong fluctuations and conspicuous periodicity. The sporocarps are more or less ephemeral. The longevity of the sporocarps differ from species to species and is influenced by many factors such as predation, incidence of parasitic moulds, weather conditions, moisture content in the substrate etc. On the basis of potential longevity these fungi are classified into five types of sporocarps, viz. Ephemeral, Short-living, Moderates, Durables and Perennials. Cord forming fungi are the fungi forming network of filamentous aggregation of mycelium, covering ample area in the litter. These cord forming fungi (CFF), are potential channels for the movement of nutrient (translocation) between any two or more colonized food bases. They contribute in the nitrogen and other element economy of the forest floor. The sporocarp formation in macrofungi shows periodicity, more strongly related to the climatic conditions and moisture content of the substrate. Fluctuations are found in sporocarp productivity from year to year, and can be distinguished as quantitative (yield) and qualitative fluctuations, even a species may be absent in a certain year. The unnatural factors causing the fluctuations have been studied; these may be environmental factors, population dynamics of mycelia, including local extension and colonization (Arnolds 1995). The factors responsible for the decrement of macrofungi are adequately well known, but on individual species level the knowledge is still insufficient. More extensive work in this field is needed. The factors are described in detail by Arnolds (1989), in the Red data list of macrofungi; a list of the factors with brief description is given below. 1.

Succession: Spontaneous succession of plant communities to climax leading to infrequent earlier succession stages of plants and the associated fungi, e.g. Spreading of scrubs and forest in the coastal dunes leads to decrease of open sand and dune grassland with many characteristics fungi, e.g. Geastrum spp. and Tulostoma spp.

2.

Natural decline of plant species: Increase in the mortality rate of a plant species leads to decrease in associated fungi.

3.

Destruction of habitats: Destruction of many fungal habitats due to human developmental activities, such as town building, industry erection, road and other transportation development activities, etc.

4.

Alteration of habitats: Drastic transformation of landscapes and plant communities by various forms of agriculture and other human activities, such as pumping of groundwater from coastal dunes, drainage of bogs and boggy forests, etc. had an enormous direct impact on the landscape and mycoflora. Besides it has number of unintended side- effects on

Diversity and Conservation of Fungi

near-by semi-natural eutrophication.

287

biocoenoses,

mainly

by

drainage

and

5.

Air pollution: The emission of chemicals and gases such as 03, SO2, NO2 and NH3 has increased tremendously in the 19th century, causing drastic changes in terrestrial and aquatic ecosystem and thereby affecting mycoflora.

6.

Changes in management or influence of human: Environmental conditions, decline or altered plant communities, influence of human activities, etc have changed a lot but the fundamental objectives of biocoenoses have not changed. The mycoflora is greatly influenced by such developments.

7.

Side effects of agricultural measures: Intensification and industrialization of agriculture.

Edible Fungi: More than 5000 species of fungi are known to be edible and are consumed in different parts of world. Only 20-25 out of these are commercially cultivated while rest are collected from wild and consumed. The most expensive edible fungus is a wild collected fungus: truffles. Picking wild edible mushrooms for private consumption or sale in local market is traditionally a small scale activity, and in recent periods commercial harvesting for local and export purpose has increased dramatically. Commercially cultivated edible fungi are not threatened but the wild edible fungi are much affected by human activities, especially picking. There exists a long tradition of mushroom picking in central and northern European countries. According to Kardell et al (1980), in Sweden about 40% of the population collects wild mushrooms at least once a year and the annual consumption of wild mushrooms in Sweden is estimated at 10 x 106 kg/year. Picking of wild edible mushrooms is not popular in India in contrast to western countries, but still a big amount of mushrooms are picked for domestic use by many tribes and communities in rural part of India. Harvesting wild mushrooms can threaten the species as it may directly affect production of sporocarps in subsequent years, either by damaging or exhausting the mycelia; shifting competitive relations with other species; or by causing reproductive failure due to decreased spore production. Also harvesting may indirectly affect productivity owing to trampling (compacting soils) or racking the forest floor. While as per Arnolds (1988) there is no significant effect on fruiting due to picking activities as studied in saprotroph Marasmius oreades. Similarly Norvell (1992) stated that the Oregon Mycological Society failed to get negative effects of picking on the fruiting of Cantharellus cibarius in a mature coniferous stand. The fruiting initiation and development is more dependent on environmental factors than mycelial growth. A long term study dealing with decline of macrofungi is the need, such as Ricker and Peer (1988) who reported a strong decrease since 1937 in sporocarp numbers for Cantharellus cibarius, Boletus edulis, B. subtomentosus, Rozites caperatus, Lactarius volemus and many other ectomycorrhizal fungi.

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Wood Decaying Fungi These are the major contributors of the lignin decomposition in the forest or in the plantation. They are the main problem for the tree plantation and timber industry. These include large number of genera and species from Polyporaceae. Besides their destroying nature as wood rotting fungi, they also have some useful applications. These fungi are fairly responsible for the maintenance of carbon balance in nature, due to the degradation of cellulose and lignin. These wood decaying fungi (WDF) are used commercially in many industries. These fungi may have vital role in maintaining natural dynamics of the forest by attacking old matured plants and thereby their removal, giving a wide scope of growth to the young plants. Many wood decaying fungi, especially polypores are proved to be medicinally important. The knowledge of these properties is well known to many tribal people. The medicinal properties such as anti-inflammatory, antitumor, anti-HIV, anti viral, hypoglycemic and many such activities of Ganoderma lucidum, Lentinus edodes (Sasaki et al 1971, Song et al 1998, Mizuno 1996, Wasser and Weis 1999) are known, and many such activities of other WDF make the destroyer a healer. Many WDF have close relationship with termites and other insects and are proved to be a niche or food source. These WDF are mainly threatened by the destruction of forest, as the major habitat is decreasing at a very fast rate and many species may be potentially threatened to be extinct. Measuring diversity For measuring overall biodiversity of fungi a large number of habitats are to be sampled. Donnell et al (1994) suggested that at least 30 discrete microhabitats can be recognized in a forest. The data of declining or threatened fungal species is often summarized in Red Lists, wherein species are classified into four classes ranging from (presumably) extinct to potentially threatened, occasionally fifth class is assigned to a taxa in which degree of threat is unknown. Some Red lists of macromycetes have been published from northern, western and central Europe. According to Rossman (1994), because of the integral role of fungi in the ecosystem process (e.g. nutrient cycling, plant growth, food source), sensitivity to pollution and disturbance, fungi (including lichen forming species) lend themselves to measuring and monitoring biodiversity. And if we are ever to collect, identify, and name these fungi, this will have to be done before their habitats and hosts disappear. Conservation is defined as preservation from harm or decay; protection from loss or being used up. Earth’s biodiversity is rapidly decreasing in response to pollution and habitat destruction as forests are logged; prairies converted to subdivisions, and wet lands drained for development. The 1991 Red List for the former Federal Republic of Germany, for instance, lists 1,037 species of threatened macrofungi (mushrooms, puff balls, etc.), 35% of all the larger fungi. No similar comparison is available for microfungi. Other than forests, many other niches can be listed as habitats of different fungal organisms. To summarize, the major threat caused to fungi and its existence is indirect and is by destruction or disturbing the

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habitats or the niches of the fungi. And if we are to conserve fungi, conservation or modeling of the habitat is the primary need. The concern about the loss of biodiversity has fostered a number of initiatives to discover, describe, and classify (name) the world’s species. One component in this process is Bioinformatics, which utilizes the catalogues of museum collections to provide baseline data on where species occur, and their abundance. Surveys of poorly known regions, or poorly known groups of organisms are another component. There should also be attempts to study the interactions among groups of organisms in order to identify critical ‘keystone’ species responsible for controlling the functioning and structure of ecosystems. With the loss of each species, we lose potential sources of new medicines, chemicals, and food. The air pollution is harmful to the symbiotic fungi that act as extensions of trees root systems by absorbing the minerals trees need for growth. The loss of these symbiotic fungi has resulted in the death of millions of trees. Despite their importance, none of the 711 federally listed endangered and threatened species under the jurisdiction of the U.S. Fish and Wildlife Service are fungi. CONSERVATION OF FUNGI The causes threatening fungi are more or less same as those for other groups of organisms like plants. Some measures can be listed as stated by Arnolds (1989): i). Protection of areas with a valuable mycoflora, either by law or by entrusting them to the care of an organization for nature conservation. ii). Creation of appropriate habitats for macrofungi. iii). Management of nature reserves and other important areas. iv). Decrease of environmental pollution. The causes threatening macrofungi may be same as that to the green plants but the relative importance of various factors and habitat are different. It is essential to recognize the hierarchy among the factors, and it is also important to save the areas with high mycological value. The creation of nature reserves and long lasting satisfactory management should be carried out. Strategies for conservation of fungi Scanty attention has been paid for conservation of fungi in most countries; a basic need for general public awareness is required specifically foresters, those working in nature, trekkers, tourists, etc. Along with awareness some pronouncement should be made such as: 1.

Correct taxonomic identification, making of inventories (checklists).

2.

Mapping programme.

3.

Categorizing for Rarity, Endangerment and Distribution and according to the categorization these are then listed in RED data list.

4.

According to the cause and threats to the organism the habitat modeling is done and the changes are recommended for its conservation. Conservation of entire habitat and thereby ‘in situ’ conservation of the fungus

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5.

Minimum disturbance in existing forests, essentially for both necrotrophic and biotrophic species.

6.

Special attention should be paid to conserve the ‘Type Localities’ for species, as preservation of localities according to habitat description and vascular plant data (host/substratum) may not always cover conservation of fungi.

7.

The other way of conservation is ‘ex situ’ in the form of ‘Herbaria’ or ‘Culture Collection’ and regular revision and duplication should be done. Exceptionally some obligate phytotrophs of flowering plants cannot be conserved ‘in vitro’.

8.

Some ectomycorrhizal fungi can be conserved in vitro in vegetative mycelial stage. Only; their reproductive structures can be obtained ex situ but need understanding of their nutritional, physiological and environmental requirements.

Generating Data Huge data is needed to be generated along with the correct taxonomic identification, preparation of inventories, mapping of the organisms. There is a very urgent need to generate the data very quickly. More accurate data collection on geology and ecology should be built up. Co-operation between amateurs and professional mycologists is needed to build such data. The collected data should be presented in systematic, convenient, re-accessible form to build a database and should be processed and presented in user friendly format. This is done under the branch of information technology called Bioinformatics. For e.g. a website developed by Cornell University which provides the data developed at regional level in US [(http://mycology.cornell.edu/fguide.html) Mycological Resources on the Internet: Regional inventories and guides]. Commissions are already established in some part of world like; European Council for Conservation of Fungi (ECCF) established by the 9th Congress of European Mycologists in 1985. ECCF has a representative of all countries, it publishes a newsletter and Red lists at irregular intervals. The World Conservation Union in 1990, confirmed the need for the conservation of fungi, by establishing a committee to stimulate conservation of fungi worldwide, including a newsletter ‘Fungi and Conservation Newsletter’. Some commissions are the current need of each country and that too at a level managing the diversity and conservation of fungi. Such commissions may be a part of Municipal Corporations or government district administration. Some judicatory sections should be made as the one made in Poland by the Government, Council for Nature Conservation, following the proposal made by Polish Botanical Society. The section says ‘It is prohibited to destroy fruit bodies of any fungal species, which are not gathered for consumption, in forest, meadows, grassland, etc. or to disturb the forest litter, whole gathering of mushrooms’. In general mycologists have to educate the public, scientists, foresters and supervisors of nature reserves, that fungi play an important role in nature, that fungi are declining and that too at an alarming rate and that measure are necessary for their

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conservation. The decline of fungi is transitional and not a local problem restricted to any area, locality or country. The study of the vast array and multifarious aspects of fungus conservation is not for the faint-hearted but a need and pleasure for those seeking intellectually rewarding and also relevant endeavor. This is high time to form small regional groups of committees to international ones to get the protection and conservation of fungi till the area of affect. Role of Mycologists 1.

Generating data of the diversity of fungi from the selected area by taxonomically correct identification.

2.

Preparation of inventories and processing them to categorise the organisms according to their rarity, endangerment and distribution (RED data list).

3.

Habitat modeling should be done so as to enhance the habitat for conservation of the concern fungal organism.

4.

The date should be generated for small areas (locally) and should be complied together to build a database for regional and national levels which further should be shared. Such work is being done in many parts of world and the information is shared through the internet.

5.

Apart from the regular conservation practices, creating literacy or awareness about fungi, the important role played by these organisms and then soliciting for their conservation. The awareness should be brought out from a grass root level i.e. layman, school and college students, postgraduate level, academic staff, government employees to public, private and corporate staff.

To inspire interest at school or college level, mycologists can assign a small experiment for locating and identifying a fungal organism from the area. With the help of microscopic and macroscopic morphological characters, tissue organization, etc. the organism can be identified up to generic level by using appropriate keys. The study area may be from market, fields like cropland, grassland, or woodland and in house fungi locating them in spoiled food or food articles, damp places along the wall or refrigerator, washing machine, kitchen gardens etc. This type of experiment will be useful to encourage school and college students to take interest in understanding the role of fungi in their day to day life and making them aware of how to identify them by sample collection, isolation and culture processes. At post graduate level the job may be emphasized at higher level so that more ecological niches are exploited to study fungal organisms extensively namely soil, air, water, wood, plant surfaces (phylloplane, rhizoplane), dung inhabiting and natural and biotically disturbed woodland/ grassland. Inventorisation and documentation of the species in such habitat will be useful in long term basis for preparing floristic inventories and listing rare species from the study area. This data will ultimately be useful for conservation planning so as to monitor rare and

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endangered organisms and thereby the species which may be useful for commercial applications. The isolated fungi need to be screened for their secondary metabolites, some of these may be useful for commercial exploitation. This part of work has to be done on priority basis. Taxonomic identification, preparing inventories and flora, rapid screening of species, assigning rare, endangered species, preparing RED data list from different habitats needs to be done. Isolating different metabolites and characterizing them and ultimately exploiting some of them at commercial scale are required. Overall mycologists play a key role and all the organizations ensuring conservation should take maximum help of mycologists in their day to day programs. This will finally open doors to amateur mycologists in the field of newly arising scientific era of Biodiversity and Conservation. Mycologists should be positive in approach in establishing collaboration with those involved in land management and especially with groups concerned with conservation of other organisms. There is certainly no excuse for mycologists being short of sound arguments for inclusion of fungi in conservation schemes. There is lack of experts needed to make serious contributions to knowledge of species sufficiently quickly to conserve those species. The taxonomists are scarce because of shift in academic programs towards molecular systematics and ecology (Molina et al 2002). In addition to this, for several years now the funding agencies around the world have been operating similarly biased/partial funding policies. The major issue to really emphasize is to conserve Mycologists. In many part of world mycologists are an endangered species. Fungal conservation can only occur if mycologists are conserved. The future of mycologists depends on their input in conservation.

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Chapter - 20

Lichens - Structure, Reproduction, Ecological and Economic Importance

The occurrence of lichens is known to man since long time, and they were thought to be specialized life forms, distinct from plants. Swedish scientist Schwendner (1868) first studied in detail the nature of lichens and explained that they are formed as a result of close association between a fungus and an alga. In the lichen thallus, fungus is the major partner and it is called mycobiont. The thallus of lichens is formed by the hyphae of the fungal partner. The algal partner occurs in the thallus either as a separate zone or distributed randomly throughout the thallus. The algal partner is described as phycobiont. In lichens, the commonly occurring algal member belongs to either chlorophyceae or cyanophyceae families. As the cyanophyceae is now considered as cyanobacteria, some lichenologists prefer to describe the autotrophic partner as photobiont rather than phycobiont. The association of autotrophic with hetrotrophic mycobiont is described variously by different scientists. Schwendner, who first studied the nature of lichen thallus in detail, described the relationship as helotism. His view was based on the nature of lichen thallus in which algal partner is found inside the thallus formed by fungal partner as a slave, and feeds the major partner through its photosynthetic activity. The famous German mycologist Anton von de Bary (1889) described the association as symbiosis. Reinke (1896) described the association as mutualism or consortium. Elenkin (1904) described it as endosaprophytism. Some lichens may have more than one algal partner. Based on this character, the association is described as polysymbiosis or parasymbiosis by well known Indian lichenologist Awasthi. However, the association between the two partners in lichen thallus is considered as symbiosis by many scientists. The two partners of a lichen thallus can occur independently, and when the conditions are not favourable for independent growth they form a close association to form the lichen. Hence, Ahmadjian (1962) emphasized that the association should be treated as symbiosis. The mycobiont takes food from algal partner and gives it protection against adverse environmental conditions. The mycobiont provides water and different minerals to the phycobiont. Hence both the organisms are benefitted. In most of the lichens, the fungus belongs to Ascomycotina group. Even among this group, the fungus mainly belongs to the classes Discomycetes and Loculoascomycetes. The fungi belonging to classes Hemiascomycetes, Plectomycetes and

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Laboulbeniomycetes do not form lichens. Among the other fungi, some fungal species belonging to the class hymenomycetes of Basidiomycotina, especially those belonging to the orders Agaricales and Aphyllophorales also form lichens. In a few lichens, the fungal partner appears to belong to mycelia sterilia group or Deuteromycotina. So far there is no evidence about the fungi belonging to Mastigomycotina and Zygomycotina forming lichens. Usually most of the lichen forming fungi do not occur in free living condition but mainly occur in lichens only. The algal partner of lichens mainly belongs to two groups of algae viz. chlorophyceae and cyanophyceae. Only about 24 genera of algae are recognized as forming lichens. Among them, the genus Trebouxia belonging to Chlorophyceae is the most common algal partner in most of the lichens. The other genera of green algae forming lichens include Trebouxia, Pseudotrebouxia, Coccomyxa, Myremeci, Trentepohlia etc. The cyanophycean algae forming lichens mainly belongs to the genera Caulothrix, Gloeocapsa, Nostoc, Scytonema and Stigonema. An algal species may occur as phycobiont in more than one lichen species and some lichens may have more than one algal species in their thallus. When more than one phycobiont is present in the lichen thallus usually one belongs to class Chlorophyceae which can carry out photosynthesis, and the other belongs to class cyanophyceae which can carry out photosynthesis as well as nitrogen fixation. When lichen thallus has only one phycobiont belonging to chlorophyceae it is described as chlorophycophilous lichen. When the lichen thallus contains one chlorophycean and one cyanophycean algae, it is described as diphycophilous lichen. The algae that occur in lichens can also occur in free living condition. Only the genus Trebouxia was adapted to only symbiotic form of life. There are no major morphological differences between algal species occurring in lichens and those occurring in free living conditions. However, the growth of algal species that adapted to symbiotic life is very slow even in the medium having rich nutrients. OCCURRENCE About 15,000 to 20,000 species of lichens have been described so far. They are distributed throughout the world in all hospitable habitats, and even in relatively adverse conditions. They are found in tropics, subtropics, temperate and even in Polar Regions. Their incidence extends from plains to mountain regions. They are most abundant in tropical rain forests and evergreen forests in temperate regions, in very cold regions like Greenland, Iceland and Alaska. Lichens form major part of the vegetation. They are also found in desert areas on exposed rocks. Some lichens are found in land waters and sea waters also. However, lichens cannot tolerate chemical pollution and, hence, are not generally found in and around industrialized cities and towns. These areas are described as ‘lichen deserts’. Lichens generally grow on stems, branches and bark of forest trees, on wood, ground, and surface of rocks etc. Depending on the substrates, lichens are described variously. 1.

The lichens that grow on tree trunks are described as Corticolous lichens, e.g. Usnea, Parmelia etc.

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2.

The lichens that grow on wood are called Lignicolous lichens, e.g. Calicium, Chanotheca, etc.

3.

`Those that grow on ground are called Terricolous lichens, e.g. Cladonia, Lecidea, etc.

4.

Those that grow on rocks are called Saxicolous lichens, e.g. Dermatocarpon, Verrucaria etc.

The scientists who carried out extensive research on lichens in India include D. Awasthi, K.P. Singh, Upreti, Ajay Singh, Hariharan, Makhija, Behera, Patwardhan, Joshi, Mathur, Manoharachary and others. In India Parmelia, Cladonia, Lecanora, Dirinaria, Heterodermia, Graphis, Pyxine etc. are common. Most of the lichens are ascolichens. THALLUS STRUCTURE Morphology: Basing on morphology the lichens are recognized as belonging mainly to three groups, viz. crustose, foliose and fruiticose lichens (Plate 20.1). The thallus with scales is often recognized as a fourth group called squammulose lichens. 1.

Crustose lichens: These are thin and flat. Thallus is strongly attached to the substratum. They are usually found growing on bark of trees, ground and surface of rocks, e.g. Glyphis, Graphis, Lecanora, Verrucaria, Rhizocarpon etc.

2.

Foliose lichens: Thallus in these lichens possesses small leaf like branches that grow parallel to the substratum. From the lower surface of the thallus special structures called rhizenes are produced and penetrate the substratum. They help to keep the thallus attached to the substratum and absorb water and minerals from the substratum. Foliose lichen thallus is divided into a number of branches which are small and measure 0.1 to 2 mm in width. Some branches are large up to 10 to 20 mm. These appear like leaves. The edges of these structures may be smooth or rough with scales e.g. Parmelia, Pyxine, Dirinaria, Parmelinella, Phaeophyscia, etc.

3.

Fruiticose lichens: Thallus is extremely branched and appears like a small bush. The branches grow from the substratum as cylindrical structures with a number of side branches. The central branch is called axis. In Usnea, which grows on the tree trunks, the branches hang down from the substratum. In some species these branches may grow up to 5 to 10 feet. Rhizenes are usually absent and the inner cortex of the axis is hollow. In some species, the central part of the branches is like a central cord, e.g. Usnea, Cladonia, Ramalina etc.

4.

Squammulose lichens: The thallus is covered with a number of scales or squammules. The scales are usually 1-10 mm long and rolled over. In these forms the morphology may be quite varied. In the thallus, upper cortex and rhizenes are usually absent. In some species of Cladonia the basal part is squammulose and upper part is fruiticose.

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Plate 20.1. Lichens of various types. Foliose lichens - A. Dirinaria applanata, B. Parmelinella simplicitor, C. Phaeophyscia pyrrhophora. Fruticose lichen - D. Usnea ghattensis. Crustose lichens - E. Lecanora fimbriatula, F. Glyphis cicatricosa.

Internal structure The anatomy of the lichen thallus shows mainly four parts. These are upper cortex, algal zone, medulla and lower cortex. In the anatomy of foliose lichens all these four parts are commonly found. In fruiticose lichens the main axis and branches are found, and hence in these forms lower cortex is absent. Medulla forms the central part. In crustose lichens also inner cortex is not clearly formed as the inner surface of the thallus is closely and strongly attached to the substratum. The algal cells in the thallus maybe found scattered in the thallus or may form a clear zone below the upper cortex. The lichens in which the algal cells are randomly distributed in the thallus are called homoiomerus lichens, and the lichens in which the alga occurs in a clear layer or zone are called heteromerous lichens. Upper cortex: This is the upper layer of the thallus. Fungal hyphae form a clear thick multilayered epidermis like structure. It is thick and protects the thallus. It also helps in absorption of water. The fungal hyphae form a clear tissue called prosenchyma. Some may have intercellular spaces, and in the others the hyphae are closely woven. In some lichens like Roccella, there are small pores in the upper cortex which help in respiration. Hence these are called breathing pores. Algalzone: In heteromerous lichens, there is a clear layer or zone of algal cells below the upper cortex. In homoiomerous lichens the algal cell are distributed randomly throughout the thallus. Medulla: This is the middle part of the lichen thallus. In this layer fungal hyphae are loosely woven and hyphal branches randomly expand to all parts. It helps in translocation of water and minerals. This layer forms the central part of fruiticose lichens which have round axis.

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Lower cortex: This layer is clearly seen in foliose lichens only. It is closely attached to the substratum. This layer is formed by tightly woven fungal hyphae. From the lower side of this layer special absorptive hyphae called rhizenes are formed and penetrate the substratum. They help the lichen thallus to closely adhere to the substratum and also in absorption of water and minerals. This layer is not usually seen in crustose lichens whose thallus is tightly adhered to the substratum on lower side. In fruiticose lichens also this layer is not found. NUTRITION AND GROWTH The major partner of lichens, the mycobiont is heterotrophic in nutrition. The minor partner of lichens, the phycobiont is autotrophic organisms which can carry out photosynthesis. Hence, the algal partner helps the fungal partner in nutrition. However, the method of nutrient transfer from algal cells to fungal hyphae is not clearly understood. In some lichens the fungal hyphae sends in haustoria into algal cells to absorb the nutrients. The fungi get vitamins essential for growth like thiamine, protein and others from algal cells. The carbohydrates found in algal cells formed through photosynthesis are transferred to fungal hyphae. The type of carbohydrate formed in photosynthesis varies in the algae with species. Nostoc forms glucose, Trebouxia produces sorbitol. The fungal hyphae store the carbohydrates in the form of mannitol. When photobiont is a cyanophycean member it fixes atmospheric nitrogen in the form of ammonia, and supplies it to the fungal partner. The fungal hyphae absorb water and mineral from the surroundings through upper cortex and from substrate through rhizenes and supply them to the algal partner. The lichens grow very slowly. The growth rate is usually about 1 mm to 1 cm in a year. This type of slow growth of the lichens is attributed to the slow growing nature of its fungal partner which forms the thallus partner. REPRODUCTION Lichens reproduce mainly through vegetative methods. On the thallus special structures are formed in which both mycobiont and phycobiont are present. These are subsequently separated from the main thallus, disperse and form new thalli. Such structures with both mycobiont and phycobiont are called diaspores. The reproduction through diaspores is described as vegetative reproduction. Asexual and sexual methods of reproduction are mainly found only in mycobiont of the lichen. The phycobiont usually do not show independent reproduction in nature, but can be induced to reproduce in laboratory cultures. A. Vegetative reproduction: It occurs mainly through fragmentation of the thallus, and formation of diaspores like soredia, isidia and cephalodia. 1. Fragmentation: When thallus fragments because of physical forces or other reasons, each fragment can form a new lichen thallus. It mainly occurs in crustose lichens, which grow strongly adhering to the substratum. When the central part of the thallus disintegrates, the peripheral parts dissociate and form new lichen thalli. It may occur sometimes in foliose lichens also. 2. Soredia (singular: Soredium): These are specialized vegetative reproductive structures on the upper cortex as small round particles or bud like

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structures. In these structures fungal hyphae occur as a network on the inner algal cells. Soredia are formed in groups. Each group is called soralium. They break through upper cortex and appear as distinct pustules. In favourable conditions the Soredia disperse, germinate and produce new thalli. 3. Isidia (singular: Isidium): These are formed on the upper cortex as small cylindrical root like structures growing upwards. In these structures both algal cells and fungal hyphae are present. At the base of these structures there is a constriction, and hence they can easily separate, disperse and form new thalli. 4. Cephalodia (singular Cephalodium): These are gall like structures on the upper cortex of lichen thallus. These structures are formed with both mycobiont and phycobiont. The fungal hyphae surround a group of algal cells. Usually these algal cells belong to a different organism other than the phycobiont present in the normal thallus. For example, in Peltigera aphthosa, the phycobiont in the thallus is a chlorophycean member, and in the gall like structures the phycobiont is a cyanophycean member. Hence, this lichen is described as diphycophyllous. Some scientists consider them as part of the thallus. However, others consider them as diaspores, as they can separate from the mother thallus and form new thalli. B. Asexual reproduction: It occurs in only mycobiont. The asexual reproductive structure is most commonly a pycnidium. It is a flask shaped structure with a narrow ostiole. The fungal hyphae lining the inner surface of pycnidium produce long vertical pycnidiophores. These stalks bear single celled spores at their tips. Under favourable conditions they disperse and form independent fungal colonies on different substrata. When these growing fungi come in contact with a suitable algal partner they form lichens. C. Sexual reproduction: It also occurs only in mycobiont. Most of the lichens have ascomycetous fungi as mycobiont. Hence, they mainly produce ascocarps. Most commonly formed ascocarps are apothecia, and to some extent pseudothecia. The apothecia form on the thallus of foliose lichens. It resembles in most aspects the apothecia formed by Discomyetous fungi. The presence of an algal layer is the special character of lichen apothecia. In lichens showing sexual reproduction ascogonium is the female gametangium, and the male gametangium may be either antheridium or spermagonium. The ascogonium has a globose basal region and it bears a long, narrow structure called trichogyne at the apical region. The trichogyne penetrates the upper cortex and exposes on the surface. It receives the male nucleus either from antheridium by gametangial contact or from a spermatium in spermatization process. The male nucleus positions itself besides the female nucleus, thus forming a dikaryon. Then ascigerous hyphae develop and produce the asci, and ascocarp is formed as in normal apothecial fungi. In some lichens the sexual reproduction may occur through somatogamy also. In Basidiolichens, basidiocarps form like in normal fungi. Among the Basidiolichens, the corticoid lichens produce bracket like fruit bodies on tree trunks. Clavarioid lichens produce erect, branching fruit bodies on wood. The agaricoid lichens rarely produce fruit bodies. The basidiospores disperse and produce primary mycelium, secondary mycelium and tertiary mycelium as in normal fungi. When

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secondary or tertiary mycelium comes in contact with a suitable algal partner, they form lichen. Sexual reproduction is absent in Deuterolichens. Reproduction in phycobiont: The algal partners normally do not reproduce in nature, but they can be induced to reproduce in laboratory cultures. They show cell division and aplanospore formation. Trebouxia, the most common phycobiont in majority of lichens, produces biflagellate zoospores in the medium. The unicellular members of cyanophyceae show reproduction through cell division. The filamentous forms produce special structures like heterocysts, akinetes or hormogonia, and undergo fragmentation at these special structures. CLASSIFICATION OF LICHENS Lichens are formed as a result of close association between two organisms having different morphology and nature. Hence, it is very difficult to classify lichens satisfactorily. Some scientists have classified lichens basing on phycobiont, and a few are based on the mycobiont. Carl von Linnaeus, father of plant taxonomy, classified lichens along with algae and recognized nine series based on the morphological forms. Famous mycologist of 19th century, Elias Magnus Fries (1831) of Sweden, classified lichens basing on mycobiont. Bold et al (1980) suggested that lichens should be classified as a separate division Mycophycophyta in plant kingdom because they are formed by close association of a fungus and alga. Zahlbruckner (1926) of Australia classified lichens and recognized two subclasses, Ascolichens and Hymenolichens based on fungal partner. The classification proposed by Zahlbruckner was followed by the famous plant taxonomists, Engler and Prantle in their book ‘Naturalichen flanzen familien’. Hence the Zahlbruckner’s classification of Lichens came into prominence and some followed it also. However many mycologists are of the view that recognizing lichens as separate group is unnatural, and they should be classified along with fungi. Hensen and Jahns (1974), Poelt (1973), and others classified lichens along with fungi. Alexopoulos and Mims (1979) in their popular text on mycology broadly followed Poelt in classification of lichens. According to this system, lichens are classified into three divisions viz. Ascolichens, Basidiolichens and Deuterolichens. 1. Ascolichens In this division the mycobiont belongs to Ascomycotina. The fruit bodies formed are either apothecium or pseudothecium. The asci formed in apothecia may be unitunicate or bitunicate. Based on the nature of fruit bodies and asci, three groups are recognized in ascolichens. Group 1: Lichens containing unitunicate asci in apothecia: In this group of lichens the apothecia formed resemble those formed in Discomycetous fungi. Unitunicate asci form an open hymenium. Three orders are recognized in this group. They are Caliciales, Leconorales and Graphidiales. Group 2: Lichens with bitunicate asci either in apothecium or hysterothecium: The mycobiont belongs to the class Loculoascomycetes. The presence of bitunicate asci in apothecium is the special character of this group. In hysterothecium, the

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hymenium is completely covered in the early stages and gradually opens up along a suture on the upper surface exposing the hymenium with bitunicate asci. The mature fruit body is boat shaped. A single order Arthomales is recognized in this group. Group 3: Lichens containing bitunicate asci in pseudothecium: These lichens are generally called pyrenolichens. The ascocarps morphologically resemble perithecia. However, they lack a clear peridium. Hence these are considered as pseudothecia and the lichens are classified along with loculoascomycetes. Three orders are recognized in this group. They are Dothidiales, Verrucariales and Pyrenulales. 2. Basidiolichens The lichens formed by basidiomycetous fungi are relatively few. Among these, two genera viz. Cora and Omphalina are important. The genus Cora belongs to the family Corticiaceae of the order Aphyllophorales. The genus Omphalina belongs to the family Tricholomataceae of the order Agaricales. Some species in the family Clavariaceae also form lichens. Basing on the characters of the mycobiont, the basidioliches are classified into two orders viz. Agaricales and Aphyllophorales. 3. Deuterolichens Some fungi forming lichens do not produce any spores either asexual or sexual. They are called sterile fungi and treated as belonging to a separate group mycelia sterilia. The lichens formed by these fungi are described as Deuterolichens. They are relatively very few. Hawksworth et al (1983) of Commonwealth Mycological Institute (CMI, now CABI) did not consider lichens as separate plants. They were considered as fungi and placed them in 16 orders in ascomycotina. The orders containing the lichenized fungi were: l. Arthoniales, 2. Caliciales, 3. Dothidiales, 4. Graphidiales, 5. Gyalectales, 6. Helotiales, 7. Leconidales, 8. Leconorales, 9. Opegraphales, 10. Ostropales, 11. Peltigerales, 12. Pyrenulales, 13. Peddusariales, 14. Sphaeriales, 15. Scieroscyphales and 16. Verrucariales. From the above account it is clear that the classification of lichens is not yet stabilized. SOME IMPORTANT GENERA OF LICHENS 1. Cladonia: It is the most common crustose lichen with about 350 species. It is worldwide in distribution. Among the crustose lichens, Cladonia rangifer grows luxuriantly in cold temperate regions, and is described as ‘reindeer moss’. It grows on stems, bark, wood and ground. It belongs to the family Cladoniaceae of the order Leconorales in ascolichens. Apothecium is the common fruit body produced by it. 2. Parmelia: It is the most important foliose lichen with about 550 species. They are worldwide in distribution. In India about 125 species of the genus are recorded. It belongs to the family Parmeliaceae, order Leconorales in ascolichens. 3. Usnea: It is the most common example of fruiticose lichens. About 600 species are recognized in the genus. This lichen is worldwide in distribution, and most commonly seen growing on trees in temperate forests. They grow hanging

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down from the tree branches. The body comprises of a main axis with a number of branches. The branches are covered with scales and the surface becomes rough. The thallus contains usnic acid which shows antibacterial properties. Taxonomically it belongs to the family Parmeliaceae. 4. Cetraria: It also belongs to the family Parmeliaceae. About 40 species are recognized in the genus. It is mainly seen in very cold regions. Cetraria islandica is the most common species in the genus. In India it occurs in only Himalayan region. Morphologically the lichen may be in foliose or fruiticose form. 5. Peltigera: It belongs to the family Peltigeraceae, order Peltigerales in ascolichens. About 30 species were recognized in the genus. Peltigera polydactyla and P. canina are important species. P. canina is described as ‘dog lichen’. It is used in the treatment of rabies spread by dog bite. These lichens are most commonly seen in subtropical regions growing on ground, sand dunes, rocks and other substrates. They are mostly foliose forms. The thallus appears as a small bush with many branches. One of the most important characters is the presence of two forms of phycobionts in the thallus. Among the two algal species, one belongs to chlorophyceae, usually Coccomyxa, and the other to cyanophyoeae, usually Nostoc. Hence, the lichen is described as diphycophilous. Sexual reproduction occurs through formation of apothecia. These fruit bodies appear as red structures on the upper cortex. The thallus contains a number of dyes or pigments, and these are usually present at the tips of the fungal hyphae. IMPORTANCE OF LICHENS Lichens are very important both ecologically and economically. 1. Ecological importance In xerosere plant succession, saxicolous lichens play an important role as primary colonizers. Lichens can grow in habitats where other organisms cannot grow, and make the habitat suitable for the growth of the others. Among the lichens that can grow in different habitats those growing on exposed rocks are very important in plant succession. They play an important role in weathering of rocks and soil formation. The lichens carryout weathering of rocks by two means a. Biogeophysical weathering, b. Biogeochemical weathering. Saxicolous lichens, especially crustose forms, produce rhizenes from their lower cortex and these rhizenes penetrate the crevices of the rock. The crustose lichens expand in humid conditions and contract during dry conditions. During the expansion and contraction, the substrate undergoes weathering. Because of these actions, new crevices form and existing crevices become large; ultimately resulting in surface of substrate rock breaking into small pieces and undergo further biogeophysical weathering. When lichens are growing on rocks, various chemicals such as oxalic acid, lichenic acid etc. are formed which help in biogeochemical weathering. Carbon dioxide released during respiration of lichens dissolves in water and releases hydrogen ions that help in weathering of rocks. Oxalic acid dissolves iron oxides. The lichens growing on calcium deposits accumulate calcium oxalate around the fungal hyphae or on upper cortex. In addition to this, the lichens release

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complex chemical substances into the substratum and these form chemical complexes with iron, maganesium, calcium, aluminium etc. to form metal complexes, and change the chemical nature of the substrates. These changes help in weathering of the substrate rocks. In most of the lichens the reserve food material is calcium oxalate and it helps in weathering of rocks. Due to the effect of lichens basalt, mica, quartz, granite and other minerals get dissolved to form soil. This process is very slow. After the substrate rock undergoes some physico-chemical weathering, soil formation gradually begins. Below the lichen thallus, humus layer forms. The lichens accumulate dust and other particles from the environment. This particulate matter accumulates in humus and its capacity to store moisture increases. Slowly the conditions become hospitable for the growth of the other organisms. Lichens not only help in soil formation but also play an important role in stabilization of soil. There is a strong relationship between soil pH and lichen growth. Because of it, many lichens can grow even in desert areas. Among the desert lichens, some squamulose lichens like Catapyrenium, Heppia, Peltula, Psora etc. can grow even at temperatures of 50°C or more for long periods. These strongly attach to the soil and help in stabilization of soil. 2. Economic importance of lichens Lichens are very important economically as food and fodder, in industrial production of various drugs, dyes, cosmetics etc. a. Lichens as food: Lichens grow luxuriantly at very cold temperatures of Polar Regions. Eskimos of Greenland and Alaska use lichens as feed for their animals. They also use a number of lichens in preparation of different food items. Cladonia rangifera and other lichens are very popular as animal feed in tundras, cold areas of Europe and North America. They are called ‘reindeer moss’. Species of Cladonia, Cetraria are also used as animal feed. Species of Bryoria and Usnea which grow on stems and bark are highly palatable for animals. Species of Lecanora esculenta and others growing on rocks and sand dunes in Lybian desert are grazed by goats. Lecanora esculenta, Cetraria islandica, Dermatocarpon miniatum and other lichens are extensively used in preparation of food items. These lichens contain carbohydrate lichenan as reserve food material in high quantities and also contain 5 – 6% proteins. However, due to lichen acids, they taste sour and bitter. Hence, they are used after cooking or overnight soaking. The people of western Canada collect Bryoria fremontii, soak overnight in water, and cook before eating. In Japan, people use Umbilicaria in salads or fried to eat. In Egypt, the lichen Evernia prunastri is used in bread making. Some Parmelia species are used as food in some areas of South India. They are described as ‘rock flower’. b. Lichens in drug preparations: Some lichens are used in the preparation of drugs. The lichen Lobaria pulmonaria resembles lungs in its morphology and was used to treat pulmonary diseases. Parmelia sulcata was used in the treatment of brain infections. The thallus of Peltigera canina was powdered, mixed with black pepper powder and taken as medicine for treatment of rabies. Parmelia saxatilis was used in

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the treatment of epilepsy. The species of Usnea, Cladonia and other lichens contain ‘usnic acid’ which has antibacterial properties. Hence, they are used in the treatment of diseases caused by Gram positive bacteria. A number of antibiotics and other drugs are now extracted extensively from lichens as a result the number of lichens has decreased drastically. Even now, the lichens are used in the treatment of various diseases by rural people and tribals. Water extract of Usnea species is used in the treatment of cough. Cetraria islandica is used in the treatment of diabetes and pulmonary infections. Peltigera canina and species of Umbilicaria are used to treat jaundice. Usnic acid extracted from lichens is used in the treatment of tuberculosis and skin diseases. It is popular as ‘Usno’ in European countries. In Japan, soric acid from species of Cetraria, Cladonia and Parmelia is used in cancer treatment. Some lichens are used to control plant diseases also. Sodium usnate can control tomato canker disease. Lecanoric acid and usnic acid can control tobacco mosaic disease. Water extract of lichens like Nephroma, Hypogymnea and Platismatia etc. can control the growth of wood rotting fungi. c. Lichens in preparation of dyes: In earlier days when the dyes from coal tar did not become popular, a number of dyes were extracted from lichens. Some are still prepared. The dye orchil extracted from species of Roccella, Lecanora and others are used for dying the silk and wool garments. Orcinol, the dye used for staining chromosomes in biology laboratories is prepared from purple lichen Roccella tinctoria and Lecanora parella. The litmus dye is also extracted from R. tinctoria. d. Lichens in preparation of perfumes: Aromatic biochemical compounds present in lichen thallus are used in preparation of a number of perfumes. Evernia prunastri, Pseudoevernia furfuracea and other lichens are collected in large quantities, dried and exported to major centres of perfume industry like Paris, New York and other places. It is a very profitable trade. Lichen compounds are also used in soap industry for preparing perfumed soaps. A number of perfumes are extracted from species of Ramalina and Cladonia. e. Lichens as pollution indicators: Lichens are very sensitive to chemical pollution. They do not usually grow in polluted environments. Hence they are not commonly found in towns and cities highly polluted due to heavy and rapid industrialization. These areas are commonly described as ‘lichen deserts’. Hence lichens are used as biological indicators of level of chemical pollution. Lichens are used to detect pollution due to sulphur dioxide, heavy metals and other pollutants. The level of chemical pollution due to toxic metals like lead, nickel, zinc, chromium, mercury, fluorine etc. is detected by lichens. f. Other uses of Lichens: The chemicals extracted from lichens like Lobaria pulmonaria and Cetraria islandica are used in leather industry for tanning. The species of Cladonia, Usnea, Ramalina and other lichens are used in production of alcohol. Lichens, like species of Cetraria and Lecanora are used to detect copper and lime deposits. g. Harmful lichens: Some lichens are harmful to man in various ways. In Canada, the wood cutters and other workers of wood industry are prone to skin rash called cedar poisoning. It is due to the exposure of these people to lichens growing

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on wood. Exposure to some species of Usnea, Evernia and other lichens cause sensitization leading to an allergic condition called eczema or skin rash. The lichens growing on stems and bark sometimes interfere with the physiology of the host plants. Lichens growing in temples often spoil the stone carvings The Usnea species described as ‘old man’s beard’ often cause forest fires because they are highly inflammable.

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Chapter - 21

Mushroom – Cultivation and Application

There are more than 2000 edible fungi throughout the world. Mushrooms are the fleshy, macroscopic spore bearing structures of some fungi belonging to Basidiomycotina and Ascomycotina. Cultivation of mushrooms started around thousand years ago in China. In India, N.N. Newton (1886) was the first to exhibit mushrooms at the annual show of Agriculture, Horticulture Society of India. In 1961, ICAR in collaboration with the state government of Himachal Pradesh, started a scheme on ‘Mushroom cultivation’ at Solan. Later, slowly mushroom cultivation and research on cultivated mushrooms has been carried out in various Universities and Horticultural Institutes in India. Mushrooms are a good source of valuable protein and can fill the protein gap to a large extent in our country and also earn foreign exchange to the country.

Plate 21.1. Common edible mushrooms. 1. Button mushroom Agaricus bisporus. 2. Oyster mushroom (Pleurotus sajor-caju). 3. Paddy straw mushroom (Volvariella volvacea).

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The most commonly cultivated mushrooms species (Plate 21.1) include Agaricus bisporus (white button mushroom), Pleurotus spp. (Oyster mushroom), Volvariella spp. (Paddy straw mushroom) and Lentinus edodes (Shitake mushroom). Recently some other mushrooms like black ear mushroom (Auricularia polytricha), nameko mushroom (Pholiota nameko) enokitake mushroom (Flammulino velutipes), giant fungus (Stropharia rugosa-annulata), truffles (Tuber melanosporum), jelly fungus (Tremella fusiformis) and milky white mushroom (Calocybe indica) are also brought under cultivation BUTTON MUSHROOM Button mushroom is also known as white button mushroom or European mushroom. The mushroom derives its name from its closed, button like fruiting body during young stage. Button mushroom is a fruiting body arising from mycelium. The fruiting body is divided into the pileus (cap) and the stipe (stalk). Pileus is the fleshy, smooth, cap like structure that bears gills underneath the cap. Stipe is the stalk, attached centrally to the pileus. Button mushroom is cultivated on small to large scales all over the world and 40% of the total world production of mushroom comes from this mushroom only. This mushroom was first cultivated in Paris (France) in 1650. Later it spread to U.K., U.S.A. and other European countries. Cultivation on commercial scale is also done in Asian countries and in some African countries. In India, button mushroom production was started by Maharaja of Patiala at Himachal Pradesh. Now button mushroom production is being done in several parts of the country. India has excellent opportunities for button mushroom production due to availability of cheap labour and plenty of agricultural wastes. Button mushroom cultivation is indoor activity and is done in farm houses. Bulk chambers are used for pasteurization of compost and its size depends on the compost to be loaded. The walls and roof of the chamber should be well insulated. In India many farms have conventional pasteurization rooms where compost is filled in trays and stacked in center of the room. The room is insulated or made of hollow cement brick wall. During pasteurization airflow of 150-200 in3 /hour per ton of fresh compost is required. Insulated growing rooms or crop rooms with a provision for forced air circulation are essential. There should be provision for humidity control. The depth of compost depends on outdoor temperature. Polythene bags or wooden trays are used for compost filling. Preparation of pure culture and spawn The pure cultures of button mushroom can be prepared either by germinating the spores or by growing pieces of inner tissue of mushroom on a suitable and sterilized culture medium. The pure culture is the one, which contains the mycelium of button mushroom only. The common media used for raising pure culture of button mushroom include Potato Dextrose Agar (PDA) medium and Malt Extract Agar (MEA) medium. The pH of these media should be 6.5 to 7. The medium is transferred to test tubes or Petriplates, sterilized with steam and used for isolation of pure culture. Pure culture can be obtained by sub-culturing pure culture obtained

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from other laboratories or by transferring the mycelium developed from single spores or multispore culture or by inoculating the pieces of tissue from a fresh mushroom (tissue culture). After inoculation the test tubes or Petriplates should be incubated at 25-27°C for growth. When the mycelium is fully grown on the medium, it can be used to inoculate spawn substrate or stored in refrigerator at 0-4°C for further use. Spawn is the planting material that consists of vegetative mycelium and its substrate. It is analogous to the seed of higher plants. Spawn preparation is the first step in mushroom cultivation. Various spawn substrates like wheat or sorghum (jowar) grains, used tea leaves, cotton waste, paddy straw, coffee pulp, ipil-ipil (Seucaura glacua) leaves are used. Wheat and jowar grains are the most commonly used substrates. Washed sorghum or wheat grains are half cooked, drained to remove excess water and dried for an hour. Chalk powder (6%) and gypsum (2%) are mixed in the grains and filled in glucose bottles or polypropylene bags (up to ⅔ of the bottle or bag). They are then plugged with non-absorbent cotton and wrapped with paper and sterilized for 2 hours at 22 psi (22 pounds per square inch ) pressure in an autoclave. After sterilization the bottles are kept at room temperature for two days to check for any fungal or bacterial growth in the spawn substrate. The bottles are then shifted to inoculation chamber and sterilized with 2% formalin. Next day before inoculation ultraviolet tube is put on for half an hour to one hour. About 1 cm of culture from a culture tube is inoculated into the spawn substrate. The inoculated bottles or bags are then incubated at 25 ± 2°C temperature in the spawn growing chamber. Once a week all bottles should be checked for their purity. The spawn prepared from the culture is called master spawn or mother spawn. This can be further multiplied at the rate of 1 bottle for 20-25 bottles by transferring few grains. Fresh spawn should be used for growing mushrooms. When the spawn needs to be stored, it can be stored at 0-4°C for 4 to 6 months. Spawn should be transported either in refrigerated vans or during night time when the temperature is low. Quality control is very important in spawn making. The pure cultures of button mushrooms can be prepared either by germinating the spores or by growing pieces of inner tissue of mushroom on a suitable and sterilized culture medium. Preparation of Compost White button mushrooms are coprophilous fungi. The first step in button mushroom growing is the preparation of compost. The main bulk material for compost preparation comes from horse dung and cereal straw. Other equally useful materials for compost making are available. Vegetable base materials like wheat, paddy, barley, rye, oat, maize stalks and other vegetable plant wastes are supplemented to prepare balanced compost. They include animal manures such as horse dung, chicken, pig, sheep, mule, yak, goat, cow, bullock and elephant manures which provide good amount of nitrogen. The nitrogen fertilizers like ammonium sulphate, urea, calcium, ammonium nitrate provide readily available nitrogen and carbohydrates. The carbohydrate sources include molasses, wet brewers grain, potato waste, molasses sugar beet pulp, apple and grape, pumic and concentrated meals such as dried breweries grains, unmolassed sugar beet pulp, corn cobs, wheat/rice

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bran, seed meals of cotton, caster, soybean, linseed etc. The minerals include potash, sugar phosphate, trace metal mixtures, gypsum and chalk powder and rectify mineral deficiencies. Various compost formulations are in use according to the availability of raw materials and supplements where they are cultivated. There are two most popular methods of composting namely long method and short method. Long method requires 26 days and 7 to 8 turnings are given at varying intervals without pasteurization. This method is more popular among small growers who cannot afford boiler for pasteurization. This method reduces the nutritional value of compost and easily attacked by weed moulds, pathogens, insects, mites and nematodes. Short method is most popular for commercial production of white button mushroom. It is completed in two phases. Phase-I is accomplished in outdoors and requires seven to 10 days, while phase-II is done in pasterurization rooms and requires three to seven days. Bulk chamber is also used for phase-II. The yield in short method is very high with efficient disease control. Good compost is dark brown in colour, with 68-70% moisture content and pH 7.2-7.8. There are different kinds of growing systems for button mushroom cultivation. They are outdoor ridge beds, shelf system, Rack system, Tray system, Bag system and Trough system. Spawning and casing Broadcasting of spawn in the compost is called spawning. This provides points of further growth of mushroom mycelium in compost. Generally grain spawn provides more points of contact for faster mycelial growth. There are three different kinds of spawning methods viz., surface spawning, layer spawning and trough spawning. Spreading of mycelium from grain spawn into the compost is called spawn running. It covers the entire compost as whitish grey thread like strands of the fully grown mycelium in about 14-18 days and turns compost colour from dark brown to light brown. Optimum temperature for spawn running is 24 ± 1°C and relative humidity is 90%. Mushroom mycelium produces CO2, which stimulates mycelial growth but at higher levels inhibits the growth. The compost fully grown with mushroom mycelium must be covered with a layer of nutritionally deficient soil to initiate fruit body formation. This process is known as casing. Casing also helps compost layer against drying out, allow gases like CO2, to escape and provide physical support to fruiting bodies. Casing layer should be of even thickness for good cropping. The water content of the casing and amount of water sprayed, affect the mycelial growth and thus yield. Various formulations are in use for casing layer. They are peat mixed in different ratios with limestone and soil and soil and sand (1:1), rotten cow dung and soil (3:1), farm yard manure and loam (1:1) and spent compost, sand and lime (4:1:1). Cow dung, farm yard manure and spent compost should be more than 2 years old. To adjust pH, lime or chalk powder is added. Mixtures like shredded tree bark and paper mill waste have also been used. The casing material must be pasteurized to eliminate harmful microbes and insect pests from casing material. This can be done either by steam or chemicals. At the time of pasteurization, casing material should be loose and moist. Steam pasteurization should be done 24 hours in advance of casing and it is the most efficient way of

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pasteurization. In chemical pasteurization formaldehyde, methyl bromide and chloropicrin are used. However, formaldehyde is most popular. Cropping and harvesting In about a week's time after casing mycelium spreads throughout the casing layer. At this stage the casing layer is transformed into reproductive stage by changing environmental conditions, especially temperature. In Agaricus bisporus, room temperature at 23 ± 2°C is maintained for eight to ten days after casing. During this time the mycelium reaches the top of casing and the temperature should be brought down to 14-18°C and CO2 level reduced to 0.05-0.8% for fruit body initiation. Relative humidity should be 90%. Sudden changes in climatic conditions result in fruit body initiation and after 96 hours pin heads appear. Few pinheads develop into fruit bodies and rest abort. The crop starts producing mushrooms in 3rd week after casing and continues for 10-12 weeks. There will be a flush at every 7-10 days interval. In India commercial farms take crops for 7-10 weeks or 5 crops per room per annum. Agaricus bitorquis, requires a temperature of 28-30°C and RH 9095% for spawning and 24 ± 2°C for pinhead formations. Mushrooms are harvested when they are still closed or at button stage and packed in perforated polythene bags. A. bisporus is susceptible for dieback disease caused by virus while A. bitorquis is resistant to it. A. bitorquis has better tolerance to CO2 and superior shelf-life of the fruit bodies. Since, this variety is grown at higher temperature it is prone to various diseases and competitor moulds. Factors affecting button mushroom production The success of white button mushroom cultivation depends on the quality of compost, casing soil, spawn and environmental factors and fruit body formation of mushrooms. During spawn run the temperature range of 24-27°C for A. bisporus and 28-30°C for A. bitorquis and relative humidity at 90-96% are required. Moisture content of compost should be 68-70% in trays and shelves and in polythene bags it should be 65-68%. During cropping the temperature range of 14-18°C for A. bisporus, 22-26°C for A. bitorquis and relative humidity 80-90% are required. Environmental or abiotic factors play a key role in button mushroom cultivation and lead to poorer yield. Brown discolouration, formation of stroma, sectors, flocks, hard caps, open veils, weepers, leakers, stinkers, scales or crocodiles, hollow core, brown pith, rose comb, purple stem etc. are some of the disorders commonly observed due to the abnormal environmental factors. White button mushrooms are also affected by living organisms especially competitor moulds, The common diseases caused by the moulds include false truffle, brown plaster mould, olive green mould, green mould, yellow mould, lipstick mould, black whisker mould, and cinnamon brown mould. Parasitic moulds live on the mushroom mycelium and cause dry bubble, wet bubble and cobweb. In addition to this bacterial blotch by Pseudomonas tolaasii, and viral diseases such as La France, Watery stipe, X-disease and die back occur. Mushroom mites, mushroom flies and nematodes feed on mushroom mycelium and affect the spawn run.

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Harvesting and packing White button mushrooms are highly perishable and become unsuitable for human consumption due to physiological changes that occur after harvesting the mushrooms. The cap size at the time of harvesting is very important to arrest the post-harvest growth and should be 30-45 mm in size. Due to the presence of an enzyme, polyphenol oxidase, the initial colour of mushrooms changes to brown after harvest. Mushrooms have high rate of respiration even after the harvest, causing rapid deterioration. Hence, proper methods of package and preservation are essential. The mushrooms are packed in polythene bags of less than 100 gauge thickness, expanded polystyrene (EPS) and plastic punnets. The preserved mushrooms are packed in polyprophylene bags, polythene bags, pouches containing aluminum foil, cans and jars. White button mushrooms are preserved for long time by canning, ordinary dehydration, freeze drying, freezing and steeping in saline preservation etc. For long term storage canning and freeze drying are the common methods. OYSTER MUSHROOM Oyster mushroom is the Pleurotus spp. It is a lignicolous fungus growing under natural conditions on trees or dead woody branches of trees as saprophytes and as primary decomposers. It is one of the choicest edible mushrooms and can be cultivated in tropics. The different species of Pleurotus grow well within a temperature range of 15 to 35°C. The different species include Pleurotus sajor-caju, P. cornucopiae, P. flabellatus, P. florida, P. ostreatus and P. eryngii. P. florida is the commonly grown species. Farm design A mushroom house with two cropping rooms, one spawn running room, one substrate preparation room each 15' x 15' size enable the grower to obtain an yield of 5-100 kg daily. Since oyster mushroom species tolerate wide range of temperatures, they can be grown in thatched huts, shed with asbestos or light roofing material and polythene tents. Brick wall with thatched roof is suitable in a rural set up. The substrate preparation room should have 1). A tub to soak minimum 15 kg straw. 2). A container to boil water for pasteurization. 3). Wire meshed side table to spread and cool the pasteurized straw and also to allow the excess water to drain off. 4). A side table with zinc sheet surface to carry out bag filling operation. This room can also be used for package work of mushrooms. Cropping and spawn running chamber should be provided with additional fine mesh shutters to prevent the entry of insect pests. Every room should have 2 to 3 ventilators, good drainage system and carpet pit to hold the disinfectant solution. The platform may be cemented or stone slabs can be spread over the floor. Raising pure culture and spawn preparation Pure culture is prepared either from a) tissue culture raised from single or multispores. For tissue culture or for spore culture a big healthy fruit body with veil still intact is selected from cropping tray. Lower portion of the stipe is cut off at the soil level with the help of a pre-sterilized knife. Then the fruit body is cleaned with a

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bit of cotton moistened in 50% ethanol to remove the soil particles, if any and finally dipped in 0.1% mercuric chloride solution for 30-60 seconds to avoid any chance of contamination. Fruit body is rinsed in three changes of sterile water to remove mercuric chloride. Small pieces of the tissue from the junction of stipe and pileus of fruit bodies are cut and inoculated onto Potato Dextrose Agar medium. These tissues develop mycelium and grow into a colony and provide the starting point for subsequent spawn preparation. In spore culture the sterilized fruit body is mounted on a sterilized Petri dish. This is covered by a sterilized beaker for 24-48 hours. After deposition of a thick spore mass in the Petri dish, the beaker and mushroom are removed and the Petri dish is covered by a sterilized lid. This spore mass is then used for raising pure culture either through single or multispores. Raising of pure culture through multispores is the best method. For spawn production jowar, wheat or maize grains can be used. They are washed in water and half boiled for 40-45 minutes. Later they are spread on news papers for about an hour which helps to break lumps and drains excess water. Calcium carbonate or chalk powder (6%) and Gypsum (2%) are mixed with half boiled grains and then filled in milk/glucose bottles up to ⅔ of the bottle. The bottle mouth is cleaned and plugged with non-absorbent cotton. Plugs are wrapped with paper and bottles are sterilized in an autoclave for 2 hours at 22 psi pressure. Sterilized bottles are transferred to inoculation chamber. A piece of pure culture is inoculated aseptically into the bottles. Later the bottles are transferred to the spawn growing chamber where the temperature is maintained around 25 ± 2°C and not more than 34ºC. The fungal mycelium spreads on the grains and occupies the whole bottle. This culture in spawn bottle is called mother spawn from which 30 bottles of spawn can be raised. Care should be taken that spawn is free from contamination by mostly fungi and sometimes bacteria and virus. Spawn can be stored for two months at room temperature or in refrigeration to obtain maximum yield. Substrate and bed preparation The different species of Pleurotus can be easily grown on a variety of agro wastes like straw of wheat, paddy, ragi, stalks and leaves of maize, jowar, bajra, cotton, corncobs, waste paper, used tea leaves and oil palm mesocarp waste. Generally, wheat or paddy straw is used for cultivation as they are commonly available substrates. The selected substrates should be sterilized to kill or inactivate the organisms present in the substrate. It can be achieved by chemical sterilization where the cut straw is dipped in water present in a drum of 100 litres capacity. Then 125 ml of 40% formaldehyde with 7.5% g of bavistin is added and the mouth of the drum is closed with a polythene sheet. After 12-18 hours of soaking the straw bits are kept on clean slanting concrete floor to drain the excess of chemical solution. Then it can be used for spawning. In hot water treatment, the straw bits are boiled in hot water at 70-75°C for one hour. The substrate is then put into basket to drain water and used for spawning. In fermentation type of sterilization paddy straw bits are made into pile of 3-4' height and 5' wide and kept as such for 2-3 days. This results in microbial formation which increases the temperature of the heap and causes death of microorganisms. The fermented straw can be used for spawning after cooling. In

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steam pasteurization the straw bits are pre-wetted and are placed in wooden trays or piled into a heap. In the pasteurization room its temperature is raised to 75-80°C for 2 hours with steam. Then the straw is allowed to cool and used for spawning. Though oyster mushroom does not require other supplements, the supplements like ammonium sulphate or urea (0.5 to 1%) and lime (1%) can be used. Chicken or horse manure at the rate of 10% can be used in place of nitrogen fertilizers. Polythene bags of 60 x 30 cm are used for bed preparation. They are tied with a rubber band at the closed end. Pure spawn is taken into a tray smeared with dettol and 60gms of any pre-sterilized pulse powder is added and mixed with the spawn thoroughly. One layer of sterilized straw is placed in the polythene cover up to 5cm height and spawn is sprinkled all along the circumference of the bag. Layer after layer of spawn and straw is placed in the bag until it is full. The bed is made compact by pressing the straw bits and is closed by tying another rubber band at the open end. 20-25 holes are made to provide aeration. The bags are kept in a cropping room for 15-20 days. Harvesting and preservation The crop cycle is of 45 days and 3 crops can be harvested from each bed. Daily watering and maintenance of 75% RH should be taken care off. Darkness for a period of 10-15 days helps for quick growth of the mycelium in the beds. When the mycelium covers the beds, the polythene cover is removed. After two days of opening of the beds 1 to 10 kg of mushrooms can be produced from each bed in two to three flushes. Harvesting should be done without any damage to the pileus and kept on trays without too much heaping. Mushrooms have a short shelf life and are highly perishable. After harvesting, the fruiting body quickly looses water and hence weight, with the result it becomes deliquescent and loses its texture, becomes shiny and sheds off spores. Ultimately it develops undesirable look and emits foul smell. Hence to avoid deterioration in mushroom quality different methods of processing are required to increase the shelf life of mushrooms. In freezing method, the collected mushrooms are washed and treated with liquid nitrogen at -120°C for six minutes and stored at -25°C in deep freeze. This treatment makes the mushroom intact as such without any change in colour or quality. Such mushrooms are called frozen mushrooms. In Freeze Drying method mushrooms are frozen at -20°C and dehydrated by heating the frozen mushrooms under very low vaccum of less than one tonne for 12-16 hours. This result in loss of water up to 90% of the total weight of fresh mushroom approximately. 10 kg of fresh mushrooms are immersed in boiling water containing 1 % common salt or 1% citric acid. The boiling should be done for 4-5 minutes with constant stirring. The foam developed while boiling is to be removed. These blanched mushrooms are dried on blotting paper and preserved in steeping solution of sodium chloride (2%), citric acid, sodium carbonate and potassium metabisulphite (0.15%) and kept at 21-28°C for long term preservation. In dehydration method washed mushrooms are blanched for 3-5 minutes in live steam or boiling water and cooled immediately. Then they are dipped in solutions of 300 ppm potassium metabisulphite and 400 ppm chlorine solutions for better colour. Then they are dried under sun for 36 to 48 hours or in a mechanical drier at 50-60°C.

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Canning, packing and marketing The bleached, graded mushrooms are filled to three fourth capacities of the tins and then filled with bleaching solution containing 15g of solution and 1 g citric acid per liter of water. The filled cans are kept in boiling water bath so that a temperature of 850C is attained in the center of the can. The cans are then sealed by hand or by automatic sealers. The sealed tins are sterilized by autoclaving at a temperature of less than 118°C and then cooled by keeping them in galvanized iron tank with constant over flow of water. Packing the bleached, graded fresh mushrooms in polythene packets of 200g, 250 or 500g can be done. If dried mushrooms are to be packed the bleached mushrooms are to be dehydrated either by sun drying or oven drying at 55 to 60°C. Due to drying the mushrooms loose ten time their weight and become brittle. These can be packed in polythene packets of 200g, 250g, 400g, 500g, 1kg, 2kg, 4kg, or 5kg. Bulk packing can be done in 1 kg wooden boxes, 4kg plastic crates, plastic trays, plastic cans etc., The oyster mushroom is a perishable product which degrades quickly after harvest. Hence, it is to be marketed afresh. It has to be organized by government or through farmer cooperatives to stabilize the prices. Otherwise, the middle man in mushroom market will take the benefit. Factors affecting oyster mushroom production Oyster mushroom production is greatly influenced by abiotic factors like temperature, pH, light, RH, gases etc., and biotic factors like competitor molds and other pathogens. Optimum growth and fruit body formation requires 20-30°C, pH between 6.0-7.0, RH of 70-85% and diffused light. Poor ventilation results in the formation of abnormal fruit bodies. During spawn run oyster mushroom can tolerate high carbon dioxide (20%) concentration and during cropping less CO2 concentration (0.6%). Weed moulds or competitor moulds such as species of Penicillium, Aspergillus, Trichoderma, Mucor, Rhizopus etc., and pathogens like species of Cladobotryum and Verticillium are prevalent in the substrates. Bavistin spray (0.01%) is more effective in controlling weed moulds. Flies can be controlled by spraying Nuvan (0.1%). PADDY STRAW MUSHROOM Paddy straw mushroom (Volvariella volvacea) is mainly grown in tropical and subtropical countries of Asia on paddy straw substrates. Hence it is referred to as tropical mushroom, warm mushroom or paddy straw mushroom. It is commonly known as Chinese mushroom, since it was first cultivated in China about 300 years ago. The genus Volvariella consists of about hundred species distributed all over the world. Many cultivated forms of Volvariella belong to V. volvaceae, V. esculenta and V. diplasia. Paddy straw mushroom is a fast growing mushroom and requires only 8-10 days from spawning to harvesting. In India it can be cultivated as gap filling crop in the period when oyster mushroom and white button mushroom cannot be grown. The fruit body of paddy straw mushroom consists of six stages. They are pin head, tiny button, button, egg, elongation and mature stages. The button stages and

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the egg stages are ovoid in shape. At these stages, the mushrooms have great demand in the market. The button stage is covered by a universal veil. In the egg stage pileus protrudes out of the veil and the stipe is covered by veil. The veil covering the stipe is called volva. In the elongation stage the upper part of the stipe elongates. On the lower part of the pileus lamellae can be seen from the margin extending till the stalk. Growth requirements Paddy straw mushroom grows well on paddy straw. In Hong Kong cotton waste composting method is widely used. Substrates having glucose and polymers of glucose support the growth of this mushroom to a great extent. As organic nitrogen supports the growth of this mushroom, it is necessary to supplement the straw, cotton or other cellulose substrates with organic nitrogen. Addition of vitamins like ascorbic acid, pyridoxines, riboflavin, thiamine, growth promoters like gibberellic acid and also L-amino benzoic acid gives significant yield. The mushroom requires 30-35°C temperature for mycelial growth and 28-30°C for fruit body formation. The pH required is 6-7 and RH is 80-90%. Diffused light for 8-10 hours per day should be provided for fruit body formation. Good ventilation provides more O2 and removes excess CO2 during fruit body formation. Raising pure culture and spawn preparation Pure cultures can be obtained by two methods. These cultures can be grown on culture media such as potato dextrose agar medium and malt extract agar medium. Tissue culture method is the most widely used one for large scale production of spawn. In this method the cleaned fruit body is dipped in 0.1% mercuric chloride solution for 30 seconds and then washed 4-5 times with sterile distilled water to remove excess chemical. The fruit body is dried superficially with sterile tissue paper and cut length wise. At the inner surface from the point of junction of pileus and stipe small bits of tissue are taken and placed on the agar surface of the test tube. The entire process should be carried out under sterile conditions. The test tubes are then incubated at 30-35°C for 6 to 7 days. White mycelium comes out of the tissue. Contaminated tubes are discarded and healthy ones can be used directly in spawn substrates. In spore culture method spores of paddy straw mushroom are collected by taking spore prints from which the spore masses are transferred on to an agar slant under sterile conditions. In taking a spore print a mature and opened mushroom is cut at the upper end of the stalk and placed on a clean paper. After 10 minutes spore print is seen. This spore print is discarded and the mushroom is again placed in a sterile Petri plate and covered with a clean beaker. In about 20-30 minutes the spores are shed on the Petri plate and are ready for inoculation. The inoculated tubes are incubated at 30-32°C for 4 to 5 days for mycelial growth. The cultures thus obtained can be used directly for inoculating the spawn substrate. A variety of following substrates are used for the preparation of paddy straw mushroom spawn. 1.

Cereal grains like sorghum, wheat, rye etc., are boiled for 20-30 minutes to increase the moisture content of the grains to 40-50%. After drying for few hours the grains are mixed with 2% calcium sulphate and 0.5%

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calcium carbonate to adjust the pH of the substrate to 7.5 and to keep the grains dry and separate. 2.

In paddy straw substrate, the paddy straw is cut into 2.5 to 5 cm long bits, soaked in water for 2–4 hours. The excess water is drained out and mixed with 1 % calcium carbonate and 1 to 2% rice bran.

3.

Used tea leaves are collected and washed thoroughly with water, drained and mixed with 2% calcium carbonate.

4.

Card waste grade of cotton waste from textile industry is soaked in water for few hours and drained. Further 2% calcium carbonate is added.

Any of these four spawn substrates are filled in bottles or polypropylene bags (300 g per bottle or bag) and plugged with non-absorbent cotton. Polypropylene rings are used to provide mouth for bags and plugged with cotton. They are then sterilized in an autoclave at 22 1b pressure for about 2 hours and then cooled to room temperature. These bottles and bags are inoculated with paddy straw mushroom mycelium from culture media under sterile conditions and incubated at 30-35°C for about three weeks. Such a culture is called ‘master culture or master spawn’. From these bottles spawn is inoculated into other bottles or bags to form commercial spawn which can be used directly by the grower. Spawn of 12-20 days old is most ideal for cultivating the paddy straw mushroom. Substrate preparation and cultivation of mushrooms A variety of waste materials like paddy straw, cotton waste, oil palm bunch, water hyacinth, banana leaves, saw dust and sugarcane bagasse are used for cultivation. The most widely used ones are paddy straw and cotton waste. Common methods for growing paddy straw mushroom include: 1) Outdoor cultivation by using paddy straw: In this method clean fresh dried paddy bundles (32), each 800g to 1kg are made by tying each of them at one or two places. The bundles are steeped in water for 24-48 hours. After draining the excess water the bundles are dipped in boiling water for half an hour. Excess water is drained off by spreading the bundles on slope. The beds are to be laid on raised (75-90cm) wooden or concrete platforms. Four bundles are to be placed length wise with butts on one end and loose ends on the other. Another four bundles are kept in line with first four bundles but with butts on the other side. This forms the first layer with eight bundles and is spawned at 3-4 inches away from the edges. Another layer of 8 bundles are placed in the same manner as the first layer and spawned. Similarly third and fourth layers of straw are placed one after the other. The whole bed is pressed tightly and covered with a polythene sheet and supported by a bamboo framework. The polythene sheet helps to increase both temperature and humidity. Such beds are made in east-west directions under the shade created by trees or creepers. After four days the sheet is removed and slight watering is done on 6th day. 2) Cage culture of paddy straw mushroom (indoor cultivation): In this method paddy straw bundles are arranged in wooden cages (1m x 50cm x 25cm). Sixty bundles of fresh dry paddy straw bundles are soaked in boiling water for 20-30 minutes. After cooling, excess water is drained. Six layers each with ten straw

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bundles and spawn are placed one after the other in the wooden cage. The beds are sprayed with 0.1% Malathion and 0.2% Dithane Z.78. The beds are then covered with polythene sheet and are disinfected with 4% formalin. The spawned cages are kept in a room or shed with around 30°C temperature for the better growth of the mycelium. Pinheads appear after 10-15 days of spawning. 3) Cotton waste composting method: The method is widely used in Hong Kong for commercial production of mushrooms. The yields are 25-40% higher in cotton waste compost than in paddy straw. The compost is prepared by using cotton waste obtained from textile industry, rice or wheat bran (4%) and limestone (1 to 3%) to adjust pH. The mixture is sprayed with water and a stack of 1 to 1.5m height is made which is left for 2-4 days for fermentation. One turning is to be given in between this period. The compost is filled in beds made of iron frame lined with polythene sheets of 4mm and steam is sent through the compost in order to raise the temperature to 66°C for 2-4 hours. Then fresh air is let in to cool it to room temperature. The spawn is mixed with the compost and incubated at 32-34°C. 4) Poly bag method: Finely chopped paddy straw and small pieces of waste paper bits (1:1) are soaked separately in water containing formalin and bavistin (125ml of 35-40% formalin and 7g bavistin in 100 liters of water) for 16-18 hours. These soaked pieces are spread on a cemented floor or wire mesh for half an hour; then mixed thoroughly with spawn and filled in polypropylene bags with few holes. The bags are tied with thread and incubated. After ten days the bags are cut open and incubated for mushrooms to appear. Regular spraying of water should be done. Harvesting and Marketing The paddy straw mushrooms appear in about ten days of incubation. They are picked at egg stage, before the volva breaks and are not allowed to grow to their maximum size. During cropping, fresh air, diffused light and 80-90% RH are to be provided. 70-90% of the expected yield is obtained from the first flush itself, it comes in about 10 days after spawning. After 3 to 5 days second flush appears which gives only 10- 30% of the total crop. In between flushes thorough watering is necessary. In a period of 15-20 days paddy straw mushrooms of about 2.4 to 7.3 kg/m are obtained. The mushrooms picked at button stage have shelf life of about 6-8 hours at room temperature. They can be stored in perforated polythene bags for 4 days at 10-18°C. The paddy straw mushrooms can be stored for a week by bleaching them in 10% brine solution and by air drying. The mushrooms can be preserved by sun drying or in a mechanical drier at 50-55°C for 5-6 hours. The fresh mushrooms packed in polythene bags are to be marketed within 6-8 hours after packing. In China fresh mushrooms are transported in wooden cases having three compartments filled with mushrooms along with ice. In Taiwan and Thailand they are packed in bamboo baskets. At the center of the basket, an aeration channel is provided and dry ice is wrapped in paper and placed on the top of the mushrooms.

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Biotic factors affecting paddy straw mushrooms These mushrooms are attacked by various weed moulds and insect pests. Button rot disease is caused by competitor moulds like Coprinus spp., Rhizopus stolonifer, Penicillium spp., Psathyrella spp. Trichoderma spp., Aspergillus spp., and Sclerotium spp. Rhizoctonia solani reported on the substrate reduces the formation of sporophores while Podasora faurelli inhibits completely the growth of the mushroom mycelium. Mites also damage the buttons of mushrooms. They can be prevented either by regulating environmental factors like temperature, RH and ventilation or by maintaining strict hygiene or spraying with chemicals like Zineb (0.2%) and Captan.

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Chapter - 22

Mycorrhiza

Several symbiotic groups, phosphorous solubilizers, plant growth promoters and other such beneficial important microorganisms are reported from different soils. Balanced microbial systems contribute to the sustainability in agriculture, forestry and range management. In this regard mycorrhiza has a substantial role. The term mycorrhiza (fungus root) was coined by Frank (1885). Harley and Smith (1983) have defined mycorrhiza as an association between fungal hyphae and roots of higher plants concerned with absorption of mineral substances from the soil. Brundrett (2004) has defined as a symbiotic association for one or both partners between a fungus and a root of living plant that is primarily responsible for nutrient transfer. Mycorrhizas were earlier classified into arbuscular, ectomycorrhizal and orchid mycorrhizal association based on the relative location of fungi in the roots. The basic types of mycorrhizas now established are: MYCORRHIZA TYPES Ectomycorrhiza (EM) Ectomycorrhia is characterized by a fungal sheath or mantle that encloses the root forming Hartig net, which is plexus of fungal hyphae between epidermal and cortical cells. Ectomycorrhizal roots are generally short, swollen, dichotomously branched and with distinctive colours of white, black, orange, yellow and olive green. These are common association for forest trees and for shrubs particularly in sub arctic and temperate regions. Many of the host plants that show ectomycorrhizal association belong to the families Pinaceae, Fagaceae, Betulaceae and Myrtaceae. The fungi that form ectomycorrhizal associations are Basidiomycetes with representatives from twenty five families. Some Ascomycetes and two species of Zygomycetes are also reported to form ectomycorrhizas. These fungi can be cultured in laboratory medium. Some of the examples of ectomycorrhizal fungi are Boletus edulis, Amanita muscaria, Suillus sp., Russula senecis, Pisolithus tinctorius, Lactarius deliciosus, Laccaria laccata, Rhizopogon roseolus and Paxillus luteolus (Plate 22.1). Ectendomycorrhiza Ectendomycorrhiza are variants of ectomycorrhiza and also display morphological characteristics of the endomycorrhiza. The association appears to be an intermediate type. These types are found in Pinus and Larix species.

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Plate 22.1. Fruiting bodies of ectomycorrhizal fungi. 1. Amanita muscaria. 2. Suillus sp. 3. Russula senecis

Arbuscular Mycorrhiza (AM) Arbuscular mycorrhiza is the most common mycorrhizal association. They have a widespread distribution throughout the plant kingdom and form mutualistic relationship with most of the vascular plants. Families that rarely form arbuscular mycorrhiza include Cruciferae, Chenopodiacaeae, Polygonaceae and Cyperaceae. Families that do not form arbuscular mycorrhiza incluede Pinaceae, Betulaceae, Fumariaceae, Commelinaceae, Utricaceae and Ericaceae. The fungal partner belongs to Glomeromycota. The fungus forms vesicles within or between cortical cells that act as storage or reproductive organs. Arbuscules are formed within the cortical cells and these provide a large surface area of contact between host and fungus. The genera, which form arbuscular mycorrhizal (AM) fungal association are Acaulospora, Ambispora, Archeospora, Dentiscutata, Fuscutata, Diversispora,

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Entrophospora, Geosiphon, Gigaspora, Glomus, Intraspora, Kuklospora, Otospora, Pacispora, Paraglomus, Racocetra, Quatunica, Centrospora and Scutellospora (Plate 22.2) (Schenck and Perez 1990; Schüßler et al., 2010), etc.

Plate 22.2. Arbuscular mycorrhizae (AM fungi). 1. Vesicles (X100); 2. Spores of Glomus versiforme (X100); 3. Spore of Entrophosphora sp (X100); 4. Sporocarp of Sclerocystis microcarpus (X100); 5. Safflower plants inoculated with Glomus mosseae in pot; 6. Safflower plants inoculated with G. mosseae in field.

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Orchidaceous mycorrhiza Orchidaceous mycorrhiza is found in the family Orchidaceae. The fungal partner generally is species of Ascomycetes or Basidiomycetes. In nature orchid embryos normally become infested with mycorrhizal fungi. Thin walled hyphae enter the protocorm through epidermis and anastomose repeatedly with the cortical cells to form pelotons or hyphal coils which provide a large surface area of contact. Ericoid mycorrhiza Ericoid mycorrhizal association involves mostly Ascomycetes fungi but sometimes Basidiomycetes also. Hosts include the members of Ericales, which grow on peaty and acidic soils. These fungi are present in the young cortical cells of the host with branched and coiled vegetative mycelia but never penetrate the stele. They lack Hartig net and instead of forming arbuscules, ericoid mycorrhizal fungi form hyphal coils, which are intracellular. Culturing of these fungi in laboratory medium is rare. Monotropoid mycorrhiza Monotropoid mycorrhiza occurs in family Monotropaceae which lacks chlorophyll and is totally dependent on the mycorrhizal fungi for supply of carbon. The fungal partner is basidiomycetes member. The hyphae of the sheath ramify in the surrounding humus. Hartig net is also produced. Fungal pegs achieve a close contact with cortical cells of the host. The contents of the fungal pegs are ultimately released into the sac enclosed by the host membranes. Arbutoid mycorrhiza Arbutoid mycorrhizal associations are found with members of Arbutoideae and Pyrolaceae. The fungal partners are basidiomycetes. Long roots have very sparse infections but intercellular hyphae also form a Hartig net. Short roots have a thicker outer sheath and well developed Hartig net between the outer cortical cells. The fungus also penetrates and it forms coils within the cells of the outer cortex. For identification of mycorrhizal fungi and to explore the structural and regulatory genes in both fungus and plant that permit mycorrhiza formation besides morphological, anatomical, histochemical and biochemical tools, molecular and genetical tools are also used. Of the seven types of mycorrhizas, the two prevalent mycorrhizal types are the ectomycorrhizas common with woody species related to forestry and the arbuscular mycorrhizas more often associated with the herbaceous plants with relevance to range, horticultural, ornamental, medicinal and crop plants. A large proportion of land area in India shows clear evidence of soil degradation which in turn is affecting the countries productive resource base which is because of salinity, alkalinity, soil erosion, water logging etc. The fundamental problem that the country is facing today is the rapidly increasing pressure of population on the limited resources of land. In order to meet the pressure of population it is essential to efficiently manage the agriculture inputs for sustaining high crop productivity on long term basis with minimum damage to environment. In

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order to reduce cost of agrochemicals and harm rendered by them biofertilizers are used. Mycorrhiza helps the plants to acquire not only mineral nutrients from the soil, especially immobile elements such as P, Zn, Cu but also mobile ions such as S, Ca, K, Fe, Mg, Mn, Cl, Br and N. They also help in increasing the extent of soil particle aggregation. Further, most of the economically important plants have been found to be mycorrhizal, and the subject is currently gaining much attention in agriculture, horticulture and forestry. It is also known that Glomalean fungi existed four hundred million years ago and helped in the colonization of the land by primitive plants. Thus the primary land plant establishment was also due to mycorrhiza. The mycorrhizal symbiotic association appears to have evolved as survival mechanism for fungi and higher plants thus making each to survive in the existing environment of low temperature, soil fertility, drought, diseases, extreme environments and other stress situations. Mycorrhizas offer first line biological defense to host plants against stress for crops and forest trees. BENEFITS DERIVED FROM MYCORRHIZA BY HOST PLANTS Mycorrhizal association helps in increased nutrient and water uptake by absorption through improved absorptive area, translocation of elements to host tissues and their accumulation. Due to the unique ability of mycorrhiza to increase the uptake of ‘P’ by plants, mycorrhizal fungi have the potential for utilization as a substitute for phosphatic fertilizers. Mycorrhizal fungi improve host nutrition by increasing the ‘P’ delivery and other minerals to roots and plants. Ectomycorrhizal fungi permeate the F and H horizon of forest floor and thus minerals get mobilized in these zones followed by their absorption before they reach sub soil system. AM fungi are known to degrade complex minerals and organic substances in soil and thus make essential elements available to host plants. Mycorrhizal association is known to offer resistance in host plants to drought and plant pathogens. Mycorrhizal association increases the tolerance of the plant to adverse conditions and helps in the production of growth hormones like auxins, gibberellins and growth regulators such as vitamin B. Mycorrhizal fungi contribute to organic matter turnover and nutrient cycling in forest and crop land ecosystems. Mycorrhiza help in soil aggregation, soil stabilization and add strength to soil fertility. Mycorrhizas are symbiotic not parasitic, therefore they live hand in hand with other living organisms and are non-pollutants. MORPHOLOGICAL DIVERSITY IN AM FUNGI Morphological characters that are stable and discrete are used in identification and classification of AM fungi. Some of the important morphological characters considered are: Hyphal characters Vegetative hyphae have been differentiated functionally into infective, absorptive and runner hyphae. Fungal hyphae may be long ‘H’ shaped parallel connections as in Glomus, constricted hyphae near branch points as in Acaulospora and Entrophospora and coiled, irregularly swollen hyphae with lateral projections or knobs in Gigaspora and Scutellospora.

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The mycelia also form specialized structures like arbuscules, vesicles (Plate 22.2) and auxillary cells. Abrupt narrowing off of branch hyphae form arbuscules in Gigaspora, and in Glomus arbuscules are formed by reduction in hyphal width. Vesicles in Glomaceaee are subglobose to elliptical wheras in Acaulosporaceae they are pleiomorphic and knobby. Auxiliary cells are found only in Gigasporineae. Subtending hyphae The stalk of the spore known as subtending hypha or sporophore has importance in identification. Subtending hypha may be absent (Acaulospora); simple, straight or recurved (Glomus); swollen and straight but often appear sessile due to detachment from saccule and two scars present on either side of the spore (Entrophospora) and sporophore bulbous (Gigaspora and Scutellospora). Spores, sporocarps and sub-cellular structures Spores may be formed singly or aggregated in loose/compact (with or without peridium) sporocarps. Spores in AM fungi may be azygospores or chlamydospores. Chlamydospores are seen in Glomus and Scutellospora. Azygospores are seen in Acaulospora, Entrophospora, Gigaspora and Scutellospora. Colour of the spores varies from hyaline, yellow, reddish-brown, orange, brown and black. Size of the spores is also variable and ranges from 50 to 250 μm (diameter) in Glomus, Acaulospora and Entrophospora, whereas in Gigaspora and Scutellospora the diameter of spore exceeds 300 μm. The shape of spores may be globose, subglobose, ovoid, pear-shaped, ellipsoid, oboviod, reniform or irregularly elongated. The cytoplasm within the spores may appear reticulate in a polygonal pattern or may be vacuolated because of the presence of many lipid droplets of variable sizes. Seven kinds of wall layers have been described in AM spores namely evanescent, unit, laminated, membranous, coriaceous, amorphous and expanding. Spores of different genera also differ in number of wall layers. Spores of Gigaspora and Glomus have 1-2 wall layers, whereas Acaulospora, Scutellospora and Entrophospora have more than three wall layers. ECOLOGICAL DIVERSITY IN AM FUNGI Ecological diversity provides useful information regarding biogeographical distribution, dispersal patterns, and competitive interactions by members, organisms in plant communities and soil microorganisms. The biogeographical distribution, dispersal patterns and competitive interactions of AM fungi with plant communities and soil microorganisms are as follows: Biogeographical distribution The available information indicates that there is more or less uniform distribution of AM fungi, though some may predominate in certain areas with broad ecological range. AM fungi are mostly present in the top 15-30 cm of soil and their numbers decrease markedly below top 15 cm of soil. The distribution of species of

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AM fungi varies with climatic and edaphic conditions for example, Gigaspora and Scutellospora are common in tropical soils whereas, Acaulospora species favour soils of pH below 5. Dispersal patterns AM fungi are indigenous to soil throughout the world. Many AM species are present in most of the continents of the world. Active dispersal of AM fungi is usually by spread from one living root to another through AM propagules like mycelia and spores that can be moved by biotic and abiotic agents. Dispersal of spores over greater distances is dependent upon passive dispersal by wind and water especially in arid environment. Animal dispersal of AM spores is well documented and occurs through ingestion and egestion of spores. Interactions with plant communities AM fungi are geographically ubiquitous commonly associated with plants in agriculture, horticulture, pastures and tropical forests. More than 80% of all terrestrial plant species form mycorrhizal associations. Arbuscular mycorrhiza is the most common mycorrhizal association and has a widespread distribution throughout the plant kingdom forming mutualistic relationship with most of the vascular plants. Families that rarely form arbuscular mycorrhiza include Cruciferae, Chenopodiacaeae, Polygonaceae and Cyperaceae. Families that do not form arbuscular mycorrhiza incluede Pinaceae, Betulaceae, Fumariaceae, Commelinaceae and Utricaceae. The fungal partner belongs to Glomeromycota forming vesicles within or between cortical cells that act as storage or reproductive organs and arbuscules is formed within the cortical cells providing a large surface area of contact between host and fungus mycelim which is formed inside and outside the root. The genera, which form arbuscular mycorrhizal (AM) fungal association are Acaulospora, Ambispora, Aracheospora, Diversispora, Entrophospora, Geosiphon, Gigaspora, Glomus, Intraspora, Kuklospora, Pacispora, Paraglomus and Scutellospora Mycorrhizal association helps in increased nutrient and water uptake by absorption through improved absorptive area, translocation of elements to host tissues and their accumulation. The unique ability of mycorrhiza helps to increase the uptake of ‘P’ and other nutrients by plants suggesting that mycorrhizal fungi have the potential for utilization as a supplement for phosphatic fertilizers. Ectomycorrhizal fungi permeate the F and H horizon of forest floor and minerals get mobilized in these zones by hyphal network followed by their absorption before they reach sub soil system. AM fungi are known to degrade complex minerals and organic substances in soil and thus make essential elements available to host plants. Mycorrhizal association is known to offer resistance to drought, plant pathogens, tolerance to adverse conditions, release growth hormones like auxins, gibberellins, growth regulators such as vitamin B and also contribute to organic matter turnover along with nutrient cycling in forest and crop land ecosystems. Mycorrhiza are known to help in soil aggregation, soil stabilization and add strength to soil fertility. Mycorrhizas are symbiotic and hence they live hand in hand with other living organisms and are non-pollutants besides sustaining competition.

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The occurrence of AM fungi in roots has been reported from an exceptionally wide range of plants. Beside roots the colonization have been reported in other plant parts also for example, in leaves of Salvinia, in senescent leaves of Fumaria hygrometrica, in decaying peanut leaves and rhizomatous tissue of Zingiber officinale. Colonization has also been reported from scales of Colocasia antiquorum, Elettaria cardamomum, Musa paradisica and Sanseviera trifaciata, garlic and ginger. Arbuscular mycorrhizal interactions bring about certain changes in the host metabolism and physiology. These include increased production of cytokinins due to the inhibition of cytokinins as evidenced by the presence of two gibberllins like substances in culture extracts of Glomus mosseae and increased nitrate reductase activity. Mycorrhizal symbioses are important considering the fact that as 70-80% terrestrial plants are mycorrhizal, thus help in the acquisition of water and minerals, besides protection from diseases. The development and formation of mycorrhizae cause changes not only in host plant but also in the rhizosphere microbial community resulting in interaction among rhizosphere microorganisms. Rhizobacteria showing a beneficial effect on mycorrhizae are often termed as ‘mycorrhizae-helper bacteria’. Studies have shown that inoculation with PGPR and diazotrophs along with AM fungi may increase plant growth and yield. Colonization by AM fungi may modify the root exudates pattern, which may act as chemo- attractants for the soil bacteria. PGPR beneficial effect was also observed in Eucalyptus diversicola along with an unidentified bacterium resulting in 49% more shoot dry weight than the uninoculated control. The effects of combined inoculation with PGPR, AM fungi and rhizobia have been tested by many workers Extracellular metabolites produced by the above organisms could possibly be the reason for the synergistic effects. Different mechanisms allow AM fungi and PGPR to increase stress tolerance in plants. This includes the intricate network of fungal hyphae which block pest access to roots and various biocontrol mechanisms of PGPR. Inoculation of apple-tree seedlings with Glomus fasciculatum and G. macrocarpum suppressed the apple replant disease (ARD) caused by phytotoxic micromycetes. The number of colony forming units per unit soil (CFU) of phytotoxic micromycetes decreased, whereas CFU of the genus Azospirillum was higher. It may be assumed that the use of some AM fungi and such bacteria can replace the chemical treatment of the soil with ARD. AM fungi protect the host plant against root infecting pathogenic bacteria. The mechanisms involved in these interactions include physical protection, chemical interactions and indirect effects. The rhizosphere is thus influenced by the plant roots as well as by mycorrhizal fungus. The mycorrhizosphere is the zone influenced by both the root and the mycorrhizal fungus and it includes the more specific term ‘hyphosphere’ which refers only to the zone surrounding individual hyphae. Bacterial communities associated with plant roots may be affected by root-colonisation with AM fungi. This may be due to metabolic products of AM fungi and their resultant changes. The hyphal exudates might have been detrimental or stimulatory effect on rhizosphere bacteria. Rhizosphere bacteria remain in close association with AM fungi. Endosymbiotic bacteria closely related to the genus Burkholderia have been found in

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symbiotic AM fungi Gigaspora margarita, Scutellospora persica and Scutellospora castanea. PGPR and AM fungi interactions have shown synergistic effects. AM interactions bring about certain changes in the host physiology. These include increased production of cytokinins due to the inhibition of cytokinins degradation by compounds produced by the fungus or plant as a result of the interaction. The presence of two gibberllins like substances in culture extracts of Glomus mosseae and increased nitrate reductase activity has also been reported in the mycorrhizal plants.

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Chapter - 23

Sexual Reproduction of Fungi - Recent Trends

Reproduction is the formation of new individuals having all the characteristics typical of the species. Two general types of reproduction are recognized, asexual and sexual. Asexual reproduction is sometimes called somatic reproduction. Sexual reproduction on the other hand is characterized by the union of cytoplasm (Plasmogamy) and two nuclei (Karyogamy) followed by meiosis. The sexual reproduction results in a very high incidence of recombination and formation of new genotypes. This enables fungi to adapt readily to several environmental conditions. In the formation of reproductive organs, the entire thallus is converted in to one or more reproductive structures, so that somatic and reproductive phases do not occur together in the same individual. Fungi that follow this pattern are called holocarpic. In majority of fungi, however, the reproductive organs arise from only a portion of the thallus, while the remainder continues its normal somatic activities. The fungi in this category are called eucarpic. The holocarpic forms are less differentiated than eucarpic. In general, asexual reproduction is more important for the colonization and multiplication of the species because it results in the production of large numbers of individuals and the asexual cycle is usually repeated several times during the season. Many fungi exist in perfect stage but some do produce only asexual phases or mycelial phases. It is presumed that their perfect stages belong to Ascomycotina and Basidiomycotina. Till their perfect stages are discovered such fungi are considered as Fungi Imperfecti. Some fungi like rusts produce more than two morphological spore types called pleomorphism. In this system, the term teleomorph is used to describe the sexual stage of a fungus while the term anamorph is used for its asexual stage. The term holomorph is used to describe the whole fungus in all its facets, forms and potentialities. Recently, there has been a proposal to replace these terms with meiosporic forms (teleomorph) and mitosporic forms (anamorph) (Reynolds and Taylor 1993, Korf and Hennebert 1993). The fungal thallus is a remarkable entity with great potential. Generally three types of reproduction are recognized in fungi. 1. Vegetative reproduction. 2. Asexual Reproduction. 3. Sexual reproduction.

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SEXUAL REPRODUCTION It involves the union of two nuclei, gametes or sex organs. Except Fungi imperfecti, fungi reproduce sexually. There are three distinct stages in sexual reproduction. i). Plasmogamy (fusion of cytoplasm); ii. Karyogamy (fusion of nuclei); iii. Meiosis (reduction divisions of the fusion nucleus which usually takes place in quick succession). In some fungi, the plasmogamy is immediately followed by karyogamy and meiosis. However, in higher fungi, these two processes are separated by time and space resulting in the interpolation of dikaryotic phase in the life cycle. Some species produce distinguishable male and female sex organs on the same thallus. Such species are called hermaphrodite. The sex organs of hermaphrodites may or may not be compatible. In others, the male and female sex organs are produced on different individuals. Such species are dioecious. The sex organs are called gametangia and the sex cells are called gametes. Morphologically similar gametangia are called isogametangia. But in majority of the fungi they are dissimilar so that they can be differentiated into male and female gametangia. Such gametangia are referred to as heterogametangia. Male gametangium is called antheridium and the female gametangium is called either oogonium or ascogonium. There are no well differentiated gametangia in Basidiomycotina and they are completely absent in Deuteromycotina. On the basis of the sex organs, the fungi may be categorized as i). Hermaphroditic or Monoecious - In this type of fungi, each thallus bears both male and female organs; ii). Dioecious - These fungi bear male and female organs on different thalli. Sexual reproduction is effected by different methods as described below. A. Planogametic Copulation This is the fusion between the gametes, in which one or both the gametes are motile. It can be further differentiated into isogamous, anisogamous and oogamous. Planogametic copulation is the characteristic feature of Mastigomycotina. 1.

Isogamous: Both the gametes are motile and similar in size and morphology. This is common in most primitive fungi which are usually unicellular and holocarpic belonging to Chytridiales, e.g. Synchytrium.

2.

Anisogamous: The gametes are motile and morphologically similar, but they differ in size. Usually male is smaller and the female is larger, e.g. Allomyces.

3.

Heterogamous (Oogamous): The female gamete is larger and non motile and the male is smaller and motile. The male gametes enter the Oogonium and fertilize the egg, e.g. Monoblepharis.

B. Gametangial Contact This is also heterogamous union, but none of the gametes is motile. In this, the gamentangia come in contact with each other and through a narrow passage the protoplasm of male gametangium migrates/enters into the female gametangium. Gametangial contact is found in fungi like Phytophthora and Albugo.

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C. Gametangial Copulation This is the complete fusion of two gametangia which are similar in all respects. Gametangial copulation occurs in some Zygomycetes like Rhizopus and Mucor. D. Spermatization In this process the non-motile naked male gamete and female gametangium provided with trichogyne or receptive hyphae are involved. The male gametes (spermatia) are transferred to the receptive hyphae either by wind or through insects. Spermatization is found in fungi such as Neurospora and Puccinia. E. Somatogamy This is also known as Pseudomixis. In the higher fungi Basidiomycotina and Ascomycotina, there are no sex organs. The somatic hyphae anastamose with each other to bring together the compatible nuclei. Somatogamy is regarded as reduced but is a highly efficient form of sexuality. A number of terms have been proposed for copulation between two vegetative cells as pseudogamy (closely related to each other – Peniphora), pedogamy (copulation between mature and immature cells - yeasts), adelphogamy (copulation of mother and daughter cells - Zygosaccha-romyces), parthenocarpy (copulation between two cells of the female gametangium Ascobolus) and autogamy (fusion of nuclei in pairs within single cells of the female gametangium – Humania). I. Para-sexuality and heterokaryosis A. Parasexuality: About 80 percent of the fungi belong to Deuteromycotina. In these fungi, the sexual reproduction is very rare or totally absent in the cycle. These derive the benefits of sexual reproduction through another method known as parasexuality. Pontecorvo and Roper (1952) first discovered this genetic recombination phenomenon in Aspergillus nidulans and defined it as ‘a cycle in which plasmogamy, karyogamy and meiosis take place, but not at a specified time or at specified point in the life cycle of an organism’. Further, mitotic crossing over takes place. B. Heterokaryosis : It is a process by which dissimilar nuclei get incorporated into hyphae. Most of the fungi possess many nuclei in each cell of the hyphae. These nuclei may or may not be of the same genotype. Nevertheless, they perpetuate to the new cells of the hyphae. Presence of genetically different types of nuclei in the same cell is known as heterokaryosis. Although the heterokaryotic nuclei are independent of each other, the characters manifested by the organism are controlled by the number and ratio of nuclei. II. Heterothallism These fungi produce sex organs, which are morphologically indistinguishable into male and female. According to compatibility, the fungi can be classified into the following two types.

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A. Homothallic fungi: In these fungi, the gametangia or gametes are produced on the same thallus unit. B. Heterothallic fungi: In these fungi, the gametangia or gametes of the same thallus do not unite due to incompatibility. Two sexually different thalli are required for the fusion of sex organs. Heterothallic fungi are further divided into two types. i.

Bipolar Heterothallic fungi: Fungi in this category consist of two mating types. One mating type consists of A1 gene and another consists of A2. These genes are alleles. Fungi possessing these two genes are compatible.

ii.

Tetrapolar Heterothallic fungi: Fungi in this category consist of four basic groups (mating types of individuals). Compatibility is governed by two pairs of alleles A¹, A², B¹, B². According to the principle of Mendel’s dihybrid ratio fusion occurs between the sex organs of the two individuals so that the resulting Zygote get A¹, A², B¹and B² genes.

Biological implications of heterothallism Microbial populations have been believed not as independently developing cells but as a community with close communication between individual cells (Oleskin et al 2000, Voloshin and Kaprel’yants 2004). The cells communicate due to the evolutionary emergence of the resultant mechanisms allowing signals from external space to be perceived and transformed (Tarchevskii 2002). These signals are mainly chemical compounds and allow the cell to adapt quickly to external impacts by changing its chemical composition. In turn, the adapted cells generate new chemical compounds (new signals), whose synthesis is determined by the stress effect (Feofilova 2006). Very little is known regarding the chemical ‘language’ used by cells to communicate. During sexual reproduction, cells of heterothallic fungal strains communicate via specific molecules called hormones (Huxley 1935, Raper 1952, Gooday 1994). Heterothallism is associated with an important cellular process – sexual reproduction. Its different stages which are controlled by special hormones involved in the interactions between sexual partners necessitate studying intercellular interactions. Consideration of the heterothallism in filamentous fungi from this particular standpoint enables a deeper understanding of this unique phenomenon and makes it possible to draw an analogy with multicellular eukaryotes (Feofilova 2006). TAXONOMIC IMPLICATIONS OF SEXUAL REPRODUCTION In different taxonomic groups of fungi, there are differences in the duration of time intervals between plasmogamy and karyogamy on one hand and between karyogamy and meiosis on the other (Fig. 23.1). A. Lower Fungi In many of the lower Fungi (Mastigomycotina) there is simple fertilization, where plasmogamy is immediately followed by karyogamy resulting in diploid zygote nucleus, the syncaryon. The resultant diploid zygote cell develops a thick wall

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around it with food reserves in it. It thus becomes either a resting spore (as in lower Mastigomycotina like the chytrids - Synchytrium) or an oosphere (as in higher Mastigomycotina, the Oomycetes - Pythium, Albugo, Phytophthora). An oospore is sexually produced (perfect state) spore of the Oomycetes.

Fig. 23.1. Cytomorphological phases in sexually reproducing higher fungi. P - Plasmogamy, C (K) – karyogamy, R – Reduction division (meiosis)

In Zygomycotina (Mucor, Rhizopus), plasmogamy by gametangial copulation (direct fusion) results in the formation of zygospores. They are the resting structures, and are typically sexually produced (or perfect state) spores of the Zygomycetes – the Mucorales and Entompthorales. Zygospores are often large, thick-walled warty structures with large food reserves. They undergo a period of rest. Karyogamy may occur early (soon after plasmogamy) or may be delayed shortly before the germination of zygospore. B. Higher Fungi In most of the higher fungi (higher Ascomycotina and Basidiomycotina), the two processes (plasmogamy and karyogamy) have been separated in time and space. Karyogamy is delayed, not followed immediately after plasmogamy (as in lower fungi). Karyogamy occurs only when the necessity for meiosis appears in the life cycle. Plasmogamy, therefore, in these fungi results into a binucleate cell with one nucleus from each parent. Such a pair of nuclei is called a dikaryon. These two nuclei may not fuse for a long time and remain as such in the life cycle of the fungus. During this interval, as a result of growth and cell division of dikaryotic cell, the dikaryotic condition may be perpetuated from cell to cell by the simultaneous (conjugate) division of the two closely associated nuclei, and by separation of the resulting daughter nuclei into daughter cells. This conjugate division of the dikaryon thus develops into a new type of thallus whose cells are dikaryotic. This phase intruded between plasmogamy and karyogamy is called the dikaryotic phase. This phase is present in most of the Ascomycotina and Basidiomycotina members. The organs in which karyogamy takes place are called the zeugites. As karyogamy is delayed until the necessity for meiosis, these zeugites in most forms function as gonotoconts (the organs in which meiosis occurs) (Fig. 23.1). In Ascomycotina, plasmogamy results in the formation of dikaryotic hyphae called ascogenous hyphae. Terminal dikaryotic cells (ascus mother cells) of the ascogenous hyphae function as zeugites and gonotoconts, in which karyogamy and meiosis takes place respectively.

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As a result of karyogamy and meiosis these cells are converted in to asci, each ascus containing variable number of ascospores. Ascospores are thus the perfect-state spores of the Ascomycotina. Ascospores vary greatly in size, shape and colour. They are uni-or multinucleate and uni-or multicellular. Their wall may be thin or thick, hyaline or coloured, smooth or rough. In Neurospora tetrasperma the ascospores are black, thick-walled, foot-ball shaped, and ribbed. There are two nuclei in the cytoplasm with endoplasmic reticulum, swollen mitochondria and vacuoles. The wall is made up of several layers, the innermost layer is edosporium, outside which is the episporium. The ribbed layer is perisporium. In Basidiomycotina the terminal dikaryotic cells of the dikaryotic phase (developed after plasmogamy) are known as probasidia. They function as zeugites and gonotoconts. The probasidia enlarge into the basidia on which basidiospores are produced on sterigmata. Basidiospospores are the perfect-state spores of the Basidiomycotina. They are unicellular except in Dacrymycetaceae where they are transeversely septate. They vary in shape from globose, sausage-shaped, fusoid, almond-shaped. Their wall may be smooth or ornamental, and they are of different colours. The cytoplasm contains a haploid nucleus, and other organelles like mitochondria. Five layers have been distinguished in the wall of some basidiospores. Outwards from the surface of the spore protoplast, the layers are endosporium, episporium, exosporium, perisporium and ectosporium. These layers develop progressively from outside inwards. II. Sex Hormones in Fungi It was in relation to dioecious species of Achlya, namely A. bisexualis and A.ambisexualis (Saprolegniales), that the occurrence of sex hormones in fungi was first suggested by de Bary (1881). Later, Kauffman (1908) supported de Bary’s ideas on the basis of his studies on Saprolegnia hypogyna in which he concluded that certain inorganic salts caused the synthesis of hormones which induced antheridial hyphae. Couch (1926) and Bishop (1940) provided additional evidence of hormonal regulation of sexual reproduction in Dictyuchus and Sapromyces reinschii, and Raper (1954) in Achlya and other genera and species. Ende (1976) has defined a sexual hormone as ‘a diffusible substance playing a specific role in the sexual reproduction of the organism that produces it’. Machlis (1972) introduced the terms, erotactin which attracts motile gamete; erotropin that induces chemotropic growth of sexual structures and erogen when it controls the induction and differentiation of sexual structures. However, Ende (1976) does not favour the distinction of these hormones into specific categories, since a single hormone may induce more than one phenomena of sexual act. It is more or less established that sex hormones are involved in sexual processes of several groups of fungi, though only some of these have so far been definitely indentified and chemically characterized (Raper 1954, Machlis 1972, Barksdale 1960, 1963, McMorris and Barksdale 1967, McMorris et al 1975, Gooday 1974, van Den Ende 1976).

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A. Mastigomycotina Involvement of sex-hormones in Allomyces (Blasocladiales) and Achlya (Saprolegnials) has been clearly demonstrated. In Allomyces, the hormone has been identified as sirenin, whereas in Achlya as antheridiol and oogoniol. 1. Sirenin This is produced by Allomyces macrogynus and A. arbusculus, belonging to the subgenus Eu-Allomyces that exhibit isomorphic alternation of generations. Sirenin released by female gametes attracts the sperms towards female gametangia. In the above said two species of Allomyces, morphologically identical gametothalli (n) and sporothalli (2n) are formed in the life cycle. The female gametes release sirenin while still inside their gametangia. The hormone diffuses into the surrounding water. The male gamets, under the influence of sirenin begin to clump around the female gametangia. Machlis (1958) demonstrated synthesis of sirenin by female gametes and its attractive action towards the male gametes. The number of male gametes clustered around the cellophane, in laboratory bioassay tests has been shown to be proportional to the sirenin concentration. Sirenin has been isolated and chemically characterised from interspecific female hybrids of Allomyces macrogynus and A. arbusculus (Machlis et al 1968). Sirenin is an oxygenated sesqueterpene having empirical formula C15H14O2 and molecular weight 236. It may be species-specific. Two forms, D- and L-forms have been isolated, only the latter being biologically effective. Immediately after copulation, the male gametes cease to respond to Sirenin. Zoospores, female gametes and zygotes (all stages being motile) do not respond to the hormone. Zygote shows a positive chemotactic response to amino acids. 2. Antheridiol and Oogoniol Hormonal system in Achlya (Raper 1952): Two dioeceous species of Achlya viz., A. bisexualis and A. ambisexualis were used by J.R. Raper (1939-1959) for investigations on the sexual processes involved. He was able to demonstrate that, the male and female thalli, if grown independently did not form any sex organs. However, when potentially male and female thalli grew in close proximity, a system involving four distinct hormones became operative and initiated the sexual process. Raper (1952) divided the sexual process in the following four steps (Table 23.1, Fig. 23.2). Step 1. Initiation of Antheridial hyphae on male plant.: This is regulated by four hormones, known collectively as the A complex. The four hormones of this complex are, A and A2 secreted by the vegetative hyphae of female plants, and A1 and A3, secreted by the vegetative hyphae of male plant. Hormones A and A2 secreted by female plant can individually or together initiate antheridial hyphae development. Hormone A1 from male plant can not initiate, but can quantitatively augment, the activity of the two hormones (A and A2) secreted by the female plant.

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Hormone A3 from male plant decreases the response to the other three hormones (A1, A, A2). Step 2. Production of Oogonial initials on the Female Plant: While the antheridial hyphae are developing they secrete Hormone B which induces the formation of oogonial initials in the vegetative hyphae of female plant. Step 3. Chemotropic growth of Antheridial Hyphae and Delimitation of Antheridia: The oogonial initials now secrete Hormone C, which directs the growth of the antheridial hyphae to the oogonial initials and also causes the formation and delimitation of the antheridia after the antheridial hyphae make contact with oogonial initials.

Fig. 23.2. Action of sex hormones in Achlya spp. (modified from Raper 1952)

Step 4. Delimitation of Oogonia and Oospheres: Finally, the antheridia secrete Hormone D which causes the delimitation of the oogonia by the development of septa across the oogonial stalks, and further also causes the protoplasts of oogonia to cleave into oospheres. Generally two to four hours contact between antheridial

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initials and oogonial initials is needed for the delimitation of oogonia. The sequence of operation of these hormones is shown in Table 23.1. (i). Antheridiol (Hormones A and C of Raper): Further work on the Raper’s system was continued by Barksdale (1963). While working with a purified sample of hormone A and A-complex, she indicated that hormone A, in addition to the initiation of antheridial hyphae, causes also the responses attributed to Hormone C (i.e., chemotropic growth of antheridial hyphae and delimitation of antheridia) as well as those of Hormone A1, thereby reducing the number of hormones in this system. Thus, according to her, Hormone A has also some secondary functions, attributed earlier to hormones C and A1. McMorris and Barksdale (1967) isolated Hormone A in purified state from A. ambisexualis and chemically identified as a sterol to which the name antheridiol was given. It is a colourless crystal, with empirical formula C29H42O5 and molecular weight 470 (Edwards et al 1969). Table 23.1. Sex-hormones in Achlya ambisexualis (Raper 1952). Hormone

Produced by

A complex: A, A2 Female vegetative hyphae

Affects

Specific action (s)

Male vegetative hyphae

Induces formation of antheridial branches A1- may augment A, A2, A3- decreases response to A1, A, A2

A1, A3 Male vegetative hyphae Male vegetative hyphae B

Antheridial branches

Female vegetative hyphae

Initiates formation of oogonial initials

C

Oogonial initials

Antheridial branches (1). Attracts antheridial branches (2). Induces thigmotropic response and delimitation of antheridia

D

Antheridia

Oogonial initials

Induces delimitation of oogonia by formation of basal walls and differentiation of oospheres

Antheridiol thus induces the following four responses: (a) induction of antheridial hyphae, (b) stimulation of male hyphae to produce Hormone B, (c) chemotropic stimulation of the antheridial hyphae and (d) delimitation of antheridia. (ii). Oogoniol (Hormone B of Raper): This hormone is produced by the male plants only, in the presence of antheridiol in A. ambisexualis (Barksdale et al 1974). They have also reported production of oogoniol by some hermaphrodite strains without stimulus of antheridiol. McMorris et al (1975) have also isolated three chemically similar compounds from a hermaphrodite strain of A. heterosexualis which showed Hormone B activity i.e., were capable of inducing oogonial initials in female strains. These substances have been named oogoniol1, -2 and -3. They are also steroids.

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However, according to Ende (1976), hormonal regulation of sexual reproduction in Achlya ambisexualis involves only two hormones, antheridiol and oogoniol. B. Zygomycotina In heterothallic species the zygote (zygospore) formation occurs only when there is fusion of two different, but compatible mycelia (+ and – strains). The sexual act commences with the formation of progametangia, which later bear gametangia or zygophores. i). Initiation of Sexual Activity Very little is known about the primary interaction between the two mating types which results in the formation of progametangia-inducing substances. When a strain is single, such activity is absent. Burgeff (1924) postulated that each strain produced a diffusible or volatile substance which induced and directed the growth of zygophores. A more or less similar phenomenon was observed in Phycomyces blakesleeanus and Mucor mucedo. Using membrane filters with pores down to 10nm diam, Ende (1968) confirmed this observation for M. mucedo and Blakeslea trispora. Accordingly, physical contact between the two strains does not seem necessary. There should be thus a soluble substance which notifies the partners each other’s presence. These hypothetical substances were termed progamones by Plempel (1963). But these have never been isolated. Production of progametangia-inducing substances (now identified as trisporic acids), is initiated only when living, full grown mycelia of either mating type are put together in one medium (Ende et al 1970). Moreover, it has been observed that trisporic acid synthesis stops immediately when one of the partners is removed. Thus the factors, by means of which the two strains primarily interact, are very labile or are rapidly metabolized. Very little is known about the chemicals which induce trisporic acid synthesis as well as zygotropism. Perhaps they are proteinaceous in nature (Ende et al 1970). These have been shown to cross diffuse in the medium and suggested as volatile. ii). Trisporic acids (Progametangia-Inducing substances) In Mucor mucedo sexual activity appears to be independent of physical contact between the mating types. Extensive and successful studies of the regulation of the reaction were made with M. mucedo by Plempel and his associates (Plempel 1962, 1963, Plempel and Dewid 1961). Plempel’s (1963) scheme is set out in Fig. 23.3. The substances responsible for the induction of progametangia were termed gamone by Plempel (1963). It was postulated that there exist two mating-type-specific progametangia-inducing substances, one formed by the (+) mycelium and active towards the (-) mycelium, and the other produced by the (-) mycelium and active towards the (+) mycelium. He presented the hypothesis that the (+) strain produces a (+) progamone which induces the (-) strain to produce a (-) gamone which in turn, is only active upon the (+) strain. The (-) strain on the other hand produces a (-) programme which induces the production of a (+) gamone in the (+) strain, this being active only towards the (-) strain. However, despite extensive purification, Plempel

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was not able to separate the (+) and (-) progametangia inducing substances. The most purified preparation was still active toward both mating types. There were no indications of differences in specific activities of purified samples. Other workers also isolated progametangia-inducing factors from culture media of M. mucedo and Blakeslea trispora (Ende 1968, Austin et al 1969). These workers obtained two fractions upon purification both being active towards the (+) and the (-) mating types of M. mucedo. No differences were found between preparations obtained from M. mucedo and Blakeslea trispora. It was concluded that these were identical with the trisporic acids B and C (Ende 1968, Austin et al 1969). These acids have been characterized after isolation from culture liquids of B. trispora by Caglioti et al (1966). Their involvement in sexual process is given in Table 23.2.

Fig. 23.3. Plempel’s (1963) scheme of operation of sex hormones in Zygomycotina

The empirical formula of Trisporic acids being C18H26O 4 and the molecular weight 306. There are three kinds of trisporic acids – A, B and C. Trisporic acid C has the major (80 %) hormonal activity, whereas B has 15 % and the A only 1-2% of activity. Perhaps trisporic acid A lacks the functional group in the side chain. Contrary to Plempel’s results, there was no indication of specificity between two mating types. Trisporic acids B and C and their methyl esters were about equally effective in inducing progametangia in the (+) as well as (-) mating types. Trisporone has been shown to exist in (+) mycelium (Ende et al 1970). This factor is only produced during sexual act, and there was relationship between trisporone and the amount of (+) mycelium. It has been suggested that both mating types produce a precursor of the trisporic acids which are transformed into trisporic acids by the (+) mycelium or rather by a factor (enzyme?) produced by it. However, Gooday (1973) showed more or less equal involvement of both mating types in the production of trisporic acids. Trisporic acids bring about the following effects. iii). Carotene synthesis Sexuality in Mucorales is often accompanied by a considerable increase in carotene synthesis. β-carotene, the major carotenoid of Mucorales is the precursor of trisporic acid synthesis. Trisporic acid itself stimulates β-carotene synthesis in the

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zygophores. β-carotene, besides being the precursor of trisporic acid, forms sporopollenin, the resistant protective material of wall of zygophore. This substance is found also in the pollen grains of flowering plants. This is an oxidatively polymerized product of β-carotene. Table 23.2. Properties of some fungal sex hormones Sex-hormone Produced by

Empirical formula

Mol. Site of synthesis Specific action weight

Sirenin

Allomyces

C15H24O2

236

Female gametes Chemotactic attraction of male gametes

Antheridiol (sterol)

Achlya

C29H42O5

470

Female vegetative hyphae

Oogoniol (-1,-2,-3) (sterols)

Achlya

Trisporic acids (A,B,C)

Several mucorales

Ca 500 Male hyphae in Oogonia formation response to on female thallus antheridiol C18H26O4

306

(+) and (-) hyphae in collaboration

Yeast α factor Saccharomyces

Ca 1400 α-yeast cells

Saccharomyces

α-and a cells

Yeast α and a hormones (steroids)

Induction of antheridial hyphae, delimitation of antheridia and their chemotropic growth towards oogonia

Formation of zygophores of (+) (-) hyphae Elongation of a cells Elongation of cells ?

iv). Zygotropism Growth of opposite zygophores towards each other is called as zygotropism (Burgeff 1924). Progametangial growth directed towards progametangium of another mating type leads to physical contact between the mating types. The nature of the stimulus which controls zygotropism is not known. A stimulus is transmitted by the diffusion of volatile substances from one progametangium to another, causing a negative tropic response in progametangia of its own mating type, and a positive one in those of the opposite type. Plempel and Dawid (1961), also postulated that the stimulus is airborne and perhaps produced by the progametangia themselves. According to Mesland et al (1974) volatile substance is produced in the vegetative hyphae. Gooday (1973) suggested that the inducer of trisporic acid synthesis could be the volatile chemical which causes zygotropism also. Mesland et al (1974) also showed that the volatile substance obtained from (+) and (-) cultures could induce trisporic acid synthesis and also brought zygotropism in the opposite mating type.

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The over-all process of sexual act in the Mucorales including the role of trisporic acids has been shown by Gooday (1973). It may be seen that when two compatible mating types grow together, they bring about a ‘mutual biosynthesis’ of trisporic acids. They mutually ‘switch on’ the biosynthesis of each other’s trisporic acids through inducers. These inducers have not yet been chemically characterized. The trisporic acids, formed by both mating types, diffuse into the medium and induce zygophore formation. Cell contact is not necessary. The acids also stimulate greater β-carotene synthesis, to ensure their own as well as of sporopollenin synthesis. Finally the zygophores are mutually directed towards each other to form zygophore. The nature of this growth is not yet known. Perhaps it is volatile. There are indications of hormonal control of sex in homothallic Mucorales also, in which trisporic acids and their precursors are known to induce zygophore formation. C. Ascomycotina There are some reports of possible involvement of hormones in sexual act of some Ascomycotina. So far only in yeast, Saccharomyces cerevisiae, these have been chemically characterized. The conjugation tubes of some yeast are directed towards each other. Experimental evidence for such a possibility was presented by Levi (1956) for S. cerevisiae. It was shown that there were two haploid mating types, cells a and α. The α cells produced a diffusible chemical which induced the formation of copulatory processes by cells. So far no factor has been demonstrated. Physical contact is necessary. The cells under the influence of α factor stop growth and budding. These cells swell and change into giant cells of various shapes. These giant cells (normally 10 or more times larger than the normal cells) have 30 or more times greater dry weight than haploid vegetative cells. The α-factor is specific, acting only on a cell and has no effect on α cells. It inhibits DNA replication in cells. Two other hormones, called α- and a-hormone have also been shown to be involved in the sexual act of α- and a-cells. These are steroids, possibly related to ergosterol. The hormones are active on both mating types. Physical contact is not necessary, and cause swelling of cells. Effect of these hormones is less specific than the yeast α-factor. Markert (1949) has shown that a diffusible substance affected perithecial development in Glomerella. Dodge (1912) indicated hormonal involvement in chemotropic growth of trichogyne in Ascobolus carbonarius. Similar observations have also been made for trichogynes of Neurospora sitophila (Backus 1939) and Bombardia lunata (Zickler 1937, 1952). Bistis (1956 1957) showed multihormonal mechanism which controls and regulates the sexual reproduction in Ascobolus stercorarius. D. Basidiomycotina With the exception of rusts, somatic copulation is typical sexual reaction in these fungi. Purely vegetative hyphal fusions are also widespread in these fungi. Sexhormones have not been demonstrated, but there is extensive literature on chemotropism between fungal hyphae, whether sexual or vegetative, resulting in such fusions.

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The foregoing account shows that hormones regulate sexual reproduction to suggest that all sexually-reproducing fungi probably use hormonal control mechanisms to a greater or lesser extent. However, the nature of sex-hormones is fully understood only in a few cases. Those, which have so far been fully identified and chemically characterized, are presented in Table 23.2. I. Mating Types Sexual reproduction offers natural selection and adaptation of the organisms to environmental conditions as a result of spreading of favorable mutations and by weakening harmful mutations. Recent findings which specify the role of sexual reproduction in fungal pathogenicity have been attained. Nevertheless, the pathogenic fungi restrain their sexual cycles to create clonal populations instead of recombinants, to allow themselves to adjust to the new conditions in the environment and in the host such as antimicrobial therapy. Cryptococcus neoformans, a haploid organism has an established sexual cycle and mating cell types ‘a and alpha’. Production of pheromones is stimulated by nutrient constraint which induces cell-cell fusion and the ensuing dikaryons go through filamentous transition, karyogamy and meiosis in basidia and chains of very infective basidiospores develop. The ‘a’ and ‘alpha’ alleles take place in MAT (Mating Type) locus. Strains of ‘alpha’ matingtype predominate in environment and clinical isolates and, in ‘a-alpha’ coinfection model, alpha-cells exhibit more pathogenic behavior than congenic ‘a’ cells. In the most widespread pathogenic variety grubii, (serotype A) there is no differentiation in the virulence of cells of opposite mating types but, during co-infection alpha-cells simply cross the blood-brain barrier. Furthermore, alpha strains make increased quantity of melanin and urease which enhance invasion of central nervous system (Cerikcioğlu 2009). In C. neoformans a novel sexual cycle termed as same-sex (monokaryotic) mating has been discovered. Alpha-alpha cells engage in sex without an ‘a’ partner that can contribute to produce diversity and generate infectious haploid basidiospores. This process is also called as ‘parasexual’ recombination. An additional aspect for C. neoformans biological property is naturally occurring AD hybrid strains between var. grubii (serotype A) and var. neoformans (serotype D) via sexual crosses. Those strains often contain both mating types, either aADalpha or alphaADa. In Candida albicans owing to its diploid nature, most strains are a/alpha heterozygous at the mating-type locus and contain both mating-type alleles. Therefore, the tetraploid cells (a/a/alpha/alpha) produced during mating can turn to diploid state (a/a and alpha/alpha) by random chromosome loss via parasexual process but with no meiosis, inside the host. Tetraploids were noticed to be less virulent in urine infections and could be cleared more swiftly than the diploids. In C. albicans, control of white-opaque switching is believed to be regulated in part by the mating locus, indicating that switch may be involved in mating. Like these two opportunistic pathogens, in Pneumocystis jiroveci, Histoplasma capsulatum and Aspergillus spp. genetic studies are being carried out to identify genes related to mating types, sexual cycle, virulence and resistance to antifungal drugs, and the interactions between them (Cerikcioğlu 2009).

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FUNGAL MATING-TYPE LOCUS Sexual reproduction is universal across kingdoms and essential for the origins and fitness of the species. Fungi, in particular, provide as excellent models to study sexual reproduction and investigate how sex is schemed. Sex is genetically determined in fungi, governed by sex-specific region in the genome known as the mating-type locus (MAT) (Hsueh and Heitman 2008). The molecular structure of MAT was first characterized in Saccharomyces cerevisiae, in which homeodomain or α domain transcription factors are encoded by the a or α idiomorph and are critical to establish cell-type identity (Herskowitz 1989). Due to large number of fungal genome sequences now available, the structure, function, and evolution of MAT has been discovered in three major fungal lineages (Ascomycetes, Basidiomycetes and Zygomycetes) (Butler 2007, Fraser et al 2007, Idnurm et al 2008). GENETIC DIVERSITY IN MICROSPORIDIA Microsporidia are obligate intracellular pathogens mostly infecting vertebrate and invertebrate hosts. The group encompasses around 150 genera with 1200 species. The sequence divergence phylogenetic reconstructions which are only based on DNA sequence have been inaccurate for these pathogens. In a comparison of genome architecture of the microsporidia to other fungi, Rhizopus oryzae, a zygomycete fungus, shared more common gene clusters with Encephalitozoon cuniculi, a microsporidian (Lee et al 2008). This corroborates the hyphothesis that microsporidia and zygomycete fungi may share a more common ancestor than other fungal lineages (Lee et al 2009).

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Chapter - 24

Fungi in Miscellaneous Substrates

The fungi grow on a variety of ecological habitats or niches, which are named accordingly as thermophillic fungi, psychrophillic fungi, coprophilous fungi, phyllosphere fungi, soil fungi, etc. THERMOPHILLIC FUNGI The fungi have adapted to grow at varios temperatures. They can be roughly categorized as psychrophiles which grow below 10°C, mesophiles grow between 10-40°C and the thermophiles literally heat lovers grow above 40°C (20-50°C). Thermophylly and thermodurism are further two different temperature adaptations of fungi. Thermophillic denotes the range of temperature at which fungi can actively grow while the thermodurism is the increased range of temperature which fungi can withstand as dormant bodies. For example, some fungi can grow vegetatively at ordinary temperatures but the spores can withstantd increased heat. The minimum and maximum temperature ranges for growth of some known thermophillic fungi have been given in Table 24.1. Table 24.1. Minimum and maximum temperatures (°C) for the growth of some thermophillic fungi Fungi Mucor miehei Mucor pusillus Chaetomium thermophile Talaromyces dupontii Talaromyces emersonii Talaromyces aurantiacus Humicola grisea Humicola lanuginosa Malbranchea pulchella Paecilomyces sp. Stilbella thermophila Torula thermophila

Min. 25 20 27 27 30 22 24 23 27 30 24 23

Max. 57 55 58 59 60 55 56 55 56 60 55 58

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Characteristics of thermophillic fungi Most of these fungi can grow well on glucose, sucrose, maltose, mannose, galactose, xylan, starch, and lignin. Sporotrichum thermophile can decompose cellulose and pectin. Humicola insolence produces extra cellular cellulases to decompose cellulose. The thermophiles viz. Mucor pusillus, M. miehei, Talaromyces dupontii, Thermoascus aurantiacus, Humicola insolence, H. lanuginosa and Malbranchea pulchella have the capacity to decompose resinous compounds. These fungi are reported to be autotrophic for vitamins and can utilize nitrates and sulfates as the sources for nitrogen and sulphur, respectively. Most interesting part of these fungi is that how can they grow at high temperature (60°C) or why can’t they grow at ordinary temperature (15-20°C) at which so many of their relatives grow excellently. In the case of thermophillic bacteria structural proteins have been found more heat resistant than in their counterparts in mesophilic species. Survival of these fungi at very high temperature which actually causes denaturation of protein is the matter of interesting adaptations. One gene can be expressed over for lower range of temperature and another for higher range as in the case of certain bacteria which can grow at 95°C and also at 10°C. The reversal of the thermophillic strains to mesophilic and vice versa has also been found. Environmental temperature may induce physiological thermoadaptative response in the cells, which may result in the formation of thermostable chemical compounds. Garrison et al (1975) found thermo stability with ultra structural elements or organelles in the cytoplasm of some thermophiles. Like most terrestrial microorganisms, and mesophilic fungi the thermophillic fungi too have a wide distribution due to the result of the worldwide presence of self-heating organic debris. The common habitats for these fungi are self heating composts, wood-chip piles, stored grains, self heated coal refuse pipes, tobacco products undergoing heating during curing, nests of birds; streamline discharge sites, snuff and diseased animals. Most of the thermophillic fungi are found and can be isolated from composts, hay grasses, peats, manure, soil, herbivore dung and plant materials. These fungi are found universally, can be isolated from air, soil and naturally decomposing substrates. Self heating materials like dung, hay, peat or compost is incubated at the elevated room temperature. Cooney and Emerson (1964) suggested two types of incubations. 1.

Primary incubation of substrate.

2.

Secondary incubation of isolated cultures at elevated temperatures. They used yeast starch, yeast glucose, oatmeal or Czapeck’s agar media for isolation.

Useful activities of thermophillic fungi 1.

Humification: Formation of humus through conversion of organic waste materials into valuable manure. They are very active decomposers of complex organic materials.

2.

Disposal of human wastes and refuse.

3.

Preparation of compost for mushrooms: Large scale self-heating processes, in which suitable material is mixed with certain ingredients,

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decomposition takes place with simultaneous raised temperature at which various pests are killed. 4.

Processing of chocolate and tobacco: Thermophillic fungi help in developing taste and colour of the chocolate and tobacco.

5.

Penicillin production: Malbranchea pulchella can produce metabolites at high temperature.

6.

Production of high quality rubber.

7.

Silage, conversion of fodder into a better feed due to self-heating microbes.

8.

Retting of fibers: In general these fungi help in making of fibers from jute and other plants and are important in industries as biological tools in Modern developments.

Harmful activities of thermophillic fungi 1.

Spoilage of food products - food grains, potatoes etc.

2.

Diseases of domestic animals, man and bird causing mycoses. PSYCHROPHILLIC FUNGI

These fungi grow best at low temperature or restricted to cold environment. The fungi have growth maxima below 5-10°C. Sclerotinia borealis, Typhula sp., Candida gelida, species of Fusarium and Sclerotinia are known as snow moulds. a) Polar Fungi: 1.

Arctic fungi: Growth of these fungi on suitable substrate (herbs at Tundra region are best at 0°C to 10°C and these fungi can survive upto 60°C.

2.

Antarctic fungi: Mosses, lichens, fungi and fresh water algae are found growing. Dodge and Baker (1938) recorded Hormiscium sp. and Penicillium sp. while, Tubaki found Rhacodium, Cryptococcus laurentii and Candida sp. from the soils of Japanese Antarctic region.

b) Alpine Fungi: Fungi at high altitude: Cooke (1955) observed fruit bodies of Lyophyllum sp. between 1.5°C to 9°C, Xenia nigrella at 2.5°C. c) Plant Pathogens: Fungi cause diseases to grasses, trees and other plants at low temperature; Fusarium nivale can cause disease at 0°to 5°C. d) Fungi on refrigerated food: Smart (1934) reported species of Aspergillus, Penicillium, Rhizopus, Mucor and Cladosporium on frozen, packed fruits and vegetables. The species of Cladosporium, Aureobasidium, Thamnidium and Mucor are present on frozen meat.

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COPROPHILOUS FUNGI Most of the fungi except members of Mastigomycotina grow on animal dung which are known as coprophilous fungi. Herbivore dung is rich in water-soluble carbohydrates, nitrogen, vitamins, growth factors and minerals. It is a good substrate for fungal succession as in fresh dung incubated for 1-2 days a number of Mucorales including species of Mucor, Pilaira and Pilobolus grow for 10-12 days. Next group of fungi appear in second week which are commonly apothecial (Coprobia, Ascobolus, Thelebolus, Saccobolus, etc). After 14-30 days members of perithecial forms (Chaetomium, Coniochaeta, Podospora, etc.) appear. The coprophilous fungi have spore-bearing structures with phototrophic responses and violent spore discharge mechanism. PHYLLOSPHERE FUNGI Population of fungi colonizing leaf surfaces as an ecological niche is termed as Phyllosphere or phylloplane fungi. Last (1955) introduced the term phyllosphere to denote fungi on leaf surface of plants a term analogous to rhizosphere of Hiltner (1904) for outer surfaces of roots. Last (1955) further suggested that the phyllosphere region is similar to rhizosphere in its nutritionally rich habitat and provides a suitable substrate for the colonization and multiplication of microorganisms. Later on Kerling (1964) suggested the term phylloplane stressing the fact that the microorganisms grow on leaf surface and not in zone as around the rhizosphere. The phylloplane fungi can be studied by various methods, as by direct observation of plant surfaces under microscope or by culturing and isolating the fungi on suitable growth media in laboratory. FUNGI IN THE ATMOSPHERE Fungi in the atmosphere are mostly present in the ‘troposphere’ up to the height of 10 km. from the earth and rarely found in stratosphere. The air we inhale and atmosphere surrounding us is rich with a variety of organic particles in addition to inorganic substances like gases, dust and smoke. The organic particulate in the air mainly are pollen grains, bacteria, fungal and algal spores, mites, fragments of plants and insects. Among these, fungal spores are extremely variable with the changes in temperature, light, humidity, and geographical location. Generally airborne fungi produce large amount of spores which are hardier and better adapted to dispersal. The fungus Cladosporium is predominant in the air of most regions of the world. Alternaria is second most common. Curvularia and Nigrospora can make large contribution to the air spora of tropical regions. Aspergillus and Penicillium spores are widespread in air. The airborne fungi may be the cause for the spread of diseases of humans, animals and plants. They may also spoil the food and other materials (biodeterioration). They cause infectious diseases, allergic and non-allergic disorders. The microbiology of the atmosphere is also known as aerobiology which mainly deals with the study of distribution and dispersion of microbial population in the air.

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SOIL FUNGI Soil is the dynamic medium for great microbial activity, among these fungi of all groups, chytrids to agarics and of all kinds - saprobic, parasitic, symbiotic, predators, etc. occurs in the soil. Soil fungi can be categorized into two types i). Soil inhabitants and ii). Root inhabitants. The soil inhabitant fungi are free living saprophytes while root inhabitants have limited saprophytic ability. The soil inhabitants further can be termed as cellulose decomposing fungi, lignin decomposing fungi, sugar fungi, coprophilous fungi (growing on dung), etc. The root inhabitant fungi are also known as rhizosphere fungi and they can be root pathogens or symbiotic fungi like mycorrhiza. MARINE FUNGI The fungi growing in sea on various types of substrates and hosts are known as marine fungi; their study is known as marine mycology. The marine fungi are important for their taxonomic and ecological aspects. Similarly these fungi are unique for their metabolites, biochemicals and enzymes. The fungi are present in tropical, subtropical and temperate seawater and tolerate high levels of salinity. They have been reported mainly from Indian Ocean, Atlantic Ocean, Pacific Ocean, south East Asia, and Hong Kong. Fifty-four species of marine fungi are more frequent in tropics. Marine fungi grow mainly on woody debris of intertidal habitats, on mangroves, salt marshes, palms, fern and animal substrates. These fungi play role in transfer of organic matter to higher tropic levels in sea. Bauch (1936) recorded first time about the existence of marine fungi. Similarly pioneering studies of Barghoorn and Linder (1944) on the fungi of submerged wood in sea made the impact on the development of Marine Mycology. There was a rapid progress on role of fungi in marine ecosystem with the publication of book ‘Fungi in Oceans and Estuaries’ by Johnson and Sparrow (1961). The marine fungi include both lower fungi (Mastigomycotina) and higher fungi like members of Ascomycotina, Basidiomycotina and Deuteromycotina. About 500 species have been reported from all over the world. These fungi are adapted to the saline environment. Hence they require seawater for the completion of their life cycle. The members of Ascomycotina have appendages on the spores to adapt marine ecosystem. These appendages are helpful in buoyancy, entrapment and surface adherence. Following are some of the common marine fungi recorded, these are species of Acrocordiopsis, Aigialus, Bathyascus, Ligninicola, Pleospora, Massarina (Ascomycotina), Calathella, and Nia (Basidiomycotina), Ascochyta, Cirrenalia, Dictyosporium, Periconia, Trichocladium (Deuteromycotina), etc.

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Chapter - 25

Entomogenous Fungi

The word entomogenous has been derived from two Greek words, entomon meaning insects and genes meaning arising in. Thus, the etymologica1 meaning of entomogenous fungi is fungi which arise in insects i.e. Entomogenous fungi grow in or on the bodies of insects. The association or symbiosis between fungi and insects which exists in nature may be antagonistic, mutualistic or naturalistic. This association of fungi and insect is interesting, ecologically as well as economically, and may either be obligate or facultative. Antagonistic obligate symbionts either have no capacity or have a poorly developed capacity for a free living existence other than as propagules in the absence of a suitable host, e.g., Entomophthora. The antagonistic facultative symbiotic entomogenous fungi have a moderate or well developed ability for a free living existence and which are potentially symbiotic. These fungi e.g., Beauveria, Nomuraea, Metarhizium, Paecilomyces, etc., cause mild debilitating or fatal diseases in insects. The capacity of these fungi in bringing about a certain degree of natural or biological control of insect pests is directly related to human welfare and thus has attracted the attention of mycologists, microbiologists and entomologists in recent years. The benevolence of fungi as natural microbial control agent was first brought to prominence in the legends of insect pathology in 325 B.C. by Aristotle’s ‘Historia Animalium’ describing disease of honey bee. Naturalists and philosophers of succeeding generation alluded to the afflictions of honey bee, silkworm and other insects. Discovery of Cordyceps sp. by De Réaumer embarked a new faculty as an Entomomycology. De Geer was first to describe a fungus which attacks houseflies. First chapter on entomomycology, ‘Diseases of Insects’ was contributed by Kirby and Spence in 1826 in the famous book ‘Introduction to Entomology’. Pioneering experimental evidence was given by Bassi who confirmed pathogenicity of the fungus Beauveria bassiana on silkworm. Cohn named the fungus which attacks houseflies as Empusa muscae. Another landmark in entomomycology was the studies of a great microbiologist, Louis Pasteur of France, who studied a silkworm disease caused by Beauveria bassiana. First significant experimental field test of an entomogenous fungus Metarhizium anisopliae was conducted by Metchnikoff and Krassilstschik against grain- weevil and sugar beet curculio in Russia. Snow used Beauveria bassiana and B. globulifera against chinch bug, Blissus leucopterus in USA. Thaxter also studied entomogenous fungi.

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Comparatively very little work on entomogenous fungi has been carried out in India and these organisms were woefully neglected by mycologists as well as entomologists. A review of literature revealed that except for a few detailed studies, most of the work done concerned with the publication of short reports of their occurrence and identification on their natural hosts. Some of the workers also attempted to study the pathogenicity, host range and possible utility of these fungi in biological control of insect pests. Berkeley was the first to report two species of Cordyceps. Sydow and Butler reported Cephalosporium lecanii (= Verticillium lecanii) infecting coffee scale insect from Karnataka. Butler and Bisby reported Empusa muscae (= Entomophthora muscae) infecting houseflies. Hirsutella abietina infecting Pyrilla pusana was first reported from Bihar by Petch. Aschersonia coffeae was reported from Darjeeling by Sydow and Mitter. The most noteworthy report was that of Kamat et al from Bombay on green muscardine fungus Metarhizium anisopliae infecting sugarcane pest Pyrilla perpusilla. Bose and Mehta described a new species of Entomophthora, viz., E. bauhiniae infecting Bauhinia species. Srinivasan et al, succeeded in culturing Entomophthora sp. in artificial culture. Nirula studied the green muscardine fungus Metarhizium anisopliae infecting Rhinoceros beetle of coconut. Jagtap published a detailed account of M. anisopliae infecting Pyrilla purpusilla. An epizootic of Sunhemp pest caused by Beauveria sp., was reported by Ramamurthi et al. Urs et al, studied the effect of certain insecticides on B. bassiana and M. anisopliae under laboratory conditions. A very useful account of entomogenous fungi and their possible use in biological control of insect pests has been published by Narsimhan, Kamat and Rao. Srivastava and Nayak reported white muscardine disease of brown plant hopper of rice caused by B. bassiana. Phadke and Rao studied pathogenicity of Nomuraea rileyi towards three lepidopterous pests. Chowdhury and Varshney recorded Acremonium zeylanicum on Aphis brassicae. Phadke summarized the Indian work on entomogenous fungi. Agarwal et al reported white muscardine disease of insect pests of teak, viz., Hyblaea puera and Pyrausta machaeralis from Jabalpur. Several investigators studied entomogenous fungi of insect pests of crops and Forest nurseries in Jabalpur. Agarwal and Rajak reviewed the work on entomogenous fungi in the biological control of insect pests. A list of entomopathogenic fungi of insect pests occurring at Jabalpur was published by Agarwal and Rajak. Entomogenous fungi belong to Eumycota and are reported from all the subdivisions, viz., Mastigomycotina, Zygomycotina, Ascomycotina, Basidiomycotina and Deuteromycotina. Some of the important genera of entomogenous fungi include Beauveria, Nomuraea, Metarhizium, Verticillium, Entomophthora, Coelomomyces, Paecilomyces, Cordyceps, Hirsutella, Aschersonia, Aspergillus, Septobasidium, Ascosphaera, Fusarium, etc. The taxonomy of Entomophthora, a genus of Zygomycetes whose numerous species parasitize a wide variety of insects, has been reviewed by Wolf. De Hoog reviewed the taxonomy of Beauveria. Samson and Evans added two more new species of Beauveria, viz., B. velata and B. amorpha. Metarhizium and Paecilomyces have M. flavoviride, M. anisopliae, and P. fumoroseus. The genus Nomuraea is resurrected by Samson. So far, only one species of Verticillium, viz., V. lecanii has

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been described, parasitizing mainly scale insects and bugs. The genus Hirsutella has been monographed by Minter and Brady. A systematic key of the genus Aschersonia, pathogen of Coccidae and Aleyrodidae was presented by Protsenko. Skou described three species of Ascosphaera. The genus Cordyceps is well known systematically. Species of Coelomomyces characterized by their specificity for mosquitoes and chironomids have been studied by Dubitskij et al. Sweeney described a new genus Culicinomyces, pathogenic to chironomids and mosquitoes. Integumental penetration of the fungus is either at the site of certain pigmented bodies or due to chemotrophic attraction or moistened integument. The penetration also takes place through the trachea, through abrasions which expose pore canal and sense organs, through digestive tract or through wounds. Entomological syndromes are observed in four phases, viz., behavioural, external, internal and physiological. Behavioural includes general sluggishness, weakness and decreased irritability. Externally colour changes at the site of infection due to melanin reaction. Eggs turn pink-red. Hypodermal lesions also form in the body. Loss of body weight, reduction in fecundity and sterility has also been observed. Infected insects may die comparatively rapidly, occasionally in an upright position still attached to a leaf or stem. Optimum temperature, high relative humidity and exudates of insect cuticle initiate spore germination which starts with a protuberance of the spore wall. The protuberance increases and bursts out forming a germ tube which proceeds parallel to the cuticle and soon gets surrounded by a mucoid coating which helps in the adhesion of spore and germ tube with the integument. Germ tube on coming in contact with the integument forms an appressorium, an anchoring organ, which provides physical force to the pathogen and proliferates into a penetrating peg. This enters into the epicuticle and forms a penetrating plate after which fungus multiplies very rapidly both in radial and vertical directions forming isolated yeast like hyphal bodies. These hyphal bodies penetrate into procuticular layer and then to hypodermis and ultimately reaching haemocoel, thus completing pre-penetrative phase. Some pathogens produce sufficient toxin in pre-penetrative phase to cause death. Post-penetrative phase involves the production of toxins and enzymes. It was demonstrated that entomogenous fungi like Beauveria bassiana, Metarhizium anisopliae, Nomuraea rileyi, Entomophthora coronata, etc., have the ability to produce lipolytic, chitinolytic and proteolytic enzymes. Toxins secreted by entomogenous fungi in the lethal process of the disease are particularly of striking importance. In coelom the hyphal bodies circulate, germinate and spread the fungus and form chlamydospores after the death of the host. These spores germinate to form emergent hyphae that sporulate on the surface of the host. EFFECT OF ENVIRONMENTAL FACTORS Under conditions of favourable temperature, high humidity and abundant sporulation an insect disease may reach epizootic proportions. The physical phenomena of the environment influence germination, growth, pathogenicity, multiplication,

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survival and dispersal of entomogenous fungi. The effect of physical factors on entomogenous fungi has been discussed by various workers. In a population of flies infected with Entomophthora muscae, Hiestand observed that growth of the fungus was greatly accelerated by an oxygen atmosphere. Light is also a factor of some importance in relation to entomophthoraceous fungi. Voronina noted that conidia of Entomophthora thaxteriana were produced in light and the germ tubes were positively phototropic. According to Wilding, in E. thaxteriana the number of conidia discharged in constant darkness was only half of that in constant light. In larvae of Bradysia infected by E. grylli, conidial discharge was maximal 2.5 hours after the onset of the light period, although Skaife reported that conidial discharge from red locusts infected with E. grylli occurred predominantly in the evening. Information on the influence of temperature and humidity, the two most important factors in the microclimate, is very well documented. Much information is available on the effects of temperature or germination and growth of entomogenous fungi in vitro. In general, the limits for growth range between 5° and 35°C and the optimum falls between 20°C and 30°C. Temperatures which support moderate growth are adequate for infection, whereas mortality is severely affected by lower temperatures. Temperature is one of the most important external factors that influences spore germination. It not only regulates the time required for germination, but also the number of germinating spores in a population, which would be directly related to the infection rate in insect population. Cold environments are not conducive to high metabolic activity and, therefore, reduce spore germination. In general, the optimum temperature for the germination of entomogenous fungi falls between 25o and 30oC. Temperature seems to have little influence on spore survival, except at high temperatures. The effect of temperature on the pathogenicity has been studied by various workers. For entomopathogenic fungi, humidity is a major ecological constraint. It completely regulates the pathogenesis, initially at the preinfection phase, where it is needed by the pathogen for germination. Secondly it is needed at post-infection phase where secondary propagules, spores from the mummified cadaver, spread and cause epizootics. With muscardine fungi, relative humidity above 97 percent is required to obtain appreciable germination of spores. A saturated atmosphere is conducive to germination, but not necessarily to spore longevity as related to inoculum potential. The influence of relative humidity on the infectivity of muscardine fungi has been investigated by many workers. DEVELOPMENT OF ENTOMOGENOUS FUNGI FOR PEST CONTROL Entomopathogenic fungi could be manipulated in many ways to increase prevalence of disease in a pest population. The most flexibility, however, will be realized when the pathogen can be introduced in the same manner as chemical pesticides. This implies that a candidate fungus must be produced economically in large quantities. There are two production options in vivo or in vitro. Many entomopathogenic fungi are known to produce epizootics but either do not grow in vitro or can only be cultured on very complex media. Species of Entomophthora viz., E. planchoniana, E. aphidis, E. grylli, E. aulicae and E. erupta were propagated on

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mass scale on their insect hosts. Various types of investigations have been undertaken to obtain fungal spores in bulk quantities in vitro. For field use, they are produced in large quantity either by liquid or solid substrate fermentation or in stages involving both. Liquid fermentation utilizes a well established industrial technology. In solid substrate fermentation, the fungus is usually grown on a particular substrate. Often cereal grains fortified with nutrients are added. This provides a large surface area for the production of conidia. Methods have been successfully developed for producing spores of Beauveria bassiana, Nomuraea rileyi, Verticillium lecanii, Entomophthora thaxteriana, and E. virulenta. Once the production technology has been developed, it becomes necessary to devise the technology needed to harvest and stockpile the spores. A proportion of conidia of most entomogenous Deuteromycotina often survive in nature for many months, but bulk storage of propagules of most entomogenous fungi is a problem. Ways of improving storage by appropriate formulation of the product are being investigated. Survival of spores is often greatly improved by mixing with refined clays and by refrigeration. Most of the entomopathogenic fungi are considered harmless to mammals. The maximum temperature at which most of these fungi survive is 37°C. Therefore, they are unlikely to infect mammals and birds where the body temperature is higher. Only few fungi, viz., Aspergillus sp., Entomophthora coronata etc., occasionally infect mammals including man and therefore, not considered for insect control. Beauveria bassiana produces allergic responses in some cases when used widely. There are no records of allergic reactions produced by other entomogenous fungi. Entomopathogenic fungi are much more selective than most chemical pesticides, although several species have intraspecific strains which are infective for a restricted range of hosts. A strain applied for control of a certain pest is unlikely to affect beneficial organisms in the same environment. The efficacy of a fungus in pest control cannot he measured simply by comparing its impact on the target pest with that of a chemical pesticide. A fungus will almost certainly not kill its host as quickly as a chemical but its value will lie more in its persistence through reproduction and in its compatibility with other abiotic and biotic agents attacking both the target and non-target pests. Efficacy of some important entomogenous fungi which are currently in commercial use or being considered for use is discussed below. Beauveria bassiana It commonly infects insects of many orders, particularly Coleoptera and Lepidoptera. It is produced on a large scale for field use only in the USSR, USA and China. In the USSR, the fungus is used as an insecticide together with reduced dosages of chemical insecticides. In China, it is used mostly to control European corn borer. Control of 80 per cent of first generation larvae was claimed by treating 0.4 million hectare of corn. In France, B. bassiana has been tested on a pilot scale and almost completely controlled the target pest.

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Metarhizium anisopliae It is a common pathogen of soil dwelling and pasture insects. The inoculum has been produced on a large scale as ‘metaquino’ in Brazil and used against leaf hoppers. It was produced by solid substrate fermentations on boiled rice in autoclavable bags and was applied by air to 50,000 hectares in 1978. In Australia, Coles and Pinnock successfully controlled Aphodius tasmaniae. A high dose of conidia caused high mortality of the larvae of this beetle. Nomuraea rileyi It attacks lepidopteran larvae including serious pests, like cabbage looper and corn earworm. Mohamed et al applied it as an insecticide to sweet corn infected with Helicoverpa zea in field cages. Three fungal applications could not prevent economic damage. Ignoffo et al obtained similar results with Helicoverpa zea larvae on soybean after an application of conidia (2.75 × 1013 per hectare). It appears that, when applied as an insecticide, the fungus kills its host slowly to prevent crop damage. The results of other experiments, however, suggest that its application might be a useful prophylactic treatment. Hirsutella thompsonii This fungus infects only mites in tropical or subtropical regions. It is marketed in the USA as ‘Mycar’ and was first registered for use against citrus rust mite (Phyllocoptruta oleivora) on citrus crops in 1981. The product is formulated with nutrients to encourage saprophytic development in the field. More than 4500 kg of Mycar was sold in 1981 to treat over 2000 hectares of citrus plantations. Unformulated preparation of this fungus gave good control of citrus rust mite within 2-3 weeks of application during suitable environmental conditions. However, there are not many published reports on the effect of the commercial product on mite populations. Verticillium lecanii It attacks aphids and scale insects in the tropics and subtropics, and was registered for use in United Kingdom and marketed as ‘Vertalec’ and ‘Mycotal’ for the control of aphids and glasshouse whitefly, respectively. The efficacy of V. lecanii in controlling Myzus persicae has been convincingly demonstrated in several glasshouse trials. The fungus eliminated or nearly eliminated even small populations of M. persicae in 2-3 weeks, cotton aphid (Aphis gossypii) on cucumber in 28 days and maintained glasshouse whitelfy (Trialeurodes vaporariorum) populations far below the level at which economic damage occurs. Aschersonia aleyrodes It is a pathogen of whiteflies and scale insects and is being considered for the control of whiteflies in glasshouses in the Netherlands and in the UK. In the Netherlands, the fungus was applied to cucumbers in combination with the release of the parasitic wasp Encarsia formosa which caused 85 percent mortality of

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Trialeurodes vaporariorum compared with 49 per cent due to wasp alone. This fungus may have particular potential because it develops at lower humidities than others. ENTOMOPHTHORALES More than 150 species of these fungi are known to infect insects and mites, and are restricted to certain host groups. No member of this group is currently produced on a commercial scale for pest control. However, two species, Erynia neoaphidis and Neozygites fresenii were used against black bean aphid (Aphis fabae) by releasing aphids infected with these fungi which gave very encouraging results. It is believed that Entomophthorales might be used to control pest population if ways of producing an infective and stable inoculum could be found. Appreciable scientific information is available on entomopathogenic fungi as potential microbial insecticides in the frame work of integrated pest management and biological control of agricultural pests and of human and domestic animal disease vectors. The research effort has grown in importance and diversity, leading to a more thorough knowledge of fungal biological characteristics and modes of action. In the best cases, industrial pre-development actions were initiated. The future of entomogenous fungi will be a continuum of the present just as surely as the present is a continuum of the past. Quite obviously, exploratory studies must continue in all climates and especially in the tropics. Reporting of entomogenous fungi in Indian literature seems to be accidental to both mycologists and entomologists. Sincere and honest explorations have never been planned and a rich flora of entomogenous fungi awaits discovery and description. This will make a substantial contribution to our knowledge of the identity, morphology, taxonomy and ecology of entomogenous fungi as a whole. Studies on the systematics of the Entomophthorales should be continued in order to gain basic knowledge for their effective use as biocontrol agents. Studies should be conducted with the new species of Fungi Imperfecti isolated from mosquito larvae. Also, it is necessary to revise the systematics of such cosmopolitan genera as Beauveria and Metarhizium utilizing physiological as well as morphological criteria. Analysis of the interaction occurring among the pathogen, host-insect and environmental parameters is another problem that needs special attention. Ecology of entomopathogenic fungi, including both ecotype potentialities and the inoculum biodegradation, taking also into account the competition phenomena that can appear in several ways when introducing an inoculum into a biotype where the mycosis is naturally endemic is to be elucidated in detail. Such data would be needed to provide an understanding of the factors responsible for initiation and development of epizootics and the optimal conditions for mass introductions of fungi into the fields. Many investigators have attempted to study the mechanisms involved in the infection process including possible toxin production. A more thorough understanding of the mode of action of entomopathogenic fungi is needed at two levels: (a) on and in the integument by suitable enzymatic studies and (b) in the haemocoel by toxin metabolism studies. Scanning electron microscopic observations on the integument should provide a better knowledge of the behaviour of entomopathogenic fungi in the

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first phase of infection as well as contributing towards an understanding of the integumentary flora. Workers in the field of biological control have long been aware of genetic variation in entomopathogenic fungi. They have not only employed strains of different geographical origins, but also have isolated strains adapted to one host and used them to kill another. Passing the pathogen through an insect and isolating the infecting strain from the cadaver have been the general and practical methods for selecting the virulent strain. Attempts to use genetic techniques to improve entomopathogens have been, however, relatively few. Mutants of fungal strains may be useful in mode of action studies. Studies on the genetic relationships between host entomopathogenic fungi should provide a new approach to increase virulence and specificity of pathotypes as well as susceptibility of insects. Mass production technologies appropriate for developing nations with low capital investment is another problem which requires intensive research. Methods for harvesting and storage of fungal products also need research on effective use of entomogenous fungi. Studies on the safety of entomogenous fungi to non-target organisms particularly warm blooded vertebrates and beneficial insects are comparatively few. More emphasis should be given to entomogenous fungi especially those which are being seriously considered for large scale use. To devise formulations of entomogenous fungi suitable to their specific characteristics and to their target insects is another area which requires investigation. Microbial control with entomogenous fungi on account of its more persistent non-toxic effect, selective action, and low production costs shows economic advantage and potentialities over chemical control. Production and use of fungal agents in a developing country like India will conserve foreign currency currently being used to import insecticides.

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Chapter - 26

Mycotoxigenic Fungi – Mycotoxins

The word toxin means a poisonous substance produced by a microbe, a plant, an animal or any living organism. Amongst microbes, fungi are well known for elaborating several hundred toxic metabolites (Turner 1971), which play crucial role in various biological and biochemical interactions. Many definitions and terminologies were made with regard to toxins till 1960 but after this a new group of toxic fungal compounds have gained prominence when it was found that many of these have highly deleterious effects on human and animal systems. The effect could be embryotoxic, teratrogenic, carcinogenic, mutagenic and/or estrogenic, etc. Several vital organs like liver, kidney, lungs, brain, etc. get affected. These toxic compounds have created a separate niche for them in the literature under the term Mycotoxin. Most of the definitions of mycotoxin approach towards their origin and target of toxicity in the living systems. Uraguchi and Yamazaki (1978) defined mycotoxins as ‘secondary fungal metabolites capable of causing pathological changes or physiological abnormalities in man and warm blooded animals’. The definition falls short of justification because the cold blooded animals are also susceptible to such toxins. Moreau (1979) defined mycotoxins as ‘extra cellular zootoxic metabolites produced by moulds in food consumed by man and animals’. This definition also does not seem to be appropriate because mycotoxins are produced practically on all organic substrates. On the other hand, a number of mycotoxins induce a variety of physio-pathological and cytological abnormalities in plants also. International Society for Human and Animal Mycology (ISHAM) at its meeting held in New Zealand in 1982 defined them ‘a mycotoxin is a metabolite of microfungus which when ingested by a natural route in sufficient concentrations will cause ill-effects in animals (including birds and men)’. This definition includes both primary and secondary metabolites but does not include i) toxin produced by mushrooms and other higher fungi, ii) compounds toxic to only plants, microorganisms and insects, iii) compounds produced by fungal pathogens in animal. In the present context, the term ‘mycotoxin’ may be defined as ‘a group of chemically unrelated secondary fungal metabolites which are detrimental to living organisms and cause illness and death in human and animals and in certain cases are also harmful to green plants’.

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Certain toxigenic moulds produce these chemical compounds during their synthesis in food and feed materials. The toxic syndrome induced by ingestion of mycotoxin contaminated food and feeds are referred to as Mycotoxicoses. MYCOTOXINS The association of moulds with feed, food and other agricultural commodities is known since the Biblical time, however, their role in initiating diseases in human being was experienced only after wide spread outbreak of Ergotism in Europe, Alimentary Toxic Aleukia (ATA) in Russia during World War II due to consumption of mouldy grains, Red Mould disease in Japan and in other rice growing countries. Till the middle of this century mycotoxins and mycotoxicoses were the subject of attention only by a few mycologists particularly in USSR and Japan where outbreak of mycotoxicoses were more frequent. But the situation changed dramatically in 1960, when a disease of unknown etiology, tentatively termed as ‘Turkey-X Disease’ (Blount 1961) appeared in England. Sudden death of more than 100,000 young turkeys in the course of few months in the poultry farm of United Kingdom attracted immediate and serious attention towards this problem. The mortality was not confined to Turkeys alone, thousands of young ducklings and pheasants also perished in nearby farms of Kenya and Uganda (Asplin and Carnaghan 1961). These reports initiated the interests in the scientific community and created need of intensive investigations with multidisciplinary approach by the veterinarians, biologists, microbiologists, toxicologists and nutritionists. Blount (1961) and Sargeant et al (1961) recorded contamination of Brazilian groundnut meal and on analysis it was revealed that there was heavy infestation of green mould, which was later identified as Aspergillus flavus. The active ingredient was given the name Aflatoxin on the basis of producing fungus, where ‘A’ represents Aspergillus, ‘fla’, represents flavus and ‘toxin’ as poison. With the increasing input from large number of microbiologists and biochemists, the science of Mycotoxicology has made a multidimensional expansion during the last more than three decades and some active research centres have emerged in our country too. Various Universities viz. Pantnagar, Ludhiana, Bhagalpur, Warangal, Mysore have been working on the problems of important crops and also on the herbal drugs with regards to mycotoxin contamination. NIN (Hyderabad), CFTRI (Mysore), BARC (Trombay) and AIIMS (New Delhi) have also contributed facts in the area of mycotoxin research in relation to human health. Mycotoxigenic fungi Mycotoxins are produced by a large number of fungi, viz; Aspergillus, Penicillium, Fusarium, Claviceps, Alternaria, Pithomyces, Stachybotrys, Phoma and Diplodia. Of these, the species of Aspergillus, Fusarium and Penicillium are the main producers of mycotoxins. Out of about 300 mycotoxins described so far only few have been found to occur naturally. Some of the important mycotoxins producing fungi and their toxic metabolites implicated in the outbreak of mycotoxicoses are presented in Table 26.1.

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Table 26.1. Toxic metabolites of some fungi and their biological effects Mycotoxin

Mycotoxin producing fungi

Biological effects

Aflatoxin (B1, B2, G1, G2) Aspergillus flavus, A. parasiticus

Hepatotoxic, carcinogenic

Ochratoxin

A. ochraceus, A. melleus

Nephrotoxic

Citrinin

Penicillium citrinum

Nephrotoxic

Patulin

P. cyclopium, P. patulum

Cell death, immune suppression

Rubratoxin

P. purpurogenum, P. rubrum

Anaemia, jaundice, convulsions.

Citreoviridin

P. citreoviride

Cardiovascular disorder

Cyclopiazonic acid

P. cyclopium

Death of cattle

Trichothecenes

Fusarium tricinctum, Stachybotrys

Dermal necrosis, vomiting Diarrhoea

T-2 toxin

F. sporotrichioiodes

Haemorrhage

Zearalenone

F. graminearum, F. roseum

Vulvovaginitis

Fumonisins

F. moniliforme

Abortion, carcinogenic

Chemistry and Toxigenic Profiles Mycotoxins are secondary metabolites of some fungi, which are formed by successive series of enzyme-catalyzed reactions from a few biochemically simple intermediates of primary metabolisms, e.g., acetate, malonate, and certain amino acids. The main biosynthetic reactions include condensation, oxidation/reduction, alkylation and halogenation steps, which create a remarkable range of secondary compounds. The main pathways involved in the formation of mycotoxins are the polyketide route (Aflatoxins), the terpene route (Trichothecenes), the amino acid route (gliotoxins) and the tricarboxylic acid route (rubratoxins). Some mycotoxins e.g., Cyclopiazonic acid are formed by the combination of two or more of the pathways. 1. Aflatoxin: These constitute a unique group of highly oxygenated coumarine derivative heterocyclic compounds produced by toxigenic strains of Aspergillus flavus and A. parasiticus. Some other species like A. oryzae and A. niger are also reported to produce aflatoxin. The blue green fluorescence emition under UV light is characteristic feature of the aflatoxin. The blue and green fluorescence and their corresponding Rf values on chromatograms represent Afla B and G derivatives as B1, B2, G1 and G2 respectively. All these aflatoxins are toxic secondary metabolites synthesized in the cytoplasm. The exposure of the aflatoxin may be acute or chronic depending on the test systems, doses and frequency of exposures. Sensitivity towards these varies with species, age and sex of the organisms as well as diet and route of ingestion. In general aflatoxins have been found to be lethal to animals even at shortterm exposure while long-term exposure to 40—60 g/kg body weight of animals causes tumors in organelles. Of the four aflatoxins B1 is the most toxic followed by G1, B2 and G2 in the descending order. Its other derivatives M1 and M2 are also

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toxic due to presence of 2, 3 vinylether double bond in the compounds. It induces variety of symptoms i.e. loss of appetite and reduction in growth of animals. Liver, kidney, skin, intestine and genital organs show necrosis and proliferation in tissues. Development of carcinoma, tumor and mutation in the cells are common features in the affected organs. In blood, reduction in total RBC, WBC and haemoglobin contents are also evident. Biochemically aflatoxin also alter RNA and DNA metabolism. Changes in protein synthesis in the affected animals have also been studied. Natural occurrence of aflatoxins has been reported from cereals i.e. maize, paddy, wheat, sorghum, millets; pulses i.e. moong, urad, bean; dry fruits, spices and herbal drugs. 2. Sterigmatocystin: It is produced by Aspergillus spp., Penicillium and Bipolaris spp. It resembles the Aflatoxin and it is a precursor in the biosynthesis of Aflatoxin. 3. Trichothecenes: It is produced by several fungal genera (Fusarium, Cephalosporium, Myrothecium, Stachybotrys and Trichoderma). There are several naturally occurring trichothecenes in food and feed elaborated by Fusarium spp. including T-2 mycotoxin and deoxynivalenol. T-2 mycotoxin has been studied most extensively and is a natural contaminant of food and feed. 4. Citrinin: It is a methylated heterocyclic compound produced mainly by the toxigenic strains of Penicillium and Aspergillus. The toxic effect of citrinin has been studied in various laboratories on animals. The lethal doses (10-70 mg/kg B.W.) in rabbits and guinea pigs cause swelling in the kidney and acute tubular necrosis. Besides renal damage, its exposure also causes problem in respiration and the cardiovascular system. 5. Citreoviridin: Several species of Penicillium (e.g. P. citreoviride) and also a species of Aspergillus have been reported to produce citreoviridin in foods and feed stuffs. It causes paralysis, cardiomuscular disturbances and loss of eyesight in experimental animals. 6. Cyclopiazonic acid: It was originally isolated from the culture of Penicillium cyclopium. This mycotoxin has been shown to occur naturally in corn, peanuts and kodo millet. There are evidences that cyclopiazonic acid might have been involved along with the aflatoxin in the ‘Turkey X’ syndrome in England in 1960. 7. Ochratoxin: It constitutes a group of hazardous mycotoxin (ochratoxin A, B and C) which received considerable attention worldwide due to their carcinogenic, teratogenic, genotoxic and immune suppressive effect in man and animals. Porcine Nephropathy and Balkan Endemic Nephropathy have been established in man due to consumption of ochratoxin A and its congeners are produced mainly by the toxigenic strains of Aspergillus ochraceus, A. sulphureus, Penicillium viridicatum and P. cyclopium. Chemically ochratoxin A is dihydro-iso-coumarin derivative linked through a 7-carboxyl group to L-phenylalanine by an amide bond. Alterations in DNA, RNA and hematopoietic tissues are common phenomena due to ochratoxin A contaminated food. Ochratoxin B is a dechlorated derivative of ochratoxin A and having comparatively lower toxic potency, while ochratoxin C is more or less equally toxic as ochratoxin A.

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8. Zearalenone: It is naturally occurring mycotoxin, produced by Fusarium moniliforme, F. roseum, F. oxysporum, F. tritici and F. graminearum, which results in estrogenic syndromes involving swelling of vulva, rectal and vaginal prolapse, enlargement of uterus, atrophy of ovaries and abortion in animals. It has been found frequently in commercial swine feed. The low or moderate temperature and higher humidity favour growth of zearalenone. Estrogenism in swine is a common feature due to intake of zearalenone contaminated feed. Degeneration of genital epithelium and sperms by 75% has also been recorded in bulls. However, poultry birds, especially chickens are less sensitive to zearalenone. Its 800 ppm concentration does not show any marked changes in body weight of chickens, whereas, 100ppm zearalenone exposure in turkey birds caused 20% reduction in egg production. Chemically zearalenone is 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-bresorcylic acid lactone. It is a white crystal soluble in aqueous alkali, chloroform, acetonitrite, alcohols, methyl chloride and ether benzene. Zearalenone is also slightly soluble in petroleum ether and insoluble in water, carbon disulfide and carbon tetrachloride. 9. Fumonisins: These are newly discovered mycotoxins, isolated from Fusarium moniliforme, and are found to cause disease in horses. They have also been reported to have cancer promoting activity in rats. Occurrence of mycotoxins Natural contamination of food, feed and other agricultural commodities by mycotoxins is a worldwide problem more in topics than in temperate zones. In the first conference of mycotoxins held at Nairobi in 1977 a review was presented which clearly indicates that practically every edible material including fruits, vegetables, cereals, pulses, spices and plant drugs are suitable substrates for mycotoxin elaboration. India being a tropical country has great diversity in agroclimatic conditions due to the unseasonal rains, high temperature and humidity, which adversely affect standing crops and stored food materials by favouring the growth of moulds. Aflatoxins are the most significant class of mycotoxins to be encountered as natural contaminants in maize under stored as well as field conditions. Aflatoxins are also reported in rice grains, pulses, oil seeds, wheat, sorghum, pearl millet, dry fruits and spices. Aflatoxin in milk and milk products and in cattle feed are reported. Natural occurrence of mycotoxins has also been reported in large number of plant samples i.e. seeds, fruits, barks, woods and leaves used as crude herbal drugs beyond the tolerance level fixed by W.H.O i.e. ppb in Indian context. Factors Affecting Mycotoxin Production Mycotoxin production in natural substrate or in nutrient culture depends on various factors, which can be categorized under three broad headings: 1.

Physical Factors - temperature, humidity, rainfall, moisture of the substrate and duration of storage.

2.

Chemical Factors - level of CO2 and O2, chemical nature of substrate, mineral nutrients and chemical treatment.

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3.

Biological Factors - Plant varietal differences, spore load, strain differences, vectors and microbial interactions.

Importance Mycotoxins enter generally in the human and animal food chains by direct or indirect contamination. In direct contamination the food materials are subjected to growth of the toxigenic fungi. Almost all food materials are susceptible to growth of toxigenic fungi at some stage during their production, processing, transport and storage. The possible route for mycotoxin entry into human and animal foods are a) Toxigenic fungi in damaged foodstuffs e.g. cereals, oilseeds, fruits, vegetables etc. (agricultural produce), consumer foods and compounded animal feeds (secondary infection); b) residues in animal tissues and animal products e. g., milk, dairy produce, meat etc; c) fungi - ripened foods, e.g., cheeses, fermented meat products, oriental fermentations, etc; and d) fermented products, e.g., microbial proteins, enzymes, food additives, such as vitamins (Fig 26.1).

Fig. 26.1. Possible route of mycotoxin contamination and mycotoxicoses (Modified from Samajpati 2002)

Consumption of mycotoxin contaminated food/feeds by human or animal results in its accumulation and exerts a wide range of adverse effects on different vital organs. Some of the very noticeable adverse effects in several vital organs of animal are discussed below. Skin: Trichothecenes are capable of inducing dermal irritation inflammation and desquamation. Aflatoxin contaminated meals cause tumour in mouse, loss of hairs and appearance of red papillae in guinea pigs and papilloma in rabbits. Liver: It is main target organ of animals for aflatoxin and several other mycotoxins. The diagnostic symptoms include hepatocellular necrosis, fibrosis, cirrhosis and various types of lesions frequently leading to death of animals.

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Kidney: Citrinin and ochratoxin A are well known to affect kidney. Exposure to aflatoxin in rabbits degenerates epithelial cells of renal sinus and crystallization in the Bowman’s capsule. In Swiss mice degeneration of glomerular uriniferous tubules and enlargement of Bowman’s capsule is reported with citrinin. Heart: Fatty infiltration of heart muscle and sub-endocardial hemorrhage has been recorded in the monkeys, dogs and cats after application of aflatoxin and Rubratoxin. Lungs: Aflatoxin induces hemorrhage in the lungs of guinea pigs and some other mycotoxins like patulin and trichothecenes cause pulmonary congestion in intoxicated animals. Digestive Tract: Hemorrhage and necrosis are common all along the digestive tract and in the mucous membrane of the affected animals. Degeneration of columnar epithelial cells as well as numerous necrotic patches in mucous layer of Aflatoxin B1 treated rabbits was observed. Reproductive Organs: Zearalenone causes precoccious sexual development in young female swine and inhibits normal development of testes in male and also induces enlargement of vulvas, nipples and prolapse of vagina in swine. In rabbits degenerated spermatogonial cells, fibrosis of somniferous tubules, hypertrophy and hyperplasia in male and degenerated follicles and ovary in females have been observed due to Aflatoxin B1. All these changes in reproductive organs contribute to infertility. Blood Composition: Decrease in total RBC and WBC counts including hemoglobin content in guinea pigs and rabbits due to Aflatoxin B1 contaminated foods are reported. Hazards on Plant System The death of plant cells or derangement of plant physiology by fungal toxins viz. phytotoxins, vivo toxins, pathotoxins or host specific toxins is known since long back. Comparatively little work has so far been done with regard to effect of mycotoxins on plant systems. However available reports reflect that some mycotoxin viz. Aflatoxin, rubratoxin, patulin, zearalenone etc. induce a variety of physiological and biochemical disorders in various plant groups. The effects of various mycotoxins on plant systems based on experimental findings are summarized as below: 1.

Aflatoxin B1: Inhibition of seed germination in lettuce, cowpea, sorghum, maize and mung; aflaroot disease of groundnut; inhibition of chlorophyll synthesis in bhendi, maize and cucumber; reduction of respiratory rate in germinating maize seeds; suppression of synthesis of RNA, DNA and protein in germinating seeds and pollen grains; cytological abnormalities such as increase in number of anaphase chromosomes, inhibition of mitosis chromosome bridge, c-mitosis and reduction of mitotic index; inhibition of fern spore, inhibition of growth of chlorella and several moulds; inhibition of nucleic acid synthesis in bacteria.

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2.

T-2 toxin: Inhibition of seed germination of pea and mung, inhibition of root and shoot elongation, wilting, necrosis, and reduction in weight of seedlings, change in cell membrane permeability in tomato leaf, inhibition in elongation of wheat coleoptiles.

3.

Trichothecenes: Leakage of electrolytes in tomato leaves.

4.

Zearalenone: Inhibition of seed germination, loss in total chlorophyll, inhibition of protein and nucleic acid synthesis in mung and maize.

5.

Patulin: Chlorosis, necrosis and narrowing of leaves in wheat. MYCOTOXICOSES

Mycotoxicoses are known diseases of human beings developed due to the consumption of mycotoxins contaminated foods. Some of the important diseases like ergotism, alimentary toxic aleukia (ATA) of Russia, acute cardiac beriberi and aflatoxicoses (liver cancer and Reye’s syndrome) are discussed. 1. Ergotism: Ergotism is the oldest mycotoxicoses known. It causes sensation of cold in hands and feet along with subsequent intense burning sensation. In advanced cases the extremities become gangrenous and necrotic. The occurrence of ergotism reached epidemic proportions in the Middle Ages, and it was thought long ago in Europe that a trip to the distant shrine of St. Anthony would bring about relief from the intense burning sensations; hence the disease was popularly known as St. Anthony’s fire. The disease is associated with the consumption of bread made from flour of rye and other grains overgrown with toxigenic strains of the fungi Claviceps purpurea and C. paspali. The fungi develop only in the female sex organs of the grasses, producing ergots, black or dark purple compact masses of hardened mycelium. As early as in seventeenth century it was recognized that alkaloids produced by the ergot fungus were responsible for the disease, even though alkaloids had long been known as powerful oxytocies. Chemistry and mode of action: The biologically active ergot alkaloids are derivatives of d-lysergic acid. These compounds produce α- adrenergic blockade, inhibiting certain responses to adrenergic nerve activity, to epinephrine, and to 5hydroxytryptamine. They bring about marked peripheral vasoconstriction, which if not corrected can result in gangrene. The compound is also highly active in direct stimulation of smooth muscles and has been used as oxytoxies to increase the force and/or frequency of uterine contractions. Ergot alkaloids also have effects on the central nervous system, which include stimulation of hypothalamus and other sympathetic portion of the midbrain and depression of the vasomotor centre. These compounds are also centrally acting emetics. Several of the alkaloids are now widely used to treat human diseases. 2. Alimentary Toxic Aleukia (ATA): The outbreaks of ATA have been found to be associated with ingestion of over wintered grains infected with toxigenic fungi, Fusarium sporotrichioides and F. poae. The disease was first reported from Russia (60% mortalities) which was found to affect the hematopoietic system resulting in decrease of red and white blood cells and platelets in animals or humans. In Russia

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harvested crops are left in the field all winter and under conditions of repeated freezing and thawing, fungi produce several toxic metabolites. Recently, it has been found that these toxic compounds contain sufficient amount of T-2 toxin i.e. epoxytrichothecene which is assumed to be the causal agent of ATA. The clinical diagnosis: a) a mild inflammation from the mouth of the stomach occur within hours of eating contaminated bread and which develops into acute gastroenteritis; this local effect continues for several days and ends spontaneously; b) a period of several weeks follows in which there are few gross symptoms but the bone marrow undergoes destruction; after extensive damage of bone marrow hemorrhagic spots appear on the skin and there is a sharp decrease in the number of leukocytes; disturbances of the central and autonomic nervous system are apparent, especially weakness, vertigo, headache, palpitation, slight asthma and lowered blood pressure; c) severe atrophy of the bone marrow with hemorrhagic diathesis, necrotic angina, sepsis, and marked decrease in the numbers of leukocytes, granulocytes, erythrocytes, and platelets; and d) either death or a period of recovery. 3. Acute Cardiac Beriberi: In the nineteenth century epidemics of an acute heart disease broke out in rural areas of Japan. After a detailed retrospective analysis of the disease it was termed as acute cardiac beriberi (‘shoshin-kakke’). It is not an avitominosis but was termed so because of its association with the consumption of polished rice. Syndrome: pericardial distress with palpitation and tachypnea, later on the victim suffers with severe anguish, pain and restlessness and at times can become violently maniacal. The right heart is dilated and the heart sounds are abnormal. In the last stage, as the dyspnea increase, the extremities become cold and cyanotic, the pupils dilate and the person loses consciousness. The neurotoxic mycotoxins of Penicillium citroviride is now known to be related to acute cardiac beriberi. 4. Aflatoxicoses: Aflatoxin is a widespread contamination in man’s food and foodstuffs. Aflatoxin B1 is known as highly carcinogenic compound and believed to be a causative factor of human liver cancer. Hepatocellular carcinoma is unusually high in Sub-Sahara Africa and in Southeast Asia. After studying the evidences for mycotoxin contamination of man’s food supply and the geographical survey of liver cancer in the world associated with the consumption of mouldy foods indicate that aflatoxin is the main cause of this disease. The studies have been conducted in Uganda, Kenya, Thailand, Taiwan and India. There is a reasonably strong epidemiological evidence indicating aflatoxin playing an active role in hepatocarcinoma in man at least in Africa, Kenya, India and Thailand. Control Measures for Mycotoxins 1.

Prevention for the entry of mycotoxins into human and animal food chain due to consumption of contaminated agricultural commodities. This can be achieved by preventing fungal infection during cultivation, harvesting, transportation and storage.

2.

Physical methods - heating, pasteurization, sterilization, cooling, vacuum packing, canning, drying and irradiation.

3.

Chemical methods - by using propionic acid, sorbic acid and acetic acid singly or in combination.

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4.

Detoxification: Once a product is contaminated a) the mycotoxin to be removed and b) the mycotoxin to be degraded into less toxic or nontoxic compounds for making product ready for human or animal consumption. The degradation of mycotoxin could be done by physical methods i.e. irradiation (UV) and heating at 250°C; and chemical treatment with acids, alkalis, aldehydes, oxidizing agents and specifically with NaOH, Ca(OH)2, NH3 etc.

5.

Biological control of mycotoxin production has also been reported by the application of bacteria, actinomycetes, yeast and moulds.

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Chapter - 27

Interaction of Fungi with Higher Plants Some Paleobotanical Glimpses

Fungi are known to live, survive and multiply on/in diversified habitats. Out of estimated 1.5 million fungi around one lac are reported, 29,000 fungal species are reported from India. Graham (1962) and Tiffney and Barghoorn (1974), have stated that approximately 500 fossil fungal species representing 250 genera are known in the literature which are from Cretaceous and Tertiary rocks (Mesozoic and Cenozoic). Some of the earliest evidences of fungi reported are Oomycete like structures from Skillogalee Dolomite, of Precambrian (Schoff and Barghooru 1969); non septate hyphae terminated into vesicles in Palaeomyces in Early Devonion (Kidston and Lang 1921); oogonia with oospore like structures associated with Pennsylvania - upper Carboniferous (Stidd and Cosentino 1975) and the Palaeancitrus, clamp connection bearing hypha like fungus (Dennis 1970). Arbuscular Mycorrhizal fungal structures were described in the cortical tissues of underground parts as evidenced in Upper Carboniferous plant remains (Wagner and Taylor 1981). Similar structures were also reported in host root cells of Cycads collected from the Triassic peat of Antarctica (Stubblefield et al 1987). They may belong to Glomus and Sclerocystis. Hyphae with clamp connections were reported in Upper Carboniferous coal ball material indicating Basidiomycetous nature. Further some fruit bodies such as Sporocarpon, Dubiocarpon, Mycocarpon and Traquairo resembling Ascomycetous fungi were reported on plant deposits of Triassic age. Rothwell (1972) has reported Palaesclerotium from Upper Carboniferous coal-ball materials. Few fruit bodies resembling the modern earth stars and Polypores such as Geasterites from the tertiary and Fomes from Pleistocene are reported (Andrews and Lenz 1947). Microthyriaceous fungi resembling the modern Asterina, Microthyrium and others are reported from fossil angiospermus of Eocene. Meliola like Callimothallus, Microthallites, Paramicrothallites and Parmathyrites are some of the microthyriaceous fungi reported from Eocene. Paleomycologists have not given the needed importance of saprophytic fungi in the fossil record. Stubbfield et al (1985) have reported mycelial pockets in the secondary wood of Callixylon newberrys from upper Devonian. The identification of parasitic fungi has become a major problem due to the absence of symptom documentation in fossils. Dilcher (1965) has reported several epiphytic fungi in the tertiary fossil materials. Angiospermic diversity and fungal

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diversity at least during Carboniferous and Cretaceous or Tertiary seems to be similar even if one compares the modern fungi. If the rate of nucleoid substitution is approximately constant for all lineages and if fossil evidence is available to calibrate the rate of nucleotide substitutions, then the percentage, substitution between pairs of species can be used to estimate their times of divergence. Berbee and Taylor (2001) have reported an initial estimate of the divergence time for major groups of fungi based on 18 S rRNA gene sequences. Basidiomycetous fungi might have radiated after the Cretaceous. Most Glomales, Endogonales, Ascomycetes and Basidiomycetes are associated with terrestrial plants. The most discreet assumption is that radiation of these fungi followed the origin of land plants. While the date of origin of the first terrestrial plants is uncertain, microfossils from 460 Ma (Gray 1985) have been attributed to terrestrial plants. Conservatively placing the origin of land plants at 600 Ma, 140 Ma earlier than their appearance as fossils, provides an earliest possible date for terrestrial fungal radiation. Fossil spores and arbuscules from about 390 Ma represent the most recent possible date for the origin of Glomales. A 290 Ma clamp connection (Dennis 1970) provides a most recent possible date for Basidiomycota. Glomales or Endogonales divergences occurred about 600 Ma or even earlier. The tree from 18 S rRNA sequence data shows Ascomycota and Basidiomycota diverging from one another in the Paleozoic, about 500Ma. As per rough estimate 300, 000 angiospermic plant species are available. It is known that angiosperms originated just prior to the Cretaceous. Further they were well preserved in Lower Cretaceous on warts. Though fossil angiosperms described are not many, still some data of Tertiary fossils from Deccan intertrappean flora of India stands for documentation. Chitaley (1974), Prakash (1974) and Lakhanpal (1974) have published excellent reviews on fossil plants of Deccan intertrappeon beds. Fungal spores and some microfossils have been recovered from oil bearing sediments. Palaeoecological studies of fungi must deal interaction with the biotic environment provided by plants and animals. Host pathogen interaction is another aspect which does not have basic information in the form of fossil evidences. In conclusion the interaction of fungi with higher plants with reference to paleobotanical evidences need to be documented in appropriate manner by exploring more fossil fungi, chemical aspects and geological aspects.

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Chapter - 28

Keratinophilic Fungi – General Account

Keratinophilic fungi are able to decompose keratin of human and animal origin. Keratins occur in animal appendages like hair, wool, feathers, nails, hooves, horns and also in the outer layer of skin. In addition to keratin, keratinaceous material contains a large proportion of non-keratin protein, which is most susceptible to decomposition by microorganisms. The fungi capable of enzymatic degradation of keratin substrates are restricted largely to a single lineage of filamentous fungi: Ascomycete (family Gymnoascaceae). A number of species of this family are keratinophilic. They are also known from different ecological niches, which are less explored and have extreme climatic conditions. These fungi are reported from different habitats of various parts of India and reported to recycle keratinic matter added to the soil. These fungi are also known to produce nano particles. There are reports of occurrence of these fungi from polluted water bodies from some parts of world. These fungi can be indicator of pollution in water bodies. The habitats for colonization by keratin fungi include feathers, human hair, human skin, cattle sheds, animal burrows, birds’ nests, poultry sheds, garbage, barber’s dumping area, dung, soil etc. These fungi play key role in the degradation of keratinized material. They reproduce asexually by conidia or mitospores and sexually by means of sexual spores/fruit bodies like ascomata. In fungi belonging to onygenales, the asci are naked and eight ascospores are produced in each ascus. However, many keratinophilic fungi are under dormant stage till fresh keratin substrate is available. There are two ecological groups of Keratinophilic fungi. 1.

Dermatophytes and other species capable of attacking native keratin by penetration and enzyme activity.

2.

Saprophytic species that attack keratin by simple hyphal penetration and by surface growth utilizing easily decomposable compounds of keratin.

The presence of keratin degrading enzyme, keratinase has been reported. Lipases, urease, amylases, phosphatases, catalase, cellulase, raffinase, pectinase, nucleotidases, and others have been reported in Trichophyton spp., and in other dermatophytes. pH range of 4-10 and moderate temperatures along with other nutrients are of great help in the growth of keratinophiles.

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The isolation of these fungi is done using Vanbreuseghem's hair bait method. Once the fungus grows on hair bait, it can be transferred to agar medium. The keratinophilic fungi can be identified using different manuals. The keratinolytic capacity of the fungus is established by estimating the keratinase, the enzyme responsible for degrading keratin. Most of the keratinophilic fungi belong to Ascomycotina and Anamorphic fungi. The onygenales of Ascomycotina has got four families represented by Arthrodermataceae, Gymnoascaceae, Myxotrichaceae and Onygenaceae, having 37 genera and around 100 species, DNA based techniques have become handy in the identification of species though morpho-taxonomic base is essential. DERMATOPHYTES Dermatophytes are the keratinophilic fungi that cause infection and include molds and yeasts. They infect both man and animals, provided suitable and congenial conditions prevail for their growth. Animals and soil act as reservoirs of Dermatophytes and some disease causing dermatophytes are specific to geographical areas and also host/ host organs. Table 28.1. Important dermatophytes Class

Disease

Infected area

Fungus

Superficial

Black Piedra

Scalp, beard

Piedraia hortae

White piedra

Beard, scalp, pubic hair Trichosporon beigelii

Tinea nigra

Thick stratum corneum, Cladosporium werneckii, palms, feet C. mansonii

Dermatomycoses

Skin, Hair, Nails

Epidermophyton, Microsporum, Trichophyton

Tinea barbae

Beard, body hair

Trichophyton uerrucosum

Tinea pedis

Feet, soles

Trichophyton rubrum

Tinea facei

Non-bearded area of adult males, face

Trichophyton rubrum

Tinea axillaris

Groin, genital area

Trichophyton mentagrophytes

Intertriginous Candidiasis

Moist skin areas, groin, Candida albicans penis, scrotum, buttocks, under the breast axilla, vagina, anus

Otomycosis

Ear

Cutaneous mycoses

Sub-cutaneous

Mycetoma, madura Skin surface foot Sporotrichosis

Candida sp. Allescheria boydii

Skin, hands, arms, legs Sporothrix schenckii

Keratinophilic Fungi – General Account Class

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Disease

Infected area

Fungus

Rhinosporidiosis

Nasal muscosa

Rhinosporidium seeberi

Systemic mycoses Histoplasmosis

Lungs, Kidney, Bones Hisoplasma capsulatum

Blastomycoses

Lungs, Skin

Blastomyces dermatitidis

Cryptococcosis

Lungs, central nervous Cryptococcus neoformans system

Aspergillosis

Lungs, skin, body organs

Aspergillus fumigatus

Mucoromycosis

Face, Sinuses, Lungs

Absidia corymbifera

The infections on humans and animals are categorized into 1. Superficialinfection on outer surface of the skin. 2. Cutaneous – infects only keratinized tissues. 3. Subcutaneous – infections is confined to deeper layers of the skin, sometimes invading muscles, bones, lymphatic system. 4. Systemic – infection of lungs by direct inhalation of fungal spores, there by spreading to vital organs leading to death. Keratinophiles / dermatophytes that invade human tissue are (Table 28.1): i.

Primary pathogens capable of fungal infection in the healthy individuals.

ii.

Opportunistic fungi invade the compromised host tissue.

Non-hygienic conditions, sanitation, health condition, nutrition, cleanliness of surroundings, human habits and living conditions etc. play important role in the spread and occurrence of keratinophilic fungi. Some antibiotics, creams, lotions, some homeopathic/ayurvedic drugs are reported to treat some infections. However, once dermatophytes attack, it is difficult to get cured.

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Chapter - 29

Mycological Methods - Collection, Observation and Isolation

COLLECTION Beginners can take a real interest in systematic mycology to acquire a general knowledge of the various groups of fungi to provide a background. The best satisfactory way to do this is to learn about common species in one’s own neighbourood and, to do this; one must collect and study them. He or she should attempt to develop a keen eye so that smaller and critical species are not overlooked. One must know the habitats in which particular groups predominate, and recognize the range of variation within individual species in the study area. Systematic collection provides the basis of all taxonomic research. Someone working on a particular group in the laboratory or herbarium also needs to study and collect material of his/her group in the field. Only through field studies one can become familiar with the inherent variability of species and the effects of environmental factors on them. Field excursions and forays will be advantageous. Most collectors at first bring back specimens which are either too small or in too poor a condition for accurate identification. Careful searching of a very limited number of sites is often more fruitful than small visits to a large number. Both the quality of the collections obtained and the number of species found tend to be in inverse proportion to the distance travelled whilst collecting. All collectors should obtain permission to collect in an area from forest department or the private owners. As future generations will also wish to study fungi and lichens the collector should be conscious of the need for conservation. Most ephemeral fungi are unlikely to be affected by over collecting but some groups, particularly the lichens, may all too, easily be endangered by overzealous collectors. Correct identification of hosts and substrates are necessary. Fungi collected from particular hosts or substrates of the same locality may be placed in separate carefully labeled bags. Paper bags or envelopes may be used and data may be written directly on them but polythene bags or tins are more suitable for collecting damp material. Larger fleshy fungi are likely to be damaged if placed in bags and should be wrapped individually in newspaper and carefully placed in flat-bottomed marketbaskets.

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Material needs to be examined on the same day. If not possible the collected material be kept in polythene bags but thoroughly dried to prevent the growth of unwanted moulds (saprophytic fungi). Agaricales Fleshy fungi (‘macromycetes’), particularly the larger members of the Agaricales, are the most difficult groups to preserve satisfactorily. On returning from a collecting trip the material should be examined on the same day. Habit sketches, measurements, colour of different parts, colour on bruising and cutting the flesh, any exudates on cutting, odour, consistency, spore-print and chemical tests (Zoberi 1972) may be made at this stage. The blotting paper method described by Bohus (1963) may be followed for preparing specimens. Satisfactory method of preserving fleshy fungi is by freezedrying (lyophilization). Rusts and smuts Rusts and smuts are the easiest fungi to preserve. Identification of hosts is essential in these groups as identification of the fungus may prove difficult if the host is not known. Rusts and smuts are readily preserved by drying the infected leaves between sheets of newspaper under slight pressure. Ascomycotina Species of Morchella and others fleshy ascomycetes require treatment as described above for fleshy fungi. Many species occur on wood and to collect these, knife and small saw are required. For some pyrenomycetes the perithecial stromata can persist long after the spores have been discharged. Small ascomycetes grow just below the surfaces of stems, leaves and bark with only their minute ostioles protruding. These can be collected by peeling back the surface layers of the bark with a sharp finger-nail or knife. Anamorphic Fungi Coelomycetes and Hyphomycetes growing on dead and living stems, bark, leaves, litter, etc. can be collected. A hand lens can be used for observing the growth of fungi. Hyphomycetes form distinct brown, black, green, pinkish, rose or whitish felt-like growths on leaves, decaying fruits, rotting wood and bark but many are relatively inconspicuous. Material be examined with a binocular microscope, sporulation may be stimulated to develop by placing them in damp chambers. Myxomycota Myxomycetes grow on woods after heavy rain. They are most commonly encountered on rotting wood but some also occur on dung and damp straw. Specimens should be removed together with the wood on which they are growing with the aid of a sheath knife and are best pinned to cork in small boxes or tins as

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they are collected to reduce the risk of damage to their often delicate fruiting structures. Plasmodia are often conspicuous and brightly coloured but difficult to name. Soil fungi Soils are rich in fungal floras in addition to the larger basidiomycetes which produce their sporocarps on the surface of the soil. Soil mycofloras can be investigated by mounting soil on a slide and examining it directly or by a variety of isolation techniques. Waksman’s Dilution plate method and Warcup’s soil plate methods are also used. Dung fungi Many members of the Mucorales and some Ascomycetes, Basidiomycetes and Myxomycetes are known only from the dung of various animals and birds. Dung at different stages of decomposition has characteristically different mycofloras, therefore many dung types in various states should be studied. If pieces of dung from different animals are collected and placed in ‘damp chambers’ (Petri dishes lined with moist filter paper),the succession of species can be followed and many fungi not detected in the field can be found in this way. Airborne fungi Airborne fungal spores can be trapped by exposing glycerine jelly or vaseline coated slides, or Petri dishes to the air for varying lengths of time, or by more complex quantitative sampling techniques such as Anderson sampler, Tilak sampler and other methods employed to detect air-borne fungi. Fungi on man and animals Hairs, skin and nail scrapings, feathers and horns infected by dermatophytes can also be collected. These may retain their pathogenicity for several years but are rendered harmless by fumigation in Petri dishes between layers of filter paper soaked in formalin or by treatment with propylene oxide. They may then be placed in transparent paper packets or glued into small boxes. Laboulbeniomycetes are often most satisfactorily preserved in 70% alcohol. OBSERVATION As soon as the collector returns to the laboratory he/she must begin to examine the material. Examination starts with a study of the macroscopic features visible to the unaided eye, by hand lens, dissecting microscope, proceeding to high resolution microscopy and microchemical characters. All notes, drawings, photographs and microscopic preparations relevant to particular collections may either be placed with them in the herbarium or retained in a series of loose-leaf folders. All specimens which are eventually destined for a herbarium are given accession numbers. Holotype, Isotype, Neotype etc., may be made and holotype be deposited in two recognized centres.

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Microscopic examination High power, microscope providing magnifications of up to x 1000 is essential for examining both larger Hymenomycetes and minute Hyphomycetes alike. Information as to the types of microscope required, their setting up and use is provided by Laundon (1968). For routine examination a minute portion of the sporing tissue is removed under a dissecting microscope with the aid of a mounted needle, needle-knife, razor blade or scalpel point, and placed in a small drop of mounting fluid on a microscope slide; the bulky material is teased apart gently and a coverslip applied. Some light pressure on the coverslip (e.g. by tapping with the butt of a pencil) serves to disperse the structures in a thin optical plane. Warming the slide over a spirit lamp or small Bunsen flame or placing it on a warm microscope lamp case will cause any air bubbles that may be present to disappear but the mounting medium should not be allowed to boil. The most satisfactory general purpose mounting medium for fungi is Amann’s lactophenol including a stain such as cotton blue. This medium has the advantage that the slides can then be readily preserved by sealing them with nail varnish or other suitable proprietary sealant (such as DPX) but is not suitable for preparations of lichens growing on calcareous substrates. If a slide in lactophenol is to be preserved it must be warmed for an hour or so to ensure that air bubbles are completely eliminated and any excess mounting fluid thoroughly wiped off. Two layers of nail varnish are then applied, a clear one first and then a coloured layer when the first has dried. This two-coloured nail varnish procedure has the advantage that it is always readily apparent whether one or two layers have been applied. Slides preserved in this way are reasonably permanent and may be kept with the material from which they were made in specially constructed slide-boxes. Tribe (1972) recommends the use of ‘Glyceel’ instead of nail varnish, and Laundon (1971) that a layer of PVA (polyvinyl acetate) water-based glue be placed over a layer of nail varnish to make the slides more durable. A valuable method for studying the microfungi on leaf surfaces is the cellulose acetate ‘Necol’ mounting technique (Ellis 1950). An alternative to this method is to use adhesive transparent tape (e.g. cellotape) to strip off the fungus. In this latter case drops of lactophenol can be placed above and below the tape on a microscope slide and a coverslip applied. To ascertain the structure and arrangement of sporocarps accurately, vertical sections will sometimes be required. Although it is sometimes possible to prepare adequate sections by hand using a single-edged razor blade or ‘cut-throat’ razor and holding the material between some tissue such as elder pith or under the dissecting microscope, a freezing microtome provides the most satisfactory results. Microscopic examination by normal transmitted light is adequate for most purposes but dark-field and phase contrast sometimes prove of value when studying indistinct (e.g. spore ornamentation) or hyaline structures. Microscopic measurements are made with a micrometer eyepiece but before the eyepiece scale can be used it must first be calibrated. Each microscope must be calibrated individually and this is achieved by placing a graduated slide (i.e. a slide with a scale accurately divided into divisions each of which is, for example, 10 µm)

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on the microscope stage and measuring the number of divisions on the eyepiece scale which correspond to one or more divisions on the graduated slide. From this information the distance each division of the eyepiece scale represents can be very easily calculated. A separate set of calibrations is required for each eyepiece and objective used and many mycologists draw up a table to show what each eyepiece division (and multiples of it) represent with particular objectives. Measurements need only be given to the nearest 0.5 µm but the lengths and breaths and sizes of any appendages all need to be ascertained. Measurements normally include any epispore or ornamentation but where these are conspicuous these should also be measured independently. The sizes of larger structures such as globose sporocarps (e.g. perithecia, pycnidia) are difficult to ascertain from squash preparations and measurements of these structures may most satisfactorily be determined from unsquashed or microtome sectioned material. Herbaria Herbaria (sing. Herbarium) are places where dead (usually dried) material of plants is permanently preserved. The larger mycological herbaria contain many tens of thousands of specimens including type material and other important collections. Such herbaria are invaluable to systematists as they serve as reference collections and contain material which needs to be examined in the course of revisionary and monographic studies. More detailed accounts of the ways in which material at CMI is handled are provided by Ellis (1960) and Onions (1971). Major Indian Mycological Herbaria are established as HCIO (Herbarium Cryptogamae Indiae Orientalis), at Division of Plant Pathology, IARI, New Delhi and at BSI (Botanical Survey of India), Kolkata Specimens A herbarium specimen may be a single sporocarp or a portion of one (e.g. in Aphyllophorales and Agaricales), dried culture, slide, or the material on its host or substrate (e.g. leaf, stem, bark, rock, soil, paper, cloth). Most microfungi and lichens are adequately preserved simply by drying and by the time they reach the herbarium this process should have been completed. All specimens used in studies which are published need to be deposited in recognized herbaria. In addition to material cited in floristic and taxonomic studies, the ones employed in chemotaxonomical, biochemical, physiological, serological, pathological or genetic studies should also be included. Slides Slides made from a specimen are kept together with the specimen. This procedure facilitates any subsequent examination and prevents additional parts of the specimen having to be removed each time anyone wishes to check it, a consideration which is particularly important in the case of type specimens. In some cases removal of fragments from a particular collection for microscopic examination by different workers over the years has rendered them almost useless today. Slides made with lactophenol are preserved by ringing with nail varnish or glyceel and placed in

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specially made slide boxes which are kept with the specimen itself. Some herbaria maintain separate slide collections in wooden cabinets with shallow drawers made specially to contain slides and affix labels to herbarium specimens to indicate that a slide is in existence but housed separately in their ‘slide collection’. Cultures Cultures now constitute an extremely important part of mycological studies and dried cultures also have a place in the herbarium. To prepare dried cultures 1.5% tap water agar and glycerol is melted and poured on to the lid of a plastic Petri dish; the culture is removed from the other half of the Petri dish and placed in the centre of the lid or on a piece of hardboard. In the case of test tube cultures these are removed from their tubes with a scalpel and laid on molten tap water agar on a smooth piece of hardboard to which an adhesive label bearing the number of the culture has also been attached. The cultures are then allowed to dry in a drying cupboard. Fungal cultures are grown in different agar media (Potato Dextrose Agar, Czapek’s, Malt extract, Oatmeal, Maize meal, Hemp Seed, Sabouraud’s etc.). Indian mycological culture collections are established at ITCC, IARI, New Delhi; NCIM, NCL, Pune; MTCC, IMTECH, Chandigarh; NFCCI, ARI, Pune and others. Packets All the material of a particular collection is most appropriately kept in a packet of a standard size made from good quality stiff paper. At the CMI 15x 10.5 (6 x 4 in) cm packets when folded are used. Packets of this pattern are the most suitable for sticking to herbarium sheets as all four flaps may be opened to expose the contents. Very large specimens are kept separately in boxes and notes placed on appropriate sheets in the herbarium to indicate their existence. Specimens to be placed in herbarium packets at the CMI are first inserted into transparent paper envelopes. Fragile specimens may be wrapped in soft tissue paper and lichens are often best glued to 5 x 3 in blank index cards. Protective boxes (c. 5 x 5 x 0.5 cm) glued to cards or herbarium sheets are also useful for preserving material likely to be damaged by friction. All items within a particular packet should be clearly numbered individually with the herbarium accession number and (or) collector’s number. ISOLATION TECHNIQUES Isolation of plant pathogens Diseased plant materials have to be collected in fresh polythene bags. After reaching the laboratory thin transverse sections of the diseased material mounted in lactophenol is observed for preliminary identification of the pathogen. Symptoms such as vascular browning with rusts, smuts, leaf spot, blights, canker’s etc. can be noted. If necessary, microtome sections can be prepared for permanent slide preparation. The diseased plant tissue is surface sterilized in 0.01%, HgCl2 or sodium hyphochlorite followed by repeated washings by sterile water. After final washing the disease plant tissue can be dabbed in sterile blotter so that moisture can be

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removed. Later the diseased tissue is plated on 1% agar medium followed by its transfer on to 2% potato dextrose agar medium. The inoculated Petri dishes are incubated at room temperature and also under UV light. Sporulation is observed frequently. The vegetative and reproductive structures produced in the culture are compared with structures observed in thin sections. Thus the identity of the pathogen is established. In order to establish whether it is the same pathogen which is sporulating in culture is the causal agent or not, Koch’s postulates have to be followed. The diseased material is inoculated on the agar medium. The sporulating pure culture is then inoculated on to the specific host. The symptoms produced after inoculation of the culture on susceptible and specific host and compared with field specimens symptoms. Thus the pathogenicity can be established. Isolation of fungi from roots The roots are first washed in running water followed by sterile water. The root pieces are treated with 0.01% HgCl2 or sodium hyphochlorite and washed with sterile water. Such treated root pieces are plated on potato dextrose agar and observed for sporulation. Isolation of fungi from aerial plant parts The aerial plant parts such as stems, bark, petiole, wood, leaves, floral parts, fruits, seeds etc., get affected by fungal pathogens. Such diseased specimens can be collected. The diseased tissues are plated on agar plate having potato dextrose medium after treating with 0.01% HgCl2 or sodium hyphochlorite and sterile water. The inoculated agar plates are incubated at room temperature and observed for fungal growth. Purification of fungal cultures Purification of cultures of filamentous fungi may be achieved by growing the primary material on plates of plain distilled water agar with a dry surface. To avoid bacterial contamination, an antibiotic such as pimonain, PCNB, streptopenicillin etc. are used. The agar plates are incubated at room temperature of required temperature. A part of the growing fungi is cut from agar plate and inoculated into full fledged agar medium. After a period of 8-10 days the cultures are observed and identified. Single spore isolation Various techniques such as semi-mechanical, mechanical methods are available. Filtered agar is recommended when microscopical searching for spores is involved. In semi-mechanical method, fungal spore suspension is made on a sterile grid containing sterile water. This spore suspension is streaked across a marked line on a very thin plate of tap-water agar and incubated at about 24°C. Germination occurs after 12 hrs. Germinating spores are selected and agar block and such spores are transferred into an agar plate or tube. In the mechanical method the dummy microscope objective lens is replaced by a sharp edged metal tube. The agar is cut by

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lowering the cutter until it reaches the bottom of the dish. The cut agar block is reinoculated into fresh agar plate. Isolation of Conidial fungi from water The conidial fungi are known to colonize the submerged decaying leaves of angiosperms and others. Under natural conditions submerged dark brown, softened and skeletonized leaves of well aerated streams have to be collected in plastic bags. On return to the laboratory the leaves are washed and placed in a separate sterilized Petri dish having sterilized distilled water. The dishes are left for a day or two at 200C. The incubated leaves can be scanned for the fungal growth. Foam samples can be collected below waterfalls and can be scanned for conidial fungi. Quantitative techniques involve sucking the water sample, through a Millipore filter and staining the conidia by cotton blue in lactic acid followed by their counting. Conidial fungi can be cultured on malt extract agar medium. Conidial fungi in water help in recycling of organic matter, serve as bioindicators and contribute to food chain cycle. Isolation of Trichoderma Trichoderma is a very important soil fungus and is involved in biological control of soil-borne and root-borne pathogens. This fungus can be isolated from soil, seed, root, leaf surface etc., using serial dilution plate technique and direct plating of the substrate on agar media. The media such as commercial dextrose agar, special nutrient agar, cellulose agar, malt extract agar, oat meal agar, potato dextrose agar, special nutrient agar, Trichoderma selective medium etc. are used in the isolation of Trichoderma. Isolation of zoosporic fungi Zoosporic fungi mostly found in fresh water and marine water can be isolated by baiting half boiled hemp seeds, maize, chitin and keratin substrates, cockroaches, home flies, snake skin, peelings of fruits, tender grass blades, seeds of brassica, bhendi, etc. The baits can be placed in tumblers having water sample. After 24-48 hrs the baits are scanned under binocular research microscope. The colonized bait are washed in sterile water and baited with fresh bait material. These fungi grow very well on potato dextrose agar media, maize meal agar medium, hemp seed agar and oat meal agar medium. Arbuscular mycorrhizal fungi The arbuscular mycorrhizal (AM) fungi form obligate symbiotic association with roots and other underground parts of the plant. AM fungi play significant role in the transport of phosphorus and other nutrients. The AM fungi consisting of spores, sporocarps and hyphal bits can be extracted from soil using modified wet sieving and decanting techniques.

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Quantification of root colonization is done by detecting the presence or absence of colonization by hyphae, arbuscules, resting spores or sporocarp in the trypan blue stained root cortex. Isolation of Actinomycetes Actinomycetes are isolated from the soil by using differential media. Actinomycetes, in general, are slow growing and hence they are likely to be masked in culture plates when grown in ordinary nutrient media. For isolation of actinomycetes, media containing low nitrogen, such as Ken-Knight's, egg albumin, or Conn's medium are used to isolate them. The plant pathogenic actinomycetes like Streptomyces scabies, are isolated by using tyrosine-casein-nitrate agar medium. For routine culturing of actinomycetes, yeast extract or Czapek's medium is used.

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References

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Questions

Give short answers 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

What is algal hypothesis? What is biodiversity and fungal spectrum? What are Aflatoxins? What are Mycoherbicides? Describe Caterpillar fungus. What is the importance of aeromycology? What are leaf spot diseases? Distinguish between downy mildews and powdery mildews. What is meant by cultural practices? What are systemic fungicides? What are the different types of endophytic fungi? Define rhizosphere. Describe coprophilous fungi. What are seed-borne fungi? What are keratinophilic fungi? Define heterokaryosis. What is bipolar and tetrapolar heterothallism? What is meiotic recombination? How is diversity measured? Distinguish between ex-situ and in-situ conservation. Differentiate between Foliose and Squammulose lichens. What are Cephalodia? What are Ascolichens? How are Lichens used as food? What is Button Mushroom? What is Ectomycoorhiza? What is Arbutoid mycorrhiza? What is Philipps and Hayman technique?

Questions 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

393

What are sex hormones in fungi? What is carotene synthesis? What are fungal mating types? What are theromophilic fungi? What are phylloplane fungi? What is the importance of Beauvaria bassiana? What are Ochratoxins? Define dermatophytic fungi. What methods are used for isolation of water fungi? What is fungal herbarium? What is meant by microscopic observation of fungi? What is done for purification of fungal culture?

Give long answers 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

Give a detailed account of phylogeny and evolution of fungi? Define biodiversity? Give a detailed account of biodiversity of fungi? Write an essay on fungal Biotechnology of fungi? Discuss in detail abut aeromycology and its importance? Give a list of fungal diseases on plants and methods of control by cultural practices? Discuss about fungicidal control of fungal diseases in plants? Give a general account of Endophytic fungi and its importance? Elaborate on various aspects of fungal Ecology? Give a general account of sexual reproduction and genetic basis? Discuss about Fungal genetics in general? Discuss in detail the conservation strategies of fungi and reasons for the loss of biodiversity? Write an essay on general characters of lichens? Outline the economic importance of lichens? Discuss in detail the methods of reproduction in lichens? Write in detail on Button Mushroom cultivation? Give an account of cultivation of Oyster Mushroom? Outline the general features and economic importance Mushrooms? Write an essay on Mycorrhiza? What are the recent trends in Sexual Reproduction? Write about fungi in miscellaneous substrates? Give a detailed account of Entomogenous fungi? Discuss about Entomophthorates? What is Aflatoxin and elaborate them in detail?

394

Biology and Biotechnology of Fungi and Microbes 24. Wirt an essay on mycotoxins and their importance? 25. Discuss in detail about Palaeobotanical glimpses of fungi in relation to Higher plants? 26. Give a detailed account of keratinophilic fungi? 27. Write in detail about mycological methods? 28. Give an account of fungal isolation techniques? 29. Elaborate on fungal herbarium preparation? 30. Define conservation? Discuss about ex-situ and in-situ conservation?

C. MICROBIOLOGY

Chapter - 30

The Living Kingdom

In living systems, a cell is the basic functional unit carrying out the life processes. A cell arises by the division of pre-existing cell or in certain cases by sexual reproduction or by fission of pre-existing cells. Earlier biologists divided the living world into two kingdoms, the Plantae and Animalia characterized by multicellular structures with extensive tissue differentiation. As the properties of various microbial groups were established, it became evident that some of them cannot be fitted into either of the traditional kingdoms. In 1866, Haeckel, a German zoologist and one of the Darwin’s disciples proposed the establishment of third kingdom, the Protista which includes unicellular, coenocytic or multicellular organisms showing no tissue differentiation as seen in protozoa, fungi, algae and bacteria. The cellular organisms are divided into two groups, namely, prokaryotes and eukaryotes based on the type of cells they possess. Prokaryotes are simpler in structure and usually small (mean diameter 1 µm) and do not possess a distinct nucleus. On the contrary, the eukaryotes cell is larger (5 µm or more) and complex structure having distinct nucleus and other organelles like plastids and mitochondria. The distinctive features of prokaryotes and eukaryotes are given in the following Table 30.1. Table 30.1. Difference between prokaryotic and eukaryotic cells. Character

Prokaryotes

Eukaryotes

Groups where unit of structure

Bacteria, cyanobacteria, protozoa

Most fungi, algae, higher plants and animals

Diameter of cell

1 µm

10 µm

Presence of lipids

Glycerol diesters

Glycerol diesters

Presence of fatty acids

+

+

Presence of steroids

+

rare

Nuclear membrane

-

+

Endoplasmic reticulum

-

+

Mitochondria

-

+

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Character

Prokaryotes

Eukaryotes

Golgi apparatus

-

+

Chloroplasts

-

+ or -

Peptidoglycan in cell wall Locomotory organells Axial filament Number of chromosomes Arrangement of DNA Mitotic division Meiosis Ribosome size

+ Bacterial flagella/cilia in some + (in some) one Circular Absent Absent 70S

Multistranded More than one Linear Present Present 80S

Ameboid movement

-

+

Prokaryotes share many common features, such as lack of nuclear membrane, unicellular, division by binary-fission and generally small size. The various species differ amongst each other based on several characteristics, allowing their identification and classification. Bacteria were first classified as plants constituting the class Schizomycetes, which along with the Schizophyceae (blue green algae/Cyanobacteria) formed the phylum Schizophyta. Haeckel in 1866 proposed a three-kingdom system which added the Protista as a new kingdom that contained most microscopic organisms. In one of the subdivisions Monera of Kingdom Protista he included completely structure less and homogeneous organisms, consisting only of a piece of plasma. A division of various Kingdoms as proposed by various workers is given in Table 30.2. Table 30.2. Division of living Kingdoms as proposed by different workers Linnaeus 1735

Haeckel 1866

Chatton 1925

2 3 2 Kingdoms Kingdoms Kingdoms

Copeland 1938

Whittaker 1969

4 Kingdoms 5 Kingdoms

Prokaryota Monera

Monera

Protista

Protista

(not treated) Protista

Vegetabilia Plantae Animalia

Animalia

Source: Wikipedia

Eukaryota

Plantae Animalia

Plantae

Woese et. al. 1990 3 Domains Bacteria Archaea

Cavalier-Smith 1998 6 Kingdoms

Bacteria Protozoa Chromista

Eucarya

Plantae

Fungi

Fungi

Animalia

Animalia

The Living Kingdom

399

The characterization and taxonomical grouping of living organisms till early 1960’s was based on the cellular organization as revealed by microscopic (light microscope and/or electron microscope) studies. A comprehensive system of classification based on the nutrition and absorption of nutrients by the living organisms was proposed by Whittaker in 1969 (Fig. 30.1). He proposed the classification of all living organisms into five kingdoms.

Fig. 30.1. Whittaker’s five kingdom system

Carl Woese argued that the bacteria, archaea, and eukaryotes represent separate lines of descent that diverged early on from an ancestral colony of organisms. However, a few biologists argue that the Archaea and Eukaryota arose from a group of bacteria. In any case, it is thought that viruses and archaea began relationships approximately two billion years ago, and that co-evolution may have been occurring between members of these groups. It is possible that the last common ancestor of the bacteria and archaea was a thermophile, which raises the possibility that lower temperatures are extremophiles (extreme environment) in archaeal terms, and organisms that live in cooler environments appeared later. Since the Archaea and Bacteria are no more related to each other than they are to eukaryotes, the term prokaryote's refers to those surviving under extreme environmental conditions (not a eukaryote) thus limiting its value. In 1987 Carl Woese divided the Eubacteria into 11 divisions based on 16S ribosomal RNA (SSU) sequences which are still followed till today with several additions While the three domain system is widely accepted, some authors have opposed it for various reasons. With improved methodologies it became clear that the methanogenic bacteria were profoundly different and were believed to be ancesstors to ancient bacteria. Carl Woese, regarded as the forerunner of the molecular phylogeny revolution, identified three primary lines namely Archaebacteria, the Eubacteria and the Eukaryotes. The latter now represented by the nucleocytoplasmic component of the Eukaryotes. These lineages were put into the rank Domain which divided life into 3 groups (Eukaryota, the Archaea and the Bacteria). This scheme is followed by microbiologists even today (Fig. 30.2).

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Fig 30.2. Carle Woese concept for grouping of all living organisms in to three domains (kingdom) based on 16S or 18S rRNA gene sequence

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Chapter - 31

Historical Developments in Microbiology

The numerous living forms in nature range from sub-microscopic viruses and phages through the microscopic bacteria, actinomycetes, algae, fungi and protozoans to the lower animals (microfauna). Bacteria and blue-green algae are classified as prokaryotes while others are eukaryotes. In nature, these organisms interact with each others. These organisms colonize, survive and multiply in or on the above substrates in the presence of favourable environmental conditions. They play an important role in the cycling of nutrients, fixing nitrogen (nitrogen fixers) and in energy production. Natural gas (methane) is produced by methanogenic bacteria. Microorganisms play a vital role in bioremediation which are employed to consume spilled oil, solvents, pesticides, antibiotics and various toxic substances. They are employed in the synthesis of products such as alcohols, glycerine, organic acids, enzymes, acetone, drugs and other industrial products. Thus the microorganisms are of great economical value and biotechnological importance. BEGINNINGS OF MICROBIOLOGY Microorganisms have created great history. In the year 1665 a crude microscope was invented by Robert Hooke (Fig. 31.1). He was able to see many small objects with a simple lens that magnified approximately 30 times. Hooke’s discovery made the beginning of the cell theory stating that all living things are composed of cells. Later discoveries have contributed largely to understand the structure and functions based on this theory. Robert Hooke was the first person to describe microorganisms.

Fig. 31.1. Robert Hooke

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Unicellular life was first described after Hooke recorded his observations of microbial world. Antony van Leuwenhoek (Fig. 31.2) (1632-1723) was the first to observe the microorganisms.

Fig. 31.2. Antony van Leeuwenhoek

He polished grains of sand into lenses which were able to magnify 300 times and accurately described and measured bacteria and protozoa that he named them as ‘animalcules’. He was also the first person to describe spermatozoa in 1677 and was the earliest to describe red blood corpuscles. In 1680 he was elected as the fellow of Royal Society of London. He was considered as the Father of Bacteriology and Protozoology. Later scientists have shown keen interest in knowing more about these tiny living things. Until the second half of 16th century, many scientists were philosophers who believed that life forms could arise spontaneously from non-living or denovo. Among the first to dispute the theory of spontaneous generation was the Italian Scientist, Francisco Redi (1626-1697) followed by observation of John Needham (1713-1781) and Lazzaro Spallanzani (1729-1799). Modern era of vaccines and vaccination against smallpox virus was discovered in 1798 by Edward Jenner (Fig 31.3), an English physician Joseph Lister (18271912) developed a system of antiseptic surgery in 1867 to protect against organisms causing wound infections.

Fig.31.3. Edward Jenner

Historical Developments in Microbiology

403

GOLDEN AGE OF MICROBIOLOGY This period during 1854 - 1914 was referred as Golden Era of Microbiology which began with the contributions of Louis Pasteur and continued till 20th century until the advent of World War I.

Fig. 31.4. Louis Pasteur

Louis Pasteur (Fig. 31.4) (1822-1895) was the first to report the role of microorganisms in fermentation processes particularly lactic acid fermentation. He discovered that alcoholic fermentation was due to yeasts. Pasteur disproved the theory of spontaneous generation by his Swan-Neck Flask (Fig. 31.5) experiment. He introduced the terms aerobic and anaerobic fermentation for yeasts.

Fig. 31.5. Swan Neck flask devised by Pasteur

The alcohol yield was more with sugar substrate under anaerobic conditions which he termed as the Pasteur Effect. Pasteur proposed the germ theory of disease and devised the process of destroying bacteria known as Pasteurization. He developed vaccine for anthrax disease and resolved the problem of pebrine disease of silkworms. He devised a vaccine for rabbies virus in 1885. Ferdinand Cohn outlined the entire life cycle of Bacillus (vegetative cell → endospore → vegetative cell). In 1886, he studied filamentous sulphur oxidizing

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bacterium, Beggiatoa mirabilis and was first to identify sulphur granules resulted from oxidation of hydrogen sulphide. Robert Koch (Fig. 31.6) (1843-1910), a German Doctor, demonstrated that anthrax is caused by Bacillus anthracis. Koch developed solid culture media and methods for studying bacteria in pure cultures.

Fig. 31.6. Robert Koch

Mycobacterium tuberculosis was isolated by Koch. Agar was used as a solidifying agent for solid cultures in Koch’s laboratory by Hesse. Koch isolated Vibrio cholerae, the causative agent of cholera. He established Koch’s postulates proving that a specific disease is caused by a specific organism. Koch was awarded Nobel Prize for physiology /medicine in 1905. Koch’s postulates 1.

Presence of microorganism in the diseased host but not in healthy one.

2.

The microorganism should be isolated in pure culture using specific medium.

3.

Disease should be reproducible when introduced in healthy host.

4.

The same organism must be isolated from the experimentally infected host.

Koch initially used potato slices for growing bacteria and observed small masses of bacteria which he termed as colonies. Later Koch could grow bacteria on gelatin- based medium and could find the colonies of same bacterium grew together and thus a pure culture produced. Koch’s proof of germ theory was presented in 1876. Within two years, Pasteur verified the proof and reported that bacteria were temperature-sensitive because chickens did not acquire anthrax at their normal body temperature of 420C, but did so when the temperature was brought down to 370C. He recovered anthrax spores from the soil. Hesse suggested agar as solidifying agent in place of gelatin. Petridish was also invented about this time by Julius Petri, one of the assistants of Koch. The advent of World War I in 1914 signaled dramatic pause in microbiology research and brought to an end the Golden Era of Microbiology.

Historical Developments in Microbiology

405

PIONEERS OF MICROBIOLOGY Kitasato of Japan studied with Koch and cultivated successfully the tetanus Bacillus which grows exclusively in the absence of oxygen. Elie Metchnikoff, an associate of Pasteur published in 1884 an account of phagocytosis, a defense mechanism in which white blood cells of body engulf and destroy microorganisms. Thus he formulated the basic theory on which science of immunology is established (cellular immunity). He was awarded the Nobel Prize in 1908. One of his notable contributions was on the Bacillus bulgaricus therapy and his underlying concept of health. Winogradsky and Beijerinck studied the role of non-infectious soil microorganisms in nitrogen, sulphur and carbon cycling as well as process of nitrogen fixation by symbiotic and free-living bacteria. Iwanowsky and Beijerinck provided the first evidence for virus as infectious agent. In 1892, D. Ivanowsky of Russia had first shown that the causal agent of tobacco mosaic disease was filterable, but Beijerinck went much further and provided strong evidence that causal agent was filterable and it has many of the properties of living organism. He called the agent a Contagium vivum fluidum, a living germ that is soluble. He postulated that the agent must be incorporated into the living protoplasm of the cell in order to reproduce and that its reproduction must be brought about with the reproduction of the cell. This is a postulate that comes very close to our current understanding of how viruses reproduce. Beijerinck also noted that there were other plant diseases for which causal agents had not been isolated and these might also be caused by filterable agents. Soon a number of other filterable agents were shown to be the causes of both plant and animal diseases. Such agent are called filterable viruses, but as further work on these agents was carried out and the word, “filterable” was gradually dropped. Bacterial viruses were first discovered by the British scientist F.W. Twort in 1915 and independently by the French scientist F.d'Herelle in 1917, who called them bacteriophages (phage meaning ‘to eat’). In 1935, the American scientist Wendell Stanley published work describing the crystallization of tobacco mosaic virus, providing the first demonstration that some of the properties associated with living organisms can be found in agents which can be crystallized like chemicals. The crystals were shown to consist of only two components, protein and RNA. Stanley’s work can well be said to mark the beginning of molecular biology, as it has provided the first insight into the chemical nature of virus itself. Martinus Beijerinck (Fig. 31.7) (1851-1931) is known for his contributions in developing enriching culturing techniques in microbiology and introducing the concept of a virus. He was a professor at the Delft Polytechnic School, Netherland in his later years of scientific career. A student of botany, Beijerinck started his career in studying the microbiology of plants. While studying the association of microorganisms with plants, he developed enrichment culturing techniques to isolate soil microorganisms of specific characters. Beijerinck proposed selecting specific microorganism from a natural sample through the use of specific culture media and incubation conditions that favoured growth of one type of organism while

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constraining the growth of others. This was a different approach by him to modify the practice of isolating microorganisms from nature by exposing nonselective culture media to some environment and allowing chance to dictate what grew. He called his techniques as ‘selective culture technique’ which is known today as ‘enrichment culture technique’.

Fig. 31.7. Martinus Beijerinck

Beijerinck isolated the first pure culture of many microorganisms from soil and water environments which included aerobic nitrogen fixing bacteria, sulfate-reducing and sulfur-oxidizing bacteria, nitrogen-fixing root nodule bacteria, green algae and many others. His other contribution to the science of microbiology is filterable agent as the cause of tobacco mosaic disease. Beijerinck showed that the infectious agent (a virus) was not bacteria but somehow became incorporated into the cells of the host plant and required the living plant to reproduce, in essence, describe the basic tenets of virology. Subsequently the tobacco mosaic virus (TMV) was crystallized and characterized by others. The enrichment technique developed by Beijerinck is the most important tool for isolating and developing microbial pure cultures of interest today. Today’s practice of enrichment culturing by making use of physical, chemical and biological environment for enriching the population of economically and biotechnologically important microorganisms in source sample is the technique introduced by Beijerinck.

Fig. 31.8. Serger Winogradsky

Historical Developments in Microbiology

407

Sergei Winogradsky (Fig. 31.8) (1856-1953) is a Russian scientist with interests similar to that of Beijerinck. He was more interested in soil bacteria and their nutritional characterization with specific interest to study the cycling of nitrogen and sulfur compounds. Winogradsky isolated pure culture of nitrifying bacteria, showing clearly that the process of nitrification, the oxidation of ammonia to nitrate, was the result of bacterial action. Paul Ehrlich laid foundation in the era of chemotherapy i.e. the use of chemicals that selectively inhibit/kill pathogens without causing damage to the victim. Gerhard Domagk in 1935 reported that Prontosil, a red dye used for staining leather was active against pathogenic streptococci and staphylococci in mice. Domagk was awarded Nobel Prize in 1939 for the discovery of sulpha drug. Two French scientists, Jacques and Trefonel reported that the compounds Prontosil was broken down within the body of the animal to sulphanilamide (sulpha drug) which was the principle active factor Alexander Fleming (Fig. 31.9) discovered the first wonder drug penicillin in 1929 from the mold Penicillium notatum. This was the first antibiotic that kills the susceptible microorganisms and inhibits their growth. Florey and Chain in 1941 developed methods for industrial production of penicillin. The three scientists (Fleming, Florey and Chain) shared the Nobel Prize in 1945 for the discovery and production of penicillin.

Fig. 31.9. Alexander Fleming

At the time of World War II (1939-1944), Selman A. Waksman (Fig. 31.10), Rutgers University, USA, discovered another antibiotic, streptomycin along with Albert Schatz in 1944 from Streptomyces griseus, an actinomycete. Waksman received Nobel Prize in 1952 for the discovery of streptomycin used in the treatment of tuberculosis caused by Mycobacterium tuberculosis, which was discovered earlier by Robert Koch in 1882.

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Fig.31.10. Selman A. Waksman

Paul Burkholder in 1947 isolated chloramphenicol (Chloromycetin) from Streptomyces venezulae. B.M. Dugger in 1948 isolated Aureomycin from S. aureofaciens and terramycin was discovered by Finlay et. al. in 1950 from S. rimosus. Presently there are many antibiotics of which only few of them are of chemotherapeutic importance. In 1943, Luria and Delbriick demonstrated that bacteria could develop spontaneous mutations which generate resistance to viral infection. This finding has significance in microbial genetics with the use of Escherichia coli as a microbial model system furthered the knowledge of using such simple microorganism to study the general principles of biology. George Beadle and Edward Tatum using the fungus Neurospora, showed one gene code for one enzyme i.e.one gene one-enzyme hypothesis. Avery, MacLeod and McCarty working with Streptococcus pneumoniae suggested that DNA is the genetic material in cells. In 1953, Alfred Hershey and Marta Chase using bacterial viruses provided strong evidence that DNA is the substance of genetic material. Joshua Lederberg (Fig. 31.11) in 1958 received the Nobel Prize in physiology/ medicine for discoveries in genetic recombination and organization of genetic material in bacteria.

Fig. 31.11. Joshua Lederberg

Historical Developments in Microbiology

409

Electron microscopic studies reveal that the bacterial cells had few of the cellular structures typical of eukaryotic cells. They lacked cell nucleus, indicating bacterial chromosome was not surrounded by a membrane envelope. Hence bacteria have a prokaryotic cellular organization (Eubacteria and Archaea). ERA OF MOLECULAR BIOLOGY With the advent of biotechnology, Carl Woese in 1967, originated the RNA World Hypothesis and also discovered extremophiles, Archaea in 1977 by phylogenetic analysis of 16S ribosomal RNA, a technique developed by Woese is now a standard practice (Fig 31.12).

Fig. 31.12. Carl Woese

Australian scientists, Barry Marshall and Obin Warrren, showed that bacterial infections of Helicobacter pylori (syn: Campylobacter pylori) is responsible for painful ulcers in stomach and intestine. They received Nobel Prize in 2005 for their discovery Har Gobind Khorana, Indian scientist from USA, was awarded Nobel Prize in physiology/medicine in 1968 for his contribution to the elucidation of genetic code (Fig. 31.13). His research shows how messages inscribed in genes are translated into proteins. He was the first person to successfully synthesize gene in 1970 which is of immense significance in biotechnology industry. The subject proteomics is being widely used to engineer new plants and animals.

Fig. 31.13. Har Gobind Khorana

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Nuclear sequencing paved the way to microbiologists to reveal phylogenetic relationships among prokaryotes that led to evolutionary concepts in biological classification. The field of genomics in which the comparative analysis of the genes of different organisms is carried out, this knowledge is leading to major activities in various biological sciences like medicine, microbial ecology, industrial microbiology and other areas. The genomic area has given birth to proteomics, a sub-division (Study of protein expression in cells). A number of scientists have been awarded Nobel Prizes for their work in this field. DEVELOPMENTS OF SOIL MICROBIOLOGY DURING 20TH CENTURY The following century:

scientist’s have made significant contributions during 20th



Omeliansky (1902) - Found the anaerobic degradation of cellulose by soil bacteria.



Lipman and Brown (1903) - Studied ammonification of organic substrates. Developed the ‘tumbler or beaker’ technique for studying the transformations in the soil.



Hiltner (1904) laid foundation of the work on rhizosphere. The scientist’s who made contributions on rhizosphere microbiology are Starkey (1929) from USA, Lochhead (1940) and Katznelson (1946) from Canada, Rovira (1956) from Australia and Macura and Vancura (1961) from Czechoslovakia and several workers from India.



Russel and Hutchinson (1909) - Made contributions on control of soil bacterial population using protozoa.



Conn (1918) - Made direct soil examination of soil microorganisms.



Cholodny (1930) - Established Ross and Choldny contact slide technique for examining the soil microbes.



Van Niel (1931) from USA - Fundamental studies on chemoautotropic bacteria and bacterial photosynthesis.



Fred, Baldwin and McCoy (1932) – Established cross inoculation groups of root nodule bacteria.



Garret (1936) From U.K. - Investigations on soil fungi and their ecological studies.



Allen and Allen (1940) - Root nodule bacteria.



Starkey (1945) - Studied transformation by iron bacteria.



Umbreit (1947) – Problems on autotrophy.



Harley (1948) from U.K. – Demonstrated phosphate mobilization by ectomycorrhizal fungi.



Gerretsen and Mulder from Wageningen – Studied the phosphate mobilization by soil microbes and importance of molybdenum in nitrogen metabolism by microorganisms.

Historical Developments in Microbiology

411



Ruinen (1956) – Developed concept of phyllosphere.



Barker from USA (1936) – Established anaerobic fermentation by methane bacteria.



Martin Alexander (1961) – Studied microbiology of pesticide degradation.



Fritsch, Fogg and Stewart from UK and M.O.P. Iyengar from India were the foremost workers on algae in general and micro algae in particular.

Some microbiology related Nobel Laureates are given in Table 31.1. Table 31.1. Examples of some microbiology related Nobel Laureates Year

Scientist

Nature of Discovery

1905

Robert Koch

Discoveries related to tuberculosis

1907

AIphonse Laveran

Role of Protozoa in causing diseases

1908

I. Metchinkoff and Paul Ehrlich

Work on Immunity

1919

Jules Bordet

Discoveries relating to immunity

1923

Federick G. Banting

Discovery of Insulin

1926

J. Fibiger

Discovery of Spiroptera carcinoma

1928

Charles Nicolle

Work on typhus

1931

Otto Warburg

Nature and mode of action of respiratory enzyme

1933

Thomas H. Morgan

Role of chromosome in heredity

1939

G. Domagk

Antibacterial effect of prontosil

1945

Sir Alexander Fleming, B. Chain and Sir H. Florey Earnst

Discovery of penicillin and its curative effect in infectious diseases

1951

Max Theiler

Yellow fever and its control

1952

Selman A. Waksman

Discovery of streptomycin

1953

Hans Krebs and F. Lipmann

Discovery of citric acid cycle and of co-enzyme A in intermediary metabolism

1954

John F. Enders, Thomas H. Weller and Frederckh C. Robbins

Ability of poliomyelitis viruses to grow in cultures of various types of tissues

1955

Hugo Theorell

Nature and mode of action of oxidation enzymes

1958

George Beadle, Edward Tatum and Joshua Lederberg

Genes act by regulating definite chemical events and discoveries concerning genetic recombination and organization of the genetic material of bacteria (Lederberg)

1959

S. Ochoa and A. Kornberg

Mechanisms of biological synthesis of RNA and DNA

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Year

Scientist

Nature of Discovery

1960

Sir F. Macfarlane Burnet and Acquired immunological tolerance Peter Medawar

1962

Francis Crick, James Watson Molecular structure of nucleic acids, significance for and Maurice Wilkins information transfer in living material

1965

F. Jacob, A. Woff and J. Monod

Genetic control of enzyme and virus synthesis

1966

P. Rous and C.B. Huggins

Discovery of tumour inducing viruses (Rous) and discoveries concerning hormonal treatment of prostatic cancer (Huggins)

1968

R.W. Holley, H.G. Khorana

Interpretation of genetic code and its function in protein synthesis

1969

M. Delbruck, A.D. Hershey and S.E.Luria

Replication mechanism and genetic structure of viruses

1971

E. Sutherland, Jr.

Mechanisms of action of hormones

1972

G.M. Edelman and R.R. Porter

Chemical structure of antibodies

1974

A. Claude, C.de Duve and E. Palade

Structural and functional organization of cell

1975

D. Baltimore, R. Dulbecco and H.M and Temin

Interaction between tumour viruses and the genetic material of the cell

1976

B.S. Blumberg and D.C. Gajdusek

New mechanisms for the origin and dissemination of infectious diseases

1978

W. Arber, D. Nathans and H.O. Smith

Discovery of restriction enzymes and their application to problems of molecular genetics

1980

B. Benaserraf, J. Dusset and G.D. Snell

Genetically determined structures on the cell surface that regulate immunological reactions

1983

Barbara McClintock

Discovery of mobile genetic elements

1987

Susumu Tonegawa

Genetic principle for generation of antibody diversity

1988

Sir J.W. Black, G.B. Elion and G.H. Hitchings

Important principles for drug treatment

1992

E.H. Fishcher and Edwin G. Krebs

Reversible protein phosphorylation as a biological regulatory mechanism

1993

R.J. Roberts and P.A. Sharp

Split genes

1993

Kary Mullis

Invention of polymerase chain reaction (PCR)

1993

Hamilton Smith

Specificity of action of restriction enzymes to splice foreign components into DNA

1994

A.G. Gilman and M. Rodbell G-proteins and their role signal transduction in cell

1997

Stanley B. Prusiner

Discovery of prions

Historical Developments in Microbiology

413

Year

Scientist

Nature of Discovery

1999

Gunter Blobel

Proteins have intrinsic signals that govern their transport and localization in cell

2001

L.H. Hartwell, Tim Hunt and Key regulators of the cell cycle Sir Paul Nurse

2002

S. Brenner, H. Robert Orde Horvitz and J.E. Sulston

Genetic regulation of development and programmed cell death

2005

Barry J. Marshall and J. Robbin Warren

Discovery of bacterium Helicobacter pyloris and its role in gastritis and peptic ulcer disease

2006

A.Z. Fire and.Craig C. Mello RNA interference, gene silencing by double stranded RNA

2007

M. Capecchi, Oliver Smithies Gene targeting on knockout mouse using embryonic and Martin Evans stem cells and in understanding gene disease relationship

PROSPECTS AND CHALLENGES 1.

Basic understanding of diseases like AIDS, Flu (Influenza, Swine flu, Dengue), SARS etc.

2.

Microbial diversity and communities at fundamental level.

3.

Microbial ecology – role of microbes in the environment

4.

Studies on Biofilms.

5.

Intensification of researches on characterization of unculturable microbes, microbial evolution and phylogenetic relationships at molecular and genomic level

6.

Finally the microbiologist’s role in the utilization of microorganisms for the benefits of society.

Some microorganisms utilized for the production of antibiotics, enzymes and fermented foods are presented in Table 31.2. Table 31.2. Utilization of Microorganisms. Product

Microorganism

Antibiotic Bacitracin

Bacillus licheniformis

Cephalospirin

Cephalosporium acremonium

Chloramphenicol

Streptomyces venezuelae

Cycloheximide

S. griseus

Cycloserine

S. orchidaceus

Erythromycin

S. erythraeus

414 Product

Biology and Biotechnology of Fungi and Microbes Microorganism

Griseofulvin

Penicillium griseofulvum

Kanamycin

S. kanamyceticus

Linomycin

S. lincolnensis

Neomycin

S. fradiae

Nystatin

S. noursei

Penicillin

Penicillium chrysogenum

Polymyxin B

Bacillus polymyxa

Streptomycin

Streptomyces griseus

Teicoplanin

Actinoplanes teichomyceticum

Tetracyclin

S. rimosus

Vancomycin

S. orientalis

Production of enzymes Amylases (α , β)

Bacillus

Penicillin amidase

Bacillus

Glucose isomerase

Bacillus, Streptomyces

Glucoamylase

Aspergillus, Rhizopus

Amylase (α)

Aspergillus

Protease

Bacillus

Rennet (Aspartic proteinases)

Aspergillus, Mucor miehei

Pectinase

Aspergillus, Sclerotinia

Protease(Aspartic proteinases)

Aspergillus

Cellulase

Aspergillus, Trichoderma

α- Galactosidase

A. niger

Invertase

Sacharomyces

Lactase(β-galactosidase)

Kluyveromyces

Raffinase (α- galactosidase)

Sacharomyces

Fermented foods Idli: Rice, Black gram

Leuconostoc mesenteroides, Streptococcus faecalis

Kaffir Beer: Sorghum cafforum, ragi Lactobacillus delbrueckii, Saccharomyces cereviseae Kafir: Milk

Lactobacillus, yeast

Yoghurt: Milk

Streptococcus thermophilus, Lactobacillus bulgaricus

Cheese: Milk

Penicillum roqueforti, P. camemberti

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Chapter - 32

Microscopy

The development of bacteriology dates from the invention around 1590 of the compound microscope, the origin of which is generally attributed to the Hans and Zacharias Jansen of Middelburg, Netherlands. Great progress was made during the last century with the development of the water-immersion lens by Amici in 1840 and his subsequent development of the oilimmersion lens in 1869; the sub stage condenser by Abbe in 1876 which provided better illumination by bringing the rays of light into focus in the object under examination; and the introduction by Abbe and Zeiss in 1886 of lenses that were fully color corrected (apochromatic objectives with compensating eyepieces). BRIGHT FIELD MICROSCOPE The compound microscope differs from the simple system by making it possible to bring the object nearer the eye; an enlarged image of the object as produced by the objective lens is brought nearer to the eye with the aid of the eyepiece lens. The eyepiece lens acts as a simple microscope which makes it possible for the eye to focus at close distance upon the enlarged image of the object being examined (Fig. 32.1).

Fig. 32.1. Simple microscope

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The approximate magnifying power, expressed as increase in linear size, is determined by multiplying the magnifying power of the eyepiece (usually 10) by the magnifying power of the objective. Thus, when used with a 10 eyepiece, the low-power (10), high dry (43), and oil-immersion (97) objectives give linear magnifications of 100, 430, and 970 diameters, respectively (Fig. 32.2). Resolving power meant the ability to show, separately and distinctly, two points which are closely adjacent in the object. The resolving power of a microscope is determined by two factors, the wavelength of light and the numerical aperture of the lens. The wavelengths to which the eye is sensitive are fixed, and there are limits beyond which the numerical aperture of a lens cannot be increased. The numerical aperture is dependent upon the actual diameter of the objective lens in relation to its focal length and upon the refractive index of the medium between the lens and the object under examination. Immersion oil has a higher index of refraction than air, and hence higher resolving power is obtained when oil is placed between the immersion lens and the object, but a limit of attainable resolving power is soon reached beyond which increased magnification does not enable one to see still smaller objects (Table 32.1).

Fig. 32.2. Compound microscope

Resolving power = smallest visible structure =

Wave length Numerical aperture

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When the microscope is fitted with a condenser with a numerical aperture equivalent to that of the objective, the equation becomes Resolving power =

Wave length 2  numerical aperture

On substitution in the above equation we find that in blue light of wavelength 0.470 m, the resolving powers of lenses ordinarily employed in bacteriological laboratories are shown in Table 32.1. When one employs an ordinary student microscope equipped with a good condenser and an ordinary oil-immersion lens, the smallest object which can clearly be seen must have a diameter of at least 0.2 m. Table 32.1. Numerical aperture and resolving power of different lenses. Lens Low-power lens

Numerical aperture

Resolving power (m)

0.25

0.94

High-power lens

0.85

0.28

Oil-immersion lens

1.25

0.19

Research oil-immersion lens

1.40

0.17

For increased resolution it is necessary to use light of shorter wavelength, and this can be accomplished with quartz lenses which are transparent to ultraviolet light. However, the eye is not sensitive to the short ultraviolet, and it is therefore necessary to photograph the image and to observe the photographic reproduction. Using light of 0.275 m wavelength, and the resolving power becomes approximately 0.1 m. THE PHASE CONTRAST MICROSCOPE Contrast in the images of bacteria or other specimens can be enhanced over that noted in an ordinary microscope by use of the phase-contrast microscope. Light traversing two objects will emerge out of phase if one object is either thicker than or has a different refractive index from the other. When these rays are brought together out of phase, they interact to produce interference or darkening; when brought into phase, they reinforce each other and yield a brighter image. In the phase microscope a diffraction plate or coating is added within the objective and an annular diaphragm below the condenser of a light microscope. This optical arrangement is of such a nature that slight and otherwise invisible alterations of the light passing through the specimen are converted into images that can be seen. The annulus below the condenser controls the illumination on the diffraction plate where the light from the specimen is selectively modified to yield an image of greater visibility or contrast. On changing the nature of the annulus or of the diffraction plate, changes can be induced in the degree of contrast, or the contrast can be reversed, thus altering the appearance of the object under examination. Phase-contrast microscopy has definite limitations, but within these limits it is of value in enabling one to observe differences in structure of living cells that are not apparent in the ordinary light microscope.

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THE ELECTRON MICROSCOPE In electron microscope electrons can be deflected from their course by magnetic fields in a manner analogous to the deflection of light by lenses. Since electrons moving at high velocity have a very short wavelength (5  10–6 m at 60 kv. potential), the resolving power of the electron microscope is approximately 100,000 times that of a light microscope (Fig. 32.3).

Fig. 32.3. Transmission electron microscope (source: Wikipedia)

The objects seen in photographs of electronic images are shadows analogous to those on an X-ray plate and represent degrees of opacity to electrons by different parts of the object. Also the specimen under examination must be held in a high vacuum, and this may tend to distort bacteria or bacterial structures. In many instances, interpretations of the photographs (electron micrographs) are difficult to make, but notwithstanding all the difficulties encountered in electron microscopy the use of this tool has increased our knowledge of the structure of bacteria and viruses. Electron microscopy has also been of considerable value in the determination of the size and shape of virus particles. Wyckoff and others developed a thin film of metal on one side of the particles so that they cast shadows. This technique makes the particles show up more prominently. THE DARK-FIELD MICROSCOPE When a beam of light shines through a darkened room, we see light reflected in all directions by dust and other particles suspended in the air. The suspended particles may be so small as to be invisible to the naked eye, and then we see only the light reflected by the particles and not the particles themselves. This optical effect, known as the Tyndall phenomenon, can be applied to microscopy and enables one to detect the presence of particles, even though their dimensions are less than the lower limits of resolution of the microscope. This is accomplished by providing a dark background through which a beam of light passes at a right angle to the optical axis of the microscope.

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Dark-field illumination is obtained when the usual condenser of the microscope is replaced with a special dark-field condenser. The path of the rays of light through this type of condenser is such that the rays are brought into focus in the object but at such a divergent angle that none of them strikes the objective lens of the microscope. Only rays of light reflected by particles in the field of view are able to enter the objective. Dark-field microscopy is of significance for the observation of the motility of bacteria and for the identification of Treponema pallidum in material from suspected syphilitic lesions. MICROSCOPIC EXAMINATION OF BACTERIA It is necessary, because of the small size of bacteria, to use high-power objectives, particularly the oil-immersion lens, to obtain sufficient magnification and resolution for the examination of these and related microorganisms. The principle involved in the use of the oil-immersion lens is illustrated in Fig. 32.4. Microorganisms can be examined as masses in living colonies or cultures or as individuals in suspension in water or in dried stained preparations. The latter method is usually employed since the unstained cells, particularly bacterial, are generally transparent and the refractive index of protoplasm is so near that of water that it is difficult to observe such minute colorless cells. Staining is also useful in revealing flagella and capsules or structures such as granules and spores within the cell.

Fig. 32.4. Diagram showing how immersion oil preventing refraction of a beam of light when passing through glass and air

Certain staining reactions are employed to bring out differences in the staining properties of specific structures within the cell or differences between different species. If one mixes some bacteria with a drop of Congo red, nigrosin solution, or India ink, spreads a drop of the suspension on a clean slide to form a thin film, and dries it, the bacteria may be seen as colorless bodies surrounded by colored background. This is called negative staining, since the background and not the bacteria are colored. The cells appear to be larger than in ordinary stained preparations, since in the latter process there is a tendency for the cells to shrink in the fixing and staining procedures. On the other hand, the dye may pull away from

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the cells during the time that the negatively stained smear is drying and create the illusion of increased size. Negatively stained preparations are useful for certain types of work as they tend to be less tiring to the eyes, the bacteria standing out as bright spots in a dimly lighted field. (i) Hanging-drop Preparation Unstained organisms can be examined under the microscope in a drop of water or broth, generally covered with a cover glass to form a thin film. Since the cells have a refractive index near that of water, it is necessary to adjust the illumination so as to obtain as great contrast as possible between the cells and the suspension medium. One may add a drop of a dye solution or of dilute iodine to the preparation to increase the contrast, and in some instances a so-called vital stain which will penetrate living cells may be employed. It is generally preferable to examine living bacteria in what is known as a hanging drop, in order to reduce movement caused by capillary forces or by evaporation from the film under the cover glass. For this preparation a glass slide with a concave depression is required (Fig. 32.5). A thin film of petroleum jelly is applied around the edge of a cover slip, and a drop of the bacterial suspension is then placed in the center of the cover slip. The hollow-ground slide is then inverted over the cover glass in such a manner that the drop is centered over the depression in the slide. The slide is then gently pressed down on the cover glass to form a seal of petroleum jelly between it and the edges of the cover glass. The sealed preparation is rapidly inverted, and one then has a drop of the suspension hanging from the cover glass. Sealing the preparation greatly reduces evaporation from the drop and at the same time markedly reduces convection currents. The organisms are now ready for examination, preferably with the high-power lens, although the oil-immersion lens can be employed if proper care is exercised. It is generally desirable to reduce the illumination and to locate the drop with the low-power lens before shifting to the higher powers of the microscope.

Fig. 32.5. Preparation of hanging drop slide

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(ii) Motility Hanging-drop preparations are valuable for the determination of motility of bacteria. Amongst the bacteria, motility is for the most part due to the possession of whip-like organs of locomotion, flagella. Bacteria can be roughly classified into two groups, motile and nonmotile, on the basis of motility in hanging-drop preparations. Even nonmotile bacteria in a suspension are in constant motion, but one must learn to distinguish between this motion and true motility. When bacteria possess flagella, they may exhibit true motility as well as Brownian movement. True motility can be distinguished from Brownian movement by the fact that a motile organism may be observed to move from one place to another in a more or less straight line and that this movement may carry the organism partly or entirely across the field of view. Bacteria in suspension always exhibit Brownian movement and that it is necessary to distinguish between this movement and true motility. The motility of bacteria is greatly reduced or lost with increasing age of a culture and that handling of the organisms. DIFFERENTIAL STAINS i. The Gram Stain The differential staining technique is generally more difficult than simple staining and is for the purpose of differentiating between different parts or structures of the cell or between different groups of organisms. The gram stain is the most important differential stain employed in bacteriology and serves to differentiate bacteria into two main groups: the gram positive and the gram negative. Gram observed in 1884 that in tissue sections stained with crystal violet and then treated with a dilute solution of iodine in potassium iodide solution, the stain could be readily removed from the tissue by alcohol but not from most bacteria within the tissue. Gram also observed that when this procedure was applied to smears of various bacteria, certain species retained the original dye while others were rapidly decolorized by the alcohol and would take up another stain. These observations were soon confirmed, and it was found that bacteria can be divided into two general groups, gram positive and gram negative, on the basis of their behaviour in the gram stain. This division of bacteria into two groups on the basis of gram-staining properties is of great value in the identification of many bacteria, being particularly important with certain types of animal pathogens such as gram-negative and grampositive cocci. ii. Acid-fast Stain The property of acid fastness, i.e., resistance possessed by certain bacteria to decolorization of their stained cells even by mineral acids, was first noted in 1882 by Ehrlich in studies on the tubercle Bacillus. The mycobacteria and a few strains of diphtheria-like bacteria and actinomycetes are stainable with difficulty by the ordinary stains, but once stained they retain the dye quite tenaciously. They can be stained if a concentrated solution of a dye such as basic fuchsin is employed in the presence of phenol acting as an intensifier. The rate of staining is slow at room

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temperature, but staining can be accomplished in a few minutes at a temperature near the boiling point of water. The Ziehl-Neelsen technique, frequently employed for the staining of acid-fast bacteria, takes advantage of these facts. Once stained, acid-fast bacteria retain the dye even on prolonged immersion in water or alcohol acidified with a mineral acid while non-acid-fast cells are decolorized in a few seconds. In one modification of the acid-fast stain, a detergent (wetting agent) is added to the staining solution, and the acid-fast forms are stained in a few minutes at room temperature. Another modification of the staining method employs auramine as the dyestuff because it possesses the property of fluorescence. When auramine-stained cells are illuminated with ultraviolet light, they emit light of a longer wavelength, to which the eye is sensitive. Non-acid-fast bacteria stained with auramine are decolorized with acid alcohol while the acid-fast bacteria retain the auramine. The smear is examined with a microscope equipped for ultraviolet-light illumination and with a yellow filter to remove blue light entering the ocular. The field of view appears dark except for the auramine-stained acid-fast bacteria, which stand out as luminous yellow bodies in the dark background. Acid fastness was explained for many years on the basis of a high concentration of fatty or waxy material in the acid-fast forms. Earlier it has been demonstrated that acid fastness of the mycobacteria (e.g., Mycobacterium tuberculosis) is not dependent on the total fat content of the cells but appears to be due to a specific component, mycolic acid, present in the waxy material. Mycolic acid, while somewhat acid fast by itself, appears to be in combination with a polysaccharide in the cell, and this combination may possess stronger acid-fast properties than mycolic acid alone. The possession of mycolic acid by all acid-fast forms has not been demonstrated, and it is possible that acid fastness is the result of the possession of other acid-fast-staining materials or even of particular cell membrane structures. However, our knowledge of some of the simpler facts concerning bacteria and other microorganisms is still far from being complete, iii. The Spore Stain Bacterial endospores stain like the acid-fast bacteria and will retain the primary stain in the Ziehl-Neelsen stain while the originally vegetative portion (spore case or sporangium) of the cell is non-acid-fast. The Ziehl-Neelsen stain can be employed for differentiating between the spore and the sporangium or for the observation of free bacterial spores, since washing with water is ordinarily sufficient to decolorize the spore case. Treatment with acid alcohol may remove the dye from the spore and should be omitted. In general, spores are more readily stained than are acid-fast bacteria, and quite frequently an aqueous solution of malachite green is employed as the primary stain, although heat is necessary to secure good staining. The excess stain is rinsed off with water and the cells counterstained with eosin or safranin. Low permeability of the spore membrane to dyestuffs is the most common explanation of the peculiar staining property of bacterial spores. It should be pointed out at this time that endospores can be observed in ordinarily stained preparations as colorless bodies within the stained portion of the original cell, and that a thin film of dye may be deposited on the exterior of free spores.

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iv. Granules In many bacteria stained by ordinary methods, it is possible to see bodies or granules stained more intensely than the rest of the cell. When the granules assume a color different from the rest of the cell, they are spoken of as metachromatic granules. The composition and function of these granules are still matters of debate, although in many instances the granules may be reserve food materials of various types which are stored in the cells. They tend to accumulate as growth slows down and to disappear again when the cells are actively growing. These granules may be composed of fats, carbohydrates, or complex nitrogenous matter, and in the thiobacteria granules of inorganic sulfur can be present. Fat globules are present in a wide variety of microorganisms and can be recognized as highly refractile bodies which are not stained by the ordinary stains. Fat globules can also be recognized by the fact that they stain black when the cells are treated with osmic acid or will take up fat-soluble dyes such as Sudan III, the bulk of the cell not being stained. The reserve carbohydrates appear to be of two general types, glycogen and granulose. The first is recognized by the reddish-brown color which develops on treatment with iodine solution and is considered to be the same as or similar to the glycogen found in tissues such as the liver. It has not been positively identified in all bacteria showing reddish-brown granules after iodine treatment, and hence it might be better to consider these granules as glycogen-like bodies. The glycogen granules have often been mistaken for nuclei but can be differentiated by the fact that they are not removed from the cell by boiling water and can be destroyed on hydrolysis with mineral acids. The second type of reserve carbohydrate granules give a blue color with iodine and are chemically related to starch. Granulose is not as widely distributed in the bacteria as is glycogen. Both types of granules are visible in the dark field. v. Flagella Stains With most bacteria, the flagella are so minute as to be invisible on microscopic examination. Flagella have been observed on dark-field examination of certain species of bacteria, but their presence generally is recognized by the fact that a particular cell is motile. The presence of flagella can be demonstrated by special staining techniques which involve the deposition of sufficient dye on the flagella to create a body large enough to be resolved under the microscope. The staining processes employed for this purpose may also increase the intensity of staining of the individual flagella sufficiently than those near the lower limits of resolving power become apparent without marked increase in thickness, or both processes may be involved. The basis of all the flagella stains is a preliminary treatment of the cells with a mordant, which is generally a complex colloidal solution frequently containing tannates. The success of the stain depends to a considerable extent on the colloidal state of the mordant. Flagella are extremely fragile, and great care must be employed, in handling the organisms and in the preparation of the smear. The presence of organic matter and of cellular debris generally hinders the demonstration of flagella, as the foreign matter may react with the mordant and adsorb considerable amounts of

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the dye, thus interfering with the observation of the smear. Flagella can also be observed following the deposition of silver rather than of dyestuff on the mordant preparation. vi. Capsule Stains A number of bacterial species under favorable conditions develop an enclosing sheath, or envelope, which is called a capsule. In most cases the capsule is a layer of gelatinous or gummy material which is not stained readily with the ordinary stains. Capsules can be demonstrated as a somewhat more transparent layer around the cells in negatively stained preparations. To increase the contrast between the cell and its capsule, a negatively stained smear can be treated with a regular stain, which will react with the cell proper, leaving a stained cell surrounded by a clear zone in the background created by the negative stain. When Congo red is employed as the negative stain, the cells can be stained with an acidic dye in acid solution, the acid at the same time tending to fix the Congo red to the slide in its insoluble acid form. Capsules can also be demonstrated as faintly stained halos around cells stained with gentian violet or carbolfuchsin if the time of staining is prolonged and the excess stain is removed by blotting. Capsules are generally more pronounced around pathogenic forms in slides prepared directly from body fluids, capsule formation being enhanced in the body. The demonstration of capsules is also facilitated by the proteinaceous material present in body fluids. This forms a film of material in the smear which acts somewhat like the background in a negatively stained preparation. vii. Cell-wall Stain Cytoplasmic membranes and other structures can at times be observed directly either in a bright-field or in a dark-field preparation of bacteria. Knaysi developed a staining procedure which is of value in demonstrating the cell wall and the slime layer around bacteria. It consists of a heat-fixed smear of bacteria with a mixture of tannic acid and alum and staining with a drop of Ziehl-Neelsen carbol-fuchsin under a cover glass. The cytoplasm appears dark red in cells stained by this method while the cytoplasmic membrane is still darker; the cell wall stands out as a blue structure, and the slime layer is bright red. Considerable differentiation can also be observed when bacteria are examined directly in suspension in dilute (1:20,000) crystal violet (gentian violet), in a hanging drop or in a film under a cover slip. viii. Differential staining during growth The coliform bacteria ferment the lactose with the production of considerable amounts of acid, and in an acidic environment some of the eosin-methylene blue complex is taken up by the bacteria, the faintly stained cells giving rise to colored colonies. The non-lactose-fermenting bacteria do not produce acid, and their colonies remain colorless. The colored colonies of lactose-fermenting bacteria which develop on eosin-methylene blue agar can be eliminated as possible pathogens, and one can examine and identify colorless colonies of non-lactose-fermenting bacteria as possible pathogenic forms.

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Chapter - 33

Bacteria

Bacteria, are simple with a well-developed cell structure which is responsible for many of their unique biological properties. Many structural features are unique to bacteria and are not found among archaea and eukaryotes. Bacteria can be manipulated experimentally due to their simplicity in structure. The hereditary material is not a true nucleus and is a double stranded DNA. Genetic material is not enclosed by nuclear membrane. Such organisms are called prokaryotes (pre-nucleus). Bacteria are included in Kingdom Monera proposed by Whittaker. Some microbiologists call it as prokaryotae Bacteria are considered as homogenous group of primitive forms of microorganisms. However, the morphological, physiological and biochemical characteristics are found to be varied and distinct from those of other forms under the plant Kingdom. This group of organisms deserve separate kingdom. Most bacteria have distinctive cell shapes, which remain more or less constant, although shape is influenced to some extent by the environment. Bacteria shaped like spheres are called cocci (singular coccus), where those shaped like rods/cylinders are called bacilli. If a rod is many times longer than it is wide it is usually called a filament. Some bacteria are shaped like spirals, and a long spiral has the shape of a helix. The shape of a cell affects its behavior and stability. When cells divide they often remain attached to each other, and the manner of attachment is usually characteristic of both the organism and the type of division the cell has undergone. Thus many coccus-shaped organisms form chains (Streptococcus) by dividing always along the same axis. The length of such chains may be short (2 to 4 cells) or long (over 20 cells). Some cocci divide along two axes at right angles to each other, leading to the formation of sheets of cells. If there is no pattern to the orientation of successive divisions, an irregular clump will be formed; such a random cluster is characteristic of Staphylococcus. Sarcina is an example of a third group of cocci, which divide in three axes and form cube-shape packets (Fig. 33.1). Rods always divide in only one plane and hence may form chains (such as in many Bacillus species). Spirally shaped organisms also divide in only one plane, but they usually separate immediately and do not form chains. Cellular components can be divided into two groups, invariant, found in all prokaryotes, and probably essential for life, and variant, found in some but not all cells, and probably involved in more specialized function. Invariant cell structures

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include the cell membrane, ribosomes, and nuclear region. Variant cell structure include the cell wall (present in most but not all organisms), flagella, pili, capsules, slime layers, holdfasts, inclusion bodies, gas vesicles, and spores (Fig. 33.2).

Fig. 33.1. Shapes of bacteria

Fig. 33.2. Structure of bacterial cell

COMPONENTS OF BACTERIAL CELL The plasma membrane, sometimes called the cell membrane, is a thin structure that completely surrounds the cell. The main components of the plasma membrane are phospholipids and proteins. The phospholipids form the basic structure of membrane. Despite its thinness, the plasma membrane functions as a tight barrier, so that the passive movement of solute molecules does not readily occur. When a bacterial cell is broken under carefully controlled conditions, the plasma membrane forms small pieces that are flat at first, but immediately become converted into spherical structures, the vesicles. It is also possible to form structures analogous to membrane vesicles from pure phospholipids, or from phospholipid-protein mixtures. Such structures are called liposomes, and it is possible to synthesize functioning liposomes with a variety of proteins embedded in them. i. Cell wall One of the most important structural features of the bacterial cell is the cell wall, which confers rigidity and shape. The prokaryotic cell wall is chemically different from that of any eukaryotic cell, and this is one of the features distinguishing prokaryotic from eukaryotic organisms. The cell wall is difficult to visualize well with the light microscope but can readily be seen with the electron microscope.

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A differential staining procedure of great value in the identification of eubacteria is the Gram stain, first developed in 1884 by a Danish physician, Christian Gram. Many modifications exist, but they all embody the same essential steps. A heat-fixed smear of bacteria is stained successively with a solution of crystal violet (or a related basic dye) and with a dilute solution of iodine. The preparation is then treated with an organic solvent, such as alcohol or acetone. The cells of some bacteria, known as Gram-negative bacteria, are rapidly and completely decolorized by the organic solvent; the cells of others, known as Gram-positive bacteria, resist decolourization. In this event, treatment by the organic solvent is followed by counter staining with a red dye such as safranin. Gram-positive cells retain the deep purple color conferred on them by the initial staining with crystal violet and iodine, whereas Gram-negative cells, which have been decolourized, accept the red color of the counter stain. As a result of this difference in color, Gram-positive and Gramnegative cells can be readily distinguished from one another in the microscope. The Gram reaction of a bacterium shows best on cells derived from a young culture, since certain bacteria are Gram positive only during the course of active growth and lose the capacity to retain the crystal violet-iodine complex after active growth ceases. This is particularly true of many spore-formers, which are strongly Gram positive when examined in young cultures but which later become Gram variable or Gram negative. Gram-positive and Gram-negative cells differ considerably in the structure of their cell walls. The Gram-negative cell wall is a multilayered structure and quite complex, while the Gram-positive cell wall consists of a single layer and is often much thicker (Fig. 33.3). The rigid layer of both Gram-negative and Gram-positive bacteria possesses peptidoglycan. This layer is a thin sheet composed of two sugar derivatives; Nacetyl-glucosamine and N-acetylmuramic acid, and a small group of amino acids, consisting of L-alanine, D-alanine, D-glutamic acid, and either lysine or diaminopimelic acid (DAP). These constituents are connected to form a repeating structure, the glycan tetrapeptide. The basic structure is in reality a thin sheet in which the glycan chains formed by the sugars are connected by peptide cross-links formed by the amino acids. In Gram-negative bacteria, cross-linkage usually occurs by direct peptide linkage of the anion group of diaminopimelic acid to the carboxyl group of the terminal D-alanine. In Gram-positive bacteria, cross-linkage is usually by a peptide interbridge, the kinds and numbers of cross-linking amino acids varying from organism to organism. The peptidoglycan structure is present only in prokaryotes, and is found in the cell wall of all species. The sugar N-acetylmuramic acid and diaminopimelic acid (DAP) are never found in eukaryotic walls. However, not all prokaryotic organisms have DAP in their peptidoglycan. This amino acid is present in all Gram-negative bacteria and in some Gram-positive species, but most Gram-positive cocci have lysine instead of DAP, and a few other Gram-positive bacteria have other amino acids. Another unusual feature of the prokaryotic cell wall is the presence of two amino acids that have the D configuration (D-alanine and D-glutamic acid). In proteins, amino acids are always in the L- configuration. In Gram-positive bacteria, as much as 90 percent of the cell wall consists of the peptidoglycan, although another kind of constituent, teichoic acid, is usually present

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in small amounts. In Gram-negative bacteria, only 5 to 20 percent of the wall is peptidoglycan, the rest of the wall consisting of lipid, polysaccharide, and protein, usually present in a layer outside the peptidoglycan layer. The peptidoglycan layer is absent in the walls of methanogenic bacteria, the halobacteria and Sulfolobus. The cell walls of these organisms apparently have different chemical constructions.

Fig. 33.3. Bacterial cell wall structure (Source: Wikipedia)

In most Gram-negative bacteria, the outer-wall layer exists as a true unit membrane. However, the unit membrane of the outer wall is not constructed solely of phospholipids. It contains additional lipid plus polysaccharide and protein. The lipid and polysaccharide are intimately linked in the outer layer to form specific lipopolysaccharide (LPS) structures. Because of the presence of lipopolysaccharide, the outer layer is often called as the lipopolysaccharide or LPS layer. Although complex, the chemical structures of some LPS layers are now understood, the polysaccharide consists of two portions, the core polysaccharide and the Opolysaccharide. The core polysaccharide consists of ketodeoxyoctonate, seven-carbon sugars (heptoses), glucose, galactose, and N-acetylglucosamine. Connected to the core is the O-polysaccharide, which usually contains galactose, glucose, rhamnose, and

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mannose as well as one or more unusual dideoxysugars such as abequose, colitose, paratose, or tyvelose. These sugars are connected in four- or five-sugar sequences, which often are branched. When the sugar sequences are repeated, the long Opolysaccharide is formed. Fatty acids frequently found in the lipid include hydroxymyristic, lauric, myristic, and palmitic acids. In the outer layer, the LPS associate with phospholipids to form the outer portion of the unit membrane. The outer layer is permeable to small molecules, but not to enzymes or other large molecules. In fact, one of the main functions of the outer layer may be its ability to keep certain enzymes, which are present outside the peptidoglycan layer. These enzymes are present in an area called the periplasmic space. These periplasmic enzymes are probably of importance in the uptake of nutrients into the cell. The outer layer in many Gram-negative bacteria possesses toxic properties. A type of toxic substance called endotoxin is either partly or equivalent to the LPS. Although Grampositive bacteria do not have a lipopolysaccharide outer layer attached to their cell wall. They generally have acidic polysaccharides called teichoic acids. Teichoic acids contain repeating units of either glycerol or ribitol. The polyol units are connected by phosphate esters and usually have other sugars and D-alanine attached. Because they are negatively charged, teichoic acids are partially responsible for the negative charge of the cell surface as a whole. Another function of certain teichoic acids is in the regulation of cell wall enlargement during growth and cell division. There is evidence that cell wall growth involves enzymes, called autolysins, and teichoic acids have been shown to regulate autolysin action, thus keeping it in balance with cell wall synthesis and uptake of nutrients into the cell. The outer layer in many Gram-negative bacteria helps in uptake of nutrients into the cell. The outer layer in many Gram-negative bacteria possesses toxic properties. A type of toxic substance called endotoxin is either partly or equivalent to the LPS. Gram-positive bacteria have very thick cell walls, which become dehydrated by the alcohol. This causes the pores in the walls to close, preventing the insoluble crystal violet-iodine complex from escaping. In Gram-negative bacteria, the solvent readily dissolves in and penetrates the outer layer, and the peptidoglycan layer also does not prevent solvent passage. ii. Ribosomes In electron micrographs of thin sections, small dark particles can often be seen within the cytoplasm. These particles, which are part of the protein-synthesizing machinery of the cell, are called ribosomes. They contain ribonucleic acid bodies. Ribosomes have characteristic sizes that are expressed by their sedimentation constants. The unit of sedimentation constant is the Svedberg, abbreviated ‘S’. Prokaryotic ribosomes have a sedimentation constant of 70 S, while those of eukaryotes have a constant of 80 S. iii. Nucleus Prokaryotic organisms do not possess a true nucleus as do eukaryotic organisms. Instead, the DNA, which contains the genetic information for the cell, is present as a naked strand, and is not surrounded by a membrane. Each DNA

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molecule consists of a single long fiber, which contains virtually all of the genetic information of the cell. Eukaryotes, in contrast, contain their genetic information in a number of separate DNA molecules, each of which is complexed with protein (forming structures called chromosomes), and surrounded by the nuclear membrane. In prokaryotes the length of the DNA molecule, when stretched out, is many times the length of the cell. For instance Escherichia coli cell is about 2 m long whereas the length of its DNA is around 1200 m. This means that the DNA is highly folded within the cell. It is sometimes possible to visualize a portion of the prokaryotic cell, called the nuclear region, or nucleoid, where the DNA is concentrated. iv. Nucleoid The bacterial chromosome remains a part of cytoplasm. This is not surrounded by a nuclear membrane. The nuclear material without a nuclear membrane is called nucleoid. The bacterial chromosome is made up of a single double stranded circular DNA. It consists of a single chromosome, which varies in size between different species of bacteria (E. coli chromosome is 4 x 106 base pairs long). The DNA is circular, tightly supercoiled and associated with proteins, which are similar to the histone proteins found in eukaryotic cells. Some bacteria also contain small molecules of extra-chromosomal DNA called plasmids. The DNA molecule is fibrillar and compactly folded without a nuclear membrane. DNA measures 5nm in length and 0.3 nm in diameter in E. coli cells. Histones are absent in bacterial DNA. v. Motility Many bacteria are motile, and this ability to move independently is usually due to the presence of a special organelle of motility, the flagellum. Bacterial flagella are long, which are free at one end and attached to the other end of the cell. A single flagellum can never be seen directly with the light microscope. Staining with the dye basic fuchsin along with tannic acid as a mordant makes them thick. Flagella are arranged differently on different bacteria. In polar flagellation the flagella are attached at one or both ends of the cell. Occasionally a tuft of flagella may arise at one end of the cell, an arrangement called ‘lophotrichous’. Tufts of flagella of this type can often be seen in the living state by dark-field microscopy. In ‘peritrichous’ flagellation, the flagella are grown all over the cell (Fig. 33.4). It is used as one of the characteristics in the classification of bacteria Bacterial flagella are composed of protein sub-units; the protein is called flagellin. The flagellin consists of sulfur-containing aromatic amino acids in smaller amounts. However, aspartic and glutamic acids occur more frequently. The shape and wavelength of the flagellum are determined by the structure of the flagellin protein, and a change in the structure of the flagellin can lead to a change in the morphology of the cell. The basal region of the flagellum is different in structure from the rest of the flagellum. There is a wider region at the base of the flagellum called the hook attached to the basal body, a complex structure involved in the connection of the flagellar apparatus to the cell envelope. The hook and basal body are composed of proteins different from those of the flagellum itself.

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The basal body consists of a small central rod which passes through a system of rings. In Gram-negative bacteria, the outer pair of rings is associated with the lipopolysaccharide and peptidoglycan layers of the cell wall, and the inner pair of rings is located within or just above the plasma membrane. Gram-positive bacteria lack the outer lipopolysaccharide layer, but the inner pair of rings are present (Fig. 33.3).

Fig. 33.4. Bacterial flagella

vi. Fimbriae and pili Fimbriae and pili are structures that are somewhat similar to flagella but are not involved in motility. Fimbriae are considerably shorter than flagella and are many in number. They may be chemically similar to flagella. Not all organisms have fimbriae, and the ability to produce them is an inherited trait. The functions of fimbriae are not known. There is evidence that they enable organisms to stick to inert surfaces, or to form pellicles or scums on the surfaces of liquids. Pili are structurally similar to fimbriae but are generally longer and only one or a few pili are present on the surface. Pili can be visualized under the electron microscope because they serve as specific receptors for certain types of virus particles, and when coated with virus can be easily seen. There is strong evidence that pili are involved in conjugation in bacteria. vii. Capsules and slime layers Some prokaryotic organisms secrete on their surfaces slimy or gummy materials, which can sometimes be seen by the use of negative stains. When the material is arranged in a compact manner around the cell surface it is called a capsule. It forms only a diffuse layer (slime layer) when it is loosely attached. Capsules and slime layers are usually compounds of polysaccharides, polypeptides, or polysaccharide-protein complexes.

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viii. Reserve materials In prokaryotic organisms, one of the most common inclusion bodies consist of poly--hydroxybutyric acid (PHB), a compound that is formed from hydroxybutyric acid units. The monomers of this acid are connected by ester linkages, forming the long PHB polymer, and these polymers aggregate into granules. The granules have an affinity for fat-soluble dyes such as Sudan black and can be identified tentatively with the light microscope after staining with this compound. Another storage product is glycogen, which is starch-like polymer of glucose subunits. Many microorganisms accumulate large reserves of inorganic phosphate in the form of polyphosphate granules. These granules are stained by many basic dyes; one of these dyes, toluidine blue, becomes reddish violet in color when combined with polyphosphate. This phenomenon is called metachromasy (color change), and granules that strain in this manner are often called metachromatic granules. Many aquatic/marine prokaryotic organisms produce gas vesicles, which confer buoyancy upon the cells. Gas vesicles are spindle-shaped structures, hollow but rigid, that are of variable lengths but constant diameter. They are present in the cytoplasm and may number from a few to hundreds per cell The membrane is composed of protein, and consists of repeating protein subunits that are aligned to form a rigid structure. The gas vesicles membrane is impermeable to water and solutes, but permeable to gases, so that it exists as a gas-filled structure surrounded by the constituents of the cytoplasm. The rigidity of the gas membrane is essential for the structure to resist the pressures exerted on it. However, the gas vesicle membrane cannot resist high hydrostatic pressure, and can be collapsed, leading to a loss of buoyancy. The presence of gas vesicles can be determined by either brightfield or phase-contrast microscopy, but their identity is never certain unless they disappear when the cells, are subjected to high hydrostatic pressure. ix. Spores Endospores are formed within the cell and are readily seen under the light microscope as strongly refractile bodies. Spores are very impermeable to dyes, and can be stained with basic dyes such as methylene blue. The structure of the spore is much more complex than that of the vegetative cell in that it has many layers. The outermost layer is the exosporium, a thin, delicate covering. Within this is the spore coat, which is composed of a layer(s) of wall-like material. Below the spore coat is the cortex, and inside the cortex is the core, which contains the usual cell wall (core wall), cell membrane, nuclear region, and so on. Thus the spore differs structurally from the vegetative cell primarily in the kinds of structures found outside the core wall (Fig. 33.5). One chemical substance that is characteristic of spores but not of vegetative cells is dipicolinic acid (DPA). This substance is probably located primarily in the core. Spores are also high in calcium ions, most of which are also associated with the core, probably in combination with dipicolinic acid. There is good reason to believe that the association of calcium and dipicolinic acid has some role in conferring the unusual heat resistance to bacterial spores.

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Fig. 33.5. Endospore of bacteria

A spore is able to remain dormant for many years, but it can convert back into a vegetative cell on germination. This process involves two steps: cessation of dormancy and outgrowth. The first is initiated by some environmental triggers such as heat. A few minutes of heat treatment at 60 to 700C will often cause dormancy to cease. The first indications of spore germination are loss in refractility of the spore, increased stainability by dyes, and marked decrease in heat resistance. The spore visibly swells and its coat is broken. The new vegetative cell pushes out of the spore coat, a process called outgrowth, and begins to divide. The spore wall and spore coat eventually disintegrate through the action of lytic enzymes. PLASMIDS These are small, circular and autonomously replicating double stranded DNA molecules present inside the bacterial cell. This is a new kind of genetic element, the sex factor. It is a small supernumerary chromosome. It may lie free in the cytoplasm. Plasmids are extra chromosomal genetic elements. These vary in size from a few to several hundred-kilo bases in length. The chromosome and the plasmid together constitute the bacterial genome. Plasmids are transferable to other cells and hence, are of great use in recombinant DNA technology. Some plasmids are capable of integrating with bacterial chromosome. Such plasmids are called episomes. The chromosome in archaebacteria, like that of the eubacteria, is a single, circular DNA molecule not contained within a nucleus, but the size of the DNA molecule is often smaller than that of E. coli. An interesting inclusion body, the gas vacuole, is found in cyanobacteria (bluegreen algae) and photoprotein-surrounded by vacuole provides buoyancy, allowing the bacteria to float near the surface of the water. Although bacteria do not have organized intracellular membranes, invaginations of the plasma membrane called mesosomes are often seen in electron micrographs. Plasmids are the extra chromosomal structures in the cells of bacteria which have the ability to self replicate. They do not combine with the genetic material of the host cell but stay independently. They are genetically modified and are used in the recombinant DNA technology. Plasmids are usually made up of double stranded

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non-chromosomal DNA, but in some cases they are circular. They make their structure circular by combining the two ends of the double stranded DNA together. These ends are combined through covalent bonds. The main functions of plasmids are as follows: 1.

Carry antibiotic resistant genes and spread them in the whole human or animal body, In this way many diseases of humans and animals can be treated.

2.

Carry those genes which are involved in metabolic activities and are helpful in digesting the pollutants from the environment.

3.

They are capable of producing antibacterial proteins.

4.

Plasmids are also able to carry the genes which are concerned with increasing the pathogenicity of bacteria which cause diseases like anthrax and tetanus.

There are different types of plasmids which are used for various purposes (Table 33.1). Table 33.1. Examples of different types of plasmids. Type

Hosts

Size (kb)/ (Number)

Fertility factor F factor

E. coli, Salmonella

95-100 (1-3) Sex pilus, conjugation

R plasmids

RP4

Pseudomonas and other Gram-negative bacteria

54 (1-3)

Sex pilus, conjugation Ampr, Kanr, Neor, Tetr

R100

E. coli, Shigella, Salmonella, Proteus, S. aureus

90 (1-3)

Chlr, Strr, Tetr, Hgr

pSJ23a

S. aureus

36

Penr, Hgr, Genr, Kanr, Neor, Eryr

Col E1

E. coli

9 (10-30)

Colicin E1 production

Col E2

Shigella

10-15

Colicin E2 production

Ent (P307)

E. coli

83

Enterotoxin production

Col V-K30

E. coli

2

Siderophore for iron uptake

CAM SAL TOL P5P4

Pseudomonas Pseudomonas P. putida Pseudomonas

230 56 75 –

Camphor degradation Salicylate degradation Toluene degradation 2,4-dichlorophenoxy acetic acid degradation

E. coli

–

Lactose degradation

Col plasmids Virulence plasmids

Metabolic plasmids

Representative

Features

Source: Prescott et al (1996, as cited by Dubey and Maheshwari 2001)

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i. Resistance (R) plasmids This type of plasmid is involved in the bacterial conjugation. They usually carry those genes which code for the resistance of antibiotics or toxins. They also code for those genes which are responsible for the production of conjugation pili. The main role of conjugation pili is to transfer the R plasmid from a donor bacterium to the recipient bacterium. This is how the other bacteria also become antibiotic resistant. The R factors spread among populations of enteric bacteria due to rapid use of antibiotics. Different R factors having different sets of resistance genes (ranging from one to eight) are required from different chemicals. Therefore, there are several genes of R- factor which confer resistance against antibiotics. An R factor consists of two segments of DNA, one the resistance transfer factor (RTF) and the second resistant determinants (r-determinants). The RTF contains genes for replication and transmission of plasmid, and the r-determinants are on another segment. Some of rdeterminants replicate autonomously. The G+C content, molecular weight and buoyant density of these two segments vary. When two bacteria one containing R plasmid and the other devoid of R conjugate the R plasmid is transferred to the latter that lacks R plasmid. ii. Fertility plasmids These plasmids carry the tra genes which are used in the process of conjugation. They are helpful in transferring the genetic material between bacteria. The plasmids of male cells that confer on their host the capability to transmit chromosomal markers but not the other properties are called sex factor or F factor. For the first time the F factor was discovered in Escherichia coli. The term sex factor is used to mediate the conjugation process in bacteria. Sometimes the F factor transfers a nonconjugative plasmid also which is present with it in a cell. A new strain was isolated from F+ cultures that underwent sexual recombination with F– cells. This new strain had recombinations rate about 103 times greater than F+ x F– cells. These strains were called high frequency recombination (Hfr) strains. The Hfr strains contained both bacterial DNA and plasmid DNA. iii. Heavy-metal resistance plasmids There are several bacterial strains that contain genetic determinant of resistance to heavy metals viz., Hg++, Ag+, Cd++, Co++, CrO4, Cu++, Ni++, Pb+++, Zn++, etc. These determinants for resistance are often found on plasmids and transposons. Bacteria that have been found resistant to heavy metals are E. coli and Staphylococcus aureus (As), Pseudomonas aeruginosa, P. fluorescens, E. coli (Cr), S. aureus, Bacillus subtilis, Alcaligens eutrophus (Cd), Shigella spp, E. coli (Hg), Pseudomonas syringae. iv. Col Plasmids The plasmids of this type produce such antibiotics which are involved in killing the other harmful strains of bacteria by staying in the host bacterial cell. The antibiotics are also called as colicin.

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There are many bacterial strains that produce proteinaceous toxins known as bacteriocins which are lethal to other strains of the same genus. Toxins secreted by the strains of E. coli are called colicins. It kills the sensitive cells. Synthesis of colicins is specified by the plasmids present in E. coli cells but not by bacterial chromosome. These plasmids associated with colicin production are called colicinogeny (Col) factor. There are several Col plasmids such as Col B, Col E, Col I, Col V which produce different types of colicins. Some Col plasmids carry fertility determinants i.e. a set of genes governing conjugation and transfer of plasmids (e.g. Col B, Col V). These can be called conjugative plasmids. The second type is the non conjugative plasmid which is nontransmissible by their own means (e.g. Col E). However, when a cell contains both the plasmids, it transfers both. v. Degradative plasmids This type of plasmid is capable of degrading or digesting the dead organic matter from dead animals or plants. They use this organic matter in the process of biosynthesis and make energy and recycle them. Extensive research has been done on degradative plasmid of Pseudomonas. The pseudomonads have been found to catalyse a number of unusual complex organic compounds through the special metabolic pathways. Anand Mohan Chakrabarty, an India-born American scientist, has isolated plasmids from a number of cultures of Pseudomonas putida which could utilize a number of complex organic chemicals such as 2,4-D, salicylate, 3chlorobenzene, biphenyls, etc. Special genes present on different plasmids confer degradations capacity to the species of Pseudomonas. For example, the camphor (CAM) plasmid of P. putida encodes enzyme for degradation of camphor; octane chromosome (OCT) plasmid degrades octane; XYL plasmid degrades xylene and toluene; NAH plasmid degrades naphthalene; SAL degrades salicylate, etc. These plasmids are transmissible between the strains of species of P. putida through conjugation. Chakrabarty has succeeded in transferring the four plasmids; OCT, XYL, CAM and NAH present in four different strains of P. putida into one strain and called it superbug (oil eating bug). The superbug can degrade the above four types of substrate. vi. Penicillinase plasmid of Staphylococcus aureus Staphylococcus aureus is a Gram-positive bacterial pathogen causing infection of skin and wounds of humans. After treatment with penicillin antibiotic, several penicillin-resistant staphylococci were developed by 1950 throughout the world. High level resistance to penicillin was possible due to secretion of an enzyme, penicillinase which degrades penicillin by hydrolyzing its -lactam ring. There is a large variety of penicillinase plasmids designated as , , , etc. on the basis of markers present on them and production of chemically different penicillinases. Penicillinase plasmids also confer resistance against kanamycin, neomycin, tetracycline, streptomycin and chloramphenicol. Molecular weight of Kanr and Netr plasmids has been found to about 15 x 106 daltons and that of Tetr, Strr and Chlr as 3 x 106 daltons

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vii. Cryptic plasmids During isolation of plasmid DNA from a large number of bacteria, it was found that every bacterium contained a low molecular weight DNA as plasmid. They do not carry any gene, therefore, they are non-functional. These plasmids have been called as cryptic plasmid. It seems that the presence of plasmids is a rule rather than exception. viii. Ti-plasmids Agrobacterium tumefaciens is a Gram-negative soil bacterium that infects over 300 dicots and causes crown gall disease at collar region. It produces tumors, therefore, it is oncogenic. The Ti- refers to tumour inducing plasmid. The size of Ti plasmid ranges from 180-205 Kb. It consists of T-DNA (transfer DNA) of about (20) Kb which comprises of two adjacent independently encoding DNA segments, the right T-DNA (TTR-DNA) and the left T-DNA (TL-DNA). T-DNA encodes enzymes for the synthesis of auxin and cytosine which interferes with plant metabolism, and develop tumour and enable the infected plant to produce a nitrogenous compound called opines. Opines are metabolized by the bacterium as a source of carbon and nitrogen.

Fig. 33.6. Ti Plasmid

In addition to T-DNA, the Ti plasmid also consists of several genes such as vir (for virulence), ori (for origin of replication), tra (for transfer), noc (for nopaline catabolism in nopaline plasmid), arc (for arginine catabolism), and occ (for octopine catabolism in octopine plasmid) genes. The Ti-plasmids are divided into the four groups: octopine Ti-plasmids (e.g. pTiB6, pTiAG), nopaline Ti-plasmid (e.g. pTiT37, pTiC58), leucinopine plasmids and succinamopine plasmids. The first two plasmids octopine and nopaline plasmids have been extensively studied (Fig. 33.6). The Ti-plasmids are divided into the four groups: octopine Ti-plasmids (e.g. pTiB6, pTiAG), nopaline Ti-plasmid (e.g.

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pTiT37, pTiC58), leucinopine plasmids and succinamopine plasmids. The first two plasmids octopine and nopaline plasmids have been extensively studied ix. Ri-plasmids The Ri (root inducing) plasmids are found in Agrobacterium rhizogenes which causes hairy root disease in plants. The Ri-plasmids are closely related to Ti plasmids. The size of Ri-plasmids varies from 190-240 Kb, thus they are large sized plasmids. On the basis of opine production in the infected plants the Ri plasmids are put into three groups: manopine Ri-plasmids (e.g. pRiTR7, pRi8196), agropine plasmids (e.g. pRiA4, pRi1855) and cucumopine plasmids (e.g. pRi2659). Viruses cause disease in a number of crop plants as well as wild plants. The viruses that infect the plants are mostly single stranded RNA viruses, and it was recognized that some plant diseases are caused by double stranded RNA viruses, single stranded and double stranded DNA viruses also. x. Virulence plasmids As the name shows, these plasmids have the ability to transform bacteria into a pathogen and they are responsible for carrying the genes which cause diseases. Plasmids in Gene Therapy Plasmids have a significant role in gene therapy. They are mostly used for the insertion of therapeutic genes in the human body to fight against diseases. They are easy to manipulate and their replication in the bacterial cell is easy. They have the ability to efficiently target the cells which are defected and trigger the therapeutic genes in them. There are no harmful effects of plasmids like the viral vectors. Plasmids in Recombinant DNA technology Recombinant DNA technology makes use of plasmids for many purposes. For the drug delivery, this technology makes use of the plasmids to insert the desired drug into the body. They are also involved in causing antibiotic resistance and are used to kill harmful bacteria from the body. Recombinant DNA technology applied plasmids for the first time on the human body for the insertion of human insulin. It gave very efficient results. The other application of plasmids is the insertion of human growth hormone in the mammalian cells of animals. CULTIVATION AND NUTRITION OF BACTERIA Culture is an active growth of microorganism under appropriate condition. A culture containing only one kind of microorganism is axenic culture. The culture obtained from single cell is called pure culture. The culture containing only two kinds of microorganism is known as two membered culture. The culture containing different microorganisms is known as mixed culture. Media is a mixture of various nutrients and is prepared as per the nutritional requirements of bacteria. They are required for the growth and maintenance of the

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organism(s). Different media are employed for the isolation, growth and maintenance of pure cultures and also for the identification of bacteria according to their biochemical and physiological properties. A culture medium must supply suitable carbon and energy sources and other nutrients and growth factors, if necessary. A single medium will not support the growth of all microorganisms. Culture media are classified on the basis of their consistency as: 1.

Liquid media: Media with liquid consistency.

2.

Solid media: If solidifying agent like agar is added in media, it confers solid consistency to media.

3.

Semisolid media: If agar or any solidifying agent is added in low concentration then it confers semisolid consistency to media.

In general, liquid media are used for obtaining pure batch cultures, whereas solid media is used for various purposes e.g. isolation of pure cultures and estimating bacterial population. Culture media may be classified into several categories depending on their composition or use. 1.

Synthetic medium: A chemically defined minimal medium that provides the required nutrients needed for the growth of organism. Chemically defined media are of value in studying the minimal nutritional requirements of microorganisms, for enrichment cultures, and for a wide variety of physiological studies.

2.

Complex medium: It is an undefined medium that contains complex materials of biological origin like serum, milk, yeast extract or beef extract, peptone etc. Complex media usually provide complete set of growth factors that may be required by an organism. Hence, it is used to cultivate unknown bacteria or bacteria whose nutritional requirements are complex.

3.

Selective medium: The medium that contain nutrients which will inhibit/ prevent the growth of certain types of bacteria and promote the growth of desired bacteria. For e.g. halophiles can grow with a very high concentration of NaCl. MacConkey's media that contain bile salt (sodium taurocholate) selectively promote the growth of Gram negative bacteria and inhibit the growth of Gram positive bacteria.

4.

Differential Medium: A differential medium is one which differentiates different types of bacteria based on their growth pattern in the medium. MacConkey's media is a good example of differential medium. It contains lactose as carbon source. It is possible to differentiate between lactose fermenting and lactose non-fermenting bacteria. Pink coloured colonies represent lactose fermenting bacteria and white colour colonies are of lactose non-fermenting bacteria. The red coloured indicator when incorporated into medium helps to detect lactose fermentation that turns pink in acidic condition. It is due to the secretion of acid by lactose fermenting organism. The lactose non-fermenting bacteria turn the red colour to white colonies.

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5.

Enrichment Medium: Enrichment medium is a medium that contains substances which encourage growth of the required organism (s) and/or inhibit the growth of other types of organisms. Some of the enrichment media are described below: a.

Selenite F-medium: This medium is used for the enrichment of the strains of Salmonella in samples of faeces Selenite inhibits many of the common enteric bacteria. The medium consists of an aqueous solution of peptone (0.5%), lactose or mannitol (0.4%), sodium selenite (0.4%) and either di-sodium hydrogen phosphate (1.0%) or a combination of di-sodium hydrogen phosphate and sodium di-hydrogen phosphate to give a final pH of 7.0. The medium should not be autoclaved, but may be steamed for 30 minutes. Incubation of the inoculated medium should be around 12 to 18 hours at 30°C.

b.

Alkaline peptone water: This medium is used for isolating Vibrio cholerae. It consists of peptone and NaCl (1% each) in water and pH adjusted to 8.5 to 9.0.

Isolation methods for bacteria Natural populations usually consist of a mixture of different types of microorganisms. For many of the microbiological investigations a pure culture is very essential. A pure culture is defined as the progeny (clone) of a single cell. A pure culture should be free from contaminants. The procedure for obtaining the pure culture is as follows. A cell must be separated from a cell population and the colony that results from its multiplication must remain separate from other cells and other colonies. When cells of a single colony are spread out on a fresh surface, many colonies are developed, each with the same shape and colour as the original. The most commonly used methods for obtaining the pure cultures of aerobic bacteria as proposed by Koch are streak plate method, pour plate method and spread plate method. The methods widely used for the isolation of bacteria are as follows (Fig. 33.7). In the spread plate method, a small sample (usually 0.1 ml) is spread over the surface of an agar plate containing an appropriate medium and the plate incubated until the colonies are visible to the naked eye. It is inferred that each colony arose from a single cell, and each colony represents a living cell. With the pour plate method, the sample is mixed with melted agar and the mixture poured into a sterile plate. The organisms are thus fixed in the agar medium and form colonies. The above methods allow for the positive identification of the organism(s). A device named Micromanipulator can be used in conjunction with a microscope to pick a single bacterial cell from a mixed culture. The micromanipulator permits the operator to control the movements of a micropipette or microprobe so that a single cell can be isolated. This technique requires a skilled operator and is meant for studies in which a clone must be obtained unequivocal.

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Fig. 33.7. Streak plate method

Enrichment Culture Methods For the study of the various physiological types of microorganisms that exist in nature, Winogradsky and Beijerinck proposed a new and profoundly important technique: the enrichment culture technique. It is essentially an application on a microscale of the Darwinian principle of natural selection. The investigator devises a culture medium of a particular defined chemical composition, inoculates it with a mixed microbial population, such as can be found in a small amount of soil or mud, and then ascertains by examination what kind of microorganisms come to predominance. Since their predominance is caused by their ability to flourish in the enrichment medium, they can be readily isolated by plating on a medium of the same composition. To take a specific example, if we wish to isolate microorganisms which can use atmospheric nitrogen, we prepare a medium that is free of combined nitrogen but which contains all the other nutrients, an energy source, a carbon source and minerals necessary for growth. This is then inoculated with soil, placed in contact with N2, and incubated under any desired set of physical conditions. Since nitrogen is an essential constituent of every living cell, the only organisms which will multiply in such a medium are those that can fix atmospheric nitrogen, if such types are present in the soil sample, they will grow. Such experiments can be varied in innumerable ways: by modifying such factors as the carbon source, the energy supply, the temperature, and the hydrogen ion concentration. For each particular set of conditions, a particular kind of microorganisms will come to predominance, provided the organism exists in the inoculum which can grow under such particular conditions. These experiments also define the medium that should be used for the

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isolation of the organisms concerned, since it is clear that the enrichment medium, whose exact composition has been set beforehand, provides the necessary nutrient environment for their development. The enrichment culture method is thus one of the most powerful experimental tools available to the microbiologist. By its use, he can isolate microorganisms with any desired set of nutrient requirements, provided that such organisms exist in nature. To isolate microorganisms capable of developing at the expense of cellulose as a source of carbon and energy, one has to prepare an enrichment medium containing cellulose as the only available source of carbon and energy, together with ammonia or nitrate as a nitrogen source, and the other necessary minerals. This is inoculated with soil and incubated until growth occurs. One can further limit the type of cellulose-decomposing microorganisms that develop by altering the physical conditions under which the enrichment culture is maintained. For example, if we wish to obtain anaerobic cellulose-decomposers, the enrichment culture has to be incubated in the absence of air. In case of cellulose-decomposers that are capable of growing at high temperatures, the cultures have to be incubated at 50°-60°C. Once the outcome of a given enrichment culture technique is known, the method also permits the isolation of a given microorganisms at will from nature. The enrichment of various organisms can be achieved by the manipulation of such environmental factors as the composition of the medium, aeration, pH, and illumination. Other factors, including temperature, osmotic pressure, and surface tension, can also be varied to give special selective advantage to different organisms. By incubating enrichment media at very high temperatures (for example, 550, or 600C), cultures of thermophilic bacteria can be obtained. Even at moderate temperatures, selective enrichment by the use of varied temperature is possible. The choice of a suitable inoculum, in which a given type of organism is already enriched in nature, is often helpful and in some instances essential for the success of enrichment. Furthermore, the inoculum can be pasteurized (two to five minutes at 80°C) to destroy bacteria that form vegetative cells and to enrich endospore-formimg members of the genera, Bacillus and Clostridium. An almost infinite number of permutations and combinations of environmental variables can be devised for the enrichment of different bacteria from nature. Thus, the enrichment culture technique offers a means, not only of isolating known bacterial species, but also for studying undescribed organisms that are capable of flourishing in any given environment. Enrichment cultures of fermentative bacteria must be incubated under anaerobic conditions, because some of these organisms are obligate anaerobes, and the presence of oxygen inhibits the growth of anaerobic bacteria. The lactic acid bacteria illustrate this point. These organisms are characterized by their remarkable resistance to lactic acid, which they themselves produce in the fermentation of sugar. To enrich for lactic acid bacteria, a poorly buffered medium containing glucose and a rich source of growth factors is used (for example, twenty grams of glucose and ten grams of yeast extract per liter). After inoculation, preferably with natural materials that are rich in lactic acid bacteria (vegetable matter, raw milk, sewage etc.), the medium is incubated under anaerobic conditions. The first enrichment culture to develop usually consists of bacteria that carry out the formic acid type of

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fermentation (Aerobater, Escherichia, and related genera) since these organisms grow very rapidly. However, as lactic acid gradually accumulates, conditions become less and less favourable for these bacteria, whereas the lactic acid bacteria continue to grow. Eventually, the acidity of the medium becomes so high that the lactic acid bacteria become predominant, and most other organisms are killed. Another good example is the enrichment of the propionic acid bacteria. These organisms produce propionic acid, acetic acid, and O2 in fermentation. If a neutral medium containing twenty grams of sodium lactate and ten grams of yeast extract per liter is inoculated with natural materials containing propionic acid bacteria like Swiss cheese and incubated at 30°C under anaerobic conditions, enrichment of these organisms is obtained. The acetic acid bacteria (Acetobacter) are adapted to environments that contain high concentrations of sugar and alcohol. They are also less susceptible than other bacteria to inhibition by acetic acid, which they produce from alcohol in respiration. To enrich Acetobacter, a complex medium containing alcohol is inoculated with materials containing these bacteria and then incubated under aerobic conditions. Fruits, flowers, and unpasteurized beer are among the favorite sources of inoculum. A medium containing forty milliliters of alcohol and ten grams of yeast extract per liter, adjusted to pH 6.0, can be used for enrichment. The Isolation of Anaerobic Bacteria Few bacteria are strict anaerobes. The isolation of pure cultures of these forms require certain modifications of the used plating technique. Provided that their sensitivity to oxygen is not too great, they can be isolated by streaking or pouring plates in the usual fashion and then placing the inoculated plates in an oxygen-free atmosphere. Some strict anaerobes are, however, very rapidly killed by exposure to air. R.E. Hungate developed a method known as Roll Tube method (Fig.33.8). For such forms, the method of choice is the use of dilution shake cultures. A series of sterile tubes about two-thirds filled with melted agar of the appropriate composition are cooled to a temperature just above the solidifying point, and the inoculum is added to the first tube. This is then well mixed, and a little of the mixture (about onetenth) is poured into the second tube, which is mixed in turn and used to inoculate the third tube. The agar is allowed to solidify, and the surface is covered with a seal of paraffin in order to minimize the access of air to column of agar. When a series of ten tubes is prepared in this fashion, the dilution is sufficient to yield one or more tubes containing well-isolated colonies. When a shake culture has developed, the tube is warmed to loosen the plug of paraffin, which is then removed with a sterile wire. A very fine sterile capillary pipette, prepared by drawing out a piece of glass tubing in the flame of a burner, is then introduced between the column of agar and the glass wall of the tube. By attaching the wide end of the pipette with a piece of rubber tubing to a source of compressed air, it is possible to blow out the entire column of agar into a sterile petridish, and then it can then be sectioned into discs with a sterile scalpel for the examination of individual colonies. By careful separation of single colonies, their suspension in suitable liquid and repeated streaking out or plating on nutrient agar of

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the same composition, it is possible to obtain pure cultures of the majority of the microorganisms under oxygen-free conditions.

Fig. 33.8. Roll tube method for isolation of anaerobic bacteria Serial dilution method for isolating anaerobic bacteria in soft agar (0.8%)

UTILIZATION OF NUTRITIONAL AND ENERGY SOURCES BY BACTERIA The bacteria utilize the CO2 from the atmosphere as source of carbon and H2, H2S and NH3 as a source of hydrogen in synthesizing food for their growth are known as autotrophs. These bacteria can be classified into two groups namely Photoautotrophs and Chemoautotrophs based on the source of energy. Photoautotrophic bacteria utilize light as a source of energy and utilize H2 and CO2 from the atmosphere in synthesizing food for growth. The sulphur bacteria take hydrogen from sulphites and thiosulphates.(Chlorobacteriaceae, Thiorhodaceae). Chemoautotrophic bacteria derive energy while oxidizing inorganic compounds such as ferrous ammonia, nitrates and nitrites. The examples are: 1.

Sulphur bacteria Beggiatoa: H2S + O  H2O + S + energy (50 K.cal) Thiobacillus denitrificans: S +H2O+H2O2  H2SO4 + energy (71 K.cal)

2..

Iron bacteria Ferrobacillus: 4 FeCO3 + 6H2O + O2  4FeOH3 + CO2 + energy (81 K.cal)

3.

Nitrifying bacteria Nitrosomonas: NH3 + 1 ½ O2  NO2– + H2O + H + energy (78.5 K.cal) Nitrobacter: NO2 + ½ O2  NO3– + energy (17.8 K.cal)

4.

Hydrogen bacteria

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Hydrogenomonas H2 + ½ O2  H2O + energy (56 K.cal) Bacteria are capable of growing at varying temeperatures. Those that grow below 100C are called as psycrophilic and those that grow up to 750C are referred as thermophilic organisms. Bacteria growing at normal ranges of temperature (20-350C) are called mesophilic. Bacteria cannot withstand high pressure, high sugar/salt or acidic conditions However, the bacteria inhabiting deep seas are capable of growing at highly saline environment are referred as halophiles. Bacteria that can withstand highly acidic conditions are known as acidophiles. GROWTH OF BACTERIA Growth refers to orderly increase in all cellular constituents. In the case of any organism growth means increase in cell number. In multinucleated cell, growth refers to the increase in cell size. In unicellular microorganisms the increase in cells, means increase in the number of individuals. Bacteria normally reproduce by binary fission, a cell division process where two identical daughter cells are formed from a cell. The bacteria are usually grown in a batch culture which is a closed system with limited amount of nutrients and specific numbers of organisms are allowed to grow for specific time period. The microbial population is monitored in terms of the logarithmic cell number at regular periods, Sampling is plotted against time intervals to get a typical bacterial growth curve which has four distinct phases (lag phase, log (exponential) phase, stationary phase and death or decline phase) (Fig. 33.9). lag

exponential

stationery

death

10 viable count

log 10 viable organisms/ ml

9 8 7 6 5 4 3 2 1 Time

Fig. 33.9. Growth curve of bacteria

i. Lag phase When bacterial culture is inoculated into a fresh nutrient medium, the microbial population remains constant for an initial period which is known as ‘Lag phase’. In

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this phase, bacteria adjust themselves to adapt to the new environment. The enzymes, coenzymes and other essential molecules are synthesized by bacterial cell during this phase. The cells are metabolically and physiologically very active but cells are not dividing. The length of the lag phase depends on the nature of medium, species of organism, and various physical and chemical growth factors. If an exponentially growing culture is inoculated into an identical medium which is kept at same temperature then no or little lag phase will be observed. But if the culture is transferred from a rich medium to minimal medium then a significantly long lag phase will be observed. This is mainly because the nutrients and enzymes necessary for growth in the new environment need to be synthesized. As a general rule, organisms will adapt to better growth conditions more rapidly than to poor conditions. ii. Exponential phase The phase during which microorganisms divide at the maximum possible rate is called Log phase (Exponential phase). The cells divide at the exponential rate. As nutrients are present in ample amount, the cells rapidly grow. Bacteria in exponential phase are preferred for physiological/genetical /molecular studies. iii. Stationary phase This phase is characterized by having same growth and death rate. In curve it appears as a straight line, like lag phase. The number of growing cells and dying cells are equal in number. The stationary phase can last for long periods of time and is dependent on type of microbe and conditions of culture medium. iv. Death/decline phase The increase in death is due to deficiency of nutrients and presence of toxicants. Bacteria commonly reproduce by binary fission. Thus, the population increases geometrically or exponentially, 1  2  4  8  16  32 ... As one cell divides into two, the number of cells is Number of Generations =

Log No. of cells – log No. of initial cells 0.301 (log 2)

The above equation applies only to actively growing cells in exponential phase but not at any other phase of growth curve. Generation time The time required for a cell to divide or time needed for the population to double under optimal conditions is known as generation time (doubling time). The generation time depends on environmental factors such as pH, temperature, O2 concentration, available nutrients and also on species.

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To calculate generation time for a microbial growth, G=t/n When optimum conditions are prevailing, E. coli grows within 20 minutes and Mycobacterium tuberculosis requires 14-15 hours. Measurement of bacterial growth Different methods using various parameters such as change in cell number, change in the turbidity and change in the amount of a cell component are usually followed. A. Determination of ‘cell number’ i. Direct methods This method refers to direct microscopic count. In this method, a known volume of cell suspension (0.01 ml) is spread over a glass-slide within a specific area (1 sq. cm). Since, it is difficult to examine the entire area (1 sq. cm), practically few microscopic areas are observed. The total number of microscopic fields per 1 sq. cm area gives total number of cells. Total number of cells /1 sq. cm = Average number of cells per field  5,000 i.e. Number of cells /0.01 ml of suspension. Multiplication of this number by 100 will give number of cells/ml of suspension. ii. Counting chamber method The Petroff-Hausser counting chamber or Haemocytometer is used in this method. A film of known depth can be introduced between the slide and cover slip. The number of cells per ml of original sample is determined by multiplying the dilution factor of the sample. iii. Coulter method In this technique electronic cell counter is used to enumerate number of cells. A light beam is directed to a photodetector instrument through capillary tube. The disturbance in light beam due to passage of bacterial cell causes reading of one unit, which is displayed by digital reader. With this method it is possible to count accurately thousands of cells in a short time (few seconds). B. Indirect method i. Total viable count This method gives the number of microorganisms in terms of colony forming units (CFU) in the diluted sample. To calculate the total viable count, multiply the number of colonies by dilution factor. Only viable cells are enumerated by this method.

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ii. Determination of cell mass In this method total nitrogen /phosphorus/other nutrients of grown cells is determined. These nutrients are the constituents of cell proteins which is one of the major component of cell and the determination of these constituents in microbial culture provides an idea of microbial growth. Indirect determination of cell mass - Turbidometric method The most widely used technique for measurement of microorganisms is by measuring its turbidity which is measured by spectrophotometer. The absorbance is measured in terms of optical density (OD). The OD does not provide information on cell numbers or cell mass but the cell number can be calculated by plotting calibration curve. It is the most common method to standardize the bacterial growth. Determination of cell activity Measurement of a specific chemical change as a result of metabolic activities of microbes can be correlated with the microbial growth. Cell metabolic activity results into formation of specific metabolite like organic acids, CO2, H2S, and enzymes. The measurement of these products is the principle of measurement of cell activity. The amount of product(s) produced is proportional to the magnitude of cell suspension. Continuous growth The technique by which microbial population is maintained in the exponential phase of growth by providing the constant environment is termed as continuous culture technique. Microbial growth in batch culture remains in exponential phase for only a few generations and then it enters in stationary phase and subsequently in death phase. However, entire population can be maintained in exponential phase of growth by allowing fresh sterile medium to the culture vessel continuously and removing already utilized medium continuously and keeping culture volume constant. In order to study the metabolism and genetical studies of an organism for experimental research or industrial processes, maintaining microbial population in the exponential phase of growth is vital. The following methods are used in continuous culture of bacteria Fermenter / Chemostat It is a continuous culture apparatus used for maintaining microbial population in the exponential phase of growth. The dilution rate is the rate at which fresh medium is being added to the culture divided by the volume of the culture vessels. It is controlled by the ‘growth-limiting nutrient’ (Fig. 33.10). Fermenter consists of a culture vessel, which can be maintained at optimum pH, temperature and proper aeration. The aeration is usually adjusted by introducing filtered air. The vessel is provided with siphon or overflow line which maintains the

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liquid level constant. The sensor detects the concentration of chemicals (growth limiting nutrients) within culture vessel. The fresh culture medium is introduced from a reservoir through a system in response to a signal given by the sensor.

Fig. 33.10. Fermenter

Maintenance of constant culture density is achieved chemostatically i.e. ‘growth response is directly proportional to concentration of growth limiting nutrients in culture vessels’. So concentration of growth limiting nutrients is kept just sufficient to allow only, desirable amount of growth. This can be done manually or automatically by using the senor. Medium contains at least one nutrient in limiting concentration, which regulates the growth rate. Volume of culture in growth chamber is kept constant by siphon overflow. When fresh medium is inoculated initially, concentration of nutrient is more and organism will grow at high rate, limiting nutrient will be consumed and a stage will come when there is no nutrient and growth will stop. Thus flow rate is adjusted in such a way that medium is added slowly but constantly with respect to rate of overflow through siphon. Thus rate of addition of fresh medium is kept constant and growth rate is also maintained constant and self-regulating. Continuous culture technique is applicable in brewing process and for producing mass cultures. It reduces the time for growth as compared to the batch system. Continuous culture is the ideal method for the production of microbial biomass. It has superior productivity.

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Turbidostat Photoelectric cell continuously monitors the turbidity of the culture in vessel and controls the dilution rate, to maintain the cell density at constant level. The turbidostat is controlled by photocell. The photocell signal operates the medium pump and is activated when the culture capacity exceeds beyond the chosen value due to increase in number of cells. Overall increase in the turbidity is detected by optical sensing device. This device controls the flow rate of fresh medium. Dialysis technique This is the process of separation of smaller molecules from solution by diffusion through semi permeable membrane. Synchronous growth The synchronous growth is growing microbes in such a way that they are all in same stage of growth phase and all will divide at the same time with respect to each other. It can be achieved by physical and Induction methods. Physical method a. Filtration The cells are filtered so that smallest cells pass through the filter. The smallest cells are not able to divide while the largest cells are ready for division. The filtrate is used to obtain a synchronous culture. b. Helmstetter-Cummings technique In this, millipore cellulose acetate filter of uniform pore size is used. A population of cells is passed through a membrane filter to trap uniform size bacteria within the filter. The filter is then inverted and fresh nutrient medium is allowed to pass through it. The loosely associated bacteria are washed from the filter. All cells of same size are in same physiological or metabolic state and are therefore of same age and will divide synchronously. c. Density gradient centrifugation Density gradient centrifugation is also used to separate the cells. A population of microbial cells is separated into fractions, each composed of the cells of the same density and at the same stage in their growth cycle. Induction method Several shock treatments induce a synchronous culture. These include variation in temperature, starvation, exposure to light in case of photosynthetic organism, sublethal treatment with drug or radiation.

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Significance of synchrony Synchrony maintains entire population uniform with respect to growth phase and facilitates the analysis of growth behaviour i.e. differentiation, organization, macromolecules synthesis of uniform culture. Thus, time course patterns of various macromolecular syntheses can be studied by removing portions of synchronously dividing culture and analysing the cells for component or the enzyme activity under investigations. Diauxic growth Bacterial growth characterized by two separate phases due to the preferential use of one carbon source over another, is called as diauxic growth. In 1942, Monod discovered the phenomenon of diauxy. In a medium containing both glucose and lactose, E. coli first uses glucose and lactose is not metabolized until all the glucose is used up. The enzymes for the catabolism of lactose are not synthesized until the glucose in the medium is exhausted. The enzymes involved in glucose metabolism are constitutive while the enzymes involved in lactose metabolism are inducible. There is no free diffusion of glucose in E. coli cell. Glucose molecule is phosphorylated to Glucose 6- phosphate by consuming ATP. During the process of translocation the intracellular concentration of cyclic AMP decreases. The enzyme, adenylate cyclase is responsible for the conversion of ATP to cyclic AMP as follows: Adenylate cyclase

ATP       AMP  Ppi

The location of the enzyme adenylate cyclase is the cytoplasmic membrane of E. coli. During the process of translocation, ATP is required at a constant rate for translocation of glucose. As the adenylate cyclase is in an inactive state, the cyclic AMP will never be formed. Thus, during the process of translocation, intracellular concentration of cylic AMP decreases. Cyclic AMP is required for transcription of lactose catalysing enzymes. Cyclic AMP forms a complex with CRP protein (Catabolite activator protein). Scarcity of glucose in medium increase intracellular ATP concentration at membrane, this subsequently increases the cyclic AMP. Factors affecting growth A. Physical factors i. Water activity – aW The water activity plays important role in growth of bacteria. If water activity of surrounding solution is lower than 0.55, it leads to distortion of the DNA, and no organism survives below this value.

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ii. Osmotic Pressure Movement of solvent molecules across a selectively permeable membrane from dilute solution to more concentrated solution is called as Osmosis. Such unidirectional flow of water through semi-permeable membrane exerts pressure, called osmotic pressure. Most bacteria grow optimally in isotonic or hypotonic environment. But some microbes grow at relatively high salt concentrations (up to 10%) and are called as osmotolerant. iii. pH pH is a measure of hydrogen ion activity of a solution. pH is defined as negative logarithm of hydrogen ion concentration pH = –Log [H+] Depending on optimum pH value of microbe they can be classified as: The microbes grow at acidic pH range (1.0 – 6.5.), e.g. Thermoplasma, Thiobacillus thioxidans, Lactobacillus acidophilus. The microbes grow at neutral pH range (6.8 - 7.4.), e.g. Staphylococcus aureus, E. Coli. The microbes grow at alkaline pH range (7.5 -14.), e.g. Vibrio cholerae, Agrobacterium sp.) Despite wide variation in habitat, internal pH of most microorganisms is close to neutrality. This may be because of the impermeability of the plasma membrane to photons. Acidophiles include many fungi and some bacteria e.g. Thiobacillus and Sulfolobus. Most of the organisms fall into the category of neutrophiles. Infact mammalian pathogens grow well in a very narrow pH range close to 7.0. Alkalophiles are usually found in basic soda lakes and high carbonate soils. Interestingly both acidophiles and alkalophiles maintain their cytoplasm at a neutral pH (pH = 7.0). iv. Temperature Growth of microbe is affected by temperature as each organism has its minimum, optimum and maximum temperature In 1966 Thomas Brock discovered microbes in the boiling hot springs of Yellowstone National Park, USA. Bacteria can grow at any temperature at which water is liquid. Microbes have also been discovered in Antarctic region Lake Vostok, a fresh water lake which is thousands of meter under the ice and temperature is near 0°C. Depending on optimum temperature required for growth, microbes are grouped as follows: (a). Psychrophiles: Microbes growing below 15°C. The habitat of psychrophiles is Arctic and Antarctic regions. Examples – Moritella, Pseudomonas and Arthrobacter.

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(b). Mesophiles: Microbes growing in the range of 20-40°C. The mesophiles are found in routine environment. Human, animal, and plant pathogens are mesophiles. Examples - Escherichia coli, Rhizobium sp., Xanthomonas citri, etc. (c). Psychrotrophic: Several mesophiles also grow at temperature below their normal range for instance at 5°C. Examples - Proteus vulgaris, Salmonella sp. and Staphylococcus sp. (d). Thermoduric: Several mesophiles can tolerate high temperature (above 60°C) However, they require mesophile range for their growth. Thermoduric microbes are cyst forming, spore forming or thick walled microbes. (e). Thermophile: Microbes grow in the range 50–110°C. The hyperthermophiles - grow at temperature of 80°C and extreme thermophiles grow at 110°C. Examples - Sulfolobus, Pyrodictium occultum, Thermoproteus, etc. (f). Barophiles : atmospheric pressure also affects growth of microbes. All microbes are adapted to grow at 1 atm. pressure. Microbes are also detected at deep sea level hydrostatic pressure (500-1000 atm.) and temperature (2–3°C.) The microbes which can tolerate such high atmospheric pressure are called as Barophiles. Examples - Photobacterium, Shewanella, Colwellia, Pyrococcus and Methanococcus jannaschii. B. Chemical Factors Oxygen Depending on O2 requirement, microbes can be classified as: i.

Aerobe – Microbes can grow in the presence of oxygen, e.g. Rhizobium, Bacillus sp.

ii.

Anaerobe – Microbes can grow in absence of oxygen, e.g. Clostridium sp.

iii.

Facultative anaerobe – Microbes can grow in both aerobic and anaerobic conditions, e.g. Pseudomonas, E. coli.

iv.

Aerotolerant – Microbes ignore O2 and grow equally well whether it is present or not, e.g. Streptococcus faecalis.

v.

Microaerophilic – They require less O2 concentration than that is present in atmosphere, e.g. Azospirillum.

Effect of O2 In aerobic system, oxygen acts as electron acceptor. Hence, it is very essential for metabolic activity. Aerobes possess two enzymes, superoxide dismutase and catalase, which destroy the toxic radical. Superoxide dismutase

2O 2 –  2H          O 2  H 2 O 2 Catalse

2H 2 O 2    2H 2 O  2O 2 

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Aerotolernt lack catalase but have superoxide dismutase, while microaerophillic microbes destroy superoxide radical by manganese ions. Strict anaerobes lack both enzymes and hence cannot grow in presence of O2. C. Nutritional factors: Carbon is an important component of all macromolecules like carbohydrate, lipid, protein and nucleic acid. Majority of microbes obtain carbon from organic molecules, while photosynthetic microbes can use CO2 as sole carbon source. The Caponiphiles are microbes which grow best at higher CO2 tension than normally present in the atmosphere. Amino acids, purines, pyrimidines, enzymes and co-factors serve as nitrogen sources. Phosphorus is present in nucleic acid, phospholipid, ATP, several co-factors and proteins. Sulphur is required for the biosynthesis of amino acids like cysteine and methionine, which are important constituents of proteins and certain vitamins. sulphates, hydrogen. Requirement of growth fators : i.

Vitamins: These are essential as coenzymes for certain enzymes.

ii.

Purines and pyrimidines: They are required for the synthesis of nucleic acid i.e. DNA and RNA.

iii.

Amino acids: These are essential for the synthesis of proteins.

iv.

Except for all bacteria, Lactobacillus requires all of the above growth factors for proper growth. These factors need to be added in the culture medium for the optimum growth of Lactobacillus.

Microbes classified on the basis of energy source and nutrition: i.

Utilize the light as energy source (Phototroph).

ii.

Derive energy from oxidation of chemical compounds (Chemotrophs).

iii.

Utilize CO2 as sole carbon source (Autotrophs).

iv.

Utilize organic compounds as carbon source (Heterotrophs).

v.

Use reduced inorganic substances (Lithotrophs).

vi.

Utilize reduced organic compounds (Organotrophs) CLASSIFICATION OF BACTERIA

Descriptions of bacteria represent their morphological characters like shape, presence of capsules, flagella spores etc. and their reaction to Gram staining. These descriptions are complemented by enumeration of physiological and biochemical characters viz., habitat; growth of cells under oxygen conditions (aerobic/anaerobic); generation of energy is derived such as from respiration, fermentation or photosynthesis; utilization of nutrients; dependence on temperature and pH for

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growth, relationship with other organisms; cell inclusions, pigmentation and capsular material; composition of cell wall constituents (peptidoglycon skeleton, lipopolysaccharides, teichoic acids); serological properties; DNA base composition (G:C contents); DNA, DNA hybridization, transformability by interspecies transfer; sequence of 16S and 5S rRNA and sensitivity to antibiotics. The binary system of nomenclature is used by generic and specific names. The classification of prokaryotic microorganisms can be distinguished as phylogenetic (or natural) and artificial (or pragmatic). It is the overall aim of bacterial systematists to group related forms i.e. those possessing common ancestors, together in a phylogenetic system. Ultimately it will be on the basis of biochemical properties such as amino acid sequences of functional enzymes or the base sequences of nucleic acids in conserved cell components like ribosomal RNA. Artificial classification This is less used than the phylogenetic system. It aims to group organisms on the basis of their similarities so that they can be recognized and identified. This system is designed for use as a determinative key. Bergey’s Manual of Determinative Bacteriology (1974) contains descriptions of morphological and physiological properties with literature citations and determinative keys for classification of bacteria. Numerical taxonomy This system started originally with Adanson and hence termed as Adansonian taxonomy and every character used for classification is given equal weight. As many diagnostic characteristics as possible are used for numerical approach and these are notated as positive (+) or negative (-) signs. Multiple correlations are worked out for computation. Every diagnostic characteristic for each strain is compared with diagnostic characteristics of all other strains. The degree of relatedness between strains is a function of the number of similar characters in proportion to the total number of characters taken into account. The similarity between pairs of strains is expressed by a similarity of coefficient (S value) as follows: S

a  b a  bcd

Where a and d are the sum total of characters that are common to a and b (a. both +ve and d both – ve), b is the sum of characters in which a is +ve and b is –ve and c is the sum of characters in which a is –ve and b is +ve. The calculations yield values between 1 and 0; S = 1 means (100%) similarity (identity) and S less than 0.0 means complete unrelatedness. The values are entered on a similarity matrix, or they can be expressed as a dendogram (similar to a phylogenetic tree). Numerical taxonomy, however, is not related to phylogeny. Bacterial phylogeny Ribosomes are meant for protein synthesis. They are functionally very conservative. This is true for ribosomal RNA (rRNA) base and its base sequence is

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not affected by degeneracy of genetic code. This RNA, therefore, has the properties of general phylogenetic marker. Determination of base sequences 16S rRNA from a large number of bacterial strains has revealed surprising similarities and differences. A catalogue of these nucleotide sequences and determination of the similarity coefficient (SAB values) have given a dendogram that must be regarded as a phylogenetic tree. The nucleotide sequence analysis of rRNA led to the accidental discovery of archaebacteria which is quite different from all other groups which are collectively designated as eubacteria. Thus both archaebacteria and eubacteria developed from primordial cells are called progenotes. There also appears to be a close relatedness between archaebacteria and eukaryotes. 1. Chemotaxonomy Taxonomy based on chemical profile of microbe is chemotaxonomy. i. Protein analysis and enzyme profiles Several sequencing studies of proteins have been performed, usually with the aim to establish phylogenetic relationship between two bacteria. The sequence determination and electrophoretic profiles (SDS-PAGE) of proteins are important techniques used in classification. Enzyme profile of bacteria is an important aspect in taxonomy particularly on thermostability of enzyme which is used in bacterial taxonomy. This technique is easy and more accurate than SDS-PAGE. ii. Peptidoglycan Peptidoglycan is present in all bacteria except mycoplasma and archaebacteria. Peptidoglycan is a major component used for the taxonomy of actinomycetes. The study of cell wall sugars (rhamnose, ribose, xylose, arabinose, mannose, glucose and galactose) is also important. iii. Cytoplasmic Membrane Quinones are an important component of cytoplasmic membrane. Bacteria contain two types of quinones – ubiquinones and menaquinones. Quinones can be used for taxonomic study of Gram-positive bacteria and actinomycetes. Phospholipids are also important components of cytoplasmic membrane. It has been shown that determination of phospholipid types divide actinomycetes into five groups. Fatty acid profiles have shown a good correlation with phylogeny of actinomycetes. iv. Outer Membrane Mycolic acids (2 alkyl, 3 hydroxl long chain fatty acid) are principle components of external membrane of Mycobacterium, Corynebacterium and Nocardia. These genera can be separated depending on their mycolic acid. Mycolic

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acid isolated from Mycobacterium has 60 to 80 carbon atoms chains, where as mycolic acid with 50 to 30 carbon atoms chains are found in Nocardia and Corynebacterium. v. Metabolites End products of metabolism are useful for taxonomic study. Chemotaxonomy was important in 1970’s and 1980’s but now is largely replaced by nucleic acid sequence analysis. 2. Molecular Taxonomy i. Mole % G + C content DNA contains four nitrogenous bases: adenine (A), guanine (G), cytosine (C), and thymine (T). In double stranded DNA, A always pairs with T by two hydrogen bonds and G with C by three hydrogen bonds. The % G + C is preserved by each organism during evolution. In bacteria G + C contents are significantly variable, ranging between 25 and 75%. The variation in same species is less than 2.5%. Identical G + C value does not prove taxonomic identity. However, differences in G + C could greatly differ in primary structure of chromosomal DNA. GC content is usually expressed as a percentage value, but sometimes as a ratio (called G+C ratio or GC-ratio). GC-content percentage is calculated as: GC  100 A T  G  C

whereas the AT/GC ratio is calculated as AT GC

It is measured by the following different methods: 1. Determination of the DNA density in cesium chloride density gradient by isopycnic ultracentrifugation. Under proper conditions, DNA density is proportional to G + C content. It does not give precise results. 2. Determination of melting temperature (Tm). G + C contents of some microorganisms are as follows (Table 33.2). ii. DNA – DNA Hybridization The quantitative measurement of DNA-DNA hybridization of two different species is used to find out the correlation between them. The DNA hybridization technique takes advantage of the fact that heat will cause a DNA double helix to separate into two identical strands of DNA. Then the DNA solution is allowed to cool. This will allow the DNA to reform (reanneal) into double helices again. If the DNA from two different organisms is put together for hybridization, the total amount of reannealing achieved will depend on how similar the DNA sequences of two

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organisms are. More the similarity in DNA sequences more will be the annealing and similarity in DNA sequence in turn is dependent on how closely related the two organisms are evolutionarily. Table 33.2. G + C content of some bacteria and protozoa. Microorganisms

% G+C

Bacteria Actinomyces

59–73

Bacillus

32–62

Clostridium

21–54

Escherichia

48–52

Mycobacterium

62–70

Mycoplasma

23–40

Pseudomonas

58–70

Protozoa Amoeba proteus

66

Paramecium

29–39

Tetrahymena

19–33

Trypanosoma

45–59

Ribotyping The coding sequences for rRNA are mostly conserved in the course of evolution. The 16S rRNA sequencing is primarily used, as it is more stable than 5S rRNA and easier to sequence than 23S rRNA. In oligonucleotide cataloguing method, 16S rRNA is obtained after hydrolysis by T1 nucleases (isolated from Aspergillus oryzae). The T1 digested extract is then subjected for PAGE to separate the 16S rRNA. For identification a primer that is complementary to conserved sequences of the 16S rRNA is used. Recently, sequencing methods are followed after polymerase chain reaction (PCR) for the amplification of 16S rRNA sequence. This technique is of importance for identifying the taxonomic position of non-cultivable microorganisms Diversity of prokaryotes Bergey’s Manual of Determinative Bacteriology (1974) classified the bacteria into 19 groups. However, it had not included the Cyanobacteria and the Archaebacteria since their establishment as separate entities were not known at the time of publication. The eubacteria was arranged according to their shape, Gram reaction and relationship to oxygen. The 19 groups in Bergey’s Manual with the addition of Cyanobacteria as group 20 in the 8th edition of the manual is given below:

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1.

Phototrophic bacteria (Anaerobic - Rhodospirillum, Rhodopseudomonas, Chromatium, Chlorobium).

2.

Gliding bacteria (Myxococcus, Beggiatoa, Simonsiella, Leucothrix).

3.

Sheathed bacteria (Sphaerotilus, Leptothrix).

4.

Budding (appendaged) bacteria (Caulobacter, Gallionella).

5.

Spirochaetes (Spirochaeta, Treponema, Borrelia).

6.

Spiral bacteria (curved bacteria) (Spirillum, Aquaspirillum, Oceanospirillum, Bdellovibriyo).

7.

Gram negative aerobic rods and cocci (Pseudomonas, Xanthomonas, Zoogloea, Gluconobacter, Azotobacter, Rhizobium, Agrobacterium, Halobacterium, Acetobacter).

8.

Gram negative facultative anaerobic rods (Escherichia, Citrobacter, Salmonella, Shigella, Klebsiella, Enterobacter, Serratia, Proteus, Yersinia, Erwinia, Vibrio, Aeromonas, Zymomonas, Chromobacterium, Flavobacterium).

9.

Gram negative anaerobes (Bacteroides, Fusobacterium, Desulfovibrio, Succinimonas).

10.

Gram negative Paracoccus).

11.

Gram negative anaerobic cocci (Veillonella, acidaminococcus).

12.

Gram negative chemolithotrophic bacteria (Nitrobacter, Thiobacillus, Siderocapsa).

13.

Methanogens (strict anaerobes - Methanobacterium, Methanothermus, Methanosarcina, Methanothrix, Methanococcus) and other Archaebacteria (aerobes - Halobacterium, Halococcus, Sulfolobus, Thermoplasma; anaerobes- Thermoproteus, Pyrodictum, Desulfurococcus).

14.

Gram positive cocci (Micrococcus, Staphylococcus, Streptococcus, Leuconostoc, Pediococcus, Aerococcus, Peptococcus, Ruminococcus, Sarcina).

15.

Endospore producing Sporosarcina).

16.

Gram positive non-sporing rods and cocci (Lactobacillus, Listeria, Erysipelothrix, Caryophanon).

17.

Actinomycetes and related forms (Corynebacterium, Arthobacter, Brevibacterium, Cellulomonas, Kurthia, Propionibacterium, Eubacterium, Actinomyces, Archina, Bifidiobacterium, Rothia, Mycobacterium, Frankia, Streptosporangia, Nocardia, Streptomyces, Streptoverticillium, Micromonospora).

18.

Rickettsias (Rickettsia, Erhlichia, Wollbachia, Bartonella, Chlamydia).

19.

Mycoplasmas (Rickettsias, Acoleplasma, Thermoplasma, Spiroplasma).

cocci

(Neisseria,

rods

and

Branhamella,

cocci

(Bacillus,

Azospirillum,

Acinetobacter,

Clostridium,

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20.

Cyanobacteria (Synechococcus, Gloeocapsa, Pleurocapsa, Gloeobacter Gloeothece, Dermocapsa, Myxosarcina, Oscillatoria, Spirulina, Lyngbya, Phormidium, Plectonema, Anabaena, Nostoc, Calothrix, Fischerella).

Since 1984, Bergey’s Manual of Determinative Bacteriology was changed and presently is Bergey’s Manual of Systematic Bacteriology. All bacteria were classified and published in 4 volumes. They were classified under prokaryotas with 4 extensive volumes as given below: Volume 1- Gram negative ordinary chemoheterotrophic eubacteria (11 classes) Volume 2- Gram positive ordinary chemoheterotrophic eubacteria (6 classes) Volume 3- Bacteria with unusual properties (9 classes) –like phototrophs, enveloped, budding, gliding types. Volume 4- Gram positive filamentous bacteria of complex morphology (4 classes) - actinomycetes and related bacteria The present volumes differ from previous volumes in that many higher taxa are not defined in terms of phenotype, but mainly on 16S phylogeny as is the case of the classes within proteobacteria below: Stanier et al. (1987) classified bacteria mainly into two groups, archaebacteria and eubacteria based on the presence and absence of muramic acid in cell wall. Archaebacteria – Muramic acid is absent in the cell wall Eubacteria - Muramic acid is present in the cell wall 1.

Archaebacteria- Methanogens; Thermoacidophiles

Halophilic;

Thermoplasma

type;

2.

Eubacteria- Gram positive; Purple bacteria; Spirochaetes; Bacterioid; Cytophaga type; Cyanobacteria; Green sulphur bacteria; Green nonsulphur bacteria; Sulphur reducers and myxobacteria; radio-resistant micrococci; Planctomyces.

The current classification is: Volume 1 (2001) – The Archaea and the deeply branching and phototrophic bacteria. Volume 2 (2005) – The proteobacteria distributed in 3 books (Introductory essays; The Gamma proteobacteria and other classes of Proteobacteria. Volume 3 (2009) – The Firmicutes. Volume 4 (2011) – The Bacteroides, Spirochaetes, Tenericutes (Mollicutes), Acidobacteria, Fibrobacteres, Fusobacteria, Dictyoglomi, Gemmatimonadetes, Lentisphaerae, Verrucomicrobia, Chlamydiae and Plancomycetes. Volume 5 (2012) – The Actinobacteria Kingdom: Prokaryotae (Divisions, orders, families, genera)

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461

Division I Gracilicutes — Prokaryotes with thinner cell walls, ordinary Gram negative bacteria The Spirochetes : Order : Spirochaetales Spirochaetaceae Spirochaeta

harmless inhabitants of water, mud and sediments

Cristispira

harmless parasites of molluscs

Treponema

inhabitants of mouth, intestinal and genital areas of human and animals, some are pathogenic, T. Pallidum causes syphilis in man

Borelia

parasites of wild rodents

Leptospiraceae Leptospira

harmless inhabitants of fresh water environments, L. interogans causes leptospirosis

Aerobic/microaerophilic, motile helical/vibrioid bacteria. Aquaspirillum

harmless saprophytes in streams and ponds.

Azospirillum

Nitrogen-fixing bacteria

Oceanospirillum

harmless saprophytes of main water.

Campylobacter

inhabitants of intestines, oral cavity and reproductive organs.

Bdellovibrio

parasite on Gram negative bacteria

Non motile (or rarely motile) curved bacteria Spirosoma

yellow pigmented

Runella

pink pigmented

Flectobacillus

pink pigmented

Microcystis

intra cellular gas vacuoles present

Aerobic rods and cocci Pseudomonadaceae Pseudomonas

inhabitants of soil and water, some are pathogenic to plants, animals and man.

Xanthomanas

plant pathogens (citrus canker, rice leaf blight).

Zoogloea

inhabitant of sewage treatment plants

Azotobacteraceae

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Azotobacter

free-living nitrogen fixer

Rhizobiaceae Rhizobium

symbiotic nodule bacteria of legumes

Bradyrhizobium

symbiotic nodule bacteria of legumes

Agrobacterium

plant pathogens causing gall and tumors.

Methylococcaceae Methylococcus

obligate methane oxidizers

Methylomonas

obligate methane oxidizers

Acetobacteraceae Acetobacter

acetic acid (vinegar) producers

Gluconobacter

sorbose, gluconic acid producer

Legionellaceae Legionella

inhabitant of thermally polluted water in air conditioning cooling towers

Nisseriaceae Nisseria

pathogenic to humans (gonorrhea and meningitis)

Facultatively anaerobic bacteria Enterobacteriaceae Escherichia

occur in colon of warm blooded animals.

Shigella

causes bacillary dysentery in human.

Salmonella

causes typhoid and paratyphoid, salmonellosis

Enterobacter

occur in sewage, meat etc.

Erwinia

soft rot of vegetables

Serratia

occur in soil, water, plant surfaces opportunistic pathogen

Proteus

occur in intestine of human, animals; opportunistic pathogen

Yersinia

causative agent of plague is Y. pestis.

Vibrionaceae Vibrio

aquatic habitat, V. cholorae causes cholera.

Aeromnas

aquatic habitat A. salmonicida causes furunculosis in Salmon fish.

Pasteurella

parasitic on mucous membranes of upper respiratory tract of mammals.

Haemophilus

H. influenzae causes meningitis in children.

Actinobacillus

occasionally pathogenic to man.

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463

Genera not assigned to any family Zymomonas

Ferments glucose to ethanol

Chromobacterium

Saprophyte of soil and water, infection to human and animals.

Gardnerella

G. vaginalis causes non-specific vaginitis.

Streptobacillus

A rat parasite (S. moniliformis) causes rat bite fever in humans.

Anaerobic curved helical rods Bacteroidaceae Bacteroides

anaerobic; B. fragilis is associated with soft tissue infections

Fusobaterium Succinomonas Wolinella Selenomonas Actinovibrio Dissimilatory sulphate or sulphur reducing bacteria Desulfuromonas

utilizes elemental sulfur

Desulfovibrio

use sulphate, thiosulphate

Desulfococcus

use sulphate, thiosulphate

Anaerobic cocci Veillonellaceae Veillionella, Acidaminococcus, Megasphaera inhabitants of oral cavity, respiratory tract, intestinal tract of humans, ruminants. rodents and pigs The Rickettsiales Rickettsiales Rickettsiaceae Rickettsia Rochalima Bartonellaceae Bartonella Anaplasmataceae Chlamydiales Chlamydiaceae

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Chlamydia, C. trachomatis causes trachoma and keratoconjuncivitis The Mycoplasmas Class Mollicutes Order: Mycoplasmatales Mycoplasmataceae Mycoplasma

M. pneumoniae causes primary atypical pneumonia in humans

Spiroplasmataceae

Urethritis in human and pneumonia in cattle.

Spiroplasma

causes plant diseases.

Endosymbionts Endosymbionts of protozoa, ciliate, flagellates, amoeba Endosynbionts of insects Endosymbionts of fungi and invertebrates other than Arthropods. Division II Firmicutes — Prokaryotes with thick and strong wall – Gram positive Grampositive cocci Aerobic/Facultatively Anaerobic cocci Deinococcaceae Deinococcus

D. radiodurans is a spoilage agent in radiated foods.

Micrococcaceae Micrococcus

harmless saprophytes of soil; found in skin of human and animals.

Planococcus

harmless saprophytes of soil and marine environments. Also found in skin of human and animals.

Staphylococcus

parasites on the skin and mucous membranes of human and warm blooded animals.

Aerotolerant fermentative cocci Streptococus

most are parasites of human and animals some ferment sugars to lactic acid

Leuconostoc

harmless saprophytes; form lactic acid, used in butter and cheese

Pediococcus

saprophytes; form lactic acid

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Anaerobic cocci Peptococcus

occur in mud, intestines, respiratory tract

Peptostreptococcus occur in human clinical specimens Ruminococcus

occur in bovine rumen

Caprococcus

occur in human feces

Sarcina

occur in soil, cereal grain, diseased human stomach.

Endospore forming rods / cocci Bacillus

most species are harmless saprophytes of soil and water; B. anthracis causes anthrax of cattle. B. thuringiensis is used as biopesticide to kill insects

Sporosarcina

soil inhabitant

Anaerobic spore forming rods Clostridium

distributed in soil, water and sediments botulism and tetanus are caused by species

Desulfotomaculum

occur in soil, water, intestines of insects

Non spore forming rods Lactobacillus

saprophytes in fermenting plant and animal products or parasites of mouth and intestines of warm blooded animal.

Listeria

L. monocytogenes pathogen of animals and humans, causes meningitis in adults; pre and post- natal disease in infants.

Erysipelothrix

parasites of mammals, birds, fish, causes erysipelas in swine, erysipeloid in humans.

Brocothrix

saprophytes of meat and meat products.

Renibacterium

parasites of salmonid fishes; cause a kidney disease.

Kurthia

harmless saprophytes in meat, meat products and animal dung.

Caryophanan

saprophytes of ruminant dung.

Non spore forming irregular shapes Aerobic / Facultatively anaerobic Non filamentous rods Corynebacterium saprophytes of water, parasites of humans, plant pathogens. C. diphtheria causes diphtheria in humans.

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Arthrobacter

saprophytes of soil

Brevibacterium

salt tolerant B. linens

Microbacterium

saprophytes in milk and dairy products

Cellulomonas

cellulose degraders

Aerobic/Facultatively anaerobic branched filamentous rods Agromyces

soil saprophytes

Arachnia

pathogenic to human and animals, causes actinomycoses

Rothia

normal inhabitant of human mouth.

Anaerobic nonfilamentous rods Propionibacterium occur in dairy products, human skin, intestines Eubacterium

occur in human oral cavity, intestines of human and animals.

Actinomyces

occur in oral cavity of human and animals; A.bovis causes actinomycosis in cattle.

Bifiidibacterium

occur in intestines of human and animals.

The Mycobacteria Mycobacterium

pathogens, causes leprosy, tuberculosis

The Nocardioforms Nocardia

saprophytes of soil and water opportunistic pathogen causing nocardiosis and actinomycetoma in humans and animals.

Psedudonocardia

occur in soil and manures.

Division III Tenericutes — Anoxygenic phototrophic bacteria Rhodospirillales Rhodospirillaceae (purple non sulfur-bacteria) Rhodospirillum, Rhodospseudomonas, Rhodomicrobium Chromatiaceae (purple non sulfur-bacteria) Chromatium, Thiocystis, Thiospirillum, Lamprocystis, Thiosarcina, Thiopedia Chlorobiaceae (Green sulphur bacteria ) Chlorobium, Prosthecochloris

Bacteria

467

Oxygenic phototrophic bacteria (Cyanobacteria or Blue Green algae) Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales, Stigonematales, Prochlorales, Prochloraceae, Prochloron Unicellular organisms containing chlorophyll b Prochlorothrix Gliding fruiting bacteria Myxobacterales (degrade cellulose, agar, chitin) Stigmatella, Chondromyces Gliding non -fruiting bacteria Sporocytophaga

forms myxospores without fruiting bodies

Capnocytophaga

occur in oral cavity of humans

Beggiatoa

aquatic environments with H2S

Cytophaga

cellulolytic organism

Herpetosiphon forms sheath Flexithrix

forms sheath

Flexibacter, Vitreoscilla, Simonsiella, Saprospira, Thiothrix The Sheathed bacteria Sphaerotilus

Sheath surrounds a chain of cells of trichome; iron deposited on sheath.

Leptothrix, Haliscomenobacter, Streptothrix, Lieskeella, Phragmidiothrix, Crenothrix, Clonothrix Budding or Appendaged bacteria i. Prosthecate Budding bacteria Hyphomicrobium

soil and aquatic environments

Anclomicrobium

aquatic bacteria form 3-8 prosthecae per cell, buds arise from cell

ii. Prosthecate non- budding bacteria Caulobacter

occur in salt water and fresh water

iii. Non prosthecate budding bacteria Blastocaulis Planctomyces

occurs in all aquatic habitats

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iv. Non prosthecate non budding bacteria Gallinonella

causes clogging in pipelines of water system

Aerobic chemolithotrophic bacteria Nitrobacteraceae Nitrate oxidizing bacteria Nitrobacter, Nitrococcus, Nitrospira Ammonia oxidizers Nitrosomonas, Nitrosococcus, Nitrosovibrio, Nitrosolobus Sulphur and sulphur compounds metabolizing bacteria Thiobacillus, Thiomicroscopira

occur in soil, water and coal mine drains

Thiobacterium, Macromonas, Thiovulum, Achromatium, Thiospira Iron or manganese oxidizers (deposition of iron or manganese oxides on slime or capsules) Siderocapsa, Siderococcus, Siderocystis, Naumanniella Archaebacteria Methanogenic archaebacteria (Methane producers) Methanobacteriales Methanobacteriaaceae Methanobacterium, Methenobrevibacter, Methanomicrobium, Methanogenium Methanothermaceae Methanococcales Methanococcaceae Methanococcus Methanomicrobiales Methanomicrobiaceae Methanomicrobium Methanosarcinaceae Methanosarcina, Methanolobus Archaebacterial sulphate Reducers Archaeoglobales Archaeglobaceae Archaeoglobus Extremely halophytic archaeobacteria (require 17-23% NaCl for growth. The cells lyse when NaCl falls below 10%) Halobacteriales

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469

Halobacteriaceae Halobacterium, Halococcus, Halomonas Thermoacidophiles Cell wall-less archaebacteria Thermoplasma grows at pH 2 and optimum temperature is 55-59ºC. Cells lyse at neutral pH. Extremely thermophilic sulphate-metabolizers Thermococcales Thermococcaceae Thermococcus, Thermaproteales, Thermoproteaceae, Thermoproteus Desulfurococcaceae Desulfurococcus Sulfolobales Sulfolobaceae Sulfolobus optimum pH is 2; temperature is 70-87°C Division IV Mendosicutes Gram positive filamentous bacteria of complex nature. Filamentous bacteria dividing in more than one plane Dermatophilus

D. congolensis is a parasite of mammals causing infection.

Frankia

Actinorhizal nodulating organism in Casuarina and Alnus.

Filamentous bacteria forming true sporangia (occur in dead plant parts, shed animal hair and soil) Streptomyces

decomposes organic matter. Streptomycin as antibiotic producer

Actinoplanes, Ampullariella, Spirillospora, Streptoverticillium, Actinopycnidium, Actinosporangium, Chainia, Elytrosporangium, Kitasatua, Microcellobiosporia Filamentous bacteria of uncertain taxonomic placement Actinomadura

soil saprophytes

Nocardiopsis

soil saprophytes

Actinopolyspora

extreme halophilosm is seen

Actinosynnema

compact hypha synnemata

Thermomonospora occurs in compost. Thermophilic and cellulolytic

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Biology and Biotechnology of Fungi and Microbes

Thermoactinomyces occur in damp hay, composts and moist grain. REPRODUCTION IN BACTERIA Bacteria reproduce by asexual and sexual methods. Asexual Reproduction Asexual reproduction is the common method of reproduction in bacteria which takes place by binary fission, budding, segmentation and endospores (Fig. 33.11).

Fig. 33.11. Binary fission (1) Bacterial cell (2) Nuclear material

Sexual Reproduction Sexual reproduction is defined as any process in which genetic material is exchanged (Fig. 33.12). Conjugation, transformation and transduction are three ways by which genetic material is exchanged in bacteria. This process contributes towards maintaining genetic variability amongst bacteria.

Fig. 33.12. Replication of bacterial chromosome

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471

Genetic recombinations Three types of bacterial genetic recombinations have been recognized for the transfer of DNA from donor cell into recipient (bacterial) cell with the recipient bacterial chromosome or plasmid in vivo. They are homologous recombination, sitespecific recombination and non-homologous recombination. Homologous recombination: This comprises the mechanism by which DNA that has been transferred into the recipient cell recombines with the host DNA by reciprocal exchange of DNA fragments. The recombined partners must possess more or less same base sequences. That exhibit maximum homology, except any mutational differences. Homologous recombination is under the control of rec A gene, mutants with a defect in this gene i.e. rec- that has lost the capacity of homologous recombination Site-specific recombination: In this foreign DNA is integrated into a particular site in the host genome. Specific site of a large double-stranded DNA. The small stranded partner loses its activity. It is independent of homologous recombination and occurs in rec- Mutants. It is a process of integration of small, double-stranded DNA segment. One example of the site-specific recombination is the integration of the bacteriophage λ. Non-homologous recombination: Recombination events between DNA segments without recognizable genetic homology are designated as non-homologous recombinations. It is an integrated form of recombination, similar to that of site-specific recombination process, involving addition of DNA instead of exchange. Non-homologous recombination is independent of rec A. The following types of DNA are able to involve in such recombinations: 1. insertion sequences (IS elements), 2. transposons (Tn elements) and 3. bacteriophage Mu. Transposons These are DNA sequences that can be integrated into the genome at number of sites not randomly. They can integrate from a plasmid to a bacterial chromosome to another plasmid or to a temperate phage. Transposons contain gene that determine recognizable characters like resistance to antibiotics and more easily recognized than IS sequences. The resistance genes in a transposon are flanked by two DNA segments with repeated base sequences in the same direction or inverted. In some transposons the inverted repeats are almost identical to known IS elements. Conjugation The transfer of genetic material from cell to cell by direct contact is known as conjugation. Lederberg and Tatum (1946) carried out experiments with two mutants of E. coli K12, each of which was auxotrophic for two different aminoacids (A and

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B). They could synthesize amino acids (C and D) {A-B-C+ D+}. The other mutant was complimentary (A+ B+ C- D-). The mutants cannot grow in minimal medium i.e. one lacking amino acids. However, mixed inoculums of both mutants on the same minimal medium produced colonies. The cells of the colonies had the hereditary ability to synthesize all the four amino acids and were of genetic type A+B+C+D+ (prototypes). They are formed at a frequency of one in one million and were called recombinants as they combined the genetic information of two reciprocally defective mutants. The use of multiple mutants in the parental organisms precludes the probability that the colonies were revertants. The possibility of simultaneous reverse mutation in two genes is of the order of 1014 – 1018 per generation. This recombination presupposes direct contact between the parental cells. In 1950 Davis supported this by his U-tube experiment wherein two mating strains were separated by sintered glass. He showed that no recombinants were produced under these conditions. Since cell contact is essential, this process of reproduction is known as conjugation. Conjugation in bacteria involves the transfer of DNA from one cell (male or donor) to another (female or recipient) (Fig. 33.13). The process of conjugation has been thoroughly studied in E. coli. A male (donor) cell contains a small, circular, piece of double stranded DNA (plasmid) in addition to the bacterial chromosome. The plasmid may lie free in the cytoplasm or get integrated with the bacterial chromosome. One well studied plasmid is F plasmid or F factor (F for fertility). F-plasmid carries genes that code for donor characteristics like sex pili. A cell which contains F plasmid is designated F+ (donor or male) while a cell which does not contain F+ plasmid is designated F– (recipient or female). When F+ and F– cells are mixed, initial contact is made through sex pili. This is followed by cell-to-cell contact. One strand F+ plasmid is transferred from the donor to the recipient cell. DNA replication occurs in both cells and the F plasmid becomes double stranded. In this process F+ cell becomes F+ or F+ cell becomes F- cell by acquiring an F– plasmid from the donor.

Fig. 33.13. Conjugation in bacteria; 1. Donor cell, 2. Conjugation bridge, 3. Recipient cell

Bacteria

473

F factor The donor state in bacteria is dependent on the presence of transferable DNA element in the cell, the sex factor F (fertility). Cells that lack F factor (F- ) can only serve as recipients. The F factor is supposed to be transferred during conjugation. Hence the recipient cells are converted to potential donor cells during conjugation. However, transfer of F factor does not necessarily involve the transfer of chromosomal characters. The F factor is classified as a plasmid (an extra chromosomal, autonomously replicated DNA element). It contains the genes responsible for the process of conjugation. The genes determine special structures on the cell surfaces, such as the F pili which are essential for conjugation. They probably facilitate the formation of conjugation bridge by which the DNA migrates into the recipient cell during pairing. Only the F factor has been integrated into bacterial chromosomes. When clones of such donor cells are used in conjugation process, the yield of recombinants is about 1000 fold higher than the ordinary F+ strains. Such cells are called Hfr (High frequency of recombinants). The integration of F factors occurs at a limited number of specific sites on bacterial chromosomes and is comparable to integration of λ phage into the host chromosome. When a population of Hfr cells mixed with an excess of F- cells, practically all the Hfr cells find F- partners and conjugate.The recombinants were examined to determine which gene had been transferred from the donor to the recipient. The analysis showed a definite time of transfer from donor to recipient for each gene. The time course of gene transfer was in agreement with the chromosomal gene sequence that had been established by genetic analysis. This advocates that a given strain of Hfr cells consists of a homogenous population in which each cell transfers in chromosome from the same origin and in the same direction. This time of transfer of each gene depends on its distance from the origin and those farthest from the origin are transferred least frequently in as undisturbed conjugation with F- recipient cells. The transfer of the complete chromosome of E. coli takes about 100 minutes at 370C. It is clear from this that the use of uninterrupted mating experiments allows the construction of gene map. The integration of F factor into the bacterial chromosome is reversible. The F factor can be excised from the chromosome so that the Hfr cell reverts to a F+ cell. This excision occurs at about the same frequency as integration. Correct excision depends on a break occurring at the same site on the chromosome as the integration site. In certain cases the break occurs at a neighbouring site and excision of a neighbouring segment of DNA remains attached to the F factor. Such an F factor containing a small piece of chromosomal DNA is called F’ factor. The origin of a F’ factor is analogous to the formation of a specific transducing phage. Transduction: Transduction is the transfer of DNA from a donor cell to a recipient cell by bacteriophages. In majority of cases only a small segment of the host DNA is transferred. Two kinds of transduction are reported. A non-specific (generalized) transduction which can transfer any part of the host DNA and a specific transduction

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Biology and Biotechnology of Fungi and Microbes

that is restricted to the transfer of specific DNA segments. In non-specific transduction the host DNA segment is integrated into the virus particle, either in addition to or in place of some of the phage genome. In specific transduction some of the phage genes are replaced by the host genes. In both the cases, the transducing phages are usually defective in some respect for e.g. they often lose the ability to lyse host cells. The transfer of genetic characters by transduction has been demonstrated in a large number of bacteria, but not all phages transduce nor can DNA be transferred by transduction in all bacteria (Fig. 33.14).

Fig. 33.14. General transduction Special transduction

Specific transduction The best example of specific transduction is phage λ. Lambda generally transduces specific genes of the gal and bio operons. It integrates into the host genome during its transition to prophage stage, specifically between gal and bio operons site. When a transducing phage infects a recipient cell with a defect in the appropriate gene, such as gal-, the intact transduced gene can exchange with the defective host gene and the resulting recombinants (tansductants) are then gal+. Large numbers of bacteria, but not all phages transduce nor can DNA be transferred by transduction in all bacteria. The transfer of bacterial genes by phage was discovered by Lederberg and Zinder in 1951. In their experiment a donor strain B+ was infected with the temperate phage P22. After lysis of host cells, the liberated phages were separated and incubated with a suspension of the recipient strain B- which was distinguished from B+ by at least one genetic character. By plating out on a selective media they found recombinants that carried the genetic marker of the donor from B+. The process that occur during the non-specific DNA transfer are rather complex. Gene transfer mediated by phage φ 80 is similar. Its DNA integrates in the neighbourhood of genes coding for tryptophan biosynthesis. Phage φ80 is particularly suitable for transfer of trp genes. Integration of phage into the host chromosome is essential for successful gene transfer by specific transduction as against the non-specific transduction (Fig. 33.15).

Bacteria

475

Fig. 33.15. Transformation of bacterial cell. A. Virulent cell; B. Avirulent cell; C. Host killed virulent cell; D. Entry of donor DNA; E. avirulent cell; F. Transformation cell; 1: Capsule; 2. DNA; 3: S gene; 4: R gene; 5. Rough wall; 6: DNA fragment

Transformation: Genes can be transferred even without cellular contact or vectors which has been extracted or otherwise. Such gene transfer by dissolved DNA which has been extracted or otherwise liberated from a donor bacterium to a recipient bacterium is called transformation. This is a well known and historically most important kind of gene transfer in bacteria (Fig. 33.15). Griffith (1928) discovered the transformation of a non-capsulated R strain of Streptococcus pneumonia into capsulated S strain. He injected mice with a small number of avirulent R cells together with heat-killed S cells. The R cells were derived from another S strain (S II) whose capsular material could be distinguished serologically from that of the heat-killed S strain (S III). Virulent cocci with capsules of the S III type could subsequently be isolated from the mice. It appeared, therefore, that the heat-killed S III cells had transferred their genetic character for capsule formation to the R cells which in turn were able to pass it on to their progeny. Subsequently Avery, Macleod and McCarty (1944) established that the transforming principle consisted of DNA. This transformation provided the decisive proof at the time for the localization of genetic information in DNA and not in protein. The potential for transformation is restricted to bacteria that are able to take up high molecular weight, double- stranded intact DNA. This ability is considered as competence. Although competent bacteria can take up any DNA, only the DNA of closely related species can lead to recombinations namely by exchange of homologous DNA fragments. Stalked bacteria There are several groups of Gram-negative, unicellular eubacteria which have the distinctive property of forming stalks (e.g. Gallionella).

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Budding bacteria A small number of unicellular eubacteria reproduce by budding instead of by the commoner method of transverse fission. One of the best-known budding bacteria is Hyphomonas, a photosynthetic organism that is metabolically similar to the simple unicellular purple bacteria (Fig. 33.16).

Fig. 33.16 Budding bacterium (Hyphomonas polymorpha)

Sheathed bacteria The best-known filamentous eubacteria are Sphaerotilus and Caryophanon. Sphaerotilus grows as a chain of cells that are surrounded by and enclosed in a delicate, smooth-walled sheath. Reproduction occurs by the liberation from the open end of the sheath of polarly flagellated swarmers. The negative Gram reaction and polar flagellation of Sphaerotilus suggest that it is related to the simple unicellular pseudomonads. BIOTECHNOLOGY OF BACTERIA Role of bacteria in industry Microorganisms have been associated with man for their mutual benefit. For example, yeasts have been known to be involved with man since time immemorial and have served a steady and useful purpose. They are involved in the fermentation processes of fruit juices, wine making, brewing and bread making etc. Pasteur showed that the process of grape juice fermentation was the result of microbial activity and the organism involved in bringing this desirable change in grape juice was identified as yeast. Pasteur’s work showed that fermentations characterized by the formation of particular products such as alcohol, acetic acid, lactic acid, butyric acid etc. were dependent on the activities of different kinds of microorganisms. This recognition of the ability of microorganisms to bring about desirable chemical changes in appreciable amounts led to the development of industrial microbiology and to the establishment of several industries (Table 33.3). Three important characteristics that have made microbes useful in industry are: 1. their ability to grow rapidly using easily available and cheap raw materials, 2. their ability to maintain physiological constancy and, 3. their ability to bring about biochemical transformations under simple cultural conditions. These properties have made the microorganisms highly preferable over synthetic processes. The wine and

Bacteria

477

beer making industries and the baking industry are mainly dependent on the activities of the microorganisms. Acetone, butyl alcohol, lactic acid, acetic acid and many other industrial products such as enzymes, vitamins, hormones etc. are manufactured with the help of microorganisms. Antibiotics are invariably manufactured with the help of the microorganisms. Table 33.3. Some industrial products from Bacillus species. Product

Application

Bacillus species

Purine nucleotides

Flavour enhancers’ medicine

B. subtilis

Riboflavin

Vitamin ingredient for health food

B. subtilis

D-Ribose

Flavour enhancer in food, health food, pharmaceuticals, cosmetics

B. subtilis, B. pumilus

Thaumatin

Sweet-tasting protein for applications in food and pharmaceuticals

B. subtilis

Polyhydroxybutyrate

Biodegradable plastics

B. megaterium

2 Acetyl-l-pyrroline

Popcorn, corn chip aroma, and flavour for B. cereus food

Poly--glutamic acid

Biopesticide, insecticide

B. subtilis

Endotoxins

Biopesticide, insecticide

B. thuringiensis, B. spharicus, B. popilliae

Streptavidin

Biotin-binding protein, applications in high density biochips

B. cereus

Organic Acids 1. Lactic Acid: There are numerous microbial species capable of producing large quantities of lactic acid. These include Lactobacillus bulgaricus, L. delbrueckii, L. leichmannii, L. casei, L. pentosus, Streptococcus lactis and Rhizopus oryzae. Lactic acid is largely used in pharmaceutical industries (e.g., Iron lactate) and in some plastic industries. It is also used in processing of food and in chemical industries. 2. Acetic Acid (Vinegar): The term ‘Vinegar’ has originated from the French word vinaigre (sour wine). Vinegar (acetic acid) is obtained by the activity of acetic acid bacteria (Acetobacter aceti). This bacterium oxidizes ethyl alcohol obtained from molasses by fermentation to acetic acid or vinegar. Acetobacter acetigenum and A. pasteurianum are the species for the production of acetic acid. Vinegar is produced by the aerobic conversion of ethanol to acetic acid by Acetobacter and Gluconobacter. Vinegar can be made from any alcoholic liquor such as wine or cider. Solvents 1. Acetone–Butanol: Industrial production of acetone butanol by a microbiological process was first started in England by the firm of Strange and

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Graham Ltd. This commercial process was developed just before World War I. Since acetone was demanded during war time (for producing explosives), the acetonebutanol industry was expanded rapidly. In addition to the wide use of acetonebutanol, it is also needed as a solvent for the rapid drying of nitrocellulose paints in the developing automobile industry. Thus, this commercial process of acetonebutanol survived even after a lack of demand for acetone after World War I. Riboflavin (a byproduct of fermentation) also helped to sustain the large scale practicability of the process. Acetone-butanol fermentation by Clostridium acetobutylicum made significant technological contributions to industrial microbiology. This was the first industrial microbiological process where the removal of contaminating microbes from the fermentor was found to be of paramount importance in the success of the process. 2. Butanediol: 2,3-Butanediol (also called diol or 2,3-butylene glycol) is a possible source for 1, 3-butadiene (used in the synthesis of rubber). It is also a source for permanent type antifreeze. Moreover, other compounds, readily derived from this mother compound include methyl ethyl ketone, methyl vinyl carbinol and methyl vinyl ketone. The last two compounds are used in the plastic industry. Majority of bacteria, except Bacillus polymyxa, produce the meso isomer with only traces of the D(-) or L(+) forms. B. polymyxa produces more than 98% of the D (-) form. Serratia marcescens gives little formic acid, and Bacillus subtilis (Ford's type) gives scarcely any glycerol. Generally, the yields of 2,3-butanediol or its oxidation product, acetone, are increased when compared to the other products under aerobic conditions. Oxygen retards ethyl alcohol formation by both Bacillus polymyxa and Aerobacter aerogenes. Amino Acids The bacteria which produce various amino acids are given in Table 33.4. L-Glutamic Acid: A derivative of glutamic acid, mono-sodium glutamate, is used to develop a flavour in food products (e.g. soups). The commercial production of this acid in the world exceeds 1,00,000 tons per annum. Presently, a one-stage process utilizing Corynebacterium glutamicum is mainly adopted to produce Lglutamic acid on a large-scale. Japanese workers have obtained high yielding mutants of this microbe. L-Lysine: L-Lysine (2,6-diaminohexanoic acid) is an essential amino acid for the nutrition of humans. It is used for supplementing cereal proteins lacking this amino acid. Thus, protein quality of certain foods (e.g. wheat-based foods) is improved resulting in an improved growth and tissue synthesis. This amino acid has also been used medically as a nutrient. Kinoshita et al. (1958) first reported the fermentative production of L-lysine using homoserine auxotrophs of Corynebacterium glutamicum. Commercial production of L-lysine has been established using this auxotrophic strain of C. glutamicum.

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Table 33.4. Different amino acids produced by bacteria Amino Acid

Bacteria

L-lysine

Corynebacterium glutamicum

L-threonine

Escherichia coli

L-leucine

C. glutamicum

L-homoserine

C. glutamicum

L-valine

C. glutamicum

L-ornithine

C. glutamicum

L-citrulline

C. glutamicum

L-proline

Serratia marcescens

L-isolecucine

Bacillus subtilis

L-arginine

C. glutamicum

L-histidine

C. glutamicum

L-tyrosine

C. glutamicum

L-phenylalanine

C. glutamicum

L-tryptophan

C. glutamicum

Enzymes Enzymes are used for a variety of purposes. They are employed in three major fields laboratory, industrial and clinical amylases, proteases are important enzyme produced by bacteria. Aspartase produced by E. coli and pullulanase produced by Aerobacter aerogenes. Amylases: Amylases play the most important part in food technology (e.g., bread - making, beer – making, etc.). -Amylases are produced by the use of fungi as well as bacteria (i.e., Bacillus amyloliquefaciens and B. licheniformis). Therefore, ccamylases are called either fungal a-amylases or bacterial -amylases according to the nature of the microbes used for their production. Bacillus licheniformis and B. amyloliquefaciens, secrete extracellular proteins which are used for several gene encoding proteins. Most alkaliphile bacilli produce various alkaline enzymes, including proteases, amylases, xylanases, pullulanases, and cellulases. Proteases: Complex mixtures of true proteinases and peptidases are usually called proteases. The concentrations of the peptidases in the production medium are low, since they are endoenzymes. Proteases, like a-amylases, are produced by bacteria (i.e. Bacillus subtilis and B. licheniformis). Food and dairy Industry The association of microorganisms with food has been known for long and methods of food preservation either by heating or heating and sealing were developed as early as 19th century: Microbes have been known to play several different roles in food industry. The microbial cells may be used as food material or

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food supplement, or their cell components, particularly the enzymes may be used as accessory agents in food industries. Microbes could also be agents of food spoilage. A number of other processes employed in food industries are dependent on microbial fermentations for preservation or for characteristic flavour development. In dairy: Microorganisms have been associated with milk and milk products in dairy industry. Milk is a complete medium for the growth and reproduction of several types of bacteria and in particular, the lactic acid bacteria. The lactic acid bacteria have the capacity to convert milk sugar into lactic acid and this lactic acid increases the acidity of milk leading to curdling of the milk casein. This phenomenon, although known and observed for several years was explained on a scientific basis only after the studies on sour milk by Pasteur. The art of cheese making developed about the same time as that of milk curdling. Cheese making also involves lactic acid bacteria and these are involved both in curdling as well as in bringing about the desirable flavours. In addition to bacteria, some molds are also known to be important in cheese making. Besides microorganisms which help in conversion of milk into various other desirable products, there are many others which cause the deterioration of milk and milk products. There are also many human diseases which are transmitted through milk to humans. In food: Yoghurt is a dairy product prepared by the fermentation of milk. According to the Russian bacteriologist Metchnikov (1908), growth of Lactobacillus bulgaricus in the colon produced a high concentration of lactic acid. This highly acidic environment does not permit the growth of proteolytic flora. Yoghurt is commercially manufactured by the fermentation of milk with two thermophilic bacteria, viz., Streptococcus thermophilus and Lactobacillus bulgaricus. The manufacture of cheese is a microbiological process. There are several hundred varieties of cheese. The particular combination of salt, incubation, temperature, pH and culture used, determine the kind of cheese manufactured. The following organisms may be used as starter culture in the pure or mixed form for curdling the milk: Streptococcus cremoris, S. lactis, S. thermophilus, Lactobacillus lactis, L. helveticus, L. bulgaricus, etc. The ripening process in the manufacture of Swiss cheese is carried out by Lactobacilli, Streptococci, and Propionibacterium. The bacteria are added to the milk prior to the curdling reaction. Lactose of the milk is attacked by Lactobacilli and Streptococci with the formation of lactic acid. Now lactic acid is further fermented by a species of Propionibacterium. The end products of this fermentation are: carbon dioxide, propionic and acetic acids and water. The presence of these two acids develop flavor in the cheese. Cheddar cheese is the most commonly sold variety of American cheeses. It is a hard curd cheese. The organisms, primarily involved, are probably Streptococcus lactis and related Streptococci and various Lactobacilli. Limburger cheese is produced by the action of the lactic acid Streptococci and a number of other bacteria like Brevibacterium linens. Brevibacterium linens is an active proteolytic bacterium and is believed to be the main organism for imparting a characteristic flavor in Limburger cheese.

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Butter is manufactured by churning pasteurized sweet or sour cream. There is a better separation of butter fat globules from the other constituents in the churning process, if sour cream is used. Natural souring of raw cream may take place, since Streptococcus lactis is present. However, no control on flavour is possible because unwanted bacteria, other than S. lactis, may be present. Butter cultures consist of a mixture of two types of microorganisms: (1) Microorganisms, (Streptococcus lactis and S. cremoris) which can attack lactose (milk-sugar) with the formation of lactic acid., and (2) microorganisms which develop, the desired aroma and flavor. Generally a mixture of Leuconostoc dextranicum and L. citrovorum is used. Single–cell proteins: For single-cell protein (SCP) production, microbes such as bacteria, yeasts, filamentous fungi and algae have been successfully employed. Some species or strains of bacteria can be used for producing SCP products. The major groups of interest are: 1.

Hydrogen – utilizing bacteria.

2.

Methane or methanol- utilizing bacteria.

3.

n-paraffins – utilizing bacteria.

4.

Photosynthetic bacteria.

1. Hydrogen-utilizing bacteria: These are aerobic bacteria. They obtain energy by the oxidization of hydrogen. They use CO2 as a source of carbon. The species of interest in this group include: Pseudomonas facilis, P. saccharophila, P. ruhlandii, P. flava, P. palleronii, Alcaligenes eutrophus and A. paradoxus. 2. Methane or methanol-utilizing bacteria: These bacteria are capable of utilizing one-carbon organic compound (i.e., methane and methanol). A number of bacteria utilize methane as a carbon and energy source for growth. These include Methanomonas methanica, M. methanoxidans, Methylococcus capsulatus, Pseudomonas methanica and others. On the other hand, methanol-utilizing bacterial species are: Methylomonas methylovora, M. clara, M. methanolica, Methylophilus (Pseudomonas) Methylotrophus, Xanthomonas spp. and others. In addition to these bacterial species, members of other bacterial genera (e.g. Bacillus, Vibrio etc.) have been reported as utilizing both methane and methanol. 3. n-paraffins-utilizing bacteria: Hydrocarbon-utilizing bacteria include Brevibacterium insectiphilium ATCC 15528, Corynebacterium spp. C. paurometabolum, Pseudomonas ligustri, P. pseudomallei, P. orvilla, Alcaligenes spp., Cellulomonas glaba, and Micrococcus cerigicans. 4. Photosynthetic bacteria: Photosynthetic bacteria may be a potential feed source for aquaculture systems or livestock. A process for biomass production using photosynthetic bacteria as a source of human food has also been developed. Fibre Industry: The separation of fibres from plants such as hemp, jute, by the dissolution of pectin that binds them is called retting. This is achieved by the bacterium Clostridium felsineum. Curing of tea and tobacco: For curing of tea, alcohol is added to crude tea leaves. Bacteria act on them and impart the peculiar taste and flavor for tea. For

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curing and ripening, molasses and alcohol are added to the raw leaves of tobacco. Bacteria act upon them and bring about the peculiar smell and taste. Role of bacteria in medicine The early recognition of the role of microbes in diseases led to the development of techniques for combating these microorganisms. One of the land marks in the history of therapeutic microbiology is the discovery by Fleming in 1928 that the substance produced by the common fungus Penicillium is inhibitory for the growth of certain groups of pathogenic bacteria. The discovery of penicillin was a major breakthrough in medical microbiology and led to the search and development of a wide variety of antibiotics. Microorganisms have practical application in medicine such as immunization against many diseases, the method of sterilization in surgery and the general sanitation followed in the hospitals etc. Antibiotics A chemical compound acting against life is called an antibiotic. It can be obtained either from natural sources (e.g. microbes) or by synthetic method. Antibiotics are widely used in different fields, such as medical, agricultural and others (Table 33.5). Antibiotics, such as tyrothricin, bacitracin, subtilin, polymycin, etc. are obtained from bacterial sources. Table 33.5. Antiobiotics Produced by Bacillus species. Class

Example

Function

Bacillus source

Cyclicoligopeptides

Bacitracin

Inhibit cell wall synthesis

B. licheniformis (Bacitracin A)

Linear or cyclic oligopeptides

Gramicidins and Interfere with tyrocidins Polymixin z, membrane function Colistins, Circulins

Basic peptides

Edines

Inhibit formation of the Brevibacillus brevis initiation complex on the small ribosome subunit

Aminoglycoside antibiotics

Butirosin complex

Affect ribosome function

Brevibacillus brevis, B. polymyxa, B. clistinus, B. circulans

B. circulans

Vitamins Vitamins are very important compounds in the diet because they are of great value in the growth and metabolism of the living cell. All the vitamins may be synthesized by prototrophic microorganisms. Vitamin B12 is an important dietary component for normal growth in human beings and domesticated animals. Its daily requirement for human beings is 0.001 mg/day. Microorganisms that may be employed in the industrial productin of vitamin B12 are Bacillus megaterium, B. cogulans, Pseudomonas denitrificans,

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483

Propionibacterium freudenreichii, P. shermanii, and a mixed fermentation of a Proteus sp. and Pseudomonas sp. Riboflavin (Vitamin B2) is an important vitamin required by man and animals. Vitamin B is obtained due to the rementation of sugars and starches by the action of Clostridium and Acetobacterium. The raw material used in the synthesis of L-ascorbic acid (Vitamin C) is LSorbose. L-Sorbose, in turn, is obtained from D-sorbitol by fermentation reaction. The bacterium used for bringing about bioconversion is Acetobacter suboxydans. Escherichia coli living in the intestine of human beings produce large quantities of vitamin K and Vitamin B complex. Biogeochemical cycles Microorganisms play a major role in biogeochemical cycling of carbon, nitrogen, sulfur and iron. As term suggests, both biological and chemical processes are involved in the cycling. Microorganisms are essential in the transformation of carbon, nitrogen sulfur and metals such as iron. Significant gaseous components occur in the carbon and nitrogen cycles and to a lesser extent, in the sulfur cycle; thus bacteria can often fix atmospheric carbon and nitrogen compounds even when they are lacking in their environment.

Fig. 33.17. Carbon cycle

Carbon cycle Carbon can be present in a variety of forms ranging from simple compounds such as CH4 to more complex macromolecules such as starch, cellulose, lignin, hydrocarbons and microbial biomass. A variety of factors control the rate of breakdown of these substrates; these include the structure of the individual components within the molecule (e.g. the major component in lignin is phenyl propane), the environmental conditions and the microbial community present. Typically, carbohydrate-rich substrates are degraded first with the more complex molecules as lignin being the most recalcitrant (resistant to degradation). The carbon

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Biology and Biotechnology of Fungi and Microbes

cycle can be divided into those processes that occur under anaerobic conditions and those that require oxygen. Organic material is broken down by fermentation or under aerobic conditions by respiration releasing CO2 or forming reduced products such as CH4. The fixation of CO2 for the production of biomass occurs through the activities of Chromatium, Chlorobium and aerobic chemolithoautotrophs (Fig. 33.17). Nitrogen cycle The nitrogen cycle involves several processes. Nitrification the aerobic process of ammonium ion oxidation to nitrite and subsequent nitrite oxidation to nitrate. Nitrosomonas and Nitrosococci, for example, play significant roles in the first step, and Nitrobacter and related chemolithoautotrophic organisms carry out the second step. Heterotrophic nitrification by bacteria and fungi contributes significantly to these processes in more acidic environments. The process of denitrification is a dissimilatory process performed by heterotrophs such as Pseudomonas denitrificans. The major products of denitrification include nitrogen gas and nitrous oxide. Nitrogen assimilation occurs when inorganic nitrogen is used as a nutrient and incorporated into new microbial biomass. Nitrogen fixation can be carried out by aerobic or anaerobic bacteria. Under aerobic conditions, bacteria such as Azotobacter and Azospirilium contribute to this process. Under anaerobic conditions, the most important nitrogen fixers are members of the genus Clostridium. Nitrogen fixation can also occur through the activities of bacteria that develop symbiotic association with plants (Fig. 33.18).

Fig. 33.18. Nitrogen cycle

Sulfur cycle Microorganisms contribute greatly to the sulfur cycle (Fig. 33.19). Photosynthetic microorganisms transform sulfur by using sulfide as an electron source. In the absence of light, sulfide can be used by Thiobacillus and other lithoautotrophs. In contrast sulfate can undergo sulfate reduction when organic reductions are present. Desulfovibrio can derive energy by using sulfate as an oxidant under these conditions. Dissimilatory reduction occurs when sulfate is used as an external electron acceptor (anaerobic respiration) to form sulfide. In comparison, the

Bacteria

485

reduction of sulfate for amino acid and protein biosynthesis is described as assimilatory reduction. When pH and oxidation-reduction conditions are favorable, several key transformations in the sulfur cycle also occur. As a result of chemical reactions. external electron acceptor (anaerobic respiration) form sulfide which accumulate at the expanse of oxidized form of sulphur. The most active agent in sulphate reduction is the bacterium Desulfovibrio desulfurians. In comparison, the reduction of sulfate for amino acid and protein biosynthesis is described as assimilatory process.

Fig. 33.19. Sulphur cycle

Biofertilizers Biofertilizers are the products containing living cells of different types of microorganisms that have an ability to mobilize nutrients from non-usable to usable from through biological processes. These broadly include nitrogen fixers (both symbiotic as well as non-symbiotic bacteria), phosphate solubilizing bacteria and fungi and mycorrhizal fungi, capable of mobilizing unavailable nutrients and transporting them to and across the plant roots. On global basis biological nitrogen fixation (BNF) is estimated to contribute 175-180 million metric tons/year globally. About eighty percent of BNF comes from symbiotic associations and the rest from free-living systems. Biological nitrogen fixation is mediated through (a) symbiotic N2-fixing Rhizobia which are the obligate symbionts in leguminous plants and actinomycetes, Frankia in Casuarina and (b) free-living nitrogen fixers such as blue green algae, Azospirillum, Azotobacter, Acetobacter diazotrophicus, Azoarcus etc. Other microorganisms that are known to be beneficial to plants are the phosphate solubilisers and plant growth promoting bacteria. In addition to supplying combined nitrogen by biological nitrogen fixation, certain bacteria affect the development and function of roots by improving minerals (NO3, PO3–3 and K+) and water uptake and thereby enhancing the crop productivity. The genus Rhizobium was the first named by Frank in 1889 (from latin meaning root living) and for many years this was a ‘Catch all’ genus for all rhizobia. Same species were later moved into new genera based on phylogenetic analyses.

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Biology and Biotechnology of Fungi and Microbes

Currently there were 4 recognised species of nodule bacteria with 11 genera, 9 belonging to α - proteobacteria, Allorhizobium, Azorhizobium, Bradyrhizobium, Devosia, Ensifer (formerly Sinorhizobium), Mesorhizobium, Ochrobactrum, Rhizobium, Methylobacterium. Besides the above genera, species of Burkholderia (B. caribensis, B. cepecia, B. polymetum, B. tuberum), Cupriovidus (C. taiwanensis) Herbasprillum (H. lucitanum), Phyllobacterium (P. trifolii) are also reported to form nodules in legumes. In some cases, these species have arisen through lateral gene transfer. Plants belonging to leguminosae have the ability to harness atmospheric nitrogen because of their symbiotic association with Rhizobium. Biological nitrogen fixation is dependent on establishment of symbiotic relationship between the legume and effective Rhizobium strain. The legume-rhizobia symbiosis culminates in the formation of nitrogen fixing root nodules (Fig. 33.20). These unique structures are agronomically significant, as they provide an alternative to the use of energyexpensive ammonium fertilizer. Not all legumes fix N. The capacity to form nodules appear to be absent from the majority of the segregate family Caesalpinaceae. On the other hand, nodulation, if not N2 fixation, with root nodule bacteria appears almost universal in the segregate family Mimosaceae and other Fabaceae although only relatively small proportion of the total number of species in these two groups has been properly examined When legumes are first introduced into soils, root nodules fail to develop because of low population of compatible and effective strains of Rhizobium in the soil. Thus addition of appropriate strains of Rhizobium by inoculation is generally essential where legumes have not been grown before and where there are no naturalised rhizobia. The aim of inoculation of legume seed is to coat the seed with sufficiently larger number of viable rhizobia of the appropriate strain to give rapid and effective nodulation of that legume in the field. Grain legumes grown in rotation or as intercrops with cereals have been reported to benefit the succeeding or intercrop. The Rhizobium-legume association can fix upto 100-200 kg nitrogen per hectare in one crop season and in certain situations can leave behind substantial nitrogen for the following crop.

Fig. 33.20. Root nodulation by rhizobia inlegumes

Bacteria

487

Stem nodulating legumes such as Sesbania rostrata, Aeschynomene sp. and Neptunia oleraccea have become popular in improving soil fertility. The N2-fixing bacteria associated with such stem nodulating legumes belong to Azorhizobium and fast growing species of Rhizobium. The N-accumulating potential of stem-nodulating legumes under flooded conditions ranges from 41-200 kg N/ha. Free-living nitrogen-fixers Nitrogen-fixing bacteria of many genera occur in high numbers in rhizosphere of a variety of plants (Tilak et al 2010) (Fig. 33.21). Some of the important nitrogenfixing bacteria include Achromobacter, Acetobacter, Alkaligenes, Arthrobacter, Azospirillum, Azotobacter, Azomonas, Bacillus, Beijerinckia, Clostridium, Corynebacterium, Derxia, Enterobacter, Herbaspirillum, Klebsiella, Pseudomonas, Rhodospirillum, Rhodoseudomonas, Xanthobacter etc. Although the list is long, bacterial species of the two genera, Azotobacter and Azospirillum have been widely used and recommended as biofertilizers to increase the crop yields. In recent years, Acetobacter diazotrophicus (now nanmed as Gluconacetobacter diazotrophicus) has gained importance as a biofertilizer for sugarcane.

Fig. 33.21. Effects of rhizosphere microorganisms in soil

Azospirillum This organism came into focus by the work of Dobereiner and associates from Brazil. Azospirilla are nitrogen-fixing bacteria with nitrogenase properties. The genus contains 5 species: A. brasilense, A. lipoferum, A. amazomnse, A. halopreference and A. irakense. The crops which respond to Azospirillum inoculation are maize, barley, sorghum, pearl millet and several other crops. Azospirillum inoculations increase grain productivity of cereals by 5-20 percent, of millets by 30

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Biology and Biotechnology of Fungi and Microbes

per cent, and of fodder by over 50 per cent. Apart from nitrogen-fixing ability, Azospirillum is known to produce phytohormones, biocontrol substance etc. Blue-green algal biofertilizer for rice A group of free-living microorganisms, commonly known as blue-green algae (BGA) or cyanobacteria has been demonstrated to be an ideal candidate as a biological nitrogen input in rice ecosystems. The ecological and agricultural importance of BGA depends upon mainly in their ability of certain species to carry out both photosynthesis and nitrogen fixation. Nitrogen-fixing BGA are either freeliving or symbiotic and can be divided broadly into 3 major groups, viz., filamentous heterocystous forms, filamentous non-heterocystous forms and unicellular forms. Some BGA have been reported to develop symbiotic association with eucaryotic algae, fungi, bryophytes, pteridophytes, gymnosperms and angiosperms. Blue-green algae have been used as biofertilizers in paddy crop in a number of countries, including India. The beneficial effect on rice has been demonstrated in many areas of the country, in terms of grain yield and N-saving. A conservative estimate shows that BGA contribute 20-30 kg. N per hectare per crop per season indicating a saving to that extent of the chemical fertilizer along with 10-20% increase in rice crop yield. A rural-orientated BGA bio-fertilizer technology for rice has been developed in our country, which involves introduction of specific strains of blue-green algae. Phosphate solubilizers Phosphorus is a key nutrient limiting the plant growth. A survey of Indian soils reveals that 98% of them need phosphorus fertilization either in the form of chemical or biological fertilizer. Though, the P content in an average soil is 0.05% but only 0.1% of the total P present in the soil is available to the plants because of its chemical fixation and low solubility. Even after the application of chemical phosphatic fertilizer, majority of the soil P reaction products are only sparingly soluble. Under such conditions, the microorganisms offer a biological rescue system capable of solubilizing the insoluble inorganic P of soil and make it available to the plants. Phosphate solubilizing microorganisms include bacteria, fungi, actinomycetes, yeasts and blue-green algae capable of dissolving inorganic phosphates. They can grow in medium having tricalcium, iron and aluminium phosphate, hydroxyapatite, bonemeal, rock phosphate and similar insoluble phosphate compounds as the sole phosphate source. These microbes not only assimilate P but in their presence, a large portion of soluble phosphate is released in quantities in excess of their own requirement. The most efficient phosphate solubilizing bacteria (PSB) belong to genera Bacillus and Pseudomonas, though species of Brevibacterium, Corynebacterium, Micrococus, Saracina and Achromobacter have also been reported to be active in solubilizing insoluble phosphates (Fig. 33.22).

Bacteria

489

Fig. 33.22. Phosphate solubilization by Pseudomonas putida and Bacillus subtilis.

Sewage treatment Sewage or wastewater is the water -borne human, domestic and farm wastes. It may include industrial effluent, sub soil or surface waters. Human wastes include faecal material. Domestic wastes include water-borne wastes such as acids, oils, greases, and animal and vegetable matter discharged by factories. Microorganisms introduced from the activated sludge carry out the oxidation of organic matters. Among the microorganisms Zoogloea ramigera, a rod shaped bacterium is found in activated sludge. In the sewage treatment process microorganisms are involved in the case of activated sludge. Zoogloea film is developed because of the presence of filamentous bacteria like Sphaetrotilus natans and Beggiatoa. Other bacterial genera that oxidize organic matter are Pseudamonas, Flavobacterium, Achrmobacter and Alcaligens. In the lower level nitrifying organisms like Nitrobacter and Nitrosomonas predominate. Carbon dioxide is obtained either from bacterial decomposition of organic matters or from the atmosphere which gives rise to dissolved CO2. The oxygen produced by photosynthesis causes an increase in the dissolved oxygen (DO) level, which favors aerobic bacteriological activity. Dominant members of the aerobic heterotrophic bacteria population are Pseudomonas, Achromobacter and Flavobacterium. The level may decrease after aerobic activities. The microbial population that degrades organic matter in Imhoff tanks occurs as dispersed gelatinous masses. Bacteria involved in the process are Pseudomonas, Nitrosomonas, Zoogloea, and Sphaerotilus. The most common treatment for metal contaminated water is microbial biofilms, Examples are many microorganisms, including Bacillus, Citrobacter and Arthrobacter. Bioaccumulation and mineral leaching is now well known. In sewage treatment processes, metals such as copper, nickel and cadmium are precipitated by bacteria, thereby purifying the waste water. Bacterial leaching of metals has been exploited and nearly 5 per cent metal production in the world comes from this 'low technology' source applicable to low grade mineral ores such as pyrite and metal sulphides. Somewhat similar technology is applicable to enrichment of uranium and gold by microbial leaching. Thiobacillus ferrooxidans and consortium of other bacteria are involved in metal sulphide leaching and iron oxidation. The Leptospirillum group (iron bacteria) also contributes to recovery of iron. The economics involved in microbial processes by mechanization and scaling up of such processes need careful review. However,

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Biology and Biotechnology of Fungi and Microbes

the science of biometallurgy remains a fascinating area of study for microbiologists and metallurgists alike. Bacteria in genetic engineering Genetic Engineering of bacteria allows a much greater range of products to be made by bacteria. Human hormones, blood proteins and proteins for vaccine development can now be made in bulk using these modified bacteria. Since the late 1970s advances in genetic engineering have produced a revolution in biotechnology and now an increasing number of products are being made by genetically engineered organisms. The greatest interest is in the production of mammalian proteins and peptides by microorganism, since many of these materials have high pharmaceutical value and are expensive or difficult to produce by other methods. If the gene or genes that code for production of a mammalian protein can be cloned in to a microorganism, and good expression of the genes obtained, then a biotechnological process for making this protein can likely be developed. Such materials produced by modified organisms include hormones, blood proteins, immune modulators (interferon) and proteins for vaccine development. A naturally occurring conjugation phenomenon in Agrobacterium tumefaciens induced crown-gall disease of plants had been ably exploited by biotechnologists to genetically engineered forging genes into dicotyledons to viral diseases, herbicides or bioinsecticides. A. tumefaciens is a soil bacterium, which infects the crown region of dicotyledonous plants (monocots are resistant) through mechanical wounds to produce crown galls. A. tumefaciens harbours large extra chromosomal genetic elements known as megaplasmids. Most of the genes required for tumour formation are located on one such 180 kb megaplasmid designated as Ti (the tumor inducing ability) of the plasmid. The Ti plasmid synthesises of two growth hormones, the IAA and cytokinins and the genes controling the synthesis of group of amino acid derivatives known as opines (nopaline and octopine). Biogas and biofuel Methane is one of the major energy sources and is widely used all over the world. Most of the methane used is produced from geological sources as a result of either specially drilled natural gas wells, or as a byproduct of petroleum wells. A mixture of organisms ferments a mixture of raw materials available and interactions result in biogas production. Biogas generated may have methane contents between 50 to 80 percent and the remaining component will be mainly carbon dioxide. Anaerobic digestion to biogas production involves activity of three groups of organisms 1)

Fermentation of organic components: Fermentative bacteria convert complex organic material into organic acids, alcohols, esters, sugars and CO2.

2)

Acid producers: The group is dependent on first and contains hydrogen and acid-producing bacteria.

Bacteria

491

3)

Methane producers: Methanogenic bacteria convert acetate and H2 into biogas, which is a mixture of methane (CH4) and CO2.

Biogas production is a convenient way of agricultural wastes disposal for more than one reason. Substrate detoxification, deodorization, inactivation of pathogens, dehelminthization occur along with biogas production and fertilizer or humus forming substance as a by-product. Emission of methane Intensive rice cultivation under heavy fertilization in the coming decades may raise the problem of increasing connection of methane and nitrous oxide, green house gases implicated in global warming. The formation of both methane and nitrous oxide in rice soil is mediated essentially by microorganisms. Mitigation options for methane promote the emission of nitrous oxide. One of the promising options for mitigation of both methane and nitrous oxide is to use nitrification inhibitors which can not only retard the emission of both green house gases but also increase the Nuse efficiency in rice. Biofuel The feasibility of using ethanol as fuel for spark ignition automobile engines, diesel-powered heavy vehicular engines, boilers, gas turbines, utility fuel cells is reasonably well-established. At the University of Florida, microbiologists have cloned and sequenced two bacterial genes that code enzymes for converting pyruvate into ethanol and used them to direct pyruvate towards alcohol production in Escherischia coli, Klebsiella oxytoca and other bacteria. Thermophilic anaerobic bacteria can ferment biomass, including cellulose, hemicellulose and other saccharides, directly to ethanol. But they produce acetic acid and other acids as co-products. Site directed mutagenesis is being used to generate strains that are efficient ethanol producers and have reduced acetic acid production. Biopesticides Commercial production of biopesticides is restricted to few bacteria, fungi and viruses. Artificially formulated media with carbon and nitrogen sources serve as substrates for some bacteria and fungi whereas, few bacteria and viruses need live insect larvae grown aseptically in large fermentors as substrate because of the obligatory nature of these insect predators. For instance, Bacillus papillae is produced on a commercial scale in the form of dust on larvae of Japanese beetles. On the other hand, B. thuringiensis and B. moritai are produced by conventional media fermentation techniques by various companies. In fact, B. thuringiensis insecticide has come to stay as an alternative to chemical pesticide in many countries. Bacillus thuringiensis is a Gram positive sporulating bacterium that produces a crystalline inclusion known as parasporal crystal in sporulating cells. The parasporal crystal contains proteins known as endotoxins that are toxic to a mixture of B. thuringeiensis spores and crystals, which are sprayed on plants. Susceptible insects feed on sprayed plant foliage resulting in gut endotoxins ingested by the inset.

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The development of B. thuringiensis strain with a spectrum of activity has been the aim of several manufacturing companies. A second current strategy has been to introduce B. thuringeiensis toxin gene into the genome of plants or plant genetic systems and Monsanto have successfully engineered B. thuringiensis toxin genes into tobacco, cotton and tomato plants and into root colonizing bacteria of the genus Pseudomonas. In the U.S.A. a transgenic Pseudomonas to which a toxic gene from B. thuringiensis has been added is being commercially distributed under the brand name 'Dagger G' to control damping off of cotton. Bacterial insecticides There are about 90 species of bacteria pathogenic to insect pests and they serve as new tools in biological control of plant pests. Among them, Bacillus thuringiensis that was first discovered in 1902 by a Japanese bacteriologist, Ishiwata from infected silkworms stands out prominent. The bacteria from a protein crystal inclusion body (molecular weight 800-900) that is an endotoxin active in inhibiting the growth of about 130 species of insects and larva. The protein crystal synthesis and spore formation in the bacterium proceed simultaneously and the two processes are in many ways interlinked. The organisms can be grown in naturally available inexpensive media (such as bran) and spores harvested to produce a mixture of spores and endotoxin crystals. Commercial preparations containing B. thuringiensis have been produced in many countries particularly in U.S.A. where they are used on several agricultural crops, trees and ornamental shrubs. A strain of B. thuringiensis that showed high toxicity to mosquito larva (Anopheles, Culex, Aedes) described as subspecies isaraelensis, has been isolated in Israel and its efficacy proved against mosquito larva. The preparation from this subspecies is not toxic to lepidopterist larvae and hence specific to mosquitoes. It has potentialities in the control of malaria in man. Other bacterial agents used against insect pathogens are Bacillus papillae, Coccobacillus acridorm and Serratia marcescens. Biological control Several microorganisms have shown potentialities as biological control agents against important plant diseases caused by soil-borne pathogens. They are Pseudomonas fluorescence against the take-all disease of wheat. Thielavioposis basicola infection of tobacco and damp off caused by Pythium in cotton; Bacillus subtilis against Fusarium wilt of corn. Pseudomonas putida against Fusarium solani wilt of beans; Bacillus sp. and Pseudomonas sp. against Fusarium oxysporum wilt of carnations. The use of Agrobacterium radiobacter in the control of crown-gall disease caused by A. tumefaciens is in vogue. Considerable damage to stone fruit, pomes fruit, other value-added plants like walnut, grapevine, Chrysanthemum and Dahlia is caused by crown- gall infection (plant cancer). This disease has been successfully controlled in Australia by using the strain 84 of the non-pathogenic A. radiobacter, which produces an unusual antibiotic called “Agrocin 84” which belongs to a new group of antibiotics known as nucleotide bacteriocins. A bacteriocin is a substance

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produced by certain bacteria and active on other strains of the same or closely related species Biodegradation Microbial methods by which biodegradation can be augmented have been explored. They are: (1). Addition of surfactants, (2) supplementation with inorganic nutrients and inoculation with biomass of enriched bacterial species known to degrade a specific compound and the method is referred to as bioaugmentation. The enzymes responsible for biodegradation are genetically encoded in bacterial plasmids. These plasmids possess broad host range and can be transferred within the same species or genera. They have been identified in species of Pseudomonas, Alcaligenes, Acinetobacter, Flavobacterium, Beijerinkia, Klebsiella, Moraxella, and Arthrobacter. Most of the novel strains have been genetically engineered from the genus Pseudomonas and have been patented for cleaning up oil spills. The DDT is converted to DDE through dehalogenation by an enzyme dehydrochlorinase as shown by several bacteria like Achromobacter, Aerobacter, Agrobacterium, Bacillus, Clostridium, Corynebacterium, Esherichia, Erwinia, Kurthia, Pseudomonas and Streptococcus. The degradation of linen is mediated by Clostridium and Escherichia. Malathion is degraded by Pseudomonas, Thiobacillus. 2,4-D , phenoxyalkanoic acids or phenoxy herbicides, are extensively investigated and well known. They are degraded by the genera Pseudomonas, Achromobacter, Flavobacterium, Coyrnebacterium, Arthrobacter and Sporocytophaga as hormone weed killers, that are active against broad-leaved weeds. Biodeterioration Bacteria can also pose significant problems for industry. The microbial deterioration of paints, textiles, concrete, metals and oils is termed biodeterioration. These breakdown processes are more common in humid environments as is usually the limiting factor in microbial biodeterioration. Many of these processes, such as the degradation of convert by Thiobacillus, render the material unusable. Microbial degradation of oils by organisms such as Pseudomonas can be used in the control of spills. Leather: It is frequently observed that yellow to red spots are produced on salted raw hides by halotolerant or halophilic bacteria viz., Micrococcus luteus, M. resens and Halococcus morrhuae. Textile: Bacterial genera such as Bacillus megaterium, Cytophaga, Sporocytophaga, Cellulomonas, Myxococcoides, Corynebacterium have been isolated from the textile showing cellulolytic activity. Jute: The bacterial species involved in the degradation of jute includes Arthobacter, Bacillus, Pseudomonas, Xanthomonas, Cytophaga etc. Paint: Bacterial genera such as Pseudomonas, Flavobacterium cause extensive damage to the painted surface.

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Food spoilage Bacteria also play a significant role in food spoilage. Foods with easily utilizable carbohydrates, proteins and fats are ideal environments for spoilage by microorganisms. For example, milk undergoes series of steps, which include increased lactic acid production. In canned foods, spoilage is typically caused by fermentative bacteria such as clostridia which alter the texture and taste of the food as a result of protein detraction. The production of CO2 and H2S during spoilage causes cans to swell. More importantly the presence of bacterial pathogens in food presents a significant problem. Major diseases associated with food poisoning are given Table 33.6. Table 33.6. Food spoilage microorganisms Pathogens

Disease

Foods

Escherichia coli

Enteropathogenic infection

Cheese, meats

Listeria monocytogenes

Listeriosis

Dairy products

Salmonella typhimurium, S. enteritidis

Salmonellosis

Meats, poultry, eggs, dairy products

Vibrio parahaemolyticus

gastroenteritis

Seafood

Clostridium perfringens

Perfringens food poisoning

Meats and poultry

C. botulinum

Botulism

Fish, meats and canned foods

Bacillus cereus

B. cereus food poisoning

Meats, rice, cereals, potatoes

Food poisoning (Caused by toxin release)

■■■

Chapter - 34

Viruses

The first viral disease of plants was discovered in 1892, when Iwanowski demonstrated that a common disease of the tobacco plant called tobacco mosaic could be transmitted to healthy plants in the sap from diseased plants, even though the sap had been passed through bacterial filters that are fine enough to remove all bacteria. Furthermore, the disease could be transmitted with sap filtrates from plant to plant in in-definite series. Yet no living thing capable of producing tobacco mosaic grew from the filtered sap of diseased plants on any culture medium in the laboratory, and nothing could be seen in the crystal-clear fluid with any microscope then available. Beijerinck, found that the filtrable, invisible and noncultivable infectious principle would diffuse through an agar gel like a fluid. He thought the fluid itself alive, and called it contagium vivum fluidum - a living infectious fluid! This concept is embodied in the very word ‘virus’, which is derived from a Latin word meaning venom (poison) It is now known that the sap from the diseased tobacco plant contained billions of particles of the virus of tobacco mosaic, the first known virus. Iwanowski had opened the world of the ultramicroscopic creatures. In 1935, Stanley crystallized the tobacco mosaic virus (TMV) and the virus was of thousands of submicroscopic nucleoprotein complexes; i.e., infective units (virions) of tobacco mosaic virus. Majority of viral diseases of other plants are now known. Pasteur studied canine rabies and reported to be as a causative agent of virus, although during his early studies he was apparently not aware of its true nature. The term virus was then commonly used for a variety of infectious agents, including bacteria. In 1898, the foot-and-mouth disease of cattle was shown by Loeffler and Frosch to be caused by an agent that, like TMV, passed through bacteria retaining filters, and was neither visible with the microscope nor cultivable in media under laboratory conditions. In 1900, Walter Reed and his associates discovered the virus of yellow fever, the first known viral disease of man. VIRUSES Viruses are not cellular in structure. They have no true nucleus, cytoplasm, cell membrane or cell wall. They multiply only within living cells. Outside living cells viruses are totally inert. Each virus particle or virion consists of only two major parts. One is a single linear molecule of either DNA or RNA. This constitutes the core or

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nucleoid of the virus. It is the active, disease-specific, host-specific, genetic, infective part of the virus. Each virion contains only one kind of nucleic acid (NA) - DNA or RNA and never both. This distinguishes all true viruses from all cellular forms, eukaryotic or prokaryotic, because all cellular organisms contain both DNA and RNA. Thus, all viruses are divided primarily into two groups: those containing DNA are called deoxyviruses and those containing RNA are called riboviruses. Surrounding the NA core is the second major portion of the virion: a protein shell or coating called a capsid. Since it is a protein, it has antigenic specificity. The capsid is made up of many identical structural units called capsomeres whose composition, numbers and forms vary with the kinds of viruses. The capsid is physiologically inert and is believed to serve only as a protective shell. The core, with its capsid, is called the nucleocapsid of the virus.

Fig. 34.1. Types of viruses. 1. Orthopoxvirus. 2. Parapovirus. 3. Rhabdovirus. 4. Paramyxovirus. 5. Herpesvirus. 6. Orthomyxovirus. 7. Coronavirus. 8 Togavirus. 9. T-even coliphage.10. Adenivirus. 11. Reovirus. 12. Papovavirus. 13. Picornavirus. 14. Parovirus. 15. Tobacco mosaic virus (Source: Johri and Lata ( 2004))

In outward form virions differ widely. They may be elongate, like a piece of insulated electric cable, or rounded, polyhedral or cuboidal. Several appear to be

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pleomorphic. In elongate forms the NA strand of the core is usually coiled like a helical spring. The capsid is closely coiled about it or is arranged as a succession of rings. Such a virion is said to have helical symmetry. In some helical viruses the whole nucleocapsid is sufficiently flexible to be secondarily coiled on itself into a spheroidal form. Many are covered externally by thin, radially projecting spikes or rods suggestive of bacterial fimbriae. A common viral form is based on cubic symmetry. Such viruses may be polyhedral or roughly cuboidal. The cores of such viruses are surrounded by well-defined capsomeres usually arranged in icosahedral symmetry. Some viruses at times are bullet shaped (Fig 34.1). The numbers of capsomeres of virions that show icosahedral symmetry are determinable and are distinctive of certain virus types. Capsomere numbers vary from about 12 to 812 or more. The symmetry and form of numerous viruses, especially the large poxviruses with lipocomplex peploes (smallpox, myxoviruses), are complex and often vague or indeterminable. Capsomeres of some helical viruses with envelopes are not sufficiently distinct to be accurately counted. Instead, the diameter of the helical nucleocapsid inside the envelope is measured as a distinguishing character. BACTERIOPHAGE Viruses that attack bacteria were first described by Twort, and were independently observed and further studied in 1917 by the French investigator, d'Herelle, who named these viruses bacteriophage (bacteria eaters). The term, phage, is commonly used for bacteriophage. Structure - Bacteriophage come in many different sizes and shapes. The basic structural features of bacteriophages (T 4) are enlisted below: 1.

Size - T4 is among the largest phages; it is approximately 200 nm long and 80-100 nm wide. Other phages are smaller. Most phages range in size from 24-200 nm in length.

2.

Head or Capsid - All phages contain a head structure which can vary in size and shape. Some are icosahedral (20 sides) others are filamentous. The head or capsid is composed of many copies of one or more different proteins. Inside the head is found the nucleic acid. The head acts as the protective covering for the nucleic acid.

3.

Tail - Many but not all phages have tails attached to the phage head. The tail is a hollow tube through which the nucleic acid passes during infection. The size of the tail can vary and some phages do not even have a tail structure. In the more complex phages like T4 the tail is surrounded by a contractile sheath which contracts during infection of the bacterium. At the end of the tail the more complex phages like T4 have a base plate and one or more tail fibers attached to it. The base plate and tail fibers are involved in the binding of the phage to the bacterial cell. Not all phages have base plates and tail fibers. In these instances other structures are involved in binding of the phage particle to the bacterium.

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VIRUS MULTIPLICATION Virus multiplication takes place in living cells and is divided into five phases (Fig. 34.2). 1. Adsorption - Viruses can enter cells via phagocytosis, viropexis or adsorption. Adsorption is the most common process and the most highly specific process. It requires the interaction of a unique protein on the surface of the virus with a highly specific receptor site on the surface of the cell. 2. Penetration - This occurs by one or more processes. 3. Enveloped viruses fuse their envelope with the membrane of the host cell. This involves local digestion of the viral and cellular membranes, fusion of the membranes and concomitant release of the nucleocapsid into the cytoplasm. Naked viruses bind to receptor sites on the cellular membrane, digest the membrane and enter into the cytoplasm intact. Both naked and enveloped viruses can be ingested by phagocytic cells. However, in this process they enter the cytoplasm enclosed in a cytoplasmic membrane derived from the phagocytic cell.

Fig. 34.2. The lytic cycle of a bacterial virus, bacteriophage T4. (Source: Kenneth Todar, www textbookofbacteriology.net)

Uncoating - During this stage cellular proteolytic enzymes digest the capsid away from the nucleic acid. This always occurs in the cytoplasm of the host cell. The period of the replication cycle between the end of the uncoating stage and maturation of new viral particles is termed the eclipse. Thus during the eclipse stage, no complete viral particles can be viewed within the cell. 4. Replication of nucleic acid. The Replication of viral nucleic acid is a complex and variable process. The specific process depends on the nucleic acid type. DNA virus replication: with the exception of the poxviruses, all DNA viruses replicate in the nucleus. In some cases one of the DNA strands is transcribed (in

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others both strands of a small part of the DNA may be transcribed) into specific mRNA, which in turn is translated to synthesize virus-specific proteins such as tumor antigen and enzymes necessary for biosynthesis of virus DNA. This period encompasses the early virus functions. Host cell DNA synthesis is temporarily elevated and is then suppressed as the cell shifts over to the manufacture of viral DNA. As the viral DNA continues to be transcribed, late virus functions become apparent. Messenger RNA transcribed during the later phase of infection migrates to the cytoplasm and is translated. Proteins for virus capsids are synthesized and are transported to the nucleus to be incorporated into the complete virion. Assembly of the protein subunits around the viral DNA results in the formation of complete virions which are released after cell lysis. The single-stranded DNA viruses first form a double stranded DNA, utilizing a host DNA-dependent DNA polymerase. They then undergo a typical replication cycle. RNA virus replication With the exception of the orthomyxoviruses and retroviruses, all RNA viruses replicate in the cytoplasm of the host cell. The exact process varies with the species of virus. The single-stranded RNA that is released after uncoating will act as either: (a) the mRNA to synthesize viral-coded proteins; or (b) a template to synthesize mRNA; or (c) a template to synthesize double stranded RNA, which is then used as a template to synthesize mRNA; or (d) a template to synthesize double-stranded DNA, which is then utilized as a template to synthesize mRNA. This latter process occurs only with the retroviruses (oncorna viruses). The replication of poliovirus, which contains a single-stranded RNA as its genome, provides a useful example. All of the steps are independent of host DNA and occur in the cell cytoplasm. Polioviruses absorb to cells at specific cell receptor sites, losing in the process one virus polypeptide. The sites are specific for virus coatcell interactions. After attachment, the virus particles are taken into the cell by viropexis (similar to pinocytosis), and the viral RNA is uncoated. The singlestranded RNA then serves as its own messenger RNA. This messenger RNA is translated, resulting in the formation of an RNA-dependent RNA polymerase that catalyzes the production of a replication intermediate (RI), a partially doublestranded molecule consisting of a complete RNA strand and numerous partially completed strands. At the same time, inhibitors of cellular RNA and protein synthesis are produced. Synthesis of (+) and (-) strands of RNA occurs by similar mechanisms. The RI consists of one complete (-) strand and many small pieces of newly synthesized (+) strand RNA. The replicative form (RF) consists of two complete RNA strands, one (+) and one (-). The single (+) strand RNA is made in large amounts and may perform any one of three functions: (a) serve as messenger RNA for synthesis of structural proteins; b) serve as template for continued RNA replication; or (c) become encapsulated, resulting in mature progeny virions. The synthesis of viral capsid proteins is initiated at about the same time as RNA synthesis.

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The entire poliovirus genome acts as its own mRNA, forming a polysome of approximately 350S, and is translated to form a single large polypeptide that is subsequently cleaved to produce the various viral capsid polypeptides. Thus, the poliovirus genome serves as a polycistronic messenger molecule. Poliovirus contains four polypeptides. 5. Maturation and Release Naked viruses maturation consists of two main processes: the assembly of the capsid, and its association with the nucleic acid. Maturation occurs at the site of nucleic acid replication. After they are assembled into mature viruses, naked virions may become concentrated in large numbers at the site of maturation, forming inclusion bodies. Naked virions are released in different ways, which depend on the virus and the cell type. Generally, RNA-containing naked viruses are released rapidly after maturation and there is little intracellular accumulation; therefore, these viruses do not form predominant inclusion bodies. On the other hand, DNA-containing naked icosahedral viruses that mature in the nucleus do not reach the cell surface as rapidly, and are released when the cells undergo autolysis or in some cases are extruded without lysis. In either case they tend to accumulate within the infected cells over a long period of time. Thus, they generally produce highly visible inclusion bodies. Enveloped viruses In the maturation of enveloped viruses, a capsid must first be assembled around the nucleic acid to form the nucleocapsid, which is then surrounded by the envelope. During the assembly of the nucleocapsid, virus-coded envelope proteins are also synthesized. These migrate to the plasma membrane (if assembly occurs in the cytoplasm) or to the nuclear membrane (if assembly occurs in the nucleus) and become incorporated into that membrane. Envelopes are formed around the nucleocapsids by budding of cellular membranes. Complex viruses These viruses, of which the poxvirus is a good example, begin the maturation process by forming multilayered membranes around the DNA. These layers differentiate into two membranes: The inner one contains the characteristic nucleoid, while the external one acquires the characteristic pattern of the surface of the virion. These form very characteristic cytoplasmic inclusion bodies. The viruses are generally released from the cell via cell lysis. The latent period: The assembly of new virions from the prefabricated parts continues inside the cell for some time after the end of the eclipse period. In the E. coli-T2 phage system the cell wall ruptures about 18 minutes after the end of the eclipse period, and the new phage units are then set free to begin the cycle anew. The cell is said to have undergone lysis from within. This signalizes the end of the latent period. The latent

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period is the time from adsorption up to, but not including, cell rupture. It includes the eclipse period. The latent cycle The period from adsorption to cell lysis comprises one lytic cycle of phase activity. As will be descried, there are other cycles of viral activity. Burst size The average number of mature virions released per cell by rupture of the cell wall at the end of the latent period is more or less constant in any given cell-virus system. It may range from about 20 to 200 or more. This number is referred to as the burst size of the cell at the end of the latent period. Cell lysis is apparently not caused by expansive pressure from within. Under the influence of the phage the cell appears to form, or to cease to inhibit, enzymic agents capable of destroying the cell wall. This causes lysis from within. Similar lytic agents have been found in phage lysates (suspensions of cells lysed by phage) of numerous bacterial species. In some respects these lytic agents resemble an enzyme called lysozyme that hydrolyzes the mucocomplex of cell walls. Some of the enzymes found in phage lysates can depolymerize capsular polysaccharides; others digest mucopeptides of cell walls and cause lysis. Lysogeny A virus may contact a susceptible cell and the viral NA may enter that cell without causing immediate lysis. In a phage infection, lysis may fail to occur or be delayed because the phage is not fully virulent for that cell; i.e., the virus NA is not able to initiate the immediate, unrestricted, vegetative replication of phage that is necessary to the lytic cycle. The phage is said to have undergone a change (reduction) to what is designated as the temperate state. When the phage becomes temperate it becomes closely associated with, or actually a segment of, the single, circular, bacterial chromosome or replicon. A phage may be invariably highly virulent in one type of cell under one set of growth conditions, yet temperate in the same type of cell under other conditions or in another type of cell. The terms virulent and temperate are applicable only to phage. Unlike the rapid and unrestrained replications of the virulent virus, the temperate viral genome is restricted to replication synchronously with, seemingly as part of, the cell genome. In this state viral proteins are not synthesized and no separate or complete virions appear. Because the temperate virus is not itself a complete virus but, can suddenly become one, and is refered as a prophage. The phenomenon has been described fully only for the bacterium-phage system. The prophage may remain in, or closely attached to, the cell genome and be distributed to the cell progeny as part of the cell genome for millions of generations. Unless some peculiar influence is brought to bear, the presence of the viral NA in the cell may never be suspected. One clue to its presence is the immunity of the cell to superinfection by that specific virus. Another clue is its inducibility. Induction If a cell containing a temperate virus is subjected to certain influences such as ultraviolet radiation or agents such as hydrogen peroxide, the prophage is

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immediately activated. It becomes vegetative and at once begins a lytic cycle as previously described. It is said to have been induced. Induction often occurs spontaneously. A bacterium containing an inducible prophage may thus be lysed at any time and, because virions are liberated, contribute to the lysis of other cells. Such bacteria are said to be lysogenic; the phenomenon of reduction of a phage to temperance and association of its replicon with the bacterial genome is called lysogeny. The term lysogeny is applicable only to phages. Lysogenic Immunity A cell containing a prophage (a lysogenic cell) is immune to super-infection by any other virion of that specific kind of virus even though an infective virion may be adsorbed and penetrates into the cell. Immunity thus conferred on a cell by viral infection appears to be caused in part by the immediate synthesis, not of lytic enzymes as in the lytic cycle, but of a repressor substance that represses vegetative replication of any secondarily entering genome of the same specific kind of virus. It appears also that any given, specific phage replicon has its own definite place or locus of attachment to or in the cell replicon. If, for a given virus, this locus is already occupied by a viral replicon, it cannot serve for the attachment of an additional replicon of that identical viral type. However, one or more viruses of some other type(s) may enter the cell and either destroy it at once or be severally reduced to the temperate state if there are specific, unoccupied loci in the cell replicon for their attachment. The immunity of lysogenic cells is called lysogenic immunity. Lysosgnic immunity to a given virus persists only as long as that provirus is present in the cell. If, as sometimes happens, the cell is ‘cured’ of its lysogenicity; i.e., if the prophage is inactivated or for unknown reasons fails to be transmitted to a daughter cell, lysogenic immunity of that cell and of its progeny to that type of virus disappears. Lysogenic immunity and resistance of a cell to viral infection are two distinct phenomena dependent on totally different mechanisms. Many strains of lysogenic bacteria are well known and are widely used in virological investigations. They are designated by adding to their specific name, parenthetically, as a suffix, the designation of the prophage that they carry; thus the lambda () phage in the lysogenic strain of Escherichia coli known as K12 is indicated as E. coli K12 (). An important factor in multiplication of viruses is host specificity. CLASSIFICATION In the beginning of 21st century, viruses were classified into four groups based on their hosts. They are: 1.

Plant viruses (include fungal, algal and bacterial viruses)

2.

Invertebrate viruses

3.

Vertebrate viruses

4.

Both invertebrate and vertebrate viruses (Binary viruses)

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Earlier Holms (1948) included all viruses in one single order called Virales and three suborders – Phaginae, Zoophaginae and Phytophaginae. Later in 1962 Lwoff, Hurne and Tournier developed a classification system for viruses, which was popularly known as LHT system. This was accepted by international community and is in agreement with International Code of Nomenclature for viruses (1996). The LHT system is based on following criteria: a.

Nature of nucleic acid (DNA or RNA).

b.

Symmetry of the virus (helical, icosahedral, cubic, cubic-tailed).

c.

Presence or absence of envelope.

d.

Number of capsomers.

Although LHT system is popular and accepted, Baltimore (1971) and Sherwood et al (1975) proposed different classification for viruses. Based on the type of nucleic acid and number of strands in the nucleic acid Baltimore (1971) divided viruses into following six classes. 1.

Double stranded DNA viruses (dsDNA) e.g: human and animal viruses.

2.

Single stranded DNA viruses (ssDNA) e.g. some bacteriophages.

3.

Double stranded RNA viruses (ds RNA)+Parvo type animal virusesStrand RNA(Reovirus)

4.

Single strand RNA viruses (+strand viruses eg: Polio virus).

5.

Single strand (–) RNA viruses (Rabdoviruses, Paramicro viruses, Orthomix viruses)

6.

RNA-DNA viruses – DNA is required for the replication of these viruses.

Casjens and King (1975) classified viruses based on the type of nucleic acid, number of strands, symmetry of protein coat and the envelope. A universal system of classification of virus as approved by International Committee on Taxonomy of Viruses (ICTV) (1999) is based on four criteria: Classification 1.

The nature of the viral genome.

2.

The strandedness of the viral genome.

3.

The facility for reverse transcription.

4.

The polarity of the viral genome.

In this classification, irrespective of their hosts, all viruses are classified into 233 genera. Out of these 204 genera are placed in 64 families and the remaining 29 are treated as unassigned or floating genera. Three orders are recognized now: (a) Caudovirales, which includes bacteriophage families Myoviridae, Siphoviridae and Podoviridae, (b) Mononegavirales, which includes four families viz., Bornaviridae, Filoviridae, Paramyxoviridae and Rhabdoviridae and (c) Nidovirales which includes two families Coronaviridae and Arteriviridae. This classification up to family level is given below.

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1. The DNA virus—The ss DNA viruses Families: Parvoviridae.

Inoviridae,

Microviridae,

Geminiviridae,

Circoviridae

and

2. The DNA viruses – The ds DNA Order – Caudovirales Families: Myoviridae, Siphoviridae, Podoviridae. Other families: Rudiviridae, Tectiviridae, Corticoviridae, Lipothrixviridae, Plasmaviridae, Fuselloviridae, Phycodnaviridae, Poxviridae, Iridoviridae, Polydnaviridae, Herpesviridae, Polyomaviridae, Papillomaviridae, Adenoviridae, Ascoviridae, Baculoviridae, Asfaviridae. 3. The DNA and RNA reverse transcribing viruses Families: Retroviridae.

Pseudoviridae,

Metaviridae,

Hepadnaviridae,

Caulimoviridae,

4. The RNA viruses—The ds RNA viruses Families: Cytoviridae, Reoviridae, Birnaviridae, Totiviridae, Partitiviridae, Hypoviridae. 5. The RNA viruses Order: Mononegavirales Families: Bornaviridae, Filoviridae, Paramyxoviridae, Rhabdoviridae. Other families: Bunyaviridae, Orthomyxoviridae and Arenaviridae. Order: Nidovirales Families: Coronaviridae, Arteriviridae. Other families: Leviviridae, Picornaviridae, Sequiviridae, Comoviridae, Polyviridae, Caliciviridae, Astroviridae, Nodaviridae, Tetraviridae, Bromoviridae, Closteroviridae, Barnaviridae, Luteoviridae, Flaviviridae, Tambusviridae, Togaviridae. 6. Naked RNA viruses and viroids. Families: Narnaviroidae, Pospoviroidae, Avsumviroidae. 7. Subviral agents Satellites, Prions. Universal system of classification as detailed above does not take cognizance of the nature of host. However, for practical purposes host based classification appears to be ideal.

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CULTIVATION OF VIRUSES (a) Chick embryo Embryo is an early developmental phase of animals characterized by having the different kind of cells. The eggs of chicken, duck and turkey are used for cultivation of viruses. The various regions of eggs are used for the viral inoculation. Allantoic cavity (fluid containing sac for embryonic waste removal), amniotic cavity (the sac provides protection to embryo.) chorioallantoic membrane (it is the region involved in gas exchange), yolk sac (provides the nourishment to embryo) and embryo. Death of the embryo is an indication of viral growth. Many times viral growth is characterized by pocks formation. The discrete opaque spots formed due to disintegration of cells are called pocks. Direct electron microscopic observation is also one of alternative for the detection of viral growth. (b) Tissue culture technique Plant viruses infect the plant cells. Cultivation of plant viruses by simple and effective way is to grow population of isolated plant cells in culture. Isolation of cells and formation of large plant tissue is possible with the help of plant tissue culture techniques. The plant cell / tissue cultures are grown in sterile chamber. After proper growth of callus (mass of plant tissues); viruses are allowed to infect the callus. All the procedures must be carried in aseptic environment in order to avoid contamination by bacteria and fungi, this can be ensured by addition of antibiotics. Plant tissue culture is also obtained as continuous culture in order to grow viruses continuously. Human immunodeficiency virus (HIV) HIV is the causal agent of dreaded disease AIDS. It belongs to the lentivirus subgroup of the family Retroviridae and thus it is a retrovirus. Three parts can be differentiated in the viral structure. 1. The outer envelope, 2. Middle icosahedral shell and 3. Central core (Fig. 34.3). The outer envelope is a bilayered lipid membrane. Projecting from the envelope spike like oligopolymers, glycoproteins gp 160 are found. The outer projecting spherical part is called gp 120 and the inner transmembrane pedicel like part is called gp 41. These two are antigenically different. The middle icosahedral shell constitutes the capsid. It is made up of protein p17, a cleavage product of the 55kDa gag gene product. The central core is cone shaped and is made up of ribonucleo proteins. The cone shaped outer layer is made up of p24 protein. The genome, RNA is associated with p7 and p9 proteins and reverse transcriptase, a characteristic enzyme present in all retroviruses. The genome of the HIV is diploid, linear, ‘+’ ssRNA. The genome consists of three structural genes viz., gag, pol, env characteristic of retroviruses and other non structural and regulatory genes specific to this virus. Gene gag codes for the core and capsid of the virus, pol gene codes for the enzyme reverse transcriptase, while the env gene codes for envelope proteins.

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Fig. 34.3. Structure of HIV and the process of its integration with host chromosome (Source: Rao 1997).

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Chapter - 35

Other Microorganisms (Actinobacteria, Archaebacteria, Mollicutes, Rickettsiae, Chlamydiae, Prions, Cyanobacteria, Protozoa)

ACTINOBACTERIA (ACTINOMYCETES) Actinomycetes are bacteria that appear in the form of mycelia. Their natural occurrence is mostly confined to soils (Plate 35.1). They are gram positive and are related to the coryneform bacteria and mycobacteria by an almost continuous sequence of intermediate forms. They are aerobic with very few exceptions. The name of this group is derived from the first-described anaerobic species Actinomyces bovis which causes actinomycosis, the ‘ray-fungus disease’ of cattle. Actinomycetes can be cultivated on simple media and can be identified by their growth in and on the surface of agar and their formation of aerial mycelia, substrate mycelia, spores, and sporangia. The proactinomycetes (Nocardia) form substrate and aerial mycelia that disintegrate into rod-shaped cells in older cultures. They do not produce true spores. Mycobacteria are invariably aerobic. Morphologically they are intermediate between the corynebacteria and the proactinomycetes (Nocardia). They do not form mycelia but grow in the form of irregularly-shaped, slightly branched cells. They are non-motile and gram positive. One way in which they differ from corynebacteria is that they are ‘acid fast’. In 1882 Ehrlich noted that tubercle bacilli (Mycobacterium tuberculosis) could not be decolourized by acid treatment after staining with aniline dyes. According to the Ziehl-Neelsen method mycobacteria and Nocardia are not decolourized by this acid treatment and are designated as ‘acid fast’. A few saprophytic mycobacteria can be decolourized with HCl-alcohol, but not with aqueous HCl. The resistance to acid is due to the high levels of mycolic acid in the cell wall, which make the cells of mycobacteria wax-like and strongly hydrophobic. Mycobacterium tuberculosis is the causative organism of tuberculosis in humans. Mycobacteria, however, are also distributed in soil. Many can flourish on paraffin and aromatic hydrocarbons. They can be cultivated from soil on simple mineral salts media in an atmosphere containing volatile hydrocarbons such as petroleum or naphthalene. They have a relatively rapid growth rate in the absence of complex nutrients. Whereas, pathogenic mycobacteria are dependent on complex media.

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Plate 35.1. Actinobacteria (Filamentous bacteria). 1. Streptomyces sp. 2. Actinomyces sp. 3. Colonies of Streptomyces sp. 4. Colonies of Streptomyces sp. 5. Nocardia sp. Source: (Chitrangada Dash)

Members of the large genus Streptomyces have permanent mycelia; their aerial mycelia are often very well developed and contain aerial hyphae (sporophores), which serve to enhance the spread of the organism by budding or conidia (Plate 35.1). The structure of the sporophores colonial morphology, colour, size, and odour are diagnostic characters that are used to differentiate the many species and strains. The fragrance which emanates from freshly ploughed soil in spring is due to streptomycetes. An oil called geosmin can be isolated from Streptomyces griseus and is responsible for this odour. It is a 1,10-dimethyl-9-decalol. Knowledge about streptomycetes has advanced considerably because of their practical importance as producers of many effective antibiotics. Streptomycin (from S. griseus), chloramphenicol (from S. venezuelae), and aureomycin and tetracyclin (from S. aureofaciens) are the most successful therapeutics among the antibiotics. Many streptomycetes degrade cellulose, chitin, and other recalcitrant natural substances. One cellulose-degrading organism, widely distributed in soil and in rotting aqueous sediments, is Micromonospora. It has flat colonies with no aerial mycelia and its spores occur singly at the ends of weakly branched sporophores. Microbispora is morphologically similar but produces aerial mycelia and paired conidia. Several actinomycetes (Actinoplanes, Streptosporangium, Ampullariella) do not produce spores directly on the aerial mycelia, but in sporangia. Streptosporangium is a cellulose-degrading aerobic streptomycete. On solid media it grows at first by a substrate mycelium, but forms aerial mycelia at a later stage. The tips of the aerial mycelia enlarge and form spherical sporangia 5-8 m in diameter; these can reach 18 m at maturity. The supporting hypha grows into the spherical end cell,

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where it assumes a helical form and liberates sporangiospores. In Streptosporangium these are non-motile. Actinoplanes grows submerged on plant remains and also produces its spores within button-like sporangiospores; a flagellar bundle confers motility on these spores. Dermatophilus congolensis is another actinomycete producing motile spores. It causes dermatitis of the dorsal skin in sheep and horses and can grow on solid media as smooth or rough colonies, forming a dense substrate mycelium. The hyphae divide longitudinally and transversely, so that up to eight parallel rows of coccoid cells are produced and liberated by autolysis of the hyphal wall. These coccoid spores are also motile by means of flagella. The spores of actinomycetes are usually not heat resistant, but they can withstand dehydration. The only thermophilic actinomycete that forms heat-resistant spores is Thermoactinomyces vulgaris. It occurs on damp haystacks and piles of organic waste, where heat is generated. In their structure and dipicolinic acid content, the spores resemble the endospores of Bacillus and Clostridium in their structure and presence of dipicolinic acid. Actinorhizal nitrogen fixation by root nodules in non-leguminous plants Actinorhizal nitrogen fixation is brought out by Frankia spp. Endosymbiotic N2- fixation by Frankia contributes to a tune of 150 – 200 kg N/ha/annum. The following plants belong to the most effective nitrogen fixers: Casuarina equisetifolia, Alnus, Hippophae, and Ceanothus. Myrica, Dryas, Elaeagnus, and Shepherdia are somewhat less effective as nitrogen fixers. The root nodules of woody plants can attain the size of tennis balls. They consist of densely packed, coral-like, branching roots that have ceased to grow. In Casuarina the nodules consist of a loose bundle of thickened rootlets whose growth is negatively geotropic. Only the outer parenchyma cells are infected by the symbionts. Infection of the roots occurs from the soil, via the root hairs, as in the leguminosae. They also share the presence of leghaemoglobin with the nodules of the leguminosae. A strain of Rhizobium was identified as the endosymbiont in a nonleguminous plant, Parasponia parviflora. This strain was transferred to bean plants, and the resulting nodules were active in N2 -fixation. ARCHAEBACTERIA Archaebacteria belong to the Kingdom Monera. Archaeal cells have unique properties distinguishing them from Bacteria and Eukaryota. The Archaea are divided into four http://en.wikipedia.org/wiki/Phylumphyla. Archaea were initially regarded as extremophiles living in harsh environments, such as hot springs and salt lakes, but they have been found in a broad range of habitats, including soils, oceans, marshlands and the human colon and navel also. They are particularly plenty in the oceans, and the archaea in plankton may be one of the most abundant groups of organisms on the planet and may play roles in both the carbon and nitrogen cycles. No clear examples of archaeal pathogens or parasites are known, but they are often mutualists or commensals. One example is the methanogens that inhabit the human and the ruminant guts, where their vast numbers

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aid digestion. Methanogens are used in biogas production and sewage treatment, and enzymes from extremophile archaea that can endure high temperatures and organic solvents are exploited in biotechnology. Archaea and bacteria are similar in size and shape. Haloquadratum walsbyi has the flat and square-shaped cells. The genetic makeup of archaea possess genes and several metabolic pathways that are more closely related to eukaryotes, notably the enzymes involved in transcription and translation. The cell membranes posses’ ether lipids. Archaea use more energy sources than eukaryotes: ranging from organic compounds, such as sugars, to ammonia, metal ions or even hydrogen gas. Salttolerant archaea (Haloarchaea) use sunlight as an energy source, to fix carbon. No known species of archaea does both the functions. Archaea reproduce asexually by binary fission, fragmentation, or budding. They do not form spores (Fig. 35.1).

Fig. 35.1. Halobacterium sp. (Source: Wikipedia)

Archaea were first classified as a separate group of prokaryotes in 1977 by Carl Woese and George E. Fox in phylogenetic trees based on the sequences of ribosomal RNA (rRNA) genes. These two groups were originally named the Archaebacteria and Eubacteria and treated as kingdoms or sub-kingdoms, which Woese and Fox termed Urkingdoms. Woese argued that this group of prokaryotes is a fundamentally different sort of life. To emphasize this difference, Woese later proposed a new natural system of organisms with three separate Domains namely the Eukarya, the Bacteria and the Archaea. The major groups of archaebacteria include (a) the methanogenic archaebacteria, (b) the archaebacterial sulfate reducers, (c) the extremely halophilic archaebacteria, (d) the cell wall less archaebacteria, and (e) the extremely thermophilic S metabolizers. Methanogenic Bacteria (Methanogens): Almost all the shapes known in the eubacteria can be found in the methanogens: cocci (Methanococcus vannielii); rods (Methanobacterium formicicum); short rods (Methanobrevibacter ruminantium, M. arboriphilicus); spirilla (Methanospirillum hungatei); coccal packets, (Methanosarcina barkeri); filaments (Methanothrix soehngenii); and even square bacteria (Methanoplanus limicola). There are mesophilic and thermophillic species (Methanobacterium thermoautotrophicum, Methanothermus fervidus). Six families are distinguished already, and the number of known species and genera is constantly increasing. The GC content varies between 27 and 61 mol%.

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Halobacteria: The genera Halobacterium and Halococcus consist of extreme halophiles. They are aerobes and heterotrophs and are found in salterns in which sea water is evaporated for the production of salt. During the mass proliferation of the carotenoid containing halobacteria, the water appears dark red. Their optimal growth range lies from 3.5-5 M NaCl. They also have the special property of being able to utilize light energy for their metabolism. Thermo-acidophilic Bacteria: This group at present contains, nonmethanogenic thermophilic archaebacteria, which do not seem to have many common features. They include autotrophs and heterotrophs, extreme acidophiles and neutrophiles, and aerobes and anaerobes. Sulfolobus acidocaldarius is found in hot acid springs and oxidizes sulphur to sulphate. The position of Thermoplasma acidophilus seems quite isolated. It lacks a cell wall, as do the mycoplasmas, but grows optimally at 59ºC and pH 1-2. Its usual habitat is in heat-generating coal slagheaps, but it has also been found in a hot spring. It has the smallest genome so far known in non-parasitic bacteria (1 x 109). It can grow heterotrophically under aerobic conditions in the presence of yeast extract. T. acidophilus differs from a group of anaerobic species collected under the name of Thermoproteales. They were isolated from hot springs, volcanoes, and from the sea floor. They are all extremely thermophilic with temperature optima of 85-105°C, and their metabolism is of the type described as ‘sulphur respiration’ i.e., they oxidize hydrogen and reduce elemental sulphur to hydrogen sulphide. The group includes facultative autotrophs (Thermoproteus tenax), obligate autotrophs (Thermoproteus neutrophilus, Pyrodictium occultum), and heterotrophs (Desulfurococcus, Thermococcus). Archaea had been identified in non-extreme environments as well. They are known to be a large and diverse group of organisms that are widely distributed in nature and are common in all habitats. This has resulted from using polymerase chain reaction (PCR) to detect prokaryotes from environmental samples (such as water or soil) by multiplying their ribosomal genes. This allows the detection and identification of organisms that have not been cultured in the laboratory. Woese demonstrated that the bacteria, archaea, and eukaryotes represent separate lines of descent that diverged early on from an ancestral colony of organisms. One possibility is that this occurred before the evolution of cells, when the lack of a typical cell membrane allowed unrestricted lateral gene transfer, and that the common ancestors of the three domains arose by fixation of specific subsets of genes. The abundant use of ether-linked lipids in their cell membranes may be a contributing factor to the ability of many Archaea to survive in extreme environments, such as extreme heat and salinity. Another unique feature of Archaea is methanogenesis (production of methane). Methanogenic Archaea play a pivotal role in ecosystems with organisms that derive energy from oxidation of methane, many of which play a critical role in the carbon cycle, breaking down organic carbon into methane, which is also a major greenhouse gas. Within prokaryotes, archaeal cell structure is most similar to that of Grampositive bacteria, largely because both have a single lipid bilayer and usually contain a thick sacculus of varying chemical composition.

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Archaea and bacteria have generally similar cell structure. Like bacteria, archaea lack interior membranes and organelles. Archaea cell membranes are usually bounded by a cell wall like bacteria, and they swim using one or more flagella. Structurally, archaea are most similar to Gram-positive bacteria. Majority have a single plasma membrane and cell wall, and lack a periplasmic space; the exception to this general rule is Ignicoccus, which possess a particularly large periplasm that contains membrane-bound vesicles and is enclosed by an outer membrane. Archaeal membranes are made of molecules that differ widely from those in other life forms, showing that archaea are related only distantly to bacteria and eukaryotes. In all organisms cell membranes are made of molecules known as phospholipids. These molecules possess both a polar part that dissolves in water (the phosphate ‘head’), and a ‘greasy’ non-polar part that does not have the lipid tail. These dissimilar parts are connected by a glycerol moiety. In water, phospholipids cluster, with the heads facing the water and the tails facing away from it. The major structure in cell membranes is a double layer of these phospholipids, which is called a lipid bilayer. Most archaea possess a cell wall with the exception of Thermoplasma and Ferroplasma. Unlike bacteria, archaea lack peptidoglycan in their cell walls. Methanobacteriales do have cell walls containing pseudopeptidoglycan, which resembles eubacterial peptidoglycan in morphology, function, and physical structure, but pseudopeptidoglycan is distinct in chemical structure. It lacks D-amino acids and N-acetylmuramic acid. Archaeal flagella appear to have evolved from bacterial type IV pili. In contrast to the bacterial flagellum, which is hollow and is assembled by subunits moving up the central pore to the tip of the flagella, archaeal flagella are synthesized by adding subunits at the base. Physiology Archaea do not carry out photosynthesis. Archaeal energy sources are extremely diverse, and range from the oxidation of ammonia by the Nitrosopumilales to the oxidation of hydrogen sulfide or elemental sulfur by species of Sulfolobus, using either oxygen or metal ions as electron acceptors (Table 35.1). Table 35.1. Nutritional types in archaeal metabolism Nutritional type

Source of energy

Source of carbon

Examples

Phototrophs

Sunlight

Organic compounds

Halobacteria

Lithotrophs

Inorganic compounds

Organic compounds and Carbon fixation

Ferroglobus, Methanobacteria, Pyrolobus

Organic compounds, carbon fixation

Pyrococcus, Sulfolobus, Methanosarcinales

Organotrophs Organic compounds

Phototrophic archaea use light to produce chemical energy in the form of ATP. In the Halobacteria, light-activated ion pumps like bacteriorhodopsin and

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halorhodopsin generate ion gradients by pumping ions out of the cell across the plasma membrane. The energy stored in these electrochemical gradients is then converted into ATP by ATP synthase. This process is a form of photophosphorylation. The proteins that archaea, bacteria and eukaryotes share form a common core of cell function, relating mostly to transcription, translation, and nucleotide metabolism. Other characteristic archaeal features are the organization of genes of related function – such as enzymes that catalyze steps in the same metabolic pathway into novel operons, and large differences in tRNA genes and their aminoacyl tRNA synthetases. Transcription and translation in archaea resemble these processes in eukaryotes more than in bacteria, with the archaeal RNA polymerase and ribosomes being very close to their equivalents in eukaryotes. Halobacterium volcanii, an extreme halophilic archaeon, forms cytoplasmic bridges between cells that appear to be used for transfer of DNA from one cell to another in either direction. Archaea reproduce asexually by fragmentation, budding; binary or multiple fission, meiosis does not occur, so if a species of archaea exists in more than one form, all have the same genetic material. Cell division is controlled in a cell cycle; after the cell's chromosome is replicated and the two daughter chromosomes separate, the cell divides. In the genus Sulfolobus, the cycle has characteristics that are similar to both bacterial and eukaryotic systems. Archaea do not form spores. Some species of Haloarchaea form thick-walled structures that are resistant to osmotic shock and survive in water at low salt concentrations. Interactions with other organisms The well characterized interactions between archaea and other organisms are either mutual or commensal. There are no clear examples of known archaeal pathogens or parasites. One example of mutualism is the interaction between protozoa and methanogenic archaea in the digestive tracts of animals that digest cellulose, such as ruminants and termites. In these anaerobic environments, protozoa break down plant cellulose to obtain energy. This process releases hydrogen as a waste product, but high levels of hydrogen reduce energy production. When methanogens convert hydrogen to methane, protozoa benefit from more energy. Archaea can also be commensals, benefiting from an association without helping or harming the other organism. Methanobrevibacter smithii is the most common archaeal member in the human flora of all the prokaryotes in the human gut. In termites and in humans, these methanogens may in fact be mutualists, interacting with other microbes in the gut to aid digestion. Archaean communities also associate with a range of other organisms, such as on the surface of corals and in the region of soil that surrounds plant roots (the rhizosphere).

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BIOPROSPECTING OF ARCHAEA Archaea recycle elements such as carbon, nitrogen and sulfur through their various habitats. Archaea carry out many steps in the nitrogen cycle. This includes both reactions that remove nitrogen from ecosystems (such as nitrate-based respiration and denitrification) as well as processes that introduce nitrogen (such as nitrate assimilation and nitrogen fixation). Researchers recently discovered Archaean involvement in ammonia oxidation reactions. These reactions are particularly important in the oceans. The archaea also appear crucial for ammonia oxidation in soils. They produce nitrite, which other microbes then oxidize to nitrate. Plants and other organisms consume the latter. In the sulfur cycle, archaea that grow by oxidizing sulfur compounds release this element from rocks, making it available to other organisms. Sulfolobus, produce sulfuric acid as a waste product and the growth of these organisms in abandoned mines can contribute to acid mine drainage and other environmental damage. In the carbon cycle, methanogen archaea remove hydrogen and play an important role in the decay of organic matter by the populations of microorganisms that act as decomposers in anaerobic ecosystems, such as sediments, marshes and sewage-treatment. Methanogens are the primary source of atmospheric methane, and are responsible for most of the world's methane emissions. As a consequence, these archaea contribute to global greenhouse gas emissions and global warming. Methane has an anthropogenic global warming potential (AGWP) of 29, which means that it is 29 times stronger in heat-trapping than carbon dioxide is over a 100year time scale. Extremophile archaea, are a source of enzymes that function under harsh conditions. These enzymes have found many uses. Thermostable DNA polymerases, such as the Pfu DNA polymerase from Pyrococcus furiosus, revolutionized molecular biology by allowing the polymerase chain reaction to be used in research as a simple and rapid technique for cloning DNA. In industry, amylases, galactosidases and pullulanases present in other species of Pyrococcus function at over 100ºC allow food processing at high temperatures, such as the production of low lactose milk and whey. Enzymes from these thermophilic archaea play a role in synthesizing organic compounds. Methanogenic archaea are a vital part of sewage treatment, since they are part of the community of microorganisms that carry out anaerobic digestion and produce biogas. In mineral processing, acidophilic archaea display promise for the extraction of metals from ores, including gold, cobalt and copper. Archaea host a new class of potentially useful antibiotics. A few of these archaeoins have been characterized, especially within Haloarchaea and Sulfolobus which may have novel modes of action. In addition, they may allow the creation of new selectable markers for use in archaeal molecular biology.

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MOLLICUTES The distinguishing feature of the mollicutes group of eubacteria is that they lack a defined cell wall. Hence, they are called ‘mollicutes’ (meaning ‘soft skin’) (Latin mollis = soft or pliable, cutis = skin). They were discovered over 100 years ago and evolved from bacteria. In 1898 the French scientists, E. Nocard and E.R. Roux, studying pleural fluids of cattle suffering from bovine Pleuropneumonia, discovered the organisms that were entirely different from other microorganisms. When cultivated on rich organic media containing about 20% of animal serum, the organisms were found in different forms as spheroid, thin branching filaments, stellate or asteroid structures and other irregular forms. Similar pleomorphic (pleo=many, morphe=forms) organisms were later isolated from other animals. Similar forms were also found growing as saprophytes in decaying organic matter. These were named as pleuropneumonia-like organisms (PPLO). The species discovered by Nocard and Roux was given the first binomal as Asterococcus mycoides by Borrel et al (1910), meaning rounded and stellate forms with radial, mold-like filaments. It was later on put under the genus Mycoplasma by Nowak (1929) and these organisms are now commonly called mollicutes or mycoplasmas. In its laboratory evolution, the mycoplasmas have become more invasive, more difficult to find and capable of causing severe diseases in humans. Diseases, like Gulf War Illness (GWI), Chronic Fatigue Syndrome (CFS), Fibromyalgia Syndrome (FMS), Multiple Chemical Sensitivity Syndrome (MCS), Rheumatoid Arthritis, acquired immunodeficiency syndrome (AIDS) etc. Class Mollicutes includes one order, Mycoplasmatales, and three families (Mycoplasmataceae, Acholeplasmataceae, and Asteroplasmataceae). When growing on agar, most species form colonies with a ‘fried-egg’ appearance because they grow into the agar surface at the center while spreading outward on the surface at the colony edges. Recently the complete genome of Mycoplasma genitalium, a parasite of human genital and respiratory tracts, has been sequenced. The M. genitalium genome is only 580 kilo bases long and appears to have 482 genes; it seems that not many genes are required to sustain a free-living existence. Mycoplasmas can be saprophytes, commensals or parasites, and many are pathogens of plants, animals, or insects. Mycoplasmas are usually more osmotically stable than eubacterial protoplasts and their membrane sterols may be a stabilizing factor. Mycoplasmas appear to be very important, even though their roles in nature and disease are not yet completely clear. They are remarkably widespread and can be isolated from animals, plants, soil, and even compost piles. Indeed, about 10% of the cell cultures in use are probably contaminated with mycoplasmas, which seriously interfere with tissue culture experiments and are difficult to detect and eliminate. Mycoplasmas cause several major diseases in livestock, for example, contagious bovine pleuropneumonia in cattle (M. mycoides), chronic respiratory disease in chicken (M. galsepticum), and pneumonia in swine (M. hyopneumoniae). M. pneumoniae causes primary atypical pneumonia in humans, and there is increasing evidence that M. hominis and Ureaplasma urealyticum are also human pathogens. Spiroplasmas have been isolated from insects, ticks, and a variety of plants. They cause disease in citrus plants, cabbage, broccoli, corn, honeybees, and other hosts. Arthropods probably often carry the spiroplasmas.

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Mollicutes are true cells. Like animal cells they do not possess demonstrable cell wall. The sole retaining structure is the cytoplasmic membrane that has a typical phospholipid protein bilayer, flexible and 7-11 nm thick. Mollicutes are simpler than the cells of higher plants and animals (Fig. 35.2). They possess all the necessary biochemical machinery to grow and multiply in the absence of other cells. The genetic machinery is in the form of DNA, RNA and ribosomes.

Fig.35.2. Structure of mycoplasma. 1. Cytoplasmic membrane. 2. Ribosomes. 3. DNA strand

They vary in size mostly from 300 nm to about 0.2 m in diameter. The volume of the smallest PPLO is about 1 x 10–3 m3. The internal structure is typical of prokaryons. The cell membrane encloses the cytoplasm that contains numerous ribosomes, fibrillar DNA, electron dense areas, soluble RNA and protein. Nuclear structures, however, are less evident than typical bacteria. Mesosomes are absent. However, cells of mollicutes divide unevenly into very minute bodies called the elementary bodies (size from about 330 m to 450 m) or minimal reproductive units. They are commonly formed inside the large bodies. These can pass through bacteria proof filters like viruses but are viable on ordinary media. These bodies are often cited as the smallest independent living entities. These represent a stage in their life cycle. They enlarge to form long filaments and mycelia and produce chains of minute spores like conidia but much smaller in size. It is thought by some, that these conidia-like bodies are liberated and each increases in size to become larger body several m in dia., inside which new elementary bodies are formed. These are released by rupture of the membrane of larger body. Growth rate of mycoplasma is very rapid, generation time is 1-3 hrs. Like viruses and animal cells, mycoplasmas are resistant to penicillin. Many other type of structures are also present. The 16 SrRNA sequencing has confirmed that the mollicutes are a coherent phylogenetic group that is closely related to the Clostridia. For many years, the yellowing of plants was thought to be caused by viruses. In 1967, however, the Japanese workers, Doi et al discovered pleomorphic mycoplasma like organisms (MLOs) in the phloem cells of plants affected by different yellowstype diseases. Since then MLOs have been found to infect over 300 plants. They occur from temperate to tropical regions but it is in the warmer areas that serious losses occur in crops like coconuts, citrus, rice, maize, cotton and potatoes. They also occur in insects and as saprophytes in soil and sewage

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Protoplasts in hypertonic or isotonic fluid are often able to grow and multiply on agar media. The colonies resemble the mycoplasmas. These cells without cell walls have been designated as stable L-forms. Stable L-forms obtained from animal and human are different from mycoplasmas. Recent genetic studies have shown that L-forms are different from mycoplasmas. For example, DNA base composition of Mycoplasma species ranges from 23 to 36 (39.41 in M. pneumoniae) mole per cent GC, whereas most other bacteria and the L-forms usually have higher per cent GC content (63-64 mole per cent GC). The mycoplasmas are distinguished by their lack of a cell wall, the outer boundary of the cells being the cytoplasmic membrane; the cells have plasticity and can assume many different shapes ranging from spheres to branched filaments. The plasticity allows many of the cells to pass through bacteriological filters even though the smallest cells are about 0.3 µm in diameter. They are susceptible to lysis by osmotic shock caused by sudden dilution of the medium with water. Mycoplasmas can be cultivated in vitro on nonliving media as facultative or obligate anaerobes. They have genomes that are about one-fifth to one-half the size of those of other bacteria capable of growing on nonliving media, which explains why these organisms have complex nutritional requirements and limited biosynthetic abilities. The colonies that are embedded in the agar surface will have a characteristic fried egg appearance. Mycoplasmas differ from the ‘L-phase variants’ that can develop from bacteria. Such variants are osmotically fragile, cell-wall-defective form may occur spontaneously (as in the genus Streptobacillus) or as the continuous exposure to sublethal levels of penicillin. They form fried-egg colonies resembling those of mycoplasmas. Moreover, penicillin-binding proteins and peptidoglycans can be demonstrated in the membranes of L-phase variants but not in the membranes of mycoplasmas. These mycoplasmas are parasites of the mucous membranes and joints of human/ animals and require cholesterol for growth. Many species of the Mycoplasma are pathogenic to animals; the species, M. pnenumoniae is the causative agent of primary pneumonia. Members of the genus Ureaplasma require urea for growth which causes urethritis in humans, pneumonia in cattle, and urogenital diseases and other animal species. These mycoplasmas do not require cholesterol for growth. They are distributed in vertebrates, in sewage, soil and possibly on plants. These organisms are unusual in that they are helical and exhibit a slow motility. How a helical shape is maintained in the absence of a cell wall, how the cells can swim while lacking flagella is unknown. The family has a single genus, Spiroplasma. The organisms are pathogenic to citrus plants. They can be isolated from plant fluids and plant surfaces and arthropods that feed on plants. The most routinely used techniques for identification of the mycoplasmas are immunological (haemaglutinin) or complement-fixing antigen-antibody reactions. These microorganisms are slow growing, therefore positive results from isolation procedures are rarely available before 30 days - a long delay with an approach that offers little advantage over standard techniques. Recently DNA probes have been applied for the detection of Mycoplasma pneumonias in clinical specimens.

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RICKETTSIAE The rickettsiae are named after H. T. Ricketts, who discovered American ‘Rocky Mountain spotted fever’. This group is also referred to as the 'spotted fever' (typhoid fever) produced by Rickettsia prowazekii. Their habitat is host vectors such as lice, fleas, ticks, mites, etc., in which they exist as harmless parasites or even symbionts. They are transferred to other animal hosts or humans by bites, scratches, or inhalation. They can be differentiated from viruses in certain respects. They contain DNA as well as RNA in a ratio of 1:3.5. The cells are surrounded by a cell wall that contains muramic acid and is lysozyme sensitive. Nuclear regions and cell walls can also be distinguished in electron micrographs of thin sections. Most rickettsiae have never been cultured except in living cells, but they can be grown in incubating chicken eggs or in experimental animals. About 109 cells can be produced in the yolk-sac of a chicken egg. The best-known pathogenic rickettsiae are those of the typhoid group. R. prowazekii is the causative agent of typhoid fever. The organism is spread by rats which remain symptomless and is transmitted from rat to rat and to humans by fleas. Coxiella burnetii, which causes Q-fever, can survive outside the host. It is transmitted by ticks to sheep, goats, cattle and can infect humans not only by tick bites but also via animal dust, infected soil, and consumption of milk. The usual pasteurization of milk (heating at 60°C for 30 min) does not affect Coxiella. CHLAMYDIAE Chlamydiae are human pathogens. Chlamydia trachomatis causes trachoma, the Egyptian eye infection that causes conjunctivitis and leads to blindness, and the venereal disease, lymphogranuloma venereum (Fig. 35.3). In both cases transmission is by contact. Chlamydia psittaci is the causative agent of ornithoses, the best known of which is psittacosis, a type of pneumonia. The main hosts of chlamydiae are birds. On the basis of their biochemical characteristics, chlamydiae belong to the prokaryotes (bacteria) and not, to the viruses. They contain RNA and DNA in a ratio characteristic of bacteria, and they synthesize substances which eukaryotic cells are unable to produce, such as muramic acid, diaminopimelic acid, D-alanine, and folic acid. These properties are in accord with their sensitivity to penicillin and sulphonamides. Their genome is very small (relative size, 0.66 x 109) and corresponds to only about one quarter of the genetic information of E. coli. Chlamydiae only grow in living cells and are cultured in chicken eggs and tissue cultures. Their dependence on the metabolism of host cells is apparently due to absence of an ATP-generating system of their own. However, they are extraordinarily permeable to ATP and CoA and therefore, be regarded as ‘energy parasites’.

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Fig. 35.3. Chalmydiae life cycle

PRIONS The prions are proteins that are transmissible to other prion proteins. The first prion protein was discovered in mammals and is referred to as the major prion protein (PrP). The word prion, coined in 1982 by Stanley B. Prusiner, is derived from the words protein and infection, in reference to a prion's ability to self-propagate and transmit its conformation to other prions. The prion protein PrP was first discovered as the infectious agent causing mammalian transmissible spongiform encephalopathies also known as ‘mad cow disease’ and scrapie in sheep. In humans, PrP causes Creutzfeldt-Jakob Disease, and variant of Creutzfeldt-Jakob Disease, GerstmannSträussler-Scheinker syndrome, Insomnia and Kuru. All known prion diseases in mammals affect the structure of the brain or other neural tissue and are currently untcontrollable and fatal. Prion proteins are also reported in a variety of other mammalian proteins. Some of these proteins have been found to cause degenerative disorders such as amyotrophic lateral sclerosis, frontotemporal lobar degeneration with ubiquitin-positive inclusions, Alzheimer's disease, and Huntington's disease. Prions are not considered living organisms as they are misfolded protein molecules. If a prion enters a healthy organism, it induces existing, properly folded proteins to convert into the misfolded prion form. In yeast, this refolding is assisted by chaperone proteins such as Hsp104p. All known prions induce the formation of an amyloid aggregates. These aggregates are fibrils, which grow at their ends, and replicate to give rise to many growing ends. The incubation period of prion diseases is determined by the exponential growth rate associated with prion replication, which is a balance between the linear growth and the breakage of aggregate (Fig. 35.4).

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Prion aggregates are extremely stable and accumulate in infected tissue, causing tissue damage and cell death (Fig. 35.4). This indicates that prions are resistant to denaturation by chemical and physical agents. Prion structure varies slightly between species. The precise structure of the prion is not known, though they can be formed by combining prion proteins, polyadenylic acid, and lipids in a Protein Misfolding Cyclic Amplification (PMCA) reaction. Proteins showing prion-type behavior are also found in some fungi. Fungal prions do not appear to cause disease in their hosts.

Fig. 35.4. Characteristic holes in the prion affected host tissues (Source: Wikipedia)

CYANOBACTERIA (BLUE-GREEN ALGAE) Blue-green algae are bacteria, also known as cyanobacteria. This group of organisms is quite similar to bacteria in metabolism and structure. It has the following basic characteristics.

Fig. 35.5. Cynobacterial cellstructure

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The cells are blue-green or violet, sometimes also red or green. The blue colour of an accessory pigment, phycocyanin, and the red colour of another accessory pigment, phycoerythrin masks the green color of chlorophyll. Both accessory pigments are associated with proteins and are located in granules 30– 40 nm in size. These granules are called phycobilisomes. The cells of cyanobacteria contain only chlorophyll a; chlorophyll b is absent. The photosynthesizing pigments in association with proteins (phycobiliproteins) are bound to lamellar structures, thylacoids, which are freely distributed in the cytoplasm of the cells (chloroplasm) (Fig. 35.5). This is in contrast to eukaryotic algae where they are enclosed in chloroplasts. The major portion of the chlorophyll and all carotenoids are found in thylacoids. DNA is located in the inner part, in the center of the protoplast similarly to bacteria. This central part is called nucleoplasm or centroplasm. Similar to bacterial cells, the cells of blue-green algae do not contain mitochondria, Golgi apparatus, an endoplasmic reticulum, or vacuoles surrounded by tonoplasts. A special kind of starchy substance serves as reserve carbohydrate. Very small bodies of this starch are found among thylacoids. Besides these bodies, bluegreen algae contain granules of arginine and asparagine polymers. Both types of cytoplasm, the chloroplasm and the centroplasm, are enclosed by a cytoplasmic membrane which is the only semipermeable barrier of cyanobacterial cells. The rigid cell walls contain murein as in bacteria. Cyanobacteria are singlecelled free - living organisms or cells held together by gelatinous sheaths in colonies or in filaments. Reproduction occurs by cell division or by spores. Sexual stages have not been observed. Cells with flagella are not known in cyanobacteria. The cells of blue-green algae are simple in shape, spherical, ellipsoidal, cylindrical, ovoidal, crescent or rod-shaped. The size of cells in each species is uniform. Nearly all cells are surrounded by slime consisting of a polysaccharide. The filamentous blue-green algae are of two types, hormogonal and pleurocapsal. The hormogonal types form chains of cells, called trichomes, and an external capsule. The chain of cells is joined with the capsule and forms a physiological unit. But a part may be separated and live alone. This is called a hormogone. The trichome with the capsule forms a filament. Filaments of cyanobactria may be ramified if one cell divides in a vertical direction. Pleurocapsal blue-green algae form short filaments consisting of single cells with very thick membranes which are associated with gelatinous sheaths. Each cell is an independent physiological unit. Some cells in the trichome of hormogonal cyanobacteria may be pale in color and miss some photosynthetic pigments. Such cells are called heterocysts. Nitrogen fixation occurs in these cells but no photosynthesis. A large cell called akinete is found in the neighbourhood of heterocysts. This serves as a resting cell. Usually hormogone fragments separate at the site of heterocysts. Some cyanobacteria are not motile; others move by gliding. The class cyanobacteria consist of the following orders: 1).

Chroococcales with the genera Synechococcus, Chroococcus, Aphanothece, Gloeocapsa, Microcystis, and Merismopedia. This order contains single-celled species or species forming colonies. Cells

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multiply by division and by forming small cell aggregates which are called nanocytes. 2).

Chamaesiphonales with the genera Cyanocysis and Chamaesiphon. These organisms are single-celled or form short filaments. The filaments are not ramified and are fastened at the basal end. Exo- and endospores are produced terminally.

3).

Pleurocapsales with the genus Pleurocapsa are cyanobacteria of the pleurocapsal type.

4).

Nostocales with the genera Oscillatoria, Lyngbya, Microcileus. Nostoc, Anabaena, Aphanizomenon. Scytonama, and Ricularia. These are cyanobacteria of the hormogonal type.

5).

Stigonematales with the genus Stigonema. The filaments of these bluegreen algae are thick and built of several cell chains. Reproduction takes place by means of hormogones.

Cyanobacteria were grouped along with the primitive archaea in the first edition of the second volume of Bergey’s Manual of Systematic Bacteriology. They were placed in section 13 with only one class Prochlorophyta. Two families (Nostocaceae and Scytonemataceae) are present in the order Nostocales while the other four orders are represented by a single family each. The importance of blue-green algae for man may be seen indirectly in the nutrition of useful animals such as fish and water birds. Some species are dangerous and produce toxic substances. Cyanobacteria live in marine and fresh water and are sensitive indicators of the quality of the water. Some species inhabit very moist soil where they penetrate deeply and influence the life of soil organisms by their ability to fix atmospheric nitrogen. Nitrogen fixing Cyanobacteria The soil inhabiting free-living blue green algae are capable of fixing atmospheric nitrogen into soil (Fig 35.6; Table 35.2). They are classified into four groups: 1.

Heterocystous aerobic nitrogen fixers – e.g. Anabaena, Aulosira, Cylindracospermum, Mastigocladus, Nostoc and Tolypothrix.

2.

Nitrogen fixing single celled aerobic colonies – e.g. Gloeocapsa.

3.

Non- heterocystous, filamentous anaerobic nitrogen fixers. These Cyanobacteria fix nitrogen in the presence of CO2 and nitrogen in the atmosphere under anaerobic conditions only. e.g. Oscillatoria, Phormidium.

4.

Non-heterocystous, filamentous aerobic nitrogen fixers. Trichodesmium is an example of this type.

Cyanobacteria fix nitrogen as symbionts inhabiting diverse plant groups from chrysophyta to flowering plants. Cyanobacterial association is found with members of Bryophyta such as Anthoceros, Phaeoceros, Dendroceros and Notothylas. Azolla

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has become an important biofertilizer in rice ecosystem mainly due to its association with cyanobacteria called Anabaena.

Fig. 35.6. Nitrogen-fixing Cyanobacteria. a Scytonema hofmannii; b. Nostoc piscinale; c. Pleurocapsa minor; d,e. Aphanizomenon gracile; f. Rivularia haematites; g.h. Stigonema ocellatum; i. Microcoleus vaginatus; j. Anabaena flos-aquae; A: Akinete; G: Gas vacuole; H: Heterocyst (Source: Van Den Hoek et al 2009)

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Table 35.2. List of Nitrogen fixing Cyanobacteria Name

Family

Chloroglea, Chroococcidiopsis, Gloeothece, Cynecococcus

Chroococcaceae

Mastigocladus

Mastigocladaceae

Microchaete, Richelia

Microchaetaceae

Aphanothece, Aulosira, Cylindracospermum, Nodularia, Nostoc, Anabaena, Anabaenopsis, Pseudoanabaena, Raphidiopsis, Richaelia

Nostocaceae

Lyngbya, Microcoleus, Oscillatoria, Phormidium, Plectonema, Pleurocapsa, Calothrix

Oscillatoriaceae

Dicothrix, Gloeotrichia

Pleurocapsaceae

Scytonema, Tolypothrix, Fritschella, Haplosiphon,

Scytonemataceae

Stigonema, Westellopsis

Stigonemataceae

Advantages Algal biofertilizer have several advantages over chemical fertilizers and are of immense use in Indian agriculture, particularly with small and marginal farmers and their economic conditions. Unlike chemical fertilizers, these are renewable as they grow, spread and stay in the soil for many years. In addition to nitrogen fixation, it helps to improve soil texture by soil aggregation. It has no pollution problems and also helps the crop to resist against fungal diseases like paddy blast. Addition of super phosphate to soil encourages the growth of blue green algae (BGA) in soil. Application of BGA 10 days after transplantation proved to give good results. Algal fertilizers are of special significance in mobilizing various micro and macro nutrients besides reclamation of usar and saline soils. Spirulina – a Single cell Protein Algae as an alternate source of food has assumed importance in addition to animal and plant based proteins. Chlorella, a member of chlorophyceae is well known for its value as single cell protein (SCP). In addition to Chlorella, the cyanobacteria called Spirulina is another algal food which has attained importance. It is a filamentous form with 60-70% proteins, 8-20% carbohydrates, 9-15% lipids and 2.5 – 4.5% nucleic acids. Spirulina protein consists of essential amino acids like lucine, isolucine, tyrosine, valine, phenylalanine in large quantities. It also contains vitamins like thiamin, riboflavin, provitamin–A, vitamin E, cobalamine, biotin and folic acid. The nucleic acid content is also relatively low and it is rich in unsaturated fats. In view of its nutritive value, researchers at Central Food Technology Research Institute (CFTRI) recommended Spirulina for heart and obese patients. A dose of 20g/ day of Spirulina provides all the required B group vitamins to the body. Spirulina is preferred over Chlorella as the latter has cell wall consisting of cellulose which requires special treatment for digestion. On the other hand Spirulina is easily digestible as the proteolytic enzymes of the host can directly act upon the

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proteins of Spirulina. The cultivation of Spirulina is also easy and even the waste water can be used for this purpose. The Spirulina thus produced may be used in pisciculture, poultry and as animal feed-which helps in obtaining higher yields. Spirulina cultivation is an age old practice in Africa and Mexico. It is cultivated in natural salty lakes with pH 9.4 -11.0 Chad lake in Africa, Texcoco lake in Mexico are world famous for Spirulina natural growth. In India, Spirulina may be grown using usar and saline soils as it does not require any fertile land or specific growth conditions. SELECTED MEMBERS OF CYANOPHYCEAE GENUS: OSCILLATORIA Class : Cyanobacteria Order : Oscillatoriales Family : Oscillaroriaceae Oscillatoria generally grow in fresh water ponds, lakes and some species grow on bark of the trees (O. agarclhii), walls and moist soils. It occurs as balls or as mats floating over the water (O. princeps). Structure: Unbranched, cylindrical, filamentous thallus is known as 'trichome'. All cells are similar in the trichome except the apical cell. The apical cell is conical or cap like and appear as calyptra. The cells are longer than width or vice versa. Sometimes they are equal in length and width. Mucilagenous sheath is rarely found in some species. Protoplasm is differentiated into chromatoplasm and centroplasm. Some species of Oscillatoria exhibit chromatic adaptation to light. Reproduction Oscillatoria reproduce by harmogones. Harmogones are broken filaments either accidentally or due to the death of few cells in the filament. Harmogones are released as a result of biconcave dead cells in the filament. GENUS : NOSTOC Class : Cyanophyceae Order : Nosctocales Family : Nostocaceae They inhabit moist soils and fresh water ponds with stagnant water. They also grow on rocks in flowing water. Some species occur in paddy field soils. Nostoc punctiformae is associated with the thallus of bryophyte Anthoceros. Nostoc also lives symbiotically in the leaves of Azolla and corolloid roots of Cycas and as a phycobiont in the lichens. Structure: Nostoc occurs generally as mucilaginous balls. Several filaments of the thallus aggregate into mucilage and form spherical or round balls. Trichomes are curved and the cells are globular or cylindrical or barrel shaped. Each trichome is

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surrounded by a hyaline or pigmented mucilage layer. Generally heterocysts are intercalary but sometimes occur terminally also (Fig. 35.6b). Reproduction: Reproduction takes place by two types of reproductive structure viz. hormogones and akinetes. Hormogones: Trichomes break at heterocysts that result in fragments. Some of the cells in the middle of the trichome die and separate the fragments. These fragments come out of the mucilage and form a new trichome. Akinetes: The immediate cells on either side of the heterocysts convert into Akinetes under unfavourable conditions. Sometimes all the vegetative cells of the trichome get converted into akinetes. Akinetes are characterized by a thick wall and germinate when the conditions are conducive to give rise to a new trichome. Heterocyst of N. commimi germinates to form a new alga. GENUS : ANABAENA Class : Cyanophyceae Order : Nostocales Family : Nostocaceae Anabaena inhabits the permanent lakes not temporary ponds. It occurs either as individual filaments or in colonies. They float on the water surface. All the cells of the filament are uniform in thickness except the apical cell which is tapered. Trichomes are straight or circular or irregularly curved which are covered by a transparent sheath. The sheath can be seen only by special methods. Cells are globular or barrel shaped and rarely cylindrical. Cells are never disc like. The cytoplasm of vegetative cells is uniform with small granules and false vacuoles. Heterocysts are like vegetative cells but little larger in size and are intercalary. Akinetes are formed singly or in a chain of few cells accompanying the heterocysts. Akinetes are larger than vegetative cells (Fig. 35.6j). Anabaena differs from Nostoc in having a transluscent mucilaginous sheath and it does not form aggregates in specific shape. Nostoc, on the other hand forms mucilage balls and trichomes are covered always by a mucilaginous sheath. PROTOZOA Protozoa are the most highly specialized, in their cell structures, modes of life, reproduction and are the most complex of all the Protista. Unless one includes the flagellate algae, protozoa are the only distinct group in the entire Kingdom Protista. There are four principal groups (classes) of protozoa, as follows (Fig. 35.7): 1.

Sarcodina: move with pseudopods, some species also have flagella (Amoeba, Radiolaria, Foraminifera)

2.

Ciliata: move with cilia (Paramaecium, Opalina).

3.

Mastigophora: move with flagella (Euglena, Trypanosoma).

4.

Sporozoa: move with pseudopods (Monocystis, Plasmodium, Noisama). Only in immature stages; the male gamete is flagellate.

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Some authors include a fifth class, Suctoria, in which young stages are ciliated while adult stages are sessile and provided with tentacles (e.g. Podophyra, Trichophyla).

Fig. 35.7. Soil Protozoa (Source: Martin Alexander 1961)

Their outer covering consists only of the cell membrane. In some groups such as the amoebas, this is a thin, limp membrane that permits amoeboid movement and phagocytosis, both motivated by intracellular cytoplasmic streaming. In some freeliving protozoa the peripheral layer of cytoplasm is condensed into a protective layer called ectoplasm. In other types, especially the large and active, flagellated and ciliated species, a thick and more or less flexible outer integument, periplast,

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plasmalemma or cortex is formed from the peripheral layers of cytoplasm. This often contains chitin. As will be seen, the cortex itself, especially of ciliates, may contain numerous organelles, such as cilia, trichocysts, mouth or cytostome, gullet or cytophage, anal pore or cytopyge or cytoproct, and contractile fibers called myonemes. Contractile vacuoles are conspicuous in species of protozoa that live in fresh water. The vacuoles appear to be regulators of intracellular water and hence of intracellular osmotic pressure. Because of the low osmotic tension of fresh water and the much higher osmotic tension of intracellular fluids, cells in fresh water tend to take in water. Like bacterial protoplasts or PPLO, the cells would burst unless the excess water taken in could be excreted. Flagella and Cilia. As is true of flagella of motile green algae, protozoan flagella act as swimming appendages. Cilia are much like flagella in structure and function but are shorter and, unlike flagella, tend to move in a coordinated and rhythmical fashion. Reproduction. As in most protists, asexual reproduction by cell fission is the most efficient and commonly seen means of increasing numbers of cells or new protozoan individuals. In many species, no other kind of reproductive process is known. In other species genetic recombination and multiplication, usually resulting in rejuvenation of the asexual processes; is commonly brought about by conjugation. Encystment. During the life cycle of many protozoa some cells may produce a thick cell wall, lose water and become dormant. During this stage metabolism is reduced to a minimum or ceases entirely, and the dormant cell resists unfavorable environmental conditions such as prolonged drought, summer heat, increased salinity or unfavorable pH. Such a stage may be formed by mature growing cells during sexual cycles of development or just after conjugation of gametes during sexual reproductive cycles. In protozoa these cells are commonly called cysts. Cells going into the cyst stage are said to be encysting. After a physiologically and genetically determined period or, depending on species and type of cyst, when growth conditions again become favorable, the cell inside the cyst wall takes in water, resumes activity, bursts the cell wall and emerges in the actively growing or, in protozoa, trophozoite stage. The encysted cell is said to have excysted. Nutrition of Protozoa. Although some types of protozoa, especially certain blood parasites, exhibit holophytic-type nutrition, protozoa typically differ from plant cells in being able to take solid food particles into the cell. All protozoa are chemoorganotrophic. Ingestion of solid foods by protozoa is accomplished by three methods: by phagocytosis, by means of cytostome and by pinocytosis. In phagocytosis, typical of amoebas and leukocytes of the blood, two or more pseudopodia are extruded like fingers around the food particle. The fingers merge into one, with the particle trapped within. The particle is passed through the cell membrane into the cytoplasm, where it is enclosed in a digestive or food vacuole. In other protozoa, most typically ciliates, food particles are wafted by cilia into a deep pouch or invagination of the cell coating called a cytophage or gullet. The food particle passes through the cell coating at the inner end of the gullet into a

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cytoplasmic digestive food vacuole. Pinocytosis is a somewhat similar process, though not dependent on cilia; the pinocytic gullets are numerous and very tiny. Protozoa commonly eat bacteria, each other and other minute organisms. Protozoa accept or reject food particles with discrimination and have quite a delicate sense of touch (tactile sense), so that they recoil on contact with hard objects, turning aside quite as though they were highly sensitive and responsive creatures. Some like Euglena have eye spots and can distinguish light of different wave lengths.

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Chapter - 36

Diseases Caused by Bacteria and other Prokaryotes

Harmful activities of bacteria Bacteria cause many diseases in plants, cattle, and human beings. These pathogenic bacteria affect their hosts by scavenging the food, destroying the cells and secreting toxins (Table 36.1). Table 36.1. Important diseases caused by bacteria in plants Name of the host

Name of the disease

Name of pathogen

Potato

Brown rot of potato

Pseudomonas solanacearum

Wheat

Yellow rot of wheat

Corynebacterium sepedonicum

Tomato

Bacterial Canker in Tomato Corynebacterium michiganensis

Cotton and root vegetables

Crown Gall

Agrobacterium tumefaciens

Citrus

Citrus Canker

Xanthomonas citri

Rice

Bacterial Blight

Xanthomonas oryzae

Cotton

Angular leaf Spot

Xanthomonas malvacearum

Pears

Fire blight

Pseudomonas solanacearum

Bacteria also cause diseases in human beings. The diseases and causal organisms are given in Table 36.2. Table 36.2. Important diseases caused by bacteria in human beings. Disease

Pathogen

Cholera

Vibrio cholerae

Typhoid fever

Salmonella typhi

Tuberculosis

Mycobacterium tuberculosis

Leprosy

Mycobacterium leprae

Syphilis (STD)

Treponema pallidum

Gangrene

Clostridium perfringens

Diseases Caused by Bacteria and other Prokaryotes Tetanus

Clostridium tetani

Weil's disease

Leptospira icterohaemorrhagiae

Brucellosis

Brucella suis

Meningococcal meningitis

Neisseria meningitidis

Anthrax

Bacillus anthracis

Rheumatic fever

Streptococcus pyogenes

Pneumonia

Streptococcus pneumoniae

Meningitis

Haemophilus influenzae

Diphtheria

Corynebacterium diphtheriae

Legionellosis

Legionella pneumophila

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Crop diseases caused by bacteria, virus and mycoplasma Bacteria and mollicutes (mycoplasma and spiroplasma) are prokaryotes and are single celled organisms. The cytoplasm in mollicutes is surrounded by cell membrane only whereas bacteria have a cell wall in addition to cell membrane. The spiroplasmas of mollicutes were often referred as mycoplasmas that cause disease in plants. On the other hand a virus is a nucleoprotein that multiplies in living cells and also has the ability to cause disease in plants and animals. Plant viruses differ from all other plant pathogens in many respects and cause typical symptoms on diseased plants. Dwarfing and stunting of the entire plant and reduction in yield occur in almost all viral diseases. Plant viruses multiply and move within the plant and once it enters the phloem, it moves rapidly in it towards growing regions. They are transmitted from plant to plant in a number of ways including vegetative propagation, mechanical transmission, through sap, seeds, pollen, dodder and insects. Management of plant viruses in most cases is achieved by spraying insecticides to control the insect vector population. BACTERIAL DISEASES 1. Wildfire of Tobacco Wildfire of tobacco occurs all over the world and causes losses in seedbed affecting all the seedlings. It causes large, irregular, dead areas on leaves in the field which ultimately fall off making the leaves commercially worthless. Symptoms The symptoms initially appear as wet rot at the margins and tips of young leaves in seedbeds. The whole leaf or parts of it may rot and fall off. Seedling may be killed in the seedbed or after transplantation. In the field, round, yellowish Spots 0.5 -1.0 cm diameter develop with brown area at the centre which enlarge rapidly and coalesce resulting in large dead areas on the leaf. Spots appear less frequently on flowers, seed capsules, petioles and stems.

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Causal organism The pathogen is Pseudomonas syringae pv. tabaci. It produces a florescent pigment and potent toxin, called tabtoxin or wildfire toxin. The bacterium over winters in plant debris in the soil, in dried diseased leaves, on seed and contaminated seedbed covers. Bacteria enter the host through stomata, hydathodes and wounds as well as insect vectors like flea beetles, aphids and whiteflies. Disease control 1.

Contaminated seed should be disinfested by soaking in a formaldehyde solution for 10 minutes.

2.

Seedbeds should be sprayed with-a copper fungicide and streptomycin.

3.

Streptomycin sprays should be at weakly intervals until plants are transplanted.

4.

Only healthy seeds should be used and preferably resistant varieties should be grown.

2. Angular leaf spot of cotton The disease is one of the most serious bacterial diseases of cotton (Gossypium malvacearum L.). This disease is also known as ‘Bacterial blight’ and ‘black arm of cotton’. This disease is a major concern in Maharashtra, Karnataka, Telangana, Andhra Pradesh and Tamil Nadu. This disease was first reported in India in 1918 from Tamil Nadu. Symptoms The bacterium attacks all the aerial parts of the plant at different stages of growth. The earliest symptom is seen on the cotyledons of the germinating seeds in the form of water soaked circular lesions which later enlarge to become irregular and brown, causing distortion and withering of the cotyledons. Severely affected seedlings wither and wilt. The water soaked spots on leaves increase in size, become angular, bound by small veinlets and turn brown to black. The infection may also spread to petioles and stems. The affected stems show cracks and gummosis. The bacterium also infects the bolls causing water soaked lesions which later turn into dark brown to black, sunken irregular spots. Infected bolls fall down prematurely. If they mature, lint is of not much commercial value. Causal organism Xanthomonas campestris pv. malvacearum is a rod shaped, gram negative, endospore forming, motile bacterium. The bacterium may remain alive in dried leaves for about 17 years. The primary infection is mainly through seed borne bacteria. The bacteria remain in the form of a slimy mass on the fuzz of the seed coat. On germination of such seeds, the bacterium moves to cotyledons. In favourable weather conditions, inoculum is spread from this source to new leaves and further spread continues. The secondary spread of the bacteria in the field may

Diseases Caused by Bacteria and other Prokaryotes

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be through wind, water and other physical and biological agents. The intensity of infection is dependent on the prevailing weather conditions. High relative humidity following rains and temperatures around 30ºC are reported to favour spread of the disease. Control 1.

Primary infection can be prevented by treating the seed with conc. HCl and with fungicides like Agrosan GN and Ceresan etc. Treating the seed with antibiotics like streptomycin eradicates the internally seed born inoculums.

2.

The secondary spread can be checked by regular sprayings with Agrymycin and Blitox.

3.

Control through development of resistant varieties provides the best and affective preventive measures.

3. Leaf spot of mango This disease was first reported in 1948 from Maharashtra. Symptoms Minute water soaked lesions appear in groups towards the tip of the leaf blade. They increase in size, turn brown to black and are surrounded by chlorotic hallows. Large necrotic patches may be formed by coalescing of several lesions. The patches are rough and raised due to heavy bacterial exudates. In the severe stage of disease, petioles, young fruits and tender shoots may also be infected and heavy defoliation occurs. Cracks may appear on the skin of the fruits, badly affected fruits drop prematurely. Casual organism Xanthomonas campestris pv. mangifera indica is gram negative, rod shaped, non-sporulating, motile with single polar flagellum. In orchards, the bacterium is a phylloplane resident throughout the year. It enters fruits through bruises and other types of injuries. Rain is a major weather factor affecting fruit infection. Control Though no specific control measure is recommended, frequent spray with plantomycin and bavistin reduces the severity of the disease. Insecticides reduce the incidence of insects like weevils, bugs and leaf webbers which sometimes act as carriers of the inoculum. 4. Wilt of potato and tomato This disease is also known as ‘bacterial brown rot’ and causes extensive damage to the potato and tomato crops. It is prevalent in several other plants also including solanaceous members.

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Symptoms The characteristic symptoms of the disease are wilting, stunting and yellowing refers to the browning of the xylem in the vascular bundle. The name ring disease is derived from the fact that a brown ring is formed in the tuber due to the discoloration of the vascular bundles. The skin of the infected tubers is often discoloured. In severely affected tubers eye buds are blackened. If the infected stems are cut across, grayish white bacterial ooze comes out of the vascular ring. When nematode infection is also associated, the stems of the tomato plants became dark and constricted, leading to toppling down of the plants. High humidity and high temperatures favours the disease severity. The causal organism Pseudomonas solanacearum is a motile, gram negative, rod shaped bacterium. The pathogen is reported to survive through soil, seed tubers and alternate hosts. Infection in tomatoes, takes place through injured roots and also through injuries on the stem and leaves. Control 1.

Since the disease is mainly soil borne, crop rotation helps in its control. In India, two year crop rotation with potato - wheat has been suggested.

2.

The disease can also be checked by adopting various field sanitation measures.

3.

Under experimental conditions, foliar sprays of agrimycin or streptomycin are effective against the pathogen.

4.

Cultivation of known resistant varieties is the better way of prevention of the disease.

5. Soft rot of potato Soft rot of potato: Bacterial soft rot of potato is caused by Erwinia carotovora in India, the disease has been investigated in some detail by Hingorani and Addy (1952). Symptoms The plants turn pale green or yellow and soon wilt and die. The affected haulms are jet black in colour at the soil level; later the discoloration extends to the old seed tuber also. The cortical tissue of the basal stem becomes black and shrivels and gives the appearance of a black leg. Sometimes the young seedlings from the diseased tuber may be destroyed soon after the emergence or they may not emerge at all. Soft rot of tubers may occur in the field if the field is moist and temperature high, or during transit and storage. In storage, bacteria cause severe rot of the tubes, sometimes resulting in almost cent percent damage. When tubers are stored under damp conditions, decay sets in and the tubers become soft with a watery mass of bacterial ooze accompanied by a sulphurous odour.

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The causal organism Two species are mainly involved in this disease viz. Erwinia carotovora sub sp. carotovora and E. carotovora sub sp. atroseptica. These are rod shaped, motile with peritrichous flagella and are gram negative, facultative anaerobes. The organism lives saprophytically in decaying plant debris and enters the tubers through strong wounds and lenticels. Diseases control Only healthy tubers are to be sown to prevent the disease. When cut tubers are sown, they should be stored at temperatures of 12-15°C, in a dark room for 4 days. This will allow cork formation at cut surfaces and will prevent entry of bacteria through cut surfaces. The cut tubers are to be treated with fungicides before sowing. 6. Scab of potato The disease is reported from all potato growing regions of the country. The greatest loss from the disease is the reduction in market value of tubers due to rough and blemished skin and deep scabs. Symptoms The symptoms are seen mostly on tubers. The lesions are of two types, the shallow and the deep scab formed on the tubers. The lesions consist of corky tissues which arise from the tuber periderm. The lesions vary widely in size and shape (Fig. 36.1).

Fig. 36.1. Potato scab disease

Causal organism This disease is caused by Streptomyces scabies. The vegetative mycelium is slender, branched, aerial, grey in colour with few or no cross walls; conidiophores are branched, septate and spirally coiled; conidia formed in chains. The pathogen spreads through soil, water and infected seed tubers. However, infected tubers role is major in the disease spread. The pathogen penetrates tissue through lenticels, wounds and stomata.

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Control 1.

Control of scab is through the use of scab free seed.

2.

Diseased / deep lesions tubers should not be used for seed.

3.

Tubers can be disinfected by dipping them for 5 minutes in 0.25% suspension of mercurial fungicides using resistant varieties for the scab disease. VIRAL DISEASES

1. Bhendi vein clearing Bhendi (Abelmoscus esculetus) is one of the most popular vegetable crops in all parts of India. Several varieties are grown in different parts of the country. The PUSA varieties are gaining popularity in recent years. Symptoms: This is the most severe and dreaded disease of bhendi crop occurring all over India (Fig. 36.2). The disease may be initiated at any stage of plant growth. This disease was first reported in India in 1924 by Kulkarni. Estimation of damage is about 94%, if the crop is infected within 35 days of sowing. The extent of damage declines with delay in infection of the crop in the field. The disease is characterized by yellowing of the entire network of veins in the leaf blade. In severe infection the younger leaves turn yellow, become reduced in size and the plant is highly stunted. Due to severe infection flowering of the plant may be restricted and the quality of the fruit declines.

Fig. 36.2. Yellow vein mosaic disease of bhendi.

Causal organism: The disease is caused by Bhendi vein-clearing virus (BVCV) transmitted by the vector white fly, Bemisia tabaci. Disease control 1.

Periodical protection of the crop with Follidol (0.3%) or other appropriate insecticides reduces the incidence of the disease.

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2.

Destructions of wild hosts also reduce the incidence of vector population. All the Bhendi varieties grown are susceptible to the disease.

3.

The improved varieties of PUSA group are relatively resistant to the virus. Resistance is controlled by a single dominant gene.

2. Leaf curl of papaya Leaf curl of papaya (Carica papaya L.) was first reported by Thomas (1939) from Madras. This disease is prevalent in several parts of India. The disease is characterized by severe curling, crinkling and distortion of the leaves accompanied by vein-clearing and reduction in the size of the leaf. Leaves become brittle and the interveinal areas are raised on the upper surface due to hypertrophy. The most prominent symptom is the rolling of leaves downward and inward in the form of an inverted cup. In some cases petiole also gets twisted. In the advanced stage of disease, defoliation takes place and flowering is arrested and finally growth of the plant is affected. Causal organism: The disease is caused by a virus, transmitted by white fly ‘Bemisia tabaci’. This disease is not mechanically transmitted but can be readily transmitted by grafting or leaf inoculation. Leaf curl virus can be transmitted to tomato and tobacco also. Host range of this virus includes papaya, tomato, tobacco, Zinnia, Althea and many other plants. Once the plant becomes affected it never recovers and if allowed to stand in the orchard it simply helps in the spread of the disease to healthy plants. Control measure 1.

Spraying with insecticides

2.

Uprooting and destroying the infected plants is the most important control measure of this disease

3. Bunchy top of banana Bunchy Top disease of banana (Musa paradisica L.) is the most serious and prevalent among viral diseases of Banana in many countries. This disease was first recorded in 1879 in Fiji and in India, from Kerala in 1940. It has slowly spread to other states in India where banana is grown (Fig. 36.3). Symptoms: The primary symptoms are seen if infected suckers are used for plantation. They put forth short, narrow leaves which are chlorotic and exhibit a rosette appearance. They are brittle with numerous dark green patches and the margins get rolled upwardly. Plants are very stunted and they fail to bear fruits. The secondary symptom in secondary infection is the premature unfurling of leaves and the development of dark green patches and streaks on the blade. The affected leaves are more rigid than the normal ones, but generally do not wilt. The diseased plants neither die nor recover from the infections. Sometimes in the affected plants bunches

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may develop at least partially, prematurely by bursting through the sides of the pseudostem. The cortical region of the corm and the roots may get decayed. Anatomical abnormalities are also found in the stem. The fibrous sheath near the phloem region is replaced by unthickned cells with chromatophores.

Fig. 36.3. Bunchy top disease of banana

Casual organisms The disease is caused by a virus, transmissible only through an insect vector, the banana aphid Pentalonia nigronervos. The disease symptoms appear in about 35 to 45 days after inoculation by the insect. Control 1.

The disease is found most commonly on all the popular varieties of Banana. No chemical method of eradication of disease is proved effective except exclusion. Systemic eradication of the diseased plants, suckers and clumps is very much essential.

2.

Injecting herbicide Agroxone (2,4–dichlorophenoxy acetic acid and MCPA (2–methyl 1–4–Chlorophenoxy acetic acid) into the diseased plant has been found effective in eradicating the disease in Australia.

4. Rice tungro Rice (Oryza sativa L.) is the staple food of people in southern and south eastern parts of India. It is cultivated under widely varying agronomic and climatic conditions of the tropical and sub tropical regions. More than 3000 varieties are under cultivation in different parts of the country. Symptoms Tungro is characterized by stunting of the plant and leaf colour ranging from various shades of yellow to orange. The discoloration and rusty blotches spread

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downwards from the leaf tip. The young leaves may show a mottled appearance and are slightly twisted, whereas the older leaves appear rust coloured. In less susceptible varieties, tungro virus infection delays flowering. Highly susceptible varieties may die before flowering due to infection and no grain formation takes place. Causal organism: This disease is caused by a virus, Tungro Virus (TV) and is transmitted by green leaf hopper Nephotetix virescens. 5. Groundnut bud Necrosis or Peanut bud necrosis Disease (PBND) Crop losses occur during kharif and summer. Virus is mainly transmitted through thrips (Thrips palmi) and cause severe reduction in yield. Early infection leads to complete failure and yield loss. Symptoms Disease symptoms appear first on young leaflets as chlorotic spots and mottling that may develop into chlorotic and necrotic rings and streaks. Initial symptoms lead to terminal bud necrosis. It occurs commonly during summer, when temperature is relatively high and Kharif season. Entire plant is killed when high temperatures prevail. Stunting due to shortening of internodes and proliferation of auxiliary shoots results as a result of severe infection. Causal organism: The pathogen is groundnut bud necrosis virus or peanut bud necrosis virus (PBNV). Disease control 1.

Peanut bud necrosis disease is managed mostly by adjusting the dates of sowing, plant population and intercropping.

2.

Varieties resistant to the virus are recommended. However insecticides are not suggested.

6. Bean common mosaic virus Infected plants are stunted and appear bushy. The leaves of infected plants develop mottle, blisters and the lamina curls downward. Infection causes the shedding of flowers and the setting of the pod is delayed. The yield is considerably reduced. Causal organism: Phaseolus virus 1 (Pierce) Smith is the common bean mosaic virus. The virus is inactivated after being exposed for 10 minutes at 62°C. The virus remains infective up to a dilution of 1,50,000. It remains infective up to 24-30 hours at room temperature. The virus is sap transmissible. The insect vectors are Aphis craccivora and A. gossypii. The virus is also carried through the seed. It has got a wide host range including several members of Fabaceae family. Control: 1.

The use of virus free seeds

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2.

The use of resistant varieties

3.

Using appropriate insecticides to control insect population in the field. PLANT DISEASES CAUSED BY SPIROPLASMAS

1. Sesamum phyllody Sesamum (Sesamum indium L.) is an oil seed crop grown in India, China, Japan, Myanmar, and in parts of Africa and the southern Mediterranean regions. Sesamum is grown in U.P., M.P., A.P., Rajasthan, Tamilnadu, Karnataka and Maharashtra states. Phyllody is one of the important diseases of Sesamum. Phyllody, a mycoplasma disease, is one of the most serious and widespread diseases in India occurring every year. Incidence of this disease in the field varies from 1 to 100%. Symptoms: Phyllody symptoms manifest mostly in the flowering stage. The floral parts are transformed into leafy structures and grow profusely. The sepals, petals, stamens and carpels turn green and leafy. The veins of the phyllody structures are thick and prominent. Ultimately the flower becomes sterile and the ovary is also malformed into an elongated shoot like structure. Thus the phylloid structure is a leafy outgrowth of every floral part. The leaves of the diseased plant are reduced in size and chlorotic with vein clearing symptoms. The internodes are shortened, branching is abnormal resulting in a plant malformed beyond recognition. Causal organism: The disease is caused by a mycoplasma. Disease cycle: The mycoplasma is transmitted from plant to plant by the insect vector Orosius albicinctus. The minimum acquisition feeding time of the vector is 8 hours and the incubation period is about 3 weeks during summer and relatively longer during winter. It is not certainly known that whether the mycoplasma is passed on from one generation to the other by this vector. Crotalaria spp. Brassia spp. Cicer arietinum, Trifolium spp. and few other plants are proved to be collateral hosts to this pathogen. Control measures: 1.

This disease can be controlled by spraying systemic and contact insecticides.

2

All the known varieties of sesamum are susceptible to the disease. Breeding for resistance is the only step to evolve resistant varieties in sesamum against this disease.

2. Potato spindle tuber disease Potato spindle tuber is one of the most destructive diseases of potatoes because it spreads rapidly.

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Symptoms: The infected potato plants appear erect, spindly and dwarfed and the tubers are elongated. The infected leaves are darker green and sometimes show rolling and twisting. The infected tubers are elongated with tapering ends, yields are reduced up to 25%. Causal Organism: The causal organism is Potato spindle tuber viroid (PSTV). This is the first recognized viroid. The pathogen is mechanically transmitted and is spread mainly by tools used to cut healthy and infected potato seed tubers and during handling and planting of the crop. It is also transmitted by several insects including aphids, grasshoppers and beetles. The viroids replicate themselves systematically throughout the plant. Control 1.

The disease can be controlled effectively by planting only disease free potato tubers in the fields.

2.

Washing of hands or sterilizing of tools after handling viroid infected plants before moving on to healthy plants.

3. Little leaf of brinjal This is one of the popular vegetables grown all over the country. Several varieties of Brinjal (Solanum melongena L.) under cultivation are prone to various diseases (Fig. 36.4).

Fig. 36.4. Little leaf disease of brinjal

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Symptoms: The disease is characterized by reduction in size of the plant, particularly the leaves, which are malformed into tiny chlorotic structures. The foliage on affected plants is clustered together and malformed beyond recognition. Such plants become mostly sterile. Causal organism: This disease is caused by a Mycoplasma and is transmitted by Hishimonus phycitis. Control measures: 1.

No effective control measures are available. However, antibiotic treatments with tetracyclines, achromycin, terramycin, aureomycin and ledermycin masked the symptoms and disease.

2.

Eradication of weed hosts and diseased brinjal plants.

3.

Controls of insect vectors by spraying insecticides are recommended in the management of disease.

4. The Sandal spike disease Sandal (Santalum album L.) is one of the most economically important forest trees in India. This species is confined to India and Indonesia. In India it is confined to only Karnataka, Kerala and Tamil Nadu. Spike disease is the most destructive of the few diseases that attack this plant (Fig. 36.5).

Fig. 36.5. Spike disease of sandal wood

Symptoms: The common symptom is 'Rosette Spike' which is characterized by severe reduction of leaf size and shortening of the internodes. This results in the crowding of leaves on the branches. All such leaves stand out stiffly on the branches like spikes. In advanced stages of disease, just before the death of the tree, leaves become yellowish and finally reddish. The flowers of the infected trees show phyllody. The ends of the roots and haustorial connections with the host plant are either damaged or die. The other form of the disease, known as ‘Pendulous Spike’is characterized by continues apical growth of shoots without proportionate thickening of shoot resulting in drooping of the shoot. Causal organism: This disease was thought to be caused by a virus, later since 1969, electron microscopic examinations have confirmed that the disease is caused

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by a mycoplasma like organisms which are found in the sieve tubes of leaves and twigs showing symptoms. Remission of symptoms and disappearance of MLO after tetracycline treatment have further confirmed mycoplasmal etiology of the disease. The spike disease is transmitted through root contacts and insect vectors. The leaf hoppers Moonia albimaculata, Coelidia indica and Nephotettix virescens are involved in the disease transmission. Control Measures: 1.

Treatment with tetracycline and systemic fungicides like benlate alone or in combination results in temporary control of the disease for 50-100 days and then the disease reappears.

2.

Resistant varieties needs to be developed.

5. Grassy shoot disease of sugarcane Sugarcane (Saccharum officinarum L.) is the important commercial crop grown all over India. This disease was first observed in India on sugarcane variety Co.419 in 1942. At present it is reported to be widespread in all sugarcane growing states viz., Andhra Pradesh, Tamil Nadu, Karnataka, Maharashtra and Uttar Pradesh. This disease is more severe on the ratoon crop.

Fig. 36.6. Grassy shoot disease of sugarcane

Symptoms: This disease is characterized by proliferation of vegetative buds from the base of the cane giving rise to crowded bunch of tillers bearing narrow leaves. These tillers bear pale yellow to completely chlorotic leaves. Cane formation rarely takes place in affected clumps and if formed the canes are thin with short internodes (Fig. 36.6). Causal organism: This disease is caused by Mycoplasma which is readily transmitted by sap inoculation and in the field it is transmitted through infected setts and perpetuates through crop ratooning. The aphids Longiunguis sacchari and Melanaphis indosacchari are the vectors for this disease.

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Control 1.

This disease is controlled by eradication of diseased stools from the field.

2.

By pretreating the setts with hot water at 52°C for one hour before planting.

3.

Treatment of sets with 500-1000 ppm ledermycin has given complete protection.

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Chapter - 37

Diversity and Conservation of Microorganisms

The importance of diversity, conservation and utilization of microorganisms is growing as the number of preserved, strains is continuously increasing by isolation and genetic manipulation. India is rich in biodiversity of microbes and one-third of global diversity exists in India. It is highly essential to produce enough food for the burgeoning population. Cost-effective agricultural production process is of utmost importance. Products of microbial origin can be integrated to curtail a part of the energy intensive supplies like chemical fertilizers and pesticides. Microbial diversity plays an important role in sustainable agriculture; hence their in-situ and ex-situ conservation is essential. They need to be preserved properly and improper preservation leads to genetic instability. Agriculture is important for India, as the majority of our rural population belongs to farmer community. Agriculture continues to play a key role to meet sustained food security for the growing population. The growth of the nonagricultural sector did not bring the much expected improvement in the socioeconomic conditions of the Indian rural population. India has a geographical area of 328.7 million hectares and land utilization statistics is available for only 305 million hectares. The cupping area has increased to 136 percent due to expansion of irrigation facilities etc. India has around 18% of world’s human population and 15% of world’s livestock with 2.3% of geographical area, 4.2% freshwater resources, one percent of forest area and 0.5% grazing lands. The net irrigated area has increased from 21 million to 60 million hectares by 2006. Degraded land is around 121 million hectares. In order to have food and nutrition security to the growing population biodiversity has a greater role. Biodiversity (flora, fauna and microbes) is dire resource material for biotechnology. BIODIVERSITY Biodiversity means the variability among living organisms from all sources including terrestrial, marine and other aquatic ecosystems and the ecological complexes of which they are part. It is a basic resource that sustains human race and it also includes diversity within species, between species and of ecosystem and

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genes. Biodiversity is the most significant global and national asset and is the enduring resource for supporting the sustained existence of human beings. It includes diversity in form, from gene to the individual organism and then on to the population, community, ecosystem and biosphere level. 5-50 million species of living form might exist on globe. India possesses immense richness of agri-diversity. Types of diversity One third of estimated global diversity exists in India, and more than ⅛ of the global diversity of microbes and flora has been reported from India. Conservation of natural resources and germplasm are important for improving productivity in agriculture. India is one the of the 12 mega diversity centres of the world which include North Eastern Himalayas and Western Ghats as biodiversity hot spots among the 25 identified all over the world. The country is endowed with enormous variability in fungi, bacteria, actinomycetes, viruses and cyanobacteria. 1. Species diversity Species diversity means the wide variety of animals, plants and microorganisms in specific area. This diversity also covers two aspects; species richness (abundance of species in one particular area), and species evenness (proportion of species in one particular area). 2. Genetic diversity Biodiversity includes genetic differences within each species, which has created great benefits to livelihood of people, especially agriculture and productions 3. Ecosystem diversity In each ecosystem, living creatures form community, interact with one another with the air, water and soil around them. They also lead to succession diversity or the natural process of succession by plant communities in forest ecosystem. 4. Taxonomic diversity The distribution of species among different groupings is taxonomic diversity. Taxonomic categories include phylum, class, order, family, genus, and species. A place with high taxonomic diversity, for example, might include species from many different families. Microbial diversity encompasses a spectrum of microscopicorganisms including, bacteria, fungi, actinomycetes, algae and protozoa. An estimated 50 percent, of all living population on earth is microbial. There may be 1.5 million species of fungi yet only about nine percent arc described and one million species of bacteria but only about 5,000 have been described. Microbial biodiversity is a vast frontier and potential gold mine for the biotechnology industry because it offers countless new genes and biochemical pathways for enzymes, antibiotics and other useful molecules. The use of DNA recombination and modern biotechnology has produced microbial strains that possess an optimal set of properties of important processes related to economical development and environmental management. The

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agriculturally important microorganisms have their own place and scope in the overall context of agro-biodiversity. 5. Microbial diversity Microbial diversity is an unseen national resource that deserves greater attention. The agriculturally important microorganisms include bacteria, mycoplasma, blue green algae (BGA), fungi, including arbuscular mycorrhizae (AM) and viruses. They are involved in organic matter decomposition, N2-fixation, solubilization and mobilization of several major and minor nutrients. Microbes also play an important role in soil structure maintenance. They contribute towards improved plant health and higher crop yield by production of growth stimulators such as plant hormones and vitamins and control of plant diseases by acting as a biocontrol agent also. Microbes are also utilized for fermentation and for value addition in food industry. CONSERVATION There is an increasing interest world-wide in the conservation of biodiversity, and its non-destructive utilization for human benefits. Convention on Biological Diversity (CBD) is an international agreement established by the United Nations with main objectives to conserve biodiversity, to enhance its sustainable use and to ensure an equitable sharing of benefits linked to the exploitation of genetic resources. Conservation of microbial diversity has been given importance in CBD. Two ways to conserve the biodiversity are in-situ and ex-situ conservation. In-situ and ex-situ conservation In-situ and ex-situ conservation, allows the population of living organisms to maintain/ perpetuate itself within their own habitat, to which it is adapted so that it has the potential for continued evolution. Moreover in-situ conservation facilitates research on species in their natural habitats. Morphological and ecological characteristics and environmental conditions can be related to the genetic variation, and thus in-situ conservation allows a better evaluation and utilization of biological diversity Ex-situ conservation In many cases, effective conservation in natural habitat is not possible due to change of climatic conditions and environmental pollution, so some species of microbes are depleted in their natural habitat. Hence, ex-situ conservation of species and genetic resources is the most effective and efficient alternate means for conserving biological diversity. Culture collections are key repositories of biodiversity and also play important roles in conservation and sustainable utilization of microbial diversity. Culture collection centres are serving in education and research through authentic identification and supply of microbes. Microbial strains in culture collections are potential genetic resources for commercial exploitation through biotechnology. It is

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rare to find a fungal natural product that is known from only one taxon or a single strain. Several taxa can produce the same metabolites, so collection of fungi from different habitat and conserving them is necessary to utilize the microorganism for industrial use. Culture collections all over the world including India for many decades have been playing a major role in preservation of microorganisms, reputed culture collection centre are manned by trained personnel and very often backed by experts. These centres are well equipped for conservation of microorganisms and represent living resources for biotechnological research and application. Usually such centres maintain available information about a culture including its use, if any and the ecological niche it was isolated from. Since human activities over the years might have disturbed habitats of some microorganisms it is possible that some of the cultures conserved in such collections may represent the only surviving members of an otherwise extinct group. At present there are about 500 culture collections in 58 countries and hold 8 15,000 cultures. Scenario in India India is endowed with a variety of climatic and ecological environments which provide ideal habitat for highly diverse group of microorganisms. Over the years, many microorganisms have been isolated from different habitats but in these studies the researchers were mainly interested in specific and limited types of organisms and never a comprehensive survey was undertaken. The Division of Microbiology at Indian Agricultural Research Institute (IARI), New Delhi, India established a facility for Rhizobium germplasm collection which has a collection of around 1100 authenticated rhizobial cultures which have been either isolated from root nodules of different legumes grown under different agroclimatic conditions of the country or obtained from various sources from India as well as from abroad. Similarly, a National Centre for Conservation and Utilization of Blue-Green Algal (BGA) Cultures has been established at this Institute and this centre has a collection of around 600 BGA culture. This centre has also a collection of different species of Azolla. A centre for collection and maintenance of mycorrhizal fungi (ecto- and arbuscular mycorrhizae) has been established recently at Tata Energy Research Institute, New Delhi, India. Institutional collections of bacteria of industrial importance were established at Institute of Microbial Technology (IMTECH), Chandigarh, India. The Indian Type Culture Collection (ITCC) established in 1936 at IARI, New Delhi holds over 4,400 cultures of fungi of agricultural importance. The pure cultures are supplied to agencies engaged in research/ production organizations for field/industrial applications on commercial basis. Screening bank of plant viruses was established in 1970 at IARI, New Delhi, which provides serodiagnostic opportunities to workers who do not have facilities to produce antisera. Subsequently in 1988, IARI established an Advanced Centre for Plant Virology to characterize different viruses and to develop diagnostic reagents. The following are the National Centres for microbial conservation 1.

ITCC — Indian Type Culture Collection, IARI, Delhi.

2.

HC1O — Herbarium Cryptogamiae Indiae Orientals, IARI, Delhi

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3.

MACS — Collection of Microorganisms, Agharkar Research Institute, Pune.

4.

MTCC — Microbial Type Culture Collection & Gene Bank, IMTECH, Chandigarh.

5.

NBAIM — National Bureau of Agriculturally Microorganisms, Kushmaur, Mau Nath Bhanjan, UP.

6.

NCDC — National Collection of Dairy Cultures, National Dairy Research Institute, Karnal.

7.

NCIM — National Collection of Industrial Microorganisms, National Chemical Laboratory, Pune.

8.

RRL — Regional Research Laboratory, Jammu.

9.

UMFFTD —Food and Fermentation Technology Division, University of Mumbai, Mumbai.

10.

VPCI —Fungal Culture Collection, Vallabhbhai Patel Chest Institute, Delhi.

11.

CMCC — Centre for Mycorrhiza Culture Collection, TERI, Delhi

12.

CCUBGA — Centre for Conservation and Utilization of Blue Green Algae, IARI, Delhi.

Important

Some strains of cyanobacteria, blue green algae have the capacity to fix atmospheric nitrogen. This property is explored for use in agriculture. The CCUBGA, IARI, New Delhi maintains more than 600 cyanobacterial isolates. HCIO (Herbarium Cryptogamie Indiae Orientalis) The unculturable fungi can be preserved in herbaria for future studies. The HCIO was created by Sir Edwin John Butler in 1905 and shifted to IARI in 1934. HCIO is our National fungal herbarium which houses nearly 48,000 diseased fungal specimens belonging to 2000 genera of various groups of fungi in our country. Approximately 3000 type specimens and also richest collection of rusts, smuts, powdery mildews and mildews are being maintained in HCIO. Type specimens are the specimens from which the description of a new species is made. So, preservation of type specimens is important for systematic studies. ITCC (Indian Type Culture Collection) ITCC is the oldest culture collection of our country established in 1935 at IARJ, New Delhi. At present ITCC maintains nearly 3000 fungal cultures of different groups which include Myxomycetes (9) Mastigomycetes (11), Zygomycetes (236), Ascomycetes (1100), Basidiomycetes (344) and Deuteromycetes (1385). The culture collection maintains plant pathogens, biocontrol agents and fungi for medicinal and industrial use. The main mandatory objectives of HCIO and ITCC are to access, preserve and maintain collection of fungi from India and abroad; to provide authenticative

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identification service; to supply culture for research institutes and industries and to conduct systematic research. Bacteriology Division of Plant Pathology, IARI maintains 96 plant pathogenic bacteria and 20 Biocontrol agents viz. Pseudomonas fluorescens, Bacillus subtilis, and Xanthomonas campestris pv. parthenii. UTILIZATION OF MICROBIAL DIVERSITY FOR SUSTAINABLE AGRICULTURE Diversity patterns of microorganisms can be used for monitoring and predicting environmental change. The untapped diversity of microorganisms is a resource for new genes and organisms of value to biotechnology. Research on identification of plant viruses could be useful for weed control which may reduce the adverse effect of organo-phosphates into the environment. Potential of Rhizobium, Azotobactcr, Beijerinckia, Azospirillum and Cyanobacteria, as biofertilisers has been exploited so these could serve as an alternative to chemical fertilizers. Bioremediation uses naturally occurring microorganisms to degrade various types of wastes. Like all living creatures, microbes need nutrients, carbon and energy to survive and multiply. Such organisms are capable of breaking down chemicals to obtain food and energy, typically degrading them into harmless substances such as carbon dioxide, water, salts, and other innocuous products. Using biotechnology techniques, bacteria can be bioengineered for the bioremediation of toxic wastes. Bacteria, in combination with yeasts and molds, are used in the preparation of fermented foods such as cheese, pickles, soy sauce, sauerkraut, vinegar, wine, and yogurt. Highly specialized groups of microorganisms play important roles in the mineralization of specific classes of organic compounds. Genetically engineered microbes can be used for the production of therapeutic drugs, such as insulin. It is estimated that annually 25.1 mt of nutrients (N, P, K) are removed from the soil by the crops. Whereas, only 15.0 mt are supplied from soil sources including organics. The fertilizer production in our country is less than the required amounts. Moreover, the fertilizer industry depends on petroleum reserves which will be exhausted in near future. To fill this gap alternate sources of nutrients have to be looked for. Organic wastes and biofertilizers are the alternate sources to meet the nutrient requirement of crops and to bridge the future gaps. Further, knowing the deleterious effects of using only the chemical fertilizers will be an environmentally benign approach to nutrient management and ecosystem function. Such integrated approach will help to maintain soil health and productivity. Tiny microorgnisms in the soil play a significant role for sustaining and improving our agricultural production. Soil micro-organisms like bacteria and cyanobacteria (blue-green algae) have the ability to use atmospheric nitrogen and supply this nutrient to the crop plants. Some of these nitrogen fixers like rhizobia are obligate symbionts in leguminous plants, while others colonize the root zones and fix nitrogen either freely or in close association with plants. A very important bacterium of the latter category is

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Azospirillum, which was discovered by Johanna Döbereiner, a Brazilian scientist in mid 1970s. The crops which respond to Azospirillum inoculation are maize, barley, oats, sorghum, pearlmillet, coarse grains, oilseeds, forage and other crops. Azospirillum applications increase grain productivity of cereals by 5-20%, of sorghum, millets like pearl millet and small millets by 30% and fodder and forage by over 50%. The third group includes free-living nitrogen fixing microorganisms like blue-green algae and Azotobacter. Mycorrhizal fungi have also been shown to have agronomical implications. Several microorganisms have been enlisted recently as endophytic organisms which are capable of entering into the host tissues and influence the plant growth Cereal nodulation The Rhizobium host plant specificity has been overcome by cellulase treatment of seedling roots in the presence of polyethylene glycol (PEG) prior to inoculation with the bacterium. By this method, clover seedlings which could not normally nodulate with Rhizobium loti were made to do so in Petri dishes and the nodules so developed were pink and showed nitrogenase activity. Extraneous source of sucrose appears to enhance nodulation in such enzyme treated seedlings. The report by Cocking and co-workers at the University of Manchester, UK prompted them as well as other workers to induce nodulation in the other plant species. Microbes as biopesticides The few biopesticide products currently in the global market are strains of Bacillus thuringiensis, baculoviruses and entomopathogenic fungi. These biopesticides are now being regulated at national level under existing Insecticides Act. The microorganisms that are exploited for insect control purposes arc relatively few in our country. The B. thuringiensis research and use have gained importance only after the ban was lifted due to their pathogenicity to silkworms. It is now being marketed in our country. However, pests like Plutella xylostella and Heliothis armigera, which have developed resistance to many pesticides could be controlled by B. thuringiensis products. Different strains of B. thuringiensis have been used against insects belonging to the orders Coleoptera, Diptera and Lepidoptera and several other pests. There is a possibility of isolation and development of novel indigenous strains of B. thuringiensis against the crop pests. Since it is a facultative pathogen, the technique of production through fermentation technology is standardised. Now this bacterium is commercially available in dust, wettable powder and flowable formulations under different trade names. The B. thuringiensis products are registered for use in all parts of the world and it amounts to more than 80 per cent of microbial insecticides sold. The baculoviruses arc currently developed for controlling specific insect pests of agriculture and forestry. These are obligate pathogens and are produced using laboratory reared host insects. There are nearly 40-45 insects pests on which viral pathogens are recorded in our country. Out of these, the baculovirus of two destructive pests Heliothis armigera and Spodoptera litura are being tried on large scale in many parts of our country. The production techniques of these viruses are

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standardized and few biotechnology firms and private entrepreneurs are manufacturing and selling the product. The field trials on various crops have resulted in adequate control comparable to synthetic chemical insecticides. Bacillus thuringiensis and baculoviruses molecular biology have led to either development of transgenic crops or recombinant strains with increased efficacy. The other group of microorganisms of importance is fungi. Besides, they cause natural epizootics; some of these could be exploited for pest control purposes. These fungal biopesticides are Beauveria bassiana, Metarrhizium anisopliae, Nomuraea rileyi and Verticillium lecani. The tendency of Trichoderma, Gliocladium, Penicillium and others which are biocontrol agents to produce potent broad-spectrum antibiotics is well known. Fluorescent pseudomonads and bacilli are good examples as plant growth promoting rhizobacteria against certain soil-borne root pathogenic fungi like Rhizoctonia solani, Fusarium moniliforme, Macrophomina phaseolina and Colletotrichum falcatum etc. Microbial nematicides So far, only three species of bacteria viz. Pesteuria penetrans, P. thornei and P. nishizawae have been identified to parasitize important group of nematodes. They are obligate and host specific parasites. Of the three species, P. penetrans has been observed parasitizing juveniles of root-knot and cyst forming nematodes. The isolates of P. penetrans have been found to be very effective in inhibiting the egg production, reducing nematode penetration and improving the plant growth. Reports indicate maximum reduction in multiplication and penetration of Meloidogyne incognita on tomato with the use of this bacterial nematicide. Similarly, nematode infestation in pigeon pea by Heterodera cajani was greatly reduced with the use of P. penetrans. Vermitechnology Earthworms are now utilized to produce organic manure to grow mushrooms, vegetables, flowers and crops profitably and as feed for fish, poultry and pigs. Earthworm technology involves three components i) vermiculture, ii) vermicomposting and iii) utilization of products like vermiprotein and vermifertilizer. Vermiculture for Indian village people will be most suited because of low investment and easy availability-of unskilled labour that can learn vermiculture within no time. Edible fungi About 10,000 edible fungi are available, of which 80 are grown in culture and few are commercialized. The commercialized ones include button mushroom, oyster and paddy straw mushroom. All the edible mushrooms contain proteins, vitamins, all essential elements, minerals, amino acids, more fibre besides having less fat and carbohydrates. Edible mushrooms are nutritive, tasty, good for health, increase immunity and add flavor.

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Chapter - 38

Molecular Methods for the Analysis of Microbial Communities

Microorganisms play vital roles in biogeochemical cycles and are responsible for the cycling of organic compounds. The knowledge of diversity exists within natural microbial communities. It has been estimated that a very low percentage (ranging from 0.001 in seawater to 15% in activated sludge) of bacteria can be cultured by standard laboratory methods (Kirk et al 2004). It is possible that this low percentage of culturable bacteria might be representative of the entire community and that the largest fraction is in a physiological state that eludes the possibility to culture them. Majority of unculturable bacteria are genetically different from the culturable ones and only the minority of the population is represented. Natural microbial communities are among the most complex, diverse and important clusters of organisms in the biosphere. The biodiversity of these communities is dependent on a variety of metabolic pathways in which they are involved. The assessment of the degree of biodiversity of natural microbial communities requires the analysis of their composition and structure, by determining the number and the relative frequency of the bacterial species in the community, the distribution of isolates within them, their physiological role in relation to the environment and the spatial-temporal dynamics as a consequence of the fluctuations of the environmental parameters. These studies require the use of specific methodologies and molecular techniques. Majority of them are based on Polymerase Chain Reaction (PCR). These studies have permitted to understand some of the complex interactions existing between the different entities of a natural microbial community (Kirk et al 2004). MOLECULAR TECHNIQUES TO STUDY NATURAL MICROBIAL COMMUNITIES Microbial diversity encompasses species richness, the total number of species present, species evenness, and the distribution of species. Methods to measure this diversity in natural environments can be categorized into two groups based on biochemical and molecular techniques. The latter methodologies overcome problems associated with non-culturable bacteria allowing identifying and studying them. Most of these molecular techniques are based on PCR amplification of target sequences. The most commonly used of which is 16S rDNA. This gene is used extensively to study bacterial diversity and allows identification of prokaryotes as well as the

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prediction of phylogenetic relationships. In these methods. The DNA is extracted either from isolated bacteria or the environmental sample, purified and the16S rDNA is amplified. The resulting amplicons can be analyzed in different ways (Kirk et al 2004). Denaturing gradient gel electrophoresis (DGGE)/temperature gradient gel electrophoresis (TGGE) Denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) were first developed to detect point mutations in DNA sequences. DNA is extracted from environmental samples and PCR-amplified. The 5′-end of the forward primer contains a 35–40 base pair GC clamp to ensure that at least part of the DNA remains double stranded. On denaturation, DNA melts in domains, which will migrate differentially through the polyacrylamide gel. In principle, DGGE can separate DNA molecules with one base-pair difference. TGGE uses the same principle as DGGE except the gradient is temperature rather than chemical denaturants. Specific DGGE/TGGE bands can also be excised from gels, reamplified and sequenced; in this way information about the specific microorganisms in the community can be obtained. Single strand conformation polymorphism (SSCP) This technique relies on electrophoretic separation based on differences in DNA sequences in single strand conformation polymorphism (SSCP). Singlestranded DNA is separated on a polyacrylamide gel based on differences in mobility caused by their folded secondary structure. When DNA fragments are of equal size and no denaturant is present, folding and mobility will be dependent on the DNA sequences (Kirk et al 2004). Restriction fragment length polymorphism (RFLP)/ amplified ribosomal DNA restriction analysis (ARDRA) One of the most used tools to study microbial diversity is Amplified Ribosomal DNA Restriction Analysis (ARDRA) that depends on DNA polymorphisms existing between 16S rDNA sequences of bacteria belonging to different species. In this approach, the 16S rDNA from bacterial isolates is amplified through PCR and further treated with a 4-base pair cutting restriction enzyme. Restriction fragments are usually separated using agarose gel electrophoresis. ARDRA banding patterns can be used to screen clones, to measure bacterial community structure and/or for detecting structural changes in microbial communities. Terminal restriction fragment length polymorphism (T-RFLP) Terminal restriction fragment length polymorphism (T-RFLP) is based on the same principle as RFLP except that one PCR primer is labelled with a fluorescent dye, such as TET (4,7,2′,7′-tetrachloro-6-carboxyfluorescein) or 6-FAM (phosphoramidite fluorochrome 5-carboxyfluorescein). This allows detection of only the labelled terminal restriction fragment (Kirk et al 2004) simplifying the banding

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pattern that, in turn, allows the analysis of complex communities as well as providing information on diversity as each visible band represents a single ribotype. The banding pattern can be used to measure species richness and evenness as well as similarities between samples. This procedure allows sampling and analysis of a large number of environmental samples. Ribosomal intergenic spacer analysis (RISA)/automated ribosomal intergenic Spacer analysis (ARISA) Similar to ARDRA and T-RFLP, RISA and ARISA provide ribosomal-based fingerprinting of the microbial community. In RISA and ARISA, the intergenic spacer (IGS) region between the 16S and 23S ribosomal genes is amplified by PCR, denatured and separated on a polyacrlymide gel under denaturing conditions. This region may encode tRNAs and is useful for differentiating between bacterial strains and closely related species because of heterogeneity of the IGS length and sequence. In RISA, the sequence polymorphisms are detected using silver stain while in ARISA the forward primer is fluorescently labelled and is automatically detected. Both methods provide highly reproducible bacterial community profiles. SNuPE (Single Nucleotide primer extension) Single nucleotide primer extension (SNuPE) is a method applied in bacterial identification. It allows the detection of the base which is present at the SNP (single nucleotide polymorphism) site of interest. In a SNuPE analysis, the first step is represented by the PCR amplification of the DNA region including the SNP site. Then an oligonucleotide that anneals to the amplified DNA region soon upstream of the SNP site is involved in an extension reaction that is catalysed by DNA polymerase. The primer is extended by a single fluorescent labeled dideoxynucleotide (ddNTP) complementary to the nucleotide present in the ‘interrogation’ site. The incorporated ddNTP acts as terminator in the extended reaction, resulting in a single base difference between the primer and the extended product. SNuPE products are analysed using laser inducing fluorescence capillary electrophoresis. The high specificity of ddNTP incorporation makes the SNuPE reaction extremely efficient to detect the nucleotide at a specific SNP site in a given strain. LAMP (Loop isothermal amplification) Loop-mediated isothermal amplification (LAMP) is a recently developed DNA amplification technique that is rapidly emerging as a powerful tool for pathogen detection and identification (Notomi et al 2000). LAMP is based on autocycling strand displacement DNA synthesis. The method uses a DNA polymerase with high strand displacement activity (Bst) and a set of two specially designed inner primers and two outer primers. Each of the inner primers contains one sense and one antisense sequences of the target DNA, one for priming in the first stage of the reaction and the other for self priming in later stages. DNA synthesis starts from the inner primers. The two outer primers, which are shorter than the inner primers and conventionally designed, initiate the strand displacement DNA synthesis. This

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displacement activity releases the newly-synthesized complementary strands which are linked to one of the outer primers at the 5’ end. These single-stranded DNAs serve as template for further amplification by the same mechanism, but because of the sense-antisense sequence derived from the outer primers, the complementary strand generated in this phase can form a dumb-bell secondary structure that allows self-primed DNA synthesis. The self-primed DNA synthesis generates stem–loop DNA that serves as starting material for the final amplification products are stemloop DNA structures with several inverted repeats of the target and repetitive structures with multiple loops. LAMP reactions are carried out at one constant temperature (between 60 and 65°C) by using only Bst polymerase. Target specificity is very high since amplification requires the annealing of six oligonucleotidic sequences on the target DNA (two sequences in each of the inner primers and one in each of the outer primers). The amplified DNA can be analyzed by agarose gel electrophoresis. FISH (Fluorescent in situ hybridization) A key technique employed to study the biology and ecology of uncultivated organisms. It is based on in situ hybridization, which allows the PCR-independent detection and localization of bacteria with a selected specificity determined by rRNA-targeted, labeled gene probes (Amann et al 1990). Because of the high number of ribosomes and rRNA molecules in most bacterial cells, the hybridized gene probes can be directly visualized in microscopic sections. Different labels coupled to a gene probe allow different types of detection (staining or epifluorescence microscopy).

In situ PCR One potential approach to characterizing the microscale genetic and taxonomic properties of natural bacterial communities might be in situ PCR, a modification of PCR in which amplification and detection of specific target nucleic acid sequences are carried out inside individual cells. In principle, individual genes, mRNA, and rRNA are all candidate targets for this technique. In this way genetic abilities and their expression, as well as taxonomic information are all potentially accessible on the individual cell level. There are two different approaches to in situ amplification of target sequences and visualization of PCR products, by either subsequent ISH (indirect in situ PCR) or by direct detection of labelled nucleotides, which have been incorporated during PCR (direct in situ PCR). In situ PCR technologies were first developed for detecting DNA or RNA viruses, and gene expression inside eukaryotic cells. Prokaryotic in situ PCR methods were firstly exploited to visualize the presence of genes and their expression inside bacterial cells. The technique was applied to some bacterial genera/species, such as Pseudomonas, Escherichia, Psychrobacter, Nitrosomonas, to detect some of them in biofilms. A wide range of methods available to study microbial diversity exist although each method has its limitations and only provides a partial picture of microbial diversity existing in a given environmental sample.

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Chapter 39

Some aspects of Applied Microbiology

Biotechnology is not just about recombinant DNA of cloning and genetics; it is equally about producing materials, like citric acid, beer, wine, bread, fermented foods such as cheese and yoghurts, antibiotics etc. It will also provide clean technology for a new millennium by providing means of waste disposal and various environmental problems. It is, in short, one of the two major technologies of the twenty-first century that will sustain growth and development in countries throughout the world for several decades to come. It will continue to improve the standard of all our lives, from improved medical treatments, through its effects on foods and food supply and into the environment. No aspect of our lives will be unaffected by biotechnology. Antibiotic 'marker' genes are used to identify and select cells which have been successfully modified. By the use of such genes, cells which have been successfully modified can grow in the presence of the particular antibiotic. The most commonly used antibiotic resistance marker genes in GM plants confer resistance to kanamycin or hygromycin while for GM bacteria the ampicillin resistance marker gene is more often used. METABOLISM Metabolism is a matrix of two closely interlinked but divergent activities. Anabolic processes are concerned with the building up of cell materials, not only the major cell constituents like protein, nucleic acids, lipids, carbohydrates etc. but also the intermediate precursors such as amino acids, purine and pyrimidines, fatty acids, various sugars and sugar phosphates. Anabolism concerns processes which are endothermic. They also invariably require a source of reducing power that may come by the degradation of the substrate. The necessary linkage between catabolism and anabolism depends upon making the catabolic processes leading to the synthesis of reactive reagents, which in turn are used to process the full range of anabolic reactions. Among the key intermediates, adenosine triphosphate (ATP) plays an important role. The potential energy is released directly or indirectly by splitting this bond in anabolic syntheses. Molecules such as ATP then provide the 'energy currency' of the cell. When ATP is used in a biosynthetic reaction it generates ADP (adenosine diphosphate) or occasionally AMP (adenosine monophosphate) as the hydrolysis product: A + B + ATP  AB + ADP + P; or A + B+ATP  AB + AMP + PPi

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(where A and B are both carbon metabolites of the cell and P is inorganic phosphate, and PPi is inorganic pyrophosphate). ADP which also possesses a 'highenergy bond', can also be used to produce ATP by the adenylate kinase reaction: ADP + ADP  ATP + AMP Phosphorylation reactions, which are very common in living cells, usually occur through the mediation of ATP: O | | || —C—OH + ATP —C—O—P—OH + ADP | | | OH The phosphorylated product is usually more reactive in one of several ways than the original compound. If we consider glucose as the usual growth substrate of a microbial cell, we can show how it is degraded into various key precursors. Their formations are linked through the process of glycolysis, which is called as the Embden-Meyerhof-Parnas (EMP) pathway (Fig. 39.1) and then by the further oxidation of pyruvate, as the endproduct of glycolysis, through the reactions of the tricarboxylic acid cycle.

Fig. 39.1. Embden Mayerhof-Parnas pathway (Glycolysis pathway)

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In addition to the glycolytic sequence, there is also an important adjunct to this which is responsible for forming pentose (C5) phosphates and tetrose (C4) phosphates. This is the pentose phosphate pathway, sometimes referred to as a 'shunt' or as the hexose monophosphate pathway (Fig. 39.2). The purpose of this pathway is two-fold to provide C5 and C4 units for biosynthesis and also to provide NADPH or biosynthesis.

Fig. 39.2. Pentose phosphate pathway

(1) Glucose-6-phosphate dehydrogenase; (2) Phosphogluconate dehydrogenase Glucose + ATP + 6NADP  Glyceraldehyde 3-P + ADP + 6 NADPH Although the EMP pathway and the pentose phosphate (PP) pathway both use glucose 6-phosphate, the extent to which each route operates depends largely on what the cell is doing. During the most active stage of cell growth, both pathways operate in an approximate ratio of 2:1 for the EMP pathway over the PP pathway. However, as growth slows down, the biosynthetic capacity of the cell also slows down and less NADPH and C5 and C4 sugar phosphates are needed so that the ratio between the pathways moves to 10:1 or even to 20:1. Although the EMP and PP pathways are found in most microorganisms, a few bacteria have an alternative pathway to the former pathway. This is the EntnerDoudoroff pathway which occurs in pseudomonads and related bacteria. The pentose phosphate pathway though still operates in these bacteria as the Entner-Doudoroff pathway does not generate C5 and C4 phosphates.

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The degradation of glucose, by whatever route or routes, invariably leads to the-formation of pyruvic acid (CH3.CO.COOH). The fate of pyruvate is different in aerobic organisms and anaerobic ones. In aerobic systems, pyruvate is decarboxylated and is simultaneously activated in the chemical sense, to acetyl coenzyme A (acetyl-CoA) in a complex reaction also involving NAD+: pyruvate + CoA+ NAD +  acetyl-CoA + CO2 + NADH This reaction is catalysed by pyruvate dehydrogenase. Acetyl-CoA, by virtue of it being a thioester, is highly reactive. It is capable of generating a large number of intermediates that progressively oxidised through a cyclic series of reactions known as the citric acid cycle. This is also known as the tricarboxylic acid cycle or the Kreb’s cycle named after its discoverer, Krebs (Fig. 39.3). pyruvate + CO2 + ATP  oxaloacetate + ADP + Pi This reaction is carried out by pyruvate carboxylase. However, insofar as oxaloacetate is also produced from the activity of the cycle, the carboxylation of pyruvate must be regulated so that acetyl-CoA and oxaloacetate are always produced in equal amounts. This is usually achieved by the pyruvate carboxylase being dependent upon acetyl-CoA . The manner in which acetate units are converted to C4 compounds is known as the glyoxylate by-pass for which two enzymes, isocitrate lyase and malate synthaser are involved.

Fig. 39.3. Tri-carboxylic acid cycle (Kreb’s cycle)

Hexose sugars can be formed by the reversal of glycolysis and C5 and C4 sugars can now be formed via the pentose phosphate pathway. Glucose itself is not an end-product of 'gluconeogenesis' but glucose 6-phosphate is used for the synthesis of cell wall constituents and a large variety of extracellular and storage polysaccharides.

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Under anaerobic conditions, the process of oxidative phosphorylation cannot occur, and the cell is deprived of its principal way of generating energy. Under such circumstances energy must be provided from the very process of degrading the original substrate. This process, known as substrate level phosphorylation, yields only about 8% of the energy that can be produced under aerobic conditions. Products of anaerobic metabolism Some of the reactions leading to the formation of reduced end-products in anaerobic micro-organisms such as: 

Glycerol, produced by yeasts when the conversion of pyruvate to ethanol is blocked.



Lactic acid, formed by lactic acid bacteria.



Formic acid, formed by enterobacteria via pyruvate-formate lyase and the formate can be converted to CO2 and H2 by formate dehydrogenase;



Ethanol, formed by yeasts (e.g. Saccharomyces cerevisiae), bacteria (e.g. Zymomonas) and by many fungi;

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2.3-butanediol, produced by various marcescens and different Bacillus spp;



Butanol with acetone and propanol or isopropanol, produced by Clostridium spp., some of which also produce butyric acid;



Propionic acid, produced by Propionibacterium.

bacteria

including

Serratia

Other products may arise from the anaerobic metabolism of compounds other than glucose, for example organic acids, such as citric acid, or amino acids and sometimes purines. Methane is perhaps the ultimate reduced carbon compound and is produced by highly specialised Archaebacteria by cleavage of acetate to CO2 and CH4 or in some cases by reduction of CO2. The secondary metabolites are usually synthesised from glucose or fatty acid catabolism. Acetyl-CoA is often used as a key starting point. They can be of considerable biotechnological importance as many of them are biologically active and few may act as antibiotics. ANTIBIOTICS Cephalosporins Cephalosporins were developed to overcome the allergic problems associated with penicillins. Cephalosporins are made from cephalosporin C, a fermented product of Penicillium chrysogenum. The product after extraction and purification, is hydrolysed, either enzymically or chemically, to the active nucleus, 7-aminocephalosporanic acid (7-ACA), which serves as substrate for the chemical synthesis of injectable, semi-synthetic cephalosporins. Cephalosporins with a 7-O-methoxy group

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(cephamycins) are produced by several Streptomyces spp. which serve as the precursor of cefoxitin and others. Aminoglycosides Streptomycin was the first aminoglycoside used for antibiotic therapy. Its activity against Mycobacterium tuberculosis initiated the widespread introduction of antibiotic treatment to combat tuberculosis. Aminoglycosides are potent antibiotics and have activity against both Gram-positive and Gram-negative bacteria as well as against mycobacteria. Aminoglycosides are bactericidal and work by binding to the 30S ribosome subunit which prevents protein synthesis. There are many aminoglycosides in medical use and are all derived from Actinomyces spp. For example; streptomycin (Streptomyces griseus), gentamycin (Micromonospora purpurea), tobramycin (S. tenebrarius), kanamycin (S. kanamyceticus) and, sisomicin (M. inyoesis). Tetracyclines Tetracyclines were the first group of antibiotics recognized to have broad spectrum activity. They act by preventing protein synthesis at the 30S ribosome interaction with tRNA. They are used for urinary tract infections, chronic bronchitis, rickettsial and chlamydial infections. They also have broad applications in veterinary use, despite the concern and known relationship of widespread use with resistance build-up. Novel applications include activity against Helicobacter pylori to combat stomach ulcers and as a prophylactic against malaria. Chlorotetracycline and tetracycline are produced by S. aureofaciens, and oxytetracycline by S. rimosus. Chlorotetracycline production is stimulated by chloride ions and tetracycline by bromide ions. The chlorination gene can be deleted making the bacterium produce only tetracycline. Tetracyclines have been modified chemically to produce products with improved activity and stability. These include doxycycline and minocycline. Macrolides Macrolides are a diverse class of antibiotics, produced by Actinomyces. Macrolides with antibacterial properties have in common a 12, 14 or 16 carbon macrocyclic lactone ring, substituted with sugar molecules. Larger ring macrolides, the polyene macrolides, can have lactone rings of 26-38 carbons. These polyenes are mainly antifungal, e.g. nystatin and amphotericin. The non-polyene macrolides are bacteriostatic. They inhibit protein synthesis by reversibly binding to the SOS portion of the ribosome. Erythromycin and its derivative clarithromycin are the most prescribed macrolides. They have a similar activity spectrum to the penicillins and are used by penicillin-sensitive people to combat Gram-positive bacteria, and in addition are used against Mycoplasma, Campylobacter, Bordetella and Legionella. Clarkhto-mycin is currently prescribed to combat Helicobacter pylori.

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Erythromycin is a 14 carbon macrolide produced by S. erythreus. Other 14 carbon macrolides include oleandomycin from S. antibioticus, pikromycin from S. felleus, megalomicin from Micromonospora inositola. Tylosin is a 16 carbon macrolide produced by S. fradiae and is produced industrially for animal use (Ratledge and Kristiansen 2001). ENVIRONMENTAL ISSUES Sanitary engineering monopolized the environmental related industrial activities. Sanitary engineering is well established for following activities: 

Catchment, treatment and distribution of drinking water.



Treatment of waste water.

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Treatment of disposal of solid waste.

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Treatment of industrial off-gases.

The new focus on the environment as a whole and on the detailed functioning of the ‘bio’ component has led to the development of new industrial activities, referred to as environmental biotechnologies. The most important issues of environmental problems now facing the world are: 

Acid rain and ozone depletion.



Enrichment of ground and surface waters with nutrients and recalcitrant pesticides.



Recovery of reusable products and energy from wastes.



Soil remediation.



Disposal of animal manures.

Industrial biotechnologists use well defined microorganisms to make products such as lactic acid, beer or monosodium glutamate. Environmental biotechnologists on the other hand, start with poor inocula and wait until desired phenomena occur. There is therefore a need to isolate, identify and characterize the microorganisms which exist and interact in soils, activated sludges, anaerobic granules etc. Only when these microorganisms function in a predictable way the environmental biotechnology becomes more realistic. New developments are concerned with the introduction of organisms and genes in mixed cultures. Practical application of these new developments is somewhat impeded by poor survival of introduced microorganisms and regulatory constraints on deliberate introduction of modified organisms in the environment. The potential is, however, enormous as advances in molecular biology now make feasible for the construction of novel genes and enzymes for the degradation of compounds. These novel genes may become incorporated in the genomes of existing microbial communities, a process called 'horizontal gene transfer'. For example, broad host range plasmids specialised in the degradation of synthetic chemicals, can be introduced into soil microbial communities, thereby enhancing their degradative capabilities.

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Anaerobic conversion of organic compounds to biogas is a stepwise process wherein different groups of bacteria operating sequentially effect full degradation of the substrates by: 

Hydrolytic acidogens: cleave polymers into short chain fatty acids;



Syntrophic acetogens: degrade the fatty acids into acetate and H2;



Methanogens: transform acetate and H2 into CH4 and CO2 (biogas). SOIL REMEDIATION

One of the major problems facing the industrialised world today is the contamination of soils, groundwater and sediments. The most common contaminants are chlorinated solvents, hydrocarbons, polychlorobiphenyls and metals. Bioremediation (the use of micro-organisms to degrade or detoxify pollutants) is becoming increasingly used in majority of cases of pollution due to hydrocarbons. However, bioremediation is not fully understood and one of the reasons is the lack of information on the role of microbes with contaminated molecules besides serving as stimulating agents when introduced into the soil. IMMUNOLOGY The study of physiological processes that generate specific responses to infection or entry of foreign substances into the body is called immunology. Immunity refers to protection from infection, tumors, etc. Innate immunity is always available, Adaptive immunity distinguishes self from non-self and involves immune system education and responses that may result in host tissue damage Immunity is of two types: Innate immunity which is not antigen-specific and receptor driven and adaptive immunity which is antigen-specific and receptor-driven Antigen: A substance that is capable of reacting with the products of a specific immune response i.e. antibody or specific sensitized T-lymphocytes. It is a self component, may be considered an antigen even though one does not generally make immune responses against those components. An antigen is able to elicit an adaptive immune response. Its chemical composition is of minor importance. Antigens include, but are not limited to, polypeptides, carbohydrates, fats, nucleic acids and (less frequently than commonly perceived) synthetic materials. A certain minimum size is required. Very small molecules only function as antigens, so-called haptens, when coupled to larger carriers. Characteristics of adaptive immunity 1.

Immune response is highly specific for the antigen that triggered it.

2.

Receptors on surface of immune cells have same specificity as the antibody/ effect or activity that will be generated.

3.

Exposure to antigen creates an immunogenic memory.

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4.

Clonal expansion and creation of a large pool of cells committed to that antigen.

5.

Subsequent exposure to the same antigen results in a rapid and vigorous response.

Antibodies: An antibody molecule (immunoglobulin) is composed of two heavy and two light chains joined by disulfide bonds. Five alternative types of heavy chains exist (μ, γ, δ, α, ε), giving rise to IgM, IgG, IgD, IgA or IgE, respectively. Light chains are either of type κ or λ. IgM always consists of five joined immunoglobulin units. Functionally, an antibody has a variable and a constant region. While the constant region is encoded in the genome, and as such determinate like any other protein, the variable region is generated by a most unusual process referred to as rearrangement, involving cutting and pasting DNA. The immunoglobulin's variable region binds antigen.

Fig. 39.4. Attachment of antibodies to antigen (protein) (Source Rao 1997)

Antibodies recognize fairly large, three-dimensional surface structures. Any non-covalent binding force such as electrostatic attraction, hydrogen bonds, Van der Waals- and hydrophobic forces. can be used to establish this contact. Antigen binding is reversible. In most cases, a biological macromolecule contains several independent structures able to elicit an antibody response which are called as antigenic determinants or epitopes. Conversely, a phenomenon known as crossreaction exists when two very different macromolecules share a certain threedimensional structure that may be bound by the same antibodies. The above statements refer to antigens bound by antibodies. Antigens recognized by Tlymphocytes and epitopes are sensed by T-lymphocytes are linear peptides having 8 to 20 amino acids.

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If a certain protease is used to digest the Y-formed antibody, two identical fragments termed Fab (fraction antigen binding) and one fragment representing the other end, containing a large part of the constant region are formed. Only after antigen-binding, rapid internalization by phagocytosis takes place that provides mechanism for rapid antigen clearance (Fig. 39.4). Bacteria, viruses and parasites in general are antigenic. After a lag phase of at least five days, which one survives with the help of innate immunity, B-lymphocytederived plasma cells will produce specific antibodies. These antibodies then bind to the pathogens. Depending on pathogen, antibodies can help by at least five different mechanisms: 

Neutralizing viruses.



Neutralizing toxins.



Targeting and enhancing complement-lysis of bacteria.

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The serum proteins form coating around bacteria (opsonization) that helps in recognition by the immune system

ADCC (antibody-dependent cellular cytotoxicity): Via their Fc-receptors, Natural killer (NK) cells are able to sense cells carrying bound antibodies, which they proceed to kill. For example, these may be virus-infected cells exposing viral envelope proteins in their cell membrane. Immunoglobulin classes (Isotypes) The plasma cells are able to produce specific antibodies because of structural features of immunoglobulins. Five classes of immunoglobulins namely, IgG, IgM, IgA, IgE and IgD are produced. They are soluble molecules with antibody function. The basic structure of immunoglobulin is a monomer, a unit made up of two pairs of identical polypeptide [light (L)and heavy (H)] chains. IgM is a pentamer consisting of five Y-formed units arranged in a circle. It is always the first immunoglobulin coming up in response to an infection, gradually declining afterwards. By its large size, IgM is mainly confined to blood plasma. It is too big to squeeze through between endothelial cells. IgG is the standard model antibody, appearing later during an immune response than IgM. Four subclasses of IgG exist namely Ig G1 - Ig G4, of which IgG1 and IgG3 efficiently activate complement. Half-life of IgG in blood is approximately 21 days, about double that of IgM. IgG attain high molar concentrations in plasma, a prerequisite for effective neutralization of viruses or toxins. IgA can be found as a monomer in the blood, but its main function is to protect ‘outer’ epithelial surfaces. IgE developed as a tool to fight parasites (worms and protozoa). Unlike the other isotypes, it is present in plasma only in small amounts as most of it is tightly bound by the high-affinity Fc-ε-receptor of mast cells, which sit in connective tissue below outer and inner surfaces, e.g., skin, gut and bronchi.

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IgD is found together with IgM on the cell membrane of newly produced B lymphocytes, and in negligible amounts in plasma. Soluble IgD is not thought to have a function in defense. Monoclonal antibodies A monoclonal antibody obviates the specificity problem, as it constitutes amplified replicas of a single antibody produced by a single B cell. However, generating a monoclonal antibody is a time-consuming and tedious procedure. An immunological assay (usually ELISA, see below) is used with the antigen, human IgM as bait to test all culture supernatants for the presence of antibody binding it. Once found, the hybridoma cell clone can be expanded and cultured virtually indefinitely, and monoclonal antibody can be purified from its culture medium in large quantities. Today, monoclonal antibodies against most diagnostically important macromolecules are commercially available. In addition, monoclonal antibodies are increasingly being used as drugs, e.g., in anti-TNFα-therapy. However, as they mostly originate from the mouse, they would elicit an immune response in humans (HAMA: human anti-mouse antibodies). Therefore, ‘humanized’ monoclonals are used, where all parts of the mouse antibody not directly required for antigen binding are replaced by their human counterparts. The following methods are in general use in studies of antigen – antibody reactions in vitro: Neutralization: In this technique when antisera first an antotoxin combines with toxin, the latter is neutralized. Inactivation of certain viruses also occurs when the antibody combines with virus coat protein and prevents its attachment to the host cell. Precipitation: Antigens and antibodies combine in groups, as they have more than one attachment and these result in precipitation of the complex. The antigens and antibodies are placed in small wells at some distance from each other on an agar plate. Both diffuse in agar and when they meet, an opaque precipitate appears. This technique can be used to compare antigenic affinity of related substances (such as from different strains of a bacterium) the formation of precipitate indicates relationship (Fig. 39.5). Agglutination: For soluble and liquid substances, a carrier support is used for exposure to antiserum. A clumping reaction occurs which can be noticed either visually or with the aid of microscope. Blood grouping is carried out with this technique using antibodies to A, B, AB and O groups. Antibodies to Rh factor are used to find out whether the blood is Rh positive or Rh negative. Very low concentrations of antibodies can be detected by this technique (Fig. 39.5). Fluorescent antibodies: In this technique, antisera are first conjugated with a fluorescent dye like acridine orange. The antisera retain the active sites where attachment to antigen occurs (epitome region). The cells are further treated with the conjugated antibodies. The presence of antigen is located by the fluorescence of the dye, which is observed under a fluorescent microscope. This technique is highly

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useful in locating bacterial cells, virus particles and other antigens in or on the host cells.

Fig. 39.5. In vitro methods for studying antigen-antibody relations (Source: Rao 1997)

Western blot (immunoblot) Western blots are used, for instance, as a confirmation test to diagnose HIV infection. HIV proteins are denatured and solubilized using the detergent SDS, separated via a polyacrylamide gel and transferred to a paper-like membrane. This blot with bound virus proteins is then subjected to basically the same steps as described above for the virus-coated plastic well in the ELISA. The membrane is first treated with diluted patient serum, then with an enzyme-linked monoclonal antibody against human antibody, finally with substrate, with washing steps in between. If the patient has antibodies against HIV, this will show in the form of colored bands on the membrane.

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ELISA: Antibody concentrations in patients sera can be measured by many methods; the most common one is ELISA (enzyme-linked immunosorbent assay) (Fig. 39.5). To ascertain a recent infection with a specific virus, a test for IgM against that virus could be performed as follows. First, the wells of a microtiter plate are coated with virus or virus protein. Then, the wells are incubated with diluted patient serum. If antibodies are present in the serum, they will bind to the plastic-bound virus proteins. After washing thoroughly, monoclonal mouse antibody against human IgM is added. This antibody is linked to an enzyme such as horse radish peroxidase. If there was anti-virus IgM in the patient's serum, the enzyme-linked antibody will bind, too. If the serum contained no anti-virus IgM, the enzyme-linked antibody will be subsequently washed away. Finally, a colorless substrate molecule is added, which is metabolized to a bright color pigment by horse radish peroxidase. The amount of color, proportionate to the amount of anti-virus IgM in the patient serum, is photometrically quantified. Color indicates the patient has IgM against the virus and no color means no anti-virus IgM is present. An analogous parallel test could be run using another monoclonal antibody against human IgG, to check whether the patient had been infected with the same virus a longer time ago. Immunofluorescence: Sometimes, for instance in autoimmune disease, it is important to test whether a patient has antibodies against certain tissue structures, without knowing the exact molecule the antibody might recognize. To assay whether a patient has anti-nuclear antibodies, cells or a tissue section are applied to a glass slide and incubated with a droplet of diluted patient serum. If antibodies are present that bind to some nuclear structure, they can again be detected using a mouse monoclonal against human antibody, in this case coupled to fluorescent dye. If the patient has antinuclear autoantibodies (ANA), the nuclei will be brightly visible in the fluorescence microscope; in the absence of ANA, they will remain dark. Immunoelectrophoresis: For an overview whether normal amounts of IgM, IgG and IgA are present in human serum, immunoelectrophoresis is informative. First, serum proteins are separated electrophoretically in a gel. Then, rabbit antihuman serum is applied to a groove running in parallel to the axis of separation. The rabbit antiserum diffuses through the gel towards the separated human proteins. Precipitation arcs form where serum proteins and antibody meet, allowing to identify three separate arcs for IgM, IgG and IgA. In case of IgA deficiency, that specific arc would be missing.

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Chapter 40

Life Cycles in Microbes and Fungi

There is no typical life cycle known in microbes. Reproductions in bacteria being mostly by cell division and in some endospores are produced. Sexual reproduction is by genetic recombination. In some genera of actinomycetes, spores are produced asexually. Multiplication of viruses takes places inside the host cell. In bacteriophages DNA has been proved as genetic material in transduction. Fungi reproduce asexually and sexually. LIFE CYCLES IN FUNGI Life cycles in fungi are broadly divided as asexual and sexual types (Fig. 40.1)

Fig. 40.1. Asexual and sexual life cycles in fungi.

In the life cycle of sexually reproducing fungi there are two important states. 1. Karyogamy and 2. Meiosis. Depending on position where these events occur, the life cycles are mainly of three types. They are 1. Haplontic life cycle. 2. Diplontic life cycle and 3. Haplo-diplontic life cycle.

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1. Haplontic life cycle: In majority of fungi vegetative structures are haploid. The important events in the life cycle of these fungi are dikaryotization and karyogamy. In the process of plasmogamy two compatible nuclei come together resulting in dikaryotization. In some fungi karyogamy may occur immediately following plasmogamy, but in some fungi, the length of dikaryotic condition varies before karyogamy. Meiosis occurs immediately after karyogamy. Depending upon the importance of dikaryotic stage, l. Three types of haploid life cycles are recognized. In zygomycota, when two compatible nuclei come together, karyogamy occurs immediately followed by meiosis. The diploid stage is confined to the zygote only, eg. Mucor. The life cycle of Mucor is shown in the Fig. 40.2.

Fig. 40.2. Life cycle in Mucor.

In majority of ascomycots, karyogamy does not occur immediately following plasmogamy. The dikaryotic condition extends but is confined to ascogenous hyphae, and in the crozier cell. Meiosis occurs immediately after karyogamy. The ascomycete life cycle in which dikaryotic condition is confined to ascigerous hyphae is shown in Fig. 40.3.

Fig. 40.3. Life cycle in ascomycetes.

In majority of Basidiomycetes, dikaryotic condition is present in entire vegetative mycelium, and karyogamy occurs in basidium and is immediately

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followed by meiosis. The life cycle of a typical basidiomycetous fungus in shown in Fig. 40.4.

Fig. 40.4. Life cycle in basidiomycetous fungi.

2. Diplontic life cycle: In fungi belonging to the class Oomycota of Zoosporic fungi, the vegetative mycelium is diploid and meiosis occurs in gametangia. Karyogamy results in restoring diploid stage. When the oospore germinates it produces vegetative mycelium which is diploid in nature, eg. Phytophthora (Fig. 40.5).

Fig. 40.5. Life cycle in Phytophthora

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3. Haplo-diplontic life cycle: In the life cycle of some fungi, haploid gametothallus and diploid sprorothallus are equally important, eg. Allomyces, and some yeasts. In these fungi both haploid and diploid stages are structurally similar. Hence life cycles of these fungi are described as isomorphic alternation of generations. The life cycle of Allomyces is shown in the Fig 40.6.

Fig. 40.6. Life cycle in Allomyces.

ASEXUAL LIFE CYCLE IN FUNGI In asexual life cycle there is no sexual mode of reproduction. No sex organs are produced. Steps like plasomogamy, karyogamy and meiosis are lacking. The reproductive units being mitospores produced by mitotic division. These include zoospores, conidia and asexual fruit bodies. Large assemblage of fungi included in this group are anamorphic fungi. Aspergillus niger though included under Ascomycota does not produce perfect stage. In some species of Saprolegnia oospores are produced without nuclear fusion and such a condition is called parthenogenetic, apogamous or amictin. Mucor ramannianus, a member of mucorales does not produce Zygospore.

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References

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Hingorani MK and Addy SK 1952 Comparative study of Erwinia carotovora and E. ardideae and E. attroseptia, Indian Phytopathology 5 40-43 Holmes FO 1948 Bergey’s Manual of Determinative Bacteriology. 6th edn (Abstract in: Phytopathology) Williams and Wilkins Co, Baltimore 38 314 Hooke Robert C 1665 Micrographia: or Some Physiological Descriptions of Miniature Bodies Made by Magnifying Glasses, London, England: Jo Martyn and Ja Allestry ICTV 1999 International committee on Taxonomy of viruses, 7th report Iwanowski D 1892 Über die Mosaikkrankheit der Tabakspflanze, Bulletin Scientifique publié par l'Académie Impériale des Sciences de Saint-Pétersbourg / Nouvelle Serie III (in German and Russian) (St. Petersburg) 35 67-70 Jenner Edward 1798 An inquiry into the causes and effects of the variolæ vaccinæ. Sampson Low, London JL Harley 1948 Mycorrhiza and soil ecology, Biological Reviews 23 127–158 Johri RM and Lata S 2004 Text Book of Microbiology, Sonali Publications, New Delhi pp 528 Katznelson H 1946 The ‘Rhizosphere Effect’ of mangels on certain groups of microorganisms Soil Science 62 343-357 Kinoshita S, Nakayama K and Kitada S 1958 Lysine production using microbial auxotroph, Journal of General and Applied Microbiology 4 128-129 Kirk JL, Beaedette LA, Hart M, Moutoglis P, Khiromonos JN, Lee H and Trevors JT 2004 Methods of studying soil microbial diversity, Journal of Microbiological Methods 58 169-188 Krieg NR and Holt JG1984 Bergey’s Manual of Systematic Bacteriology, Vol. 1, Williams and Wilkins, Baltimore Kuby J 1994 Immunology 2nd edn WH Freeman, New York Lederberg J and Tatum EL 1946 Gene recombination in E. coli, Nature 158 558 doi:10.1038/158558a0. Lehringer AL 1982 Principles of Biochemistry, New York, Worth publishers Inc Linnaeus C 1735 Systemae Naturae, sive regna tria naturae, systematics proposita per classes, ordines, genera & species Lochhead AG 1940 Qualitative studies of soil microorganisms. III. Influence of plant growth on the character of the bacterial flora, Canadian Journal of Research (C) 18 42-53 Luria SE, Darnell JE Jr, Baltimore D and Campbell A 1978 General Virology, 3rd edn, Wiley, New York Lynch JM 1983 Soil Biotechnology: Microbiological Factors in Crop Productivity, Oxford, ELBS Publ, UK Major SR, Cronan JE and Friefielder D 1994 Microbial Genetics, Jones and Barlett Publ, London Martin Alexander 1961 Soil Microbiology, New York, Wiley McElroy WD 1969 Cell Physiology and Biochemistry, Prentice-Hall of India Pvt Ltd, New Delhi

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Subba Rao NS 1999 Soil Microbiology, Oxford and IBH Publ Co Pvt Ltd New Delhi Talaro KP 2009 Foundations in Microbiology, McGraw-Hill, New York Thimann KV 1968 The Life of Bacteria, McMillan Co Ltd, New York, USA Thomas KM and Krishnaswamy CS 1939 Leaf Crinkle - A Transmissible Disease of Papaya, Current Science 8 316 Tilak KVBR, Pal KK and Dey R 2010 Microbes for Sustainable Agriculture, IK International Publishing House Pvt Ltd, New Delhi, India Tortora GJ; Funke BR; Case CL 1989 Microbiology - An Introduction, 3rd edn The Benjamin-Cummings Publ Co Inc, New York Umbreit WW 1947 Problems of autotrophy, http://www.ncbi.nlm.nih.gov/ pmc/articles/PMC180682/ - fn1 Bacteriology Review Sep 11 157–166 van Niel CB 1931 On the morphology and physiology of the purple and green sulfur bacteria. Archiv für Mikrobiologie 3 1-112 Waksman SA 1952 Soil Microbiolog,. John Wiley and Sons, New York, USA Watson JD 1976 Molecular Biology of the Gene. 3rd edn, WA Benjamin Inc, New York Whittaker RH 1969 New concepts of kingdoms of organisms, Science 163 150–60 Bibcode:1969Sci...163..150W. doi:10.1126/science.163.3863.150. PMID 5762760 Willey JM, Sherwood L, Woolverton CJ, Prescott LM 2009 Prescott’s Principles of Microbiology, McGraw-Hill Higher Education, New York Woese C, Kandler O, Wheelis M 1990 Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya, Proceedings of the National Academy of Sciences of the United States of America 87 4576–4579 Bibcode: 1990PNAS...87.4576W. doi:10.1073/pnas.87.12.4576. PMC 54159. PMID 2112744.

Questions

Give short answers 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

What is the contribution of Haeckel? Differentiate Prokaryotes from Eukaryotes. Enlist the contributions of Whittaker and Carl Woese. Enlist contributions of: i). Anton van Leeuwenhoek, ii). Edward Jenner, iii). Louis Pasteur, iv). Robert Koch, v). Joshua Lederberg. What are Koch’s postulates? What do you understand by the theory of spontaneous generation? List five antibiotics produced by the microorganisms with their names. List five enzymes produced by the microorganisms with their names. Mention the organisms that ferment the following foods: i). Idly, ii). Kafir beer, iii). Kafir milk, iv). Yogurt, v). Cheese. Mention briefly the contribution of Har Gobind Khorana. Enlist the application of simple versus compound microscope. What is the principle of electron microscope? What is the principle of Gram staining? How do you stain the tubercle bacilli? What is differential staining? How do you calculate resolving power? What oil we use for observing bacteria under immersion lens? How do you stain bacterial cell wall? Give a brief account on size, shape and arrangement of cells in bacteria. What are the different kinds of bacterial flagella? What is the significance of enrichment culture? What is generation time? How do you determine the bacterial cell number? Classify the microorganisms on the basis of their oxygen requirements for growth. Write an account of HIV virus. What is the basis of classification of actinobacteria? What are archaebacteria?

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Biology and Biotechnology of Fungi and Microbes 27. 28. 29. 30. 31. 32.

33. 34. 35. 36. 37. 38. 39. 40. 41.

Give a brief account of mycoplasma. Give a brief note on prions? Where do you put cyanobacteria in microbial taxonomy? List the families of Cyanobacteria that fix atmospheric nitrogen. Give a brief account of protozoans. Mention the causal organism for the following diseases: i). Brown rot of potato, ii). Yellow rot of wheat, iii). Crown gall of cotton, iv). Citrus canker, v). Bacterial blight, vi). Angular leaf spot of cotton. Mention five diseases caused by bacteria in human beings. Mention few virus diseases of plants. What is biodiversity? List National Centres for conservation of microorganisms in India. Give a brief account of microbial pesticides. Write briefly on: i). Single strand conformation polymorphism (SSCP); ii). SNuPE (Single Nucleotide primer extension). Differentiate anabolism from catabolism. What is the role of Acetyl Co A in Kreb’s cycle? Mention various products formed during anaerobic metabolism.

Give long answers 1.

Give a brief outline of division of living kingdoms as proposed by different workers. 2. What are the recent developments in the field of molecular microbiology? 3. How did Koch’s postulates influence the development of microbiology? 4. Give notable contributions of the following scientists that resulted in the Nobel prize award to them in microbiology: i). Otto Warburg, ii). Selman A. Waksman, iii). Kary Mullis, iv). Stanley B. Prusiner, v). S. Ochoa, vi). A. Kornberg. 5. Write in detail the contributions of M.W. Beijernick and S. Winogradsky to soil microbiology. 6. Write in details the proponents and opponents of theory of spontaneous generation. 7. Describe the significant contributions of: i). Omeliansky, ii). John Russel, iii). Harley, iv). Garrett, v). Barker, vi). Ross and Cholodny, vii). Ruinen, viii). Alexander Fleming, ix). Starkey, x). Van Niel. 8. Discuss the developments of microbiology during 1854-1914 (Golden era of microbiology). 9. Mention some significant contributions of at least six scientists in soil microbiology during 20th century. 10. Discuss briefly the following: i). Dark field microscopy, ii). Phase contrast microscopy, iii). Numerical aperture.

Questions

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11. How do you prepare the hanging drop slide? 12. Discuss the role of staining for spores, cell wall and flagella. 13. Mention the numerical aperture and resolving power of the following lenses: i). Low power, ii). High Power, iii). Oil immersion. 14. Discuss in detail the cell wall structure of Gram - positive and Gram - negative bacteria. 15. What are plasmids? Discuss in detail different types of plasmids. 16. Discuss briefly the media used in bacterial cultivation. 17. Define growth. What are the different growth phases in bacteria? 18. Write short notes on: i). Nucleus, ii). Nucleoid, iii). Generation time, iv). Fermenter, v). Numerical taxonomy, vi). Bacterial phylogeny, vii). Diauxic growth, viii). Transposons, ix). Ribosomes, x). Pili. 19. Describe briefly the methods used in molecular taxonomy. 20. Mention the basis of outline classification of bacteria. 21. What is genetic recombination? Describe briefly the various recombinations in bacteria. 22. Describe the role of bacteria in industry. 23. Discuss briefly on the following: i). Biogeochemical cycles, ii). Nitrogen-fixers, iii). Phosphate solubilizers, iv). Sewage treatment, v). Biopesticides, vi). Biodeterioration, vii). Food spoilage, viii). Biofuels, ix). Biological control, x). Biodeterioration, xi). Bofertilizers. 24. Discuss in detail the role of Ti plasmids. 25. Describe the role of fermenter in bacterial culturing. 26. Classify bacteria on the basis of their energy and nutritional requirements. 27. Write briefly on the following: i). Numerical taxonomy, ii). Bacterial phylogeny, iii) DNA-DNA hybridization, iv). Ribotyping. 28. What is virus? Give an account of its history and development. 29. Mention different types of viruses with their structural arrangement. 30. Discuss the classification of viruses as proposed by Casjens and King. 31. Describe the structure of bacteriophage. 32. Describe the methods for cultivation of viruses. 33. Write short notes on: i). Lysogeny, ii). Burst size, iii). Icosahedral symmetry, iv). Enveloped viruses, v). Complex viruses, vi). RNA virus replication. 34. Discuss the characteristics of different members of archaebacteria and their significance. 35. Discuss in detail the bioprospecting of Archaea. 36. How do you classify Archaea on the basis of their nutrition and energy sources? 37. What are the basic characters of cyanobacteria? 38. What are the different orders of cyanobacteria? 39. Write short notes on the following: i). Hormogonia, ii). Heterocyst, iii). Single cell protein, iv). Nostoc, v). Anabaena, vi). Nutrition of protozoa.

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Biology and Biotechnology of Fungi and Microbes 40. Mention briefly the following: i). Actinorhizal nitrogen fixation, ii), Mollicutes, iii). Chlamideae, iv). Rickettsiae, v). Actinomycosis, vi). Mycobacteria, vii). Sporophores, viii). Mutualism. 41. Discuss briefly symptoms and control of following diseases: i). Wild fire of tobacco, ii). Soft rot of potato, iii). Leaf spot of mango, iv). Scab of potato. 42. Give the symptoms and transmission of the following viral diseases: i). Bhendi vein clearing, ii). Bunchy top of banana, iii). Tungro disease of rice, iv). Bean mosaic virus, v). Groundnut necrosis. 43. Mention few plant diseases caused by Spiroplasma and their control measures. 44. Describe briefly the causal organism, symptoms and control of: i). Spike disease of sandal wood, ii), Grassy shoot of sugarcane. 45. Describe different types of diversity. 46. How do you conserve biodiversity? 47. Write briefly on the following: i). HClO, ii). ITCC, iii). Cereal nodulation, iv). Microbial nematicides, v). Vermitechnology, vi). Edible fungi. 48. Give an account of PCR techniques. 49. Differentiate between the following: i). RFLP vs. ARDRA, ii). DGGE vs. TGGE, iii). RISA vs. ARISA. 50. Give a brief account of EMP pathway. 51. What is the significance of PP pathway? 52. Write short notes on the following: i). Cephalosporins, ii). Aminoglycosides, iii). Tetracyclines, iv). Erythromycin. 53. Mention various biotechnological approaches that help in sanitary engineering related environmental activities. 54. What are the developments in industrial biotechnology? 55. Write an account of soil remediation.

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Glossary

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Acervulus (pl. acervuli) (L. acervus = heap) — Saucer shaped conidiomata, embedded in host tissue in which the hymenium of conidiogenous cells develop on the floor of mat of hyphae in the cavity from a pseudoparenchymatous stroma beneath an integument of host tissue which ruptures at maturity (characteristics of the order Melanconiales). It bears conidia. Acid-fast stain — A differential stain used to identify bacteria that are not decolorized by acid-alcohol. Acquired immunity — The ability, obtained during the life of the individual, to produce specific antibodies. Acrogenous — Development at the apex Acropetal — Having the youngest conidia at the apex of a chain. Activated sludge — Aerobic digestion used in secondary sewage treatment. Adenine — A purine nucleic acid base that pairs with thymine in DNA and uracil in RNA. Adenosine diphosphate (ADP) — The substance formed when ATP is split and energy is released. Adenosine triphosphatase (ATPase) — The enzyme that catalyzes the following reactions: ADP + P = ATP and ATP = ADP + P. Aeciospore (Gr. aikia = injury + spora = seed spore) — A binucleate spore produced in an aecium. Aerobe — An organism requiring O2 to grow. Aerobic respiration — Respiration in which the final electron acceptor in the electron transport chain is oxygen. Aeromycology — The study of fungal (and other) propagules in the atmosphere. Aflatoxin — C17 H10 O6, a carcinogenic toxin produced by Aspergillus flavus. Agar — A complex polysaccharide derived from a marine alga and used as a solidifying agent in culture media. Agaric — A term commonly used to describe a fungus having a cap (pileus), gills (lamellae), and a stem (stipe). Agglutination — A joining together or clumping of cells. Alcohol — An organic molecule with the functional group – OH.

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Biology and Biotechnology of Fungi and Microbes Aleuriospore — Frequently used for thick walled and pigmented, but sometimes thin walled and hyaline conidia developed from the blown end of conidiogenous cell or hyphal branch from which it secedes with difficulty. Algae — Numerous groups of chlorophyll-containing, mainly aquatic eukaryotic organisms ranging from microscopic single-celled forms to multicellular form. Alkaline — having more OH- ions than H+ ions; pH is greater than 7. Allele (allelomorph) — Any of one or more alternative forms of a given gene; both (or all) alleles of a given gene are concerned with the same trait or characteristic, but a particular allele codes for a product qualitatively and/or quantitatively different from that coded by other alleles of that gene. Allergen — A substance which provokes a hypersensitive reaction in the body. Amoeboid cell (of Ameobidiales) — Uninucleate cells formed by protoplasmic cleavage within the fungal thallus which lacks a rigid wall and is released usually encysted. Amino group — – NH2 Ammonification — Removal of amino groups from amino acids to form ammonia. Amoeba — An organism belonging to the Kingdom Protista that moves by means of pseudopods. Amphitrichous — Having tufts of flagella at both ends of a cell. Anabolism — All synthesis reactions in a living organism. Anaerobe — An organism that does not require oxygen for growth. Anaerobic respiration — Respiration in which the final electron acceptor in the electron transports chain is an inorganic molecule other than oxygen; for example a nitrate or sulfate. Anamorph (Gr. ana = anew, morph = form) — The non-sexual or asexual (usually conidial) state in the life cycle of a fungus (teleomorph). Anastomosing — Joining up, running into each other of branched paraphyses which form a network. Angiocarpous — In case of basidiocarp (basidiome) hymenial surface at first exposed but later covered by an incurving pileus margin and/or excrescence from the stipe. Animal virus — A virus that multiplies in animal tissues. Animalcules — Animal-like organisms Anisogamy — The copulation of gametes of unlike form. Annular — Arranged in or forming a ring. Anoxygenic — Not producing molecular oxygen; typical of bacterial photosynthesis. Antagonism — Active opposition; for example, between two drugs or two microbes. Antheridiol — A sex hormone (sterol) of Achlya bisexualis which induces antheridial formation in male strains of Achlya. Antibiotic — An antimicrobial agent produced naturally by a bacterium or fungus.

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Antibody — A protein produced by the body in response to an antigen and capable of combining specifically with that antigen. Anticodon — The three nucleotides by which a transfer RNA recognizes an RNA codon. Antimicrobial — An agent that kills or inhibits microorganisms. Antimycotic (antifungal) — An agent that destroys or inhibits the growth of fungi. Antiseptic — A chemical disinfectant for disinfection of the skin, mucous membranes, or other living tissues. Antiserum — A solution containing antibodies. Apoenzyme — The protein portion of an enzyme, which requires activation by a coenzyme. Apothecium (pl. apothecia) (Gr. Apo = lack, theke = sheath) — Cup-shaped, sporebearing structures produced by certain types of fungi; the cup or saucer-shaped fruiting body of the Ascomycotina. Appressorium (pl. appressoria) (L. apprimere = to press against) — An enlargement on a hypha or germ tube which attaches itself to the host before penetration takes place. Aquatic fungi — fungi living in water, especially freshwater, in contrast to marine fungi. Arbuscular Mycorrhiza (AM) — Mycorrhizal type that forms highly branched arbuscules within root cortical cells. Arbuscule — A tree-like dichotomously branched hyphal structures within the host plant cells, main site for nutrient exchange. Archaebacteria — Procaryotic organisms lacking peptidoglycan. Ascocarp (Gr. askos = sac, karpos = fruit) — The ascospore bearing, muliticellular sporocarps formed by a member of the Ascomycotina. Ascogenous hyphae (Gr. askos = sac, genesis = being produced) — Those hyphae often arising from the ascogonia, which give rise to asci. Ascogonium (Pl. ascogonia) (Gr. askos = sac, gonos = offspring) — The cell or cells of the female structure, which usually receives the male nuclei and then gives rise to the ascogenous hyphae; group of cells in Ascomycotina fertilized by a sexual act or process; female reproductive organs of the Ascomycetes which receives the antheridial nuclei in fertilization, and from which the dikaryotic hyphae emerge; (in Ascomycetes) the female gametangium; it may be unicellular or muliticellular, simple or complex in form. Ascomycotina — Major division of fungi in which spores are developed within asci (endogenously). Ascospore (Gr. askos = sac, sporos = seed) — A spore borne in an ascus; a haploid spore (meiospore) produced after sexual reproduction within the ascus of a sac fungus; usually 8 per ascus; the spores are formed by karyogamy and meiosis within an ascus. Ascus (pl. asci) — A specialized sexual reproductive cell found in the fertile area of the hymenium of all Ascomycetes.

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Biology and Biotechnology of Fungi and Microbes Asexual (L. a = away + sexus = sex) — Reproduction not involving karyogamy and meiosis. Attenuation — Lessening of virulence of a microorganism, also, a regulatory mechanism for protein synthesis. Autoclave — Equipment for sterilization by steam under pressure usually operated at 15 psi and 121ºC. Autoclaving — Sterilisation by steam under pressure [15 lb/in2 or 15 psi (per square inch)] for prescribed time period. Autotroph — An organism that uses carbon dioxide as its principal carbon source possessed by the parent. Axial filament — The structure for motility found in spirochetes. Axoneme (in cilia and flagella) — The active central unit, including the microtubules and their interconnections. Bacillus — Any rod-shaped bacterium; when written as a genus refers to rodshaped, endospore-forming, facultatively anaerobic, gram-positive bacteria. Bacteria — All living organisms with prokaryotic cells. Bacterial growth curve — A graph indicating the growth of a bacterial population over time. Bactericidal — Capable of killing bacteria. Bacteriocin — Toxin protein produced by bacteria that kill other bacteria. Bacteriophage (phage) — A virus that multiplies in bacterial cells. Bacteriostatic — Capable of inhibiting bacterial growth. Bacteroid — Enlarged Rhizobium cells found in root nodules. Basal body —A structure that anchors flagella to the cell wall and plasma membrane. Basidia — The plural form of basidium. Basidial stage (sing. basidium) — A spore stage of the rust fungi; a specialized structure in the Basidiomycetes bearing basidiospores. Basidiocarp (Gr. basidion = small base, karpos = fruit) — Sporocarps produced by members of the Basidiomycotina and which bear basidiospores. Basidiospore — A propagative cell (typically a ballistospore but in Gasteromycetes a statismospore) containing one or two haploid nuclei produced after meiosis on a basidium. Bergey’s manual — The standard taxonomic reference on bacteria. Binary fission — Bacterial reproduction by division into two daughter cells. Binomial (L. bi = two + nomial = name) — The scientific name of an organism; it is composed of two names; the first designating the genus, the second the species, or ‘trivial’. Binomial nomenclature — The system of having two names (genus and specific epithet) for each organism. Bioconversion — Changes in organic matter brought about by the growth of microorganisms.

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Biodeterioration — Any undesirable change in the properties of a material caused by the vital activities of organisms. Biogeochemical cycles — The recycling of chemical elements by microorganisms for use by other organisms. Bioinoculants — Microbial cultures used as biofertilizers and biopesticides Biomass — Organic matter produced by living organisms and measured by weight. Bioprospecting — The action of surveying natural ecosystem for economically valuable biotic products. For fungi, such products include novel edible fungi, valuable enzymes for biotechnology companies, metabolites for pharmaceutical investigations, or new biological control agents, etc. Biotechnology — The use of microbes in industry. Bipolar heterothallism (L. bi = two + Gr. polos = pole) — A condition of sexual compatibility in which there are only two mating types; also known as unifactorial heterothallism. Bitunicate (L. bi = twice, two , + tunica = covering, coat, mantle) — An ascus in which the inner wall is elastic and expands greatly beyond the outer wall at the time of spore liberation. Blastomycosis — A disease in humans caused by Blastomyces dermatidis (teleomorph Ajellomyces dermatidis) Bloom (algal) — Abundant growth of microscopic algae, producing visible colonies in nature. Budding — Asexual reproduction beginning as a protuberance from the parent cell that grows to become a daughter cell; also, release of an enveloped virus through the plasma membrane of an animal cell. Capsid — The protein coat of a virus that surrounds the nucleic acid. Capsomere — A protein subunit of a capsid. Capsule — An outer, viscous covering on some bacteria composed of a polysaccharide or polypeptide. Carbohydrates — Organic compounds composed of carbon, hydrogen, and oxygen, with the hydrogen and oxygen present in a 2:1 ratio; includes starches, sugars and cellulose. Carbon cycle — The series of processes that converts carbon dioxide to organic substances and back to carbon dioxide in nature. Carboxyl group — – COOH. Carboxysome — Procaryotic inclusion containing ribulose 1,5-diphosphate carboxylase. Carotenoid — Any of a group of red, yellow, and orange plant pigments chemically and functionally similar to carotene (yellow and orange). Carrier — An inert material used as a vehicle for the active ingredient or toxicant. Casein — Milk protein. Catabolism — All decomposition reactions in a living organism. Catalase — An enzyme that catalyzes the breakdown of hydrogen peroxide to water and oxygen.

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Biology and Biotechnology of Fungi and Microbes Catalyst — A substance that affects the rate of a chemical reaction, usually increasing the rate, but isn’t changed in the reaction. Cell wall — The semi rigid extracellular encasement, outside the cell membrane of a fungal cell that gives it a definite shape. Cell wall — The outer covering of most bacterial, fungal, algal, and plant cells: in eubacteria, it consists of peptidoglycan. Cellulase — An enzyme that breaks down cellulose, the primary component of plants. Cellulose — The inert, insoluble carbohydrate consisting of unbranched chains of beta glucose (β-1, 4-linkage), that is the principal constituent of wood. Cellulose — A glucose polysaccharide that is the main component of plant cell walls. Cephalosporin — An antibiotic produced by the fungus Cephalosporium that inhibits the synthesis of gram-positive bacterial cell walls. Chemical energy — The energy of a chemical reaction. Chemoautotroph — An organism that uses an inorganic chemical as an energy source and carbon dioxide as a carbon source. Chemotherapy — Treatment of a disease with chemical substances. Chemotroph — An organism that uses oxidation-reduction reactions as primary energy source. Chitin — A structural carbohydrate that is the principal organic component of arthropod exoskeleton. A polymer of N- acetyl glucosamine. Chitin — A glucosamine polysaccharide that is the main component of fungal cell walls and arthropod skeletons. Chloramphenicol — A broad spectrum bacteriostatic chemical. Chlorophyll a — The light-absorbing pigment in cyanobacteria, algae, and plants. Chromosome — The structure that carries hereditary information. Cilia — Relatively short cellular projections that move in a wavelike manner. Cisternae — Stacked elements of the Golgi complex. Clamp connection (also clamp) — Upon mitosis in the dikaryotic cells of certain fungi, the formation of a peculiar branching and rejoining of cytoplasm occurs, which acts as a bridge to allow passage of one of the products of nuclear division into the penultimate cell, thereby ensuring maintenance of the dikaryotic condition (of members of the Basidiomycotina). Cleistothecium (pl. cleistothecia) (Gr. kleistos = closed, + theke = case) — Completely closed ascocarp. Clone — A population of genetically identical cells derived from a single parent cell. Coccus — A spherical or ovoid bacterium. Codon — A group of three nucleotides in mRNA that specifies the insertion of an amino acid into a protein. Coenocyte — A multinucleate tube like hypha without septa with continuous cytoplasm.

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Coenzyme — A non-protein substance that is associated with and that activates an enzyme. Coliforms — Aerobic or facultatively anaerobic, gram-negative, non-spore forming, rod-shaped bacteria that ferment lactose with acid and gas formation within 48 hours at 35ºC. Colony — A clone of bacterial cells on a solid medium that is visible to the naked eye. Columella (pl. columellae) (L. columen = column) — A sterile dome like expansion at the apex of a sporangiophore. Communicable disease — Any disease that can be spread from one host to another. Compatibility — Ability to be cross-mated or cross-fertilised. Compound light microscope — An instrument with two sets of lenses that uses visible light at the source of illumination. Condenser — A lens system located below the microscope stage that direct light rays through the specimen. Conidioma (pl. conidiomata) — Any specialized multihyphal structure producing conidia, e.g. synnematal, sporodochial, acervular, or pycnidiomata. Conidiophore (Gr. phoreus = bearer + conidium) — A simple or branched hypha arising from a somatic hypha and bearing at its tip or side, one or more conidiogenous cells; sometimes used interchangeably with conidiogenous cell producing conidia. Conidium — Asexual reproductive unit in Deuteromycotina fungi; in Mitosporic fungi. Conjugation — The transfer of genetic material from one cell to another involving cell-to-cell contact. Conjugative plasmid — A plamid with genes for carrying out conjugation. Constitutive enzyme — An enzyme that is produced regardless of how much substrate is present. Contagious disease — A disease that is easily spread from one person to another. Cristae — Folding of the inner membrane of the mitochondria. Crop rotation — The practice of growing a sequence of different crops on the same land in successive years or seasons; done to replenish the soil, curb pests, etc. Crossing over — The process of exchange of chromatid segments by enzymatic breakage and reunion during meiotic prophase. Culture medium — The nutrient material prepared for the growth of microorganisms in a laboratory. Culture — Microorganisms that grow and multiply in a container of culture medium. Curd — The solid substance that forms when milk turns sour; the solid part of milk that separates from the liquid in the making of cheese. Cyanobacteria — Oxygen-producing photoautotrophic prokaryotes; formerly called blue-green algae.

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Biology and Biotechnology of Fungi and Microbes Cyclosporine — A drug that suppresses the T-cell response. Cystidium (pl. cystidia) — A sterile structure, frequently of distinctive shape, occurring at any surface of a basidiomata, particularly the hymenium from which it projects. Cytochrome oxidase — an enzyme that oxidizes cytochrome c. Cytoplasm — In a prokaryote, everything inside the plasma membrane; in a eukaryote, everything inside the plasma membrane and external to the nucleus. Cytosine — A pyrimidine nuclei acid base that pairs with guanine. Darkfield microscopy — A microscope that has a device to scatter light from the illuminator so that the specimen appears white against a black background. Decolorization — the process of removing colour/stain. Decomposer (reducer) — An organism that breaks down organic wastes and remains of dead organisms into simpler compounds such as carbon dioxide, ammonia and water. Dehydration — The removal of water. Denitrification — The reduction of nitrates to nitrites or nitrogen gas. Deoxyribonucleic acid (DNA) — The molecule that carries the genetic information in most living systems. Deoxyribose — A five-carbon sugar contained in DNA nucleotides. Dermatitis — Inflammation of skin evidenced by itching, redness and various skin lesions. Detergent — Any substance that reduces the surface tension of water. Diagnosis — Identification of a disease. Dicaryon (dikaryon) — A pair of haploid nuclei that occur in a cell, the nuclei undergo simultaneous division (conjugate division) upon formation of each new cell. Typified in the diploid phase (dikaryophase) of Basidiomycetes. Dikaryotisation — the conversion of homokaryon into a dikaryon typically by the fusion of two compatible homokaryons, illegitimate the sporadic occurrence of diaryon in non—compatible di—mon mating. Dipicolinic acid — Chemical substances found in bacterial endospores and not in vegetative cells. Diplanetic (L. di = two; Gr. planos = wandering) — Having a succession of two morphologically different zoospore stages separated by a resting stage (in the Oomycetes). Diplococci — Cocci that divide and remain attached in pairs. Diploid (Gr. diplos = double) — Having a double set of genes and chromosomes, one set from each parent. Disaccharide — A sugar consisting of two monosaccharides. Disease — Any change from a state of health. Disinfectant — An agent, such as heat, radiation, or a chemical, that destroys, neutralizes, or inhibits the growth of disease-carrying microorganisms commonly used inanimate objects.

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Diversity — Variety in morphology, ecosystem, soil, water, marine and other habitats and also their interrelationships and ecosystem complexes, variation among genera, species and gene pools. Dolipore septum (L. dolium = large jar, + pore) — A septum with central pore surrounded by a barrel shaped swelling of the septal wall and covered on both sides by a perforated membrane termed the septal pore cap; common in may Basidiomycetes. Dysentery — A disease characterized by frequent, watery stools with blood and mucus. Ecological niche — The environment favorable to an ecotype. Ecosystem — A dynamic system of plants, animals, and other organisms, living together with the nonliving components of the environment, functioning as an interdependent unit, an ecological niche. Electron acceptor — An ion that picks up an electron that has been lost from another atom. Electron microscope — A microscope that uses a flow of electrons instead of light to produce an image. Electron transport chain — A series of compounds that transfer electrons from one compound to another, generating ATP by oxidative phosphorylation. Electron — A negatively charged particle in motion around the nucleus of an atom. Electrophoresis — The separation of substances by their rate of movement through an electric field. ELISA (enzyme-linked immunosorbent assay) — A group of serological tests that use enzyme reactions as indicators. Endemic disease — A disease that is constantly present in a certain population. Endospore — A resting structure formed inside some bacteria. Enrichment culture — A culture medium used for preliminary isolation that favors the growth of a particular microorganism. Enterotoxin — An exotoxin that causes gastroenteritis; produced by Vibrio, Escherichia coli etc. Entner-Doudoroff pathway — An alternative pathway for the oxidation of glucose to pyruvic acid. Entomophilous — Fungi colonized by insects. Enzyme — A protein that catalyzes chemical reactions in a living organism. Epidemic disease — A disease acquired by many people in a given area in a short time. Epidemiology — The science dealing with when and where diseases occur and how they are transmitted. Eubacteria — Procaryotic organisms which possess petidoglycan cell walls. Eucarpic (Gr. eu = normal, karpos = fruit) — A condition in which only part of assimilative thallus is converted into reproductive structure(s). Eucaryote — A cell with DNA enclosed within a distinct membrane-bounded nucleus.

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Biology and Biotechnology of Fungi and Microbes F factor: Fertility factor; a plasmid found in the donor cell in bacterial conjugation. Fairy rings — Fungus rings, which are generally formed in the members of Basidiomycetes (some 60 recorded species), are very frequent in grass and grass land, and not uncommon in woods. Fermentation — The enzymatic degradation of carbohydrates in which the final electron acceptor is an organic molecule, ATP is synthesized by substrate-level phosphorylation and oxygen is not required. Flagella — Thin appendages that arise from one or more locations on the surface of cell and are used for cellular locomotion. Flagellum (pl. flagella) (L. flagellum = whip) — A long, whip like, motile, cylindrical extension of a eukaryotic cell, bound by a plasma membrane and containing an axoneme; longer and fewer in number than cilia, they propel the cell in a liquid medium by undulations. Two types can be distinguished by electron microscopy the whiplash with a smooth continuous surface (as in Chytridiomycota) and the tinsel characteristic of Hypochrytridiomycota, with the surface covered with hair-like processes (mastigonemes or flimmers). Flavoprotein — Protein with flavin coenzyme that functions as an electron carrier in respiration. Fluorescence — The ability to give off light of one colour when exposed to light of another colour. Fluorescent microscope — A microscope that uses an ultraviolet light source to illuminate specimens that will fluoresce. Foliose — A lichen thallus which has broad lobes free from the substrate. Foot cells — The base of the conidiophore of Aspergillus species where it merges with the hypha and resembles the heel and toes of a foot. Fossil — Any remains, impression, or trace of an animal or plant of a former geological age, such as mineralized skeleton, a footprint, or a frozen mammoth. Fungicide — Chemical used to control fungal diseases. Despite the name, most fungicides only slow down or prevent the spread of disease; only a few actually kill the fungus. Gametangium (pl. gamentangia) (Gr. gamete = germ, angeion = case, vessel) — A differentiated cell that produces discrete gametes or whose undifferentiated protoplast functions instead of discrete gametes. Gamete (Gr. gamete = germ) — A differentiated ‘germ-cell’ (egg or sperm); a reproductive cell whose haploid nucleus, containing a single copy of chromosome, is capable of fusion with that of a gamete of the opposite mating type. Gelatin — The product obtained by boiling collagen Gene — A segment of DNA or a sequence of nucleotides in DNA that codes for a functional product. Generalized transduction — Transfer of bacterial chromosome fragments from one cell to another by bacteriophage. Genetic code —The code in which information for the synthesis of protein is contained in the nucleotide sequence of a DNA molecule (or in certain viruses of a RNA molecule)

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Genetic engineering — Manufacturing and manipulating genetic material in vitro. Genetic recombination —The process of joining pieces of DNA from different sources Genetics — The science of heredity. Genome — The genetic material of an organism (eg. the chromosome(s) in a bacterial cell or the DNA or RNA in a virion Genotype — The genetic makeup of an organism. Genus (pl. genera) — The first name of the binomial scientific name; the taxon between family and species. Germ theory — The principle that microorganisms cause disease. Germ — Part of an organism capable of developing. Gill fungi — Mushrooms; members of the family Agaricaceae of the class Basidiomycetes, which produce fruiting bodies consisting of a stripe that supports a pileus whose lower surface has radially arranged lamellae bearing the hymenium. Globulin — The protein type to which antibodies belong. Glycerol — An alcohol; C3H5 (OH)3. Glycolysis — The main pathway for the oxidation of glucose to pyruvic acid. Golgi complex — An organelle involved in the secretion of certain proteins. Gram-stain — A differential stain that divides bacteria into two groups, grampositive and gram-negative. Guanine — A purine nucleic acid that pairs with cytosine. Habitat — The natural environment or substrate in which a population or individual lives; includes not only the place where a species is found, but also the particular characteristics of the place, e.g. climate or the availability of suitable food and shelter that makes it especially well suited to meet the life cycle needs of that species. Not necessarily synonymous with ecological niche. Hallucinogenic — Mind-altering. An urge that produces mood changes or changes in perception. Halophile — An organism that grows in high concentration of salt. Hartig net — The intracellular hyphal network formed by an ectomycorrhizal fungus in the surface layers of a root; the effective interface between the symbionts. Haustoria — Specialized host penetrating hyphae of parasitic fungi. Helicospore (Gr. helic = helix; spora = seed, spore) — Non-septate or septate spore, with a single (usually elongated) axis curved through at least 180 degree but may describe one or more complete rotations, in two of three dimensions; any protuberances other than secular less than ¼ spore body length. Herbarium — A collection of dried plants or fungi the place in which such a collection is stored. Often also used for dried reference collections of fungi, especially when curated along with plant specimens. Heredity — The transmission of genetic characters from parents to offspring, and the effects of this transmission. 2. Transfer of genetic information from parent cell to progeny (refer Genetics). Heterocyst — A large cell in certain cyanobacteria; site of nitrogen fixation.

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Biology and Biotechnology of Fungi and Microbes Heteroecism (Autoecious) (Gr. heteros = other, different + oikos = home, i.e. host) — The necessasity of two species for the completion of the life cycle of certain parasitic fungi. Heterokaryon (Gr. heteros = different from, karyon =nucleus) — A cell which contains genetically different nuclei or a thallus made of different cells, in which mating is prevented between strains. Heterothallic (Gr. heteros = different from, thallus = shoot) — The condition of being self sterile, i.e. male and female organs are developed on one individual as in case of Ascobolus magnificus, requiring a partner for sexual reproduction. Heterotroph —An organism that uses organic compounds for most of its carbon requirements Histones — Proteins associated with DNA in eukaryotic chromosomes. Holobasidium (pl. holobasidia) (Gr. holos = entire + basidion = a small base) — A single-celled basidium although typically club shaped. Holobasidia may resemble tuning forks in some taxa, while in others the basidium may become divided by adventitious septa. A single celled structure not divided by septa which ultimately gives rise to basidia (refer Phragmobasidium). Holoblastic — When both outer and inner walls of the conidiogenous cell contribute to the formation of the conidium. Holomorph — All manifestations of a genotype; in a fungus this frequently means anamorph plus teleomorph. Homothallism (Gr. homo = same + thallus= shoot, thallus) — The condition exemplified by the homothallic species, i.e. the condition of being self fertile, able to reproduce sexually without a partner. Host — An organism infected by a pathogen. Humus — Organic matter that remains in soil following partial decomposition. Hymenium (pl. hymenia) (Gr. hymen = membrane) — A palisade like layer of asci or basidia, including any sterile cells such as paraphyses or cystidia. Hyperplasia (Gr. hyper= over + plasis = molding, formation) — An overgrowth resulting in an abnormal increase in the number of cells. A condition in which a tissue is enlarged or overdeveloped as a result of excessive multiplication of cells, as in crown gall caused by Agrobacterium tumefaciens, or witches broom, leading to the enlargement of an organ or tissue owing to an increase in the number of cells. Hyphomycetes — Conidial anamorph producing exposed conidiophores, not enclosed in any protective structure. ICBN — International Code of Botanical Nomenclature. Immunization — A process that produces immunity. Incubation period — The time interval between the actual infection and first appearance of any signs or symptoms of disease. Infection — Growth of microorganisms in the body. Ionization — Separation of a molecule into groups of atoms with electrical charges. Isogametangia (sing. isogametangium) (Gr. ison = equal + gametes = husband + angeio = container) — Gamentangia, presumably of opposite sex, that are morphologically indistinguishable.

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Isolation — 1. The process of separating a microorganism, a fungus or virus from a substratum or host into pure culture. 2. Maintenance of healthy plant material at its sites, away from sources of inoculums, or maintenance of stocks of infected material at sites remote from potential hosts. Jelly fungi — 1. A term sometimes applied to the Tremellales. 2. Usually saprobic, wood-inhabiting Basidiomycetes with gelatinous basidiomata; Orders: Tremellales, Auriculariales (Phragmobasidiomycetes); Dacrymycetales (Holobasidiomycetes). Keratinophilic — Fungi that are capable of decomposing keratin and causing superficial mycoses in humans. Kingdom — One of the primary divisions of life forms in the traditional plant and animal kingdoms. It is the highest taxonomic category of which five are currently recognized (Monera, Protista, Fungi, Plantae, Animalia). Kingdom — The highest category in the taxonomic hierarchy of classification. Koch’s postulates — Criteria used to determine the causative agent of infectious diseases. Kreb’s cycle — A pathway that converts two carbon compounds to carbon dioxide, transferring electrons to NAD + and other carriers. L form — A natural bacterium with defective cell wall. Lag phase — The time interval in bacterial growth curve with no growth. Lichens — Symbiotic association of a photobiont and mycobiont growing on rocks, twigs, bark, petioles, leaves etc. Life cycle (life history) — 1. The complete succession of changes undergone by an organism during its lifetime. A new cycle occurs when an identical succession of changes begin. 2. The succession of stages following a particular phase of development, or spore which culminates with the production of the same phase or spore form. 3. (in fungi) The stage or series of stages, frequently characterized by different spore states (refer states of fungi) between one spore form and development of the same spore form again. 4. Characteristic structure of plants, animals or fungi in each development stage. Ligase — An enzyme that joins together pieces of DNA. Lignin — 1. An amorphous substance that gives wood its rigidity. 2. The second most abundant constituent of wood after cellulose. It is the thin cementing layer between the wood cells. A polymer of phenylpropanoid units is an important constituent of wood, very resistant to biodegradation, but degraded by many Basidiomycetes. Lipase — An exoenzyme that breaks down fats into their component fatty acids and glycerol. Litter (also leaf litter) — Duff, the uppermost slightly decayed layer of organic material on a forest floor. 2. Surface layer of the forest floor consisting of freshly fallen leaves, needles, twigs, stems, bark and fruits. Loculoascomycetes — A subclass for Ascomycotina, having bitunicately discharging asci, producing ascospores which are generally septate, and born in unwalled locules (pseudothecia) in ascomata, with an ascolocular ontogeny. Lyophilisation (freeze-drying) — 1. A technique used in the preservation of plant tissues, microorganism, etc., where by water is removed under vacuum while the

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Biology and Biotechnology of Fungi and Microbes tissue remains in the frozen state. Synonymous with freeze-drying. 2. Rapid freezing of a material at low temperature followed by rapid dehydration by sublimation in a high vacuum. A method used to preserve biological specimens or to concentrate macromolecules with little or no loss of activity. Lysogeny — A state in which phage DNA is incorporated into the host cell without lysis. Lysozyme — An enzyme capable of lysing bacterial cell walls. Lytic cycle — A sequence for replication of phages that results in host cell lysis. Macrocyclic — 1. Describes rust fungi which produce all five developmental stages basidiospore, spermatia, aeciospores, teliospores and uredinospores. 2. The two primary spore stages are present (of rusts) (telial with teliospores and aecia with aeciospores). Mastigonemes (Gr. mastix = whip + nema = skein, thread) — One of the numerous small, hair-like projections on a hairy flagellum of the Chromista; also known as flimmers. Meiospore (Gr. meion = less + spora = spore). — A uninucleate, haploid spore arising directly by meiosis. Mesophile — Organism whose optimum temperature for growth falls in an intermediate range of approximately 25 to 40o C. Microorganism — A living organism too small to be seen with naked eye; Microtubule — The structure of the proteins comprising eukaryotic flagella and cilia. Mitochondria — Organelles containing the respiratory ATP synthesizing enzymes. Mitosis (pl. mitoses) — The division of the cell nucleus, often followed by division of the cytoplasm of the cell. Mitospore — 1. A spore from a mitosporangium 2. Any non-basidiosporous propagule (of Basidiomycetes). 3. A uninucleate, haploid or diploid spore arising by mitosis. Mixed culture — A culture containing more than one kind of microorganism. Molecular biology — The science dealing with DNA and protein synthesis of living organisms. Molecule — A combination of atoms forming a specific chemical compound. Monomers — The units that combine to form polymers. Morphology — The external appearance. Mortality — The state of being subject to death; death, especially on a large scale. Most probable number (MPN) — A statistical determination of the number of coli forms per 100 ml of water or food. Motility — The ability of an organism to move itself. Mushroom (toadstool) — 1. A fleshy basidioma, usually stalked and with a cap (pileus) beneath which gills or fleshy tubes are covered with or lined with the hymenium, edible or poisonous, especially of a Basidiomycete of the family Agaricaceae. 2. Caesar’s Amanita caesarea. Chinese straw mushroom. Mutagen — An agent in the environment that brings about mutations.

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Mutation — Any change in the base sequence of DNA. Mutualism — A symbiosis in which both organisms are benefitted. Mycelium (pl. mycelia) (Gr. mykes = mushroom , fungus) — The filamentous vegetative portion of a fungal thallus especially excluding the fruiting structure or reproductive phase of the life cycle. Mycologist — A person studying fungi, their various morphology, taxonomy, origin evolution, reproduction, dispersal, like cycle, growth requirement, occurrence, distribution economic importance and other aspects. Mycology (Gr. mykes = mushroom, fungus + logos = discourse) — The branch of biology that deals with the scientific study of fungi. Mycorrhiza — fungus growing in symbiosis with plant roots. Mycosis — A fungal infection. Mycotoxin — A toxin produced by a fungus. The term is usually reserved for fungal metabolites that are toxic to man and/or animal and are produced by molds growing on foodstuffs, e.g. aflatoxin, ergot alkaloids. Myxomycota — The slime molds; organisms having a noncellular and multinucleate creeping amoeboid vegetative phase and a propagative sporeproducing stage with brightly colored spore bearing capillitia. Free-living, unicellular or plasmodia, non-flagellate in single or multi-celled phagotrophic stages, mitochondrial cristae tubular, with single to many spored, spore wall of cellulose or chitinous, spores germinating to produce one or two flagellate cells. Nitrogen cycle — The series of processes that converts nitrogen (N2) to organic substances and back to nitrogen in nature. Nitrogen fixation — The conversion of nitrogen (N2) into ammonia. Nomenclature — The system of naming things. Nuclear envelope — The double membrane that separates the nucleus from the cytoplasm in the eukaryotic cell. Nucleic acid — A macromolecule consisting of nucleotides; for example RNA and DNA. Nucleoid — The region in a bacterial cell containing the chromosome. Nucleoprotein — A macromolecule consisting of protein and nucleic acid. Nucleoside — A compound consisting of a purine or pyrimidine and pentose sugar. Nucleotide — A compound consisting of a purine or pyrimidine base, a five carbon sugar, and a phosphate. Nucleus — The part of a eucaryotic cell that contains the genetic material; also, the part of an atom consisting of the protons and neutrons. Numerical taxonomy — A method of comparing organisms on the basis of many characteristics. Nutrient broth (agar) — A complex medium made of beef extract. Objective lens — In a compound light microscope, the lens closest to the specimen. Obligate anaerobe — An organism that is unable to use oxygen. Obligate parasite — An organism that is incapable of living as a saprophyte and require a living host for completion of life cycle.

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Biology and Biotechnology of Fungi and Microbes Oidium (pl. oidia) (Gr. oidion = small egg) — A thin walled free, flattened, asexual spore or a fertilizing agent. It is a hyphal cell derived from the fragmentation (centripetally) of a somatic hypha into its component cells or from an oidiophore. Ontogeny — Development of the individual. Oogamy (Gr. oon = egg) — A type of heterogamy in which plasmogamy takes place between a large non motile egg and small motile male gamete or cytoplasm from antheridia. Oogonium (Gr. oon = egg + gennao = I give birth) — A specialized sexual structure, formed as a female gametangium, by fungus-like organisms in Phylum Oomycota, that contains one or more discrete gametes. Oomycota — Phylum of Protoctistan fungi with biflagellate zoospores; zoogamous, with non-motile gametes; have cellulose and glucan cell walls, and diploid vegetative thalli. Ooplasm (of Peronosporales) — The protoplasm, at the centre of the oogonium, which becomes the oosphere. Ooplast (Gr. oon = egg + plastes + molder) — Membrane bound cellular incision in the oospore of the Saprolegniaceae. Oosphere (Gr. oon = egg, sphaira = sphere) — A large naked non-motile sphere of protoplasm which functions as the egg or female gamete in the oogonium, the only one having many functional nuclei. Operon — The operator site and structural genes it controls. Organelles — Membrane-bounded structures within eukaryotic cells. Oxidation — the removal of electrons from a molecule or the addition of oxygen to a molecule. Oxidative phosphorylation — The synthesis of ATP coupled with electron transport. PAGE — Polyacrylamide gel electrophoresis, See electrophoresis. Paleozoic — The geological era covering the period from 600-225 million years ago, includes the Cambrian, Ordovician, Silurian, Devonian, Carboniferous and Permian periods. Parasexuality (Gr. para = beside + sex) — A process in which plasmogamy, karyagamy and haploidisation take place in a sequence but not at specified points in the life cycle of an individual. It is a partial genetic recombination. Pasteurization — The process of mild heating to kill particular spoilage organisms or pathogens. Pathogen — A disease causing organism. Penicillin — A group of closely related powerful antibiotic substances produced by Penicillium notatum and P. chysogenum (Hyphomycetes), active against Gram negative bacteria and of low toxicity to man. Discovered by Alexander Flemming. Pentose phosphate pathway — A metabolic pathway that can occur simultaneously with glycolysis to produce pentoses and NADH without ATP production. Peptide bond — A bond joining the amino group of one amino acid to the carboxyl group of a second amino acid with the loss of a water molecule.

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Peptide — A chain of two, three or more amino acids. Peptidoglycan — The structural molecule of bacterial cell walls consisting of the molecules N-acetylglucosamine, N-acetylmuramic acid, tetra peptide side chain, and peptide side chain. Peptone — short chains of amino acids produced by the action of acids or enzymes on proteins. Perithecium (pl. perithecia) (Gr. peri = around, theke = case) — Subglobose or flask-like ascocarp sometimes limited to ascohymenial types formed from the development of an ascogonium (not of stromatic origin), but now widely used as a general term regardless of the ontogenetic type. Peritrichous — Having flagella distributed over the entire cell. Peroxide — An oxygen oxide consisting of two atoms of oxygen. pH — The symbol for hydrogen ion concentration; a measure of the relative acidity of a solution. Phage — See Bacteriophage. Phagocytosis — The process in which particulate matter is ingested by microorganisms Phase-contrast microscope — A compound light microscope that allows examination of structures inside cells through the use of a special condenser. Phenol — C6 H5 OH; carbolic acid. Phialide (adj. phialidic) (Gr. phiale = drinking vessel) — A type of terminal conidiogenous cell that blastogenously produces conidia through a special opening where neither wall contributes towards formation of the conidium; conidia are produced basipetally with no detectable increase in length and its apex does not become smaller in diameter. A collarette is often present at the apex of the phialide. Phosphate group — A portion of a phosphoric acid molecule attached to some other molecule. Phospholipid — A complex lipid composed of glycerol, two fatty acids, and a phosphate group. Phosphorylation — The addition of phosphate group to an organic molecule. Photoautotroph — An organism that uses light for its energy source and carbon dioxide as its carbon source. Photohetrotroph — An organism that uses light for its energy source and an organic carbon source. Photophosphorylation — The production of ATP by photosynthesis. Photosynthesis — The light-driven synthesis of carbohydrate from carbon dioxide. Phototroph — An organism that uses light as its primary energy source. Phyllosphere — Microorganisms inhabiting the surface of plant leaves Phylogeny — The evolutionary history of a group of organisms. Phylum (pl. phyla) — A taxonomic classification between kingdom and class. Pinocytosis — The engulfing of small molecules by in folding the plasma membrane.

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Biology and Biotechnology of Fungi and Microbes Plant growth promoting rhizobacteria (PGPR) — Bacteria inhabiting plant roots helping in improving the plant growth Plant virus — A virus that multiplies in plant tissues. Plantae — A kingdom composed of multicellular eukaryotes with cellulose cell walls. Plaque — A clearing in a confluent growth of bacteria due to lysis by phages. Plasma membrane — The selectively permeable membrane enclosing the cytoplasm of a cell; the outer layer in animal cells, internal to the cell wall in other organisms. Plasmid — A small cyclic DNA molecule in bacteria replicating independent of the chromosome. Plasmodium — A multinucleated mass of protoplasm; when written as a genus refers to the etiology of malaria. Plasmogamy (Gr. plasma= a molded object + gamos = marriage, union) — 1. The fusion of cytoplasm of two protoplasts, often sexual in nature and followed by karyagamy. 2. The initiation of the diploid phase in the life cycles of certain fungi; nuclei or different mating types come together and divide conjugately as dicaryons, but do not fuse until later. Plasmolysis — Loss of water from a cell in a hypertonic environment. Plate count — A method of determining the number of bacteria in a sample by counting the number of colony-forming units on a solid culture medium. Plectomycete — A member of the Plectomycete group of primitive or reduced Ascomycetes forms that have a fructification without an ostiole (cleistothecium), the entire interior of which is irregularly penetrated by ascogenous hyphae, with the result that the generally spherical asci, without accompanying paraphsyses or other threads, lie scattered irregularly in pseudoparenchymatous tissue composed of the ascogenous hyphae. Pleomorphic — Having many shapes. Pleomorphism (polymorphism) — 1. The occurrence of several forms in the life cycle (for example, many rust fungi are pleomorphic in that they produce as many as five different spore forms in their complete life cycles). 2. An inherent variability in size and shape (in general), e.g. among the cells in a pure culture or clone of a given organism. Polymer — A molecule consisting of a sequence of similar units or monomers. Polymerase — An enzyme that synthesizes specific polymers. Polymorphism (having many forms) — The existence within the population of two or more discrete, genetically determined forms other than variations in sex or maturity and apart from rare mutant forms. Polypeptide — A chain of amino acids. Poly-β-hydroxybutyric acid — A fatty acid storage material unique to bacteria. Pour plate method — A method of inoculating a solid nutrient medium by mixing bacteria/fungi/ actinomycetes in the melted medium and pouring the medium into a Petri plate to solidify.

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601

Precambrian — Geological era from the earliest days of the earth until 600 million years ago, at the end of which the earth’s atmosphere is believed to have attained a level of oxygen capable of supporting muliticellular, eukaryotic organisms. Prokaryote — A cell whose genetic material is not enclosed in a nuclear envelope. Promoter site — The starting point on DNA for transcription of RNA by RNA polymerase. Prontosil — An early sulphonamide, first used clinically in 1930’s. Its activity was due to its breakdown in vivo to sulphanilamide. It has been superseded by the more effective, less toxic modern sulphonamide Prophage — Phage DNA inserted into the host cell’s DNA. Prosthetic group — A tightly bound nonpolypeptide structure required for the activity of an enzyme or other protein; a tightly bound, specific non-polypeptide unit required for the biological function of some proteins. Protein — A large molecule containing carbon, hydrogen, oxygen, and nitrogen (and sulfur); some have a globular structure and others are pleated sheets. Proteolytic — An enzyme capable of hydrolyzing a protein. Proteomics — Study of protein expression in cells Protista — The kingdom to which protozoans belong; unicellular, eukaryotic organisms. Proton — A positively charged particle in the nucleus of an atom. Protoplast — A gram-positive bacterium without a cell wall; a plant cell lacking a cell wall. Protozoa — Unicellular eukaryotic organisms belonging to the Kingdom Protista. Pseudothecium (Gr. pseudo = false, theke = sheath or cover) — An ascostromatic ascoma having asci in numerous walled locules, as in Loculoascomycetes. Psychrophile — An organism that grows best at 15°C and does not grow above 20°C. Puff-ball — A Gasteromycetes basidioma in which the basidiospore mass (gleba) is enclosed by a papery peridium at maturity; an ostiole allows compression or wind suction to disperse. Basidiocarp in the Lycoperdales (Calvatia gigateum) and Tulostomateles have an opening for dischargevof spores; the basidiomata of the Sclerodermales have no ostiole. Purines — The class of nucleic acid bases that includes adenine and guanine. Pycnidiospore (Gr. spore = seed, spore) — The conidia borne in pycnidium, the asexual fruitbody. Pycnidium (pl. pycnidia) (Gr. pyknos= dense) — An open-pored, round or flask shaped tiny, fruiting structure of the fungus in which asexual spores called conidia are produced. Pycniospore (Gr. pyknos = concentrated + spora= seed, spore) — A spore (spermatium) borne in a pycnium in the Uredinales, which sometimes act as a male cell. Pycnium (pl. pycnia) (Gr. Pyknos = dense) — Stage 0 (in the Uredinales), consisting of the male fertilizing element (pycniospore) and the female element, the flexuous hyphae.

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Biology and Biotechnology of Fungi and Microbes Pyocyanin — Blue-green pigment produced by Pseudomonas aeruginosa, that is an antibiotic and antifungal agent. Pyrimidines — The class of nucleic acid bases that includes uracil, thymine and cytosine. Quaternary ammonium compound (quat) — A cationic detergent with four organic groups attached to a central nitrogen atom; used as a disinfectant. Quorn — The trade name under which mycoprotein is manufactured from Fusarium graminearum by Marlow Foods. R genes — Genes carried on the R factor that code for enzymes that inactivate certain drugs. Recalcitrant — Being resistance to degradation. Receptor — An attachment for a pathogen on a host cell. Recipient cell — A cell that receives DNA from a donor cell in recombination. Recombinant DNA — A DNA molecule produced by recombination. Recombinant RNA technology — Techniques used to make RNA molecules. Recombination — The process of joining pieces of DNA from different sources. Reduction — The addition of electrons to a molecule; the gain of hydrogen atoms. Replica plating —: A method of inoculating a number of solid minimal culture from an original plate of complete medium; mutant colonies that don’t grow on the minimal media can be selected from the original plate. Repressor — A protein that binds to the operator site to prevent transcription. Reproduction — The bringing forth of new individuals having all the characteristics typical of the species through sexual, asexual and vegetative methods. Resistance (R) factor — A bacterial plasmid carrying genes that determine resistance to antibiotics. Resistance — The ability to ward off diseases through non-specific and specific defenses. Resolution — The ability to distinguish fine detail with a magnifying instrument. Respiration — An ATP-generation process in which chemical compounds are oxidized and the final electron acceptor is usually an inorganic molecule; also, the process by which living organisms produce carbon dioxide. Restriction enzymes — Enzymes that cut double-stranded DNA leaving staggered ends. Rhizomorph (Gr. rhiza = root, morphe = form) — A thick microscopic strand of somatic hyphae that have lost their individuality, with whole mass behaving like and somewhat resembling a root tip. Rhizomorphs often are enduring structures that can remain dormant under adverse conditions. They are formed by the longitudinal joining of hyphal strands into a bundle; it has a hard outer covering, often dark colored, grows from the apex, having a well defined apical meristem and frequently differentiated into a rind of small dark colored cells surrounding a central core of elongated colorless cells. They have specialized tissues for absorption and water transport (Armillary spp.). Rhizosphere — The region in soil where the soil and roots make contact.

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Riboflavin — B vitamin that functions as a flavoprotein. Ribonucleic acid (RNA) — The class of nucleic acids that comprises messenger RNA, ribosomal RNA and transfer RNA. Ribose — A five-carbon sugar that is part of ribonucleotide molecules and RNA. Ribosomal RNA (rRNA) — The RNA molecules that form the ribosomes. Ribosomes — The sites of protein synthesis in cell composed of RNA and protein. RNA-dependent RNA polymerase — An enzyme that synthesizes a complementary RNA from an RNA template. Rust — Plant diseases caused by pathogenic fungi of the order Pucciniales (previously also known as Uredinales). Rusts are so named after the reddish rusty looking sori.; black stem rust of wheat by Puccinia graminis var. tritici; white pine blister rust caused by Cronartium ribicola. Saccharide — A simple sugar, combination of sugars, or polymerized sugar Sugar general formula (CH2O) n. Sanitation — Removal of microbes from eating utensils and food preparation areas. Saprophyte — An organism living on dead organic matter, using it as food, and commonly causing its decay (saprobe is the preferred usage for fungi). It is a nonparasitic nutritional mechanism. Sarcina — Agroup of eight bacteria that remain in a packet after dividing. Saturation — The condition in which the active site on an enzyme is occupied by the substrate or product at all times. Scanning electron microscope — An electron microscope that provides three dimensional views of the specimen magnified about 10,000 times. Schaeffer-Fulton stain — An endospore stain that uses malachite green to stain the endospores and safranin as a counterstain. Sclerotium (pl. sclerotia) (Gr. Skleron = hard) — A compact mass of hardened mycelium with a thick pigmented outer rind that serves as a dormant stage in some fungi; Sclerotia are also the reddish hardened ovaries of a grain that are filled with mycelia of the fungus Claviceps purpurea Septate (L. septum = hedge) — With more or less regularly occurring cross walls forming divisions in a spore or hypha. Serial dilution — The process of diluting a sample several times. Serum — An amber-coloured, protein-rich liquid which separates out when blood coagulates. Sewage — Domestic waste water. Sexual reproduction — Reproduction that requires two opposite mating strains, usually designated as male and female. Simple stain — A method of staining microorganisms with a single basic-dye. Single cell protein (SCP) — A food substitute consisting of microbial cells. Slide agglutination test — A method of identifying an antigen by combining it with a specific antibody in a slide. Smut — A disease caused by a member of Ustilaginaceae in cereals, like wheat, sorghum. The grains get filled up with black spore mass. A smut spores also called

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Biology and Biotechnology of Fungi and Microbes as ustilospore, ustospores, chlamydospores, brand spores and resting spores. In covered smut the mature spore mass keeps for a time within a covering of the host (or fungal) tissue, till after the sours becomes free from the host. Covered smut of barley caused by Ustilago segetum (syn. U. hordei); oats by U. kolleri; and sorghum by Sporisorium sorghi. Soil — A mixture of solid inorganic matter, water, organic matter, and living organisms. Solvent — A dissolving medium. Specialized transduction — The process of transferring piece of cell DNA adjacent to a prophage to another cell. Species — The most specific level in the taxonomic hierarchy. Bacterial species: A population of cells with similar characteristics. The second name in scientific binomial nomenclature. Spermatisation (Gr. sperma = seed) — 1. Plasmogamy by the union of a spermatium with a receptive structure. 2. (in certain higher fungi) The union of a spermatium with a female reproductive structure. Different patterns of spermatisation are in Ascomycetes. Spheroplast — A gram-negative bacterium lacking a complete cell wall. Spirillum — A spiral or corkscrew-shaped bacterium. Spirochete — A corkscrew-shaped bacterium with an axial filament. Spontaneous generation — The idea that life could arise spontaneously from nonliving matter. Spontaneous mutation — A mutation that occurs without mutagen. Staphylococci — A broad sheet of spherical cells. Stationary phase — The period in a bacterial growth curve when the number of cells dividing equals the number dying. Statismospore — A spore is not forcibly discharged, e.g. Gasteromycetes in which spores are liberated in a global mass. STD — Sexually transmitted disease. Stem cells — Fetal cells that give rise to bone marrow, blood cells, and B and T cells. Sterile — Free of microorganisms. Steroids — A specific group of chemical substances, including cholesterol and hormones. Strain — A group of cells all derived from a single cell. Streak plate method — A method of isolating a culture by spreading microorganisms over the surface of a solid culture. Streptococci — Cocci that remain attached in chains after, cell division. Structural gene — A gene that codes for the amino acid sequence of a protein (as an enzyme) or for a ribosomal RNA or transfer RNA. Substrate — Any compound with which an enzyme reacts. Substrate-level phosphorylation — The synthesis of ATP by direct transfer of a high-energy phosphate group from an intermediate metabolic compound to ADP.

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Sulfahydryl group — - SH. Sulfonamides — Bacteriostatic compounds that interfere with folic acid synthesis by competitive inhibition. Superoxide dismutase — Enzyme that destroys 02 Susceptibility — The lack of resistance to a disease. Swan-Neck Flask — Apparatus used by Pasteur to repute the doctrine of Abiogenesis. Each spherical flask had a long narrow neck which was bent downwards then upwards, thus preventing the access of air-borne microbes to the sterilized contents of the flask Symbiosis — The living together of two different organisms. Symptom — A change in body function that is felt by a patient due to a disease. Synergistic effect — The principle whereby the effectiveness of two drugs used simultaneously is greater than either drug used alone. Synthetic drug — A chemotherapeutic agent that is prepared from chemicals in a laboratory. Taxol — An antitumor diterpenoid used in treatment of some cancers. Originally obtained from the bark of pacific yew (Taxus brevifolia), but also produced by the endophytic fungus, Taxomyces. Taxon (pl. taxa) — The unit of classification. Taxonomy — The science dealing with the description, identification, naming, and classification of organisms. Teichoic acid — A polysaccharide found in gram-positive cell walls. Temperate phage — A bacteriophage existing in lysogeny with a host cell. Tetracyclines — Broad spectrum antibiotics that interfere with protein synthesis. Tetrahedron — A four-sided solid structure. Thallus — The entire vegetative structure or body of a fungus, lichen, or alga. Thermophile — An organism whose optimum growth temperature is between 50°C and 60°C. Thylakoid — A chlorophyll-containing membrane in a chloroplast. Tolerance — A state of immunological non-responsiveness to self antigens. Toxigenic — Toxin producing organisms. Toxin — Any poisonous substance produced by a microorganism. Trace element — A chemical element required in small amounts for growth. Transamination — The reversible exchange of amino groups between different amino acids. Transcription — The process of synthesizing RNA from an r DNA template; the process of transcribing RNA, with existing DNA serving as a template, or vice versa. Transduction — Transfer of DNA from one cell to another by a bacteriophage. See also generalized transduction, specialized transduction. Transfer RNA (tRNA) — The class of molecules that brings amino acids to the site where they are incorporated into proteins.

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Biology and Biotechnology of Fungi and Microbes Transformation — The process in which genes are transformed from one bacterium to another as ‘naked' DNA in solution Translation — The use of RNA as a template in the synthesis of protein. Transposon — A small piece of DNA that can move from one region of the DNA molecule to another. Tretic (of conidiogenesis) — The sort of conidiogenesis in which each conidium (teratoconidium, poroconidium, and porospore) is delimited by an extension of the inner wall of the conidiogenous cell. Teratoconidia are solitary or in acropetal chains; mono-, poly-, (of conidiogenous cells) producing teratoconidia by the extrusion of the inner wall through one or several channels, respectively. Turbidity — The cloudiness of a suspension. Ultrastructure — Fine detail not seen with a compound light microscope. Ultraviolet (UV) radiation — Radiation from 10 to 390 nm. Uracil — A pyrimidine nucleic acid base in RNA that pairs with adenine. Urediniospore — A binucleate spore borne in a uredinium and capable of infecting the same host on which it originated, usually echinulate. Vaccination — The process of conferring immunity using a vaccine. Vaccine — A preparation of killed, inactivated, or attenuated microorganisms or toxoids to induce artificially acquired active immunity. Vacuole — An intracellular inclusion, in eukaryotic cells, surrounded by a plasma membrane containing raw food; in prokaryotic cells, surrounded by a proteinaceous membrane containing gas. Variation — Differences in the frequency of genes and traits among individual organisms within a population. Vector — An arthropod that carries disease-causing microorganisms from one host to another. Vegetative — A mode of reproduction by somatic means without mitosis and meiosis. Reproducing by fragmentation, fission, budding, sclerotia, rhizomorph. Vegetative cells — Cells involved with obtaining nutrients Vibrio — A curved or comma-shaped bacterium. Virion — A fully developed complex viral particle. Viroid — An infectious piece of ‘naked’ RNA. Virus — A submicroscopic, parasitic, filterable agent consisting of a nucleic acid surrounded by a protein coat. Visible light — Radiation from 400 to 700 nm, which the human eye can see. Volutin — Stored phosphate in a prokaryotic cell. Volva (L. volvere = to roll) (of Agarics and Gasteromycetes) — The cup-like remnant of lower part of the veil found around the base of the mature stipe (stalk) or receptacle; sometimes called the universal veil, which is the preferred usage, e.g. in Amanita spp. Wilt — A common symptom of disease due to loss of turgor and resulting in subsequent drooping and collapse of the foliage or succulent tissues, eg. vascular wilt.

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Yeast — A unicellular fungus belonging to the Phylum Ascomycetes. Zoospore — A motile mitospore, a naked spore (motile by means of one to many flagella) produced within a sporangium. It is an asexual spore formed by some fungi that usually can move in an aqueous environment via one or more flagella. Zygomycotina — A subdivision of fungi characterized by the formation of a thickwalled diploid resting spore formed after the fusion of two gametangia. Zygospore (Gr. zygos = yoke, sporos = seed) — A thick walled diploid, sexual resting spore resulting from the fusion of gametangia (in the Zygomycetes). Zygote (Gr. zygos = yoke) — A diploid cell formed by the union of two gametes, a fertilized egg.

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