Fungi And Allied Microorganisms 0070700389, 9780070700383

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Fungi And Allied Microorganisms
 0070700389, 9780070700383

Table of contents :
Cover
Half Title
About the Author
Title Page
Copyright
Contents
Chapter 1: Introduction
1.1 What are Fungi?
1.2 Various Names Used for Fungi
1.3 Numerical Estimates of Fungi
1.4 Why Should we Study Fungi?
1.5 Hypha and Mycelium
1.6 A Fungal Cell (Ultrastructure)
1.7 Fungal Growth
1.8 Haustorium
1.9 Nutrition
1.10 Reproduction
1.11 Some Important Indian Journals of Fungi
1.12 Some Important Foreign Journals of Fungi
1.13 Some International Journals Which also Publish Fungal Research
Test Your Understanding
Chapter 2: History of Mycology (Chronology of Major Events)
2.1 Global Developments of Mycology
2.2 Some Major International Contributors of 21st Century
2.3 Development of Mycology in India
2.4 Some other Indian Contributors and their Major Contributions
Test Your Understanding
Chapter 3: Fungal Cell: Structure and Composition
3.1 Cell Envelope
3.2 Cytoskeleton
3.3 Nucleus
3.4 Mitochondria
3.5 Hydrogenosomes: Hydrogen – Generating Organelles
3.6 Microbodies
3.7 Plasmids
3.8 Mycoviruses
3.9 Reserve Materials
3.10 Vacuoles
Test Your Understanding
Chapter 4. Classification of Fungi
4.1 What is Classifi cation?
4.2 Whether there Exist only Two Kingdoms?
4.3 Why is the Fungal Classification so Much Variable?
4.4 Groups of Uncertain Affinity
4.5 Criteria Used in Fungal Taxonomy
4.6 Recommendations of International Committee
4.7 Botanical Ranks of Nomenclatural Hierarchy
4.8 Important Systems of Classification of Fungi
4.9 Some Older Contributions from 1623 to 1926
4.10 Classification Proposed by H.C.I. Gwynne-Vaughan and B. Barnes (1937)
4.11 Classification Proposed by Lilian E. Hawker (1966)
4.12 Classification Proposed by G.C. Ainsworth (1973)
4.13 Classification Proposed by Constantine J. Alexopoulos and Charles W. Mims (1979)
4.14 Classification Proposed by P.M. Kirk, P.F. Cannon, J.C. David and J.A. Stalpers (2001)
4.15 Outline of Latest Classification Proposed by John Webster and Roland W.S. Weber (2007) in their Book Introduction to Fungi
Test Your Understanding
Chapter 5: Myxomycota
5.1 Introduction
5.2 Acrasiomycetes
5.3 Hydromyxomycetes
5.4 Myxomycetes
5.5 Life-Cycle of a Typical Myxomycete
5.6 Stemonitis
5.7 Plasmodiophoromycetes
5.8 Plasmodiophora brassicae
Test Your Understanding
Chapter 6: Eumycota
6.1 What are Eumycota?
6.2 Characteristic Features
6.3 Occurrence
6.4 Vegetative Plant Body
6.5 Branching
6.6 Fine Structure of a Cell of Eumycota
6.7 Haustorium
6.8 Hyphal Aggregations
6.9 Hyphal Aggregations into Reproductive Structures
6.10 Vegetative Reproduction
6.11 Some Asexual and Sexual Spores
6.12 Sexual Reproduction
6.13 Modes of Sexual Fusion
6.14 Classification of Eumycota
Test Your Understanding
Chapter 7: Mastigomycotina (General Account and Chytridiomycetes)
7.1 General Characteristics of Mastigomycotina
7.2 Classification of Mastigomycotina
7.3 Chytridiomycetes
7.4 Chytridiales
7.5 Synchytriaceae
7.6 Synchytrium Endobioticum
7.7 Blastocladiales
7.8 Monoblepharidales
Test Your Understanding
Chapter 8: Hyphochytridiomycetes
8.1 What are Hyphochytridiomycetes?
8.2 General Characteristics
8.3 Classification
8.4 Rhizidiomyces
8.5 Phylogeny of Hyphochytridiomycetes
Test Your Understanding
Chapter 9: Oomycetes
9.1 What are Oomycetes?
9.2 General Characteristics
9.3 Classification of Oomycetes
9.4 Order Saprolegniales
9.5 Saprolegnia
9.6 Achlya
9.7 Peronosporales
9.8 Family Pythiaceae
9.9 Pythium
9.10 Phytophthora
9.11 Family Peronosporaceae (Downy Mildews)
9.12 Peronospora
9.13 Sclerospora
9.14 Plasmopara
9.15 Family Albuginaceae
9.16 Albugo
Test Your Understanding
Chapter 10: Zygomycotina (Zygomycetes)
10.1 What are Zygomycotina?
10.2 General Characteristics of Zygomycetes
10.3 Classification
10.4 Mucorales
10.5 Mucoraceae
10.6 Rhizopus
10.7 Mucor
10.8 Pilobolaceae
10.9 Pilobolus
10.10 Economic Importance of Mucorales
10.11 Heterothallism in Mucorales
Test Your Understanding
Chapter 11: Trichomycetes
11.1 What are Trichomycetes?
11.2 General Characteristics
11.3 Classification
11.4 Harpellales
11.5 Asellariales
11.6 Eccrinales
11.7 Amoebidiales
Test Your Understanding
Chapter 12: Ascomycotina (Ascomycetes) (General Account )
12.1 What are Ascomycotina?
12.2 General Characteristics of Ascomycotina
12.3 Economic Importance
12.4 Somatic Structures
12.5 Asexual Reproduction
12.6 Sexual Reproduction
12.7 Methods of Bringing Compatible Nuclei Together
12.8 Compatibility
12.9 Ascus Development
12.10 Asci and Ascospores
12.11 Ascocarps
12.12 Classification
12.13 Differences from Phycomycetes
Test Your Understanding
Chapter 13: Hemiascomycetes
13.1 What are Hemiascomycetes?
13.2 General Characteristics
13.3 What are Yeasts?
13.4 Classification
13.5 Endomycetales
13.6 Saccharomycetaceae
13.7 Saccharomyces cerevisiae
13.8 Economic Importance of Yeasts
13.9 Taphrinales
13.10 Taphrina
Test Your Understanding
Chapter 14: Loculoascomycetes
14.1 What are Loculoascomycetes ?
14.2 Habitat
14.3 Somatic Structure
14.4 Asexual Reproduction
14.5 Sexual Reproduction
Test Your Understanding
Chapter 15: Plectomycetes
15.1 What are Plectomycetes?
15.2 General Characteristics
15.3 Classification
15.4 Eurotiales
15.5 Eurotiaceae
15.6 Aspergillus
15.7 Penicillium
15.8 Difference between Talaromyces and Eupenicillium
15.9 Difference between Aspergillus and Penicillium
15.10 Role of Aspergillus and Penicillium in Biotechnology
15.11 Mycotoxins from Aspergillus and Penicillium
Test Your Understanding
Chapter 16: Laboulbeniomycetes
16.1 What are Laboulbeniomycetes?
16.2 A Brief Background
16.3 Occurrence
16.4 Thallus
16.5 Sex Organs
16.6 Perithecia, Asci and Ascospores
Test Your Understanding
Chapter 17: Pyrenomycetes
17.1 What are Pyrenomycetes?
17.2 Distinguishing Features
17.3 Classification
17.4 Erysiphales
17.5 Erysiphe
17.6 Phyllactinia
17.7 Sphaerotheca
17.8 Sphaeriales
17.9 Sordariaceae
17.10 Neurospora
17.11 Clavicipitaceae
17.12 Claviceps
Test Your Understanding
Chapter 18: Discomycetes
18.1 General Characteristics
18.2 Classification
18.3 Pezizales
18.4 Ascobolaceae
18.5 Ascobolus
18.6 Morchellaceae
18.7 Morchella
18.8 Pezizaceae
18.9 Peziza
18.10 Pyronemataceae
18.11 Pyronema
18.12 Tuberales
18.13 Tuber
Test Your Understanding
Chapter 19: Basidiomycotina (General Account )
19.1 What are Basidiomycotina?
19.2 General Characteristics
19.3 Mycelium and Dikaryotization
19.4 Clamp Connection
19.5 Dolipore Septum
19.6 Asexual Reproduction
19.7 Sexual Reproduction
19.8 Basidium
19.9 Basidiospore
19.10 Discharge Mechanism of Basidiospores
19.11 Buller Phenomenon
19.12 Differences from Ascomycotina
19.13 Classification
Test Your Understanding
Chapter 20: Teliomycetes
20.1 General Characteristics
20.2 Classification
20.3 Uredinales
20.4 Puccinia
20.5 Puccinia graminis (Life Cycle)
20.6 Control Measures of Rusts
20.7 Annual Recurrence of Rust in India
20.8 Comparison Between Black, Orange and Yellow Rusts
20.9 Uromyces
20.10 Phragmidium
20.11 Ravenelia
20.12 Melampsoraceae
20.13 Melampsora
20.14 Ustilaginales
20.15 Ustilago
20.16 Graphiolaceae and Graphiola
Test Your Understanding
Chapter 21: Hymenomycetes
21.1 What are Hymenomycetes?
21.2 Holobasidiomycetidae
21.3 Agaricales
21.4 Edible and Poisonous Mushrooms
21.5 Fairy Rings
21.6 Classification of Agaricales
21.7 Agaricaceae
21.8 Agaricus
21.9 Aphyllophorales (= Polyporales)
21.10 Polyporaceae
21.11 Polyporus
Test Your Understanding
Chapter 22: Gasteromycetes
22.1 What are Gasteromycetes?
22.2 General Characteristics
22.3 Classification
22.4 Lycoperdales
22.5 Lycoperdaceae
22.6 Lycoperdon
Test Your Understanding
Chapter 23: Anamorphic Fungi (Deuteromycotina or Deuteromycetes )
23.1 Anamorphic Fungi and their General Characteristics
23.2 Types of Fructifications
23.3 Parasexuality in Anamorphic Fungi
23.4 Classification
23.5 Recommendations of ICBN about Nomenclature of Anamorphic Fungi
23.6 Delimitation of Taxonomic Entity of Anamorphic Fungi
23.7 Blastomycetes
23.8 Sporobolomyces
23.9 Candida
23.10 Cryptococcus
23.11 Hyphomycetes
23.12 Alternaria
23.13 Cercospora
23.14 Curvularia
23.15 Pyricularia
23.16 Helminthosporium
23.17 Drechslera
23.18 Fusarium
23.19 Coelomycetes
23.20 Colletotrichum
23.21 Phyllosticta
23.22 Phoma
23.23 Phomopsis
Test Your Understanding
Chapter 24: Economic Importance of Fungi
24.1 Introduction
24.2 Negative Aspects of Fungi
24.3 Positive Aspects of Fungi
24.4 Future Expectations from Fungi and Mycologists
Test Your Understanding
Chapter 25: Fungi and Biotechnology
25.1 Biotechnology and its Relation with Fungi
25.2 Fermentation Technology
25.3 Enzyme Technology
25.4 Production Technology of Alcoholic Beverages
25.5 Cultivation of Mushrooms and other Macrofungi
25.6 Single-Cell Protein
25.7 Fungi in Food Processing Industry
25.8 Production of Primary Metabolites by Fungi
25.9 Production of Secondary Metabolites by Fungi
25.10 Role of Biotechnology in Selection and Mutation of Fungal Strains
25.11 Role of Biotechnology in Genetic Recombination and Gene Cloning
25.12 Gene Cloning and Future of Fungal Biotechnology
Test Your Understanding
Chapter 26: Mushroom Cultivation
26.1 Mushrooms and Mycophagy
26.2 Food Value of Mushrooms
26.3 Edible and Poisonous Mushrooms
26.4 Commercial Cultivation of Mushrooms
26.5 Cultivation of White Button Mushroom on Commercial Basis
26.6 Mushroom Growing in Laboratory
26.7 Cultivation of Shiitake (Lentinus elodes)
26.8 Paddy Straw Mushroom
26.9 Oyster Mushroom
26.10 Commercial Production of Some Other Macrofungi
26.11 Mushroom Parasites
26.12 Mushroom Dishes
Test Your Understanding
Chapter 27: Single-Cell Protein
27.1 What is Single-Cell Protein?
27.2 Why do we Need to Produce SCP?
27.3 Microorganisms used for SCP-Production
27.4 Composition of Single-Cell Proteins
27.5 Advantages and Disadvantages of Using Microorganisms for Animal or Human Consumption
27.6 Mycoprotein
27.7 Single-Cell Protein from Cyanobacteria
27.8 Single-Cell Protein from Algae
27.9 Single-Cell Protein from Organic Wastes
27.10 SCP From Petroleum Hydrocarbons and other Related Substrates
Test Your Understanding
Chapter 28: Heterothallism in Fungi
28.1 What is Heterothallism?
28.2 Heterothallism in Mucorales
28.3 Heterothallism in some other Lower Fungi
28.4 Hormonal Basis of Sex and Heterothallism in Lower Fungi
28.5 Heterothallism in Ascomycetes
28.6 Heterothallism in Basidiomycetes
Test Your Understanding
Chapter 29: Sex Hormones and Pheromones in Fungi
29.1 Hormones, Sex Hormones and Pheromones
29.2 Some Earlier Studies on Sex Hormones and Pheromones in Fungi
29.3 What has Finally Been Established with Sex Hormones in Fungi?
29.4 Sex Hormones Isolated from Lower Fungi
29.5 Some Extensively Studied Sex Hormones of Lower Fungi
29.6 Sex Pheromones in Higher Fungi
Test Your Understanding
Chapter 30: Mycorrhizae
30.1 What are Mycorrhizae?
30.2 Nature of Mycorrhizal Relationship
30.3 Types of Mycorrhizae
30.4 Mycorrhizosphere and “Mycorrhization-Helper Bacteria” (MHB)
30.5 Significance of Mycorrhizae
Test Your Understanding
Chapter 31: Lichens
31.1 What is a Lichen?
31.2 Components of Lichens
31.3 A Brief History
31.4 Occurrence
31.5 Classification
31.6 Lichen Thallus (Morphology and Anatomy)
31.7 Interaction Between Phycobiont and Mycobiont
31.8 Tissue Types in Lichens
31.9 Attachment Organs in Lichens
31.10 Propagules Associated with Lichen Thallus
31.11 Vegetative Reproduction
31.12 Asexual Spores
31.13 Sexual Reproduction of Mycobiont
31.14 Reproduction in Phycobiont
31.15 Economic Importance
Test Your Understanding
Chapter 32: Bacteria
32.1 What are Bacteria?
32.2 Major Characteristics
32.3 A Brief History
32.4 Occurrence and Distribution
32.5 Classification
32.6 Morphology of Bacterial Cell
32.7 Structures External to the Bacterial Cell Wall
32.8 Bacterial Cell Wall: Ultrastructure and Composition
32.9 Cytoplasm and Cytoplasmic Inclusions
32.10 Mycoplasmas and L-forms of Bacteria
32.11 Nutrition in Bacteria
32.12 Gram Reaction
32.13 Growth
32.14 Cell Division (Binary Fission)
32.15 Spore Formation
32.16 Genetic Recombination (Sexual Reproduction)
32.17 Economic Importance
Test Your Understanding
Chapter 33: Viruses
33.1 What are Viruses?
33.2 Major Distinguishing Features
33.3 How do Viruses Differ from Bacteria and Mycoplasmas?
33.4 Some Common Human Viral Diseases
33.5 A Brief History
33.6 Nature and Origin
33.7 Classification
33.8 Size and Symmetry of Viruses
33.9 Three Dozen Interesting Facts about Viruses
33.10 Chemical Composition
33.11 Morphology (Virus Organization and Structure)
33.12 Symptoms of Viral Infection in Plants
33.13 Transmission
33.14 Control of Viral Diseases of Plants
33.15 Viral Vaccines
33.16 Plant Viruses
33.17 Animal Viruses
33.18 Bacterial Viruses (Bacteriophages)
33.19 Life-Cycle or Replication of Bacteriophage
33.20 Mycoviruses
33.21 Cyanophages
33.22 Insect Viruses
33.23 Satellite Viruses and Satellite Nucleic Acids
33.24 Viroids
33.25 Prions
33.26 Swine Flu: Some Basics We Should all Know and Follow
Test Your Understanding
Chapter 34: Plant Diseases and their Control (General Account)
34.1 Plant Pathology and its Objectives
34.2 What is a Plant Disease?
34.3 Causes of Plant Diseases
34.4 Classification of Plant Diseases
34.5 Symptoms of Plant Diseases
34.6 Pathogenesis or Process of Infection
34.7 Enzymes, Toxins and Growth Regulatory Substances
34.8 Control of Plant Diseases
Test Your Understanding
Chapter 35: Selected Diseases Caused by Fungi, Bacteria and Viruses
(A) Some Diseases Caused by Fungi
35.1 White Rust of Crucifers
35.2 Late Blight of Potato
35.3 Green-Ear Disease and Downy Mildew of Bajra
35.4 Downy Mildew of Pea
35.5 Powdery Mildew of Pea
35.6 Powdery Mildew of Cucurbits
35.7 Loose Smut of Wheat
35.8 Covered Smut of Barley
35.9 Whip Smut of Sugar Cane
35.10 Black Rust or Stem Rust of Wheat
35.11 Yellow Rust or Stripe Rust of Wheat
35.12 Brown Rust or Orange Rust of Wheat
35.13 Rust of Linseed
35.14 Paddy Blast or Blast Disease of Rice
35.15 Tikka Disease of Groundnuts
35.16 Red Rot of Sugar Cane
35.17 Wilt of Arhar
35.18 Early Blight of Potato
(B) Some Diseases Caused by Bacteria
35.19 Tundu Disease or Yellow Ear Rot of Wheat
35.20 Citrus Canker
35.21 Brown Rot of Potato
(C) Some Diseases Caused By Viruses
35.22 Leaf Roll of Potato
35.23 Tobacco Mosaic Virus (TMV)
35.24 Leaf Curl of Papaya
35.25 Vein-Clearing of Bhindi
Test Your Understanding
Appendix 1: Some Common Culture Media and Mounting Media for Fungi
Appendix 2: Countrywise List of Institutions with Signifi cant Collections of Fungi, Fungus-Related Websites, and Websites Related to Lichens, Bacteria and Viruses
Appendix 3: Classification of Fungi
Appendix 4: Glossary of 210 Mycological Terms
Appendix 5: Answers to Questions
Appendix 6: 136 Recommended Readings
Index
Plates

Citation preview

Series on Diversity of Microbes and Cryptogams

ABOUT THE AUTHOR O P Sharma, with his 36 research articles published in national and international journals, 30 books written for university students, and 40 years of teaching experience, is an able researcher, established Indian author , and an e xperienced teacher. His areas of research include pollen morphology, angiosperm’s anatomy and mycology , with special focus on Indian Cyperaceae (with particular interest on Cyperus). Over a dozen of Dr Sharma’s books have been published through internationally known publishers. He has also re vised Economic Botany, an internationally renowned text by the late Professor Albert F Hill (Harv ard University, USA), a publication of McGra w-Hill, New York. Encouraging re views of his books ha ve been published in reputed scientif c journals, and his books on Practical Botany have received appreciations from some eminent botanists including J D Dodge (England), T Christensen (Denmark), J M Herr (USA) and C R Metcalfe (England). Immediately after he completed his postgraduation (MSc) in Botan y in f rst division from CCS Uni versity, Meerut, and thereafter obtaining a PhD from the same university, Dr Sharma started his teaching career in 1967 as a faculty member of Botany Department, Meerut College, Meerut, and retired from acti ve services as a Reader from the same department in 2007. Besides attending several national and international workshops, symposia and conferences during the four decades of his teaching career, Dr Sharma is still enjoying his post-retirement innings as an active author. Recently, Dr O P Sharma re vised some of his widely circulated books published through McGra w-Hill Education, which include Plant Taxonomy (released f rst in 1993 and reprinted 19 times), Textbook of Algae (released f rst in 1986 and reprinted 20 times), and Textbook of Fungi (released f rst in 1989 and reprinted 17 times).

Series on Diversity of Microbes and Cryptogams

O P Sharma Reader (Retired) Department of Botany Meerut College, Meerut

Tata McGraw Hill Education Private Limited NEW DELHI McGraw-Hill Off ces New Delhi Ne w York St Louis San Francisco A uckland Bogotá Car acas Kuala Lumpur Lisbon London Madr id Me xico City Milan Montreal San Juan Santiago Singapore Sydne y Tokyo Toronto

Tata McGraw-Hill Published by Tata McGraw Hill Education Private Limited, 7 West Patel Nagar, New Delhi 110 008. Series on Diversity of Microbes and Cryptogams: FUNGI AND ALLIED MICROBES Copyright © 2011 by Tata McGraw Hill Education Private Limited No part of this publication may be reproduced or distrib uted in an y form or by an y means, electronic, mechanical, photocopying, recording, or otherwise or stored in a database or retrieval system without the prior written permission of the publishers. The program listings (if any) may be entered, stored and executed in a computer system, but they may not be reproduced for publication. This edition can be exported from India only by the publishers, Tata McGraw Hill Education Private Limited. ISBN (13): 978-0-07-070038-3 ISBN (10): 0-07-070038-9 Vice President and Managing Director—McGraw-Hill Education: Asia Pacific Region: Ajay Shukla Head—Higher Education Publishing and Marketing: Vibha Mahajan Manager—Sponsoring: SEM & Tech. Ed.: Shalini Jha Editorial Executive: Smruti Snigdha Development Editor: Renu Upadhyay Executive—Editorial Services: Sohini Mukherjee Senior Production Manager: P L Pandita Dy Marketing Manager: SEM & Tech Ed.: Biju Ganesan Senior Product Specialist: John Mathews General Manager—Production: Rajender P Ghansela Asst. General Manager—Production: B L Dogra Information contained in this w ork has been obtained by Tata McGraw-Hill, from sources belie ved to be reliable. However, neither Tata McGra w-Hill nor its authors guarantee the accurac y or completeness of an y information published herein, and neither Tata McGraw-Hill nor its authors shall be responsible for an y errors, omissions, or damages arising out of use of this information. This work is published with the understanding that Tata McGraw-Hill and its authors are supplying information but are not attempting to render engineering or other professional services. If such services are required, the assistance of an appropriate professional should be sought. Typeset at Bharati Composers, D-6/159, Sector -VI, Rohini, Delhi 110 085, and printed at Rashtriya Printers, M-135, Panchsheel Garden, Naveen, Shahdara, Delhi-110 032 Cover: Rashtriya Printers

The McGraw-Hill Companies

CONTENTS

Preface

xix

1.

Introduction 1.1 What are Fungi? 1 1.2 Various Names Used for Fungi 1 1.3 Numerical Estimates of Fungi 1 1.4 Why Should we Study Fungi? 2 1.5 Hypha and Mycelium 2 1.6 A Fungal Cell (Ultrastructure) 3 1.7 Fungal Growth 4 1.8 Haustorium 5 1.9 Nutrition 5 1.10 Reproduction 6 1.11 Some Important Indian Journals of Fungi 10 1.12 Some Important Foreign Journals of Fungi 11 1.13 Some International Journals Which also Publish Fungal Research Test Your Understanding 11 2. History of Mycology (Chronology of Major Events) 2.1 Global Developments of Mycology 13 2.2 Some Major International Contributors of 21st Century 15 2.3 Development of Mycology in India 16 2.4 Some other Indian Contributors and their Major Contributions Test Your Understanding 17 3. Fungal Cell: Structure and Composition 3.1 Cell Envelope 18 3.2 Cytoskeleton 22 3.3 Nucleus 22 3.4 Mitochondria 23

1

11 13

16 18

vi

3.6 3.7 3.8 3.9 3.10 4.

4.10 4.11 4.12 4.13 4.14

5. 5.1 5.2 5.3 5.4 5.6 5.7 5.8 6. 6.2 6.3

Contents 3.5 Hydrogenosomes: Hydrogen – Generating Organelles 23 Microbodies 24 Plasmids 25 Mycoviruses 25 Reserve Materials 25 Vacuoles 26 Test Your Understanding 27 Classif cation of Fungi 4.1 What is Classif cation? 28 4.2 Whether there Exist only Two Kingdoms? 28 4.3 Why is the Fungal Classif cation so Much Variable? 29 4.4 Groups of Uncertain Aff nity 30 4.5 Criteria Used in Fungal Taxonomy 31 4.6 Recommendations of International Committee 31 4.7 Botanical Ranks of Nomenclatural Hierarchy 31 4.8 Important Systems of Classif cation of Fungi 31 4.9 Some Older Contributions from 1623 to 1926 32 Classif cation Proposed by H.C.I. Gwynne-Vaughan and B. Barnes (1937) 33 Classif cation Proposed by Lilian E. Hawker (1966) 33 Classif cation Proposed by G.C. Ainsworth (1973) 34 Classif cation Proposed by Constantine J. Alexopoulos and Charles W. Mims (1979) 34 Classif cation Proposed by P.M. Kirk, P.F. Cannon, J.C. David and J.A. Stalpers (2001) 35 4.15 Outline of Latest Classif cation Proposed by John Webster and Roland W.S. Weber (2007) in their Book Introduction to Fungi 35 Test Your Understanding 37

28

Myxomycota Introduction 39 Acrasiomycetes 39 Hydromyxomycetes 40 Myxomycetes 41 5.5 Life-Cycle of a Typical Myxomycete 42 Stemonitis 47 Plasmodiophoromycetes 48 Plasmodiophora brassicae 49 T est Your Understanding 53

39

Eumycota 6.1 What are Eumycota? 54 Characteristic Features 54 Occurrence 54 6.4 Vegetative Plant Body 55

54

Contents 6.5 6.7 6.8 6.10 6.12 6.14 7. 7.2 7.3 7.4 7.5 7.6 7.7 7.8 8.

vii

Branching 55 6.6 Fine Structure of a Cell of Eumycota 55 Haustorium 55 Hyphal Aggregations 56 6.9 Hyphal Aggregations into Reproductive Structures 57 Vegetative Reproduction 59 6.11 Some Asexual and Sexual Spores 60 Sexual Reproduction 65 6.13 Modes of Sexual Fusion 66 Classif cation of Eumycota 67 T est Your Understanding 68 Mastigomycotina (General Account and Chytridiomycetes) 69 7.1 General Characteristics of Mastigomycotina 69 Classif cation of Mastigomycotina 69 Chytridiomycetes 70 Chytridiales 71 Synchytriaceae 73 Synchytrium Endobioticum 73 Blastocladiales 79 Monoblepharidales 79 T est Your Understanding 80 Hyphochytridiomycetes 8.1 What are Hyphochytridiomycetes? 81 General Characteristics 81 Classif cation 82 Rhizidiomyces 82 8.5 Phylogeny of Hyphochytridiomycetes 83 Test Your Understanding 83

81

Oomycetes 9.1 What are Oomycetes? 84 9.2 General Characteristics 84 9.3 Classif cation of Oomycetes 85 9.4 Order Saprolegniales 86 9.5 Saprolegnia 87 9.6 Achlya 91 9.7 Peronosporales 95 9.8 Family Pythiaceae 97 9.9 Pythium 97 9.10 Phytophthora 101 9.11 Family Peronosporaceae (Downy Mildews) 107

84

8.2 8.3 8.4

9.

viii 9.12 9.13 9.14 9.15 9.16 10.

10.3 10.4 10.5 10.6 10.7 10.8 10.9

Contents Peronospora 107 Sclerospora 108 Plasmopara 109 Family Albuginaceae 111 Albugo 111 T est Your Understanding 117 Zygomycotina (Zygomycetes) 10.1 What are Zygomycotina? 118 10.2 General Characteristics of Zygomycetes 118 Classif cation 119 Mucorales 119 Mucoraceae 121 Rhizopus 121 Mucor 126 Pilobolaceae 130 Pilobolus 130 10.10 Economic Importance of Mucorales 132 10.11 Heterothallism in Mucorales 133 T est Your Understanding 133

11. Trichomycetes 11.1 What are Trichomycetes? 134 11.2 General Characteristics 134 11.3 Classif cation 135 11.4 Harpellales 135 11.5 Asellariales 135 11.6 Eccrinales 136 11.7 Amoebidiales 136 T est Your Understanding 136 12. Ascomycotina (Ascomycetes) (General Account ) 137 12.1 What are Ascomycotina? 137 12.2 General Characteristics of Ascomycotina 137 12.3 Economic Importance 138 12.4 Somatic Structures 138 12.5 Asexual Reproduction 139 12.6 Sexual Reproduction 140 12.7 Methods of Bringing Compatible Nuclei Together 141 12.8 Compatibility 143 12.9 Ascus Development 143 12.10 Asci and Ascospores 145 12.11 Ascocarps 146

118

134

Contents 12.12

ix

Classif cation 147 12.13 Differences from Phycomycetes 147 Test Your Understanding 148

13.

Hemiascomycetes 13.1 What are Hemiascomycetes? 149 13.2 General Characteristics 149 13.3 What are Yeasts? 149 13.4 Classif cation 150 13.5 Endomycetales 150 13.6 Saccharomycetaceae 151 13.7 Saccharomyces cerevisiae 151 13.8 Economic Importance of Yeasts 157 13.9 Taphrinales 159 13.10 Taphrina 159 T est Your Understanding 160

149

14.

Loculoascomycetes 14.1 What are Loculoascomycetes ? 161 Habitat 161 Somatic Structure 161 Asexual Reproduction 162 Sexual Reproduction 162 T est Your Understanding 163

161

Plectomycetes 15.1 What are Plectomycetes? 164 General Characteristics 164 Classif cation 165 Eurotiales 165 Eurotiaceae 166 Aspergillus 166 Penicillium 171 15.8 Difference between Talaromyces and Eupenicillium 175 15.9 Difference between Aspergillus and Penicillium 175 15.10 Role of Aspergillus and Penicillium in Biotechnology 175 15.11 Mycotoxins from Aspergillus and Penicillium 176 T est Your Understanding 177

164

14.2 14.3 14.4 14.5 15. 15.2 15.3 15.4 15.5 15.6 15.7

16.

Laboulbeniomycetes 16.1 What are Laboulbeniomycetes? 178 16.2 A Brief Background 178 16.3 Occurrence 179 16.4 Thallus 179

178

x 16.5

Contents Sex Organs 179 16.6 Perithecia, Asci and Ascospores 179 T est Your Understanding 180

17.

Pyrenomycetes 17.1 What are Pyrenomycetes? 181 17.2 Distinguishing Features 181 17.3 Classif cation 182 17.4 Erysiphales 182 17.5 Erysiphe 183 17.6 Phyllactinia 187 17.7 Sphaerotheca 189 17.8 Sphaeriales 191 17.9 Sordariaceae 191 17.10 Neurospora 192 17.11 Clavicipitaceae 194 17.12 Claviceps 195 T est Your Understanding 197

181

18. Discomycetes 18.1 General Characteristics 198 18.2 Classif cation 199 18.3 Pezizales 199 18.4 Ascobolaceae 199 18.5 Ascobolus 200 18.6 Morchellaceae 202 18.7 Morchella 203 18.8 Pezizaceae 205 18.9 Peziza 205 18.10 Pyronemataceae 207 18.11 Pyronema 207 18.12 Tuberales 208 18.13 Tuber 210 Test Your Understanding 212

198

19. 19.2 19.4 19.5 19.6 19.7 19.8

Basidiomycotina (General Account ) 213 19.1 What are Basidiomycotina? 213 General Characteristics 213 19.3 Mycelium and Dikaryotization 214 Clamp Connection 215 Dolipore Septum 216 Asexual Reproduction 217 Sexual Reproduction 217 Basidium 217

Contents

xi

19.9

Basidiospore 220 19.10 Discharge Mechanism of Basidiospores 222 19.11 Buller Phenomenon 222 19.12 Differences from Ascomycotina 222 19.13 Classif cation 223 Test Your Understanding 224 20. Teliomycetes 20.1 General Characteristics 225 20.2 Classif cation 225 20.3 Uredinales 225 20.4 Puccinia 227 20.5 Puccinia graminis (Life Cycle) 228 20.6 Control Measures of Rusts 233 20.7 Annual Recurrence of Rust in India 235 20.8 Comparison Between Black, Orange and Yellow Rusts 235 20.9 Uromyces 235 20.10 Phragmidium 236 20.11 Ravenelia 236 20.12 Melampsoraceae 236 20.13 Melampsora 236 20.14 Ustilaginales 237 20.15 Ustilago 238 20.16 Graphiolaceae and Graphiola 244 Test Your Understanding 245

225

21.

Hymenomycetes 21.1 What are Hymenomycetes? 247 21.2 Holobasidiomycetidae 247 21.3 Agaricales 247 21.4 Edible and Poisonous Mushrooms 249 21.5 Fairy Rings 249 21.6 Classif cation of Agaricales 249 21.7 Agaricaceae 250 21.8 Agaricus 250 21.9 Aphyllophorales (= Polyporales) 254 21.10 Polyporaceae 255 21.11 Polyporus 255 Test Your Understanding 256

247

22.

258

Gasteromycetes 22.1 What are Gasteromycetes? 258 22.2 General Characteristics 258 22.3 Classif cation 259

xii 22.4 22.5 22.6

Contents Lycoperdales 259 Lycoperdaceae 259 Lycoperdon 259 Test Your Understanding

261

23. Anamorphic Fungi (Deuteromycotina or Deuteromycetes ) 262 23.1 Anamorphic Fungi and their General Characteristics 262 23.2 Types of Fructif cations 263 23.3 Parasexuality in Anamorphic Fungi 264 23.4 Classif cation 264 23.5 Recommendations of ICBN about Nomenclature of Anamorphic Fungi 265 23.6 Delimitation of Taxonomic Entity of Anamorphic Fungi 266 23.7 Blastomycetes 266 23.8 Sporobolomyces 266 23.9 Candida 267 23.10 Cryptococcus 268 23.11 Hyphomycetes 269 23.12 Alternaria 269 23.13 Cercospora 271 23.14 Curvularia 271 23.15 Pyricularia 272 23.16 Helminthosporium 272 23.17 Drechslera 273 23.18 Fusarium 273 23.19 Coelomycetes 274 23.20 Colletotrichum 275 23.21 Phyllosticta 276 23.22 Phoma 277 23.23 Phomopsis 277 T est Your Understanding 277 24. Economic Importance of Fungi 24.1 Introduction 279 24.2 Negative Aspects of Fungi 279 24.3 Positive Aspects of Fungi 283 24.4 Future Expectations from Fungi and Mycologists 285 Test Your Understanding 285

279

25. Fungi and Biotechnology 25.1 Biotechnology and its Relation with Fungi 287 25.2 Fermentation Technology 288 25.3 Enzyme Technology 292

287

xiii

Contents 25.4 Production Technology of Alcoholic Beverages 293 25.5 Cultivation of Mushrooms and other Macrofungi 294 25.6 Single-Cell Protein 295 25.7 Fungi in Food Processing Industry 295 25.8 Production of Primary Metabolites by Fungi 296 25.9 Production of Secondary Metabolites by Fungi 298 25.10 Role of Biotechnology in Selection and Mutation of Fungal Strains 301 25.11 Role of Biotechnology in Genetic Recombination and Gene Cloning 302 25.12 Gene Cloning and Future of Fungal Biotechnology 302 Test Your Understanding 304 26.

Mushroom Cultivation 26.1 Mushrooms and Mycophagy 305 26.2 Food Value of Mushrooms 305 26.3 Edible and Poisonous Mushrooms 306 26.4 Commercial Cultivation of Mushrooms 306 26.5 Cultivation of White Button Mushroom on Commercial Basis 306 26.6 Mushroom Growing in Laboratory 307 26.7 Cultivation of Shiitake (Lentinus elodes) 309 26.8 Paddy Straw Mushroom 310 26.9 Oyster Mushroom 310 26.10 Commercial Production of Some Other Macrofungi 310 26.11 Mushroom Parasites 311 26.12 Mushroom Dishes 311 Test Your Understanding 311

305

27.

312

Single-Cell Protein 27.1 What is Single-Cell Protein? 312 27.2 Why do we Need to Produce SCP? 312 27.3 Microorganisms used for SCP-Production 313 27.4 Composition of Single-Cell Proteins 313 27.5 Advantages and Disadvantages of Using Microorganisms for Animal or Human Consumption 27.6 Mycoprotein 314 27.7 Single-Cell Protein from Cyanobacteria 314 27.8 Single-Cell Protein from Algae 314 27.9 Single-Cell Protein from Organic Wastes 314 27.10 SCP From Petroleum Hydrocarbons and other Related Substrates 315 Test Your Understanding 315 28. Heterothallism in Fungi 28.1 What is Heterothallism? 316 28.2 Heterothallism in Mucorales 316 28.3 Heterothallism in some other Lower Fungi 318

313

316

xiv

28.5

Contents 28.4 Hormonal Basis of Sex and Heterothallism in Lower Fungi 318 Heterothallism in Ascomycetes 318 28.6 Heterothallism in Basidiomycetes 319 Test Your Understanding 320

29. Sex Hormones and Pheromones in Fungi 29.1 Hormones, Sex Hormones and Pheromones 321 29.2 Some Earlier Studies on Sex Hormones and Pheromones in Fungi 321 29.3 What has Finally Been Established with Sex Hormones in Fungi? 322 29.4 Sex Hormones Isolated from Lower Fungi 322 29.5 Some Extensively Studied Sex Hormones of Lower Fungi 323 29.6 Sex Pheromones in Higher Fungi 325 Test Your Understanding 327

321

30.

Mycorrhizae 30.1 What are Mycorrhizae? 328 30.2 Nature of Mycorrhizal Relationship 328 30.3 Types of Mycorrhizae 328 30.4 Mycorrhizosphere and “Mycorrhization-Helper Bacteria” (MHB) 333 30.5 Signif cance of Mycorrhizae 333 Test Your Understanding 334

328

31.

Lichens 31.1 What is a Lichen? 335 31.2 Components of Lichens 335 31.3 A Brief History 336 31.4 Occurrence 336 31.5 Classif cation 337 31.6 Lichen Thallus (Morphology and Anatomy) 338 31.7 Interaction Between Phycobiont and Mycobiont 340 31.8 Tissue Types in Lichens 340 31.9 Attachment Organs in Lichens 341 31.10 Propagules Associated with Lichen Thallus 341 31.11 Vegetative Reproduction 343 31.12 Asexual Spores 344 31.13 Sexual Reproduction of Mycobiont 345 31.14 Reproduction in Phycobiont 346 31.15 Economic Importance 347 Test Your Understanding 348

335

32.

349

Bacteria 32.1 What are Bacteria? 349 32.2 Major Characteristics 349 32.3 A Brief History 350

Contents

xv

32.4 Occurrence and Distribution 350 32.5 Classif cation 351 32.6 Morphology of Bacterial Cell 352 32.7 Structures External to the Bacterial Cell Wall 355 32.8 Bacterial Cell Wall:Ultrastructure and Composition 357 32.9 Cytoplasm and Cytoplasmic Inclusions 360 32.10 Mycoplasmas and L-forms of Bacteria 363 32.11 Nutrition in Bacteria 363 32.12 Gram Reaction 364 32.13 Growth 365 32.14 Cell Division (Binary Fission) 365 32.15 Spore Formation 365 32.16 Genetic Recombination (Sexual Reproduction) 369 32.17 Economic Importance 372 Test Your Understanding 375 33. Viruses 33.1 What are Viruses? 377 33.2 Major Distinguishing Features 378 33.3 How do Viruses Differ from Bacteria and Mycoplasmas? 378 33.4 Some Common Human Viral Diseases 378 33.5 A Brief History 378 33.6 Nature and Origin 380 33.7 Classif cation 380 33.8 Size and Symmetry of Viruses 382 33.9 Three Dozen Interesting Facts about Viruses 383 33.10 Chemical Composition 384 33.11 Morphology (Virus Organization and Structure) 385 33.12 Symptoms of Viral Infection in Plants 387 33.13 Transmission 387 33.14 Control of Viral Diseases of Plants 389 33.15 Viral Vaccines 389 33.16 Plant Viruses 389 33.17 Animal Viruses 391 33.18 Bacterial Viruses (Bacteriophages) 391 33.19 Life-Cycle or Replication of Bacteriophage 393 33.20 Mycoviruses 395 33.21 Cyanophages 395 33.22 Insect Viruses 396 33.23 Satellite Viruses and Satellite Nucleic Acids 396 33.24 Viroids 397

377

xvi 33.25

Contents Prions 398 33.26 Swine Flu: Some Basics We Should all Know and Follow 399 Test Your Understanding 400

34. Plant Diseases and their Control (General Account) 401 34.1 Plant Pathology and its Objectives 401 34.2 What is a Plant Disease? 401 34.3 Causes of Plant Diseases 401 34.4 Classif cation of Plant Diseases 402 34.5 Symptoms of Plant Diseases 402 34.6 Pathogenesis or Process of Infection 404 34.7 Enzymes, Toxins and Growth Regulatory Substances 407 34.8 Control of Plant Diseases 407 Test Your Understanding 409 35. Selected Diseases Caused by Fungi, Bacteria and Viruses (A) Some Diseases Caused by Fungi 410 35.1 White Rust of Crucifers 410 35.2 Late Blight of Potato 412 35.3 Green-Ear Disease and Downy Mildew of Bajra 413 35.4 Downy Mildew of Pea 413 35.5 Powdery Mildew of Pea 414 35.6 Powdery Mildew of Cucurbits 416 35.7 Loose Smut of Wheat 417 35.8 Covered Smut of Barley 418 35.9 Whip Smut of Sugar Cane 418 35.10 Black Rust or Stem Rust of Wheat 419 35.11 Yellow Rust or Stripe Rust of Wheat 420 35.12 Brown Rust or Orange Rust of Wheat 421 35.13 Rust of Linseed 422 35.14 Paddy Blast or Blast Disease of Rice 423 35.15 Tikka Disease of Groundnuts 423 35.16 Red Rot of Sugar Cane 424 35.17 Wilt of Arhar 425 35.18 Early Blight of Potato 425 (B) Some Diseases Caused by Bacteria 426 35.19 Tundu Disease or Yellow Ear Rot of Wheat 426 35.20 Citrus Canker 427 35.21 Brown Rot of Potato 427 (C) Some Diseases Caused By Viruses 428 35.22 Leaf Roll of Potato 428 35.23 Tobacco Mosaic Virus (TMV) 428

410

Contents

xvii

35.24 Leaf Curl of Papaya 429 35.25 Vein-Clearing of Bhindi 429 Test Your Understanding 430 Appendix 1: Some Common Culture Media and Mounting Media for Fungi 431 Appendix 2: Countrywise List of Institutions with Signif cant Collections of Fungi, Fungus-Related Websites, and Websites Related to Lichens, Bacteria and Viruses 435 Appendix 3: Classif cation of Fungi 439 Appendix 4: Glossary of 210 Mycological Terms 444 Appendix 5: Answers to Questions 450 Appendix 6: 136 Recommended Readings 454 Index

459

PREFACE

Fungi and Allied Microbes is the second of the Series on Diversity of Microbes and Cryptogams. In this I have discussed classif cation, history of mycology, and structure and composition of fungal cell in the earlier few chapters, followed by chapterwise discussion of almost all fungal di visions and classes ( Chapters 5–23) and then some selected general topics like Economic importance, Fungi and biotechnology, Mushroom cultivation, Single-cell protein, Heterothallism, Sex hormones and pheromones in fungi, and Mycorrhizae ( Chapters 24–30), followed by a detailed discussion of Lichens, Bacteria, Viruses and Plant diseases (Chapters 31-35) at the end. In the approach used and rationale behind the arrangement of chapters and selection of topics, I ha ve been mainly guided by the syllabi requirements of the Indian Uni versities. In the recent years, exceptional advances have been done in some specif c areas of research, particularly the molecular cell biology of tw o yeasts ( Saccharomyces and Schizosaccharomyces). The researches on these tw o model organisms have a bearing far beyond mycology, and have brought almost a revolution in fungal biotechnology, some major aspects of which have been brief y discussed in Chapter 25 (Fungal Biotechnology) of the book. The molecular phylogeny and computer-aided comparison of homologous DN A have been instrumental in clarifying the relationships of man y fungi, especially anamorphic fungi, pro viding an opportunity to discuss some of their aspects with their se xually reproducing relatives, and I have tried to incorporate these de velopments in Chapter 23 (Anamorphic Fungi). Recent investigations have also suggested to discuss some well-known fungal groups (e.g., Oomycota, Myxomycota and Plasmodiophoromycota) outside the fungi. However, in this book I have discussed them with fungi because they have been, and continue to be studied by mycologists and are still the syllabi requirements of most of the uni versities, especially of India. In brief, I may mention that I have tried to make proper use of molecular insights in discussing development, taxonomy, ecology, and pathogenicity of discussed fungi. Additional information is presented in the appendices at the end of the book. These include: • Some common culture media and mounting media for fungi (Appendix 1); • Countrywise list of institutions with signif cant collections of fungi, fungus-related websites, and websites related to lichens, bacteria and viruses (Appendix 2); • A detailed outline of classif cation of fungi proposed in the 9th edition (2001) of Dictionary of Fungi by Kirk et al. (Appendix 3); • Glossary of 210 mycological terms (Appendix 4); • Answers to chapter-end questions of all chapters (Appendix 5); and • 136 Recommended Readings, o ver 90 percent of which ha ve been published globally only during the last tw o decades (Appendix 6). Chapter 4 on classif cation of fungi includes an outline of the latest available classif cation proposed by Webster and Weber (2007) as well as botanical ranks of nomenclatural hierarchy as given by Kirk et al. (2001). Deuteromycotina has

xx

Preface

been named and discussed as ‘ Anamorphic Fungi’. Chapter 32 (Bacteria) includes Ber gey’s new classif cation of bacteria published in May 2001 besides giving the latest available details of bacterial (i) cell wall, (ii) pili, (iii) sheaths, (iv) prosthecae, (v) nucleoid, (vi) ribosomes, (vii) plasmids, (viii) endospores, and (ix) xeospores. Chapter 33 (Viruses) gives the latest details of (i) def nitions of viruses up to 2009, (ii) Baltimore scheme of virus classif cation, (iii) recommendations of ICNV (International Committee of Nomenclature ofViruses) up to 2000, (iv) mycoviruses, (v) cyanophages, (vi) insect viruses, (vii) satellite viruses, (viii) satellite nucleic acids, (ix) viroids, (x) prions, and (xi) swine f u. Two chapters at the end of the book pro vide a general account of plant diseases and their control ( Chapter 34) and selected 25 plant diseases caused by fungi, bacteria and viruses (Chapter 35). All chapters end with suff cient number of chapter-end questions, with the answers of majority of them gi ven at the end of the book in Appendix 5. Some major highlights of this book are as follows: • Chapterwise coverage of important general topics of fungi and allied microorganisms: – History of Mycology – Fungal Cell: Structure and Composition – Classif cation of Fungi – Heterothallism in Fungi – Sex Hormones and Pheromones in Fungi – Mycorrhizae – Lichens – Bacteria – Viruses – Selected Plant Diseases caused by Fungi, Bacteria and Viruses • Five application-based chapters: – Economic Importance – Fungi and Biotechnology – Mushroom Cultivation – Single-Cell Protein – Plant Diseases and their Control • Examination preparation tools to help students while preparing for their examination: – 42 website addresses related to fungi, lichens, bacteria and viruses in Appendix 2 – 19 chapters discussing almost all recognised classes and divisions of fungi – 210 mycological terms explained in the glossary in Appendix 4 – 387 chapter-end questions with answers of most of them at the end of the book in Appendix 5 – 25 tables of comparison spread in the entire text – List of 23 cultural media and mounting media used for studying fungi in Appendix 1 • Rich pedagogy: – List of 42 institutions of 14 countries with signifcant collections of fungi along with their website addresses in Appendix 2 – 288 well-labelled line diagrams to make the text clear to the reader – More than 40 electron micrographs supplied specially for this book by eminent mycologists of the world • Up-to-date as per details given in: – Botanical Code of Greuber et al. (2001) – Classif cation of fungi suggested by (a) Kirk et al (2001) in the 9th edition of Dictionary of Fungi, and (b) Webster and Weber (2007) in the 3rd edition of Introduction to Fungi

xxi

Preface – –

Classif cation of bacteria as given in Bergey’s classif cation published in May 2001 Def nitions, classif cation and some other details given in Principles of Microbiology by Sumbali and Mehrotra (2009) With all these inclusions, the book should cater to all needs of the readers, especially undergraduate and postgraduate students of Botany, Agriculture, Mycology and Plant Pathology of all universities the world over, in general, and of Indian universities, in particular. It should also be useful for students preparing for AIPMT, CPMT, IFS, IAS, PCS, NET, SLET and several other major competitive examinations. A number of experts took pains to critically review and examine the manuscript. My heartfelt thanks go out to all those whose names are given below: V Kumaresan J K Misra N S Atri Umesh B Kakde

Mahatma Gandhi Government Arts College, Puducherry Sri Jai Narain PG College, Lucknow, Uttar Pradesh Punjab University, Chandigarh, Punjab Government of Maharashtra’s Ismail Yusuf College, Mumbai, Maharashtra

I shall be failing in my duties if I do not duly ackno wledge the exceptional help and literary support given to me for this project by my dear student Dr M U Charaya, Professor of Botan y, CCS University, Meerut. He deserves all my praise, love and support, and I wish a bright future for him. Dr Charaya also contrib uted Chapter 30 (Mycorrhizae) for this book. For teaching me some parts of fungi at the postgraduate level, I wish to place on record my most sincere gratitude to Drs R Shiam, P K Dublish and Lokendra Singh. Without complete devotion and full cooperation of my wife, Dr (Mrs) Kanti D Sharma, PhD, this project could not have been completed. I e xpress my deep feelings of lo ve for her . My grandchildren (K uhu, Karan and Krish)—from whom, though unwillingly, I stole a lot of time during the preparation of this book, time that the y could have spent with me happily—deserve a special mention at this junction. I wish they become worthy citizens of our great country. Comments and suggestions aimed for the improvement of this book, shall be duly acknowledged and may also be sent to [email protected] or directly to me. Finally I express my gratitude to the publishing team of Tata McGraw-Hill who made this book see the light of day. O P Sharma + 91 98375 66555 0121-2401400

1

C H A P

INTRODUCTION

T E R

1.1

WHAT ARE FUNGI?

Fungi (L. fungus, mushroom) are achlorophyllous, heterotrophic (saprophytic, parasitic, symbiotic or hyperparasitic), eukaryotic and spore-bearing organisms, surrounded by a well-defined cell wall made up of chitin, with or without fungal cellulose, along with many other complex organic molecules. Fungi usually obtain food by absorption, except for a few lower groups where they take in food by ingestion. According to Kirk et al. (2001) fungi are “Eukaryotic organisms without plastids, nutrition absorptive (osmotrophic), never phagotrophic; lacking an amoeboid pseudopodial phase; cell walls containing chitin and b - glucans; mitochondria with flattened cristae and peroxisomes nearly always present; Golgi bodies or individual cisternae present; unicellular or filamentous and consisting of multicellular, coenocytic haploid hyphae (homo- or heterokaryotic); mostly non-flagellate, flagella when present always lacking mastigonemes; reproducing sexually or asexually; the diploid phase generally shortlived, saprobic, mutualistic, or parasitic”. The scientific study of fungi is called mycology (Gr. mykes, mushroom or fungus; logos, discourse). According to Alexopoulos and Mims (1979), the Italian botanist Pier’Antonio Micheli (1729) deserves the honour of being called the ‘founder of mycology’ because of his researches on fungi, which he published in Nova Plantarum Genera. Elias Fries (1794-1874), for his deep mycological understanding in earlier times, has been named the ‘Linnaeus of Mycology’, by Professor Eriksson Gunnar (1978) of Sweden.

1.2

VARIOUS NAMES USED FOR FUNGI

Various names used for fungi by different workers from time to time are Carpomycetes, Eumycota, Eumycophyta, Eumycetes, Fungales, Hysterophyta, Inophyta, Mycota, Mycetes, Mycetoideum, Mycetales, Mycetalia, Mycophyta, Mycophytes and Mycophycophytes, as mentioned by Kirk et al. (2001) in 9th edition of Dictionary of Fungi.

1.3

NUMERICAL ESTIMATES OF FUNGI

According to the estimates of Eizabeth Tootill (1984)published in The Penguin Dictionary of Botany, the fungi contain about 5100 genera and about 50,000 recognized species. ‘However it has been estimated that the actual number

2

Fungi and Allied Microbes

of species may be between 100,000 and 250,000’ (Tootill,1984). Webster and Weber (2007), however, mentioned that “about 80,000 to 120,000 species of fungi have been described to date, although total number of species is estimated at around 1.5 million (Hawksworth, 2001; Kirk et al., 2001)”.

1.4

WHY SHOULD WE STUDY FUNGI?

The study of fungi is important for common man as well as for experts (Gray, 1959; Christensen, 1965). Fungal saprophytes, along with bacteria, decay the complex plant and animal materials into simple form which is absorbed easily by the green plants. In the absence of this decaying process the future generations of green plants would not be able to survive for too long (Burges, 1958). A good symbiotic relationship exists between the majority of green plants and fungi (Webster, 1980), because the latter infect the roots of the former under the mycorrhizal system. Food, timber and textiles, the three basic needs of human-being, are rotted by the fungi, making their study essential for us. Thousands of the diseases of plants (Brooks, 1953; Mehrotra, 1980) and animals (Ainsworth and Austwick, l973) are caused by the parasitic fungi. On the contrary, the fungi are needed as essential items in brewing and baking industries, cheese making, wine making, preparation of many acids and in the production of antibiotics as well as many other drugs. Yeasts are the sources of vitamins of B-complex group. Further, many fungi are used as basic tools in scientific investigations of physiology, genetics, microbiology and biochemistry. Uses of Neurospora in genetical experiments, Gibberella fujikuroi in the discovery of gibberellins and yeasts in our knowledge of respiration process are well-known even to young students of biology. Study of yeasts is now a major aspect of research in brewing science and in food technology. The metabolic versatility of fungi is now exploited by fermentation industry, to make antibiotics and several other substances of interest to medicine, agriculture and chemical industry. Recent researches in recombinant DNA technology have led to fungi being used to produce hormones and vaccines.

1.5

HYPHA AND MYCELIUM

The fungi reproduce by means of one or more types of fungal spores. Germinating spores On being detached the spores are readily dispersed by wind and germinate on a suitable substratum by means of one or more tube-like outHypha growths, called germ tubes (Fig. 1.1). Each germ tube elongates to form long, fine, branched filaments, called hyphae (Fig. 1.1). Each hypha (Gr. Germ tube hyphe, web) divides by transverse walls or septa (L. septum, partition) and results into the formation of uninucleate or multinucleate cells. Such hyphae are called septate (Fig. 1.2A). But in many fungi hyphae do not develop the cross walls or septa, as in Phycomycetes. Such hyphae are called non-septate (Fig. 1.2B). Many nuclei are also present in Spore such hyphae. The unicellular and multinucleate condition of the hyphae is called coenocytic (Gi. koinos, common; koitos, couch). Kirk et al. (2001) defined hypha as “one of the filaments of a mycelium”. A mass of more or less loosely interwoven hyphae, constituting the vegetative body of most of the true fungi, is called mycelium (Gr. mykes, Fig. 1.1 Different stages of a germinating mushroom or fungus). The mycelium, if growing in between the host fungal spore cells, is called intercellular. But if it penetrates into the cells, it is called intracellular. The mycelial portion remaining inside the substrate is called vegetative mycelium, whereas the part that

3

Introduction

Nonseptate hyphae

Septate hyphae

Spore

A

Fig. 1.2

B

A, Septate hyphae; B, Non-septate hyphae of Phycomyces blakesleeanus

extends into the air and is responsible for spore production is called reproductive mycelium (Fig. 1.3). In many Basidiomycetes a small hyphal outgrowth develops into a short branch just behind the septum. This outgrowth becomes curved and its tip comes in contact with the cell on the other side of the septum. This all develops a communication link between the cell contents of two adjacent cells, leaving a characteristic clamp, called clamp connection (Fig. 19.2).

1.6

A FUNGAL CELL (ULTRASTRUCTURE)1

Substrate

Reproductive mycelium

The vegetative fungal cell (Fig. 1.4) has almost the same general ultrastructure as that of other eukaryotes. Some of its details are given below: Vegetative mycelium 1. The fungal cell remains surrounded by a cell wall made up of elongated microfibrillar units, which consist of chitin or fungal cellulose. Fig. 1.3 Vegetative and reproductive According to Bracker et al. (1976) the chitin microfibrils are formed mycelia of a fungus by the enzyme chitin synthetase. 2. The cell wall is followed by plasmalemma. It regulates the movement of soluble substances into and out of a hypha. 3. In between the plasmalemma and cell wall, or at the surface of the plasmalemma, some membranous structures have been reported in some fungi. They have been named lomasomes and plasmalemmasomes. A lomasome is the ‘membranous vesicular material embedded in the wall external to the line of the plasmalemma’, whereas a plasmalemmasome represents the ‘various membranous configurations which are external to the plasmalemma, often in a pocket projecting into the cytoplasm, and less obviously embedded in the wall material’ (Greenwood, 1970). 4. The cytoplasm contains a well-developed endoplasmic reticulum.

1

For details, see Chapter-3 (Fungal cell: Structure and Composition).

4

Fungi and Allied Microbes

Dictyosome

Vacuole

Fig. 1.4

Endoplasmic reticulum

Nucleus

Lipid body

Mitochondria

Ribosome

Cytoplasmic vesicle

Cell wall

Microbody

Hyphal apex of Pythium ultimum (diagrammatic) as viewed under electron microscope (after Grove et al., 1970)

5. The cells contain either many smaller vacuoles or a single large vacuole, each of which remains surrounded by a vacuolar membrane or tonoplast. 6. Mitochondria and many food particles made up of glycogen and lipids are also present. Structurally, the fungal mitochondria resemble those of green plants. 7. Other cell organelles and inclusions include ribosomes, vesicles, microtubules, microbodies and crystals. Golgi bodies are not reported in all fungal cells. Ribosomes are numerous, minute, almost of uniform size and distributed throughout the cytoplasm and also on endoplasmic reticulum. 8. Plastids or starch grains are absent. 9. All fungi lack chlorophyll. However, some other pigments have been reported to be distributed either in the cell wall, or irregularly in the cytoplasm, or just near the oil globules. Matsueda et al. (1978) isolated a novel fungal pigment, neocercosporin (C29 H26O10, m.p.237°C) from Cercosporina kikuchii. It is a reddish violet pigment. 10. The cells are uninucleate or multinucleate, and the nuclei are either globose or ellipsoid. The nuclear membrane is porous and consists of an outer and an inner layer of electron-dense material and a middle layer of electrontransparent substance. 11. A well-developed nucleolus, consisting mostly of RNA, and distinct chromatin strands, which become organized into chromosomes during nuclear division, are also present in the nucleus. The genetic information of the fungus is contained in the DNA of the chromosomes. Instructions are sent out into the cytoplasm by the messenger RNA, specially for the synthesis of enzymes and other proteins by the ribosomes. 12. The unit membrane of all the membrane-bound structures (like mitochondria, nucleus, etc.) consists of phospholipids along with proteins. The membranes are of uniform thickness, of about 0.02 m.

1.7

FUNGAL GROWTH

Most fungi do not require light for their vegetative growth. However, it is needed by many species for sporulation and spore dispersal (Buller, 1950). The optimum, minimum and maximum temperatures for growth vary from species to species but in most fungi the minimum is 2-5°C, the optimum 22-27°C, and the maximum 35-40°C. However, few species may survive at 0°C, and a few thermophilic species may endure a temperature of 60°C. Fungi generally require an acidic medium for normal growth, with a pH of approximately 6. The optimum pH for the growth of most of the fungi is 5-6.5, although a few fungi grow much below pH 3 and a few others even above pH 9 (Ingold, 1967). Oxygen supply is also an important factor for fungal growth. A majority of the fungi are aerobic and stop growing in the absence of oxygen. Like all other living organisms, water is an essential requirement for all fungi.

5

Introduction

Regarding the longevity in fungi, the fungal colonies are known to grow continuously for 400 years or more if favourable conditions are available (Alexopoulos and Mims, 1979).

1.8

HAUSTORIUM

In many parasitic fungi (e.g. Melampsora lini and Albugo candida) the lateral branches of hyphae penetrate into the host cells and enlarge in the form of knob-like, elongated or branched absorptive organs called haustoria (L.haustor, drinker). The haustoria originate commonly on the hyphae of obligate parasites. However, they are also produced on the hyphae of some facultative parasites Haustoria obtain nourishment from the protoplasts of the host cells. Electron microscopic studies of Coffey et al. (1972) and Coffey (1975) suggest that a penetrating fungal haustorium does not puncture the plasma membrane of the host. It simply invaginates into it. Moreover, the fungal wall around the haustorium also remains intact and unruptured. A haustorium, therefore, mainly functions as an organ of increasing the absorptive area of the fungus. Honneger (1992) described three main types of fungus-plant cell interactions (Fig. 1.5): (1) Wall-to wall apposition with no penetration; (2) intracellular haustoria where the fungus penetrates into the plant cell, with or without the formation of special sheath, neckband, or collar; (3) intraparietal haustoria where penetration is restricted to the wall layers. Wall-to wall apposition

Simple

Intracellular haustoria

Appressorium

With Without Sheath

Papilla

Collar

Intraparietal haustoria

Type 1

Fig. 1.5

1.9

Type 2

Type 3

Various types of fungus-plant cell interactions in parasitic and mutualistic symbiosis. (after Honneger, 1992)

NUTRITION

Fungi lack the green pigment, chlorophyll, and therefore remain unable to manufacture their own food material. The majority of the known fungi obtain food either from living organisms as parasites or from dead organic substances as saprophytes. Parasitic as well as saprophytic fungi may be either obligate or facultative. The living organism (either plant or animal) infected by a parasite is called a host. Besides the parasitic or saprophytic nature of fungi, many of them also remain in symbiotic relationship in the form of lichens or mycorrhyzae. Alexopoulos and Mims (1979) mentioned that probably all fungi require C, O, H, N, P, K, Mg, S, B, Mn, Cu, Zn, Fe, Mo and also Ca in the form of essential elements to fulfil their nutritional requirements. Glucose, nitrogenous and ammonium compounds and nitrates form the best food for many fungi. Some compounds, functioning as vitamins, are also synthesized by them. Excess food is stored in the form of glycogen or lipids.

6

Fungi and Allied Microbes

Food molecules of smaller size, specially in the form of solution, are easily absorbed by the fungi. But larger-sized food molecules are first broken into smaller-sized molecules by some extracellular enzymes, secreted by the fungus, and then absorbed.

1.10

REPRODUCTION

Fungi reproduce asexually as well as sexually. In the asexual reproduction, certain types of spores are formed without involving the fusion of nuclei or sex cells. But in sexual reproduction, formation and fusion of two types of sex cells or gametes take place. Some prefer to recognize a third category of reproduction, in which certain part of the vegetative plant body separates and develops into the new individual of the same species. This category is called vegetative reproduction. However, none of the categories of vegetative reproduction involves the fusion of sex cells or gametes. On the basis of the involvement of the entire thallus or a part of the thallus in the formation of reproductive organs, all fungi may be grouped into following two categories: (a) Holocarpic fungi: In genera such as Synchytrium the entire thallus converts into one or more reproductive bodies. Therefore, the vegetative and reproductive phases do not occur together. Such fungi are called holocarpic. (b) Eucarpic fungi: In majority of the fungi, only a part of the thallus develops into the reproductive organs, and the remaining part of the thallus continues to function its normal somatic activities. Such fungi are called eucarpic.

1.10.1

Asexual Reproduction

Alexopoulos and Mims (1979) put the asexual reproduction in fungi into four categories: (a) Fragmentation: In this method a part or fragment of the vegetative plant body detaches and develops into a new individual, as in a majority of the filamentous fungi. (b) Fission: It is a process in which splitting of the somatic cell results into the formation of two cells, as in yeast. (c) Budding: In this method a small outgrowth or bud is produced from the parent cell. The young bud later on separates from the parent cell and behaves as a new individual, as in Saccharomyces. (d) Spore formation: Various kinds of spores form the most common means of reproduction in majority of fungi. The spores Sporangium Zoospores vary in shape (oval, globose, helical, oblong, needle-like), size (from very minute to large), colour (orange, yellow, brown, red, black or even transparent or hyaline), number (from one to many Sporangiospores thousands in a spore-containing body), arrangement as well as in the way of their formation in different fungi. The structure responsible for spore production is called sporophore. Zoosporangium Some of the asexual spores are briefly described in the forthcoming Columella account: In many fungi the terminal end of the sporophore develops into a sac-like structure, called sporangium. Sporangiophore The sporangium-bearing sporophore is called sporangiophore. A B The sporangiophore extends into the sporangium in the form of a sterile inflated end, called columella (Fig. 1.6A). The motile Fig. 1.6 A, A sporangium and sporanflagella-bearing spores are called zoospores and the sporangium giospores; B, A zoosporangium and zoospores containing them is called zoosporangium (Fig. 1.6B). On the contrary, non-motile, aflagellated zoospores are called aplanospores or sporangiospores. For detailed treatment of fungal zoospores, refer Chapter 6 (Eumycota; Article No 6.11).

7

Introduction

In many fungi (Aspergillus, Penicillium) the spores are not contained within membranes, i.e. they are borne free. Such a sporophore is called conidiophore and the spores are called conidia (Fig. 1.7A). The shape, size, structure, colour and arrangement of conidia are different in different genera and serve as satisfactory points in fungal taxonomy. Conidia Conidia

Conidiophore

Phialide Vesicle

Conidiophore Foot cell D A Gelatinous material

Conidia

Conidia Phialide Metula Ramus

Conidiophore B Conidiophore

E Macroconidium

Conidia Bicelled conidia

Conidiophore Microconidia

C

Fig. 1.7

F

G

Conidia and conidiophores of some fungi. A, Aspergillus; B, Penicillium; C, Hormodendrum; D, Trichoderma; E, Gliocladium; F, Trichothecium; G, Microconidia and a macroconidium of Microsporum

8

Fungi and Allied Microbes

Conidiophore may be unbranched (Aspergillus, Fig. 1.7A) or branched (Penicillium, Fig. 1.7B), and may (Aspergillus) or may not (Penicillium) contain a vesicle at the end. At the tip of the vesicle or branched conidiophore are present many small, flask-shaped structures, called phialides. The tip of each phialide functions as a growing point and cuts conidia, which remain arranged basipetally, i.e. youngest at the base and oldest at the top on a phialide. In some genera (Hormodendrum), however, the conidia develop from the conidiophore by the process of budding. The first-formed spore on the conidiophore develops secondary spores by budding. The ultimate arrangement of conidia is therefore acropetal, i.e. oldest at the base and youngest at the top (Fig. 1.7 C). The type and arrangement of conidiophores and conidia, and the body containing these structures, in some other fungi are given below: 1. In Trichoderma, the conidia form globular clusters on the conidiophores (Fig. 1.7D). A 2. In Gliocladium, the groups of conidia on different phialides get surrounded by a mass of slime or gelatinous material (Fig. 1.7E). Conidia Coremium 3. In Trichothecium, bicelled conidia are formed (Fig. 1.7F). Conidiophore 4. In Microsporum (Fig. 1.7G) and Fusarium more than one type of conidia are formed. Of these, some are small, unicellular and are called microconidia, whereas others are large, multicellular and are called macroconidia. 5. In Ceratocystis ulmi and Arthrobotryum, group of conidiophores cement together to form an elongated, spore-bearing structure, B called synnema or coremium (Fig. 1.8A). 6. In Cystopus, the mass or cluster of conidia developing on short Fig. 1.8 A, Coremium of Arthrobotstalks or conidiophores represent a sorus (Fig. 1.8B). ryum; B, A sorus of Cystopus 7. In Colletotrichum and Marssonina, short conidiophores develop from a cushion-like flat mass or mat of hyphae. These Macroconidia conidiophores remain closely packed together to form an erumpent bed-like mass. This body is called acervulus (Fig. 1.9A). Conidia 8. In Fusarium (Fig. 1.9B) and Epicoccum the compact mass of conidiophores develop on a cushion-like mass of hyphae or stroma. Such acervulus-like body is called sporodochium. 9. In Septoria, Endothia and many other DeuteromyB Pycnidiospores A cetes, a well-developed globular body develops. It Ostiole remains embedded in the substratum and has a mouth opening or ostiole. This body is called pycnidium, and the conidia borne in the pycnidium are called pycnidiospores (Fig. 1.9C). In some yeasts and many other fungi the vegetative portion of the thallus develops into spore-like bodies, called thallospores, which may be of the following types: 1. The buds formed in Saccharomyces (Fig. 1.10A), Cryptococcus and Candida are simplest type of thallospores, called blastospores.

C

Fig. 1.9

A, An acervulus of Colletotrichum; B, A sporodochium of Fusarium; C, A pycnidium (diagrammatic) with pycnidiospores

9

Introduction

2. In genera such as Ustilago the hyphal cells are converted into thick -walled, round, resting thallospores, called chlamydospores (Fig. 1.10B). Depending upon their position on the hyphae the chlamydospores may be terminal, intercalary or lateral. 3. In genera such as Geotrichum and Oospora the hyphae get segmented to form rectangular and thick-walled cells, called arthrospores (Fig. 1.10C). Terminal chlamydospore Arthrospores

Buds

Intercalary chlamydospore

A

Fig. 1.10

1.10.2

B

A, Budding cells of Saccharomyces; B, Terminal and intercalary chlamydospores (diagrammatic); C, Arthrospores of Geotrichum

Sexual Reproduction Paraphysis

The sexual reproduction involves the fusion of two compatible nuclei. The spore that develops because of this fusion and subsequent reduction division is called sexual spore. The fusing nuclei contain X number of chromosomes and the fusing cells are called gametes. Ascospores The process of sexual reproduction is completed in three distinct Ascus phases: (i) fusion of two protoplasts of two gametes (plasmogamy), (ii) fusion of two nuclei of both the fusing gametes (karyogamy) to form a diploid zygotic nucleus, and (iii) formation of four haploid spores by Ascus the process of reduction division (meiosis). All steps of sexual fusion, starting from the initiation of sex organs or gamete formation up to the completion of karyogamy, are controlled by the secretion of some sexual hormones. For details of sex hormones, refer to Chapter 29 (Sex Hormones Fig. 1.11 Asci and ascospores (diagrammatic) and Pheromones in Fungi) Fungal sex organs are called gametangia. Morphologically indistinguishable gametangia and gametes are called isogametangia and isogametes, respectively. Similarly, morphologically different gametangia and gametes are called heterogametangia and heterogametes, respectively. In heterogametangia, the male gametangium is called antheridium, which contains antherozoids, whereas the female gametangium is called oogonium, which contains the female gamete.

10

Fungi and Allied Microbes

Compatible nuclei of two different gametes are brought together, in the process of plasmogamy, with the help of some general methods like gametangial contact (gametangy), gametangial copulation (gametangiogamy), spermatization, somatogamy and planogametic copulation. These methods are described, in detail, with the fungal genera in which they occur and also in brief in Chapter 6 (Eumycota). The sexual spores, when formed within a sac, are called ascospores, and the sac or the body containing these spores is called ascus (Fig. 1.11). The asci and ascospores are the characteristic features of Ascomycetes. They remain enclosed in a definite type of fruiting body, called ascocarp, which may be of any of the four types, i.e. cleistothecium, apothecium, perithecium and pseudothecium. In Basidiomycetes, generally four sexual spores develop from the end of club-shaped structure, called basidium. These spores are called basidiospores. A basidiospore develops on a spore-supporting process, called sterigma (Fig. 1.12A). In bracket fungi, puff balls and mushrooms the basidia, basidiospores and other mycelial parts remain organized in the form of highly developed fruiting bodies (Fig. l.12B,C). Pileus Basidiospores

Sterigma

Iamellae

Basidium Annulus A Stalk Basidiospores

Basidium

Paraphysis B

Fig. 1.12

C

Diagrammatic representation of basidia, basidiospores and fruiting body of higher Basidiomycetes. A, A basidium and basidiospores; B, Section of lamella; C, A well-developed fruiting body.

In a majority of Deuteromycetes true sexual cycle is absent. They show the phenomenon of parasexuality, under which plasmogamy, karyogamy and haploidization take place in a definite sequence, but not at the specified points in the life-cycle of the fungus.

Introduction

1.11 1. 2. 3. 4. 5.

1.12 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

1.13

1. 2. 3. 4. 5. 6. 7. 8. 9. 10.

SOME IMPORTANT INDIAN JOURNALS OF FUNGI Indian Phytopathology, New Delhi, India. Indian Journal of Mycology and Plant Pathology, Udaipur, India. Indian Journal of Mycological Research, Kolkata, India. Indian Journal of Plant Pathology, Lucknow, India. Kavaka, Chennai, India.

SOME IMPORTANT FOREIGN JOURNALS OF FUNGI Experimental Mycology, New York, USA. Instituto de Mycologia Publications, Recife, Brazil. International Journal of Mycology and Lichenology, Braunschweig. Mycologia, Bronx, New York, USA. Mycological Research (Transactions of British Mycological Society), Cambridge, U.K. Mycopathologia (Mycopathologia et Mycologia Applicata), Dordrecht, Netherlands. Mycotaxon, Ithaca, New York, USA. Phytopathology, Lancaster, USA. Phytopathologische Zrschrift, Halle, East Germany. Review of Plant Pathology (Review of Applied Mycology), Kew, Surrey, England. Studies in Mycology, CBS, Baarn, Netherlands. Sydowia, Annales Mycologici, Vienna, Austria.

SOME INTERNATIONAL JOURNALS WHICH ALSO PUBLISH FUNGAL RESEARCH American Journal of Botany, Texas, USA. Annals of Applied Biology, London, U.K. Annals of Botany, Oxford, U.K. Botanical Gazette, Chicago, USA. Botanical Review, New York, USA. Current Science, Bangaluru, India. Japanese Journal of Botany, Tokyo, Japan. Nature, London, U.K. Persoonia, Leiden, Netherlands. Science, Washington, USA.

11

12

Fungi and Allied Microbes

TEST YOUR UNDERSTANDING 1. The number of fungal species so far described is about: (a) 8 to 800 (b) 800 to 8000 (c) 8000 to 80000 (d) 80000 to 120000 2. What are fungi? Explain in about 100 words. 3. Study of fungi is also called ______ . 4. Which of the following names have never been used for fungi? (a) Myxophyceae and Phycology (b) Carpomycetes and Eumycetes (c) Inophyta and Hysterophyta (d) Mycophyta and Mycota 5. What is the major difference between hypha and mycelium? 6. Write a note on the ultrastructure of a fungal cell. 7. All fungi lack ______ . 8. What is haustorium? Describe its functions. 9. Give an illustrated account of methods of asexual reproduction in fungi. 10. What do you mean by holocarpic and eucarpic fungi? 11. Name any two important Indian and two foreign journals of fungi.

2

C H A P T

HISTORY OF MYCOLOGY (CHRONOLOGY OF MAJOR EVENTS)

E R

2.1

GLOBAL DEVELOPMENTS OF MYCOLOGY

To give even a brief description of the entire history of mycology is neither possible nor within the scope of this book. It is so because many references of fungi could be traced in Greek and Roman classics, and some observations of fungi being the causal agents of many plant and animal diseases are available in literatures related to Buddha (400 B.C.) and Vedas (1200 B.C.). However, a few major selected events, related to history of this fascinating field of organisms, are listed below: • Although Romans are credited to have done first drawings of a fungus, and they were also able to distinguish between boleti, fungi, suilli and truffles, there was practically no development of our knowledge about the fungi during 300 A.D. to 1500 A.D. • Clusius (1526 A.D.-1609 A.D.) described and presented illustrations of fungi in 1601 in his “Rariorium Plantarum Historia”. This book has an appendix of 28 pages, which may be treated as the first monograph on fungi. • G. Bauhin’s (1623), “Pinax Theatri Botanici” describes about 100 species of fungi which are now grouped under Agaricaceae, Boletaceae, Clavariaceae, Lycoperdaceae and Pezizaceae. • J.F. van Sterbeeck (1675) authored the first book devoted to fungi titled as “Theatrum Fungorum.” It was written in Dutch. He tried to distinguish between edible and poisonous fungi in this book. • Antonie Van Leeuwenhoek (1632-1723) was the first to use simple microscope to observe bacteria, yeasts and protozoa. • Robert Hooke (1635-1703) made first illustrations of microfungi in his Micrographia in 1665. He observed that the flesh of mushrooms consisted of an infinite company of small fragments. This British physicist and mathematician improved and used compound microscope, which later on proved revolutionary in the development of biology. Hooke studied Mucor and Phragmidium in detail. • M. Malpighi (1620-1694) delineated molds (Mucor, Rhizopus, Penicillium and Botrytis) in his Anatome Plantarum. • J.P. de Tournefort (1694) wrote Element de Botanique in which fungi have been arranged in six groups, viz., Fungus, Boletus, Agaricus, Lycoperdon, Coralloides and Tuber. • P.A. Micheli’s (1679-1737) Nova Genera Plantarum was published in 1729. It had description of large number of fungi. Published in Latin, Micheli also saw ascospores within asci. He was from a very poor family, self-educated but an outstanding student of fungi. He was the first person to culture molds by placing spores of Mucor and Aspergillus on freshly cut pieces of some fruits. Micheli named some genera including Polyporus, Tuber, Mucor and Aspergillus.

14

Fungi and Allied Microbes

• Linnaeus (1707-1778) made important contribution in the field of fungi in his Species Plantarum published in 1753. He described several fungi and included them all under class Cryptogamia of plant kingdom. He included fungi in his Latin binomial system, and his work is considered as the starting point for the nomenclature of lichens and Myxomycetes • Mathieu Tillet (1714 - 1791) laid the actual foundation of the fact that the development of mycology is closely related with the subject of Plant Pathology. Tillet (1755) provided experimental proof that bunt of wheat is contagious and this disease can be prevented by seed treatments with saltpeter solution and lime. • C.H. Persoon (1761-1836) made notable contributions in the systematics of fungi in his Synopsis Methodica Fungorum published in 1801. Persoon, along with Fries and Schweinitz, laid foundations for the modern classification of fungi. Being originally of South America, and educated in Germany and Holland, Persoon went to Paris in 1851 and lived there for rest of his life. Some of the other major contributions of Persoon are “Observations Mycologicae” (1796), “Tentamen Dispositionis Methodicae Fungorum” (1797), and “Mycologia Europaea” (1822). Persoon divided fungi in 2 classes and 6 orders. • E.M. Fries (1794-1878), a Swedish mycologist, is popularly called “Father of Systematic Mycology” or “Linnaeus of Mycology”. His most notable contribution is his Systema Mycologicum published in three volumes between 1821-1832. Fries described all the fungi known at that time in these volumes. Some of the other major contributions of Fries include his Elenchus Fungorum (1828), Lichenographia Europaei (1874) and Econes Selectae Hymenomycetum (1877). • C.de la Tour (1836) started working on biochemical aspects of fungi. Their role in various processes of industrial importance was recognised by Tour along with T. Schwann (1837) and F.T. Kutzing (1837). All these workers announced independently that yeast was associated with alcoholic fermentation. • Because of the great contributions of H.A. de Bary (1831-1888), a German mycologist, he is called “father of modern mycology”. His major contributions include “Untersuchungen uber die Brandprize” (1853), “Die Mycetozoen” (1864) and “Morphologia and Physiologie der Myxomyceten” (1866). He is also known for discovering heteroecism in rusts e.g. Puccinia graminis tritici. • L.D. Schweinitz (1780-1834) is called father of American Mycology. He collected over 4000 species of fungi from various parts of USA and described majority of them in his two famous works entitled “Synopsis Fungorum Carolinae Superioris” (1822) and “Synopsis Fungorum in America Boreali Media Degentium” (1832). • A.C. Josef Corda’s famous six volumes of Icones Fungorum Hucusque Cognitorum were published from 18371854. • Fungal infections in several animals, including man, were observed and described in detail by several mycologists, including (i) aspergillosis in birds in 1832 by Richard Owen, (ii) ringworm of man by D. Gruby (1844), and (iii) candidiasis in humans by Candida albicans (F.T. Berg, 1844). In a series of papers between 1841 and 1844, D.Gruby, an Austrian, described four fungi associated with mycosis of man. These were Trichophyton schoeleinis, T. mentagrophytes, Candida albicans and Microsporum audounii. • Classic experiments of Pasteur, describing origin of living organisms from inanimate objects, dominated the biological thinking of the world around 1860. • Mordecai Cubitt Cooke (1825-1914) has been a great British mycologist and authored many books including “Mycographia Handbook of British Fungi”, “Handbook of Australian Fungi” and “Fungoid Pests of Cultivated Plants” during 1857 and 1906. A cryptogamic journal Grevillea was started by Cooke in 1872. • Monumental work of P.A. Saccardo (1845-1920), an Italian mycologist, was published during 1882 and 1925 in 25 volumes of his Sylloge Fungorum Omnium Hucusque Cognitorum. These volumes of Sylloge described majority of the genera and species of fungi known at that time. Saccardo’s other major contribution include 38 volumes of “Fungi Italic”. • L.R. Tulasne (1815-1885) is called “Reconstructor of Mycology”. His “Fungi Hypogaei” was published in 1851. “Selecta Fungorum Carpologia” (1861-1865) is the world famous mycological contribution of Tulasne brothers, L.R.Tulasne and Charles Tulasne.

History of Mycology (Chronology of Major Events)

15

• Some other major contributions include (i) Les Hymenomycetes d’ Europe of Patovillard (1887), (ii) Kryptogamen Flora of Robenhort (1884-1920), (iii) British Fungal Flora of Massee (1892), and (iv) Enumeratio Systematica Fungorum of Oudeman (1924). • Arthur H.R.Buller (1874-1944) is well-known for his researches on spore discharge and sexuality in higher fungi. His major writings include “Researches on Fungi” published in six volumes. “Buller’s phenomenon”, a term given for Buller’s discovery of “the dikaryotization of a homokaryon by a dikaryon” is widely known in fungi. • P. Sydow (1851-1925) is recognised for his five volumes of Thesaurus Litteraturae Mycologicae et Lichenologicae published during 1908-1918. • A.F. Blakeslee (1904) discovered the phenomenon of heterothallism in Mucorales. • R.J. Sabouraud (1864-1938) is a widely known name for his various studies on fungi responsible for causing diseases in man, animals and plants. A dermatologist of Paris, Sabouraud’s researches on fungi pathogenic to man are detailed in Les Teignes published in 1910. • Discovery of penicillin in 1929 by Alexander Fleming (1881-1935) proved revolutionary for all of us. Since then, large numbers of antibiotics have been produced from fungi and other organisms in different parts of the world. Fleming’s discovery of penicillin from Penicillium opened a new line of therapeutic value of fungi. • Genetical studies on Neurospora by Dodge and Shear in a series of papers during 1927 and 1942 contributed laws of heredity, genetic control of enzymes and several other phenomena in fungi. • Researches of several mycologists during 20th and ongoing 21st centuries have seen major development in our knowledge of cytology, genetics, sex, physiological specialization as well as industrial exploitation of fungi. These have resulted in manufacture of cheese, commercial production of enzymes, food spoilage, rotting of textile fibers, and thousands of such articles, which have changed the entire scenario of these organisms for the entire humanity, in general and botanists in particular.

2.2 2000 2001

2002

2003

2004 2005 2006 2007

SOME MAJOR INTERNATIONAL CONTRIBUTORS OF 21st CENTURY* Beaks, G.W.; Cairney, J.W.G.; Glass, N.L.; Humber, R.A.; Kerry, R.A.; Kues, U.; Maheshwari, R.; Mishra, J.K.; Molina, R.; Reijnders, A.F.M.; Rogers, J.D. Barr, M.E.; Benny, G.L.; Bonner, J.T.; Braselton, J.P.; Carlile, M.J.; Cannon, P.F.; Cavalier-Smith, T.; Cerda- Olmedo, L.; David, J.C.; Fell, J.W.; Gooday, G.W.; Goodwin, S.B.; Hawksworth, D.L.; Heath. I.B.; Kirk, P.M.; Lumbsch, H.T.; O’Donnel, K.L.; Pegler, D.N.; Redhead, S.A.; Richardson, M.T.; Seifert, K. A.; Wells, K. Agerer, R.; Bartnicki-Garcia, S.; Blinder, M.; Brown, J.K.M.; Cairney, J.W.G.; Casselton, L. A.; Christensen, M.J.;Gow, N.A.R.; Heath, M.C.; Hilbert, D.S.; Hu,G.G.;Janex-Favre, M.C.; Klich, M.A.; Kumar, J.; Liroux,P.; Line, R.F.; Mendgen, K.; Mass, M.O.;Nash, T.H.; Parguey-Leduc, A.; Pitt, H, J.I.; Sharma, R.; Soll, D.R.; Stringer, J.R.; Weber, R.W.S. Alderman, S.C.; Anikster, Y.; Berbee, M.L.; Chaverri, P.; Dick, M.W.; Erikson, O.E.; Evans, H.C.; Gams, W.; Green, F.; Hell, I.R.; Harrington, T.C.; Karaffa, L.; Kinloch, B. B.; Kurtzman, C.P.; Landvik, S.; Leuchtmann, A.; Mitani, S. Amano, J. P.; Baver, R.; Beever, R.E.; Brasier, C.M.; Callaghan, A.A.; Chang, S.T.; deHoog, G.S.; Elad, Y., Geiser, D.M.; Liu, Y.J.; Lowry, D.S.; Peterson, R.L.; Samson, R.A.; Seaward, M.R.D.; Vellinga, E.C. Agrios, G.N.; Akins, R.A.; Asiegbu, D.O.; Batra, R.; Kolmer, J.A.; Magliani, W.; Silar, P.; Taylor, J.N. Samuels, G.J.; Webster, J. Weber, R.W.S.; Webster, J.

* For bibliographical details of these contributors, refer “Introduction to Fungi”(3/ed.) by John Webster and Roland W.S. Weber (2007).

16

2.3

Fungi and Allied Microbes

DEVELOPMENT OF MYCOLOGY IN INDIA • Besides some records of diseases of plants caused by fungi available in Vedas (1200 B.C.) and observations of Buddha (400 B.C.), a vast literature of last 150 years is available regarding the development of mycology in India. Most of the work in mycology in India has been done on the floristic and taxonomic side, and over 10000 species of fungi have been reported from different parts of the country. • D.D. Cunningham and M.J. Barkley, the two Britishers, collected many fungi from our country during their stay from 1871 and 1886 and made notable investigations in Mucorales, Ustilaginales and Uredinales. • Lt. Col. K.R. Kirtikar was, however, the first Indian to initiate work on fungal collection and identification in 1885. He studied many fleshy fungi, and his studies mark the beginning of Indian initiative in fungi. • Sir Edwin John Butler (1874-1943) is regarded as “Father of Indian Mycology”. Butler was actually the first person to initiate and organise large scale mycological and phytopathological research in India, and was the principal architect of mycology and plant pathology in this country. He established the Imperial Agricultural Research Institute at Pusa, Bihar, which was later on named as Indian Agricultural Research Institute (IARI) and shifted to New Delhi. Butler was appointed as first Imperial Mycologist of IARI in 1905. He published a book entitled “Fungi and Diseases in Plants” (1918), which is still supposed to be a real classic. He began a mycological herbarium “Herbarium Cryptogamae Indiae Orientalis”, which later on became the part of Division of Mycology and Plant Pathology of I A R I, New Delhi. Butler worked in detail on Pythium and also discovered the genus Allomyces in 1911. For his contributions, Butler was elected as Fellow of the Royal Society (FRS), London in 1926. Butler, along with his associates (particularly Bisby, H. Sydow and P. Sydow), compiled information on Indian fungi in the form of “Fungi of India” by Butler and Bisby, a monumental piece of work. Published first in 1931, Fungi of India has later on been revised and updated by several Indian Mycologists, including Mundkur (1938), Ramkrishnan and Subramanian (1952) and Vasudeva (1960). Fungi of India has also been compiled in two volumes by Bilgrami, Jamaluddin and Rizwi in 1979.Along with S.G. Jones, Butler also publihed Textbook of Plant Pathology. Born in 1874, Sir E.J Butler passed away on April 4, 1943. • K.C. Mehta and his associates deserve special mention for their contribution towards our knowledge of the recurrence of wheat rusts in plains of India. ICAR has publised their work in the form of a monograph entitled “Further Studies in Cereal Rusts in India” in 1940. Born in 1892 in Amritsar, Mehta joined Agra College in 1914, did his Ph.D. and D.Sc. from University of Cambridge, and has been a reputed Indian plant pathologist. • B.B. Mundkur (1896-1952) did extensive contribution to Indian fungi. In 1947, he founded Indian Phytopathological Society under the Chairmanship of Prof. S.R. Bose. Prof. Mundkur is known for his contributions towards Indian rusts and smuts. Along with M.J. Thirumulachar, Mundkur published a detailed account of Indian Ustilaginales in 1952. Mundkur also published “Fungi and Plant Diseases” in 1949. • S.R. Bose (1885-1970) worked on Polyporaceae of Bengal, published in 11 parts and 143 supplements between 1918-1947. In the later part of his career, Bose worked on physiology of skin diseases and deep mycoses in culture and worked as Head of Medical Mycology at Calcutta School of Tropical Medicine.

2.4

SOME OTHER INDIAN CONTRIBUTORS AND THEIR MAJOR CONTRIBUTIONS 1. P. Bruhl and J. Sen Gupta (1927) – Indian Myxomycetes. 2. H. Chaudhary(1927) – Aquatic fungi; Handbook of Indian Water Moulds (1947), Aspergilli and Penicillia from Punjab. 3. S.L. Ajrekar and K.D. Rajulu (1931) – Mucorales, Pythium and Phytophthora. 4. B.N. Uppal and J.H. Weston (1936) – Sclerospora.

History of Mycology (Chronology of Major Events)

17

5. T.S. Ramakrishnan (1931-1954) – Rusts from South India. 6. S.N. Das Gupta and R. John (1953) – Indian Blastocladiales, Monoblepharidales and Lagenidiales. 7. A.K. Kar (1949- 1970) – Fungi of West Bengal. 8. K.S. Thind (1942-1980) – Myxomycetes, Thelephoraceae, Clavariaceae and Helotiales of Himalayas. 9. B.S. Mehrotra (1952-1970) – Mucorales from Allahabad. 10. S.T. Titak (1958-1970) – Ascomycetes. 11. R.L. Munjal (1960-1975) – Hyphomycetes of India. Besides the above-mentioned, some other worthmentioning Indian contributors* of fungi include A. Mahadevan (physiology and biochemistry), A.P Misra (Graminicolous Helminthosporia), Bharat Rai (ecology of rhizosphere, litter and phylloplane fungi), C.V. Subrahmanian (Hyphomycetes), J.N. Mitter (Fungi of Allahabad), J.N. Rai (soil fungi), K.G. Mukherji (taxonomy and ecology of fungi), K.S. Bhargav (ecology of soil fungi), K.S Bilgrami (physiology and biochemistry of fungi), O.P. Sharma (Graphiola), P.D.Sharma (taxonomy and ecology of rhizosphere, litter and phylloplane fungi), R.K. Saxena (fungi causing storage diseases), R.N. Tandon (physiology and biochemistry), R.R. Misra (taxonomy and ecology of fungi), S.P. Raychaudhary (fungal, viral and mycoplasmal diseases), S. Sinha (phyllosphere) and T. Sreemulu (aeromycological studies).

TEST YOUR UNDERSTANDING 1. Write an essay on the major events of history of mycology. 2. Name the author of the following: (a) Pinax Theatri Botanici (b) Micrographia (c) Species Plantarum 3. Who is popularly called “Father of Systematic Mycology” or “Linnaeus of Mycology”? 4. Alexander Fleming (1881-1935) discovered _________ in _________. 5. Name any five major international contributors of fungi belonging to 21st century. 6. Give a detailed account of the development of mycology in India with special reference to the contributions of E.J. Butler, B.B. Mundkur and K.C. Mehta.

* Names arranged alphabetically.

3

C H A P T

FUNGAL CELL: STRUCTURE AND COMPOSITION

E R

In animals, a typical cell consists of a nucleus with associated cytoplasm bounded by a plasma membrane or cell membrane. In plants, however, a cell wall also surrounds the cell membrane. All other structures are almost the same as that of an animal cell. A typical eukaryotic cellular organization (Fig. 1.3) exists in a fungal hypha but it is very difficult to decide what constitutes a fungal cell in a mycelium. Fungal hyphae differ considerably in diameter even within the same species. Clear differences can be observed in the hyphae of different species. The hyphae of majority of the higher fungi are divided into compartments by cross walls or septa. The length of the apical compartment of the hypha, including the hyphal tip, varies in different species and may reach upto 400 µm (e.g. Aspergillus nidulans) with the subsequent compartments having a length of upto 50 µm. The breadth of the hyphae also varies between 3 µm (Aspergillus nidulans) to 10 µm (Neurospora crassa) or more. In most of the lower fungi, the hyphae are not subdivided into compartments. Majority of the yeasts have a well-defined cell. In Saccharomyces cerevisiae (Fig. 13.1) the cells are usually oval and attain a diameter of 5-10 µm whereas in Schizosaccharomyces pombe cells are short rods with a diameter of about 4-5 µm.

3.1

CELL ENVELOPE

3.1.1 Plasma Membrane Not only in fungi, but in all plants and animals all cells are bounded by plasma membrane, also called cell membrane or plasmalemma. Much information is still not available on the differences in plasma membrane of fungi and other organisms. A major difference, however, is the presence of ergosterol as a major sterol of the plasma membrane of fungi while that of cholesterol in the membranes of other organisms i.e. plants and animals. Due to the presence of ergosterol in the fungal cell membrane, the fungi are more susceptible than plants and animals to some polyene antibiotics and to inhibitors of ergosterol biosynthesis, which are therefore highly useful in treating fungal infections to plants and animals.

3.1.2 Glycocalyx Outside the plasma membrane, the fungal cells possess a macromolecular coating known as glycocalyx. In Myxomycetes the glycocalyx of the plasmodium is in the form of a sheath chiefly made up of a polysaccharide. This polysaccharide is slimy in nature and is a galactan which is a polymer of galactose. Some of the hydroxyl groups in this galactose are replaced by sulphate or phosphate. Differing from that of Myxomycetes, the glycocalyx in most of the other fungi is in the form of a firm and well defined cell wall. The slimy coating of the hyphal wall of some fungi is due to the presence of some extracellular polysaccharides.

19

Fungal Cell: Structure and Composition

3.1.3

Major Components of Hyphal and Yeast Walls

Electron microscopy and many available latest techniques revealed that fungal cell walls exhibit a dynamic structure rather than an inert coat to the cell. Its construction can change in response to environmental stresses such as osmotic shock. According to Gooday (1994) the major components of the fungal walls are polysaccharides: (i) chitin, glucans and mannoproteins in Ascomycetes, Basidiomycetes and mitosporic fungi, (ii) chitosan, chitin and polyglucuronic acid in Zygomycetes, (iii) chitin and glucan in Chytridiomycetes, (iv) chitin and cellulose in Hyphochytridiomycetes, and (v) cellulose and other glucans in Oomycetes. Cell wall proteins and glycoproteins, specific to each group of fungi, are also present. Chitin (Fig. 3.1) a polysaccharide containing nitrogen. This is the main substance in most fungal cell walls. Chitin also occurs as a major constituent of the exoskeleton of insects and other arthropods. Chemically, chitin is a linear polymer of acetylated amino sugar N-acetylglucosamine. Its subunits remain linked by b -(1Æ 4) glycosidic bonds. Physically, Chitin is very strong “with a tensile strength much greater than artificial materials such as carbon fibres and steel” (Carlile et al. 2001). The strength of chitin is due to extensive hydrogen bonding, along the chains, which provide them rigidity. Crystallization of the chitin chain results in the formation of microfibrils. In most hyphal walls, the chitin microfibrils are randomly arranged. Microfibrils range from 10-25 nm in dameter and approximately 1 µm long. In majority of Zygomycetes, the hyphal walls contain chitosan (Fig. 3.1) along with chitin. Chitosan is a chitin, of which most of the acetyl groups are removed enzymatically during synthesis. It is a polymer chiefly of b -(1Æ 4) glucosamine. Cellulose (Fig. 3.1) is present in the hyphal walls of Oomycetes. It is a linear b -(1Æ 4) glucose polymer. It is actually the main constituent of plant cell walls. b -(1- 3) glucan is a major and most abundant component of the walls of most of the fungi, except Zygomycetes. Its long chains form microfibrils like that of chitin. b -(1Æ 3) linkage of glucan provides it a helical structure. In mature cell walls, b- (1Æ 6) glucan is also associated with b -(1Æ 3) glucan. b-(1Æ 6) glucan is as much as 10% of the total cell wall glucan in Saccharomyces cerevisiae. Some other glucans reported in fungal walls are a- (1Æ) glucan as in Paracoccidioides brasiliensis; schizophyllan, a b -(1Æ 3) glucan in Schizophyllum commune; pullulan, a linear glucan as in Aureobasidium pullulans; and nigeran, a hot water soluble glucan as in some species of Penicillium and Aspergillus. According to Carlile et al. (2001) cell walls of yeasts (e.g. Saccharomyces cerevisiae) and some other fungi contain as much as 50% mannoproteins. These are elaborated in ER and Golgi bodies, and are secreted into the wall via usual secretion pathway.

H O

CH2OH O H OH H H

O H

H

NHCOCH3

H

NHCOCH3

OH

H

H

H O CH2OH

n

Chitin

H O

CH2OH O H OH H H

O H

H

NH2

H

NH2

OH

H

H

H O CH2OH

n

Chitosan

H O

CH2OH O H OH H H

O H

H

OH Cellulose

Fig. 3.1

H

OH

OH

H

H

H O CH2OH

n

Structural formulae of the principal fibrous components (chitin, chitosan

Cell walls of yeasts and some and cellulose) of fungal cell walls hyphal fungi contain two specific types of glycoproteins. These are glycosylphosphatidylinositol proteins or GPI – proteins (e.g. a-agglutinin) and proteins characterized with internal repeats of amino acid sequences or Pir proteins of cell walls.

20

Fungi and Allied Microbes

These are a class of hydrophobic proteins reported in some Basidiomycetes, Ascomycetes, Zygomycetes and some mitosporic fungi during last two decades between 1990 and 2010. They were first reported from Schizophyllum commune, a basidiomycetous fungus. These are highly hydrophobic to run on electrophoretic gels. These small secreted proteins have eight cysteine residues attached in a specific manner in their amino acid sequences: Xn - C- Xn - C - C - Xn - C - Xn - C - Xn - C - C - Xn - C – Xn (where Xn is a variable number of amino acids, and C is cysteine). Hydrophobins have also been reported in Agaricus bisporus, Magnaporthe grisea and Ophiostoma ulmi. Melanins are dark pigments found in walls of some fungi. They consist of branched polymers derived from some phenolic metabolites, e.g catechol, tyrosine and dihydroxynaphthalenes. Some probable roles of melanins in fungal walls include (i) to resist enzymatic lysis, and (ii) to work as photoprotective.

3.1.4 Structure of Cell Wall As is apparent from the above-mentioned components, the fungal cell wall is not a simple lining outside the plasma membrane. It is a complex dynamic assemblage of many components. Electron microscopy has revealed that fungal cell wall is made of layers. Recent findings have shown that these layers are not distinct. They are interconnected as a single massive multimolecular complex according to Carlile, et al. (2001). These workers have suggested a model of the cell wall of yeasts and hyphae of other fungi. As detailed in this model (Fig. 3.2) the chitin microfibrils form the innermost layer of wall just close to plasma membrane. b (1Æ 3) glucan fibrils remain attached to these microfibrils by glycosidic linkages. These glucan fibrils are mainly in the inner part of the wall. Towards the outer side the cell surface is formed by b (1Æ 6) glucans covalently bound to the b (1Æ 3) glucan fibrils. The GPI – mannoproteins are either anchored in the membrane, coming out through the wall, or bound to b (1Æ 6) glucans in the outer part of the wall. It appears that Pir-cell wall proteins are attached to b (1Æ 3) glucans. Some mannoproteins also seem to be linked to chitin microfibrils. These mannoproteins also extend out through the wall. It can thus be concluded that fibrous chitin and b (1Æ 3) glucan are more concentrated in the innermost part of the wall. Other components, such as, b (1Æ 6) glucan, proteins and mannoproteins are concentrated in the outermost part of the wall. All components are, however, cross-linked and interconnected as is shown in Fig. 3.2. Diagrammatic representation of principal regions of the cell wall of Neurospora are shown in Fig. 3.3.

Cell wall mannoproteins GPI-CWP

b(1 Æ 6)-glucan

Cell wall mannoproteins GPI-CWP

Pir-CWP

b(1 Æ 6)-glucan

Cell wall proteins with internal repeats i.e. Pir-CWP

3-D network of locally aligned b(1 Æ 3)-glucan chains

Chitin Microfibrils

Fig. 3.2

Chitin Microfibrils

Chitin Microfibrils

Chitin Microfibrils

Model of interrelationships of major components of the cell wall of Saccharomyces cerevisiae (GPI – CWP = Glycosylphosphatidylinositol cell wall proteins; Pir-CWP = Cell wall proteins with internal repeats) (Adapted from Smits, et al. 1999)

21

Fungal Cell: Structure and Composition

(a) Outermost layer of mixed amorphous glucans

Fig. 3.3

3.1.5

(c) Principally protein layer

Plasmalemma

(b) (d) The reticulum, The innermost proteinaceous glucans merging region embedded with into protein chitin microfibrils

Principal regions of cell wall of Neurospora, a diagrammatic representation.

Septa of Fungal Hyphae and Yeasts

Hyphae are divided into compartments by cross walls or septa in Ascomycetes, Basidiomycetes and mitosporic fungi. Usually the septa are perforated. A single central pore is present in each septum of Ascomycetes and mitosporic fungi. The pore helps in the easy passage of nuclei and other cytoplasmic inclusions between different cells of hyphae. Close to the pore are observed some membrane bound crystalline organelles in the cytoplasm of over four dozen fungal species. These are known as Woronin bodies. They attain a diameter of about 0.1 µm. Woronin bodies block the pore and stop leakage of protoplasm in case the cells are damaged by some accident or other unfavourable conditions. A protein of hexagonal crystals has been reported from the Woronin bodies of Neurospora crassa. Its molecular weight is 19000. Either a single micropore (e.g. Candida albicans) or numerous minute septal perforations (e.g. members of Saccharomycetales) are present. Micropore of C. albicans attains a diameter of 25 nm. Nuclei and mitochondria cannot pass through the micropore. However, it allows cytoplasmic continuity between the adjacent compartments of the hyphae. Simple septum with a single central pore (Fig. 3.4A) is present in Botrytis cinerea, majority of other mitosporic fungi, Ascomycetes and some Basidiomycetes. This allows easy passage of nuclei and mitochondria. Woronin bodies can block this pore in case of any accident. Cell wall

Septum Pore cap

Septum

Pore

Woronin bodies

re Po Pore

Perforations Rim

Septum

A

Fig. 3.4

B

C

A –C. Simple septum (A), dolipore septum (B) and multiperforate septum (C).

Septum

22

Fungi and Allied Microbes

Dolipore septa (Fig. 3.4B) are common in Basidiomycetes. These are the most complex septa. Around their pore is present a prominent rim. A membranous structure (pore cap) is present on either side of the pore. Pore cap is in continuation with the endoplasmic reticulum, and it has its own pores which allow the cell organelles to pass through. Multiperforate septa (Fig. 3.4C) are seen in some Ascomycetes and mitosporic fungi. Septum is crossed by very fine perforations which allow the cytoplasmic continuity between the compartments. Cell organelles can not pass through these perforations because they are very fine and thin. Septa of Schizophyllum commune are most intensively studied. In this fungus, a middle layer of chitin is covered by a layer of chitin and glucan. The septal swelling of glucan forms the prominent rim of the septum. In Candida albicans, the septum is made up of two chitin-rich septal plates, which remain apart by a thin amorphous layer. A very small micropore is present in this septum. Organelles can not pass through this micropore. During budding, the septum formation is seen in Saccharomyces cerevisiae. It allows the separation of bud from the parental cell. Two cells in this yeast are initially separated by a chitin-rich primary septum. Glucan-rich secondary septa develop on both the sides of the primary septum. When two cells are separated, a chitinous bud scar of the left-over material of the primary septum can be seen on the parental cell.

3.2

CYTOSKELETON

The soft hyphal wall at the apex is protected internally, and studies show that this is mediated by the cytoskeleton. Two main components of the cytoskeleton are microtubules and actin filaments. They are abundant in filamentous fungi and yeasts (Heath, 1995). Microtubules are oriented longitudinally and help in long-distance transport of organelles, e.g. secretory vesicles (Seiler et al., 1997) or nuclei (Steinberg,1998). They also help in positioning of nuclei, vacuoles and mitochondria according to Steinberg (1998). The actin filaments are found in the centre of the Spitzenkorper in the form of discrete subapical patches. (Ultrastructural studies suggest that the cytoplasm of the hyphal apex is occupied mostly by secretory vesicles and microvesicles. In Ascomycotina and Basidiomycotina, the secretory vesicles are arranged as a spherical shell around the microvesicles, and the entire formation has been called as apical body or Spitzenkorper by Bartnicki Garcia, 1996). At the hyphal apex, the soft wall is assembled on an internal scaffold made up of actin and other structural proteins, e.g. spectrin. According to Heath (2001), the “rate of hyphal extension might be controlled, and bursting prevented by the actin/spectrin cap being anchored to the rigid, subapical wall via rivet-like integrin attachments which traverse the membrane and might bind to wall matrix proteins” (Fig. 3.5).

Spectrin scaffold

Spitzenkörper Chitosome Secretory vesicle Microtubules Secretory vesicles Chitosomes Actin filaments Plasma membrane Cell wall Integrin molecule

Fig. 3.5

3.3

NUCLEUS

Diagrammatic representation of the internal scaffold model of the tip growth of fungi proposed by Heath (1995)

One or many nuclei may be present in each hyphal component of fungi. Yeasts have a single nucleus in each cell while as many as 50 or even more nuclei may be present in the apical compartment of Aspergillus nidulans. One to many nuclei per compartment are present in members of Basidiomycetes. In the vegetative phase of most of the filamentous fungi the nuclei are haploid. But in Oomycetes they are diploid. As far as the size is concerned, the nuclei of most fungi are small and attain a diameter of only 1-2 µm (Carlile, et al., 2001). In comparison to other eukaryotic organisms, the fungal nuclei have smaller chromosomes.

Fungal Cell: Structure and Composition

23

Acording to Turner (1993) haploid chromosome number of some higher fungi varies from 6 (Schizophyllum commune) to 20 (Ustilago maydis). It is 7 in Neurospora crassa, 8 in Aspergillus nidulans and 16 in Saccharomyces cerevisiae. In humans, it is 23. Chromatin structure of majority of the above-mentioned higher fungi resembles that of higher eukaryotic organisms. In the nucleosomes of these investigated higher fungi as many as 140 base pairs of DNA are wrapped around a disc made up of four different histones or basic proteins each present as two molecules. The chromosomes of Saccharomyces cerevisiae and Aspergillus nidulans contain a single linear double-stranded DNA molecule, as probably is the case with majority of the other fungi. Approximately 60% of the genes from filamentous fungi that have been sequenced contain introns, DNA sequences which are transcribed but not translated. Usually less than 100 base pairs are present in these DNA sequences. According to Carlile et al. (2001) “the gene density of Saccharomyces cerevisiae is very high, with one gene being encountered for every two kilobases (kb) of DNA in contrast to the human genome where the figure might be as low as one gene in 30 kilobases (kb)”. Complete sequencing of the genome of S. cerevisiae has been completed in 1996. A total of 6000 genes are present in the genome of this yeast. Complete genome sequencing has also been worked out for Candida albicans and Schizosaccharomyces pombe.

3.4

MITOCHONDRIA

Mitochondria are round or rod-shaped (Fig. 1.4) organelles of all eukaryotic cells in which reactions of the Kreb’s cycle and electron transfer chain take place. They have a smooth outer membrane and an inner membrane which is folded into cristae. The enzymes related with electron transfer and ATP production are located on the inner membrane. Tubular cristae are present in mitochondria of Oomycetes and slime moulds, but in other fungi the cristae are plate like. The size, form and number of mitochondria vary during cell cycle and also in response to different environmental conditions. In Saccharomyces cerevisiae, each cell contains either one or a few well-branched mitochondria. A large mitochondrion of this yeast can also divide into many smaller mitochondria. In the scarcity or complete absence of oxygen the membrane systems of the mitochondria of S. cerevisiae become less complex and electron-transfer enzymes (e.g. cytochromes) disappear. In Physarum polycephalum, the mitochondria divide by binary fission. Mitochondria of fungi contain DNA, which may form a nucleoid as the center of each mitochondrion. A nucleoid of the mitochondria of Physarum polycephalum consists of one or more molecules of mtDNA (mitochondrial DNA). A single molecule of mtDNA consists of a circle of double-stranded DNA. Linear molecules of mtDNA are observed in some other investigated fungi (e.g. Hansenula markii). The mtDNA constitutes the mitochondrial genome. It contains the genes for tRNA (transfer RNA) and rRNA (ribosomal RNA) of the mitochondria. It also contains the genes for some enzymes involved in oxidative phosphorylation. The size of the mitochondrial genome of different species of fungi is different as mentioned in Table 3.1. Total sequencing of genome of some fungi has been completed. These include a strain each of Aspergillus nidulans and Schizosacccharomyces pombe. In S. pombe the genome is only a little larger than the human mitochondrial genome (Table 3.1). In Saccharomyces cerevisiae, however, the genome is much larger than that of human genome as is evident from Table 3.1. Detailed study of the genome of a strain of S. cerevisiae shows that genes account for 62% intergenic regions, 22% for introns and for 16% of the mtDNA.

3.5

HYDROGENOSOMES: HYDROGEN – GENERATING ORGANELLES

Mitochondria are absent in some anaerobic rumen chytrids (e.g. Neocallimastix hurleyensis). These are the fungi found in the rumen (a specialized region of gut) of some ruminant animals, e.g. sheep, cattle, etc. Virtually anaerobic conditions exist in the rumen of these animals. Instead of mitochondria, these anaerobic rumen chytrids contain some hydrogen – generating organelles called hydrogenosomes. They are also found in many anaerobic protozoa. Hydrogenosomes are a major site of fermentative metabolism in these organisms. They finally lead to the production of CO2, hydrogen and

24

Fungi and Allied Microbes

Table 3.1

Size of the mitochondrial genome of humans, some fungi and higher plants as mentioned by Hudspeth (1992): S.No. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Group and species

DNA (thousands of base pairs)

Human Schizosaccharomyces pombe Phycomyces blakesleeanus Aspergillus nidulans Schizophyllum commune Achlya ambisexualis Dictyostelium discoideum Neurospora crassa Physarum polycephalum Saccharomyces cerevisiae Ustilago cynodontis Higher plants

17 17-22 26 32 50 50-51 55-60 62 69 74-85 77 160

acetate. All these are formed from malate, which is the major substrate for the hydrogenosomes. Hydrogenosomes supply energy for the motility of zoospores because they are present quite close to the flagellar bases. Several evidences of the evolutionary origin of hydrogenosomes suggest that they are derived from mitochondria.

3.5.1 Similarities Between Hydrogenosomes and Mitochondria 1. 2. 3. 4.

Like mitochondria, the hydrogenosomes are also surrounded by two membranes. Similar to the crystae of mitochondria, the hyrogenosomes may also have inner membrane invaginations. Both mitochondria and hydrogenosomes play a prominent role in the energy generation of the cell. Malic enzyme is a key component of the metabolism in both mitochondria and hydrogenosomes.

3.5.2 Dissimilarities Between Hydrogenosomes and Mitochondria 1. DNA is absent in hydrogenosomes while it is present in mitochondria. 2. Hydrogenosomes lack components of protein-synthesizing machinery while such components are present in mitochondria. 3. Hydrogenosomes also lack components of mitochondrial electron transport chain. 4. With reference to the energy generation of the cell, the hydrogenosomes use protons as electron acceptors to give hydrogen while mitochondria use oxygen to give H2O.

3.6

MICROBODIES

Microbodies are the cytoplasmic organelles of eukaryotic cells including those of fungi. They are bounded by a single membrane. Their types include glyoxysomes and peroxisomes. The major group of microbodies in the fungal cell are peroxisomes. Peroxisomes contain oxidizing enzymes and catalase. In each peroxisome is present at least one oxidase enzyme which produces hydrogen peroxide, along with peroxidase, which decomposes hydrogen peroxide, either to produce oxygen or to oxidize a substrate. In this way, it carries out respiration. It, however, does not conserve energy as ATP and thus differs from a mitochondrion. In Saccharomyces cerevisiae, peroxisomes also have a major role in the b - oxidation of fatty acids to produce acetyl – CoA. Peroxisomes also provide a site for the enzymes of glyoxylate cycle.

Fungal Cell: Structure and Composition

25

These enzymes are nedded for conversion of two carbon compounds into carbohydrate. An essential role of microbodies is in the utilization of many available sources of nitrogen and carbon. Only because of peroxisomes, many species of yeast can grow on unusual substrates, such as uric acid, fatty acids, alkanes and alkylamines. Fungal cytologists believe that new microbodies arise by division of the pre-existing microbodies. Woronin bodies (Fig. 3.4) are a specialized group of microbodies in many filamentous fungi. Hexagonal crystals present in Woronin bodies play a definite role in plugging septal pores to seal the damaged hyphae after some accidents.

3.7

PLASMIDS

Plasmid is a genetic element containing nucleic acid and is able to replicate independently of its host chromosome. Plasmids are actually pieces of DNA. They are very common in bacteria and are infrequent in fungi and other eukaryotic organisms. The plasmid of Saccharomyces cerevisiae is the 2 µm DNA which occurs in its nucleus. Its circular doublestranded DNA molecule is made up of approximately 6200 base pairs. The role of these 2 µm DNA pieces in the life of the host is still not clear. Plasmids often carry genes determining antibiotic resistance. Two plasmids of different lengths are present in another yeast, Kluveromyces lactis. Plasmids in Podospora anserina originate from mitochondrial DNA and are responsible for causing senescence. Studies suggest that if the plasmids are allowed to propagate continuosly, they may delay the senescence in the progeny. Since fungal plasmids have concern only with their own propagation, they are sometimes called the pieces of “selfish DNA”. Plasmids are highly useful in recombinant DNA technology.

3.8

MYCOVIRUSES

For details of mycoviruses, see Chapter 33, Viruses (Article No. 33.20).

3.9

RESERVE MATERIALS

Four common reserve materials of fungi are (i) glycogen, an a-glucan, (ii) trehalose, a non-reducing disaccharide, (iii) sugar alcohols e.g. mannitol alcohols, and (iv) polyphosphate (Fig 3.6 A-D). Similar to other eukaryotic organisms, fungi have the ability to accumulate lipids as a carbon reserve. In slightly mature and older cells oil droplets are also present.

3.9.1

Glycogen

Glycogen (Fig. 3.6A) is a storage polysaccharide, a, 1Æ 4 linked with frequent a, 1Æ 6 branches. It exists as small granules in the cytoplasm of fungi and animals as well as in blue – green algae and bacteria but not in plants or eukaryotic algae. Inside the cells, the glycogen is in the form of insoluble granules of complex tertiary structure with a molecular weight of over 100 million. On the other hand, the extracted glycogen is water soluble and has a molecular weight ranging between 1-10 million. Being a polymer of D-glucose molecules in the a-configuration, glycogen is an a - glucan. Glucose molecules are mostly linked to two others by a (1Æ 4) glycosidic bonds, thus forming chains. Usually, every 10th glucose molecule is linked to a 3rd glucose molecule by an a (1Æ 6) glycosidic bond, and this results into chain branching. Glycogen granules constitute approximately 10% of the dry weight of a fungus.

3.9.2 Trehalose Trehalose (Fig. 3.6B) is a disaccharide reserve material found commonly in several fungi. Its molecule is made up of two glucose molecules in a - configuration, joined together by a glycosidic bond present between the two C –1 atoms. Thus, trehalose is a non – reducing disaccharide. Being a reserve material, trehalose has a major role of protection against environmental stresses.

26

Fungi and Allied Microbes

CH2OH

O

O

5 4 OH 3

1

CH2OH

2

HOCH

OH O CH2OH

A

O

5 4OH 3

1 2

5 4OH 3 O

OH Glycogen

B

Fig. 3.6

O

HCOH

1 2

HCOH

O

CH2OH

OH

Mannitol

C

CH2OH

CH2OH 5 4 OH HO 3

HOCH

CH2 O

O 1 2

O 1 2

O OH OH Trehalose

5 OH 4 3 OH

O –

O—P— –

O

O

O

—O—P— —O—P—O –

O

n Polyphosphate

O





D

A-D. Molecular structure of common reserve materials of fungi. A, Glycogen; B, Trehalose; C, Mannitol, a sugar alcohol; D, Polyphosphate.

3.9.3 Sugar Alcohols or Polyols Polyhydric alcohols or polyols are also the common reserve materials of fungi. Some of the common sugar alcohols or polyols are (i) glycerol (a 3-carbon compound), (ii) erythriol (a 4-carbon compound), (iii) ribitol and arabitol (5-carbon compounds), and (iv) mannitol (a six carbon compound). Mannitol (Fig. 3.6) is found commonly in many fungi. High molecular weight polysaccharides also with lipids are common carbon reserves of most fungi.

3.9.4 Polyphosphate Polyphosphate and basic amino acids are the other commonly occurring stored metabolites of fungi . Polyphosphates are stored in the vacuoles along with phosphates, and are, therefore, discussed under vacuoles in Article 3.10.

3.10

VACUOLES

Fungal cells usually contain many vacuoles, which are easily visible under light microscope. Hyphal tips lack vacuoles. A vacuole is actually a liquid-filled space in a cell, surrounded by a membrane, called tonoplast.

Role of Vacuoles 1. Vacuoles in fungal cells serve as a store for metabolites and cations. 2. They also serve as regulators for pH and ion homeostasis in the cytoplasm. 3. Vacuoles contain several lytic enzymes (e.g. proteases, nucleases, phosphatases and trehalase) and thus play a role similar to that of animal lysosomes. 4. Tonoplast, the membrane of vacuole, has a proton- pumping ATPase like that of plasma membrane. It keeps the interior of vacuole acidic by energising the transport of solutes.

Fungal Cell: Structure and Composition

27

5. The amino acids stored in the vacuoles are mobilized to function as sources of nitrogen when nitrogen is available in short supply. 6. The vacuoles also store phosphate and polyphosphate (Fig. 3.4D). Polyphosphate work as a supply of phosphate for metabolism. It also exerts a much lower osmotic pressure than an equivalent amount of phosphate. It is therefore highly important in maintaining water relations of the vacuole. 7. The hydrolytic enzymes of the vacuoles also serve in the recycling of the cell contents during growth and further development. 8. In senescent cells, hydrolytic enzymes of the vacuole help in the autolysis of cell components. Using techniques of fluorescence microscopy, it has been observed in several species of Zygomycetes, Ascomycetes, Basidiomycetes and mitosporic fungi that “vacuoles are interconnected by a pleiomorphic motile tubular system, that undergoes peristaltic-like dilations and contractions. This results in transport of vacuolar contents over considerable distances along hyphae” (Carlile, et al. 2001).

TEST YOUR UNDERSTANDING 1. In the fungal cell wall, chitin is a: (a) glycoprotein, (b) polysaccharide, (c) protein, (d) lipopolysaccharide. 2. Fungal viruses are transmitted by: (a) insect vectors, (b) hyphal anastomosis, (c) air, (d) all of these. 3. The polyene antibiotics kill fungi by their interaction with: (a) protein synthesis, (b) microtubules (c) DNA, (d) plasma membrane. 4. Describe the structure and composition of fungal cell. 5. Write a brief note on glycocalyx. 6. Outside the plasma membrane, the fungal cells possess a macromolecular coating known as _____ . 7. Describe briefly chitin, chitosan and cellulose. 8. Explain briefly any five components of fungal and yeast walls. 9. Differentiate between: simple septa, dolipore septa and multiperforate septa. 10. A total of about ________ genes are present in the genome of Saccharomyces cerevisiae. 11. What are hydrogenosomes? How are they similar and different from mitochondria? 12. Write a note on plasmids? 13. Describe in brief the reserve materials found in fungi.

4

C H A P T

CLASSIFICATION OF FUNGI

E R

4.1

WHAT IS CLASSIFICATION?

The word ‘classification’ may be defined as the scientific categorization of the organisms in a hierarchical series of groups. In spite of the existence of many varieties, biological strains and physiological or cultural races, the species is generally considered as the smallest group. More similar species are grouped together into a genus, similar genera are grouped into a family, families into an order, orders into a class, similar classes into a division, divisions into a kingdom, and kingdoms into a domain or superkingdom.

4.2

WHETHER THERE EXIST ONLY TWO KINGDOMS?

Generally, it is believed that there exist only two kingdoms, i.e. Animals plant kingdom and animal kingdom, and all living organisms Pigmented Origin come within their purview. If we go back in the history, Lin- of life life forms Ferns Plants Seed naeus (1753) proposed that if a thing simply existed then it was a plants mineral, if it lived then it was a vegetable, and if it also had some Algae Fungi Mosses senses then it was an animal. If such an assumption is applied to Bacteria fungi, they are neither minerals nor have any discernable senses, and hence they are naturally vegetables. This idea actually gave Fig. 4.1 A two-kingdom system for the classification rise to the notion that all living things belong to one of the two of living organisms great kingdoms, i.e. plants and animals (Fig. 4.1), and fungi belong to plant kingdom. G.C. Ainsworth (1973), while discussing the status of fungi, put forward a promising aspect of all living organisms before the future biologists about existence of only two kingdoms, i.e. plant and animal kingdoms. The same has also been questioned by Copeland (1956), Barkley (1968), Stafleu (1969), Whittaker (1969) and others. Barkley (1968) suggested a four-kingdom system, whereas Whittaker (1969) suggested a five-kingdom system and raised fungi to the status of separate kingdom. Cytochrome-C1 studies of different organisms, made by Nolan and Margoliash (1968), also suggest that fungi form a phylogenetic line distinct from animal and plant kingdoms. Cooke (1977) has been quite straight forward in assigning fungi a separate and ‘new kingdom’. He strongly questioned the validity of teaching fungi along with plant sciences in many universities and mycological institutions of the world. 1

Cytochrome-C is a ‘component of the terminal respiratory chain of enzymes in aerobic organisms’ (Ainsworth, 1973).

29

Classification of Fungi

According to him, there is no scientific reason why this should be so; for mycology is as far removed from botany, as botany is from zoology, bacteriology or virology. The ‘five-kingdom system’ based on the level of organization and mode of nutrition of the living things, provides fungi a status of an independent kingdom (Fig. 4.2). There exist three fundamental modes of nutrition among the living organisms, i.e. photosynthesis (in which the organic matter is manufactured), absorption (in which the soluble organic compounds are absorbed or taken into the cell), and ingestion (in which the solid food particles are taken in). From the organization point of view there exist unicellular forms where there is no well-defined internal structures, and multicellular forms which show clear division of labour in their body. Absorption Multicellular organisms having uninucleate or multinucleate cells

Photosynthesis

Fungi

Plantae

Unicellular organisms having definite nuclei and some with chloroplasts (e.g. Euglena, Amoeba) Unicellular organisms having no definite nuclei and chloroplasts (e.g. bacteria, blue-green algae)

Ingestion Animalia

Myxomycota Protista Monera Origin of life

Fig. 4.2

A five-kingdom system for the classification of living organisms.

Recent trend is to divide Eukaryota into three kingdoms viz. Eumycota (True fungi), Protozoa and Chromista. Both Protozoa and Chromista contain mainly organisms not studied by mycologists. They were earlier grouped together under the name Protoctista by Kirk et al. (2001). Recently, Webster and Weber (2007) treated all Eukaryota as consist of three kingdoms viz. Protozoa, Straminipila and Fungi (Eumycota). An overview of all eukaryotic organisms, as suggested by Barbee and Taylor (1999) and also followed by Webster and Weber (2007) is highlighted in Fig. 4.3. It also suggests the phylogenetic relationships of fungi and fungus-like organisms with other groups of Eukaryota. Whether there exist only two (plant and animal) or more kingdoms is an important, yet unsettled fundamental aspect, and is to be decided by the future ‘biologists’, but recent phylogenetic, morphological, taxonomical, nutritional and biochemical studies indicate that fungi should be treated as separate taxonomic unit of the living organisms. Some promising evidences of this aspect can be gathered from the fossil records, but unfortunately much is not known about the fossil fungi.

4.3

WHY IS THE FUNGAL CLASSIFICATION SO MUCH VARIABLE?

No one knows how long fungi have inhabited the earth. No correct picture is available to us even about how, when or where the fungi evolved or originated? The details of the fungal phylogeny are by and large based on simple guesses, which might even be altogether wrong. We have to depend upon and accept the theories put forward by various present or past mycological authorities of the world. Therefore, if we believe the theory put forward by many earlier mycologists that evolution of fungi has taken place by loss of chlorophyll from the algae, they naturally belong to plant kingdom. But, if we believe the theory of many present-day mycologists that fungi and flagellates had a common ancestory, then ‘the fungi are neither plants nor animals: they are fungi’, in the words of Alexopoulos and Mims (1979). The fungal classification is highly variable because different details have been given about their origin and evolution.

30

Fungi and Allied Microbes

STRAMINIPILA

a ot PROTISTA yc m o l EUMYCOTA (True Fungi) u a ) th ta ae alg rin co A alg etes nd my by LI wn AE a d T o A o a s r /L a ot (re myc es a hor ete IM (b AN ot ioyc ta ta PL AN yc yta telio cet diop myc yta ta hytr m co co h h o m y o s o i m m y y p p c io io o m id m m os do om eo yc ho do dia od icty yxo lasm cras ytr sid co go ar ng iat ha om yp ng h h i a s i y Gi A M P D R K B C A K D P O H Z e)

PROKARYOTES

Fig. 4.3

4.4

Phylogenetic relationships of fungi and fungus-like organisms studied by mycologists, with other groups of Eukaryota. (Based on comparisons of 18Sr DNA sequences suggested by Barbee and Taylor, 1999).

GROUPS OF UNCERTAIN AFFINITY

The position of ‘Slime moulds’ has always been shifting between plants and animals in different systems of classification. Mycologists treat them under Myxomycetes in fungi and zoologists still treat them under Mycetozoa. The same is also true with Acrasiomycetes (cellular slime moulds) and Hydromyxomycetes. Plasmodiophoromycetes have been considered and discussed along with slime moulds in most of the earlier standard mycological contributions, but in many recent works they are ranked and treated along with Phycomycetous fungi. Fraser and Buczacki (1983) studied the molecular weights of ribosomal RNA of three members of this group (i.e. Plasmodiophora, Sorosphaera and Spongospora) and compared their data with the representatives of other fungal groups. They suggested that ‘Plasmodiophorales are quite distinct from Myxomycetes, Acrasiales, Trichomycetes, Oomycetes, Ascomycetes and Basidiomycetes’. Recently, Kirk et al. (2001) treated Plasmodiophoromycetes under an independent phylum Plasmodiophoromycota of kingdom Protozoa. Because of the ‘miscellaneous assemblage’ (Ainsworth, 1973) of Phycomycetes, they have all been considered in a single class by some but in many separate classes (Chytridiomycetes, Hyphochytridiomycetes, Oomycetes, Zygomycetes, Trichomycetes) by other workers. Ascomycetous fungi on the basis of characters of asci, and Basidiomycetous fungi on the basis of the structure of basidiocarp and many field and other characters were also assigned different taxonomic positions in different systems of classification of fungi. Deuteromycetes or Fungi Imperfecti, characterized by having only asexual spores, also have a highly uncertain taxonomic position. They have been variously ranked as an independent ‘Class’ (Stevenson, 1970), or “Form-Class” of Ascomycetes, or ‘Subdivision’ (Ainsworth, 1973; Alexopoulos and Mims, 1979; Webster, 1980) by different mycologists. Recently, in the 8th edition of Dictionary of Fungi, Hawksworth et al. (1995) treated these fungi under an informal group Mitosporic Fungi, but in the 9th edition of Dictionary of Fungi, Kirk et al. (2001) treated these fungi under a phylum rank taxa i.e., Anamorphic Fungi. Using DNA sequencing makes it now possible to place these remaining taxa with the groups of teleomorphic fungi from which they are or once were derived.

Classification of Fungi

4.5

31

CRITERIA USED IN FUNGAL TAXONOMY

Seven major characters that are used in fungal taxonomy are: (i) morphological characters, (ii) host specialization, (iii) physiological characters, (iv) cytological and genetical characters, (v) serological characters, (vi) biochemical characters and (vii) numerical taxonomy.1 Kuraishi et al. (1985) emphasized the importance of ubiquinone systems in the fungal taxonomy. They determined the ubiquinone (coenzyme Q) systems of 218 species belonging to Ascomycetes, Basidiomycetes and Deuteromycetes, and mentioned that ‘ubiquinone systems are very useful in the classification of fungal taxa’. Recently, DNA sequencing techniques are also used while classifying fungi (Fig. 4.3).

4.6

RECOMMENDATIONS OF INTERNATIONAL COMMITTEE

The committee on the International Rules of Botanical Nomenclature recommended the use of following ‘suffixes’ for the divisions and other major categories of fungi: Divisions should end in — mycota Subdivisions should end in — mycotina Classes should end in — mycetes Subclasses should end in — mycetidae Orders should end in — ales Families should end in — aceae No standard endings have been proposed for genera and species. The latter are broken down sometimes into varieties, forms and physiological races. The generic name is a noun and its first letter is always a capital letter. The species is generally an adjective, and is not to be capitalized. Names of the genera, species, varieties or forms are generally of Greek or Latin language.

4.7

BOTANICAL RANKS OF NOMENCLATURAL HIERARCHY

The current Botanical Code of Greuter et al. (2000) for principal, secondary and some other ranks, as also published in 9th edition of Dictionary of Fungi by Kirk et al. (2001), is mentioned in Table 4.1:

4.8

IMPORTANT SYSTEMS OF CLASSIFICATION OF FUNGI

It is not only difficult but impossible, and equally undesirable, to put before the readers all systems of classification of fungi proposed so far by different mycologists, since the beginning. The standard, for which this book has been written, 1 About numerical taxonomy, Dayal (1975) mentioned that it employs highly standard methods to determine as many phenotypic properties of as many specimens as possible of a group of organisms to be classified. Numerical taxonomy is really a creature of computer age for it would be a difficult science indeed without a computer. Percentage similarities (%S) of the organisms are calculated by the following formula: Nsp %S = ________ × 100 Nsp + Nd

Or

Nsp+Nsn %S = ____________ × 100 Nsp+Nsn+Nd

where Nsp = number of similar positive matches (i.e. both positive); Nsn = number of similar negative matches (i.e. both negative); Nd = number of dissimilar matches (i.e. one positive, one negative).

32

Fungi and Allied Microbes

Table 4.1 S. No. 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.

Principal, secondary and some other ranks in the botanical nomenclatural hierarchy RANKS

Example

Domain Kingdom Subkingdom Phylum (Division) Subphylum (Subdivision) Class Subclass Order Suborder Family Subfamily Tribe Subtribe Genus Subgenus Section Subsecton Series Subseries Species Subspecies Variety . Subvariety Form Subform Special form Physiological Race Individual

Eukaryota Fungi * Basidiomycota *-mycotina Teliomycetes *-mycetidae Uredinales *-ineae Pucciniaceae *-oideae Puccinieae *-inae Puccinia Puccinia (Hetero-Puccinia) * * * Puccinia graminis Puccinia graminis subsp. graminis P.graminis var. stackmanii * * * Puccinia graminis f.sp. avenae P.graminis f. sp. avenae Race *

* Not essential for this example

has further compelled the author to be still more restrictive. In most of the ‘standard mycological books’ only one system of classification is mentioned in detail. It is generally the system that has either been proposed or followed by the authors of these books. But in the syllabi of different Indian universities, for which this book is prepared, different systems of classification are required. A pick-and-choose policy was therefore applied. Few important older systems and some largely accepted recent systems of classification of fungi are therefore mentioned in the present text.

4.9

SOME OLDER CONTRIBUTIONS FROM 1623 TO 1926

Few worth-mentioning contributions before Linnaeus (1753) are those of Gaspard Bauhin (1623) and Pier A. Micheli (1719). Bauhin described about 100 fungi and placed them in some ‘groups’, whereas Micheli first used the microscope to study these organisms. Some of the generic names used by Micheli (1719) are in use even today (Clavaria, Lycoperdon, Geaster). Carl Linnaeus, the ‘Father of Botany’, in his Species Plantarum (1753) placed all fungi in 24th class ‘Cryptogamia’. C.H. Persoon (1801) in his Synopsis Methodica Fungorium, and E. Fries (1821) in his Systema Mycologicum also made some contributions in the field of taxonomy of fungi. Tulasne (1861), de Bary (1887), Guilliermond (1913) and Gawmann (1926) also proposed their own systems of classification of fungi.

33

Classification of Fungi

4.10

CLASSIFICATION PROPOSED BY H.C.I. GWYNNE-VAUGHAN AND B. BARNES (1937)

Gwynne-Vaughan and Barnes (1937) divided the fungi into three main classes (Phycomycetes, Ascomycetes and Basidiomycetes) on the basis of septation of mycelium and characters of spores. The representatives of the fourth class (Deuteromycetes or Fungi Imperfecti) do not show any sexual reproduction, reproduce by conidia and resemble the Ascomycetes. According to them Myxomycetes are not fungi. FUNGI

Phycomycetes (aseptate mycelium)

Septate mycelium

Ascomycetes (Endogenous ascospores present)

4.11

Basidiomycetes (Exogenous basidiospores present)

Deuteromycetes (Ascospores and basidiospores lacking)

CLASSIFICATION PROPOSED BY LILIAN E. HAWKER (1966)

Hawker (1966) preferred to divide all fungi into Lower Fungi and Higher Fungi. The former includes only the members which are either uninucleate or contain aseptate vegetative mycelium, whereas the later includes all members having regularly septate vegetative mycelium. All lower fungi are treated under Phycomycetes whereas all higher fungi are separated into two classes on the basis of the presence of characteristic endogenous ascospores (Ascomycetes) or exogenous basidiospores (Basidiomycets). Myxomycetes (slime moulds) have been shown to be originated from groups of aquatic flagellates by Hawker (1966). It has also been mentioned that there exist ‘structural resemblances between fungi and Myxomycetes (slime moulds) and some Protozoa’. But these important fungal members have not been discussed at all by Hawker (1966). Deuteromycetes (Fungi Imperfecti) have been treated by Hawker (1966) as a Form-Class of Ascomycetes. He stated that many species of Ascomycetes ‘are known only in the imperfect form and comprise the Form-Class Deuteromycetes or Fungi Imperfecti’. FUNGI

Higher fungi Classes

Lower fungi (Phycomycetes) Groups

Biflagellatae Aplanatae Ascomycetes Class Classes Classes Oomycetes

Uniflagellatae

1. Chytridiomycetes 2. Hyphochytridiomycetes 3. Plasmodiophoromycetes

1. Zygomycetes 2. Trichomycetes

1. Heterobasidiomycetidae

Basidiomycetes Subclass 2. Homobasidiomycetidae Contd..

34

Fungi and Allied Microbes

Contd..

Subclasses

1. Hemiascomycetidae

2. Euascomycetidae

3. Loculoascomycetidae

4. Laboulbeniomycetidae

Series 1. Plectomycetes

2. Hymenoascomycetes Sub-series 1. Pyrenomycetes

4.12

2. Discomycetes

CLASSIFICATION PROPOSED BY G.C. AINSWORTH (1973)

The well-known general-purpose classification for fungi proposed by Ainsworth (1966), and followed by himself in Dictionary of Fungi (1971) and also in The Fungi: An Advanced Treatise (Ainsworth, 1973), may be considered as an ideal scheme of classification that reflects natural relationship. In this system the fungi with plasmodia or pseudoplasmodia are classified in the division Myxomycota, whereas most of the remaining, usually filamentous fungi which do not have any plasmodium or pseudoplasmodium, are classified in division Eumycota. John Webster (1980) also ‘chosen to adopt the scheme proposed by Ainsworth (1973)’. The author has also followed this scheme to discuss fungi in the present book. Its details are given on next page.

4.13

CLASSIFICATION PROPOSED BY CONSTANTINE J. ALEXOPOULOS AND CHARLES W. MIMS (1979)

Following the suggestions of Whittaker and Margulis (1978), about the existence of Superkingdom as the largest taxonomic rank, Alexopoulos and Mims (1979) placed all fungi, including the slime moulds, in Kingdom Myceteae of Superkingdom Eukaryonta. Kingdom Myceteae, as proposed by Alexopoulos and Mims (1979), includes 3 Divisions, 8 Subdivisions, 11 classes, 1 Form-Class, 3 Subclasses and 3 Form-Subclasses. Their proposed classification of fungi is given below in tabulated form. Kingdom MYCETEAE (fungi, including slime moulds) Divisions I. GYMNOMYCOTA Subdivisions

II. MASTIGOMYCOTA Subdivisions

III. AMASTIGOMYCOTA

Acrasiogymnomycotina Plasmodiogymnomycotina Haplomastigomycotina Diplomastigomycotina Class

Classes

Acrasiomycetes Protosteliomycetes

Class

Classes

Oomycetes

Subdivisions

Myxomycetes

Subclasses

1. Chytridiomycetes 2. Hyphochytridiomycetes 3. Plasmodiophoromycetes Contd. on page 36

Classes

ZYGOMYCOTINA (zoospores absent; perfect-state spores are zygospores) Classes

Plasmodiophoromycetes (presence of parasitic plasmodium within host cells)

Subdivisions

Classes

Classes

DEUTEROMYCOTINA BASIDIOMYCOTINA (zoospores and perfect-state spores (zoospores and zygospores absent; perfect-state spores are basidiospores) like zygospores, ascospores are basidiospores absent)

Myxomycetes (presence of freeliving saprobic plasmodium)

ASCOMYCOTINA (zoospores and zygospores absent; perfect-state spores are ascospores)

Labyrinthulales (net-plasmodium is present)

EUMYCOTA (absence of plasmodium or pseudoplasmodium)

1. Hemiascomycetes (ascogenous 1. Teliomycetes (parasitic on vascu- 1. Blastomycetes (true mycelium is 1. Chytridiomycetes (posteriorly 1. Zygomycetes (mostly saprobic; if parasitic the hyphae and ascocarps absent; lar plants; basidiocarp absent; tepoorly developed or absent; budding uniflagellate zoospores mycelium is immersed thallus yeast-like or mycelial) liospores are either scattered cells with or without pseudomycontain whiplash-type in host tissue) 2. Loculoascomycetes (ascowithin host cell or grouped in celium) flagella) 2. Trichomycetes (atgenous hyphae and ascocarps sori) 2. Hyphomycetes (true mycelium well2. Hyphochytridiomycetes tached to the cuticle or present; thallus mycelial; asci 2. Hymenomycetes (mostly saprodeveloped budding cells absent; (anteriorly uniflagellate digestive tract of arbitunicate; ascocarp as ascosphytic; basidiocarp present and of mycelium is either sterile or bear zoospores contain tinselthropods; mycelium not troma) gymnocarpous or semiangiocarspores; pycnidia or acervuli absent) type flagella) immersed in host tissue) 3. Plectomycetes (ascogenous hypous type; basidiospores ballisto- 3. Coelomycetes (true mycelium well3. Oomycetes (biflagellate phae and ascocarps present; spores) developed; budding cells absent; zoospores, of which posterior ascospores aseptate; asci 3. Gasteromycetes (mostly saprospores develop in acervuli or flagellum is whiplash type and unitunicate) phytic; basidiocarp angiocarpous; pycnidia) anterior flagellum of tinsel type) 4. Laboulbeniomycetes (asci arbasidiospores are not ballistoraged as a basal or peripheral spores) layer within ascocarp; ascocarp of perithecium type; exoparasites on arthropods; asci inoperculate) 5. Pyrenomycetes (ascocarp typically perithecium; not exoparasites on arthropods; asci inoperculate with an apical pore or slit) 6. Discomycetes (ascocarp apothecium; not exoparasites on arthropods; asci inoperculate or operculate)

Classes

MASTIGOMYCOTINA (zoospores present; perfect-state spores are typically oospores)

Acrasiomycetes (free-living assimilatory phase of amoebae unite as a pseudoplasmodium before reproduction)

Classes

MYXOMYCOTA (Presence of plasmodium or pseudoplasmodium)

FUNGI Divisions

CLASSIFICATION OF FUNGI PROPOSED BY G.C. AINSWORTH (1973)AND FOLLOWED ALSO IN THIS BOOK

Classification of Fungi

35

36

Fungi and Allied Microbes

Contd..

1. Ceratiomyxomycetidae 2. Myxogastromycetidae 3. Stemonitomycetidae Zygomycotina

Ascomycotina

Classes 1. Zygomycetes 2. Trichomycetes

Class Ascomycetes Subclasses

Basidiomycotina Class Basidiomycetes Subclasses

Deuteromycotina Form-Class Deuteromycetes FormSubclasses

1. Hemiascomycetidae 1. Holobasidiomycetidae 1. Blastomycetidae 2. Plectomycetidae 2. Phragmobasidiomycetidae 2. Coelomycetidae 3. Hymenoascomycetidae 3. Teliomycetidae 3. Hyphomycetidae 4. Laboulbeniomycetidae 5. Loculoascomycetidae

4.14

CLASSIFICATION OF FUNGI PROPOSED BY P.M. KIRK, P.F. CANNON, J.C. DAVID AND J.A. STALPERS (2001)

See details in Appendix 3.

4.15

OUTLINE OF LATEST CLASSIFICATION PROPOSED BY JOHN WEBSTER AND ROLAND W.S. WEBER (2007) IN THEIR BOOK INTRODUCTION TO FUNGI (3RD EDITION)

Webster and Weber (2007) mentioned that “Fungi in the widest sense, as organisms traditionally studied by mycologists, currently fall into three kingdoms of Eukaryota, i.e. the Eumycota which contains only fungi, and Protozoa and Chromista (= Straminipila)”. Both Protozoa and Straminipila “contain mainly organisms not treated by mycologists and were formerly lumped together under the name Protoctista” by workers such as Beaks (1998)and Kirk et al. (2001). About Protozoa, Webster and Weber (2007) mentioned that at present it can only be said that “they are a diverse and ancient group somewhere between higher Eukaryota and Prokaryotes” as has also been suggested by Kumar and Rzhetsky (1996). An outline of the classification proposed by Webster and Weber (2007) is undermentioned:

KINGDOM PROTOZOA

Classes:

1. 2. 3. 4.

Acrasiomycetes Dictyosteliomycetes Protosteliomycetes Myxomycetes

Classification of Fungi

Orders:

1. Plasmodiophorales 2. Haptoglossales (Oomycota ?)

KINGDOM STRAMINIPILA

Classes:

1. Labyrinthulomycetes 2. Thraustochytriomycetes

Orders:

1. Saprolegniales 2. Pythiales 3. Peronosporales

KINGDOM FUNGI (EUMYCOTA)

Class:

Chytridiomycetes

Classes:

1. Zygomycetes 2. Trichomycetes

Classes:

1. 2. 3. 4. 5. 6.

Archiascomycetes Hemiascomycetes Plectomycetes Hymenoascomycetes Pyrenomycetes Loculoascomycetes

Classes:

1. 2. 3. 4. 5.

Homobasidiomycetes Homobasidiomycetes: Gasteromycetes Heterobasidiomycetes Urediniomycetes Ustilaginomycetes

37

38

Fungi and Allied Microbes

TEST YOUR UNDERSTANDING 1. Ainsworth (1973) divided fungi into how many divisions? Give an outline of his classification upto the level of classes, mentioning at least one characteristic feature of each class. 2. Who proposed (a) four - kingdom system, and (b) five – kingdom system of classification and when? 3. Recent trend is to divide Eukaryota into three kingdoms. What are they? 4. Metasporic fungi and Anamorphic fungi are the names given to which fungi? 5. In the botanical nomenclatural hierarchy, which of the following is the lowest rank? 6. Give an outline of the system of classification of fungi given by Gwynne-Vaughan and Barnes (1937). 7. Plasmodium or pseudoplasmodium is present in which of the following? (a) Myxomycetes, (b) Acrasiomycetes, (c) Myxomycota, (d) All of these th 8. The 2001 (9 edition) of the famous book “Dictionary of Fungi” has been authored by whom?

5

C H A P T

MYXOMYCOTA

E R

5.1

INTRODUCTION

Slime moulds and many similar organisms are kept under Myxomycota. Earlier biologists were perplexed in assigning them a proper taxonomic position. The main question has always been whether to treat them with plants or animals? But ‘today practically all botanists consider them related to fungi’ (Smith, 1955). Because of the presence of creeping, acellular somatic phase the slime moulds were treated under animals by De Bary (1887), Lister (1925), Bessey (1950), Olive (1970) etc. But the structure of their reproductive bodies, production of spores which remain covered with definite walls, and many similar characters bring these organisms more closer to plants. According to Webster (1980), Myxomycota are possibly ‘more closely related to Protozoa’. In his general-purpose classification of fungi, Ainsworth (1973) divided division Myxomycota into four classes, i.e. (i) Acrasiomycetes (ii) Hydromyxomycetes (iii) Myxomycetes (iv) Plasmodiophoromycetes. As mentioned earlier in Chapter 4, the classification of fungi suggested by Ainsworth (1973) has been followed in this book. However, in the latest classification of fungi proposed by Kirk et al. (2001) , all these organisms have been treated under kingdom Protozoa (see Appendix-3). Independent phylum ranks have been given to Acrasiomycota, Myxomycota and Plasmodiophoromycota. And, phylum Myxomycota in this system has been divided into three classes viz. Dictyosteliomycetes, Myxomycetes and Protosteliomycetes.

5.2

ACRASIOMYCETES

5.2.1

Characteristic Features

1. Members exist as amoeboid cells or myxamoebae. In the later stages many myxamoebae form stellate pseudoplasmodial masses, as in Dictyostelium. Myxamoebae ingest bacteria or other prey as their food. 2. Because of their structure they have been named ‘cellular slime moulds’ by Bonner (1967). 3. Some species form groups or aggregated masses or myxamoebae. Aggregation of myxamoebae in Acrasiales result into the formation of pseudoplasmodium.

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4. After migration the process of cellular rearrangement and differentiation takes place. This results in the formation of a multicellular stalk and a globose sorus. 5. Stalk consists of thousands of myxamoebae. 6. The globose sorus contains many spores. 7. New myxamoebae are formed on germination of spores. Raper (1973) divided Acrasiomycetes into three sub-classes, i.e. Protostelidae, Acrasidae and Dictyostelidae. Important genera of Acrasiomycetes are Dictyostelium (Fig 5.1 A), Polysphondylium, Protostelium (Fig. 5.1 B-C) and Acrasis.

Spore Apophysis

Sorus

C

Stalk

Disc A

Fig. 5.1

5.3

B

D

A-D. Some Acrasiomycetes and Hydromyxomycetes. (A, Dictyostelium; B-C, Protostelium; D, Labyrinthula)

HYDROMYXOMYCETES

5.3.1 Characteristic Features 1. Most of the representatives are aquatic, and occur parasitically on many marine algae and angiosperms like Zostera and Spartina. 2. The thallus consists of a network of branched tubes called filoplasmodium or net-plasmodium. 3. Many spindle-shaped cells remain arranged in the form of colonies. 4. Labyrinthula macrocystis is found in the form of spindle-shaped cells having terminal branched pseudopodia. It occurs in the air spaces of Zostera marina. 5. Cells show typical eukaryotic structure (Stey, 1968), but also contain an unusual structure called bothrasome (Porter, 1969) or sagenetosome (Perkins, 1974).

Myxomycota

41

6. Reproduction takes place by biflagellate zoospores. Labyrinthula reproduces also by cyst formation and congregation. Important genera of Hydromyxomycetes are Labyrinthula (Fig. 5.1 D) and Labyrinthuloides.

5.4

MYXOMYCETES

The Myxomycetes are commonly known as ‘true slime moulds’ or ‘Plasmodial slime moulds’. Professor Constantine J. Alexopoulos, because of his valuable and monumental contributions (Alexopoulos, 1960, 1962, 1963, 1966, 1973, 1978; Gray and Alexopoulos, 1968; Martin and Alexopoulos, 1969; Alexopoulos and Mims, 1979) may rightly be called the Father of Modern Myxomycetes. Ainsworth (1973) treated Myxomycetes as a class of division Myxomycota whereas Alexopoulos and Mims (1979) considered it as a class of subdivision Plasmodiogymnomycotina of division Gymnomycota. Myxomycetes is one of the three classes of phylum Myxomycota of Kingdom Protozoa. The other two classes included under Myxomycota are Dictyosteliomycetes and Protosteliomycetes (Kirk et al. 2001). The term ‘Myxomycetes’ (Gr. myxa, slime; myketes, mushrooms or fungi) was first used by American scientist Thomas H. Macbride (1899). Inspite of various treatments of slime moulds along with animals (De Bary, 1887; Lister, 1925; Olive, 1970) as well as plants (Macbride, 1899; Martin, 1932, 1949, 1960; Smith, 1955), Alexopoulos and Mims (1979) mentioned that ‘the true relationships of the Myxomycetes continue to remain obscure’. Some notable contributions on the Indian Myxomycetes have been made by Thind (1977), Singh et. al., (1979), Shekhon and Thind (1980), Lakhanpal and Mukherji (1981), Lakhanpal and Sood (1981) and Nanir (1984, 1985). Thind (1977) has authored “The Myxomycetes of India” while Lakhanpal and Mukherji (1981) have written Indian Myxomycetes. Myxomycetes: A Handbook of Slime Mould’s has been authored by Stephenson and Stemphen (1994).

5.4.1 Characteristic Features 1. Myxomycetes occur commonly in cool and moist shady places on decaying woods, leaves, animal dung, plant debris and other organic substrata. 2. They are represented by about 450 species, the majority of which are universally distributed (Alexopoulos & Mims, 1979). Kirk et al. (2001), however, mentioned that class Myxomycetes of phylum Myxomycota contains 5 orders, 13 families, 62 genera and 798 species. 3. The vegetative phase is in the form of a free-living plasmodium, which is simply a multinucleate, naked and acellular mass of protoplasm. 4. The plasmodium in different members may be a very small, microscopic body called aphanoplasmodium, or consists of networks or large sheets called phaneroplasmodium. 5. The plasmodial protoplasm shows a rythmic reversible streaming. 6. Under favourable conditions of moisture and temperature etc., the plasmodium gives rise to sporophore or fructifications of different shape. 7. A specialized layer (hypothallus) is deposited by the plasmodium just beneath the developing sporophores. 8. The hypothallus may be membranous, spongy, horny or disc-like in form. 9. Wind helps in spore dispersal. 10. Spore germination results into the formation of myxamoebae or zoospores. 11. The zoospores are uniflagellate or biflagellate. 12. The flagella are generally anteriorly attached and of whiplash type. 13. Asexual reproduction of plasmodium and myxamoebae may be by fragmentation and fission. 14. At the time of sexual reproduction the zoospores or myxamoebae may fuse to form zygotes. 15. The development of zygote results into the formation of plasmodia.

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5.4.2 Classification of Myxomycetes Alexopoulos (1973) divided Myxomycetes into three sub-classes: 1. Ceratiomyxomycetidae: Spores borne singly and externally on the individual stalks. 2. Myxogastromycetidae: Spores borne in large masses and internally in sporophores; development of sporophore is myxogastroid. 3. Stemonitomycetidae: Spores borne in large masses and internally in sporophores; development of sporophore stemonitoid. Ceratiomyxomycetidae is a very small subclass, consisting of only one order (Ceratiomyxales), one family (Ceratiomyxaceae) and only one genus (Ceratiomyxa), represented by only three species. The organisms included in the remaining two subclasses (Myxogastromycetidae and Stemonitomycetidae) are commonly known as Myxomycetes. Members of both these subclasses contain endospores, which develop within a fruiting body or fructification. A thin membrane (peridium) usually encloses the spores. Alexopoulos and Mims (1979) called them ‘endosporous Myxomycetes’. Myxogastromycetidae includes four orders (Echinosteliales, Liceales, Physarales and Trichiales) whereas Stemonitomycetidae includes only one order, i.e. Stemoninales. Kirk et al. (2001) included 5 orders in class Myxomycetes. These are Echinosteliales, Liceales, Physarales, Stemonitales and Trichiales.

5.5

LIFE CYCLE OF A TYPICAL MYXOMYCETE

The life-cycle pattern of a typical Myxomycete (Fig.5.2 A-K) includes the stages like spores, myxamoebae, zoospores, zygote, plasmodia and sporophores. The haploid spores germinate and release either myxamoebae or flagellated zoospores. Myxamoebae fuse in pairs. The stages of myxamoebae and zoospores are inter-convertible (Fig. 5.2 A-C). After copulation, karyogamy takes place and both these stages result into the formation of diploid zygote. The diploid zygotic nucleus undergoes many mitotic divisions to form a multinucleate plasmodium having diploid nuclei. The plasmodium changes itself into one or more fruiting bodies or sporophores. Meiosis takes place at this stage. Many uninucleate haploid spores are formed, and thus the life-cycle is completed. Although, many details are still unknown, a brief description of all these stages is given below: The spores (Fig. 5.2 A) are unicellular, usually globose structures surrounded by a thick wall. They are haploid bodies formed after meiosis. In a majority of the species the cell wall is spiny (Fig. 5.3 A), warty (Fig. 5.3 B), punctate or reticulate (Fig. 5.3 C), but in some genera the wall is smooth (Fig. 5.3 D) as in Badhamia. Much is still not known about the chemical composition of the cell wall of most Myxomycetes. Some contradictory reports suggest the presence cellulose or chitin. Studies of McCormick et al. (1970) suggest that the chemical composition of the spore walls of Physarum polycephalum is absolutely different from the walls of both fungi and Protozoa. The spore wall is mostly bilayered as in Didymium nigripes but is trilayered in Physarum gyrosum. The wall is variously coloured but in some species it is hyaline. The coloured spores may be yellow, grey, purple, violet, brown or even black in different species. Various cell organelles like mitochondria, Golgi-bodies, ribosomes, vacuoles, endoplasmic reticulum and centrioles have also been observed with the help of electron microscope. The spores are generally uninucleate, but in some species 2-8 nuclei are also present (Gray and Alexopoulos, 1968). The spores germinate either by the formation of a pore, or by the splitting or cracking of the spore wall (Fig. 5.4 A). Water, temperature, pH of the medium, thickness of wall and its chemical composition are some of the factors responsible for spore germination. The spores germinate either into one or more flagellated swarm cells (zoospores, Fig. 5.4 B) or non-flagellated myxamoebae (Fig. 5.4 C). The zoospores are generally biflagellate bodies having both the flagella of unequal length (Fig. 5.4 B). In many cases, however, uniflagellate cells are also seen (Cohen, 1959). Haskins (1973) reported 3-4 or more flagella in a few cases. In the biflagellate condition, both the flagella are of whiplash type and

43

Myxomycota

Mature spore (n)

Germinating spore (n) A Mature sporangium B Myxamoeba (n) Swarm cell(n)

K

MEIOSIS

C

PL

Young sporangium (2n)

C1

PL

O

O PH

PH

AS

E

AS

E

Myxamoebae

J Sporulation Sclerotium

D

PLASMOGAMY

DI

HA

Swarm cells D1

I KARYOGAMY

H

Young plasmodium (2n) G

E Zygote (2n)

Mature plasmodium (2n) F

Fig. 5.2

A–K. Sequence of events in the life-cycle of a typical myxomycete (diagrammatic)

show 9 + 2 arrangement of fibrils (Aldrich, 1968). According to Ross (1957) the zoospores may be ‘briefly flagellate’ (for 1-5 hr), ‘flagellate’ (for a few to several days) or ‘completely flagellate’ (for many weeks) in different species. In many cases the flagella are not developed on the germinated naked protoplasts of the spore. These are called myxamoebae. The stages of myxamoebae and zoospores are readily interconvertible. Both feed on bacteria, fungal spores, yeasts, organic matter, etc. Because of the sticky nature, the posterior end of swarm cell is important for catching the food (Koevenig, 1961). In many Myxomycetes, the myxamoebae either develop flagella after some time and change into zoospores, or they divide and redivide to form many smaller haploid myxamoebae. On the contrary, a zoospore may change into a myxamoeba by withdrawing its flagella.

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Fungi and Allied Microbes

Spiny spore

A

Fig. 5.3

Warty spore

Reticulate spore

B

Smooth spore

C

D

Spore types of Myxomycetes. A, Spiny; B, Warty; C, Reticulate; D, Smooth.

Both the stages, i.e., flagellated zoospores or non-flagellated myxamoebae, may function as gametes (Alexopoulos and Mims, 1979; Webster, 1980). Sexual fusion has been observed only in some of the Myxomycetes. Most of the investigated species are homothallic. According to Winstead (1970) some species are also heterothallic, but according to Therrien, et al. (1977) a few species (Didymium iridis) are homothallic, heterothallic as well as apogamic. Fusion takes place either between two zoospores (Fig. 5.5 A) or between two myxamoebae (Fig. 5.5). The zygote is formed after the processes of plasmogamy and karyogamy. For some time the zygotes bear two sets of flagella and keep on moving. According to Alexopoulos and Mims (1979) two compatible cells first come in contact with each other and keep on moving for several hours before the actual fusion of their haploid nuclei. The zygote (Fig. 5.2 E) keeps on moving for some time. Its diploid nucleus undergoes repeated mitotic divisions resulting into a multinucleate amoeboid structure. The zygote thus transforms into a plasmodium. According to Alexopoulos and Mims (1979) these mitotic divisions do not involve the centrioles, and are therefore intranuclear or closed.

Spore wall Uniflagellate swarmer

Biflagellate swarmer A B Pseudopodium Myxamoebae

C

Fig. 5.4

Germination of spore in Physarum polycephalum. A, Splitting of the spore wall; B, Uniflagellate and biflagellate swarmers; C, Myxamoebae.

Plasmodium is an amoeboid, multiFusing nucleate and naked mass of protoplasm (Fig. 5.2) having many swarmers diploid nuclei (Therrien, 1966). It is irregular in shape, and Fusing myxamoebae B A remains surrounded by a thin plasma membrane and a gelatinous sheath. This ever-changing and ever-flowing multinucleate body continues to feed on bacteria, myxamoebae and other fungal cells, etc. Mature plasmodia organise themselves into fan-shaped network of veins or channels, and develop into Fig. 5.5 A, Fusion between two zoospores of Reticfructification of different shapes and structures in different ularia lycoperdon; B, Fusion between two species. According to Indira (1964), diploid zoospores or myxmyxamoebae of Physarum polycephalum. amoebae may also develop from a mature plasmodium, but according to Webster (1980) this needs confirmation. The plasmodia are generally yellow or white. But many species possess plasmodia of different colours, ranging from white, grey, violet, black, green, blue, orange to even red (Alexopoulos and Mims, 1979). This colour is because of the

Myxomycota

45

presence of some pigments like pteridines, flavones, polyenes and polypeptides, but much is not known about these pigments. The function of the plasmodial pigments is also not known. The plasmodium is naked because it has no cell wall. However, recent electron microscopic studies have shown the presence of a gelatinous sheath consisting of microfibrils. The plasmodial protoplast also contains many granules, vacuoles and other cell organelles. The protoplasmic granules keep on moving with a great speed in the veins of the mature plasmodium. According to Alexopoulos and Mims (1979) the protoplasm first flows from one direction of the plasmodium, comes to a momentary stop in another side after about 50 or 60 sec. and then begins to flow in another direction. Detailed studies on the protoplasmic streaming in Myxomycetes have been made by Gray and Alexopoulos (1968). Alexopoulos and Mims (1979) recognized the following four types of plasmodia: It is a primitive type of microscopic and homogeneous plasmodium with no veins. It does not show rhythmically reversible streaming of protoplasm. A protoplasmodium developes into a single sporangium. Protoplasmodia are characteristics of Echinosteliales. The aphanoplasmodia are inconspicuous, flat and transparent structures having a thin open network of plasmodial strands. They show rhythmically reversible streaming of protoplasm. In these plasmodia the gelified and fluid regions of the veins are not conspicuously differentiated. Aphanoplasmodia are found in Stemonitomycetidae. Such plasmodia are very large, massive, fan-shaped, reticulate structures. They are yellow and contain very granular protoplasm. The gelified and fluid regions of the veins are conspicuously differentiated. They show very clear rhythmically reversible streaming of protoplasm. Phaneroplasmodia are characteristics of Physarales. Rammeloo (1976) observed in some Trichiales a fourth type of plasmodium, which shows some characters of aphanoplasmodium and some of phaneroplasmodium. Much is not known about such plasmodia. Normally the plasmodium sporulate into fruiting bodies. But if the conditions are not suitable for sporulation, the plasmodium may give rise to sclerotia (Fig. 5.2 H). These are dark, hard, irregular and horny structures. According to Jump (1954) the cytoplasm of the sclerotia is divided into many multinucleate cells called macrocysts. Alexopoulos and Mims (1979) named them spherules. The sclerotia develop into fresh plasmodia on return of favourable conditions. The entire plasmodium gets converted into one or more fruiting bodies. The important factors responsible for sporulation are light, temperature, moisture and pH. In nature, the sporulation mostly occurs at night. The following types of the fruiting bodies or sporophores are produced by the Myxomycetes: Generally numerous sporangia are produced from a plasmodium. In a few cases, however, only one sporangium is produced. A sporangium may be sessile or stalked, globose, cylindrical or cup-shaped (Fig. 5.6 A-G). At the base of each sporangium persists a thin cellophane like, membranous sheath, called hypothallus. Each sporangium contains an outer layer, called peridium. Structures like columella and capillitia are also present in some. Spores develop in the sporangium. A plasmodiocarp retains the branching habit of the plasmodium. It develops by the concentration of the protoplasm around some of the prominent veins of the plasmodium, as in Hemitrichia. It resembles sessile sporangium. These are the cushion-shaped fruiting bodies of some of the Myxomycetes. An aethalium is a group of sporangia which have not separated individually, whereas others consider that several sporangia fuse and form an aethalium. The aethalia in some Myxomycetes (Reticularia lycoperdon) attain the size of a hen’s egg. The aethalia of Fuligo septica are the largest sporophores of any Myxomycete(Sunhede, 1974). In some cases many sporangia grow very close to each other and the fruiting body appears like that of a single sporophore. Such false aethalia are called pseudoaethalia.

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Fungi and Allied Microbes

A

B

E

C F G D

Fig. 5.6

Sporangia of some Myxomycetes. A, Physarum; B, Arcyria; C, Dictydium; D, Undehisced sporangia of Trichia; E, Dehisced sporangium of Trichia; F, Sporangium of Comatrichia; G, Sporangium of Stemonitis.

Various parts of a sporangium may be a plasmodial sheath or hypothallus, stalk, peridium, columella, pseudocolumella, capillitium, pseudocapillitium and spores. However, all these parts are not present in the sporophore of all the species or genera. Some lack stalk, others lack columella, still others the pseudocolumella, and so on. Hypothallus is thin, transparent, cellophane-like layer or crust-like calcareous deposits at the base of the sporophore. Stalk may or may not be present in the sporangia. If present, it varies in height, colour, surface structure etc. A B C Peridium is the outer membranous covering or wall of the sporangium. It is calcareous in some but proteinaceous in others. However, Fig. 5.7 A–C, Some basic types of capillitia. much is still not known about its chemical composition. Columella is either the part of the stalk extended into the spore-containing body, or it develops from the base of sporangial sac. Pseudocolumella is a calcareous rod, present in the centre of the spore-containing body. Capillitia are sterile, uniform, thread-like structures found intermingled with the spores. Some basic types of capillitia are shown in Fig.5.7 A-C.

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Myxomycota

Pseudocapillitia are sterile but irregular, thread-like or plate-like structures found intermingled with spores. At the time of spore formation the vacuoles or vesicles of the sporangium fuse and form a vacuolar network. This results in cleavage of sporangial protoplasm. Many irregular and uninucleate masses of protoplasm are ultimately formed. Some wall material gets deposited around such irregular masses, which function as spores (Bechtel, 1977). Spines, warts or other wall ornamentations develop but their exact mode of formation is not fully known. The young spores are therefore uninucleate and diploid. Position of meiosis is different in different members. According to some workers the plasmodial nuclei are diploid, whereas the spores are haploid bodies. But according to Aldrich and Carroll (1972) meiosis takes place 12-24 hrs after cleavage. Alexopoulos and Mims (1979) mentioned that meiosis occurs 18-30 hrs after cleavage, resulting into a quadrinucleate stage. Three of the four nuclei abort, ultimately forming a uninucleate haploid spore. The haploid spores so formed are again able to germinate into myxamoebae, and thus the life-cycle is completed.

5.6

STEMONITIS

Stemonitis belongs to class Myxomycetes, order Stemonitales and family Stemonitidaceae. This family contains 16 genera and 197 species (Kirk et al.2001). The plasmodium of (Fig. 5.8 A,B) Stemonitis is a typical aphanoplasmodium. At maturity it is branched and show a network of very fine, transparent strands. Its protoplasm is not very granular and its veins are not clearly differentiated. Rapid and rhythmically reversible streaming is shown by the protoplasm of the plasmodium.

Aphanoplasmodium

Spores Capillitium Sporangium

B Sporangia

Stalk A

Fig. 5.8

C

D

A–D. Some stages of the life-cycle of Stemonitis. A, A part of plasmodium; B, Spores and capillitium; C, A bunch of sporangia attached to the substratum; D, A single sporangium.

Immediately prior to fruiting, the plasmodium starts becoming flat having many fan-shaped structures (Fig. 5.8C). Many sporangia start developing soon under favourable conditions. Each sporangium (Fig. 5.8D) is a cylindrical to fasciculate, stalked and fan-shaped body. Columella is seen throughout the length the sporangium. Numerous threads radiating from all parts of columella form the capillitium. These threads combine into a loose network. Their ultimate branches unite to form a superficial net attached to the sporangial walls. If lime is present, it is never on the capillitium. Lime is confined mainly to the stalk, columella, hypothallus and sometimes also to the base of the peridium. Spores are

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Fungi and Allied Microbes

violet-brown in colour. Each spore starts germinating into 1-4 naked myxamoebae or swarm cells. The myxamoebae fuse in pairs to form diploid zygotes. The zygote develops into a plasmodium after nuclear divisions and subsequent growth. The exact stage of the occurrence of meiosis is not clearly known but it perhaps occurs during spore formation in the sporangium.

5.7

PLASMODIOPHOROMYCETES

5.7.1 Characteristic Features 1. Members are obligate endoparasites, attacking many plants of economic importance like cabbage and potato. Some species also attack many aquatic pteridophytes (Isoetes), angiosperms (Juncus), some algae (Vaucheria) as well as some fungi (Saprolegnia and Pythium). 2. The infection results into the hypertrophy (abnormal enlargement of host cells) and hyperplasia (abnormal multiplication of host cells) in the host. Disruption in the vascular elements of host results into its general stunting. 3. A characteristic cruciform-type of nuclear division is seen only in Plasmodiophoromycetes and in no other fungi ( Braselton et al., 1975; Alexopoulos and Mims, 1979). 4. The life-cycle includes two distinct plasmodial phases. 5. The plasmodium is parasitic within the cells of the host plant. 6. Biflagellate zoospores contain flagella of unequal length. The flagella are anterior and of whiplash type. 7. The first plasmodial phase of the life-cycle is a zoosporangial plasmodium. 8. The second plasmodial phase gives rise to resting spores. 9. The wall of the resting spores contain either chitin or cellulose. 10. In some species sexual fusion is observed before the development of resting spore plasmodium.

5.7.2 Classification Plasmodiophoromycetes contains only one order (Plasmodiophorales) and one family (Plasmodiophoraceae) according to Karling (1968). Waterhouse (1973) recognized 9 genera and one doubtful genus (Membranosorus) in this class i.e. Woronina, Plasmodiophora, Tetramyxa, Sorosphaera, Spongospora, Ligniera, Sorodiscus, Polymyxa and Octomyxa. Fraser and Buczacki (1983) studied the taxonomic affinities of three members (Plasmodiophora, Sorosphaera and Spongospora) of Plasmodiophorales by measuring molecular weight of their ribosomal ribonucleic acids. They compared their data with that of the representatives of other fungal groups and suggested that ‘Plasmodiophorales are quite distinct from Myxomycetes, Acrasiales, Trichomycetes, Oomycetes, Ascomycetes and Basidiomycetes’. Kirk et al. (2001) treated Plasmodiophoromycetes under phylum Plasmodiophoromycota of kingdom Protozoa. According to these workers Class Plasmodiophoromycetes includes only 1 order (Plasmodiophorales), 2 families (Plasmodiophoraceae and Endemosarcaceae), 15 genera and 47 species.

5.7.3 Still-Unsolved Questions Life-cycle in Plasmodiophoromycetes has always been and still is a much debated point. It is not yet fully solved because different workers have suggested the position of fusion and meiosis in the life-cycle at different stages, i.e.(i) between primary zoospores or amoebae, and (ii) between secondary zoospores or amoebae (Waterhouse, 1973). Some other stillunanswered questions of the life-cycle of Plasmodiophoromycetes are: 1. Origin of cystogenous plasmodia. 2. Nuclear condition of cystogenous plasmodia. 3. Exact structural differences between cystogenous plasmodia and sporangiogenous plasmodia.

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Myxomycota

4. Possible role of environment on the structure of plasmodia. 5. Confirmation of the haploid nature of sporangiogenous plasmodia and diploid nature of cystogenous plasmodia. 6. Whether or not the cyst formation is preceded by meiosis? Detailed discussion of the possible life-cycle of Plasmodiophora brassicae is given below.

5.8 5.8.1

PLASMODIOPHORA BRASSICAE Systematic Position

According to Ainsworth (1973) Division Class Order Family

5.8.2

– – – –

Myxomycota Plasmodiophoromycetes Plasmodiophorales Plasmodiophoraceae

According to Kirk et al. (2001) Kingdom Phylum Class Order Family

– – – – –

Protozoa Plasmodiophoromycota Plasmodiophoromycetes Plasmodiophorales Plamodiophoraceae

Occurrence

Plasmodiophora brassicae is an obligate endoparasite in the roots of Cruciferaceous plants, attacking both cultivated as well as wild members. It causes diseases like club-root disease or finger-and-toe disease of brassicas. The disease is of universal occurrence. Commonly attacked plants include cabbage, rape, mustard, turnip, etc.

5.8.3

Symptoms of Club Root Disease

It is very difficult to distinguish between the infected and healthy plants if their above-ground parts are observed. The infected plants show following symptoms: 1. Wilting of the leaves is the first symptom. 2. Plants appear yellow, stunted and show retardation in growth. 3. Roots are hypertrophied and sometimes become 10-12 times enlarged (Fig. 5.9) to form club-shaped malformations. 4. Infected root hairs are also hypertrophied. Their tips are also expanded and form club-shaped swellings. In severe infection the expanded tips of root hairs become lobed and branched. Structures like root buds are also formed.

5.8.4

Infected swollen roots

Fig. 5.9

Club root disease of cabbage caused by Plasmodiophora brassicae (after Woronin, 1878)

Life Cycle

All details of the life-cycle of Plasmodiophora brassicae are not yet clear. Tommerup and Ingram (1971) studied its life-cycle in callus tissue culture in Brassica napus, and also confirmed their tissue-culture studies with separate studies on clubbed roots in soil. Their interpretations of the possible life-cycle have been accepted by Webster (1980) and are mentioned below and presented in Figures. 5.10 and 5.16. 1. The enlarged swollen clubbed roots of the infected plants contain many spherical haploid spores, called resting spores. Electron microscopic studies of Williams and McNabola (1967) suggest that these resting spores contain spiny walls, and are hyaline and uninucleate (Fig. 5.11 A).

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Fungi and Allied Microbes

Primary plasmodium 2. Host roots get decayed and the resting spores are (multinucleate) released into the soil (Fig. 5.11 A). Germination of Plasmogamy of Zoosporangia resting spores starts in situ. protoplasts (multinucleate) 3. Each liberated resting spore swells and releases a Infection to host Zoospores uninucleate and biflagellate zoospore on germination (uninucleate) (Fig. 5.11 B, C). Germination Plasmogamy 4. Each zoospore (Fig. 5.11 C) is uninucleate and naked mass having two flagella of unequal length and whip- Resting spore (n) Plasmodium (n) lash type. Earlier it was thought that zoospores are (binucleate) uniflagellate bodies but electron microscopic studies Meiosis of Kole and Gielink (1962) have confirmed the presSecondary plasmodium (n) ence of two flagella. The flagella show the usual 9 + (multinucleate) Zygotic 2 arrangement of fibrils. nuclei (2n) 5. The zoospores penetrate the root hairs and the young Karyogamy epidermal cells of the host. Process of the zoospore penetration of the cabbage Fig. 5.10 Life-cycle of Plasmodiophora brassicae root hair has been described by Aist and Williams in callus tissue culture (redrawn after (1971). According to them a cyst zoospore gets atTommerup and Ingram, 1971) tached to the root-hair wall of the host. It is soon followed by the inactivation of its flagella, retraction of Zoospore Resting Germinating axonemes and encystment of the zoospore. A tubular spore spore cavity (rohr) appears within the encysted zoospore (Fig. 5.12 A). Webster (1980) used the term tube for rohr. A light staining plug fills the end of the rohr towards the wall of the host. A dark-staining rod stachel or stylet (Webster, 1980) is present within the tubular A B C cavity or rohr. Rohr invaginates and forms a bulbous structure (Fig. 5.12 B). The latter adheres to the cell Fig. 5.11 Plasmodiophora brassicae. A, A resting wall of the host with the help of adhesorium. Simulspore; B, Germination of a resting spore; taneously, the stachel punctures the host cell wall and C, A zoospore (after Karling, 1942) through this the protoplast of the zoospore enters into the host cell (Fig. 5.12 C). The zoospore protoplast is spherical and amoeboid in the host cell. These amoeboid zoospores are called myxamoebae. Many such uninucleate, amoeboid protoplasts of the parasite are thus released into the infected cell of the host.

Zoospore Lipid Cyst vacuole Sac Stylet Adhesive material Tube Plug Host wall A

Fig. 5.12

Zoospore

Stylet

Adhesorium

Adhesorium Adhesive material Host wall B

Stylet Host wall C

Plasmodiophora brassicae, showing the penetration process of zoospore into the root hair wall of cabbage (after Aist and Williams, 1971)

Myxomycota

51

6. Plasmogamy of these haploid amoeboid protoplasts results into Fusing the formation of multinucleate primary plasmodia (Fig. 5.10). gametes Alexopoulos and Mims (1979) called them sporangiogenous plasmodia. During this phase the nuclear division is cruciform, which is a characteristic feature of Plasmodiophoromycetes. 7. Cleavage of multinucleate primary plasmodia takes place and results into the formation of zoosporangia. These zoosporanGametes gia are thin-walled and multinucleate and may form within 4 A B C days of infection. 8. In each zoosporangium develop many uninucleate and haploid Fig. 5.13 A, Zoospores now behaving zoospores (Fig.5.10). as gametes; B, Copulation; C, 9. At the time of the liberation of zoospores, the zoosporangium Plasmogamy (based on Karling) gets attached to the cell wall of the host. A pore develops beHypertrophied Plasmodia tween the zoosporangium and the cell wall, and through this pore host cell the zoospores are liberated. According to Webster (1980) the behaviour and the exact nature of these ‘released zoospores is not known but it is possible that they function as gametes and fuse in pairs’. 10. Two zoospores, which now perhaps behave as gametes, come together and undergo plasmogamy (Fig. 5.13 A, B). According to Tommerup and Ingram (1971) the plasmogamy is not immediately followed by karyogamy. So the zoospores remain binucleate (Fig. 5.13). 11. Binucleate zoospores are released, and are able to reinfect the roots, where they develop into binuclete plasmodia. Some workers have used the words ‘sexual reproduction’ for their fusion and ‘zygote’ for their fusion product. 12. The binucleate plasmodia are able to penetrate deep into the root cortex. But the details of their exact mode of penetration and transference up to the deep cortex still need more investigations. Fig. 5.14 T.S. of young cabbage root Specialized feeding structures like haustoria are also not known infected with Plasmodioin the plasmodia. phora brassicae. Note the plasmodia in the cortex and 13. Each of the binucleate plasmodium enlarges and undergo rehypertrophy of the host cells peated mitotic nuclear divisions to form a multinucleate body. It is called the multinucleate secondary plasmodium. So far Resting spores(n) there is no karyogamy. Many haploid nuclei are present in Nuclei (n) such plasmodia. 14. The host cells containing such enlarged multinucleate plasmodia get hypertrophied (Fig. 5.14). Webster (1980) mentioned that the multinucleate secondary plasmodia may penetrate in the medullary ray cells and even in the vascular cambium of Host cell Mature plasmodium the host. A B 15. The haploid nuclei of these multinucleate secondary plasmodia get associated in pairs. Plasmodiophora brassicae. A, 16. Karyogamy takes place at this stage and many zygotic nuclei Fig. 5.15 A plasmodium after meiosis; B, are formed. Buczacki and Moxham (1980) and Braselton and Resting spores within a host cell Short (1985) worked on the karyogamy of Plasmodiophora (based on Karling, 1942) brassicae. According to Braselton and Short (1985) the hap-

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loid chromosome number in P.diplantherae is 9, which is the lowest haploid number for Plasmodiophoromycetes. In P.brassicae it is N = 20. 17. Meiosis takes place soon after the formation of diploid zygotic nuclei. 18. The mature plasmodia now contain many haploid nuclei (Fig. 5.15 A). Soon the cleavage of the cytoplasm takes place and many haploid resting spores are formed within the host cell (Fig. 5.15 B). On being liberated these resting spores are able to reinfect the host. Thus, the life-cycle is completed, the different stages of which are depicted in Fig. 5.16. Resting spores masses

B Germinating A resting spore Resting spore Primary zoospore P

Karyogamy, meiosis Multinucleate plasmodium

Zoospore Root hair surface

C Attachment of zoospore to root hair

D

Adhesorium Zoospore cyst E Entry into root hair F

O

Amoeba In root cortex

Hypertrophied cell of cortex

In soil

Root hair

In root hairs

Zoosporangial plasmodium (primary plasmodium) Enlarging plasmodia

N

G Root hair Young zoosporangia

Cortical cell

H

Young binucleate plasmodia

Mature and discharged zoosporangia

M I J Released secondary Quadriflagellate Fusing gametes zoospores (2) zygote (plasmogamy) L

K

Fig. 5.16 A-P, Different stages of life-cycle of Plasmodiophora brassicae

Myxomycota

53

TEST YOUR UNDERSTANDING 1. Slime moulds are kept under _______ . 2. Ainsworth (1973) divided Myxomycota into Acrasiomycetes, Hydromyxomycetes, Myxomycetes and _______ . 3. Myxomycetes are commonly known as _______ . 4. Write at least five characteristic features of Myxomycetes. 5. Draw the pictorial life-cycle of a typical myxomycete. 6. Explain the following terms: (a) Protoplasmodium (b) Phaneroplasmodium (c) Aphanoplasmodium 7. Write a note on the life-cycle of Stemonites. 8. Club-root disease is caused by _______ . 9. Draw different stages of the life-cycle of Plasmodiophora brassicae.

6

C H A P

EUMYCOTA

T E R

6.1

WHAT ARE EUMYCOTA?

Eumycota are commonly called true–fungi. The terms Eumycota, Eumycophyta or Eumycetes have been used for fungi by different workers. Kirk et al. (2001), however, did not use the word Eumycota, and discussed all fungi under three independent kingdoms (viz, Chromista, Fungi, and Protozoa). They further mentioned that “some authors use Eumycota as the kingdom name which has the advantage of avoiding confusion with fungi” (e.g. Barr,1992). Ainsworth (1973), however, divided all fungi into two divisions, viz. Myxomycota (presence of plasmodium) and Eumycota (absence of plasmodium), and since Ainsworth’s classification has been followed in this book, the details mentioned in this chapter follow Ainsworth (1973).

6.2

CHARACTERISTIC FEATURES

Eumycota are unicellular or filamentous fungi which do not possess plasmodia or pseudoplasmodia. Most of the fungi belong to Eumycota. According to Fuller and Tippo (1954) there are approximately 75,000 known species of true fungi, but this number is now much larger because of the discovery of many new records in different parts of the world during the last six decades. All Eumycota have a definite cell wall throughout their somatic phase. They are heterotrophic in their mode of nutrition. Some are saprophytic and others are parasitic. Photosynthetic pigments are absent. Parasitic Eumycota attack both plants as well as animals. The plants parasitized by these fungi may range from simplest algae to most highly advanced angiosperms. The plant body consists of colourless filaments, called hyphae. However, variously coloured hyphae are also present. The colour is owing to the presence of some pigments. The mass of hyphae is called mycelium. Most of the true fungi thrive only in the presence of free oxygen, and they are therefore aerobic. However, a few are also facultative anaerobes. Some types of spores are present in all Eumycota. In a majority of genera the spores are present within sporangia of different shape. Many fungal spores are the common contaminants of our daily food as well as environment. They are also responsible for a large number of common as well as dangerous diseases of other plants and animals, including man. True fungi are harmful as well as beneficial to the human life.

6.3

OCCURRENCE

Eumycota occur universally. They are found at almost all places where some organic material, living or dead, is present. A majority of them are aerobic, but some are also facultative anaerobes. True fungi occur in or on the soil, air, water, rocks,

Eumycota

55

dead as well as living plants and animals, on all types of foodstuffs like fruits, bread, jams, pickles, clothes, timbers, plastics and many other similar materials. Many Eumycota are even coprophilous, i.e. found on animal dung. Piontelli et al. (1981) recorded 1267 fungi from the faecal samples of 60 horses and Blackwell and Rossi (1986) reported 20 fungi growing ectoparasitically on termites. Rao and Manoharachary (1985) made an interesting study of pollen fungal associationship. On the pollen grains of 50 plants they reported 24 fungal species, mainly belonging to Alternaria, Aspergillus, Curvularia, Fusarium, Penicillium, Phoma and Rhizopus. Through this study they showed that ‘pollens can be a suitable habitat for fungal colonization’. Kost (1984) reported some Basidiomycetes on mosses. Hundreds of eumycotaceous fungi have also been reported growing endophytically from the 36 tropical plants belonging to Pteridophyta, Araceae, Bromeliaceae, Orchidaceae and Piperaceae (Drefuss and Petrini, 1984).

6.4

VEGETATIVE PLANT BODY

In most of the Eumycota the thallus is differentiated into a vegetative part and a reproductive part. Such fungi are called eucarpic (Gr. eu, good; karpos, fruit). But in some fungi (Synchytrium) there is no such differentiation, and the entire vegetative thallus gets converted into one or more reproductive structures. Such fungi are called holocarpic (Gr. holos, whole; karpos, fruit). The vegetative filaments of eumycotous fungi are called hyphae (Gr. hyphe, web), and the collective mass of hyphae constituting the thallus or body of a fungus is called mycelium (Gr. mykes, fungus or mushroom). The mycelium may attain a length from a few microns to many metres in different members. The hyphae are non-septate in Oomycetes and Zygomycetes, but are septate in higher groups like Ascomycotina, Basidiomycotina and Deuteromycotina. Many nuclei are generally present in the non-septate hyphae, and such a condition is called coenocytic. Multicellular mycelium contains either uninucleate, binucleate or multinucleate cells. The mycelium containing genetically identical nuclei is called homokaryotic. But if it contains the nuclei of different genotypes it is called heterokaryotic. The cell containing a single haploid nucleus is called monokaryotic, whereas a cell containing two genetically distinct haploid nuclei is called dikaryotic.

6.5

BRANCHING

The branching in fungi is controlled by some genetic as well as external factors (Burnett, 1976). In majority of fungi the growth of fungal hyphae is monopodial. There is generally present a single main axis having the potentiality of unlimited growth. A branch develops behind the apex with a considerable distance. There is a clear indication of apical dominance in the hyphal growth mainly because a lateral branch generally does not develop close to the main apex. Except in a few genera (e.g., Allomyces), dichotomous branching is rare. In majority of Ascomycotina, Basidiomycotina and Deuteromycotina a lateral branch generally develops close to the septum.

6.6

FINE STRUCTURE OF A CELL OF EUMYCOTA

Detailed ultrastructural studies of the cells of Eumycota suggest that they are typically eukaryotic (Fig. 6.1). The chloroplasts are, however, absent. For details, refer chapter 3 (Fungal Cell: Structure and Composition).

6.7

HAUSTORIUM

The mycelium of parasitic Eumycota either grows in between the cells of the host (intercellular) or penetrates into them (intracellular). If the mycelium is intercellular the fungus absorbs its food with the help of some specialized structures,

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Fungi and Allied Microbes

Plasmalemma

Hyphal wall

Fig. 6.1

Vacuole

Endoplasmic reticulum

Mitochondrion

Ribosomes

Nucleus

A typical fungal cell based on electron micrograph (diagrammatic).

called haustoria (L. haustor, drinker). In a majority of the cases the haustoria are knob-like (Fig. 6.2 A). But in many genera these specialized organs may also be either elongated (Fig. 6.2 B) or well-branched (Fig. 6.2 C). For more details, refer Article 1.8 in Chapter 1. Haustorium

Hypha

Host cells Hyphae Haustoria

Host cell wall Host protoplasm A

Fig. 6.2

6.8

B

C

Types of haustoria. A, Knob-Iike; B, Elongated; C, Well-branched.

HYPHAL AGGREGATIONS

Alexopoulos and Mims (1979) mentioned that mycelium in most of the true fungi gets organized into loosely or compactly woven fungal tissues, called plectenchyma. The plectenchyma may again be of two types:

Prosenchyma

The hyphae remain arranged loosely but parallel to one another. They may therefore be clearly recognized (Fig. 6.3 A). The hyphae remain arranged compactly, and therefore loose their individuality. The hyphal cells are oval or isodiametric (Fig. 6.3 B).

Pseudoparenchyma A

Fig. 6.3

B

Fungal plectenchyma. A, Prosenchyma; B, Pseudoparenchyma.

57

Eumycota

However, Webster (1980) mentioned that the vegetative hyphae in different members may aggregate to form structures like mycelial strands, rhizomorphs, sclerotia etc., whereas reproductive structures may aggregate to form bodies like ascocarp in Ascomycotina, basidiocarp in Basidiomycotina, and synnemata or pycnidia in Deuteromycotina. A brief description of some of these aggregations is given below: These are actually such aggregations of fungal hyphae which are undifferentiated and have no well-developed apical meristem. The hyphae run more or less parallel to each other. Young hyphae of Agaricus bisporus, when developing on the food base, form the mycelial strands. The mycelial strands in Serpula lacrimans (Fig. 6.4) and some other species extend for several metres. Rhizomorph

The rhizomorphs (Gr. rhiza, root; morphe, shape) are thick strands of somatic hyphae having a well-developed apical meristem (Armillariella mellea). They are so named because their growing tip resembles that of root tip. In rhizomorphs the Fig. 6.5 L.S. of rhizomorph of hyphae loose their individuality. In a fully deArmillariella mellea veloped rhizomorph there is present a central core consisting of large, elongated. thin-walled cells. The central core remains surrounded by a rind or outer covering made up of small, thick-walled cells (Fig. 6.5). About 1 mm diameter of a rhizomorph may contain as many as 1000 or more hyphae. The rhizomorphs can survive for long under unfavourable conditions. In many genera, they help in spreading the fungus from one root system to another root system. A sclerotium (Gr. skleron, hard) is a hard body, made up of pseudoparenchymatous aggregations of hyphae (Fig. 6.6 AC). Because of its hard nature it is resistant to unfavourable conditions, and can survive for several years. Sclerotia are very common in genera like Claviceps, Sclerotinia and Thanatephorus. The size of the sclerotium varies from a few cells in some genera to as large as that of the size of man’s head (e.g. Polyporus mylittae, Webster, 1980). The sclerotia may germinate either with the help of the development of their mycelium, or by the development of conidia. or by the formation of basidiocarps or ascocarps. Sometimes the roots of many trees, having sheathing mycorrhizas, get surrounded by the aggregation of mycelium. Such an aggregation of mycelium is called mantle or sheath.

6.9

Hyphal strand

Fig. 6.4

Formation of mycelial strands in Serpula lacrimans.

Conidia Sclerotium

Sclerotia

A

Fig. 6.6

B

C

Sclerotium of Claviceps purpurea. A, Head of rye plant (Secale cereale) having sclerotia; B, A sclerotium; C, T.S. of young sclerotium.

HYPHAL AGGREGATIONS INTO REPRODUCTIVE STRUCTURES

Various types of reproductive structures are formed by the aggregation of hyphae in higher fungal groups such as Ascomycotina, Basidiomycotina and Deuteromycotina.

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Fungi and Allied Microbes

In Ascomycotina the characteristic spores (ascospores, plate 4) are formed in some cylindrical sacs or asci, which are grouped together in a characteristic fruiting body, called ascocarp. The following types of ascocarps are formed in Ascomycotina: A cleistothecium (Gr. kleistos, closed; theke, case) is a fruiting body which is closed from all sides. There is no special opening (Fig. 6.7 A, B). The cleistothecia are common in Erysiphales and Eurotiales. Appendages Cleistothecium

Cleistothecium

A

B Ascus Ascospores

Perithecium Apothecium

C

Pseudothecium

E

Fig. 6.7

D

Fruiting bodies of Ascomycotina. A, A cleistothecium of Erysiphe; B, A section through a cleistothecium of Erysiphe graminis; C, An apothecium of Ascobolus immersus; D, L.S. of perithecium of Sordaria fimicola; E, L.S. of Immature pseudothecium of Leptosphaera acuta.

59

Eumycota

An apothecium (Gr. apotheke, store house) is an open cup-shaped or saucer-shaped fruiting body of many Ascomycotina including Pezizales and Helotiales (Fig. 6.7C). A perithecium (Gr. peri, around; theke, a case) is a closed fruiting body having a pore or ostiole at the top. It has a wall of its own (Fig. 6.7 D). The asci in perithecia are unitunicate, i.e. they contain single ascus wall. Perithecia are common in Sphaeriales and Hypocreales. The perithecia with double ascus wall (i.e. bitunicate) are called pseudothecia. They are common in Loculoascomycetes (FIg. 6.7 E). In Basidiomycotina the characteristic spores (i.e. basidiopores) are produced on basidia. Many basidia are grouped in the form of fruiting bodies of many fungi such as mushrooms, bracket fungi, puff balls, etc. In Deuteromycotina the mycelial aggregation may result into the formation of many specialized structures like synnemata, pycnidia, etc.

6.10

VEGETATIVE REPRODUCTION

Eumycota reproduce vegetatively by fragmentation, fission, budding or even by formation of oidia. In this most common method of vegetative reproduction the vegetative plant body breaks into one or more fragments, each of which is capable to develop into a new individual. Sometimes, a somatic hypha gets fragmented into its component cells. Such thin-walled hyphal cells are called oidia. An oidium (G. oidion, small egg) normally behaves as a spore. In Coprinus lagopus the oidia develop on well-defined oidiophores (Fig. 6.8 A). In yeasts and some other unicellular true fungi, a vegetative cell splits into two cells (Fig. 6.8 B), each of which develops into individual cells. In budding, a small outgrowth or bud is produced from a parent cell (Fig. 6.8 C). Budding is of common occurrence in yeasts.

Water Oidium Oidiophore

Daughter cells

Cell

B Bud A

Bud

Daughter nuclei

Daughter cells

C

Fig. 6.8

A–C. Some methods of vegetative reproduction. A, Oidia and oidiophore of Coprinus lagopus; B, Fission in yeast; C, Budding in yeast.

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6.11

SOME ASEXUAL AND SEXUAL SPORES

All Eumycota contain some or other types of spores. Some of them are discussed below.

6.11.1

Zoospores

The zoospores (Gr. zoon, animal; spora, spore or seed) are motile asexual spores having one or more flagella. They may be haploid or diploid. According to Lange and Olson (1983) three essential features of a fungal zoospore are: (i) “The zoospore is formed inside a sporangium; (ii) the zoospore is discharged from the sporangium to become a free-swimming stage; (iii) after a motile period, the zoospore encysts on a suitable substrate or a host”. Therefore, a fungal zoospore “is the propagule for the spread of the organism” (Lange and Olson, 1983). According to these workers, true zoospores are formed in a zoosporangium, by mitotic nuclear divisions, and directly give rise to a vegetative thallus. The size of the zoospore is also variable as shown in the undermentioned table: Table 6.1 Size of fungal zoospore S. No. 1. 2. 3. 4. 5. 6.

Name of the Fungus

Size

Synchytrium endobioticum Olpidium brassicae O. radicale Sclerophthora Phytophthora (Photoplate-2A) Pythium

3 mm 4 mm 7-8 m m 9-12 m m 9-13 m m 6-16 m m

In majority of the uniflagellate as well as biflagellate fungi the flagellar length of the zoospore is, however, close to 20 m m. According to Webster (1980) Eumycota zoospores are of following three kinds: (i) Posteriorly uniflagellate zoospores having whiplash flagellum. (ii) Anteriorly uniflagellate zoospores having tinsel flagellum. (iii) Biflagellate zoospores. Sparrow (1960) divided biflagellate zoospores again into two categories, i.e., biflagellate heterokonts (i.e., having one flagellum of whiplash type and another of tinsel type) and biflagellate isokont (i.e., having both the flagella of whiplash type). Such type of zoospores are found in Chytridiomycetes. In Blastocladiella emersonii (Fig. 6.9 A) the tadpole-like zoospores have a pear-shaped head and a long flagellum. The head region contains a crescent shaped nuclear cap which surrounds the nucleus. The nuclear cap remains filled with ribosomes. A single mitochondrion is generally present. The kinetosome consists of nine fibrils, each consisting of a triplet of microtubules. These triplets remain arranged in the manner of a cart wheel. The main shaft of the flagellum is axoneme which has a typical 9 + 2 arrangement of flagellar microtubules. All the 11 microtubules remain surrounded by a flageller sheath. Some Gamma particles are also present at the anterior side of the zoospore. Cantino, et al. (1963), Hill (1969), Fuller (1966, 1976), Sparrow (1973) and Lange and Olson (1979, 1983) are some of the workers who have reviewed the fine structure of this kind of zoospores. Webster (1980) mentioned that such zoospores are found only in Hyphochytridiomycetes and no other fungi. Fuller (1966, 1976) studied the zoospores of Rhizidiomyces under electron microscope (Fig. 6.9 B). The axoneme of the single anteriorly placed flagellum bears a series of fine mastigonemes throughout its length. A mastigoneme has a wider basal shaft and a ter-

61

Eumycota

minal part. The terminal part is about one-fifth of the diameter of the basal shaft. The basal shaft also contains some transverse or spiral bands made up of alternating dark and light material. Two terminal extensions are present in some mastigonemes. The nucleus is anteriorly situated while the nuclear cap is not membrane bound. Many ribosomes are present near the posterior region of the nucleus. Mitochondria, microtubules, vacuoles, lipid granules, dictyosomes and endoplasmic reticulum are also present.

Nuclear cap Gamma particle Nucleus Ribosomes Nucleolus Head Mitochondrion

The zoospores having two flagella are characteristically found in Oomycetes. Out of the two flagella, one is of whiplash type while another is of tinsel type. Such zoospores are called heterokont. However, in some fungi, both the flagella of biflagellate zoospores are of whiplash type, and such zoospores are called isokont. In case of the heterokonts, the whiplash flagellum is directed backward while the tinsel type is directed forward. Colhoun (1966), Ho, et al. (1968) and Reichle (1969) have studied the Phytophthora zoospore while Lunney and Bland (1976) have studied the biflagellate zoospore of Pythium aphanidermatum. The zoospores are biflagellate, ovoid and possess a longitudinal groove. Both the flagella develop from within the groove. Both are unequal in size and have a typical 9 + 2 arrangement of microtubules. On each tinsel type of flagellum develop series of mastigonemes or flimmer hairs. The whiplash flagellum may also contain fine hair-like appendages (Vujicic, et al., 1968).

Lipid sac

Lateral mastigonemes Axoneme

Head

A

6.11.2

B

Ultrastructural Zoospore Types

Lange and Olson (1979) recognized five major ultrastructural zoospore types. Some of their main features are undermentioned:

Fig. 6.9

(A) Type 1 (i) (ii) (iii) (iv) (v)

A, Posteriorly uniflagellate zoospore of Blastocladiella emersonii as seen under electron microscope (diagrammatic representation); B, Anteriorly uniflagellate zoospore of Rhizidiomyces as seen under electron microscope (diagrammatic representation).

The ribosomes are distributed evenly. Electron dense bars may or may not be found associated with kinetosome. Cytoplasmic microtubule flare out from kinetosome. Either the functional or both the kinetosomes remain surrounded by a system of props. Mitochondria are either found throughout the zoospore (Fig. 6.10 A) or remain confined to only posterior region. (vi) Some members show petal-like configuration (Fig. 6.10 B) of mitochondria, e.g., Synchytrium endobioticum, Phlyctochytrium dichotomum, Olpidium pendulum, etc.

(B) Type 2 (i) Nuclear cap region remains filled with ribosomes. (ii) Nucleus is either centrally or anteriorly located. (iii) Nucleus has no special association with functional kinetosome.

Lipid body

F

Mitochondria

Lipid body

Endoplasmic reticulum

Mitochondrion

Lipid body

Dictyosome

C Ribosome filled nuclear cap

G

Nucleus

Tinsel flagellum

Lipid body

Ribosomes

Osmiophilic vacuole

Nucleus

Microtubules

D

A-G, Diagrammatic representation of ultrastructural details of five major types of zoospore types as recognized by Lange and Olson (1979). A-B, Type 1; C-D, Type 2; E,Type 3; F, Type 4; G, Type 5 (Redrawn from Lange and Olson, 1979).

E

Nucleus

B

Mitochondrion

Functional kinetosme

Nucleus

Nucleus

Mitochondria

Fig. 6.10

Kinetosome

Ribosome-filled nuclear cap area

Endoplasmic reticulum

Nuclear cap area

A

Nucleus

Mitochondria Nuclear cap area Nuclear cap area

Functional kinetosome

Lipid body

Mitochondria

62 Fungi and Allied Microbes

Eumycota

63

(iv) The cysternae of the endoplasmic reticulum are associated with nuclear cap area. (v) Either a single mitochondrion (Fig. 6.10 D) or many mitochondria (Fig. 6.10 C) remain associated with nuclear cap area, e.g., Rhizophidium sphaerotheca, R. patellarium, Chytridium olla, etc.

(C) Type 3 (i) (ii) (iii) (iv) (v)

Nuclear cap area is either partially encapsulated or traversed by endoplasmic reticulum (Fig. 6.10 E). The ribosomes are confined only to nuclear cap region. Kinetosomes remain surrounded by striated disc. Dictyosome is well-developed. Microbodies and lipid bodies have no direct association, e.g., Monoblepharella, Oedogoniomyces, etc.

(D) Type 4 (i) (ii) (iii) (iv) (v) (vi) (vii)

The ribosomes are aggregated in a nuclear cap (Fig. 6.10 F). A nuclear cap envelope is also present (Fig. 6.10 F). Posterior region contains a single mitochondrion. Nucleus is located in the posterior region. From the kinetosome extends a striated rootlet at right angle. Dictyosomes are absent. Gamma-like bodies and osmiophilic gamma are found, e.g., Allomyces, Blastocladiella, Coelomomyces, etc.

(E) Type 5 (i) (ii) (iii) (iv) (v) (vi) (vii)

6.11.3

An anteriorly attached tinsel flagellum is found. Ribosomes are centrally located and not bounded by any bounding membrane (Fig. 6.10 G). No special association is shown by lipid bodies and micro-bodies. Crystae of mitochondria are large, swollen and undulating. Dictyosome is well-developed. Microtubules are located almost parallel to the plasmalemma. Osmiophilic vacuoles are present, e.g., Rhizidiomyces apophysatus, Hyphochytrium catenoides, etc.

Sporangiospores

Many unicellular, smoothwalled, globose or ellipsoidal and non-motile asexual spores are developed in sac-like sporangia in may Zygomycetes. These are called sporangiospores. Because of their non-motile nature they are also called aplanospores. The number of sporangiospores in a sporangium may vary from one to several thousands (Webster, 1980) in different species.

6.11.4

Conidiospores or Conidia

Conidia are the asexual spores found in many Ascomycotina and Deuteromycotina (Photoplate-3). They develop from conidiogenous cells. The development of the conidia, when there is no enlargement of the conidial initial, is called thallic. But, if the conidial initial enlarges considerably before the septum formation, the development is called blastic.

6.11.5

Chlamydospores

The chlamydospores (Gr. chlamys, mantle; spora, seed or spore) are thickwalled, terminal or intercalary structures of the mycelium filled with reserve food material. They remain surrounded by a hyaline or coloured wall. Thickwalled dikary-

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otic spores of Ustilaginales are also named as chlamydospores. Smut spores of many species have been investigated (Photoplate-6) in detail by Kakishima (1980, 1982).

6.11.6

Ascospores

The characteristic sexual spores of Ascomycotina, formed because of nuclear fusion, followed by meiosis, are called ascospores (Photoplate-4). Meiosis results into the formation of four haploid nuclei. They divide mitotically to form eight ascospores. The ascospores develop in a cylindrical sac called ascus. They are generally eight in each ascus. The shape, size and colour of ascospores are different in different species. They may be oval or globose in shape but may also be elliptical, cylindrical, needle-shaped or even sousage-shaped. According to Webster (1980) the ascospores may be unicellular or multicellular and uninucleate or multinucleate. They remain surrounded generally by a multilayered wall. Structures like mitochondria, endoplasmic reticulum, lipid granules and vacuoles are also present in ascospores.

6.11.7

Basidiospores

The basidiospores (Gr. basidion, small base; spora, seed or spore) are the characteristic sexual spores of Basidiomycotina. These are unicellular spores with smooth or variously ornamented wall. Generally, they are globose but they may even be fuscoid, sausage-shaped or flattened. The basidiospores vary greatly in colour. They may be brown, pink, black, purple, yellow, creamy or whitish in colour. Some are even colourless. A basidiospore is attached at the tip of a sterigma. Its point of attachment with the sterigma is called hilum. Each basidiospore usually contains a single haploid nucleus. According to Pegler and Young (1971) the basidiospore wall consists of five layers, i.e., ectosporium, perisporium, exosporium, episporium and endosporium from outside within. Of these, the size and form of the basidiospore is determined by the episporium, while exosporium is responsible for its ornamentations. A typical development of basidiospore is shown in Fig. 6.11 A-F. Basidiospores Diploid probasidium

Chiastic

A

Fig. 6.11

6.11.8

B

C

or

Sterigmata

Basidium

Stichic

D

E

F

A-F. Development of basidiospore (diagrammatic). A-E, Meiosis (C, chiastic and D, stichic); F, Basidium (metabasidium) with four basidiospores on sterigmata (after Kirk et al., 2001).

Oospore

The oospores (Gr. oon, egg; spora, seed, spore) are also sexually produced spores, formed by the fusion of two unequal sized gametangia. Kirk et al. (2001) defined oospore as “the resting spore from a fertilized oosphere”, as in Oomycetes. Oosphere in Oomycetes is the female gamete, or the egg of the oogonium.

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Eumycota

6.11.9

Zygospore

Sporangium

The union of two, almost equal-sized gametangia under the process of sexual reproduction results into the formation of zygospore (Gr. zygos, yoke; spora, seed or spore). These are, therefore, sexually produced spores (Fig. 6.12 A-C). The zygospores are large, thickwalled, warty bodies filled with reserve food materials, The zygospores are characteristic features of Zygomycetes. Kirk et al. (2001) defined zygospore as “a resting spore resulting from the conjugation of isogametes (in Zygomycetes), from the fusion of like gametangia”.

6.12

Zygospore

A Zygospores

SEXUAL REPRODUCTION

Except Fungi Imperfecti, which have now been treated as AnamorB C phic Fungi by Krik et al. (2001), almost all true fungi reproduce sexually. They produce gametes. The gametes of opposite sex fuse Fig. 6.12 A-C, Formation of zygospores. under the processes of plasmogamy and karyogamy, and a zygote, with its nucleus having diploid number of chromosomes, is formed. The diploid nucleus of the zygote undergoes reduction division to reduce the number of chromosomes to the haploid number. In all lower fungi (Phycomycetes) and a few Ascomycetous fungi, sexual reproduction takes place by the fusion of two protoplasts, called gametes. In many genera the fusion takes place between two gametes of equal size (Fig. 6.13 A). Such a gametic union is called isogamy (Fig. 6.13 B). Two fusing gametes are generally flagellated (zoogametes), but in some

Anisogametes Isogametes

A

Fusing isogametes

B

C

Antheridium Antherozoid Oogonium Egg Fusing Anisogametes

D

Fig. 6.13

E

F

Sexual reproduction in fungi. A, Isogametes; B, Fusion of isogametes; C, Anisogametes; D, Fusion of anisogametes; E-F, Oogamy.

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Fungi and Allied Microbes

cases they may be non-flagellated (aplanogametes). In some genera the fusion takes place between two motile gametes of unequal size. Such a gametic fusion is called anisogamy (Fig. 6.13 C. D). Of these two fusing gametes the smaller one is the male gamete and the larger of the two is the female gamete. Many Phycomycetous and Ascomycetous genera show oogamy, where the male gamete is smaller, flagellated and motile, and the female gamete is larger, non-flagellated and immobile. The male gamete is called antherozoid, and the female gamete is called egg (Fig. 6.13 E, F). In the isogamous and anisogamous Phycomycetous fungi the gamete-containing body is called a gametangium. In oogamous species, however, the female gametangium is called oogonium. and the male gametangium is called antheridium. Fusion in all cases results into a zygote. Diploid zygotic nucleus divides meiotically at different stages in different genera. The majority of the Ascomycotina show plasmogamy between a small male gamete (spermatium) and a large female gamete. Motile gametes are absent.The male-gamete-containing body is called spermatangium or antheridium, whereas the female-gamete-containing body is called ascogonium. Plasmogamy is not followed by karyogamy. Ascus is formed. Karyogamy and meiosis take place in the ascus. In Basidiomycotina gametic union takes place but well-defined sex organs are absent. Plasmogamy takes place between protoplasts of two vegetative cells, or between the protoplasts of a vegetative cell and a spore. The plasmogamy is not immediately followed by karyogamy, as in Ascomycotina. Basidium is formed. Karyogamy and meiosis take place only in the basidium.

6.13

MODES OF SEXUAL FUSION

Some common methods of sexual fusion, under which the compatible nuclei are brought together, through the process of plasmogamy, are mentioned below.

6.13.1

Gametangial Contact

The male and female gametangia of many Eumycota come in contact with each other (Fig. 6.14 A). Either a pore or a fertilization tube develops at the point of contact. The nucleus or many nuclei of the male gametangium pass either through the pore or through fertilization tube, and bring about the sexual fusion. Antheridium

B

Fertilization tube Oogonium

Egg

Zygospore

C

Suspensor

A Trichogyne Spore

Somatogamy

Spore

Spermatium

D

Fig. 6.14

E

F

Some ways of sexual fusion. A, Gametangial contact; B-C, Gametangial copulation; D-E, Spermatization; F, Somatogamy.

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Eumycota

6.13.2

Gametangial Copulation

The gametangia come in contact through their tips. The entire contents of one gametangium are transferred into the other gametangium through a pore in many true fungi. But in some cases there is an amalgamation of the contents of both the gametangia by the dissolution of their contact walls. In such cases the ultimate result is the formation of a common cell (Fig. 6.14 B, C).

6.13.3

Spermatization

In some Eumycota uninucleate, non-motile male gametes (spermatia) are carried up to the receptive organ (trichogyne) of female gametangium through various agencies like wind, water, and insects (Fig. 6.14 D, E). The contact wall between the spermatium and the receptive organ dissolves, and the contents of spermatium pass into the receptive structures.

6.13.4

Somatogamy

Well-organized sex organs are absent in many true fungi. In such cases the sexual fusion is brought about by the anastomosis of somatic hyphae belonging to two different parents (Fig. 6.14 F).

6.14

CLASSIFICATION OF EUMYCOTA

Ainsworth’s (1973) classification has been followed in this book. He divided the division Eumycota into following 5 sub-divisions and 17 classes: Division EUMYCOTA Subdivisions Motile cells or zoospores present; Oospores present (Mastigomycotina)* Classes 1. Chytridiomycetes* 2. Hyphochytridiomycetes* 3. Oomycetes*

Perfect state spores are zygospores (Zygomycotina)* Classes 1. Zygomycetes* 2. Trichomycetes*

Motile cells absent

Perfect state present

Perfect state absent (Deuteromycotina)*

Zygospores absent

Perfect state spores are ascospores (Ascomycotina)* 1. Hemiascomycetes* 2. Loculoascomycetes* 3. Plectomycetes* 4. Laboulbeniomycetes* 5. Pyrenomycetes* 6. Discomycetes*

*Discussed in detail in independent chapters (refer Chapters 7-23)

Classes 1. Blastomycetes 2. Hyphomycetes 3. Coelomycetes

Perfect state spores are basidiospores (Basidiomycotina)* Classes 1. Teliomycetes* 2. Hymenomycetes* 3. Gasteromycetes*

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TEST YOUR UNDERSTANDING 1. Eumycota are commonly called _______ . 2. Write brief notes on the following: (a) Sclerotium (b) Rhizomorph (c) Mantle 3. How will you differentiate between? (a) Cleistothecium and apothecium (b) Perithecium and pseudothecium 4. What are zoospores? Describe three kinds of zoospores found in Eumycota. 5. Describe major zoospore types recognized by Lange and Olson (1979). 6. Give an illustrated account of various modes of sexual fusion in Eumycotaceous fungi. 7. Give an outline of classification of Eumycota as proposed by Ainsworth (1973). 8. Perfect state spores are _______ in Ascomycotina and _______ in Zygomycotina.

7

C

MASTIGOMYCOTINA

H A

(GENERAL ACCOUNT AND CHYTRIDIOMYCETES)

P T E R

7.1

GENERAL CHARACTERISTICS OF MASTIGOMYCOTINA 1. The sub-division Mastigomycotina of division Eumycota (Ainsworth, 1973) includes all such eumycotaceous fungi which produce flagellated cells during their life-cycle. 2. A majority of them are filamentous and contain coenocytic mycelium. However, many genera are unicellular and some show the pseudosepta formation. 3. Some unicellular forms also bear rhizoids. 4. They show centric nuclear division. Their centrioles remain functional during nuclear division. 5. The mode of nutrition is typically absorptive, because a majority of Mastigomycotina contain some or other type of haustoria. 6. All Mastigomycotina produce zoospores. 7. The sexual reproduction takes place by many different methods, but the perfect-state spores of almost all Mastigomycotina are typically oospores.

7.2

CLASSIFICATION OF MASTIGOMYCOTINA

In the Ainsworth’s (1973) classification, followed also in this book, Mastigomycotina have been ranked as a sub-division of division Eumycota, and divided as under: MASTIGOMYCOTINA (containing zoospores and oospores) Classes

Chytridiomycetes (with posteriorly uniflagellate zoospores containing whiplash type of flagella)

Hyphochytridiomycetes (with anteriorly uniflagellate zoospores containing tinsel type of flagella)

Oomycetes (biflagellate zoospores, of which posterior flagellum is whiplash type and anterior flagellum is tinsel type)

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Fungi and Allied Microbes

Kirk et al. (2001), however, placed these fungi under a separate kingdom Chromista, divisible further into three phyla viz., 1. Hyphochytridiomycota, 2. Labyrinthulomycota, and 3. Oomycota, while many of these fungi under Phylum Chytridiomycota of kingdom Fungi divisible further into 2 classes viz. Chytridiomycetes and Rumpomycetes.

7.3

CHYTRIDIOMYCETES

7.3.1 General Characteristics 1. They occur commonly in aquatic habitats, although many inhabit the soil. Genera like Olpidium, Physoderma and Synchytrium occur parasitically on many plants of economic importance to man, whereas some species of Coelomomyces are parasitic on mosquito larvae and are thus helpful in controlling malaria. According to Sparrow and Lange (1977) ‘Sphagnum bogs support a diversified and interesting flora of Chytridiaceous zoosporic Phycomycetes1. Some are also parasitic on algae, e.g. Phlyctochytrium planicorne on Vaucheria geminata. 2. The presence of posteriorly uniflagellate zoospores is the distinguishing feature of the class. 3. The flagella are of whiplash-type. 4. The cell wall mainly consists of chitin, but in some genera the cellulose is said to be present. 5. The vegetative plant body is coenocytic. However, a septum develops at the base of each reproductive body. 6. Some genera lack mycelium, and are unicellular and holocarpic. 7. In genera such as Rhizophidium, a few rhizoids develop from the unicellular thallus. Such rhizoids fix the thallus on the substratum and absorb the food. In some species the rhizoids become well-branched to form rhizomycelium. The rhizoids and rhizomycelium generally lack nuclei. 8. The asexual reproductive body is the sporangium. Its entire protoplast gets cleaved into many small compartments. Each such compartment contains one nucleus and develops into a uninucleate zoospore. Zoospore structure has been considered as one of the most relevant and authentic character in classifying Chytridiomycetes. Lange and Olson (1979) stated that ‘the ultrastructural organization of zoospore is the most valid taxonomic character which we have at present’. Some ultrastructural characteristics of the zoospore, according to Lange and Olson (1979), are mentioned (Fig. 6.10 A-G) below: (i) Zoospores of Chytridiales are spherical to ovoid or oval in shape, whereas these of Blastocladiales and Monoblepharidales are obpyriform to elongate. Their size varies from 1.5 mm to 10 mm. (ii) The whiplash type of the flagellum is usually 20 mm in length. (iii) The nucleus is located in the anterior (e.g. Olpidium brassicae), central (e.g. Phlyctochytrium palustre) or posterior (e.g. Olpidium pendulum) portion of the zoospore. (iv) The shape of the nucleus is generally spherical to spheroid. (v) The ribosomes are either evenly distributed in the zoospores (e.g. Synchytrium endobioticum) or they are loosely aggregated without a bounding membrane and are not delimited by organelles. In Chytridium and many other genera the ribosomes are aggregated in a nuclear-cap area. (vi) The mitochondria are (i) without any specialized organization, (ii) located at the periphery of the nuclear cap area, (iii) located only in the posterior portion of the zoospores, or (iv) arranged in a petal-like configuration. In many species of genera like Coelomomyces, Catenaria and Rhizophydium, however, only a single mitochondrion is present. (vii) A unique ‘microbody-lipid globule complex’ is present in posteriorly uniflagellate zoospores. 1 ‘Phycomycetes’ is one of the three universally recognized classes of Eumycota (the other two being Ascomycetes and Basidiomycetes). Ainsworth (1973) call Phycomycetes as’a miscellaneous assemblage’, and following the researches of Sparrow (1959) he preferred to accomodate all the constituents of Phycomycetes in ‘a series of classes: Chytridiomycetes, Hyphochytridiomycetes, Oomycetes, Zygomycetes and Trichomycetes’.

Mastigomycotina (General Account and Chytridiomycetes)

71

(viii) The endoplasmic reticulum may be either rough or smooth, i.e. with or without associated ribosomes, respectively. Lange and Olson (1979), reported seven types of organization of endoplasmic reticulum in the investigated 50 species of uniflagellate Phycomycetes. (ix) Dictyosomes have been reported in many species of Monoblepharella, Chytridium etc. (x) Some special type of vesicles, vacuoles and inclusions (e.g. gamma-body, contractile-like vacuoles, osmiophilic vacuoles, paracrystalline inclusions, microfibrillar inclusions) have been reported by different workers. But much is still not known about these bodies. (xi) Rhizoplasts, rootlets and electron dense bars, having close structural association with the functional kinetosomes, have been reported in the zoospores of many species (Olpidium brassicae). Five different types of such structures have been reported by Lange and Olson (1979). (xii) Many cytoplasmic microtubules have been reported by Lange and Olson (1979). (xiii) Functional and non-functional kinetosomes are found in the zoospores of Chytridiomycetes. (xiv) The plasmalemma of the zoospores is attached with the kinetosomal-axonemal microtubules through a system of election-dense props. 9. Sexual reproduction in Chytridiomycetes varies from simple isogamy to oogamy through anisogamy. In genera like Olpidium and Synchytrium conjugation of two isogamous planogametes takes place, but in some species of Blastocladiales conjugation takes place between two anisogamous planogametes. In Monoblepharidales, however, fertilization takes place between a non-motile egg (female gamete) and a motile antherozoid (male gamete).

7.3.2

Classification

Sparrow (I960) divided Chytridiomycetes into the following three orders: The thallus is generally not differentiated into a well-organized vegetative system. It is microscopic and either gets converted completely into a reproductive body (holocarpic) or changes into a simple rhizoidal system (eucarpic) and one or more reproductive bodies. Eucarpic thalli with one reproductive body are called monocentric, whereas those containing many reproductive bodies are called polycentric. Zoospores contain a well-differentiated oil globule. They show monopolar type of germination. The thallus consists of a well-organized mycelium. Resting structure is a thick-walled resting spore. Zoospores do not contain a well-developed oil globule. They show bipolar type of germination. Sexual reproduction is by isogamy or anisogamy. The thallus consists of a well-organized mycelium. Resting structure is an oospore. Zoospores show bipolar type of germination. Sexual reproduction is oogamous.

7.4

CHYTRIDIALES

7.4.1 General Characteristics 1. Members are commonly called chytrids. They are found in aquatic as well as terrestrial conditions. Aquatic chytrids occur parasitically on algae, Myxomycetes as well as many vascular plants. Soil-inhabiting chytrids are found parasitically on many vascular plants. Pathogens mentioned in parenthesis are serious destructive agents of highly important crops such as maize (Physoderma zeaemaydis), potato (Synchytrium endobioticum) and cabbage (Olpidium brassicae). Some chytrids grow saprophytically on plant and animal remains. 2. Many forms are endobiotic, i.e. live completely within the cells of the host (Synchytrium) whereas some are also epibiotic, i.e. having their reproductive bodies on the host surface (Rhizophydium). 3. Nutritional requirements of the majority of Chytridiales include mineral salts and carbohydrates in the form of sugar, starch or cellulose.

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4. The cell wall consists mainly of chitin. It is a polymer of N-acetylglucosamine. 5. Thallus structure shows considerable variation. In genera like Synchytrium, the thallus lacks rhizoids, and the entire cytoplasmic contents are reproductive in function (holocarpic, Fig. 7.1A). However, in a majority of Chytridiales, the thallus is differentiated into a vegetative part and a reproductive part. Such thalli are called eucarpic. Rhizoids are the integral part of the thallus in eucarpic thalli. The eucarpic thallus with a rhizoidal system having only a single reproductive structure is called monocentric (Fig. 7.1B). The monocentric thalli may be endobiotic (i.e. having the reproductive as well as rhizoidal parts inside the host cell; Fig. 7.1B) or epibiotic i.e. having the reproductive part on the host surface while the rhizoidal part inside the host (Fig. 7.1 C). On the contrary, such eucarpic thalli, which bear more than one reproductive structures, are called polycentric (Fig. 7.1 D).

Sporangia/gametangia

Host cell A Reproductive part

Sporangium

Host cell

B

Rhizoidal part

Reproductive part Rhizoidal part Host cell

D

C

Fig. 7.1

Types of thalli in Chytridiales. A, Holocarpic endobiotic; B, Eucarpic endobiotic; C, Eucarpic epibiotic; D, Eucarpic polycentric.

6. Asexual reproduction takes place with the help of zoospores, which are posteriorly uniflagellate. The flagellum is of whiplash type, and generally it remains coiled round the zoospore like a watch spring in many species. According to Karling (1977) and many other workers there is present a second non-functional centriole in the zoospore. Olson and Fuller (1968), while working on the zoospores of Phlyctochytrium kniepii and P. punctatum, suggested the biflagellate origin of the uniflagellate fungal zoospore. 7. The zoospore-containing body is a spherical or pear-shaped sac, called zoosporangium. At the time of the discharge of zoosporangium the zoospores come out through one or more exit papillae or discharge tubes. At the tip of exit papillae or discharge tube there is present a lid of circular cap in some species. This lid is called operculum and such species are called operculate chytrids (e.g. Chytridium). Many chytrids do not possess operculum.

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Mastigomycotina (General Account and Chytridiomycetes)

These are called inoperculate chytrids (e.g. Olpidium). The zoospores of inoperculate species are discharged either through a pore in the sporangial wall or by the dissolution of discharge tubes or exit papillae. 8. The size of the zoospores is almost constant in a given species, but their number in a zoosporangium is highly variable and may be anywhere between 1 and 2 to several thousands in different species (Webster, 1980). 9. Sexual reproduction has not been observed in a majority of the already studied chytrids (Alexopoulos and Mims, 1979). A thick-walled resting spore or resting sporangium is formed after sexual reproduction. However, many workers have shown the asexual formation of resting spores.

7.4.2

Classification

Sparrow (1960) divided Chytridiales into a number of families, the important ones are Olpidiaceae (Olpidium), Synchytriaceae (Synchytrium), Rhizidiaceae (Rhizophlyctis), Cladochytriaceae (Cladochytrium), Chytridiaceae (Chytridium) and Megachytriaceae (Nowakowskiella). Discussion of all these families is beyond the scope of this book, and moreover the life-histories in Chytridiales are so much variable that ‘no typical example could be selected as a general illustration’ according to Alexopoulos and Mims (1979). However, Synchytriaceae is discussed in some detail.

7.5

SYNCHYTRIACEAE

1. Members are microscopic, unicellular, holocarpic and lack a true mycelium. 2. Unicellular thallus divides into many compartments at the time of reproduction. These compartments function as sporangia or gametangia. 3. The sporangia are inoperculate. 4. Many sporangia or gametangia remain enveloped in a common membrane to form a structure, called sorus. 5. Asexual reproduction is with the help of uniflagellate zoospores whereas sexual reproduction takes place with the help of uniflagellate gametes. Sparrow (1960) recognized three genera, viz. Synchytrium, Endodesmidium and Micromyces. Singh and Pavgi (1979) reported Johnkarlingia brassicae, a new genus and new species of Synchytriaceae growing parasitically on the roots of cauliflower (Brassica oleracea var. botrytis) and cabbage (Brassica oleracea var. capitata). Kirk et al. (2001) mentioned that family Synchytriaceae of Chytridiales is represented by 5 genera and 136 species. Of all the reported genera, Synchytrium is the most studied genus. According to Alexopoulos and Mims (1979) it is represented by more than 200 species. The best-known species is Synchytrium endobioticum, and its life-history is discussed below.

7.6 7.6.1

SYNCHYTRIUM ENDOBIOTICUM Systematic Position

According to Ainsworth (1973) Division Subdivision Class Order Family Genus

– – – – – –

Eumycota Mastigomycotina Chytridiomycetes Chytridiales Synchytriaceae Synchytrium

According to Kirk et al. (2001) Superkingdom Kingdom Phylum Class Order Family Genus

– – – – – – –

Eukaryota Fungi Chytridiomycota Chytridiomycetes Chytridiales Synchytriaceae Synchytrium

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Fungi and Allied Microbes

7.6.2 Occurrence Synchytrium endobioticum is the causal organism of the most serious disease of potato, called black wart disease or potato wart disease. It occurs in almost all potato-growing regions of Warty outgrowth the world (Webster, 1980). It occurs as an obligate parasite in the Potato tuber epidermal cells of many angiospermic plants like potato (Fig. Leaf 7.2A), tomato, cucurbits (Fig. 7.2 B), etc. Tendril Early infection shows rapid cell division in the host tissue. In the later stages the cells become enlarged due to hypertrophy and assume the shape of galls. Many galls become confluent and form the characteristic warty outgrowths. Dark-brown, warty, cauliflower-like outgrowths are formed in the diseased tubers (Fig. 7.2 A). Though the disease is very serious, and the Galls yield as well as quality of the potato tubers is affected adversely, many immune potato varieties have been developed (Hampson, Fruit 1977). A B Some other species, with their hosts mentioned in parentheses, are Synchytrium rhytzii (on Peristrophe bicalyculata), S. Fig. 7.2 A, Wart disease caused b y Synchytrium trichosanthoides (on many cucurbits), S. sisamicola (on Sesamendobioticum on Solanum tuberosum; B, um indicum), S. mercurialis (on Mercurialis perennis), S. taraxS.trichosanthoides infection on cucurbit. aci (on Taraxacum officinale) and S. fulgens (on Oenothera). Synchytrium aureum has been reported from 198 hosts belonging to 123 genera of 34 families (Kirk et al., 2001).

7.6.3 Start of Infection Synchytrium endobioticum is a unicellular, endobiotic and holocarpic fungus found in the epidermal cells of the host. In the spring season large number of uniflagellate zoospores (Fig. 7.3 I) are released from the infected parts. Such zoospores keep on swimming in the soil water for about 2 hrs. They come to rest either on the surface of a potato ‘eye’ or on the stolon of the plant or even on the young tubers. Such uniflagellate zoospore dissolves a very small pore in the epidermal wall and penetrates in the host (Fig. 7.3 A,B). Its flagellum is left outside. The zoospore inside the host epidermal cell is uninucleate and amoeboid in shape (Fig. 7.3 C). It absorbs the food from the surrounding protoplast and increases in size. Simultaneously, its nucleus also increases in size, as well as the entire structure gets surrounded by a golden brown thick wall. It is now called prosorus (Fig. 7.3 D). Ultrastructural studies of Lange and Olson (1981) suggest that the encysted spore contains a prominent lipid body (Plate 1A) and a crescent-shaped nucleus on one of its side. At the time of penetration (Plate 1B), cyst wall becomes flattened and tightly appressed to the host surface. Penetration takes place through the host cell wall, and after the completion of the penetration process the wall of encysted zoospore remains outside the host cell. The fungal thallus starts to develop freely inside the host cell immediately after the penetration. The fungus shows following drastic changes in its ultrastructural organization at this stage (Lange and Olson, 1981): 1. Single centrally-located lipid body gets divided into many smaller lipid bodies. 2. Nucleolus gets enlarged and becomes very prominent (Plate 1C). 3. Nucleus also becomes large and centrally-located. 4. Mitochondria become peripheral in position. 5. Endoplasmic reticulum becomes quite extensive. 6. Just near the plasma membrane are seen many electron opaque spherical bodies.

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Mastigomycotina (General Account and Chytridiomycetes)

This results also into the following: 1. Host cell, bearing the fungus, becoms greatly enlarged. 2. Surrounding epidermal and cortical cells of the host also divide irregularly. 3. The ultimate result is the formation of tumor-like or wart-like bodies. 4. The infected host cell dies ultimately Germinating prosorus

Nuclear division

Mature prosorus

Zoospore

D

E

F

Host cell

Sorus

ASEXUAL REPRODUCTION

C Penetrating zoospore

G

Zoospore B Host A

Zoosporangium or gametangium

H I

Zoospores

S Zoospores

J Gametes Fusing gametes

Ruptured sporangium

K Karyogamy

SEXUAL REPRODUCTION

R

L Zoospore primordia

HAPL

S

Q

I OS

ASE OPH

DIPL

Penetrating zygote Host

?

M

I

ME

P

Fig. 7.3 A-S

ASE

OPH

O Resting sporangium (2n)

N

, Life-cycle of Synchytrium endobioticum (based on Curtis, 1921)

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7.6.4 Prosorus Germination and Zoosporangium Development After maturation the zoospore inside the host cell changes into a thick-walled structure, called prosorus (Fig. 7.3D). However, Lange and Olson (1981) preferred to name it as zoosporangium (Photoplate 1 D). The mature prosorus starts germinating within the dead host cell. At the time of its germination the fungal protoplast ruptures the wall and migrates into the upper half of the host cell (Fig. 7.3 E). It remains surrounded by a thin hyaline membrane. Its nucleus undergoes repeated mitotic divisions (Fig. 7.3 F) to form as much as 32 nuclei. At this stage the entire multinucleate prosorus gets divided into 4-9 multinucleate chambers with the help of thin hyaline walls (Fig. 7.3 G). The nuclei in all these multinucleate chambers keep on dividing repeatedly to form as many as 200-300 nuclei. Each of such multinucleate chamber represents a sporangium. The group of sporangia is called a sorus. Electron microscopic studies of Lange and Olson (1981) suggest that a young zoosporangium (Photoplate 1D) contains a single central nucleus with a prominent nucleolus, many mitochondria, numerous lipid bodies and a variety of electron-dense structures of different shape and size. These studies also confirm the formation of many daughter nuclei, followed by the division of single sporangium (Fig. 7.3 D) into several zoosporangia, as shown by Curtis (1921). Group of zoosporangia is bounded by a common wall, and a wall also develops around each zoosporangium. According to Lange and Olson (1981), ‘no information is at present available on the ultrastructural details of the process’ of segmentation of the sporangial content into several smaller sporangia.

7.6.5 Is it a Zoosporangium or Gametangium? As mentioned earlier, each multinucleate chamber of the sorus contains 200-300 nuclei (Fig. 7.3 H). At the time of cleavage all these nuclei get surrounded by dense cytoplasmic contents. According to Curtis (1921) they develop into uninucleate and uniflagellate zoospores (Fig. 7.3 I, Photoplate 1E), if water is in abundance. But, if there is scarcity of water, these uniflagellate bodies are released and function as gametes (Fig. 7.3 J). In the former condition the body containing asexual zoospores is called a zoosporangium. But, if these uniflagellate structures fuse (Fig. 7.3 J) and behave as gametes, the body containing them is called a gametangium (Fig. 7.3 H). Miss Curtis also mentioned that if these motile bodies are released immediately after their formation they function as zoospores. And if their release is delayed they function as gametes. That the zoospores may also act as isogametes has also been confirmed by the studies of Lange and Olson (1981).

7.6.6 Ultrastructure of Zoospores According to Lange and Olson (1978), the zoospore of Synchytrium endobioticum shows following characters: 1. It is uniflagellate, spherical to elongate in shape and attain a diameter of approximately 3 m m. 2. Ribosomes are evenly distributed. 3. An anteriorly located, large, lipid globule is present. 4. Mitochondria surround the nucleus from all sides. 5. Cytoplasmic microtubules remain associated with the functional kinetosome. 6. Most of the cell organelles are partially encapsulated by an extensive system of endoplasmic reticulum. 7. A contractile vacuole-like structure is also present.

7.6.7 Fate of Zoospores The released uninucleate and uniflagellate zoospores (Fig. 7.3 I) keep on swimming in the film of water in the soil. They may reinfect (Fig. 7.3 A) the same host and thus again repeat all the same processes. Thus, the asexual cycle is completed.

Mastigomycotina (General Account and Chytridiomycetes)

7.6.8

77

Gametangium

As mentioned earlier, multinucleate chambers of prosorus function as gametangia if conditions of drought are persisting. Now, instead of zoospores, the motile uninucleate cells of the gametangium behave as planogametes (Fig. 7.3 J). These gametes are slightly smaller in size than the zoospores. According to Miss Curtis (1921), two planogametes coming from different gametangia copulate (Fig. 7.3 K) in the water film present either on the host surface or in the soil. The copulation is isogamous, and the gametes belonging to two gametangia of the same sorus may fuse. Karyogamy (fusion of two nuclei) and plasmogamy take place and a diploid biflagellate zygote is formed (Fig. 7.3 L). No one has so far studied the actual gametic fusion in any species of Synchytrium under electron microscope (Lange and Olson, 1981).

7.6.9

Zygote and Resting Sporangium

Diploid and biflagellate zygote keeps on swimming on the host surface or in the soil water for some time. Finally, it settles on the host surface and penetrates (Fig. 7.3 M) an epidermal cell exactly in the same way as described for the zoospore penetration. It migrates towards the bottom of the infected epidermal cell of the host. As a result the host epidermal cells become hypertrophied and divide repeatedly (hyperplasia). The infected host cell gets burried deeply within the tissue. In this position the diploid zygote enlarges in size, gets enclosed by a thick and reticulately ornamented bilayered wall and is now called resting sporangium (Fig. 7.3 N, O). Because it remains in the resting stage throughout the winter season, some prefer to call it resting spore or winter spore.

7.6.10

Ultrastructure of Resting Sporangium

According to Lange and Olson (1981), the surface of the resting sporangium contains some dense fibril-like structures (Plate 1F, G). It remains surrounded by a thick sporangial wall. There is present a large centrally located nucleus with a prominent nucleolus. The cytoplasm contains many membrane-bound lipid bodies and osmiophilic bodies. The osmiophilic bodies are identical with the chromatin granules described by light microscopy. These ultrastructural studies of Lange and Olson (1981) also confirm the findings of Curtis (1921) that the thick wall of the resting sporangium is made up of dead cytoplasm, vacuolar region and the cell wall of the host cell along with the true fungal wall.

7.6.11

Germination of Resting Sporangium

The resting sporangia are released and keep on moving in the soil water for about 2 months. According to Curtis (1921), many granules develop in the cytoplasm of the released sporangium. These granules are the primordia of the future zoospores (Fig. 7.3 P). Actual process of meiosis has not so far been observed in any species of Synchytrium (Lange and Olson, 1981). But it is believed that the diploid nucleus of the resting sporangium divides repeatedly, and its first division is a reduction division. Many haploid nuclei are thus formed. The multinucleate protoplast of the resting sporangium undergoes cleavage to form many uninucleate, daughter protoplasts, each of which metamorphoses into a uninucleate and uniflagellate haploid zoospore. The haploid zoospores are liberated by the rupturing of sporangial wall (Fig. 7.3 Q, R) and again infect the host. Thus, the life-cycle is completed (Fig. 7.5). Some stages of the resting spore, germinating resting spore, an infected host cell containing prosorus showing extrusion of vesicle, cleavage of vesicle, extruded zoosporangia, zoospores, hypertrophied potato cells and young resting sporangium of S.endobioticum are shown also in Fig. 7.4.

7.6.12

Control of Potato Wart Disease

Hampson (1977) and Nohr and Mygind (1977) worked on the control of potato wart disease. Many wart resistant varieties of potato have been developed, and the best way to control this disease is to use these varieties as seed. The use of

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Resting spores

Resting spore Vesicle Sporangium

B A

Zoosporangia

Prosorus

E

C

D F Zoospores

G Rosette of hypertrophied potato cells

Young resting sporangium

H

Fig. 7.4 A–H, Synchytrium endobioticum. A, Resting sporangium; B, Germinating resting spore showing vesicle formation containing a single spor angium; C, Section of an infected cell containing prosor us; D, Showing cleavage of vesicle forming zoosporangia; E, Two extruded zoosporangia; F, Zoospores; G, Hypertrophied potato cells; H, Young resting sporangium.

fungicides in the soil before plantation has also provided fruitful results in checking the menace of this disease. Complete destruction of infected crops may also check the disease to some extent. The governments of many developed countries have introduced the legislation to use only the approved immune varieties for planting and to prohibit the sale and movement of diseased seeds. Fungicides like mercuric chloride, copper sulphate or ammonium thiocyanate at the rate of about 2.5 tonnes/ha (1 ton/ acre) are also used (Webster, 1980) in America and some European countries.

79

Mastigomycotina (General Account and Chytridiomycetes)

Zoospores (n)

7.7

BLASTOCLADIALES Infection to host MEIOSIS ?

7.7.1

General Characteristics

Prosorus (n) Resting sporangium (2n)

Zoosporangium n( )

1. Most of the members occur saprophytically in water or or Gametangium n( ) soil. Some are found in mud or inhabit plant or animal debris. However, Coelomomyces is an obligate parasite Infection to host Zoospores (n) of mosquito larvae and other invertebrates. Isogametes (n) Zygote (2n) 2. The thallus is eucarpic in all the Blastocladiales except Coelomomyces. Copulation of isogametes (n) 3. The cell wall in a majority of members consists of chitin. However, proteins, glucans and ash are also reported Fig. 7.5 Probable graphic life-cycle of Synchytrium in the wall of some genera. endobioticum 4. All Blastocladiales produce zoospores either in thinwalled zoosporangia or thick-walled resting sporangia. The brown colour of the resting sporangia is due to the presence of melanin and carotene. 5. The zoospores are posteriorly uniflagellate and possess a prominent nuclear cap, made up of a mass of ribonucleic acid. 6. The zoospores also contain a peculiar structure, called side-body. Its function is still not known. It is a doublemembraned structure situated just beneath the cell membrane on the posterior side of the zoospore. 7. The sexual reproduction is isogamous in some and anisogamous in other investigated species. In majority of the species, however, sexual reproduction has not been observed. 8. The gametes, if present, are uniflagellate. According to Kirk et al. (2001) Blastocladiales of class Chytridiomycetes includes 5 families, 14 genera and 179 species. The families are Blastocladiaceae, Coelomomycetaceae, Catenariaceae, Physodermataceae and Sorochytriaceae. Important genera Allomyces, Blastocladiella, Blastocladiopsis, Microallomyces and Coelomomyces.

7.8

MONOBLEPHARIDALES

7.8.1

General Characteristics

1. This is the smallest order of aquatic Chytridiomycetes. Its members are found in shallow waters or on submerged fruits and twigs of the deciduous trees. 2. Plant body is eucarpic and filamentous. The hyphae are branched and coenocytic. 3. The cell wall contains microfibrils of chitin. 4. Because of the presence of many vacuoles, the cytoplasm appears alveolated. 5. Asexual reproduction takes place by zoospores, which are uninucleate and posteriorly uniflagellate bodies. A crown of lipoid granules is seen at the anterior end of the zoospore. It also contains a distinct conical nuclear cap. 6. The zoospores develop in terminal, cylindrical or flask-shaped zoosporangia. 7. The sexual reproduction is oogamous, and takes place by the fusion of non-flagellated female cells and flagellated spermatozoids.

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8. The spermatozoids are posteriorly uniflagellate and develop in the antheridium, whereas the female gamete or egg develops in the oogonium. According to Kirk et al. (2001), Monoblepharidales of class Chytridiomycetes includes 4 unigeneric families (viz, Gonapodyaceae, Harpochytriaceae, Monoblepharidaceae and Oedogoniomycetaceae). Monoblepharidales includes only 4 genera (Monoblepharis, Monoblepharella, Gonapodya and Oedogoniomyces).

TEST YOUR UNDERSTANDING 1. The subdivision of Eumycota which contains zoospores and oospores is known as _______ . 2. Chytridiomycetes, Hyphochytridiomycetes and Oomycetes are all placed under: (a) Ascomycotina (b) Basidiomycotina (c) Zygomycotina (d) Mastigomycotina 3. Kirk et al. (2001) placed phyla Hyphochytridiomycota and Oomycota under kingdom _______ . 4. Write any five characteristic features of Chytridiomycetes. 5. What causes wart disease of potato? Describe the general symptoms of this disease. How this disease can be controlled? 6. Describe life-history of Synchytrium endobioticum. 7. Synchytrium zoospores are: (a) Uniflagellate (b) biflagellate (c) multiflagellate (d) none of these.

8

C H A P T

HYPHOCHYTRIDIOMYCETES

E R

8.1

WHAT ARE HYPHOCHYTRIDIOMYCETES?

Ainsworth (1973) placed all such Mastigomycotina (zoospores and oospores-containing fungi) under class Hyphochytridiomycetes which possess “anteriorly uniflagellate zoospores”. Chytridiomycetes, the another class of sub-division Mastigomycotina possess “posteriorly uniflagellate zoospores” while Oomycetes, the third class of this subdivision, possess biflagellate zoospores. Alexopoulos and Mims (1979), however, treated Hyphochytridiomycetes as a class of sub-division Haplomastigomycotina of division Mastigomycota of kingdom Mycetae along with two more classes (Chytridiomycetes and Plasmodiophoromycetes) in this sub-division. Zoospores of Hyphochytridiomycetes contain tinsel-type of flagellum while that of Chytridiomycetes contain whiplash type of flagellum. In Oomycetes, however, posterior flagellum is of whiplash type while the anterior flagellum is of tinsel type . Recently, Kirk et al. (2001) included all Hyphochytriomycota under kingdon Chromista containing only 1 class (Hyphochytriomycetes), 1 order (Hyphochytriales), 2 families (Hyphochytriaceae, Rhizidiomycetaceae), 6 genera and 23 species.

8.2

GENERAL CHARACTERISTICS

The general characteristics of Hyphochytridiomycetes are mentioned below: 1. Hyphochytridiomycetes or Hyphochytriomycetes are aquatic fungi, found either in fresh water or in marine conditions. Six genera and 21 species of this class have been reported growing parasitically on freshwater and marine algae and aquatic Phycomycetes. 2. The plant body is thalloid and holocarpic (that is having all the thallus used for fruiting body) or eucarpic (that is having only part of the thallus used for fruiting body). 3. The thallus is either monocentric (that is having one centre of growth and development) or polycentric (that is having many centres of growth and development and more than one reproductive organ). 4. Thallus may or may not bear rhizoids. 5. The vegetative system is rhizoidal or hypha-like with intercalary swellings . 6. The cell wall contains chitin or chitin with cellulose. 7. At the time of reproduction, thallus becomes converted into a sporangium or resting spore. 8. The sporangia are inoperculate.

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9. The zoospores are either completely differentiated inside the sporangium, or the entire protoplasm remains undifferentiated and extrudes through the sporangial opening and thus the zoospores differentiate outside the sporangium. 10. The zoospores bear a single, anterior, tinsel-type flagellum with mastigonemes. 11. Sexual reproduction is by copulation of identical isogametes, and known only in a few species (e.g. Anisolpidium ectocarpii). Fusion results in the production of a zygote which functions directly as a zoosporangium on germination.

8.3

CLASSIFICATION

Hyphochytridiomycetes contains only one order Hyphochytriales (syn. Anisochytridiales), divisible further into three families, viz., Anisolpidiaceae, Rhizidiomycetaceae and Hyphochytriaceae (Sparrow, 1973, and Karling, 1978). Anisolpidiaceae and Hyphochytriaceae are not known in India. Kirk et al. (2001), however, recognized only two families, viz., Hyphochytriaceae and Rhyzidiomycetaceae. In India, the best and only known species of Rhizidiomycetaceae is Rhizidiomyces apophysatus, a few details of the life-cycle of which are given below. Sparrow (1973) included only three genera under family Rhizidiomycetaceae. These are Rhizidiomyces, Rhizidiomycopsis and Latrostium.

8.4

RHIZIDIOMYCES

Rhizidiomyces is the only known genus of RhizidiomyZoospores Zoospore mass cetaceae reported from India, and one of its commonest with beating species is R.apophysatus (Fig. 8.1 A-H). Its thallus is euA flagella Zoospore carpic, monocentric and epibiotic (i.e. living on the surgermination Flagella and infection face of another organism). It bears a system of branched B rhizoids, which keep the thallus sunken in various subH strata, e.g. soil, pollen grains, algal cells or oospores of Zoospore aquatic moulds. Host wall Emerging Apophysis Its zoospores are anteriorly uniflagellate, which differprotoplasm entiate in the vesicle. They are released by the dissolution C of vesicle and settle on various substrata like soil, pollen G grains, etc. The flagellum of the zoospore is absorbed Young and body encysts and becomes somewhat rounded. The sporangium D cyst or thallus enlarges during germination and produces Exit tube a germ tube. The latter penetrates the host and becomes Exit papilla well-branched inside the host. The germ tube swells to F form a globular apophysis inside the host (Fig. 8.1 A-C). E Rhizoids seem to arise from the swollen apophysis. The globular portion of the thallus outside the host grows and Sporangium develops into a papillate sporangium (Fig. 8.1 D). The papillate sporangium develops into an exit tube or discharge tube (Fig. 8.1 E). Its tip swells and develops into a vesicle, into which multinucleate protoplast migrates Fig. 8.1 A-H, Life-cycle of Rhizidiomyces apophysatus. (Fig. 8.1 F, G), which soon changes into many uninucleate portions, each differentiating into uninucleate, uniflagellate zoospore. (Fig. 8.1 G, H). The vesicle dissolves, making the uniflagellate zoospores free-swimming in the surrounding water or moist surface of the host, thus completing the

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Hyphochytridiomycetes

asexual cycle (Fig. 8.1 A-H). The zoospores are pear-shaped, and each contains a single, tinsel-type of flagellum with mastigonemes (Fig. 8.2 A, B). The sexual reproduction has not been reported in R. apophysatus.

Mastigonemes Subsurface tubules

Basal body

Centrioles

Rhizidiomyces zoospores are most extensively studied bodies (Fig. 8.2 A, B). They attain a diameter of about 5 m m. Series of fine mastigonemes are present along the entire length of axoneme of the flagellum. A large and well-organised nucleus is present in the anterior part of the body. The flagellum develops from the kinetosome quite close to the nuclear membrane. A nuclear cap is absent. Body of the zoospore also contains several lipid bodies, microtubules, vacuoles, dictyosomes, endoplasmic reticulum and numerous ribosomes.

Dictyosome

Axoneme Nucleus Mitochondrion ER Ribosome containing region Vacuole B

8.5

PHYLOGENY OF HYPHOCHYTRIDIO MYCETES

A Zoospore

Hyphochytrids or Anisochytrids could not be placed in any other major class of fungi due to the differences in molecular weights of their ribosomal RNA (Lovett and Haselby, 1971). According to these workers, “the differences in moFig. 8.2 Rhizidiomyces apophysotus. A. An lecular weights of other fungi “may be correlating” with the entire zoospore showing axoneme and mastigonemes; B. L.S. zoospore. monophyletic origin of Chytridiomycetes, Zygomycetes, Ascomycetes and Basidiomycetes and independent origins of Myxomycetes and Oomycetes”. Hyphochytrids are also placed outside the main phylogenetic line of fungi but nearer to Oomycetes by Bartnicki-Garcia (1970). Le John (1972) studied regulation of glutamic dehydrogenase activity of these fungi and suggested that hyphochytrids or anisochytrids “are predecessors of both Chytridiomycetes and Oomycetes”. However, there exit several differences between Chytridiomycetes and Hyphochytridiomycetes with regards to their (i) nature of flagella, (ii) cell wall composition, (iii) pathways of lysine synthesis, and (iv) position of flagellum on the zoospores. All these characters justify the placement of hyphochytrids in the form of an independent class Hyphochytridiomycetes under sub-division Mastigomycotina of division Eumycota.

TEST YOUR UNDERSTANDING 1. Hyphochytridiomycetes is a class of Mastigomycotina which contains _______ and _______ . 2. Zoospores in Hyphochytridiomycetes are: (a) aflagellate (b) anteriorly uniflagellate (c) anteriorly biflagellate (d) multiflagellate. 3. Write any five distinguishing features of members of Hyphochytridiomycetes. 4. Write brief scientific note on Rhyzidiomyces. 5. Describe in brief the phylogeny of Hyphochytridiomycetes.

9

C H A

OOMYCETES

P T E R

9.1

WHAT ARE OOMYCETES?

Ainsworth (1973) placed all such Mastigomycotina under class Oomycetes, which contain biflagellate zoospores. Of the two flagella the posterior one is of whiplash-type, whereas the anterior one is of tinsel type. Oomycetes usually lack chitin in their cell wall. Sexual reproduction is oogamous. Although Smith (1955) discussed these fungi under subclass Biflagellatae of class Phycomycetae, but in almost all later contributions (Talbot, 1971; Sparrow, 1973; Muller and Loeffler, 1976; Alexopoulos and Mims, 1979; Webster, 1980) the biflagellate zoosporic fungi have been discussed under class Oomycetes. Because of the aquatic occurrence of majority of Oomycetes they have been assigned the name ‘water moulds’ by Alexopoulos and Mims (1979). Kirk et al. (2001), in the 9th edition of Dictionary of Fungi, discussed all Oomycetes under phylum Oomycota of kingdom Chromista of superkingdom Eukaryota. They mentioned further that these are aquatic or terrestrial freshwater or marine, saprobic or parasitic members. Thallus is mainly aseptate. Their assimilative phase is diploid (as in plants); zoospores with unequal flagella, a tinsel flagellum diverted with 2 rows of mastigonemes formed, and a whiplash flagellum is smooth or with fine flexous hairs backwards; with protoplasmic and nucleus-associated microtubules. Their cell walls are a glucan cellulose, rarely with minor amount of chitin.

9.2

GENERAL CHARACTERISTICS

The general characteristics of Oomycetes are: 1. Although Oomycetes occur in a variety of habitats, a majority of them are aquatic fungi, and live parasitically on algae, water moulds, aquatic insects and other animals as well as plants. Some higher forms grow in the soil, e.g. some Saprolegniales and Peronosporales. 2. The mycelium is well-branched, filamentous, coenocytic and grows abundantly in the substratum. However, some Oomycetes are unicellular. 3. The cell wall shows the presence of cellulose, which is very rare in most of the other fungi. According to BartnickiGarcia (1970) the oomycetous cell wall is mainly composed by cellulose b-glucan. The chitin is absent or rarely present. 4. Majority of Oomycetes are eucarpic, i.e. develop reproductive bodies in some parts of the thallus, and the thallus continues to function as a somatic body. The reproduction takes place by asexual as well as sexual methods.

Oomycetes

85

5. Zoospores are produced by almost all Oomycetes. They are biflagellate bodies, having one whiplash flagellum directed backward and another tinsel-type of flagellum directed forward. The flagella are attached anteriorly or laterally. The zoospores are pyriform or reniform bodies and are devoid of cell wall. According to Lange and Olson (1983), the biflagellate zoospores of Oomycetes are larger than the uniflagellate zoospores of Chytridiomycetes. A few species of Saprolegniales (Aplanes) and Peronosporales (Peronospora) do not produce zoospores. Some details of the ultrastructural characteristics of biflagellate zoospores (Lange & Olson, 1983) are mentioned below: (i) The nucleus is pyriform, with a beaked portion just adjacent to the kinetosomes. (ii) Near the beaked or tapered end of the nucleus are present two basal bodies, from which develop two flagella. (iii) Many microtubules and fibers radiate from the basal bodies into the cytoplasm. (iv) Cytoplasm contains many mitochondria, lipid bodies, one or two Golgi-bodies, ribosomes and many electrondense bodies. Many Oomycetous zoospores also contain contractile vacuoles. (v) Rumposomes are absent. (vi) Mitochondria of all Oomycetes have closely packed swollen and undulating tubular cristae. 6. The zoospores are produced inside zoosporangium. In some higher forms the sporangia are borne on specialized reproductive hyphae, called sporangiophores. 7. Many Oomycetes produce only one kind of zoospores, which germinate directly into new plants. Such species are called monoplanetic and this phenomenon is called monoplanetism. But in genera such as Saprolegnia, Leptolegnia etc. two kinds of zoospores are produced in succession. These zoospores are called primary and secondary zoospores, the genera or species producing these zoospores are called diplanetic, and this phenomenon is called diplanetism. 8. Many Oomycetes produce non-motile asexual spores (conidia), generally at the tip or side of an unbranched or branched hypha. The branch bearing conidia is called conidiophore. 9. Sexual reproduction is oogamous, taking place by gametangial contact, and results in the formation of thick-walled resting spore or oospore. In Lagenidiales the fusion takes place between two holocarpic thalli of different sizes, but in a majority of members of Saprolegniales, Peronosporales and Leptomitales the fusion takes place between an antheridium and a globose oogonium having an egg. 10. The flagellated gametes are not formed in Oomycetes. Fertilization takes place by fertilization tube produced by the antheridium. 11. Meiosis, in all the Oomycetes investigated so far, is gametangial. The somatic nuclei are, therefore, diploid in nature. On the basis of this point alone they have been placed in a separate sub-division Diplomastigomycotina.

9.3

CLASSIFICATION OF OOMYCETES

Ainsworth (1966) divided Oomycetes into the following four orders: (i) Lagenidiales, (ii) Leptomitales, (iii) Peronosporales, and (iv) Saprolegniales. However, Sparrow (1976) divided Oomycetes into the following six orders: (i) Eurychasmales, (ii) Saprolegniales, (iii) Lagenidiales, (iv) Peronosporales, (v) Thraustochytriales, and (vi) Labyrinthulales. Kirk et al. (2001) divided Oomycetes into 8 orders, 24 families, 82 genera and 650 species. The orders are Leptomitales, Myzocytiopsidales, Olpidiopsidales, Peronosporales, Pythiales, Rhipidiales, Saprolegniales and Sclerosporales.

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9.4

Fungi and Allied Microbes

ORDER SAPROLEGNIALES

9.4.1 Distinguishing Characteristics 1. Except some soil-inhabiting species, majority of Saprolegniales are aquatic, occur in clear waters, and are thus often termed ‘water moulds’ (Webster, 1980). Many of them are saprobic, but a few species are even parasites. Species of Saprolegnia grow in water commonly on the body of dead insects, fishes and fish eggs. Saprolegniales also parasitise some algae and fungi. Some species are also marine. 2. The mycelium is coarse, stiff and sparsely branched. 3. The thallus is holocarpic and endobiotic in Ectrogellaceae but eucarpic in a majority of other members. 4. Members are generally coenocytic. Thallus is, however, always septate in Leptolegniellaceae. 5. Some cellulose is present in the cell wall of Saprolegniaceae. 6. Zoospores always develop within a zoosporangium. 7. Zoospores are typically biflagellate with their anterior flagellum tinsel-type and posterior whiplash-type. 8. In some Saprolegniales two types of zoospores are formed, i.e. primary and secondary. The primary zoospores are generally pear-shaped with the apically attached flagella, whereas the secondary zoospores are bean-shaped with two laterally attached flagella. 9. Sexual reproduction is oogamous.

9.4.2 Classification of Saprolegniales Dick (1973) divided Saprolegniales into five families (Saprolegniaceae, Thraustochytriaceae, Ectrogellaceae, Haliphthoraceae and Leptolegniellaceae). Kirk et al. (2001), however, recognized only 2 families (Saprolegniaceae and Leptolegniaceae) containing 20 genera and 132 species. Only Saprolegniaceae is discussed in some detail.

9.4.3 Family Saprolegniaceae 1. They are more or less universally distributed aquatic fungi, found commonly in many types of temporary or permanent fresh-water reservoirs. Some species occur in moist soils. 2. The vegetative thallus is filamentous and eucarpic. The mycelium is coenocytic. The septa, however, develop at the base of reproductive organs. 3. The cell wall consists of cellulose b-glucan. 4. Zoosporangia are long, cylindrical, terminal and brown. 5. The majority of Saprolegniaceae show the phenomenon of diplanetism. However, the zoospores of Pythiopsis show monoplanetism, and of Dictyuchus show polyplanetism. 6. The dimension of the encysted zoospore is 8-15 mm (Dick, 1973). 7. Sexual reproduction is oogamous and takes place by gametangial contact. The majority of the species are hermaphrodite and homothallic, i.e. develop compatible oogonia and antheridia on the same thallus. However, heterothallism was shown by Raper (1957) in two species of Achlya. 8. Meiosis is gametangial, and takes place in antheridia and oogonia, just before the gamete formation. According to Dick (1973) Saprolegniaceae includes only 14 genera, viz. Saprolegnia, Achlya, Aphanomyces, Dictyuchus, Leptolegnia, Pythiopsis, Thraustotheca, Sommerstorffia, Geolegnia, Brevilegnia, Calyptralegnia, Plectospira, Aplanopsis and Scoliolegnia. Kirk et al. (2001) included 16 genera and 105 species under Saprolegniaceae. Life-cycles of Saprolegnia and Achlya are discussed here in some details.

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Oomycetes

9.5

SAPROLEGNIA

9.5.1 Systematic Position

According to Ainsworth (1973) Division



According to Kirk et al. (2001)

Eumycota

Superkingdom



Eukaryota

Subdivision



Mastigomycotina

Kingdom



Chromista

Class



Oomycetes

Phylum



Oomycota

Order



Saprolegniales

Class



Oomycetes

Family



Saprolegniaceae

Order



Saprolegniales

Genus



Saprolegnia

Family



Saprolegniaceae

9.5.2 Occurrence It is represented by about 30 species. A majority of them occur regularly as saprophytes on various types of substrata found in water, and hence are called water moulds. They grow saprophytically on dead bodies of many insects, tadpoles, fishes and other animals found in water. S. parasitica and S .ferax occur parasitically on fishes as well as on their eggs and cause diseases. Saprolegnia actually loves to grow on aquatic substrata rich in proteins. A highly destructive epidemic disease of fishes (Salmon disease) is caused by S. parasitica. Some species also occur on moist soil. Saprolegnia in India is represented by seven species, viz. S. diclina, S. ferax, S. litoralis, S. luxurians, S. monilifera, S. parasitica and S. unispora (Srivastava, 1985).

9.5.3 Laboratory Culture Saprolegnia can be grown in the laboratory if some substrata, such as sterilized split hemp seeds, ant eggs, dead flies (Fig. 9.1), pieces of solid white of egg, pieces of meat etc., are placed in samples of pond water in shallow petri-dishes for few days. It develops on these substrata as fringes of colourless hyphae, which first appear as tiny tufts of cotton fibres.

9.5.4 Somatic Structure The vegetative mycelium is profusely branched and coenocytic. However, the septa develop just below the reproductive organs. The vegetative phase of the thallus consists of following two types of hyphae: Rhizoidal hyphae, which are short, enter into the substratum and serve to anchor the organism and to absorb the food material. External hyphae, which are well-branched (Fig. 9.7 A) and remain on the outside of the substratum. They form the visible part of the fungus, develop in all directions, and produce the reproductive organs in the later stages. The main component of the hyphal wall is glucans, but according to BartnickiGarcia (1970) cellulose (cellulose b-glucan) is also present. The hyphal wall encloses vacuolated cytoplasm having many nuclei. Glycogen and oil globules constitute the reserve food.

Fig. 9.1

Saprolegnia and other related water moulds growing on dead insect.

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Fungi and Allied Microbes

9.5.5 Vegetative Reproduction The vegetative hyphae break into small pieces or fragments of different length. Each hyphal fragment develops into a new mycelium. Sometimes the hyphal tips become swollen irregularly. Each of such swollen portion contains many nuclei, abundant food material, and gets separated by a septum. Such swollen bodies are called gemmae. Some prefer to call such bodies as chlamydospores. On being detached, such ovoid, rounded or irregular-shaped bodies germinate into new mycelium.

9.5.6 Asexual Reproduction It takes place by means of biflagellate zoospores produced in club-shaped or pear-shaped zoosporangia (Fig. 9.7 A-H). The zoosporangia develop at the tips of the hyphae. The hyphal tips are somewhat pointed in the vegetative condition (Fig. 9.2 A). Vegetative At Protuberant Zoosporangia hypha Proliferated tip zoosporangium the time of the development of zoosporangium the pointed hyZoospores phal tip starts swelling, and becomes club-shaped with a rounded tip (Fig. 9.2 B). The cytoplasm gets accumulated in this region, and its central vacuole becomes less clearly visible. The entire structure gets separated by a basal septum (Fig. 9.2 C). In terms of sporangial tip the septum is convex, i.e. it bulges into the Second sporangium (Webster, 1980). zoosporangium Numerous nuclei are present in this region. Alexopoulos and Mims (1979) mentioned that just before the septum formation many nuclei, from the somatic hyphal region, move towards the sporangium. Cleavage of the cytoplasm takes place. The ultimate result is the formation of many uninucleate pieces of cytoplasm. At this stage the central vacuole is invisible, and a flattened proA B C D E F tuberance develops at the sporangial tip (Fig. 9.2 C). The zoospores are now almost fully differentiated and also show slight Fig. 9.2 Saprolegnia. A, A vegetative hypha; movement. They also show some change in their shape. Webster B-D, Development of zoospores; E, Libera(1980) mentioned that a pressure is now built up within the spotion of zoospores; F, Development of a new rangium. Because of such a pressure the septum now becomes sporangium within the old empty one. concave, i.e. it is now pushed into the hyphal lumen beneath the sporangium (Fig. 9.2 D). Such a pressure within the sporangium, might be caused by the increase in osmotic pressure of the sporangial content (Webster, 1980).

The zoospores are liberated by the breaking of the protuberant tip of the sporangium (Fig. 9.2 E). An opening develops in the tip region. Liberation of the zoospores from the zoosporangium is backward, i.e. their blunt posterior end emerges out of zoosporangium first (Webster, 1980). Saprolegnia is peculiar in its sporangial growth. Immediately after the discharge of all the zoospores the septum at the base of the zoosporangium shows renewed growth. It develops into a new sporangial apex inside the old sporangial wall (Fig. 9.2 F). This develops into a new zoosporangium inside the older one. The spores of this new zoosporangium are discharged through the pore of the older or parental empty zoosporangium. In some species of Saprolegnia this process is repeated several times, and thus many empty zoosporangial walls are seen inside each other. The liberated zoospores are pear-shaped, uninucleate (Fig.9.3 A) and bear two apically attached flagella. One flagellum is of whiplash type, whereas the another is of tinsel type. Each zoospore also bears a contractile vacuole. These zoospores are also called primary zoospores.

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Oomycetes

The primary zoospores keep on swimming in the surrounding water for less than 1 min to over 1 hr in different species. After the swimming period is over, the zoospores withdraw their flagella and become non-motile. The cytoplasm of each deflagellated zoospore gets surrounded by a distinct firm membrane (Fig. 9.3 B). These zoospores now undergo a resting period of 2-3 hr. After the resting period is over this encysted primary zoospore germinates (Fig. 9.3 C) to release a new secondary zoospore (Fig. 9.3 D). Structurally, the secondary zoospore is different from that of primary zoospore. Its shape is variable and shows amoeoid changes. In general, the secondary zoospores are bean-shaped or kidney-shaped (Fig. 9.3 E) bodies having two laterally attached flagella. The primary zoospores are pear-shaped bodies having two apically attached flagella. Electron microscopic studies of Holloway and Heath (1977) showed that primary cysts are smooth (Fig. 9.4 A), whereas the secondary cysts contain many small double-headed hooks (Fig. 9.4 B, C). In S. ferax (Fig. 9.4 B) the hooks on the secondary cysts are short and develop singly, whereas in S. parasitica (Fig. 9.4 C) they are very long and develop in tufts. According to the electron microscopic studies of Hallett and Dick (1986) the aquatic species normally have ornamented zoospore cysts, whereas most soil-inhabiting species contain unornamented cysts. In four species of Saprolegnia they reported unornamented cysts. The secondary zoospores keep on swimming for several hours. They swim three times faster than the primary zoospores (Salvin, 1941). The secondary zoospores show chemotactic movement (Webster, 1980). After some time they stop swimming, withdraw their flagella, get enclosed by a distinct firm membrane and assume almost spherical shape (Fig. 9.3 F). These encysted secondary spores may now be called secondary cysts. Each secondary cyst germinates by producing a germ tube (Fig. 9.3G).

9.5.7

Saprolegnia and Phenomenon of Diplanetism

Secondary cyst

Primary zoospore

A B Primary cyst

C

Secondary zoospore

D

E

F

G

Fig. 9.3

Structure and germlnation of zoospores in Saprolegnia. A, Primary zoospore; B, primary cyst; C-D, Germination of primary cyst; E, Secondary zoospore; F, Secondary cyst; G, Germination of secondary cyst. Primary cyst

Secondary cyst

B Biheaded hooks A

Cyst membrane C

Fig. 9.4

Ultrastructure of zoospores (diagrammatic) of Saprolegnia. A, Primary zoospore of S. ferax; B, Secondary cyst of S. ferax showing short biheaded hooks; C, Secondary cyst of S. parasitica showing biheaded hooks in tuft (after Manton et. al., 1951).

Saprolegnia is dimorphic because it produces two types of zoospores in succession, i.e. primary and secondary zoospores. This phenomenon of successive production of two types of zoospores by a single organism is called diplanetism. A dimorphic species in which two swarming periods occur, is called diplanetic. Saprolegnia is said to be diplanetic because of the presence of two separate motile stages in the asexual reproduction. According to Alexopoulos and Mims (1979) diplanetism is the rule in Saprolegnia. Fischer and Werner (1958), however, showed that under certain enviromental conditions not only two but a few more motile stages and cysts may be formed in Saprolegnia. Owing to the presence of several periods of motility, Webster (1980) suggested the use of the term polyplanetic instead of diplanetic.

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Fungi and Allied Microbes

9.5.8 Sexual Reproduction The sexual reproduction (Fig. 9.7 I-O) is oogamous. A majority of the species are homothallic or monoecious, whereas few species are heterothallic or dioecious. Usually the oogonia are globose, but in some species they are oblong. At maturity a septum separates the oogonium from the remaining vegetative hypha. The oogonia remain surrounded by a smooth thick wall, but in some species the wall may be spiny or papillate. The oogonia develop singly and terminally but rarely they are also intercalary. Multinucleate oogonium gets cleaved into a number of uninucleate oospheres or eggs. Usually the oospheres are 4-10 in an oogonium, but rarely they may reach up to 32. Each oosphere is dark, uninucleate, and contains a single large or many small oil globules. There is no periplasm. Antheridial Oogonium Furrowed The oogonium starts to develop by the swelling of the tip branch cytoplasm of a hyphal branch. The swollen portion ultimately becomes Oogonium spherical (Fig. 9.5 A) or globose. Many nuclei of the hyOospheres pha along with some cytoplasmic contents migrate into this Antheridium swollen portion, which finally gets separated by a septum (Fig. 9.5 B, C). In the multinucleate swollen oogonial portion the nuclear divisions continue for some time. According A to Alexopoulos and Mims (1979) meiosis takes place in the B oogonium and thus the oospheres in the oogonium are hapOospheres C loid. After some time many nuclei degenerate except those Oogonium D which are included in the formation of eggs. Cleavage furAntheridium rows start to develop (Fig. 9.5 C) from the central vacuole region of the oogonium and start to radiate outwards. Many uninucleate portions are thus formed. They ultimately beOospheres come round and function as eggs or oospheres (Fig. 9.5 D). The antheridia are multinucleate, Somatic Fertilization elongated and smaller than oogonia. Usually, they develop hypha tube from the same hyphal branch (Fig. 9.6 B) which bears the E F oogonium, but they may also develop on a different hyphal branch of the same thallus. In S. litoralis the antheridial Fig. 9.5 Saprolegnia litoralis. A-D, Development of branch develops from the oogonial stalk, showing androgyoogonium (note the young antheridial branch nous condition (Fig. 9.5 B). If the antheridia develop on the in B, Furrowed cytoplasm of oogonium in C, same hypha as the oogonium, they are called monoclinous. and formation of oospheres in D); E, TermiBut if they develop on different hyphal branches, they are nal oogonium with antheridia; F, Separate called diclinous. fertilization tube for different oospheres. The mature antheridia become attached to the oogonium (Fig. 9.5 E) and each of them gets delimited by a septum. Each antheridium is multinucleate, tubular and elongated body filled with dense cytoplasmic contents. Formation of well-organized sperms is not clearly known in Saprolegnia. Many haploid gamete nuclei are formed in the antheridium by meiosis (Alexopoulos and Mims, 1979). Just before fertilization, one or more antheridia become closely attached to the oogonial wall (Fig. 9.5 D, E). At the point of contact a tube originates from the antheridium and penetrates into the oogonium. This is called fertilization tube (Fig. 9.5 E, F). Inside the oogonium the fertilization tube may give out some slender branches, one each for each oosphere. If more than one antheridia are in contact with the oogonium, one fertilization tube approaches only one oosphere (Fig. 9.5 F). Through the fertilization tube many male nuclei migrate from the antheridium towards oospheres. A male nucleus enters into each oosphere, comes near the female nucleus, plasmogamy and karyogamy take place (Fig. 9.5 F), and a diploid zygotic nucleus is formed. A thick wall now develops around each oosphere. Thick-walled oosphere having diploid zygotic nucleus is called oospore (Fig. 9.7 N).

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Oomycetes

A

Lipid droplets B Ooplast

Centric

Arrangements of ooplast and lipid droplets in oospores of Saprolegnia. A, Centric; B, Subcentric; C, Subeccentric; D, Eccentric.

The fertilized oosphere (oospore) undergoes a series of changes. Its wall gets thickened and oil globules become clear. Mature oospores contain a membrane-bound vacuole-like body, called ooplast, which remains surrounded by cytoplasm containing various organelles. The position of ooplast in the oospore is used in the identification of species. Four types of the oospore (Fig. 9.6 A-D) have been distinguished: (a) Centric: These have a central ooplast surrounded by one or two outer layers of small lipid droplets as in S.frax; (b) Sub-centric: These have many layers of small lipid droplets on one side of ooplast and only one layer or none at all on the other, as in S.unispora; (C) Subeccentric :These have small lipid droplets which fuse into many large ones all grouped to one side and their ooplast contracting the plasma membrane on the opposite side, as in S.eccentrica; and (d) Eccentric: These are similar to subeccentric type except that there is only one large lipid body (Fig. 9.6 D).

Primary cyst

Germinating cyst

Secondary zoospore

E D Primary zoospore

F

Secondary cyst

C G Zoosporangium

Germinating cyst

H

Somatic hypha B

A Germinating oospore

O

sis Meio

I Oospores

The oospores undergo a prolonged resting period. After the resting period is over the oogonial wall is disintegrated and the oospores are liberated. Each oospore germinates by producing a germ tube, which soon develops into a well-developed mycelium, and thus the life-cycle is completed (Fig. 9.7 A-O).

(2n) ase loph (n) Dip ase loph Hap

N

J

ion

Oospheres

M

9.6

Eccentric

Subeccentric

Fus

Fig. 9.6

Subcentric

D

C

ACHLYA

K L

9.6.1 Systematic Position

According to Ainsworth (1973) Same as for Saprolegnia

According to Kirk et al. (2001) Same as for Saprolegnia

Fig. 9.7

A-O. Life-cycle of Saprolegnia (based on findings of Seymour 1970, and Fuller, 1978).

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Fungi and Allied Microbes

9.6.2 Occurrence Achlya is represented by over 50 species, of which A.ambisexualis, A.bisexualis, A.caroliniana and A.racemosa are common. A majority of the Achlya species are aquatic. According to Johnson (1956) some species are common also in damp humus soil as well as in waterlogged plant debris like broken twigs. Some species occur on dead bodies of insects, whereas a few occur as parasites on fishes.

9.6.3 Laboratory Culture Achlya can be cultured in the laboratory by placing substrates like cut hemp-seeds or twigs or dead fly in a petri-dish having pond water. Within a few days the hyphae of Achlya appear on these substrates.

Zoosporangium Hyphal tip

9.6.4 Somatic Structure The mycelium is well-developed, branched, dense and generally white-coloured. The hyphae are stout, aseptate and multinucleate, showing typical coenocytic condition. Some hyphal branches penetrate into the substratum and absorb the food. The nuclei remain arranged peripherally in the cytoplasm around the central vacuole.

9.6.5 Vegetative Reproduction It takes place by fragmentation as well as by gemmae formation. Fragmentation takes place by breaking the mycelium in some fragments, each of which develops into new mycelium. At the time of gemmae formation the cytoplasm of hyphae becomes dense in certain portions. Such dense portions are later on cut off by septa. They develop either singly or in chains, and are located either terminally, or are intercalary in position. These thick-walled resistant bodies survive during the temporary unfavourable conditions. Gemmae serve as the means of perennation. Some workers prefer to call the gemmae as chlamydospores.

Germinating cyst

Septum

H

G

B

Secondary cyst A Primary zoospores

Secondary zoospores

Zoosporangium

F

Secondary zoospore Primary cyst

Empty primary cyst

Full primary cyst

E

9.6.6 Asexual Reproduction It takes place by zoospores formed in zoosporangia. The ‘zoosporangial development in Achlya is similar in all respects to that in Saprolegnia’ (Webster, 1980). The zoosporangia develop singly and terminally on the vegetative hyphae. Many zoosporangia develop if the conditions are favourable for their growth. At the time of the development of a zoosporangium the cytoplasm flows into the hyphal tip, where it keeps on accumulating (Fig. 9.8 A). After some time a basal septum is formed (Fig. 9.8 B), which stops the cytoplasmic flow into the hyphal tip. At this stage the young zoosporangium contains many nuclei and grey-coloured cytoplasm. Generally, the zoosporangium is slightly larger in diameter than that of the parent hyphae.

D C

Fig. 9.8

Development of zoosporangium in Achlya. A, Accumulation of cytoplasm in the hyphal tip; B, Septum formation; C, A cluster of primary zoospores at the zoosporangial mouth; D, Some complete and some empty primary cysts; E, Release of secondary zoospores from primary cysts; F, Secondary zoospores; G, Secondary cyst; H, Germination of secondary cyst.

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Oomycetes

In a majority of species it tapers slightly towards base as well as towards apex. Aggregation of cytoplasm around these nuclei results in the formation of primary zoospores. The primary zoospores lack flagella in the soil-dwelling species, whereas in other species they are pyriform and biflagellate bodies. Johnson (1956), however, mentioned that the presence of flagella in the primary zoospores is not confirmed. The primary zoospores discharge through an apical pore of the zoosporangium, come out in the form of a group, remain clustered (Fig. 9.8 C) at the zoosporangial mouth and get encysted. These encysted zoospores (or primary cysts) are also called cystospores. Within few hours each primary cyst ruptures and forms a small pore, through which a secondary zoospore is released (Fig. 9.8 D, E). Each secondary zoospore is a reniform body, having two unequal flagella attached laterally. Sometimes the secondary zoospore may again undergo the same process and develop into tertiary zoospore. The latter are identical with that of secondary zoospores. The secondary or tertiary zoospores keep on swimming for some time, get themselves encysted and germinate into new mycelia (Fig. 9.8 F-H). Because of the repeated zoospore formation (i.e. primary, secondary and tertiary zoospores) Achlya shows the phenomenon of polyplanetism.

9.6.7

Sexual Reproduction

The sexual reproduction is oogamous. A majority of the species are homothallic (monoecious), i.e. bear male and female sex organs on the same mycelium (A.racemosa, A. colorata). Some species, however, are heterothallic or dioecious (A.bisexualis, A.ambisexualis). Detailed investigations on the morphology of sex organs and the cytology of sexual reproduction have been made by Raper (1939, 1950), Dick (1969, 1972,1973) and Dick and Win-Tin (1973).

(A)

Oogonium

Sexual Reproduction in Homothallic Species

The sexual reproduction in homothallic species is controlled by some hormones (Raper, 1950; Webster, 1980). Antheridia and oogonia in A.racemosa and A.colorata develop on the same mycelium. The oogonia are either terminal or intercalary on the main hypha or on the short lateral branches (Fig. 9.9). They are either globose or pyriform in shape, and remain surrounded by thin, smooth and hyaline walls. In A. colorata the oogonial wall bears blunt rounded projections (Fig. 9.9 D-E). Each oogonium remains filled with dense homogeneous contents. The young oogonial initial is multinucleate and its nuclei keep on dividing for some time (Fig. 9.9 A). A large central vacuole is also present. After some time some of the nuclei degenerate and the swollen portion is cut off by a cross wall at the base (Fig. 9.9 B). Cleavage furrows start to develop from the centre towards outside, and the entire oogonial cytoplasm divides into uninucleate portions, which round off to form oospheres (Fig. 9.9 C, D) or eggs. The number of eggs in each oogonium varies between 1 and 10 but they are generally 2-6 in number. There is no residual cytoplasm in the oogonium. The antheridia develop on the antheridial or male branches, which arise from the lower portion of the

Oospheres

Antheridium

Oogonial initial

D B

A

C

Germinating Oospores

Germ tube Oospores Zoosporangium Germ tube Oospore

Antheridium

E

Fig. 9.9

F

G

Sexual reproduction in Achlya colorata. A, Oogonial initial; B-E, Development of oogonium; F, Germinating oospore; G, Germ tube with a terminal zoosporangium.

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Fungi and Allied Microbes

same oogonium-bearing hypha. Each antheridial branch is thin and multinucleate, with an inflated tip. The club-shaped, multinucleate, inflated tip portion is cut off by a septum and represents an antheridium (Fig. 9.9 C, D). The antheridia grow towards oogonia chemotropically and ultimately get themselves attached with the oogonial wall. It has been observed that the portion of the oogonial wall that remains in contact with the antheridium ultimately becomes thin. Fertilization takes place by the formation of fertilization tube, which develops from each antheridium, penetrates the oogonial wall and reaches up to an oosphere or egg. In some species the fertilization tube may be branched within the oogonium. But when it penetrates the oosphere wall the male nucleus fuses with the single egg nucleus and the fertilization is completed. Fertilized eggs are called oospores. The oospore gets surrounded by a thick wall and contains many oil globules. According to Dick (1971) mature oospores contain a vacuole-like, membrane-bound body, called ooplast. The cytoplast surrounding this ooplast contains various organelles. A Brownian movement of the particles can be seen in the ooplast. The oospores do not germinate immediately. They require a maturation period of a few weeks to many months. Each oospore germinates by the formation of a slender germ tube. In A. colorata the germination of oospores starts while they are still within the oogonium. The young germ tubes repture the oogonial wall (Fig. 9.9 F) and develop into young mycelial strands. Sometimes the germ tube gives rise to a terminal zoosporangium (Fig. 9.9 G). Genetic evidence of Mullins and Raper (1965) and Barksdale (1966) indicates that the germination of oospore results in the formation of diploid progeny. This conclusion was reached because, on germination, an oospore never produce both male and female thalli. It develops either into a male or a female thallus.

(B)

Sexual Reproduction in Heterothallic Species

Antheridia and oogonia in species like A. bisexualis, A. ambisexualis and A. heterosexualis develop on different thalli, showing heterothallic nature. According to Raper (1952, 1954, 1957) the development of sex organs in these species in controlled by some hormones secreted by male and female plants. If grown separately, neither the female thalli form oogonia nor the male thalli form the antheridia. Both of them form only asexual reproductive bodies, i.e. sporangia in such condition. But if the male thalli are grown together with the female thalli in the same dish, the antheridia develop on the male filaments and the oogonia develop on the female hyphae within 2-3 days. This indicates that the presence of male and female strains in the same dish causes a mutual stimulation for the development of sex organs. This mutual stimulation is because of the production of some diffusible substances, i.e. hormones. The hormones produced by the female plants stimulate the production of antheridia on the male plant and vice versa. This type of mixed sexuality is called gynandromixis. According to Raper (1939), production of following 4 hormones govern the formation of antheridia and oogonia in A. Antheridial hyphae ambisexualis: Oogonial initial It is secreted by the vegetative female hyphae. It induces the development of antheridial branches. McMorris and Barksdale (1967) purified Hormone A of A. bisexualis and named it antheridiol with its empirical formula C29H42O5. It is secreted by the antheridial branches. It initiates the formation of oogonial initials on the female branches. McMorris et al. (1975) isolated three chemically similar compounds (oogoniol-1, oogoniol-2 and oogoniol-3) from a strain of A. heterosexualis. All these oogoniols are steroids and show Hormone B activity. It is secreted by the oogonial initials. It first stimulates the antheridial initials to grow towards the oogonial initials and then also results in the delimitation of antheridia.

MALE THALLUS

A

Oogonium

Oogonial branch

B

Fig. 9.10

FEMALE THALLUS

Antheridial branch

Achlya ambisexualis. A, Male and female thalli growing together; B, Fertilization.

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Oomycetes

It is secreted by the antheridial branches only after the latter had established contact with the oogonial initials. Hormone D initiates the formation of a septum at the base of the oogonium. The formation of oospheres is also initiated because of this hormone. As mentioned earlier, the sex organs in heterothallic species develop only when male and female plants are grown together in the same petri-dish or same medium for a few days, and this happens because of mutual stimulation. In A. ambisexualis many long, branched antheridial hyphae develop on male thallus only in response to the presence of the female thallus (Fig. 9.10 A). Antheridial development on the male plant induces the initiation of oogonia on female plant present nearby. Antheridia are attracted by the oogonial initials towards them. This initiates the formation of a septum at the base of oogonium and delimitation of oospheres in the oogonium. According to Barksdale (1966) gametes are differentiated in A. bisexualis after meiosis. Fertilization (Fig. 9.10 B) and structure of oospore in heterothallic species is almost the same as in homothallic species. Oospores in A. ambisexualis germinate by producing multinucleate germ tubes. This germ tube either develops into a coenocytic mycelium or into a germ sporangium.

A

Fucosterol HO OH

O O B HO

O

OH HO OH

C

(c)

Steroid Sex Hormones in Achlya

Steroid sex hormones in Achlya have been discovered by Carlile (1996). Some sterols include fucosterol, antheridiol and oogoniol as depicted in Fig. 9.11 A-C.

9.7

Antheridiol

HO

Fig. 9.11

O

Oogoniol

Fucosterol (A) which is precursor to the sex hormone, Antheridiol (B) and Oogoniol (C).

PERONOSPORALES

9.7.1 Characteristic Features 1. Most of the Peronosporales are obligate parasites (Albuginaceae and Peronosporaceae) of higher plants but some are aquatic or semi-aquatic facultative parasites (Pythiaceae). Some genera survive also saprophytically on dead or decaying vegetation and a few survive easily in soil or in mud. Thus, Peronosporales include aquatic, amphibious and terrestrial species. They cause serious diseases of a number of important crop plants, such as dampingoff (Pythium debaryanum), white rusts (Albugo candida), downy-mildews (Peronosporaceae) and late-blight of potato (Phytophthora infestans). 2. The mycelium is delicate, well-branched, coenocytic, and intercellular (Peronosporaceae) or intracellular (majority of Pythiaceae). The intercellular mycelium runs through the intercellular spaces and only their haustoria enter the host cell. But the hyphae of the intracellular mycelium rarely develop the haustoria. 3. The haustoria may be lobed, extensively branched, or in the form of minute spherical or cylindrical ingrowths. 4. The cell wall is fibrillar and consists of polysaccharides, lipids and proteins in Phytophthora (Hunsley, 1973) and Pythium (Sietsma et al. 1975). About 90% of the wall is constituted by glucans, and it also contains amino acid hydroxyproline. 5. Asexual reproduction takes place by sporangiospores produced in sporangia. 6. The sporangia are generally globose, spherical or pear-shaped. However, they are simply inflated lobes of mycelium in many species of Pythium. In Peronosporaceae the sporangia develop on well-branched sporangiophores, whereas in Albuginaceae they develop in chains.

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Fungi and Allied Microbes

7. The sporangia in genera such as Albugo and Phytophthora develop zoospores. However, many Peronosporales show a tendency of sporangial germination by a germ tube instead of formation of zoospores (Peronospora and many downy mildews). 8. The zoospores are kidney-shaped, monoplanetic and laterally biflagellate. The posterior flagellum is of whiplash type and the anterior one of tinsel type. 9. Sexual reproduction is oogamous. Majority of the Peronosporales are homothallic. But some also show heterothallism and relative sexuality (Webster, 1980). Sterols are essential nutritional requirement for sexual reproduction. 10. Meiosis takes place in the gametangia, resulting in the formation of haploid antheridial nuclei and oospheres (Sansome and Sansome, 1974). 11. The oogonium is characteristically differentiated into ooplasm and periplasm, and it contains a single egg (Webster, 1980). The periplasm gets deposited around the fertilized oospore in the form of its wall material, and is responsible for the external thickenings and ornamentations of the oospore wall. 12. The antheridium is a club-shaped body, generally developing just below the oogonium. It soon cuts off by a septum and is of limited growth. 13. The antheridial tip becomes flattened against the oogonial wall and develops a fine penetration tube or fertilization tube. 14. The antheridial contents pass through the fertilization tube into the oosphere, brings about the fusion of functional male and female nucleus, forms the diploid oospore and completes the fertilization. This type of fertilization is called paragynous. Few Peronosporales such as Phytophthora show amphigynous type of fertilization. 15. The oospore wall is hyaline or brownish and smooth or ornamented. It germinates either by a germ tube (Peronospora) or by the production of a large number of zoospores (Albugo).

9.7.2 Classification of Peronosporales Waterhouse (1973) divided Peronosporales into three families: (i) Pythiaceae, (ii) Peronosporaceae, and (iii) Albuginaceae. Ko et al. (1978) recognized a fourth monotypic family Peronophythoraceae in Peronosporales, represented by only one genus, Peronophythora with a single species P. litchii. It is a link between Pythiaceae and Peronosporaceae, resembling Pythiaceae in possessing inconspicuous periplasm, and Peronosporaceae in having determinate sporangiophore. Kirk et al. (2001) also discussed Peronosporales under class Oomycetes but included only 2 families (Albuginaceae and Peronosporaceae) in this order. They discussed Pythiales as a separate order of Oomycetes, including 2 families (Pythiaceae and Pythiogetonaceae).

9.7.3 Differences between Pythiaceae, Peronosporaceae and Albuginaceae Table 9.1 S.No 1. 2.

Differences between Pythiaceae, Peronosporaceae and Albuginaceae Pythiaceae Nonobligate parasites, saprobes. Sporangiophores or conidiophorcs generally undifferentiated from the mycelium. They are branched, indeterminate, resuming growth after the production of a sporangium or conidium.

Peronosporaceae Obligate parasites. Sporangiophores or conidiophores differentiated from the mycelium. They emerge singly or in tufts from the host epidermis generally via stomata; with determinate growth and no subsporangial regrowth.

Albuginaceae Obligate parasites. Sporangiophores are clavate, unbranched, having a basipetal chain of deciduous sporangia, forming dense subepidermal clusters of white creamish sori on the host.

Contd..

97

Oomycetes

Contd..

3.

4.

9.8

Oogonial periplasm is either absent or present in the form of a thin layer. Haustoria are either branched or absent.

Periplasm is well-developed and persistent.

Periplasm persistent and conspicuous.

Haustoria varied and usually branched.

Haustoria knob–like.

FAMILY PYTHIACEAE

Pythiaceae occur parasitically on plants or animals, but according to Hawker (1966) all are also able to grow saprophytically. Some are truely aquatic, occurring parasitically on algae or small protozoa, whereas many are found in soil, growing saprophytically on plant or animal debris. Their other distinguishing points are mentioned in Table 9.1. Kirk et al. (2001) discussed Pythiaceae under an independent order Pythiales containing one more family Pythiogetonaceae. According to these workers, 11 genera are included under Pythiales, of which 10 belong to family Pythiaceae and only 1 genus (Pythiogeton) belongs tto Pythiogetonaceae. Waterhouse (1973) recognized only six genera in Pythiaceae (Trachysphaera, Diasporangium, Pythium, Pythiogeton, Phytophthora and Sclerophthora). The life-histories of only Pythium and Phytophthora are discussed.

9.9 9.9.1

PYTHIUM Systematic Position

According to Ainsworth (1973) Division Sub-Division Class Order Family Genus

-

Eumycota Mastigomycotina Oomycetes Peronosporales Pythiaceae Pythium

According to Kirk et al. (2001) Superkingdom Kingdom Phylum Class Order Family

-

Sporangial initial

9.9.2

Occurrence

Pythium, the largest genus of Pythiaceae, is represented by 127 species (Kirk et al. 2001). Many species occur only in aquatic situations as saprophytes, whereas some may be weak parasites of aquatic plants or animals. A majority of the species are soil-inhabitants and a few occur even in mycorrhizal association. Some serious diseases of seedlings, such as damping-off, pre-emergence killing, foot-rot and root-rot are caused by species of Pythium. Pythium occurs more commonly in cultivated than in natural soils (Webster, 1980). Damping-off is caused by P. debaryanum, P. aphanidermatum and P. ultimum. Some Pythium species are also marine (Decock, 1986). Structure and life-history of Pythium debaryanum, the most common species, are discussed below:

Eukaryota Chromista Oomycota Oomycetes Pythiales Pythiaceae Mycelium Host cells

Fig. 9.12

Mycelium of Pythium in the rotting tissue of a seedling possessing sporangil initials and absence of haustoria.

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Vesicle

9.9.3 Somatic Structure The mycelium (Fig. 9.12) is well-developed and consists of fine, well-branched, hyaline, intercellular or intracellular hyphae (Fig. 9.13 A and Fig. 9.15 A) giving the appearance of a white fluffy mass. Generally, the lateral branch of the mycelium contains a slight constriction at its base. The hyphae do not produce any haustoria. The hyphal wall consists of cellulose (Alexopoulos and Mims, 1979), impregnated with chitin. The cytoplasmic contents are granular and contain oil droplets and glycogen. The older parts of the mycelium contain vacuolated cytoplasm. The young hyphae are coenocytic but cross-walls develop in the mature hyphae. Mitochondria, dictyosomes, endoplasmic reticulum and ribosomes are also seen if viewed under electron microscope.

9.9.4 Asexual Reproduction

Mature zoospores Vesicle

Tube

C

D

E Zoospores Encysted zoospores

Zoosporangium

F

B

Somatic hypha

G

Germinating zoospore

A

The asexual stage is constituted by sporangia, which may be interFig. 9.13 Asexual reproduction in Pythium debaryacalary or terminal. They may be globose (P. debaryanum, Fig. num. A, Somatic hypha; B, Zoosporangium; 9.13 B; P. proliferum), filamentous (P. gracile, P. monosperC, Vesicle formation; D, Zoospore formamum) or with inflated lobes (P. aphanidermatum). A sporantion; E, Liberated zoospore; F, Encysted gium contains a hyaline papilla. zoospore; G, Germinating zoospore. At the time of the sporangial development, the terminal or Zoospores intercalary portion of a hypha enlarges, becomes spherical and starts to function as a sporangial initial. It later on gets cut off by a cross-wall, thus enclosing numerous nuclei. In P. debaryanum a short tube develops from the sporangium (Fig. 9.13 C). A bubble-like vesicle is formed at the tip of this tube. At this stage the cleavage of the sporangial protoplast takes place. It flows rapidly into the vesicle through the tube (Fig. D C 9.13 C), and the zoospores are differentiated in the vesicle (Fig. 9.13 D) within 15-20 minutes. Newly formed zoospores keep on moving very rapidly inside the vesicle. This continues for a few minutes. The wall of the vesicle bursts suddenly like a soap bubble and the zoospores are liberated in all directions. The zooF spores are reniform or kidney-shaped and biflagellate bodies having their both the flagella attached on their lateral side (Fig. 9.13 E). After some time the zoospores become deflagellated, get encysted (Fig. 9.13 F) and each of them germinates by a B A germ tube (Fig. 9.13, G) into a new somatic hypha (Fig. 9.13 A). This young hypha infects a fresh seedling. E In P. aphanidermatum, however, a long tube develops from the sporangium (Fig. 9.14 A). The cytoplasm of the sporanFig. 9.14 A-F, Stages of the zoospore fromation gium moves into the vesicle, leaving the former into an empty in Pythium aphanidermatum condition (Fig. 9.14, B). The cleavage of the cytoplasm into uninucleate portions begins within the sporangium but completes within the vesicle (Fig. 9.14 C). The flagella start developing within the vesicle (Fig. 9.14 D). Rupturing of vesicle results in the liberation of zoospores (Fig. 9.14 E, F), which after some time cast-off their flagella, get encysted, and each germinates by means of a germ tube as in P. debaryanum.

99

Oomycetes

In some Pythium species the hyphal portions give rise to some intercalary, thick-walled and globose resting spores, called gemmae or chlamydospores. They germinate by producing long tubular hyphae.

9.9.5 Sexual Reproduction The sexual reproduction is oogamous. The two sex organs generally develop in close proximity on the same hypha. A majority of the species are homothallic. The antheridia generally develop below the oogonia (Fig. 9.15 B). However, certain species are heterothallic, e.g. P. heterothalicum and P. sylvaticum. Antheridia Oogonium

A

Germinating oospore

F

Empty antheridium

Fused nucleus

is

Diplophase os

Haplophase

M

ei

Oospore

B

Antheridia Functional female nucleus

E

Fusion

Oosphere C Oosphere Male nucleus

Fig. 9.15

D

Fertilization tube

Sexual reproduction in Pythium debaryanum. A, Somatic hyphae; B, Gametangial contact; C, Gametangia after meiosis (note 4 nuclei in each antheridium); D, Plasmogamy; E, Nuclear fusion and formation of oospore; F, Oospore germination

The oogonium in P. debaryanum generally develops at the tip of the hyphal branch, but sometimes it is also intercalary. It is spherical or globose and smooth-walled (Fig. 9.15 B). The oogonium gets cut off from the parent hypha by a septum in its early stages of development. At first, the swollen portion of the young oogonium remains filled with hyaline contents containing ribosomes, endoplasmic reticulum, dictyosomes, mitochondria, several vacuoles and nuclei. But later on its contents get differentiated into a central multinucleate ooplasm (Webster, 1980) and a peripheral multinucleate periplasm. The ooplasm gives rise to egg. The periplasm does not take part in egg formation.

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Different workers have suggested different views about the type of the nuclear division. According to many early workers the antheridial nuclei divide only mitotically during the egg development. It happens because the vegetative hyphae arc haploid, and the meiosis occurs at the time of oospore germination. But Sansome (1961, 1963) opined that in P.debaryanum meiosis occurs in oogonia and antheridia. According to him certain nuclei of oogonium degenerate, only 1-8 survive, enlarge and undergo meiosis in P. debaryanum. Therefore, an oogonium contains up to 32 nuclei in later stages. Of these 32 or less nuclei of ooplasm only one survives and all others degenerate. This surviving nucleus of ooplasm forms the egg. The nuclei of the periplasm also degenerate. The antheridia in P. debaryanum develop near the oogonia, generally on the same hypha (Fig. 9.15 B). They are smaller than oogonia and club-shaped or somewhat elongated bodies. One to six antheridia may remain attached on a single oogonium, even developing from the neighbouring hyphae and a few even from the oogonial stalk. In P. ultimum a single antheridium develops for one oogonium. At the time of antheridial development, a hyphal branch starts to function as a male branch. Its tip gets slightly inflated. The inflated portion gets separated by a cross wall and functions as an antheridium. The young antheridia are multinucleate. All antheridial nuclei, except one, degenerate. The surviving antheridial nucleus undergoes meiosis and forms four nuclei at the time of fertilization.

9.9.6 Fertilization Pythium exhibits an example of gametangial contact. The antheridia are applied to the wall of the oogonium and become flattened. From each antheridium develops a fine fertilization tube that penetrates the oogonial wall and periplasm, and reaches up to the egg (Fig. 9.15 C, D). Meiosis takes place in antheridium as well as oogonium in the mean time, and all haploid nuclei, except one, degenerate in both the gametangia (Fig. 9.15 D). Through the fertilization tube the functional male nucleus passes into the oosphere, reaches up to the functional female nucleus, fuses with it, and forms a diploid zygotic nucleus. The haploid oosphere thus changes into a diploid oospore, which is a smooth but thick-walled uninucleate structure. During this process the entire contents of the antheridium pass into the oogonium, and therefore the antheridium becomes empty after fertilization process (Fig. 9.15 E). Zoospores

9.9.7 Germination of Oospore In P. debaryanum and many other species the oospores require a resting period of several weeks before germination. At relatively high temperature of about 28°C the oospore geminates by putting out a germ tube, which soon develops into a well-developed vegetative mycelium (Fig. 9.15 F). But at lower temperatures between 10-17°C, a short germ tube (of 5-20 mm) is given out, at the tip of which develops a vesicle. The contents of the oospore pass into this vesicle through the small tube and get differentiated into many zoospores (Fig. 9.16). Webster (1980) mentioned a third category in which the oospore in some species develops a short germ tube containing a sporangium at its tip. The details mentioned above indicate that the vegetative mycelium in P. debaryanum is diploid and the reduction division takes place in the two gametangia.

Vesicle

Oospore

9.9.8 Damping-off It is a seedling disease caused by several species of Pythium, including P. debaryanum, P. aphanidermatum, P. arrhenomanes and P. ultimum. Seedlings of tobacco, cucurbits and other vegetables, many Poaceae and a variety of other hosts are invaded. According to Mehrotra (1980), damping-off of seeding occurs in almost every greenhouse of the world, and the damage in some areas can be 100% (Rangaswamy, 1984). Damping-off shows the following symptoms:

Fig. 9.16

Oospore germinating into a vesicle in Pythium ultimum (after Drechsler).

Oomycetes

101

1. In the pre-emergence phase the young seedlings are killed even before emergence from of the soil. It happens because of the killing of young radicle and plumule. 2. In the post-emergence phase the young seedlings become soft, and water-soaked lesions appear on their stem. It causes the stem to ultimately collapse or topple over to the ground. 3. The rotting ultimately involves even foliage and root system. 4. Rotting, toppling and killing of the young seedlings spread so rapidly that within 3-4 days the entire bed of seedlings gets damaged. Damping-off can be controlled partly by adopting the following measures: 1. By providing adequate drainage in the soil, without allowing irrigation or rain water to stagnate. 2. By spraying the nursery beds with 1% Bordeaux mixture, Blitox, or Wetcop, or 0.1% Ceresan. 3. By giving steam-treatment or pasteurization treatment to the soil of the greenhouse. 4. By fumigating with chloropicrin or methyl bromide. 5. By using chemicals, such as chloroneb (1,4-dichloro 2-5 dimethoxy-benzene) on the seedlings of tomato, bean, pepper, etc.

9.9.9

Other Diseases Caused By Pythium

This common disease of cucurbits is caused by Pythium aphanidermatum, P. butleri, and some other fungi such as Fusarium and Phytophthora. It occurs on bottlegourd (Lagenaria vulgaris), cucumber (Cucumis sativus), bitter gourd (Momordica charantia) and other cucurbits like muskmelon and watermelon. The main symptom is the appearance of a watery soft rot on the parts of the fruit touching the soil surface. The disease can be controlled partly by keeping the fruits away from the soil surface. This disease of papaya (Carica papaya) is caused by Pythium aphanidermatum, specially during rainy season. Its main symptom is the appearance of spongy, water-soaked patches on the stem, immediately at the soil line. The basal portion of the stem is girdled by the infection and gets rotted. This might result in the toppling of the entire tree. Stem rot can be controlled by growing the plants in well-drained soil. Affected plants should be removed and burnt. Spraying of Bordeaux mixture is also effective. The rhizome rot of ginger (Zingiber officinale) is caused by Pythium myriotylum, P. aphanidermatum, P. gracile, and some species of Fusarium and Pellicularia. The basal parts of the plant become watery and soft, and the leaves become pale or yellow. Ultimately, the rhizome starts rotting and changes into a pulpy mass. It can be controlled by treating the rhizome and the soil with copper fungicides. Selection of healthy seed pieces might also be effective.

9.10

PHYTOPHTHORA

9.10.1 Systematic Position

According to Ainsworth (1973) Same as of Pythium.

According to Kirk et al. (2001) Same as of Pythium.

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Fungi and Allied Microbes

9.10.2 Occurrence Phytophthora (Gr. phyton, plant; phthora, destruction) is represented by 65 species (Kirk et al., 2001). It is widespread and includes important pathogens of apple and pear (P.cactorum and P.syringae) causing fruit rot. It causes ink disease of chestnut (P.cambivora), gummosis and foot rot of citrus (P.citrophthora), downy mildew of lime beans (P.phaseoli), blight of potato and tomato (P. infestans), foot rot of tobacco (P. nicotianae), etc. (Kirk et al., 2001). The most common species is P. infestans, the causal organism of late blight of potatoes. Pregnant women have been warned recently by American press and a well-known British geneticist against the use of blighted potatoes. Such potatoes might cause serious birth defects like incomplete development of the spinal cord of the foetus. They have also advised (i) not to peel potatoes without wearing gloves; (ii) not to inhale the steam of boiling potatoes, and (iii) not to use decayed, discoloured or bruised potatoes. Decock (1986) reported one marine species of Phytophthora growing on decaying seaweeds, and Rajalakshmy et al. (1985) reported P. meadii causing leaf-fall disease of rubber plants in South India. Goodwin (1997) published the details of population genetics of Phytophthora while Phytophthora Diseases Worldwide has been published by Erwin and Ribeiro (1996) Undermentioned details mainly belong to the life-history of Phytophthora infestans,the most common species.

9.10.3

Somatic Structure

Haustorium

Host cell wall Somatic hypha A

Intercellular hypha

Mycelium B Papilla

H

Zoospore Basal plug

G

Haustorium Papilla C

F Basal plug

Zoosporangium

Sporangia

Sporangiophore Host cell Guard cell E Stoma D

Fig. 9.17

Phytophthora infestans. A, Somatic hypha; B-C, Intercellular mycelium with haustoria; D, Sporangiophores emerging through a stoma; E, Sporangiophores with disseminated sporangia; F, Sporangium showing cleavage; G, Zoospores; H, Germinating zoospore infecting the host.

The mycelium is tubular, coarse, hyaline, branched and coenocytic (Fig. 9.17 A). The mycelium is generally intercellular, but the haustoria are formed and penetrate the host cells. A hyphal branch generally shows a constriction at the point of its origin. The hyphae are 3-8 mm in breadth. The hyphal wall consists mainly of glucans. The cellulose is either feebly present or absent. The cytoplasm contains mitochondria, endoplasmic reticulum, ribosomes, dictyosomes, many oil globules, large vacuoles and many nuclei. A part of the intercellular hypha bulges into the host cell wall in the form of a slender, lateral outgrowth, which develops into a haustorium (Figs. 9.17 B, C, 9.18). The penetrating bulge first enlarges into a club-shaped head containing a narrow constricted region, called stalk. This hyphal bulge or the young haustorium invaginates the plasma membrane of the host. The haustorium remains surrounded by an extra-haustorial sheath, an extra-haustorial membrane and the cytoplasm of the host cell (Fig. 9.18). In P. infestans the haustoria have finger-like protuberances (Webster, 1980).

9.10.4

Asexual Reproduction

The internal mycelium generally comes out through the stomata in the form of tuft of sporangiophores (Fig. 9.17 D) in P. infestans. The sporangiophores may also come out by piercing through the epidermal cells, or through lenticels of tubers, or also through the injured portions of the infected part. The sporangiophores are hyaline, freely-branched and indeterminate. Their production depends on high humidity. A sporangium develops at the tip of each branch of sporangiophore. The first sporangium on each sporangiophore is therefore terminal, but the developing sporangiophore branch may push this sporangium to one side and proceed to form further sporangia. The sporangiophore is, therefore, sympodially branched.

103

Oomycetes

Each sporangium is a thin-walled, hyaline, multinucleate, pear-shaped or oval body, and contains a terminal papilla or beak-like projection (Fig. 9.17 E). It remains separated from the sporangiophore by a thickening of the wall material, which develops into a basal plug (Fig. 9.17 E). The mature sporangia (Plate 2 A) are detached and dispersed by wind, rain splashes, contact or even slight movements. They remain viable only for a few hours. The germination of sporangium is governed by temperature and moisture. The sporangia show indirect germination in low temperatures and wet conditions by first producing zoospores, which are soon liberated and infect the host. But in high temperatures and dry conditions the sporangia show direct germination, where the whole sporangium starts to behave as a single spore and quickly germinates by producing a germ tube, which penetrates the host.

Host cell wall

Host cell plasma membrane

Fungal cytoplasm

Host cell cytoplasm Haustorium Host cell microbody Host cell tonoplast Extra haustorial membrane

Nucleus

Hyphal plasma membrane

Fig. 9.18

Host cell vacuole Mitochondria

Haustorium wall

Host cell dictyosome

Extra haustorial matrix

Phytophthora infestans, showing the ultrastructure of the haustorial apparatus (based on Hohl and Stossesl).

In the presence of moisture and at temperatures below 15°C the sporangium starts to function as a zoosporangium (Fig. 9.17 F). Cleavage of the cytoplasmic contents results in the formation of uninucleate zoospore initials (Photoplate 2). Golgi apparatus helps in the formation of cleavage vesicles (Webster, 1980). Each uninucleate zoospore initially develops into a laterally biflagellate reniform zoospore (Fig. 9.17 G; Photoplate 2). The optimum temperature for indirect germination is 12°C (Alexopoulos and Mims, 1979). The zoospores are released either by the breakdown of the papilla, or by the first formation of a vesicle from the papilla and then passing of the zoospores into it before release. According to Webster (1980) the zoospores are attracted chemotactically to the host tissue. Liberated zoospores swim in water film on the host, come to rest by withdrawing their flagella, get encysted, and each produces a fine germ tube. The latter penetrates the host generally through the stoma (Fig. 9.17 H). But in many cases the germ tube comes in contact with the leaf epidermal cell. At the point of contact develops a small swelling or appressorium, which provides the way to the penetrating germ tube into the host epidermis. The germ tube develops into intracellular or intercellular, coenocytic mycelium within the host tissue. The sudden disastrous effects of Phytophthora infection are observed because a number of asexual Anterior Water expulsion vacuole generations may be produced in one growing season if favourable Straminipilous flagellum conditions persist for the growth of the pathogen. In high temperatures and dry conditions the sporangium starts to behave as a single spore and germinates directly by forming a multinucleate germ tube. The zoospores are not formed. In P. infestans each sporangium shows direct germination into a multinucleate germ tube (Fig. 9.20 A-C) above 20°C. However,the optimum temperature for direct germination is 24°C (Alexopoulos and Mims, 1979). The resorption of flagella preceds direct germination. A new inner wall (germination wall) develops between the plasma membrane and the sporangial wall. The germ tube comes out in the form of a vesicle or bulge and develops into well-developed mycelial branch. Longitudinal section of a zoospore of Phytophthora, as suggested by Dick (2001), is shown in Fig. 9.19.

Dorsal

Ventral Kinetosome boss Kinetosome Ventral groove

Nucleus

Whiplash flagellum Posterior

Fig. 9.19

Diagrammatic illustration of L.S. of a zoospore of Phytophthora (Modified from Dick, 2001).

104 9.10.5

Fungi and Allied Microbes

Sexual Reproduction

According to Galindo and Gallegly (1960), Phytophthora is heterothallic and requires two mating types for sexual reproduction. However, Webster (1980) mentioned that P. cactorum and P. erythroseptica are homothallic species, whereas P. infestans and P. palmivora are heterothallic. The sexual reproduction in heterothallic species is controlled by hormones and chemicals. The sexual reproduction is oogamous. The two sex organs (antheridia and Germ tube oogonia) develop as the terminal hyphal swellings which are later on separated by a septum from the remaining part of their respective hyphae of different strains. In P. infestans (Fig. 9.21 A-D) the oogonium punctures the antheridium (Fig. 9.21 A), passes through it and comes out in the form of a globose structure (Fig. 9.21 B) above the antheridium. Around the base of the mature oogonium, the antheridium is thus present in the form of a funnel-shaped collar. C Such an antheridial arrangement is called amphigynous. A B P. erythroseptica (Fig. 9.21 E-I) and P. capsici also show amphigynous condition like P. infestans. Here the oogonial hyphae also penetrate the antheridium (Fig. 9.21 E), grow through it and come out in the form of a spherical Fig. 9.20 A-C. Phytophthora infestans, showing direct oogonium (Fig. 9.21 F-H). The antheridium persists as a collar at the oogonial germination of sporangium base. In P. capsici, P. erythroseptica and P. infestans the antheridia as well as oogonia are multinucleate bodies. The oogonial protoplast is differentiated into an outer periplasm and a central ooplasm. All the nuclei of the ooplasm, except one, move towards the periplasm. The remaining ooplasm nucleus matures into an egg or oosphere nucleus. In P. cactorum (Fig. 9.21 J-N) the antheridia are not punctured or penetrated by oogonia. Here the antheridia remain attached laterally to the oogonium. This arrangement of antheridium is called paragynous. Here the antheridial and oogonial initials (Fig. 9.21 J) get inflated (Fig. 9.21 K) and the antheridium gets laterally attached to oogonium. According to Blackwell (1943) the antheridium has about 9 nuclei and the oogonium about 24. A septum develops at the base of each sex organ (Fig. 9.21 L). Some of the nuclei of both the sex organs are degenerated, leaving only 4-5 in the antheridium and 8-9 in the oogonium. The surviving nuclei also show some divisions. Large vacuoles develop in the protoplasm. The oogonial protoplasm gets differentiated into ooplasm and periplasm. At this stage the oogonium presses into the antheridium (Fig. 9.21 M). A fertilization tube develops and one of the antheridial nucleus enters into the oogonium through this tube (Fig. 9.21 N). The fusion results in the formation of an oospore. The protoplasm and the remaining antheridial nuclei degenerate.

9.10.6 Fertilization Alexopoulos and Mims (1979) mentioned that fertilization has not been so far observed in P. infestans. Hemmes and BartnickiGarcia (1975), of course, observed the fertilization tube entering into the oogonium in P. capsici but they also did not observe the actual fertilization event. However, it does not mean that fertilization does not take place. It, of course, does take place, perhaps in the following manner: At the time of fertilization the oogonial wall bulges into the antheridium at one point. This point represents the receptive spot. The contact wall near the receptive spot dissolves, and through this opening the antheridium pushes a short fertilization tube. The latter penetrates the periplasm, reaches up to the ooplasm, opens its tip and brings the male nucleus near the female nucleus. The male and female nuclei fuse, and this completes the fertilization process. A thick wall is secreted around the fertilized egg, which is now called oospore or resting spore. Parthenogenetic development of oospores, in the absence of antheridia, has also been observed in P.infestans.

105

Oomycetes

Antheridia

Oogonium

Oogonium

Oospore

A

B

D

C

Antheridia

Empty antheridium

Oogonia

Oogonial hypha

F

E

Oogonium Antheridia H Oogonial hyphae

Fertilization I tube

G Oogonium

Septum

Antheridium

L K

J Periplasm

Female nucleus Male nucleus

Ooplasm

Fertilization tube M

Fig. 9.21

N

Sexual reproduction in Phytophthora. A-D, In P. infestans; E-I, In P. erythroseptica; J-N, In P. cactorum (A-D, Modified after Smoot et al.; E-I, Modified after Webster; J-N, Modified after Blackwell).

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Fungi and Allied Microbes

9.10.7 Germination of Oospore For germination, the oospores require a maturation period lasting several weeks or months (Webster, 1980). Each oospore in P. infestans germinates by producing a germ tube, and at the tip of the germ tube develops a sporangium. The multinucleate sporangium produces many uninucleate biflagellate zoospores like that of asexual reproduction. These zoospores get encysted and germinate into new somatic hyphae.

9.10.8 Where does Meiosis occur in Phytophthora? The exact stage of the occurrence of meiosis in Phytophthora is still not known (Alexopoulus and Mims, 1979). Webster (1980) mentioned that meiosis occurs at the time of the germination of diploid oospore, and the sporangium present at the tip of the germinating oospore thus bears haploid zoospores. On the contrary, many workers (Elliott and Macintyre, 1973; Mortimer and Shaw,1975; Sansome, 1976) reported that the meiosis occurs in the gametangia (antheridium and oogonium) during gamete formation. Therefore, the zoospores arc diploid. According to Day (1974) the place of meiosis in the life-cycle of Phytophthora and many other Oomycetes is still not clear.

9.10.9 Differences from Pythium Table 9.2 S.No. 1. 2. 3. 4. 5. 6. 7.

8. 9.

Differences between Phytophthora and Pythium

Phytophthora

Pythium

Average width of the hyphae is 6 mm, with a maximum of 14 mm (Waterhouse, 1973). Hyphall wall contains little amount of protein.

Average width of the hyphae is 5 mm, with a maximum of 10 mm (Waterhouse, 1973). Hyphal wall contains greater amount of protein (BartnickiGarcia, 1968). Haustoria are always present. Haustoria are absent. Sporangia develop on specialized aerial hyphae, called Sporangiophores are indistinguishable from the somatic sporangiophores. hyphae of the mycelium. Sporangia are always terminal. Sporangia are either terminal or intercalary. Sporangia are limoniform, obpyriform or ovoid. Sporangia are hyphal, spherical and rarely ovoid. Zoospores are fully differentiated within the Zoospores are not differentiated inside the sporangium; sporangium itself; vesicle is formed only rarely. undifferentiated sporangial contents are extruded into a vesicle in which the zoospores are differentiated. Appresoria may be formed. Appresoria are not formed. Oogonial wall is brownish, rough, warted, and never spiny. Oogonial wall is hyaline, smooth and spiny.

9.10.10 Leaf Blight of Colocasia Phytophthora colocasiae is the causal organism of the leaf blight and corm rot of Colocasia antiquorum (arvi, family Araceae). Leaves as well as inflorescence are attacked, and the blight appears in the form of many characteristic large concentric rings (Fig. 9.22 A.B). Clear, yellow liquid oozes from the infected portion, and ultimately the central portions become dry and drop off. Holes are thus formed in the leaves. To control the Colocasia-blight, healthy corms should be sown. Spray of Bordeaux mixture, Blitox-50 or Dithane Z-78 also provide satisfactory results. Certified blight-resistant varieties, if available, should be used as seed.

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9.11

These are obligate parasites of many higher plants, and are responsible for a group of diseases, called downy mildews. The mycelium is coenocytic and intercellular. The sporangiophores or conidiophores are well-branched and tree-like, with determinate growth. At the tip of each sporangiophore or conidiophore is present a single sporangium or conidium. The periplasm is well-developed and persistent. The haustoria are generally well-branched or lobed. Waterhouse (1973) described 7 genera (Basidiophora, Sclerospora, Peronospora, Plasmopara, Pseudoperonospora, Bremia and Bremiella). Kirk et al. (2001) included 8 genera and 208 species under Peronosporaceae. Some life-history details of Peronospora, Sclerospora and Plasmopara are given here in this account.

9.12 9.12.1

A

Flower

Infected parts

B

Fig. 9.22

Phytophthora colocasiae on Colocasia antiquorum. A, On leaf; B, On inflorescence

PERONOSPORA Systematic Position

According to Ainsworth (1973) Division Sub-Division Class Order Family Genus

9.12.2

Infected parts

Leaf

FAMILY PERONOSPORACEAE (DOWNY MILDEWS)

– – – – – –

Eumycota Mastigomycotina Oomycetes Peronosporales Peronosporaceae Peronospora

According to Kirk et al. (2001) Subkingdom Kingdom Phylum Class Order Family

– – – – – –

Eukaryota Chromista Oomycota Oomycetes Peronosporales Peronosporaceae

Life History

Peronospora is by far the largest genus of Peronosporaceae represented by over 75 species. Commonly called “downy mildews”, its species occur on clover (P. trifoliorum), crucifers (P. parasitica), tobacco (P. hyoscyani sp. tabacina), etc. (Kirk et al. 2001). Some of its other species with the diseases caused by them in the brackets are P. pisi (downy mildew of Pisum sativum), P. brassicae (downy mildew of crucifers), P. lathyri-patustris (downy mildew of Lathyrus sativus), P.spinaciae (downy mildew of Spinacea oleracea), P. viciae (downy mildew of Pisum sativum, Vicia hirsuta and Lens esculenta) and P. farinosa (downy mildew of beetroot and spinach), The fungus remains associated on the upper surface of the leaves of many crucifers and other hosts in the form of yellowish patches. White sporangiophores are seen of the lower surface of leaves. The stems of the diseased host become swollen, thickened, distorted, and bear a whitish or yellowish-white fur of sporangiophores. The infected shoots show deformed growth and become pale green in colour. Their leaves appear shorter than the normal. The mycelium of Peronospora parasitica is well-developed, branched, intercellular and coenocytic. The haustoria are well-branched and lobed (Fig. 9.23 A). Haustoria are completely or partially surrounded by a sheath, secreted by the host. The haustoria are finger-shaped in P. effusa, and elongate or richly-branched in P. calotheca.

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Sporangia Lobed haustorium Asexual reproduction takes place by sporangia developing on sporangiophore. The sporangiophores come out through stomata either singly or in groups. Each sporangiophore is a stout hyphal branch showing repeated dichotomous branching (Fig 9.23 B). The ultimate branches are curved with generally pointed tips. A sporangium develops at the tip of each of these curved branches. The sporangia are oval or elliptical in shape. They are deciduous and Host cells discharged by wind or rains (Hawker, 1966). The discharged sporangia germinate by the formation of an apical germ tube on falling on a suitable host. The Sporangiophore A B zoospores are not formed. Oogonium Receptive Antheridium Sexual reproduction is oogamous, and takes place papilla Oospore Septum in the adverse environmental conditions by producMale nucleus ing antheridia and oogonia. The sex organs develop as terminal swellings of closely adjoining hyphal tips. Both the sex organs are multinucleate when young. Fertilization Female nucleus Slightly mature sex organs get separated from the reOogonium Periplasm Ooplasm tube maining mycelial portion by a septum (Fig. 9.23 C). E C D Antheridium is tubular paragynous, multinucleate when young, and get separated by septum. Accord- Fig. 9.23 A-E, Peronospora parasitica. A, Part of mycelium with lobed haustorium; B, A sporangiophore showing ing to Sansome and Sansome (1974) the antheridium dichotomous branching; C, Sex organs; D, Process is diploid. Its nuclei divide first reductionally and of fertilization showing fertilization tube; E, Oospore then ordinarily. All the haploid nuclei, except one, showing fusing male and female nuclei. degenerate at maturity, leaving the antheridium as a uninucleate structure. Oogonium is globose, and its protoplast gets differentiated into central ooplasm and peripheral periplasm. Sansome and Sansome (1974) opined that the oogonium is diploid. The reduction of chromosomes occurs in the oogonium. Nuclei are evenly distributed in the ooplasm and periplasm in the early stages. But later on all the nuclei, except one, migrate towards periplasm, where they all finally degenerate. The uninucleate ooplasm functions as an oosphere or egg. Fertilization is affected by the development of a fertilization tube (Fig. 9.23 D) by the anteridium. The tube pierces the oogonial wall, enters into the ooplasm, dissolves its tip, and finally discharges the male nucleus into the oosphere. The male and female nuclei fuse and develop into diploid oospore (Fig. 9.23 E). Under suitable conditions the oospore germinates by producing a germ tube, which brings about fresh infection. Sansome and Sansome (1974) opined that the mycelium, thus formed by the germination of the oospore, would be diploid because the reduction of chromosomes occurs in the sex organs.

9.13 9.13.1

SCLEROSPORA Systematic Position

According to Ainsworth (1973) Division Class Order

– – –

Eumycota Oomycetes Peronosporales

According to Kirk et al. (2001) Superkingdom Kingdom Phylum

– – –

Eukaryota Chromista Oomycota

109

Oomycetes

Family Genus

9.13.2

– –

Peronosporaceae Sclerospora

Class Order Family Genus

9.14.1

Oomycetes Sclerosporales Sclerosporaceae Sclerospora

Life History

The Sclerospora of Peronosporales (Ainsworth, 1973) or of order Sclerosporales (Kirk et al. 2001) is represented by 13 species (Waterhouse, 1964), of which the common Indian species are Sclerospora graminicola, S. philippinensis and S. sorghi. S. graminicola causes green-ear disease of Bajra (Pennisetum typhoides, Fig. 9.24 A), whereas S. sorghi causes downy mildew of Jowar (Sorghum vulgare) and S. philippinensis, the downy mildew of Makka (Zea mays). The genus is confined to Gramineae, and its main hosts are maize, sorghum, sugarcane and millets. According to Kirk et al. (2001), genus Sclerospora of family Sclerosporaceae contains only 3 species. The mycelium is well-developed, branched, intercellular, coenocytic, and contains digitate or vesicular haustoria. Asexual reproduction takes place by sporangia, developing exogenously on long, stout sporangiophores. Upper part of the sporangiophore is more or less dichotomously branched, the tips of which represent sterigmata. Waterhouse A (1973) has used the term conidiophore instead of sporangiophore. According to her the conidiophores have a basal “cell” differing in length and the Fig. 9.24 bulbosity of the base as well as in the length of their sterigmata and shape of conidia in different species. The conidia are smooth, colourless, spherical to cylindrical or oval (Fig. 9.24 B). Sexual reproduction is oogamous.The oogonial wall is ornamented and the oospore wall is smooth. The oogonia are sometimes deeply coloured, and some are even brown. Green-ear disease has been discussed in detail in Chapter 35 (Article 35.3).

9.14

– – – –

Conidia Conidiophore

B

Sclerospora graminicola. A, Green ear disease of Bajra (Pennisetum typhoides); B, Conidiophore with conidia.

PLASMOPARA Systematic Position

According to Ainsworth (1973) Same as of Peronospara.

According to Kirk et al. (2001) Same of Peronospora. Plasmopara viticola is the causal organism of the “downy mildew of grapes” (Vitis vinifera). 109 species of this fungus have so far been reported, of which P. viticola (Vine downy mildew) and P.halstedii (downy mildew of sunflowers) are common species (Kirk et al. 2001). Some other common species include P. nivea causing downy mildew of carrot, P. pusilla causing downy mildew of Geranium pratense, and P. pygmaea forming yellow patches on the leaves of Anemone nemorosa.

110 9.14.2

Fungi and Allied Microbes

Life History

The mycelium of P. viticola is well-branched, intercellular coenocytic and contains knob-like haustoria (Fig. 9.25 A). In the substomatal cavities of the host the hyphae form mycelial pads. From the latter develop the upright group of hyphae, which emerge through the stomata and function as sporangiophores. Each sporangiophore branches randomly and not dichotomously, and its later branches emerge at right angles or nearly so. The ultimate branchlets of sporangiophores are very short, truncate (Fig. 9.25 B) and often called sterigmata. At the tip of each of the sterigma develops a sporangium. The sporangia are ovate and hyaline. Each sporangium germinates (Fig. 9.25 C) by producing biflagellate zoospores (Fig. 9.25 D), which germinate and enter the host tissue (Fig. 9.25 E), causing fresh infections.

Sporangia

Sporangiophore Mycelium Germinating sporangium

Zoospores

A Mycelial pad

Host cell

C

B

Zoospore Germinating zoospore

Oogonium F

D

Antheridium

is

ios

Me

E

Diplophase Haplophase Oogonium Antheridium

G

J

Oospore

Fertilization tube

n sio

Fu H

I

Fig. 9.25

A–J, Showing life-cycle stages of Plasmopara viticola

111

Oomycetes

The sexual reproduction is oogamous. The oogonium and antheridium lie just adjacent to each other (Fig. 9.25 F). The oogonium is spherical and remains differentiated into ooplasm and periplasm. At maturity the ooplasm develops into an egg (Fig. 9.25 G). The antheridium is clavate or club-shaped structure. Alexopoulos and Mims (1979) mentioned that both the sex organs are diploid bodies, and the reduction division takes place before gamete formation. Fertilization tube is formed (Fig. 9.25 H). In P. viticola the oospore germination also results in the formation of diploid beflagellate zoospores (Fig. 9.25 I-J), which germinate and infect the host tissue.

9.15

FAMILY ALBUGINACEAE

Similar to Peronosporaceae, the members of Albuginaceae are also obligate parasites having intercellular mycelium. But they differ from those of Peronosporaceae in possessing clavate and unbranched sporangiophores. The sporangia are deciduous and are produced in basipetal chains. The groups of sporangia and sporangiophores form subepidermal clusters of white or creamish sori of the host. Albuginaceae contain well-developed and persistent periplasm. All the species investigated so far contain knob-like haustoria (Waterhouse, 1973). The fungi included under Albuginaceae are called ‘white rusts’. Albuginaceae contains only a single genus, Albugo.

9.16 9.16.1

ALBUGO Systematic Position

According to Ainsworth (1973) Division Sub-Division Class Order Family Genus

9.16.2

– – – – – –

Eumycota Mastigomycotina Oomycetes Peronosporales Albuginaceae Albugo

According to Kirk et al. (2001) Superkingdom Kingdom Phylum Class Order Family

– – – – – –

Eukaryota Chromista Oomycota Oomycetes Peronosporales Albuginaceae

Occurrence

Albugo is represented by about 44 species (Kirk et al. 2001), which are widespread and cause the diseases ‘white rust’ or ‘white blisters’ of many vascular plants. All species are obligate parasites. Albugo candida (= Cystopus candidus) is the commonest species throughout the world, occurring on a number of species of Cruciferae, causing ‘white rust of Crucifers’. Its common hosts are cabbage, turnip, Indian mustard (sarson), Shephered’s purse, horse-radish, etc. Some other common species of Albugo are A. bliti on Amaranthaceae, A. ipomoea-panduratae on Convolvulaceae and A. trapogonosis on Compositae (Waterhouse, 1973). Alexopoulos and Mims (1979) mentioned that A. occidentalis occurs on spinach, and A. portulacae on Portulaca.

9.16.3 White Rust of Crucifers Discussed in detail in Chapter 35, Article No. 35.1.

112 9.16.4

Fungi and Allied Microbes

Somatic Structure

The mycelium is well-developed, branched, intercellular, aseptate and coenocytic (Fig. 9.26 A). The hyphall wall consists of cellulose. The protoplasm of the hyphae is granular and contains many nuclei, oil globules and glycogen. The nuclei are about 2.5 mm in diameter. The ultrastructure of the vegetative hypha reveals the presence of many mitochondria, endoplasmic reticulum and ribosomes. From the plasma membrane of the hyphae develop a system of unit membranes and tubules towards the fungal cytoplasm. This system represents lomasomes. From the intercellular mycelium develop many spherical or knob-like haustoria, which penetrate into the host cell (Fig. 9.26 B) and absorb the food. Glycogen bodies

Mycelium

Nuclei Vacuole Oil drops

Haustorium

A

B

Fungal cell wall

Host cell

Host cell wall Lomasomes Fungal plasma membrane Fungal cytoplasm Sheath

Host cell vacuole

Fungal cell wall

Lomasomes Fungal cytoplasm

Fig. 9.26

Host cell Host vacuole cytoplasm

C

Host plasma membrane

Albugo, A, Diagrammatic structure of the mycelium; B, A part of the intercellular mycelium showing knob-like haustoria; C, Diagrammatic representation of the ultrastructure of a haustorium of Albugo candida (C, after Berlim and Bowen, 1964).

9.16.5 Ultrastructure of Haustorium Berlin and Bowen (1964) and Coffey (1975) studied the ultrastructure of the haustorium of Albugo candida (Fig. 9.26 C). The haustoria are spherical or somewhat flattened structures, made up of haustorial head of about 4 mm in diameter and a slender neck-like stalk of about 0.5 mm width. Lomasomes are more developed than that of intercellular hyphae. The fungal cytoplasm remains surrounded by the plasma membrane and fungal cell wall. In the haustorial head, the cytoplasm contains many mitochondria, endoplasmic reticulum, ribosomes and lipid granules. A collar-like sheath surrounds the haustorial base. This sheath is the extension of the host cell wall and does not generally surround the remaining portion of the stalk and haustorial head. The haustorium is separated from the host plasma membrane by an encapsulation made up of extra-haustorial matrix. Hence, the haustorium remains surrounded completely by host plasma membrane. The plasma membrane of the parent hypha is continuous throughout the haustorium. The fungal cell wall is discontinued (Berlin and Bowen, 1964) in the distal region of the stalk of the haustorium (Fig. 9.26 B). However, Coffey (1975) showed that the fungal cell wall remains as a continuous layer throughout in the stalk as well as in the haustorial head in A. candida.

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9.16.6

Asexual Reproduction

After attaining a certain maturity the intercellular mycelium forms a sort of palisade of hyphae below the host epidermis. The tips of these hyphae develop into short, erect, thick-walled and club-shaped structures, called sporangiophores (Waterhouse, 1973; Webster, 1980). Instead of sporangiophores and sporangia, some workers prefer to call them conidiophores and conidia. Many such sporangiophores lie perpendicular to the host surface, and they are so closely packed that they appear as a palisade-like layer beneath the host epidermis (Fig. 9.27 A). The apical end of the sporangiophore is multinucleate, thin-walled and contains dense cytoplasm. It enlarges or swells. A deep constriction appears below the swollen end, which results in the formation of a multinucleate and spherical or oval sporangium. In the same manner another sporangium is formed by the tip of the sporangiophore. The process is repeated several times and a chain of sporangia develops on the sporangiophore in basipetal succession, i.e. youngest sporangium at the base and oldest at the top of the chain. In between the successive sporangia develop Sporangia

Ruptured host epidermis

Sporangiophores Oldest sporangium

Mycelium

Haustorium

A Sporangia Disjunctor

Host cell

Youngest sporangium

Somatic hypha Sporangiophore

Haustorium

B

Asexual Reproduction

I

Germinating sporangium C

Germinating zoospore

D

H E Zoospores

Deflagellated zoospore Zoospore

Fig. 9.27

G

F

Asexual reproduction in Albugo candida. A, Mycelium with chains of sporangia and sporangiophores; B, Sporangiophore with chains of sporangia (note disjunctor in between the sporangia); C-E, Germination of sporangia and release of zoospores; F, Group of biflagellate zoospores; G-H, Germination of zoospore; I, Formation of intercellular mycelium.

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Fungi and Allied Microbes

a mucilaginous pad or disc, called disjunctor (Fig. 9.27 B). The pressure of the developing sporangia first raises the epidermis of the host and finally ruptures it (Fig. 9.27 A). The sporangia are now visible on the host surface as a white powdery mass. The sporangia are smooth, colourless or nearly so, multinucleate, and spherical or shortly ellipsoid, cylindrical, 12-25 mm in diameter (Waterhouse, 1973). According to Alexopoulos and Mims (1979) the sporangia are generally globose, but may be cuboid or polyhedral. On the basis of the form of sporangium, Waterhouse (1973) divided the genus Albugo into two groups: 1. Aequales : Sporangia with an uniformly thin wall. 2. Annulati: Sporangia with an internal equatorial thickening on the wall. Sporangia are dispersed from the white rusty pustules or blisters of the pathogen, by the shrinking and drying of the mucilaginous disc. The sporangia get separated and first become accumulate in the space between sporangiophores and host epidermis, and are then blown away by wind or washed away by the rain water. After landing on a suitable host the sporangia start germination within a few hours under suitable conditions. Detailed structures of mycelium, sporangiophores and chains of sporangia formed under ruptured epidermis of the host has been shown in Fig. 9.28. Under moist condition and at low temperatures (approx. Sporangia 10° C) the sporangium behaves as a zoosporangium. Ruptured It swells by absorbing some water, and its multinucleepidermis ate protoplast divides to form 4-12 polyhedral daughter protoplasts, each of which ultimately develops into a Sporangiophores zoospore. Side by side a papilla also protrudes from the sporangium (Fig. 9.27 C). The papilla enlarges (Fig. 9.27 D) and the young 4-12 zoospores are released (Fig. 9.27 E) in a sessile vesicle (Vanterpool, 1959). The zoospores are about 8 per sporangium Mycelium (Webster, 1980). The zoospores are first released in a group (Fig. 9.27 F). The entire process of differentiation and final release of zoospores from the sporangium takes Fig. 9.28 Albugo candida on Capsella bursa-pastoris showplace only within 2 min. (Webster,1980). ing details of mycelium, sporangiophores and chains of sporangia under ruptured epidermis. Each zoospore (Fig. 9.27 F, G) is a biflagellate structure. Of the two flagella, one is short and of tinsel type, whereas the other is large and of whiplash type. The released zoospores settle on some suitable host, become deflagellated, and each of them secretes a wall. The encysted zoospore forms a germ tube (Fig. 9.27 H), which penetrates the host epidermis and develops into a fresh mycelium (Fig. 9.27 I). Under dry conditions and at high temperature the sporangium germinates directly into a germ tube, without forming any zoospore. However, this type of direct germination of sporangium is uncommon in Albugo.

9.16.7

Sexual Reproduction

The sexual reproduction is oogamous. The sex organs (antheridia and oogonia) develop quite deep into the tissues of the stem or petiole by the penetration of the hyphae, generally towards the end of the growing season of the host. Both the sex organs develop near each other. The presence of sex organs in the host is externally indicated by hypertrophy and deformation of the particular organ. The oogonia develop in the intercellular spaces of the host by the inflation or swelling of the female hyphae. Each such inflated hyphal tip is multinucleate and gets separated by a cross wall just below the inflation. The separated swollen portion represents the oogonium. The cytoplasm within such young oogonia is uniformly vacuolate and the nuclei are evenly

115

Oomycetes

distributed throughout (Fig. 9.29 A). In the later stages its central portion becomes dense, round, and represents ooplasm. The nuclei keep on dividing in the ooplasm and migrate towards its periphery. In the peripheral region of the ooplasm the spindles of many dividing nuclei are so oriented that one of the daughter nucleus remains in the region of the denser ooplasm, whereas the other moves towards the outer region. At this stage, the outer peripheral region of the oogonium Oogonium

Periplasm A

L Zoospore

Antheridia

K J

Ooplasm B

Zoospores Fertilization tube Vesicle

Male and female nuclei

Tube I

Periplasm Sexual Reproduction

Ooplasm

C Antheridium

Oospore Zygotic nucleus

Zoospores

Sessile vesicle

D Oospore Haustorium

H Vesicle

Host cell

G Oospore

Fig. 9.29

F

Mycelium

E

Sexual reproduction in Albugo candida. A, Young sex organs before any differentiation; B, Oogonium showing differentiation of ooplasm and periplasm; C, Oogonium just after fertilization; D-E, Different stages of oospores; F-G, Germination of oospore; H, Formation of sessile vesicle; I, Tube at the base of vesicle; J, Zoospore; K-L, Germination of zoospore.

116

Fungi and Allied Microbes

becomes more vacuolated and represents periplasm (Fig. 9.29 B). The ooplasm represents the egg. Many nuclei are present in the egg when it is first delimited from the periplasm (Fig. 9.29 B). But soon all egg nuclei, except one, disintegrate in A. candida (Alexopoulos and Mims, 1979). However, according to some other workers (Webster,1980) all the egg nuclei migrate to the periphery and are included in the region of the periplasm. Whether all but one nuclei in the ooplasm disintegrate or all of them except one migrate from the ooplasm towards periplasm, ultimately only one functional nucleus survives in the egg in A. candida. The antheridia are elongated, club-shaped and multinucleate bodies, each developing at the end of a male hypha lying very close to an oogonium (Fig. 9.29 A). The swollen antheridial tip is soon cut off by a septum. Out of its many nuclei only one remains functional.

9.16.8 Where does Meiosis occur in Albugo? Thirumalachar et al. (1949) reported meiosis in the zygotic nucleus in some species of Albugo. Smith (1955) also reported that the nuclear divisions in the sex organs (antheridia and oogonia) are all mitotic, and meiosis is seen in the diploid zygotic nucleus at the time of its germination. However, Stevens (1899) stated that two nuclear divisions of the gametangial (antheridia and oogonia) nuclei represent meiosis. In all the recently investigated species of Albuginaceae as well as other Oomycetes (Alexopolous and Mims, 1979), meiosis occurs in gametangia and not in the zygotic nucleus. Findings of Sansome and Sansome (1974) in Albugo candida also suggest the occurrence of meiosis in gametangia (oogonium and antheridium), confirming the old view of Stevens (1899). The zygotic nucleus, therefore, divides mitotically and not meiotically.

9.16.9

Fertilization

In A. candida, a slender fertilization tube is formed by the antheridium at the place of its contact with the oogonium. This tube grows through the oogonial wall, and passing through the periplasm it penetrates deeply into the egg. Through this fertilization tube the functional male nucleus enters the egg, fuses with the functional female (Fig. 9.29 C) nucleus and results in the formation of diploid zygotic nucleus (Fig.9.29 D). The fertilized egg secretes a thick wall around itself (Fig. 9.29 D, E) and changes into an oospore.

9.16.10 Oospore The oospore is uninucleate. It remains surrounded by a thick ornamented wall. The oospore wall consists of two or three layers, the outermost of which is thick, warty, tuberculate or with many other patterns. In A. candida the outermost layer (exospore) is warty and brown. The inner two (mesospore and endospore) are thin layers (Fig. 9.29 D). The single oospore nucleus is diploid.

9.16.11 Germination of Oospore Before germination the oospore undergoes a long resting period of several months (Webster, 1980). During this period the host tissue first shows hypertrophy and then might decay, setting ultimately the oospores free. The zygotic diploid nucleus shows repeated divisions to form as many as 32 nuclei. Some workers still believe that diploid zygotic nucleus divides meiotically at this stage, but majority of the workers (Sansome and Sansome, 1974; Alexopoulos and Mims, 1979; Webster, 1980) support the view that this zygotic diploid nucleus divides only mitotically, forming only diploid nuclei. At the time of its germination in the following spring, the outer oospore wall bursts and the inner endospore comes out in the form of a thin spherical vesicle (Fig. 9.29 F, G). According to Vanterpool (1959) the vesicle may be sessile (Fig. 9.29 H) or develop at the end of a wide cylindrical tube (Fig. 9.29 I). Inside the vesicle hundreds of zoospores are extruded in a mass. However, Webster (1980) mentioned that only 40-60 zoospores are differentiated. The vesicle is soon dissolved and the zoospores swim freely in all directions. Each of the zoospores so formed is uninucleate, reniform and biflagellate (Fig. 9.29 J). On reaching a suitable host, their flagella are withdrawn. They get encysted (Fig. 9.29 K) and germinate by procuring germ tubes (Fig. 9.29 L). The mycelium, so formed, would therefore be diploid.

Oomycetes

TEST YOUR UNDERSTANDING 1. Kirk et al. (2001) discussed Oomycetes under phylum Oomycota of kingdom _______ . 2. According to Ainsworth (1973) all Oomycetes contain: (a) uniflagellate zoospores (b) biflagellate zoospores (c) multiflagellate zoospores (d) no zoospores. 3. Majority of Oomycetes are aquatic fungi and live parasitically on: (a) algae (b) water moulds (c) aquatic insects (d) all of these. 4. Write any five general characteristics of Oomycetes. 5. What do you mean by the term diplanetism? 6. How can you culture Saprolegnia in laboratory? 7. Describe asexual reproduction in Saprolegnia. 8. Explain phenomenon of diplanetism with reference to Saprolegnia. 9. Explain the role of hormones in the formation of sex organs in Achlya. 10. Write any five distinguishing features or Peronosporales. 11. Tabulate three differences between Pythiaceae, Peronosporaceae and Albuginaceae. 12. The common disease, “damping-off”, of seedlings is caused by _______ . 13. Describe in brief the life-history of Pythium debaryanum. 14. Give an illustrated account of asexual reproduction in Phytophthora. 15. The disease “Late blight of potato” is caused by _______ . 16. What do you mean by downy mildews? Downy mildew pea is caused by _______ . 17. Green-ear disease of Bajra (Pennisetum typhoides) is caused by _______ . 18. Downy mildew of grapes (Vitis vinifera) is caused by _______ . 19. What causes “white rust of crucifers”? 20. Describe in brief the asexual reproduction in Albugo candida.

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10

C H A P T

ZYGOMYCOTINA (ZYGOMYCETES)

E R

10.1

WHAT ARE ZYGOMYCOTINA?

Subdivision Zygomycotina of division Eumycota includes the fungi that do not have motile cells or zoospores during any stage of their life-cycle, and the perfect-state spores are present in the form of thick-walled resting spores, called zygospores (Ainsworth, 1973). Of the two classes (Zygomycetes and Trichomycetes) included under Zygomycotina, Zygomycetes includes mainly saprobic members. In parasitic members of Zygomycetes the mycelium is immersed in the host tissue. Members of Trichomycetes remain attached to the cuticle or digestive tract of arthropods or gut of earthworms, and their mycelium is not immersed in the host tissue. Kirk et al. (2001), however, discussed these members under phylum Zygomycota of kingdom Fungi. They also divided Zygomycota into same 2 classes (Zygomycetes and Trichomycetes) as of Ainsworth’s (1973) system of classification.

10.2

GENERAL CHARACTERISTICS OF ZYGOMYCETES

1. The majority of the members are saprobic. Some occur on dung, showing coprophilous nature, and some members attack other fungi. A few Zygomycetes are weak parasites, attacking plants and animals. 2. Most Zygomycetes produce a well-developed and branched mycelium, consisting of coarse, grey or white, coenocytic hyphae. A few members, however, contain a highly reduced mycelium, having septa at definite intervals (Alexopoulos and Mims, 1979). 3. Cells contain all typical cellular organelles including mitochondria, nuclei, ribosomes, lipid granules and endoplasmic reticulum. 4. Cell wall is mainly composed of chitosan-chitin (Hesseltine and Ellis, 1973). Chitin has been reported in Mucorales and Entomophthorales but not in Zoopagales. 5. Centrioles are absent. 6. Motile cells or zoospores are absent. 7. Asexual reproduction takes place by non-motile aplanospores (Hesseltine and Ellis, 1973), produced in very large number within the sporangia. 8. Some reproduce by chlamydospores and a few also by oidia.

Zygomycotina (Zygomycetes)

119

9. Many Zygomycetes reproduce by ‘modified sporangial units functioning as conidia, or by true conidia’ (Hesseltine and Ellis, 1973). In Entomophthorales, the sporangium gets reduced and functions as a single conidium. Conidia are borne singly or in chains in Zoopagales (Hesseltine and Ellis, 1973). 10. In Mucorales appendaged sporangiospores are also formed. Sporangiospores and appendaged sporangiospores develop in sporangia, merosporangia, sporangiola, or also as one-spored sporangia or conidia in Mucorales (Hesseltine and Ellis, 1973). 11. Sexual reproduction takes place by gametangial fusion. Two fusing gametangia may arise from the same mycelium or from different mycelia. 12. Gametangial fusion results in the production of a thick-walled resting spore, called zygospore. The zygospore develops within a zygosporangium. Only because of the production of zygospore the name ‘Zygomycetes’ has been given to the class. Each zygospore remains surrounded by a very thick, pigmental and sculptured wall, which is highly resistant to desiccation and other unfavourable factors. 13. At the time of germination of the zygospore a hypha emerges and bears a terminal sporangium. It is believed that meiosis occurs during germination. 14. Regarding the nutrition in Zygomycetes, no vitamins or growth factors are required by primitive Mucorales (Hesseltine and Ellis, 1973). Only inorganic nitrogen with minerals and sugars are required. Higher forms like Pilobolus require growth factors such as ferrichrome (coprogen). Entomophthorales require complex nutrition medium.

10.3

CLASSIFICATION

Hesseltine and Ellis (1973) divided Zygomycetes into three orders: (i) Mucorales, (ii) Entomophthorales, and (iii) Zoopagales, but also mentioned that ‘possibly a fourth order, Endogonales, should also be recognized. But, Benjamin (1979) divided Zygomycetes into following seven orders, and the same classification has also been followed by Alexopoulos and Mims (1979), except in the order Harpellales: (i) Mucorales, (ii) Dimargaritales, (iii) Kickxellales, (iv) Endogonales, (v) Entomophthorales, (vi) Zoopagales, and (vii) Harpellales. Webster (1980) mentioned that Zygomycetes comprise only two orders: Mucorales and Entomophthorales. Kirk et al. (2001) divided Zygomycetes into 10 orders, 32 families, 124 genera and 870 species. The orders of Zygomycetes are Basidiobolales, Dimargaritales, Endogonales, Entomophthorales, Geosiphonales, Glomales, Kickxellales, Mortierellales, Mucorales and Zoopagales. Only Mucorales are discussed here in some details.

10.4 10.4.1

MUCORALES General Characteristics

1. Most of the genera are terrestrial saprophytes, living on a wide variety of organic substrates, including bread, cooked food, dung and decaying animal and plant matter. A few genera are obligate parasites on other Mucorales and mushrooms, and a few are weak parasites causing diseases on higher plants and animals. Some Mucorales are among the most common fungi encountered, when isolating micro-organisms from soil, air, dung, or decaying plant material (Hesseltine and Ellis, 1973). Some occur in mycorrhizal association with many higher plants. 2. Mucorales occur much earlier than the other fungi on decaying organic matter. So, they utilize most simple carbohydrates very efficiently. Due to this, they are commonly called ‘sugar fungi’. 3. Economically, Mucorales are of definite importance for human beings. (For details, refer Economic Importance of Mucorales, under Article No. 10.10).

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4. The mycelium consists of stout, well-branched, coenocytic hyphae. The septa, however, develop at the base of the reproductive organs. Septa, having a median plug, are formed regularly in some advanced families of Mucorales (Hesseltine and Ellis, 1973). 5. Nuclei, mitochondria, ribosomes, lipid granules, endoplasmic reticulum and all other typical cellular organelles are present in the hyphae. Typical dictyosomes and centrioles are not generally reported. 6. Motile zoospores are absent. 7. Asexual reproduction takes place by unicellular, non-motile sporangiospores (aplanospores), produced in very large number in sporangia. The latter develop on sporangiophores. A flat or bulged septum cuts the sporangium from the sporangiophore. In the presence of bulged septum the sporangiophore contains an inflated head, called columella. The columella extends into the sporangium, and is therefore, a vesicle or central sterile region continuous with the sporangiophore into the sporangium. 8. Besides the usual occurrence of sporangium, some other spore-producing asexual bodies found in Mucorales are: (i) Merosporangia : Sporangia which lack columella. (ii) Sporangioles : Sporangia in which spores are greatly reduced in number. (iii) Sporangiola : Spores produced singly without a well-defined sporangial membrane. (iv) Chlamydospores : Thick-walled hyphal cells containing dense cytoplasm. (v) Oidia : Thin-walled globose cells which bud like yeasts and detach as individual units. (vi) Trophocytes : Large, variously shaped, swollen cells which give rise to sporangiophores. 9. Sexual reproduction takes place by the fusion of two multinucleate gametangia, usually of equal size. Mucorales contain homothallic as well as heterothallic species. Gametangial fusion results in the formation of a zygote, containing many diploid nuclei. 10. The zygote develops a thick wall and changes into a resting spore, called zygospore. The zygospore wall is heavily pigmented, and ranges from light yellow to black. The zygospore germinates to produce a germ tube. 11. In many species of Mucorales the two fusing gametangia develop on the zygophores arising from the same mycelium. Such species are called homothallic. But in some Mucorales two distinct types of mycelia (+ and -strains) are required to form zygospores. Such species are called heterothallic. The phenomenon of heterothallism in Mucorales was first discovered by Blakeslee (1904). 12. In some Mucorales, zygospore-like structures (azygospores) develop without the fusion of gametangia.

10.4.2

Classification

Martin (1961) distinguished nine families in Mucorales, i.e. Mucoraceae, Pilobolaceae, Thamnidiaceae, Choanephoraceae, Piptocephalidaceae, Kickxellaceae, Cunninghamellaceae, Mortierellaceae and Endogonaceae. Hesseltine and Ellis (1973), however, divided Mucorales into 14 families, i.e. Choanephoraceae, Cunninghamelaceae, Dimargaritaceae, Endogonaceae, Helicocephalidaceae, Kickxellaceae, Mortierellaceae, Mucoraceae, Pilobolaceae, Piptocephalidaceae, Radiomycetaceae, Saksenaeaceae, Syncephalastraceae and Thamnidiaceae. Kirk et al. (2001) divided Mucorales into 12 families, 47 genera and 130 species. The families are Chaetocladiaceae, Choaephoraceac, Cunninghamelaceae, Gilbertellaceae, Mucoraceae, Mycotyphaceae, Phycomycetaceae, Pilobolaceae, Radiomycetaceae, Saksenaeaceae, Syncephalastraceae and Thamnidiaceae. Recent molecular evidence (Voigt et al., 1999; O’ Donnell et al., 2001) has suggested that the “traditional classification adopted above is highly artificial, and is maintained only for practical reasons” (Kirk et al., 2001).

Zygomycotina (Zygomycetes)

10.5 10.5.1

121

MUCORACEAE Distinguishing Characteristics

1. The cell walls contain chitin microfibrils, chitosan as well as many polysaccharides, such as glucosamine and galactose, proteins, purines, lipids, magnesium, calcium, etc. (Bartnicki-Garcia and Jones, 1968). 2. All Mucoraceae have large multi-spored sporangia. 3. Sporangia contain well-defined columellae. 4. Sporangioles are absent except in Backusella. 5. Sporangiophores never end in sterile spines. 6. Sporangia are pyriform, and the sporangial wall is thick and persistent. 7. Zygospores are common in almost all. According to Hesseltine and Ellis (1973), Mucoraceae is the largest family of the order, containing 20 genera. Its common genera are Mucor, Rhizopus, Absidia, Phycomyces, Actinomucor and Circinella. Kirk et al. (2001), however, included only 18 genera and 59 species in Mucoraceae. Rhizopus and Mucor are discussed in some details.

10.6

RHIZOPUS

10.6.1 Systematic Position

According to Ainsworth (1973)

According to Kirk et al. (2001)

Division – Eumycota Kingdom – Fungi Sub-Division – Zygomycotina Phylum – Zygomycota Class – Zygomycetes Class – Zygomycetes Order – Mucorales Order – Mucorales Family – Mucoraceae Family – Mucoraceae Genus – Rhizopus Genus – Rhizopus Invi et al. (1965) recognized 14 species of Rhizopus. But according to Hesseltine and Ellis (1973) at least 120 species and varieties have been described. Kirk et al. (2001) recognized only 10 species of Rhizopus. Of these, R. stolonifer (syn. R. nigricans) is most common, and can be considered as a type-species representing the genus. Some details, largely of R.stolonifer, are given here.

10.6.2 Occurrence Rhizopus stolonifer occurs very frequently on bread, and is therefore commonly called ‘bread mould’. It is so frequent a contaminant of laboratory cultures of bacteria and fungi that it is considered a weed of laboratory. Rhizopus occurs world-wide in soil, on decaying fruits, dung and vegetation. R. stolonifer also behaves parasitically, causing rot of sweet potato or fruit rot of apple, strawberry and tomato (Webster, 1980). Some Rhizopus species cause ‘mucormycosis’ in domestic animals, and a few are reported from human lesions. Almost all Rhizopus species occur saprophytically.

122 10.6.3

Fungi and Allied Microbes

Laboratory Culture

Since almost all Rhizopus species are saprophytes, the fungus can be grown on dead organic materials such as bread and butter by keeping them in dark and damp atmosphere. By first exposing a moistened piece of bread in a petri-dish for about 24 hr at room temperature, and then covering it for a few days, Rhizopus appears in the form of white tuft of mycelium (Fig. 10.1 A).

Vacuole

Rhizopus Bread

Nuclei Petri-dish

Hyphal wall

Cytoplasm (granular) A

Fig. 10.1

10.6.4

Cytoplasm (vacuolate) B

A, Rhizopus on moistened bread; B, A part of vegetative mycelium.

Somatic Structure

Sporangium

Sporangiophores

Spores

Young mycelium of R.stolonifer consists of many well-branched, white, tubular or filamentous hyphae, which are multinucleate and without cross walls, i.e. coenocytic (Fig. 10.1 B). They provide a cottony appearance when young. But later on the mycelium soon enters the reproductive phase, and becomes differentiated into three different types of hyphae, i.e. rhizoids, stolons and sporangiophores (Fig. 10.2). Rhizoids are the repeatedly branched hyphae that penetrate the substratum. They arise in the form of a cluster towards lower side from each node of the stolon. Stolons are the hyphae that grow horizontally above the substratum for some distance and then bend down into the substratum. The part bending down functions as a node and forms a tuft of rhizoids. The hyphae of the stolon are therefore aerial and unbranched. Sporangiophores are the erect, aerial, unbranched and negatively geotropic hyphae, which grow upwards in tufts at the point where the stolons form rhizoids. They are reproductive in function. Each sporangiophore bears a terminal sporangium.

10.6.5

Internal Structure of Hypha

Columella

Stolon

Rhizoids

The hyphae (Fig. 10. 1 B) are tubular structures and the hyphal wall is composed of fungal chitin. The ultrastructure of the hyphal wall shows that it is microfibrilFig. 10.2 Fruiting mycelium of lar, and the microfibrils run parallel to the surface. It is lined internally by a thin Rhizopus stolonifer. plasma membrane. The protoplast is granular and encloses many nuclei, glycogen and oil droplets, ribosomes, endoplasmic reticulum, mitochondria and many small vacuoles. In the older hyphae the smaller vacuoles coalesce and form a large central vacuole.

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10.6.6

Asexual Reproduction

It takes place generally by the formation of sporangiospores and rarely by chlamydospores.

Sporangium and Sporangiospores

Vesicle

Young sporangium

Vacuolate layer

Sporangiospores are unicellular, multinucleate, non-motile spores, produced in round black bodies, called sporangia. A sporangium develops singly and terminally on the erect and phototrophic sporangiophore. The sporangiophores develop D in tufts from the mycelium. At a stage when a number of such pin-head-like black-coloured sporangia are present Columella C A B (Fig. 10.2), the entire mycelium appears blackish and hence Sporangiophore a popular name black-mould is also given to the funSporangium gus. Spores Development of sporangium starts by the swelling of the tip of the sporangiophore into a knob-like vesicle Columella (Fig. 10.3A). The cytoplasm along with many nuclei from the sporangiophore flows into the swollen vesicle. The swollen portion represents the young sporangium. The proInvaginated Columella columella toplasmic contents of the young sporangium soon become Sporangiophore differentiated into two zones, i.e. outer peripheral multinuE F G cleate dense region, and the central less denser region with comparatively fewer nuclei. Two portions get separated by a layer of vacuoles (Fig. 10.3B). Fusion and flattening of these vacuoles results in the formation of a cleft in beH I tween two zones (Fig. 10.3C). A wall is then secreted in this region of cleft. This wall thus finally differentiates Chlamydospores outer sporangiferous zone and central columella in the young sporangium (Fig. 10.3E). The columella (L. coluJ mella, small column), therefore, remains in continuity with that of the protoplast of sporangiophore. Cleavage in the peripheral sporangiferous zone results in the formation of Fig. 10.3 Rhizopus stolonifer. A-E, Development of sporangium and differentiation of spores; F, Columella many multinucleate segments (Fig. 10.3D). These segments and attached spores; G, Invaginated columella; secrete wall around each of them and metamorphose into H, Spores; I, A germinating spore; J, Chlamyunicellular, globose or oval, multinucleate, non-motile dospores. sporangiospores, also called aplanospores. Dehiscence of sporangium and dispersal of spores in R. stolonifer take place by a special technique. Ingold and Zoberi (1963) described this technique as under: The columella in this species is very large (Fig.10.3E). The sporangium wall dries out after the aplanospores are mature. At the same time the columella collapses in such a manner that it appears like an inverted cup-like dish (Fig. 10.3 F, G) balanced at the end of a stick (= sporangiophore). Simultaneously, the sporangial wall breaks up into many fragments and the dry spores are liberated in the currents of the wind. A spore (Fig. 10.3H) germinates by producing a germ tube (Fig. 10.3I), that develops into a fluffy, well-branched, white, aerial mycelium.

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Fungi and Allied Microbes

Chlamydospores Rarely, the mature hyphae become transversely septate, and some of these cells get surrounded by thick walls, contain sufficient reserve food, and represent chlamydospores (Fig. 10.3J). These are very resistant to unfavourable conditions. They germinate by producing fresh hyphae.

10.6.7

Sexual Reproduction

Rhizopus reproduces sexually by the process of conjugation, which results in the formation of zygospores. A majority of the species, including R.stolonifer, are heterothallic. However, species such as R.sexualis are homothallic. In the heterothallic species the zygospores are formed only when two compatible strains of different mating types are mated together. Blakeslee (1904) named these two compatible strains as + and - strains because it could not be made possible to designate them male and female, mainly because of the very close external similarity between both of them. In the homothallic species the zygospores are formed in the mycelium derived from a single sporangiospore.

Sexual Reproduction in Rhizopus stolonifer, a Heterothallic Species In this heterothallic species the two fusing mycelia belong to two different mating types. One belongs to + (plus) strain and the other to -(minus) strain. Sexual reproduction takes place only when a + strain hypha and a - strain hypha come in contact with each other. Two compatible hyphae are attracted towards each other. They are capable of developing into progametangia and called zygophores. According to Burgeff (1924) and Mesland et al. (1974), following three reactions take place in Mucorales when two compatible strains approach each other: 1. Telemorphotic reaction: It involves the initiation of club-shaped, aerial zygophore formation. According to Gooday (1973,1974) and Van den Ende (1976) the zygophores in both the strains (+ and -) are induced by trisporic acids B and C. These trisporic acids are produced when cultures of the + and - strains are in continuous diffusion contact with each other on the media. 2. Zygotropic reaction: Under this reaction the zygophores of + and - mating partners are directed to grow towards each other. According to Banbury (1955) the zygophores of opposite strains show a mutual attraction, whereas that of the similar strains show mutual repulsion. 3. Thigmotropic reaction: It controls the stages occurring after the contact of zygophores. The stages like gametangial fusion and septation are controlled by thigmotropic reaction. Under zygotropic reaction the zygophores of opposite strains approach towards each other (Fig.10.4A), and from each develops a copulation branch (Fig. 10.4 B). The two copulating branches are called progametangia. The progametangia of opposite strains adhere together by their tips (Fig. 10.4 B, C). They begin to enlarge because of the flow of the cytoplasm and nuclei into them (Fig. 10.4D). Owing to the enlarged size of the progametangia, the zygophores are pushed apart. The tip of each progametangium is soon cut by a septum (Fig. 10.4E). The small terminal cell so formed is called a gametangium whereas the long tubular part is called a suspensor (Fig. 10.4E). The gametangium has densely granular multinucleate protoplast, whereas the suspensor has a more vacuolated protoplast. The protoplasm of each gametangium constitutes the aplanogamete. The size of both the gametangia and the number of nuclei there in increase. The gametangia of the fusing pairs are generally equal in size, but they may also be unequal. A large pore develops in the adjoining wall of the two gametangia (Fig. 10.4F), which allows both the gametangial protoplasts (aplanogametes) to fuse and form a zygospore (Fig. 10.4G). Alexopoulos and Mims (1979) mentioned the name ‘prozygosporangium’ to this fusion cell formed by the fusion of two gametangia. It gets surrounded by a thick warty wall after some time, and is now called ‘zygosporangium’. According to them the zygosporangium contains a single ‘zygospore’. Many of the nuclei belonging to opposite strains (one + with one -) pair and fuse to form many diploid nuclei in the combined protoplast. The nuclei, which do not fuse in pairs, ultimately degenerate. The zygospore soon becomes surrounded by a thick, black, warty wall (Fig. 10.4G). The zygospore wall is made up of two layers, of which the outer dark, thick and warty layer is called exine, and the inner thin layer is called intine. The zygospores undergo a resting period of about a week to 5-9 months. Therefore, the

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Zygomycotina (Zygomycetes)

zygospore is the only diploid structure in the life-cycle. Segregation of strains takes place during meiosis. According to Cutter (1942) half of the resultant nuclei belong to + strain and the remaining half to - strain. Germination of zygospore starts by the development of a lateral crack in its wall. The inner thin intine comes out in the form of a hypha-like germ tube, which is also called promycelium (Fig. 10.4H). The early divisions are meiotic, resulting into a number of haploid nuclei in the protoplasm of germinating zygospore. The young germ tube functions as a sporangiophore and develops a germ sporangium at its tip (Fig. 10.4I). Webster (1980) mentioned that this sporangium is of usual columellate type. Many germ spores or meiospores remain filled in this sporangium. According to Gauger (1961) the germ sporangia of R.stolonifer contain either all + or all - spores, or a mixture of both. These spores germinate to form fresh mycelium (Fig. 10.4 J ,K).

Zygophores Progametangia

C

(+) (–)

(+)

A

(–) B

Gametangia

D

E

Suspensor

Suspensor

Fusion cell

F Zygospore G Zygosporangium Columella Meiospores J Promycelium Germinating zygospore

H

Fig. 10.4

I

Germinating zygospore

K

A-K, Sexual reproduction in Rhizopus stolonifer, a heterothallic species.

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Fungi and Allied Microbes

Sexual Reproduction in Rhizopus sexualis, a Homothallic Species It is a homothallic species. The two fusing zygophores develop in the same mycelium derived from a single sporangiospore. The two zygophores come in contact through their tips, and their walls become flattened (Fig. 10.5 A-C). Each cell becomes distended to form a progametangium. A septum develops in the tip region of each progametangium (Fig. 10.5D), which separates the terminal gametangium and the lower suspensor. In spite of the presence of septum, the cytoplasm remains continuous between gametangium and suspensor through a series of plasmodesmata. Food material from the mycelium keeps on flowing into the developing zygophore through their plasmodesmata. Many nuclei (150-300, Webster, 1980) in both the gametangia assemble near the flattened walls. The fusion walls of the two gametangia dissolve (Fig. 10.5E), and their cytoplasm gets intermixed. At this stage the nuclei become arranged in the periphery of the cytoplasm. The gametangial nuclei fuse in pairs immediately. Webster (1980) mentioned that the fusion nuclei then divide quickly. Fusion of the two gametangia results into a zygospore (Fig. 10.5F). The wall of the zygospore becomes dark owing to deposition of melanin (Webster, 1980). Further developments are similar to that in R. stolonifer. The zygospore germinates by producing a germ tube, bearing a sporangium at its tip. The spores of this sporangium, on liberation, germinate into a fresh mycelium.

10.7

Zygophores

A

B

Gametangium Suspensor C

D Zygospore

E

Fig. 10.5

F

A-F, Sexual reproduction in Rhizopus sexualis, a homothallic species.

MUCOR

10.7.1 Systematic Position

According to Ainsworth (1973) Same as of Rhizopus.

According to Kirk et al. (2001) Same as of Rhizopus.

10.7.2 Differences from Rhizopus Mucor (Fig. 10.6) resembles with Rhizopus in almost all major characteristics, except that of following differences mentioned in Table 10.1. Table 10.1 Major differences of Rhizopus from Mucor No. 1. 2.

Rhizopus

Mucor

Rhizoids (absorptive hyphae) or holdfasts are present. Stolons are present.

Rhizoids or holdfasts are generally absent, or less specialized. Stolons are absent, and thus there is no differentiation of stolons and holdfast in the mycelium. Contd..

Zygomycotina (Zygomycetes)

127

Contd..

3. 4. 5.

Food material is absorbed mainly by rhizoids. Sporangiophores develop in well-organized groups mainly against the rhizoidal hyphae. Spores remain adhered to columella and are not easily disseminated.

Food is mainly absorbed by the entire mycelial surface. Sporangiophores arise singly, and not in groups. Spores easily blown away by wind.

Sporangium Mycelium

Sporangiophore Globules of liquid

A

Dehisced sporangium

Immature sporangium

Frill

Columella

Sporangiophore B

Fig. 10.6

C

Mucor mucedo showing mycelium and young sporangiophores (A), immature sporangium (B), and dehisced sporangium showing columella (C).

Some other important aspects of the life-history of Mucor are given below.

10.7.3 Occurrence Mucor is a cosmopolitan saprophytic fungus living on dead organic matter, specially on the dung of horses and cattles, e.g. M. mucedo. Its many species are widespread in soil, e.g. M. racemosus and M. spinosus. It also spoils human foods like bread, jams, jellies, pickles and cheese. Some of its species cause the disease ‘mucormycosis’ in man and some domestic animals. M. rouxii is used in industries to breakdown starch into sugar. Some of the common Indian species are M. indicus, M. hiemalis, M. mucedo and M. javanicus.

10.7.4 Culture Like Rhizopus, Mucor can also be cultured very easily in the laboratory by exposing a piece of moist bread to atmosphere for 24 hr and then covering it for a few days. After 2-3 days, a white fluffy growth of mycelium appears on the bread.

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Fungi and Allied Microbes

10.7.5 Somatic Structure Same as that of Rhizopus, except the differences mentioned in Table 10.1.

10.7.6 Asexual Reproduction All the details of asexual reproduction are similar to that of Rhizopus. It takes place by the formation of sporangiospores or chlamydospores. Sporangiospores (aplanospores) are the non-motile spores formed in sac-like bodies, called sporangia. Each sporangium develops at the tip of the long, erect, generally unbranched hyphae, growing vertically from the mycelium. These erect, sporangium-bearing hyphae are called sporangiophores (Fig. 10.7A). Sporangiophores arise singly from the mycelium and not in groups. Occasionally, the sporangiophores are branched, as in M. racemosus (Fig. 10.7B) and M. plumbeus (Fig. 10.7C). Through the sporangiophore the cytoplasm and nuclei flow into the tip, i.e. sporangium, and divide into many multinucleate, elliptical sporangiospores or aplanospores. Mucor hiemalis contains uninucleate spores (Webster, 1980). Sporangial wall becomes dark at maturity. In some species the sporangial wall bursts, releasing the spores into the air. However, in many other species the sporangial wall dissolves, leaving the spores attached to the columella in a sticky mass, and the spore dispersal is achieved by insects crawling over the fungus. In M. plumbeus the sporangial wall breaks into pieces. Under suitable conditions the dispersed spores germinate to form a mycelium of branched, multinucleate, coenocytic hyphae. Instead of forming a filament, spores of Mucor rouxii show yeast-like budding in the liquid medium under anaerobic conditions, especially in the presence of CO2. However, if oxygen is available in plenty, the spores again revert to filamentous growth (Bartnicki-Garcia and Nickerson, 1962; Lara and Bartnicki-Garcia, 1974; Bartnicki-Garcia, 1978). Such yeast-like cells have much thicker walls, and also have five times more mannose content in their cell walls than that of filamentous stage of the fungus. Such species (M. rouxii), which exist in more than one form (Yeast-like as well as filamentous), are called dimorphic. These are formed when some of the hyphae break up by transverse walls into chains of thick-walled cells (Fig.10.7B). These cells round off and represent chlamydospores. In some species (M. racemosus) the formation of chlamydospores (Fig. 10.7B) is a useful diagnostic feature.

10.7.7

Sexual Reproduction

It is essentially similar to that of Rhizopus. Mucor genevensis and many other species are homothallic (Fig. 10.7D), and therefore the mycelium is self-fertile (i.e. progametangia from the same mycelium come together, fuse and form zygospores). However, Mucor mucedo and many other species are heterothallic (Fig. 10.7 E), and sexual reproduction will take place only when two physiologically different and compatible mycelia (of + and - strains) are present together. Side branches develop from each of these hyphae of + and - strains. Cytoplasm and nuclei flow into the tips of these branches, which ultimately swell up and represent the progametangia. Two progametangia of opposite strains come in contact with each other through their tips. The tip of each progametangium separates by a septum into a gametangium and suspensor. The walls at the point of the contact of the gametangia of opposite strains break down, and the nuclei of + and - strains fuse to form diploid nuclei. Multinucleate zygospores are formed. In M. hiemalis, the diploid nuclei quickly undergo meiosis, and hence the mature zygospore contains only the haploid nuclei. The zygospores develop thick warty wall, and are set free by the disintegration of parent mycelia. In M.mucedo, the zygospore wall is rich in ‘sporopollenin’ (Gooday et al., 1973). The zygospores germinate when conditions are favourable. Zygospore germination results in formation of a single sporangium. Meiosis occurs and haploid spores are formed. All the spores from one sporangium will be of one mating strain (either + or -) and can develop into new mycelia of + or - strains. These may again fuse and undergo the same process of sexual reproduction. In some species such as M. azygospora, M. bainieri and M. hiemalis sometimes the gametangial copulation does not take place. Here one or both gametangia may develop parthenogenetically into a structure, which resembles morphologically a zygospore. Such structures are called azygospores. The azygospores, therefore, develop on a single suspensor-like cell.

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Zygomycotina (Zygomycetes)

Sporangium (+ or –)

Spores Sporangium

Asexual Reproduction

Spores (+ or –)

Mucor mycelium (+ or –)

Sporangiophore Spores (+ or –)

Mycelium A Sporangial wall Sporangiospores

Progametangium (–) Progametangium (+) Gametangium (–) Sexual Sporangium Reproduction (+ or –) Gametangium (+)

s

iosi

Me

Zygospore (+ or –)

D

Remnants of sporangial wall Spores

Sporangium (+)

Columella

Chlamydospores

Branched sporangiophore

B

Asexual Reproduction

Mycelium (+)

Mucor

Sporangium Spores (+)

C

Mycelium

Progametangium (+)

Asexual Progametangium Sporangium Repro(–) (–) duction Spores (–) Sporangium Spores Gametangium (+) (–) Sexual Reproduction Sporangium (–)

osis

Mei

Fig. 10.7

Spores (+)

Branched sporangiophore

Zygospore (+ –)

Gametangium (+) E

Mucor. A, Mycelium with sporangiophores and sporangia; B, Branched sporangiophore containing chlamydospores in M.racemosus; C, Branched sporangiophore in M.plumbeus; D, Graphic life-cycle of a homothallic species; E, Graphic life-cycle of a heterothallic species.

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10.8 10.8.1

Fungi and Allied Microbes

PILOBOLACEAE Distinguishing Characteristics

1. Only three genera (Pilobolus, Pilaira and Utharomyces) and 13 species are included in Pilobolaceae (Kirk et al. 2001), and all of them always found growing on dung of herbivorous animals. 2. Sporangia of all members contain dark-coloured, persistent walls. 3. Sporangia are columellate. 4. Many spores are present in a sporangium. 5. Specialize liberation mechanism of spores present. 6. Sporangiophores are large, elongate and phototrophic, i.e., grow towards sunlight. 7. At the base of the vegetative mycelium are found some swollen structures called trophocysts, except in Pilaira. 8. In Pilobolus the sporangiophores are swollen. Its sporangia are always shot off the sporangiophore. 9. Zygospores smooth, borne on tongs – like or aposed suspensors. 10. All members are heterothallic.

10.9 10.9.1

PILOBOLUS Systematic Position

According to Ainsworth (1973) and Kirk et al. (2001) Same as that of Rhizopus, except that the family is Pilobolaceae.

10.9.2

Sporangiophore

Spores

Sporangium

Sub-sporangial vesicle Sporangiophore

Occurrence

Pilobolus is a coprophilous fungus, i.e. found on the dung of many herbivorous animals, specially on horse dung. Out of its 7 reported species, some common species are P. crystallinus, P. kleinii and P. longipes.

Horse dung

10.9.3 Culture For culturing Pilobolus, bring fresh horse dung into the laboratory and incubate it in a large glass dish on a window-still. The fungus appears within a few days. After 4-7 days characteristic bulbous sporangiophores also appear on the dung (Fig. 10.8A). Hesseltine et al. (1952,1953) discovered a factor (coprogen) in dung necessary for the growth of Pilobolus. Coprogen is an organic iron derivative.

Sporangium

Trophocyst A

Fig. 10.8

B

Pilobolus. A, Sporangiophores growing on horse dung; B, A fully developed sporangiophore.

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Zygomycotina (Zygomycetes)

10.9.4

Somatic Parts

The mycelium consists of short, coarse hyphae, which are whitish, well-branched, aseptate and multinucleate. Some of the hyphae penetrate into the substratum, draw nourishment from there, and thus function as rhizoids. The hyphal cytoplasm contains many oil globules and glycogen bodies. The swollen parts or trophocysts appear yellow because of the presence of carotene.

10.9.5 Asexual Reproduction It takes place by the spores present in the sporangia, which develop on positively phototropic sporangiophores. A sporangiophore (Fig. 10.8 B) has four parts, viz. basal swollen trophocyst, long sporangiophore proper, swollen subsporangial vesicle and the terminal thick and dark-coloured sporangium. The swollen trophocyst is coloured and rich in carotene. The columella is conical and remains covered by spores. Sporangiophore

Sporangiophore

Sporangium

Glass window Discharged sporangia

A Columella

Globules of liquid

Trophocyst B

Sporangium

Trophocyst C

Sporangium Light rays

Sporangium (partially dehisced)

Globules of liquid

Spores

Ocellus Ocellus E

Subsporangial vesicle

Sporangiophore

Ocellus

Sporangiophore D F

Fig. 10.9

Pilobolus. A, Growth of sporangiophores towards light; B-E, Development of sporangiophore and sporangium; F, L.S. of sporangiophore, showing the path of light rays.

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Buller (1934) reported that the sporangia are thrown by special mechanism (Fig. 10.9A) even up to as far as 2 m, and therefore the fungus has been named ‘hat-thrower’. The discharged sporangia stick to grasses, which are eaten by the herbivorous animals. The fungal sporangia thus reach the alimentary canal of the animals. Here the sporangial wall gets dissolved and the spores are released. These spores pass out along with the dung or faeces of the animal, germinate and develop into mycelium. The spores germinate best above pH 6.5, and the mycelial growth is best at pH 7.0. After about 4 days the swollen segments or trophocysts are formed at the dung surface (Fig. 10.9B). From each trophocyst develops a young sporangiophore (Fig. 10.9B). In the late afternoon the sporangiophores are seen to bend towards the sunlight. In the same night the tip of the sporangiophore enlarges to form a sporangium (Fig. 10.9C). The subsporangial vesicle, present at the base of the sporangium, enlarges and gets swollen between midnight and the early morning hours. The photo-sensitive nature of the young sporangiophores is mainly because of the presence of flavin than a carotenoid. Fully developed sporangiophores (Fig. 10.9 D-F) show highly phototropic nature. The light which falls over a sporangium and sub-sporangial vesicle is brought to a focus at a point beneath the swollen vesicle (Fig. 10.9 F). In this region, where the light gets focused beneath the subsporangial vesicle, carotene-rich cytoplasm gets accumulated. This band of cytoplasm is called ocellus (Fig.10.9E). The conical columella (Fig. 10.9F) remains separated from the spores by a pad of mucilage. The subsporangial vesicle contains a liquid under pressure. The excreted drops of this liquid remain generally adhered to the sub-sporangial vesicle. Usually about mid-day the subsporangial vesicle explodes, its liquid contents are thrown out and the entire sporangium is projected, or rather, thrown forward in the direction of the light. According to Buller (1934) the Pilobolus, under this mechanism, forcibly discharges or shoots its sporangia vertically upwards to a height of nearly 2 m and horizontally for up to 2.5 m. According to Page (1964) the sporangia are apparently propelled from the subsporangial vesicle by a jet of liquid. The discharged sporangia fall on the nearby herbs, which are again eaten by herbivores, and the same process is again repeated.

10.9.6 Sexual Reproduction All known Pilobolaceae are heterothallic. The sexual reproduction takes place by the fusion of gametangia, which are present at the terminal part of suspensors. The suspensors of the fusing gametangia remain curved like a pair of tongs (Fig. 10.10). The gametangial fusion results in the formation of zygospore. Other details are like that of Mucor.

10.10

Zygospore

Suspensors

ECONOMIC IMPORTANCE OF MUCORALES

1. Soft rot of sweet potato, and the ‘leak’ of peach, raspberry, strawberry and Fig. 10.10 Fusing gametangia many other fruits are caused by some species of Rhizopus. of Pilobolus curving like a pair of tongs. 2. Stored grains are always found infected by species of Absidia, Mucor and Rhizopus. 3. Rhizopus is a common spoiling agent of bread. 4. The disease ‘mucormycosis’ and some other fungal diseases in man and animals (Ajello, 1976) are caused by many species of Mucor, Rhizopus and Absidia. 5. Large amounts of lactic acid and fumaric acid are formed by various species of Rhizopus such as R. oryzae and R. stolonifer. Some other Mucorales are known to produce citric acid, succinic acid and oxalic acid. 6. Some Rhizopus and Mucor species are used in making alcochol. 7. Many oriental foods like ‘tempeh’ and ‘sufu’ are prepared from species of Actinomucor and Rhizopus (Lockwood, 1975). 8. b-carotene is prepared in large amounts from species of Blakeslea. 9. In the manufacture of cortisone, Rhizopus stolonifer is used.

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Zygomycotina (Zygomycetes)

10. Members of family Piptocephalidaceae of Mucorales occur parasitically on other fungi, and primarily on other Mucorales. Such fungi which occur on other fungi are called mycoparasites. In the study of physiology of parasitism such organisms are used as ideal organisms.

10.11

HETEROTHALLISM IN MUCORALES

For details refer Article No. 28.2 in Chapter 28 (Heterothallism in Fungi).

TEST YOUR UNDERSTANDING 1. The perfect state spores of Zygomycetes are present in the form of _______ . 2. Write any five general characteristics of Zygomycetes. 3. Write one sentence each about (a) merosporangia, (b) sporangiola, (c) oidia, 4. Explain in brief the phenomenon of heterothallism in Mucorales. 5. The common name “bread mould” is often given to _______ . 6. Describe in brief the sexual reproduction in Rhizopus stolonifer. 7. Give any four major differences between Rhizopus and Mucor. 8. Draw graphic life-cycle of a homothallic species of Mucor. 9. A common coprophilous fungus, found on horse dung, is _______ . 10. Give a brief account of economic importance of Mucorales.

(d) trophocytes.

11

C H A P

TRICHOMYCETES

T E R

11.1

WHAT ARE TRICHOMYCETES?

As mentioned already under chapter Zygomycotina Article 10.1, Trichomycetes is a class of this subdivision of Eumycota along with one more class (Zygomycetes). Besides the characters such as absence of motile cells or zoospores and presence of zygospores as the perfect state spores, members of class Zygomycetes are mostly saprobic fungi, while those of class Trichomycetes are generally parasite and found attached to the cuticle or digestive tract of animals, like arthropods or guts of earthworms. In parasitic Zygomycetes the mycelium remains immersed in the host tissue while in members of Trichomycetes the mycelium is not immersed in the host tissue. J.F. Manier (1969) of France and R.W. Lichtwardt and his associates of University of Kansas (USA) have been the pioneer workers of these fungi (Manier and Lichtwardt, 1968; Lichtwardt, 1973, 1976, and; Moss and Lichtwardt , 1977). As many as 38 genera and approximately 140 species of these fungi have alredy been repoted. Kirk et al. (2001) treated Trichomycetes as a class of phylum Zygomycota of kingdom Fungi. It contains 3 orders, 6 families, 55 genera and 218 species, according to these workers.

11.2

GENERAL CHARACTERISTICS

1. Trichomycetes are parsitic fungi found within the digestive tract of living arthropods and guts of earthworms. The hosts of Trichomycetes include marine, freshwater and terrestrial arthropods. Amoebidium is an ectozoic trichomycete growing on the exoskeleton of arthropods. 2. Amongst the commonest hosts of these fungi include insects (aquatic larvae of Diptera, beetles), Crustaceans (e.g. crabs, amphipods) and millipedes. 3. According Lichtwardt (1976) arthropod hosts also benefit from the presence of trichomycetous fungi, and thus the relationship between the two is mutualistic. 4. The thallus consists either of branched septate filaments growing attached on the cuticle of the host by a holdfast, or it consists of unbranched coenocytic filaments. 5. In the unbranched coenocytic members, the septa are formed only to delimit the reproductive units. 6. In the branched multicelluar members, the “septa are perforated with a flared border around the pore, which usually remain plugged” (Lichtwardt, 1976). 7. Mycelium in these fungi is not immersed in the host tissue.

135

Trichomycetes

8. Motile cells or zoospores are absent. Asexual reproduction takes place by characteristic trichospores, sporangiospores, arthrospores or amoeboid cells. Trichospores are non-motile, nonflagellated exogenous spores with one or more appendages. Trichospores in Harpella have four appendages which are slightly curved or coiled. Ellipsoidal trichospores of Stachylina have only one appendage. 9. Sexual reproduction takes place by biconical zygospores, and has been reported only in members of Harpellales. The zygospores are formed without conjugation in Genistellospora of Harpellales. 10. Sometimes, the zygospores also bear appendages and resemble with asexual spores i.e trichospores.

11.3

CLASSIFICATION

Lichtwardt (1976) divided Trichomycetes into four orders, mainly on the basis of their asexual reproductive spores. These are Harpellales, Asellariales, Eccrinales and Amoebidiales. Harpellales produce trichospores, Asellariales produce arthrospores, Eccrinales produce sporangiospores, while Amoebidiales produce amoeboid cells. Kirk et al. (2001) recognized only 3 orders (Asellariales, Eccrinales, Harpellales) under Trichomycetes. They considered order Amoebidiales under kingdom Protozoa. A few more characteristics of these orders are listed below.

11.4

HARPELLALES

1. Septate thalli are branched or unbranched and found attached to the hindgut line of young insects. 2. The characteristic trichospores are present. They possess one or more appendages. 3. Appendages function like that of flagella. However, 9+2 structure of flagella is absent. 4. Each trichospore is an exogenously produced, dehiscent structure. 5. Sexual reprodurtion takes place by biconical zygospores. Harpellales contains two families, viz (i) Harpellaceae, which “possess unbranched thalli attached to the peritrophic membrane of the midgut” of insects, e.g. Harpella (Fig. 11.1), and (2) Genistellaceae, which “possess branched thalli attached to the cuticle lining the hindgut” (Lichtwardt, 1976), e.g Genistellospora.

11.5

ASELLARIALES

Trichospores

Apical spore body

Trichospore

Generative cells (3)

Holdfast

Fig. 11.1

Harpella melusinae showing its thallus attached by a holdfast to the peritrophic membrane from the mid-gut of a larva of Simulium, a blackfly.

1. The thalli are branched and septate. 2. They remain attached to the hindgut lining of isopods or insects. 3. Asexual reproduction takes place by characteristic arthrospores. 4. Sexual reproduction has not been reported. This small order of class Trichomycetes consists of only one family Asellariaceae, bearing three genera,of which most common member is Asellaria.

136

11.6

Fungi and Allied Microbes

ECCRINALES

1. The thalli are coenocytic and usually unbranched. If branched, the branches develop only at the base. 2. Thalli remain attached to the chitinous lining of the hindgut or foregut of the insects, such as isopods, amphipods, decapods and millipedes. 3. Asexual reproduction takes place only by sporangiospores. 4. The sporangiospores are produced endogenously. They are produced singly in a series of terminal sporangia. 5. Each sporangiospore may be uninucleate or multinucleate and usually remains surrounded by thick walls. 6. Sexual reproduction has not been reported in any member of Eccrinales. Order Eccrinales is divided into three families. These, along with an example of each of them, are Palavasciaceae (e.g. Palavascia), Parataeniellaceae (e.g. Parataeniella) and Eccrinaceae (e.g. Enterobryus).

11.7

AMOEBIDIALES

1. The thalli are unbranched, aseptate and multinucleate. 2. They are found in the hindgut or also on the external parts of aquatic insects and crustaceans. 3. Asexual reproduction takes place only by amoeboid cells. 4. The entire thallus transforms into a multiamoeboid or multispored sporangium. 5. The sporangium releases amoeboid cells or amoeboid spores, each surrounded by a rigid wall. 6. Encystment of these amoeboid cells produce cystospores, each of which develops into a new thallus. 7. Sexual reproduction has not been reported in any member of Amoebidiales. Amoebidiales contains a single family, Amoebidiaceae, which has only two genera, viz., Amoebidium and Paramoebidium

TEST YOUR UNDERSTANDING 1. 2. 3. 4.

What are Trichomycetes? Trichomycetes are generally found attached to the digestive tract of animals life _______ or guts of _______ . Write any five general characteristics of Trichomycetes. According to Ainsworth (1973), Zygomycetes and Trichomycetes are the two classes of subdivision _______ of division _______ . 5. Harpellales, Asellariales, Eccrinales and Amoebidiales are orders of class _______ .

12

C H A P T

ASCOMYCOTINA (ASCOMYCETES) (GENERAL ACCOUNT)

E R

12.1

WHAT ARE ASCOMYCOTINA?

The subdivision Ascomycotina of Ainsworth’s (1973) classification is equivalent to class Ascomycetes of the older classifications and phylum Ascomycota of kingdom Fungi of latest classification of Kirk et al. (2001). Ascomycotina includes only such fungi in which the zygospores are absent and the perfect-state spores are the ascospores. The Ascomycetes and the Basidiomycetes are sometimes combindly called ‘higher fungi’. Webster (1980) mentioned that Ascomycetes is the largest class of fungi, including more than 15,000 species. Some of the commonly-known Ascomycetes are yeasts, black moulds, green moulds, powdery-mildews and morels. The characteristic ascospores are present in a sac-like body called ascus and therefore these fungi are also commonly called ‘sac fungi’ (Gr. askos = sac).

12.2

GENERAL CHARACTERISTICS OF ASCOMYCOTINA

1. Ascomycotina occur in almost all climatic conditions, and in a wide variety of habitats, i.e. in soil, on dung (coprophilous), in marine as well as fresh water, as saprophytes of animal and plant remains, and also as parasites on plants as well as animals. Mycelia of most parasitic species grow within the host tissue, but powdery mildews grow superficially upon the host showing ectoparasitic nature. Few Ascomycetes are entirely hypogean, i.e. grow and develop only under ground. Marine Ascomycetes are either saprobic or parasitic on marine angiosperms or large algae. Doebbeler (1985) reported many Ascomycetes growing on mosses. For collecting plant-parasitic Ascomycetes, the best time is early spring. 2. The mycelium is well-developed, profusely branched and septate. Each segment of the hypha contains several nuclei. However, yeasts are single-celled organisms. 3. In each septum or cross wall of the mycelium there is present a simple central pore. According to Moore (1965) the pore is wide enough to allow mitochondria, nuclei and other cytoplasmic contents to pass from cell to cell. 4. The cell walls contain chitin in the form of microfibrillar skeleton in filamentous members. Mannose, glucose, aminosugars and protiens, along with many surface enzymes, have also been reported in different members. Because of the presence of enzymes the cell wall is not a functionally inert coating in Ascomycetes. 5. The chief distinguishing character of all Ascomycotina is the presence of a sac-like body, called ascus (pl. asci). It contains sexually produced spores, called ascospores. 6. The ascospores are formed after karyogamy and meiosis. In an ascus, the number of ascospores is usually 8. 7. The ascospores are always endogenous in origin, and are also called perfect-state spores.

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Fungi and Allied Microbes

8. The asci are usually grouped to form a definite type of multicellular fruiting-body called ascocarp. The ascocarps remain enveloped in a sheath of sterile hyphae. 9. The ascocarps are either cup or saucer-shaped (apothecium, e.g. Discomycetes), flask-shaped (perithecium, e.g. Pyrenomycetes), or closed, spherical and indehiscent (cleistothecium, e.g. many Plectomycetes). 10. Flagellate cells are completely absent in the life cycle of all Ascomycetes. 11. Asexual reproduction takes place by the imperfect stage, present in the form of non-motile, exogenously produced spores, called conidia, developing on conidiophores. In some members pycniospores, oidia or chlamydospores are formed. (Classifications of Ascomycetes suggested by various workers are based mainly on perfect stages, i.e. ascospores, asci and ascocarps. Imperfect stages, i.e. conidia, are not considered. All such fungi, where only imperfect stages are known, are placed under a separate class of imperfect-fungi i.e. Deuteromycetes, now called Anamorphic fungi (Kirk et al. 2001). Their perfect stages (i.e. asci and ascospores) have either lost during their evolutionary development, or have not so far been discovered. Grouping of these imperfect Ascomycetes is a big taxonomic problem for mycologists, and has still not been solved completely).

12.3

ECONOMIC IMPORTANCE

Ascomycetes are of tremendous importance to man. Some are detrimental while others are beneficial. Some aspects of detrimental nature of these fungi are listed below: 1. Species of Aspergillus and Penicillium cause great demage to food, and also to some common goods such as leather. The fabrics containing cellulose are destroyed by Chaetomium. 2. They attack many crop plants, as well as timber and ornamental plants, causing diseases such as powdery mildew, apple scab, foot-rot of cereals and chestnut blight. 3. They cause many diseases of domestic animals as well as man. ‘Aspergillosis’ is caused by Aspergillus fumigatus, A. flavus and A. niger. Its infection in lungs shows symptoms similar to those of tuberculosis. Tokelan disease, histoplasmosis and ringworm are some other diseases caused by Ascomycetes. 4. Claviceps purpurea contains the sclerotia with many such alkaloids that, if consumed, are deadly poisonous to animals and human-beings. On the contrary, it is also used as a medicinal drug. Some of the beneficial aspects of Ascomycetes are mentioned below: 1. Many yeast species are well-known for their fermenting activities in brewing and baking industries. 2. Antibiotics, such as penicillin from Penicillium notatum and P. chrysogenum, are produced from many members. 3. Many industrial products, such as citric acid, oxalic acid, gluconic acid, vitamins and glycerol, are prepared by ascomycetous members. 4. Some of the morels and truffles are edible, and well-known throughout the world for their flavour delicacy. 5. In processing soybeans Aspergillus wentii is used in Japan.

12.4

SOMATIC STRUCTURES

Except that of yeasts (e.g. Saccharomyces) and few other forms, majority of Ascomycetes consist of well-developed, profusely branched and septate mycelium. The cells are either uninucleate or multinucieate. The ‘yeasts’ are either unicellular, or form a sort of chain of cells forming false mycelium or “pseudomycelium”. Actually, the so-called transverse wall is an incomplete septum having a central perforation, allowing the cytoplasmic streaming and the movement of nuclei and other organelles from cell to cell. In between the cells the septum originates on

139

Ascomycotina (Ascomycetes) (General Account)

the inner side of the tubular wall in the form of an annular outgrowth of the wall material. These outgrowths grow inwards leaving a perforation or central pore. The septa in Ascomycetes are, therefore, incomplete. Sometimes the septal pores remain blocked by some electron dense, membrane-bounded bodies called Woronin bodies. These are actually rounded or elongated and highly refractive bodies in some of these fungi. These are found particularly associated with septa. The cell wall contains chitin. The chitin is present in the form of microfibrillar skeleton. Mannose, glucose, aminosugars, proteins and some surface enzymes have also been reported in Ascomycetous cell walls. Typical cell organelles, including nuclei, mitochondria, ribosomes, endoplasmic reticulum, microtubules, vacuoles, lipid bodies, lomasomes, etc. are also present in the cell. The mycelium may be organized into tissues, where the hyphae are either loosely compacted (prosenchymatous) or closely compacted (pseudoparenchymatous). Sometimes the pseudoparenchymatous aggregations of hyphae develop into a hard resting body called sclerotium (e.g. Claviceps purpurea).

12.5

ASEXUAL REPRODUCTION

Asexual reproduction by vegetative methods takes place mainly by fragmentation, fission and budding. But by spores, it takes place by the formation of nonflagellated spores, e.g.,conidia. However, some genera reproduce by oidia or chlamydospores. Fragmentation is common in filamentous Ascomycetes. Fission is seen in unicellular Ascomycetes, especially yeasts. There occurs a simple splitting of a cell into two daughter cells (Fig. 12.1 A-D) by constriction and cell wall formation. The nucleus also divides mitotically into two daughter nuclei, each of which enters into newly formed daughter cells. Budding is the production of a small protuberance or bud from a parent cell (Fig. 12.1 E-G) as in yeasts. The bud increases in size, breaks off eventually from the parent cell and develops into a new individual (Fig 12.1 H). Sometimes chains of buds are formed, and the entire structure appears as a false mycelium or pseudomycelium. Parent cell

A

Septum

B

C

Protuberance

Daughter cells

New individual

D

Buds

Parent cell

Parent cell E

Fig. 12.1

F

G

H

A–D, Showing fission in yeasts; E-H, Showing budding in yeasts.

Conidia are the most commonly occurring spores in Ascomycetes. Conidium is a non-motile, deciduous, exogenously produced asexual spore, which develops at the free end of a special hypha called conidiophore. Usually, a conidiophore

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cuts conidia in a rapid succession, resulting into a chain of condia. The conidiophores may be unbranched (e.g., Erysiphe, Fig. 12.2 A) or branched (e.g. Penicillium, Fig 12.2 B); unicellular as in Phyllosticta (Fig. 12.2 C) or multicellular as in Penicillium (Fig.12.2 B). In Penicillium, Aspergillus and many other genera the ultimate branches of the conidiophores bear some bottle-shaped structures called sterigmata. The conidia, in majority of Ascomycetes, remain arranged on the conidiophores in a basipetal succession i.e., youngest at the base and oldest at the top of the chain. However, in some genera they develop in acropetal succession, i.e., oldest at the base and youngest at the top. On getting detached each conidium slightly swells (Fig. 12.2 D, E), gives rise to a germ tube (Fig 12.2 F, G) and develops into a young mycelium (Fig. 12.2 H). Oidia or arthropores are single-celled, thin-walled spores formed simultaneously throughout the length of a hypha (Fig. 12.3 A). They are, actually, the break up cells of the hyphae. Chlamydospores are the large, thick-walled spores (Fig. 12.3 B) formed in the mycelium. A lot of reserve food remains accumulated in chlamydospores. They are generally intercalary in position, but sometimes they may also be terminal, e.g., Taphrina deformans (Webster, 1980). Conidia Unicellular conidiophore

Conidium

Unbranched conidiophore

C

Chlamydospore Oidia

Branched conidiophore

A

B

Conidium

D E

Fig. 12.2

12.6

F

G

H

A-H. Showing unbranched conidiophore of Erysiphe (A), branched conidiophore of Penicillium (B), unicellular conidiophore of Phyllosticta (C), germination of conidium to form young mycelium (D-H).

A

Fig. 12.3

B

Showing formation of oidia (A) and chlamydospores (B).

SEXUAL REPRODUCTION

Various steps of the sexual reproduction in these fungi may briefly be summarised as under: 1. Sexual reproduction takes place by the fusion of two compatible nuclei, brought together in many different ways, such as gametangial copulation, gametangial contact, spermatization and somatogamy.

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2. In some genera definite male (antheridia) and female (ascogonia) gametangia are formed, whereas in others the fusion takes place between somatic hyphae. 3. Both the types of the species, i.e., homothallic as well as heterothallic, occur. 4. In majority of the Ascomycetes the bringing together of two compatible nuclei stimulates the female gametangium (ascogonium) to produce a number of hyphal extensions called ascogenous hyphae. 5. Pairs of nuclei migrate into these ascogenous hyphae and undergo mitotic divisions. 6. The ascogenous hyphae become septate (Fig. 12.4 A, B), and two compatible nuclei fuse in an ascus mother cell. The latter subsequently develops into an ascus (Fig. 12.4 C-E). 7. The diploid zygotic nucleus of the ascus immediately undergoes meiosis. Four haploid nuclei are produced (Fig. 12.4 F, G). Further, they divide mitotically to form eight haploid nuclei in an ascus (Fig. 12.4 H). 8. These eight haploid nuclei get enveloped individually with a portion of cytoplasm and develop into eight ascospores (Fig. 12.4 H, I). 9. In majority of the Ascomycetes the asci are produced, which may be of perithecium, apothecium or cleistothecium types. 10. The ascospores are liberated from the ascus, and germinate into fresh mycelium. Mature ascus Ascospores

Haploid nuclei Diploid nucleus Ascus Ascogenous mother hypha Terminal cell cell

A

Fig. 12.4

B

C

D

Ascus

E

F

G

H

I

A–I, Showing various stages of the development of ascus and ascospores

Some of the important aspects of the sexual process and post-fertilization stages are underdiscussed in detail.

12.7 12.7.1

METHODS OF BRINGING COMPATIBLE NUCLEI TOGETHER Gametangial Copulation

Two similar gametangia come together, touch at their tips or coil around each other and fuse. Two nuclei in the fusion cell, which come from two different gametangia, fuse, resulting into a diploid cell, which functions as an ascus (Fig. 12.5 A-F). The diploid nucleus divides meiotically to form haploid ascospores, as in many Hemiascomycetes.

142 12.7.2

Fungi and Allied Microbes

Hologamy

Gametangia

In Schizosaccharomyces octosporous two mature somatic cells function as gametangia, come together and touch at their tips. Plasmogamy and karyogamy result in the formation of a zygotic nucleus, and the resultant cell functions as ascus mother cell (Fig. 12.5 G-J).

12.7.3

Autogamy

In Penicillium vermiculatum the antheridial tip simply touches the ascogonium, and this mere touch stimulates the ascogonial nuclei to arrange themselves in functional pairs or dikaryon (Fig. 12.5 M,N). Here antheridia remain nonfunctional. Such a phenomenon of pairing of nuclei of the same gametangium is called autogamy.

12.7.5

B

D

C Ascospores

Ascus

Gametangy or Gametangial Contact

Morphologically differentiated gametangia are produced by many higher Ascomycetes. These gametangia may be uninucleate (Sphaerotheca) or multinucleate (Pyronema). The male gametangium is called antheridium, and the famale ascogonium (Fig. 12.5 K, L). A pore develops at the point of contact of antheridium and ascogonium. Through this pore male nucleus passes from the antheridium into the ascogonium. Sometimes the ascogonium contains a tubular receptive process, called trichogyne, through which it receives the male nucleus. No fertilization tube is formed.

12.7.4

A

E Gametangia

F Conjugation tube I

H G

Ascogonium

Zygote Antheridium Trichogyne J Antheridium Ascogonia

Antheridium

Antheridium L

M

N

K

Spermatization

In Neurospora sitophylla, Mycosphaerella tulipiferae and Fig. 12.5 A–F, Gametangial copulation; G-J, Hologamy; many other higher Ascomycetes, antheridia are not formed. K-L, Gametangial contact; M-N, Autogamy. The male sex cells are present in the form of minute, spherical, uninucleate bodies called spermatia. In some species the spermatia develop in pycnidium-like bodies called spermogonia, whereas in others they develop at the tip of special reproductive hyphae called spermatiophores. Ascogonia in such species are well-developed. The spermatia are detached from the parent hyphae, and carried away up to trichogyne or other receptive part of the ascogonium through wind, water or insects. The spermatium empties its contents in the receptive female organ. This fusion between spermatium and female receptive organ is called spermatization. Sometimes minute conidia (microconidia) and oidia also function as spermatia. Instead of germinating into a mycelium, they attach on female receptive organ of ascogonium, empty the contents there in, and bring about spermatization.

12.7.6 Somatogamy In some higher Ascomycetes fusion takes place between the somatic hyphae or two compatible mycelia. The nuclei of one migrate to the other, and reach up to the ascogonia through septal perforations. In such cases neither antheridia nor spermatia are formed.

Ascomycotina (Ascomycetes) (General Account)

12.8

143

COMPATIBILITY

Compatibility actually means fusing capability, or sexual nature of the mycelia. According to this, Ascomycetes fall into two distinct groups: 1. Homothallic species, in which all individuals are able to form asci themselves, i.e. without the aid of other mycelium. They are therefore self-compatible or self-fertile. The mycelium in homothallic species may, therefore, also be considered as bisexual. 2. Heterothallic species, in which two compatible individuals must be mated before asci are formed. Therefore, the mycelium in these species is unisexual. Or, it may also be said that sexually it is self-sterile, and for sexual reproduction it requires the aid of another compatible mycelium of a different mating type. The two compatible mycelia may be named as + and - strain mycelia. There can therefore be a fusion only between a + ascogonium and - antheridium, or a - ascogonium and + antheridium. This phenomenon is called heterothallism, and it was first discovered in Mucorales by Blakeslee (1904). For other details of compatibility in Ascomycetes, heterothallic Ascomycetes, bipolar heterothallism and tetrapolar heterothallism, refer Article No 28.5 in Chapter 28 (Heterothallism in Fungi).

12.9

ASCUS DEVELOPMENT

Ascus develops after fertilization by direct or indirect method.

12.9.1 Direct Development of Ascus In lower Ascomycetes plasmogamy is followed immediately by karyogamy, resulting in the formation of a diploid nucleus. The cell containing this diploid nucleus develops directly into an ascus. This nucleus divides first meiotically and then by ordinary divisions, resulting into eight haploid nuclei, which change into same number of ascospores. This type of development is seen in Schizosaccharomyces, Saccharomyces, Dipodascus, Eremascus , etc.

12.9.2 Indirect Development of Ascus In higher Ascomycetes the ascus development is indirect. Here the two gametangia (antheridium and ascogonium) contact with each other. The male nuclei from the antheridium pass through the trichogyne into the ascogonium, and get themselves paired with the female nuclei present there in. But there is no fusion of male and female nuclei at this stage. These paired nuclei are called dikaryons (Fig. 12.6 A ). At this stage, when many dikaryons are present in the ascogonium, the wall of the ascogonium gives rise to many papilla-like outgrowths, which develop into short ascogenous hyphae (Fig. 12.6 B). The paired nuclei or dikaryons migrate into these ascogenous hyphae. Young ascogenous hyphae are aseptate but later on they become multicellular and profusely branched. Only rarely they are unbranched. Ascogenous hyphae remain intertwined with one another (Fig. 12.6 C). A majority of their cells are binucleate. Of these two nuclei of the cells of ascogenous hyphae one is of antheridial origin and the other is of ascogonial origin (Smith, 1955). From the stalk of the ascogonium develop many sterile hyphae to form a pseudoparenchymatous hollow apparatus (Fig. 12.6 C). Some of the sterile hyphae form a thick peridium around the ascocarp, and many develop into paraphyses in the fruiting body (Fig. 12.6 C). The asci develop at the tips of of ascogenous hyphae (Fig. 12.6 C). The terminal cell of each ascogenous hypha, contains two nuclei. It recurves and forms a crozier or hook (Fig. 12.6 D). Both the nuclei of crozier divide simultaneously to form four nuclei (Fig. 12.6 E). Wall forms in such a way that a uninucleate tip cell, binucleate penultimate cell and a uninucleate basal cell are formed (Fig. 12.6 F). This penultimate binucleate cell becomes the ascus mother cell. Of the two nuclei in this ascus mother cell, one is of antheridial origin and other is of ascogonial origin. It enlarges and develop into an ascus.

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Ascogonium

Sterile hyphae Trichogyne

Sterile hyphae

Antheridium Ascogenous hyphae Ascogonium Antheridium

B

A Paraphyses

Peridium

Trichogyne Ascospores Ascus

Ascogenous hyphae

1st ascus Antheridium C Penultimate cell

Crozier Ascogenous hyphae

Tip cell

Ascogonium

New crozier

Basal cell D

Fig. 12.6

E

F

Ascus initial

G

H

2nd ascus

I

J

Indirect ascus development. A, Formation of dikaryons in ascogonium; B, Development of ascogenous hyphae; C, A hypothetical ascocarp in vertical section; D-J, Stages of ascus development.

The two nuclei of this ascus mother cell fuse and form a diploid nucleus. This diploid nucleus divides first meiotically and then ordinarily or mitotically, forming four haploid nuclei (Fig. 12.6 G, H) according to Olive (1965). One more mitotic division results in the formation of eight haploid nuclei (Fig. 12.6 I). All these nuclei get surrounded by some cytoplasmic contents and change into eight ascospores (Fig. 12.6 J). In some Ascomycetes one, two or few more mitotic divisions may result in the formation of 16, 32 or more ascospores in an ascus. In Pyronema confluens and many other Ascomycetes the uninucleate tip and basal cell unite with each other to form a binucleate cell. It develops into a new crozier cell (Fig. 12.6 G ), which ultimately develops into a new ascus (Fig. 12.6 I). The process of new crozier- and ascus-formation is repeated to form many asci. However, in a few other members the tip cell becomes shorter and finally disintegrates.

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Ascomycotina (Ascomycetes) (General Account)

Electron microscopic studies suggest that at the time of ascospore formation a double membrane (ascospore membrane or ascus vesicle) develops in the ascus, just inner to the plasma membrane (Fig. 12.7 A). The diploid nucleus divides (Fig. 12.7B) meiotically, forms four and then eight haploid nuclei. The ascospore membrane invaginates between the haploid nuclei (Fig. 12.7 C) and delimits the young ascospores (Fig. 12.7 D) from the epiplasm of the ascus. In between the two layers of the ascospore membrane the primary spore wall of the ascospore is secreted (Fig. 12.7 E). Ascospore membrane

Young ascospore Epiplasm

Primary spore wall

Vesicle

Vesicles

Ascospore Ascus Plasma membrane wall membrane Ascospore Haploid membrane nuclei

Diploid nucleus

A

Fig. 12.7

B

C

D

E

A-E, Ascospore development in Ascobolus (after Oso, 1969).

About the origin of the double membrane or ascospore membrane various views have been put forward by different workers. Some of the views are mentioned below: 1. It originates from small vesicles derived from Golgi apparatus (Bracker, 1969). 2. It arises from nuclear envelope (Oso, 1969). 3. It originates from plasmalemma (Greenhalgh and Griffiths, 1970). 4. It originates both from plasmalemma and nuclear envelope (Wells, 1972). 5. It develops from fungal mesosomes (Gil, 1973). 6. It develops from mitochondrial membranes (Carroll and Carroll, 1974). 7. It originates from myelin figures (Hill, 1975), which are the structures identical with fungal mesosomes.

12.10

ASCI AND ASCOSPORES

The asci in different Ascomycetes may be elongated, oval, globose, club-shaped, cylindrical or even tubular. The arrangement of ascospores in an ascus is also variable. They may be arranged in uniseriate, biseriate, fasciculate or irregular manner. The ascus surrounded by a single-layered wall is called unitunicate (Fig. 12.8 A) as in a majority of Plectomycetes, Pyrenomycetes and Discomycetes. On the contrary, the ascus with double-layered wall is called bitunicate (Fig. 12.8 B), as in Loculoascomycetes (Luttrell, 1973). The two layers in a bitunicate ascus differ only in the arrangement of microfibrils, according to Reynolds (1971). The outer layer is called ectoascus or ectotunica, whereas the inner layer is called endoascus or endotunica. According to some workers the wall of both unitunicate and bitunicate asci consists of both these lay-

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Ascospores ers. In the unitunicate ascus both these layers remain closely or Operculum Slit tightly pressed against each other throughout the existence of the Outer wall Pore ascus. But in bitunicate ascus both ectoascus and endoascus layers remain separated from each other. Because of these conflicting reInner ports, Alexopoulos and Mims (1979) commented that ultrastrucwall tural studies of the ascus wall ‘are badly needed’. In a study of ultrastructure, Parguey-Leduc and Janex-Favre (1984) mentioned that ‘in comparison with bitunicate Pyrenomycetes, unitunicates are characterized by the relative thickness of exo- and endoascus, the absence of a clear space between two coupled tunicae, and the absence or weak presence of fibrillar waves in the endoascus’. According to these workers the endoascus in unitunicate Pyrenomycetes may be of three types: (i) fibrillar, (ii) granular and then with parallel fibrils, and (iii) granular and then with reticulate fibrils. The dehiscence of asci is also variable in different Ascomycetes. In some genera the ascospores are liberated through an ascal pore developed at the tip of the ascus (Fig. 12.8 C), whereas in others a cap-like operculum is separated (Fig.12.8 D). The dehisA B C D E cence may also take place by the formation of a definite line at the tip, called slit (Fig. 12.8 E ). In some genera (Aspergillus), however, the ascus wall gets degenerated or dissolved, liberating the Fig. 12.8 Asci and ascospores. A, Unitunicate ascus; B, Bitunicate ascus; C-E, Variascospores in the fruiting body. Single ascospores are discharged ous types of ascal dehiscence. commonly for about 1-2 cm. But on the contrary, they are discharged up to a long distance where the ascospores stick together in the form of a group (Webster, 1980). The ascospores may be elliptical, spherical, long, narrow, club-shaped or oval in different species. Their wall is usually thin in a majority of Ascomycetes. In some species the wall of ascospores is bilayered. Generally, they are unicellular, uninucleate, and contain granular cytoplasm having many oil droplets. In some cases the ascospores are multicellular. On getting the suitable environmental conditions, the ascospore germinates by producing one or more germ tubes.

12.11

ASCOCARPS

In a majority of the Ascomycetes, except yeasts and some related fungi of Endomycetales, the asci remain enclosed by many sterile hyphae. The ascogonium, ascogenous hyphae, asci, ascospores, paraphyses and the enveloping sheath or peridium in Ascomycetes remain grouped together in the form of a fruiting body or fructification, called ascocarp.

12.11.1 Main Types The following four main types of ascocarps are met with in Ascomycotina: 1. Cleistothecium: Globose fruiting body with no special opening to the outside, as in Erysiphales, Eurotiales (Fig. 6.7 A, B). 2. Apothecium: A saucer- or cup-shaped fruiting body, as in Helotiales and Pezizales (Fig. 6.7 C). 3. Perithecium: A flask-shaped fruiting body, opening with an ostiole or pore (Fig. 6.7 D), and its asci are unitunicate, as in Pyrenomycetes (Hypocreales and Sphaeriales). 4. Pseudothecium: A perithecium with bitunicate asci (Fig. 6.7 E), as in Loculoascomycetes. Some call pseudothecium as ascostroma.

Ascomycotina (Ascomycetes) (General Account)

147

12.11.2 Sterile Threads Along with the asci, a majority of the ascocarps also contain some sterile threads. Alexopoulos and Mims (1979) mentioned following common types: 1. Paraphyses: These are long, club-shaped or cylindrical, unbranched but rarely branched, unicellular or multicellular structures, developing at the base of the fruiting body. Their tip is generally free. 2. Periphyses: These are short, hair-like structures present on the inner side of the mouth opening of perithecium. 3. Periphysoids: These are the periphyses present all along the inner side of the wall of the fruiting body, except near the ostiole. 4. Apical paraphyses: The paraphyses developing at the top of perithecial centrum are called apical paraphyses. 5. Pseudoparaphyses: The paraphyses of pseudothecial type of fruiting bodies, especially those which originate at the top and grow down towards the base of the fruiting body.

12.12

CLASSIFICATION

The main criteria of the classification of Ascomycotina have been (i) type and structure of ascus,(ii) type of ascocarp, (iii) ascocarp centrum, and (iv) all ascogenous and conidial stages of the organisms. Owing to the absence of fossil evidence and lack of our present knowledge regarding the developmental stages of this large group, the statement made by Alexopoulos and Mims (1979) about the taxonomy of Ascomycotina appears quite convincing. They mentioned that ‘it is no exaggeration to say that no two specialists agree completely on the classification of this large group of fungi.’ Ainsworth’s (1973) classification has been followed in this book. He divided the subdivision Ascomycotina into six classes. (i) Hemiascomycetes, (ii) Loculoascomycetes, (iii) Plectomycetes, (iv) Laboulbeniomycetes, (v) Pyrenomycetes, and (vi) Discomycetes. (For major points of distinction betwen these classes, see the detailed Ainsworth’s (1973) classification in Chapter 4. Their detailed treatment is made in Chapters 13-18). Kirk et al. (2001), however, treated these fungi in phylum Ascomycota of kingdom Fungi. They divided phylum Ascomycota in 6 classes, namely Ascomycetes, Neolectomycetes, Pneumocystidomycetes, Saccharomycetes, Schizosaccharomycetes and Taphrinomycetes.

12.13

DIFFERENCES FROM PHYCOMYCETES

Phycomycetes is now an obsolete term, used formerly for a class of fungi. It is now treated in “Chromista and some Fungi (Chytridiomycota and Zygomycota) by Kirk et al. (2001). Table 12.1 No. 1. 2. 3. 4.

Differences between Ascomycetes and Phycomycetes

Phycomycetes

Ascomycetes

Along with many terrestrial forms, a large number of Phycomycetes are aquatic. Usually the mycelium is aseptate and coenocytic. Somatic hyphae do not show a tendency to get organized into fungal tissues. Common method of asexual reproduction is by the formation of motile zoospores formed in zoosporangia.

Only a few forms are aquatic; the majority of them are saprophytes or parasites. Except in unicellular members (yeasts), the mycelium is always septate. The hyphae, in many members, show a tendency to aggregate into fungal tissues. Zoospores are not formed; asexual reproduction takes place mainly by exogenously produced conidia. Contd..

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Contd..

5. 6. 7.

8.

Sex organs become progressively complicated from lower to higher Phycomycetes. Plasmogamy is immediately followed by karyogamy, i.e., fusion of nuclei. Diplophase is represented by the zygospore, which is generally long-lived, i.e. requires a long resting period before germination. Well-organized fruiting bodies are not formed.

There is a progressive simplification and ultimate disappearance of sex organs from lower to higher forms. Plasmogany is not immediately followed by karyogamy in a majority of members. Diplophase is represented by the ascus; it does not require long resting period; its diploid nucleus divides soon to form haploid nuclei. Well-organized fruiting bodies or ascocarps are formed in nearly all members.

TEST YOUR UNDERSTANDING 1. Ascomycetes and _______ are sometimes combindly called “higher fungi”. 2. The characteristic ascospores are present in a sac-like body, called _______, and ascomycetous fungi are therefore also called _______ fungi. 3. Write a note on the economic importance of Ascomycetes. 4. Ascocarp is generally _______ in Discomycetes, _______ in Pyrenomycetes, and _______ in Plectomycetes. 5. What are Woronin bodies? 6. Describe briefly the various steps of the sexual reproduction in Ascomycetes with particular reference to development of ascus and ascospores. 7. Write at least two sentences each on (a) hologamy, (b) autogamy, and (c) somatogamy. 8. Give an account of various types of ascocarps found in Ascomycetes. 9. Tabulate at least five differences between Ascomycetes and Phycomycetes. 10. Write one sentence each on (a) paraphyses, (b) periphyses, (c) periphysoids. 11. Ainsworth (1973) divided Ascomycotina into six classes. These are Hemiascomycetes, Loculoascomycetes, Plectomycetes, Laboulbeniomycetes, _______ and _______ .

13

C H A P

HEMIASCOMYCETES

T E R

13.1

WHAT ARE HEMIASCOMYCETES?

These are primitive and morphologically very simple Ascomtycetes in which the mycelium is either totally absent or poorly developed, and a majority of them are yeast-like. They do not show the formation of ascogenous hyphae and ascocarps, and the asci are formed directly from zygotes or single cells. Because of their primitive nature they are also named Protoascomycetidae. Kirk et al. (2001) defined Hemiascomycetes as the “Ascomycota in which the asci are not produced in ascomata. In addition, the thallus usually comprises poorly-developed mycelium or is represented by separate cells.” They have included these fungi mainly in order Saccharomycetales of phylum Ascomycota.

13.2

GENERAL CHARACTERISTICS

1. They occur commonly on healthy or decaying fruits, specially in their sugary exudates. A majority of them are saprophytes (Endomycetales). Some occur symbiotically with insects, and a few are even parasites (Taphrinales) on vascular plants, human-beings and animals, causing many diseases. 2. The mycelium is either poorly-developed or completely absent, and the majority of members are yeast-like. 3. The asci are not borne on ascogenous hyphae. 4. The asci develop singly, usually following karyogamy. They develop either directly on the mycelium or formed from a specialized cell called ascogenous cell. 5. Ascocarps are totally absent. 6. The wall of the asci is thin. 7. The ascospores are usually released from the ascus by bursting.

13.3

WHAT ARE YEASTS?

The word ‘yeast’ is a general term with no taxonomic significance. The term has been used in a variety of ways (Flegel, 1977). According to Kreger-van Rij (1973) the ‘Yeasts are those fungi which, in a stage of their life cycle, occur as single cells, reproducing by budding or fission’. Organisms with multinucleate cells or producing black pigments are not included in the yeasts. Kirk et al. (2001) Defined yeasts as “unicellular, budding fungi”. A majority of the yeasts belong to order Endomyc-

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Fungi and Allied Microbes

etales of class Hemiascomycetes and are called ‘ascomycetous yeasts’. But according to Kreger-van Rij (1973) many yeasts belong to Basidiomycotina and are called ‘basidiomycetous yeasts’ (e.g. Filobasidium, Leucosporidium, Rhodosporidium and Sporidiobolus), and some also to Fungi Imperfecti or Deuteromycotina and are called ‘deuteromycetous yeasts’ (e.g. Bullera, Intersonilia, Trigonopsis, Torulopsis and Trichosporon). Kurtzman and Fell (1998) included 100 genera and over 700 species under yeasts, while Barnett et al. (2000) recognized 90 genera and 678 species as yeasts. For details of yeast readers may refer Guide to Yeast Genetics and Molecular Biology by Guthrir and Fink (1991) and The Yeasts by Kurtzman and Fell (1998). Only ‘ascomycetous yeasts’ are discussed in this chapter.

13.4

CLASSIFICATION

Martin’s (1961) classification, followed by Kramer (1973), recognizes three orders under Hemiascomycetes: Protomycetales, Endomycetales and Taphrinales. The members belonging to Protomycetales are parasitic on higher plants and contain their asci in a compound spore sac, called synascus. However, the asci are formed separately in Endomycetales and Taphrinales. Endomycetales are mostly saprobic, whereas Taphrinales are parasitic on vascular plants. In Endomycetales, most of which are yeasts (Kreger-van Rij, 1973), the mycelium is lacking, and zygote transforms directly into an ascus. But in Taphrinales the dikaryotic hyphae bear terminal chlamydospores or ascogenous cells, each of which develops into a single ascus. Only Endomycetales and a brief account of Taphrinales are discussed in this chapter.

13.5

ENDOMYCETALES

Because of the presence of yeasts, such as Saccharomyces and Schizosaccharomyces, some prefer to name Endomycetales as Saccharomycetales (Bessey, 1965). Recently, Kirk et al. (2001) also used the word Saccharomycetales for Endomycetates. Some general characteristics of these fungi are mentioned below: 1. Plant body in yeasts (Saccharomyces and Schizosaccharomyces) is unicellular, but in genera such as Eremascus true mycelium is present. The cell wall composition is insufficiently known (Kreger-van Rij,1973). 2. Budding in succession in some yeasts results in the formation of a sort of false mycelium, called pseudomycelium, e.g. Saccharomyces (Fig. 13.4 B). 3. Yeasts contain uninucleate cells but coenocytic mycelium is present in some Spermophthoraceae. 4. Asexual reproduction takes place by fission, budding and arthrospore formation (Alexopoulos and Mims, 1979). However, in Spermophthoraceae the asexual spores are produced in spore sacs. Some workers call these sporesacs as sporangia. 5. Sexual reproduction takes place by the fusion of two somatic hyphal cells, two ascospores or two gametangia. 6. Nuclear fusion takes place in the young ascus in a majority of the members. 7. Dikaryotic phase is absent. 8. The zygotes or single cells transform directly into asci. Kreger-van Rij (1973) divides Endomycetales into four families, viz. Ascoideaceae, Spermophthoraceae, Saccharomycetaceae and Endomycetaceae. Kirk et al. (2001) divided Saccharomycetales (equivalent to Endomycetales into 11 families, and included 66 genera and 276 species under this order. Eleven families proposed by these workers are Ascoideaceae, Cephaloascaceae, Dipodascaceae, Endomycetaceae, Eremotheciaceae, Lipomycetaceae, Metschnikowiaceae, Phaffomycetaceae, Saccharomycetaceae, Saccharomycodaceae and Saccharomycopsidaceae. Only Saccharomycetaceae is discussed here.

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Hemiascomycetes

13.6

SACCHAROMYCETACEAE

It includes most of the ascogenous yeasts. The mycelium is usually lacking or scanty, and a majority of the members are unicellular. Vegetative reproduction takes place by budding or fission of single cells. The ascospores may be round, oval, hemispherical, oblate-ellipsoidal, reniform or sickle-shaped, but never needle-or spindle-shaped (Kreger-van Rij,1973). Nearly all members show ability to ferment. Kreger-van Rij (1973) divided Saccharomycetaceae into following four subfamilies: (i) Schizosaccharomycoideae, e.g. Schizosaccharomyces (ii) Nadsonioideae, e.g. Nadsonia, Saccharomycodes (iii) Lipomycetoideae, e.g. Lipomyces (iv) Saccharomycetoideae, e.g. Saccharomyces Kirk et al. (2001), however, did not divide this family into subfamilies. They included 18 genera and 159 species under family Saccharomycetaceae. Detailed aspects of Saccharomyces cerevisiae, and some major aspects of the life-cycle of Schizosaccharomyces octosporus and Saccharomycodes ludwigii are discussed here in some details.

13.7 13.7.1

SACCHAROMYCES CEREVISIAE Systematic Position

According to Ainsworth (1973) Division Subdivision Class Order Family Subfamily Genus

– – – – – – –

Eumycota Ascomycotina Hemiascomycetes Endomycetales Saccharomycetaceae Saccharomycetoideae Saccharomyces

According to Kirk et al. (2001) Kingdom Phylum Class Order Family Genus

– – – – – –

Fungi Ascomycota Ascomycetes Saccharomycetales Saccharomycetaceae Saccharomyces

13.7.2 Occurrence Saccharomyces is represented by about 40 species (van der Walt, 1970), while Kirk et al. (2001) recognised only 10 species. Of these, S. cerevisiae is the best known (Webster, 1980). Because of its widespread use in baking and brewing industries in making bread, yeast cakes, beers, wines, etc., it is popularly called ‘baker’s yeast’ or ‘brewer’s yeast’. It occurs saprophytically in or on a sugary solution, particularly on the surface of ripe fruits, decaying vegetables and the substrata that contain sugar. It also occurs in the soil, on vegetative parts of the plants and in the nectar of flowers. For details of the taxonomy of yeasts, readers may refer “The Yeasts: A Taxonomic Study” by Kurtzman and Fell (1998).

13.7.3 Culture In laboratory, yeast can be cultured by placing a yeast-cake piece (or few grains of dried yeast) in a solution of molasses and water or sugar solution. Within a day millions of the yeast cells will develop in the solution.

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Somatic Structure

The thallus is non-mycelial, microscopic and unicellular. The cells are round, spherical, elliptical or oval. Rarely, they are elongated or rectangular. The size of the cells generally ranges between 6-8 mm × 5-6 mm (Webster, 1980). Instead of light microscopy, the transmission electron microscopy and chemical analysis of wall preparations have provided a more clear picture of the yeast cells. Some details of different parts are mentioned below. The cell wall surrounds the protoplast. It is delicate, thin, firm and chitinous. The cellulose is absent. Along with chitin the cell wall also contains polysaccharides (glycogen and mannan), lipids, phosphate and proteins, as confirmed by chemical analysis and X-ray diffraction methods. Electron microscopic studies revealed that the cell wall consists of two layers, of which outer electron-dense layer is about 0.5 mm thick and the inner microfibrill-containing layer is less dense in electrons and is of about 0.2 mm thickness. However, studies of Matile et al. (1969) suggest that cells with thick walls in S.cerevisiae contain three layers. Of these three, the outer layer consists mainly of mannan-protein and some chitin, middle layer of glucan and the innermost layer of protein-glucan (Matile et al., 1969). About 7% of the dry weight of the cell wall consists of protein (Webster, 1980) and about 30% of glucan, which is the yeast cellulose. Mannan also constitutes about 30% of the dry weight. Plasmalemma is the limiting membrane of the cytoplasm. It is characteristic in having a series of shallow invaginations or elongated pits. Inner to the plasma membrane are present almost all cell inclusions in the cytoplasm, typical to eukaryotic cells, viz. mitochondria, Golgi apparatus, ribosomes, endoplasmic reticulum, lipid granules in the form of sphaerosomes and nucleus. Mature yeast cells enclose a large, well-developed, centrally-located vacuole, surrounded by a single vacuolar membrane, called tonoplast. Water, lipid granules and granules of polymetaphosphate remain filled in the vacuole. The mitochondria may be of many different shapes, even within the same cell. They may be oval, rod-like, spherical or even thread-like. They are generally unbranched, but sometimes also branched. Nucleus is differently interpreted by different workers. Yeast nucleus can be studied best at the time of budding; it is difficult to be seen in living cells (Webster, 1980). Lindegren (1949) opined that vacuole is a part of the nucleus and on one of its side is present centrochromatin. Nuclear vacuole also contains six pairs of chromosomes. Mitochondria remain adhered mainly to the centrosome and nuclear membrane. However, electron microscopic studies suggest that nucleus and vacuoles are two separate entities (Fig. 13.1). The nucleus remains surrounded by a difinite nuclear membrane, which bears many pores. It has no contact with the vacuole. The nucleus contains a cup-shaped nucleolus and dome-shaped nucleoplasm at the time of budding (Webster, 1980). Transmission electron microscopy has been used to study details of budding yeast cell of Saccharomyces cerevisiae by Baba and Osumi (1987). They have observed a morphologically recognizable Golgi stack in this yeast (Fig. 13.2) which is unusual among Eumycota. They have shown that a vegetative yeast cell is densely packed with organelles, including mitochondria, a large central vacuole and one nucleus.

13.7.5

Chromosome Number

Generally, the vegetative cells of Saccharomyces cerevisiae are diploid. According to Ganesan (1959) the diploid chromosome number is 8, but according to Sherman and Lawrence (1974) there are present 17 linkage groups. Yeasts also show polyploidy.

13.7.6

Nutrition

Yeasts are also heterotrophic, because of the absence of chlorophyll. They live saprophytically on ripe fruits, fruit juices and other sugary solutions, and obtain nutrition in the form of simple sugars. The elements required as nutrients by the different strains of yeasts include C, H, O, N, K, P, Mg, S, Fe, Cu and Zn. They are taken from different sources, e.g. carbon from sugars or starch.

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Mitochondria Bud vacuole Bud Spindle pole body Microtubules

Dividing nucleus Nuclear membrane Pore

Vesicles Vacuole

Golgi apparatus

Vacuole membrane Cell membrane

Endoplasmic reticulum

Bud scar

Vacuolar granules Cell wall

Fig. 13.1

Storage lipid granules

Diagrammatic representation of a section of a cell of Saccharomyces cerevisiae showing budding under electron microscope.

Spindle-pole body Ribosomes Nucleus

Rough ER

Nucleolus Nuclear pore

Golgi stack

Vacuole Smooth ER

Microbody Glycogen granules Mitochondrion Polyphosphate granules

Ribosomes Cell wall Lipid droplets

Plasma membrane

Fig. 13.2

Bud scar Periplasmic space

Diagram of a budding yeast cell as seen under transmission electron microscope (modified from Baba and Osumi, 1987).

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Yeast protoplasm secretes certain enzymes, which are collectively called zymase. Zymase changes the complex sugars or starch of the substratum into simple type of sugars. These simple sugars diffuse into the cytoplasm through thin permeable cell membrane. A little amount of this diffused sugar is used as food. Under aerobic conditions the yeast cells multiply rapidly, and the remaining sugar is oxidized during the process of respiration: C6H12O6 + 6O2 Æ 6CO2 + 6H2O + energy However, under anaerobic conditions or when the oxygen supply is poor, yeast cells multiply very slowly. The cause is the conversion of large amount of sugar into CO2 and ethyl alcohol. C6H12O6 + yeast cells Æ 2 C2H5OH + CO2 + energy (enzymes)

glucose

ethyl alcohol

The ethyl alcohol and carbon dioxide, so formed, diffuse through the cell wall into the surrounding liquid. This process, in which carbohydrates (glucose, etc.) are broken by the yeasts into ethyl alcohol and carbon dioxide in the absence of oxygen, is called alcoholic fermentation. This liberated energy, during respiration, is used in many vital activities. The process of alcoholic fermentation by the yeasts is utilized in various industries in the preparation of bread, alcohol, wines, etc.

13.7.7 Asexual Reproduction in Saccharomyces and Other Yeasts Saccharomyces reproduces asexually only by budding, and therefore, it is commonly called ‘budding yeast’. But all species of Schizosaccharomyces reproduce asexually by fission and are commonly called ‘fission yeasts’. It is the most common method of reproduction in Saccharomyces (Fig. 13.3 A-D), which takes place under favourable conditions. The process of budding starts by the formation of a small outgrowth or bud at or near one pole of the cell. During the formation of the bud, the nucleus of the parent cell divides into two, and one of the daughter nuclei migrates into the bud. The bud increases in size and becomes constricted at the base. This constriction increases and ultimately brings about a division of the parent cell into two daughter cells of very unequal size. Bud

New cell Parent cell

A

Fig. 13.3

B

C

D

A-D, Process of budding in Saccharomyces cerevisiae.

According to Hartwell (1974), in a fully mature bud, first a primary septum develops at a place where the bud joins the parent cell. The primary septum is made up of chitin. This is followed by the formation of a secondary septum, made up of glucan (Hartwell, 1974). Separation of the bud leaves a scar with a convex surface on the parent cell. This is called bud scar. A concave scar is also retained by the newly formed daughter cell. It is called birth scar (Fig. 13.4A). This process of the formation of new buds from the parent cell goes on leaving the bud scars. Therefore, each cell has a number of bud scars and one birth scar according to Belin (1972). A bud scar is actually a place on a cell from where a bud has been developed. By counting the

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bud scars on a cell, one can determine the number of buds that have been produced from it. As many as 23 bud scars have been observed on a single cell of Saccharomyces cerevisiae (Webster, 1980). If the nutrient supply (sugar solution, etc.) is available in abundance, the budding process is very quick. It becomes so rapid that the daughter cell formed by the budding begins to form a new bud before becoming separated from the parent cell. It results in the formation of a chain of buds. Such chains may be branched or unbranched and give the appearance of a false mycelium, called pseudomycelium (Fig. 13.4B). Bud scar

A

Fig. 13.4

Birth scar

B

A, Yeast cell showing bud scar and birth scar; B, Showing pseudomycelium in Saccharomyces cerevisiae.

Fission is not shown by Saccharomyces. It is shown by the genus Schizosaccharomyces, and hence its species are called ‘fission yeasts’. Schizosaccharomyces has been discussed by Kirk et al. (2001) under family Schizosaccharomycetaceae of order Schizosaccharomycetales, subclass Schizosaccharomycetidae of class Schizosaccharomycetes of Ascomycota. During the process of fission, nucleus divides into two daughter nuclei, and the parent cell divides into two daughter cells by the formation of transverse wall in the centre (Fig. 13.5 A-D). Both the uninucleate daughter cells so formed are later on separated and lead an independent life. The process of fission is actually not that much simple as it appears. The nucleus of the cell divides by intranuclear mitosis. It constricts, becomes dumb-bell shaped and divides into two daughter nuclei. The cell divides into two daughter cells by centripetal development of a septum, which cuts the cytoplasm into two. Parent cell

A

Fig. 13.5

Daughter cells

B

C

D

A-D, Stages of fission in Schizosaccharomyces.

13.7.8 Sexual Reproduction in Yeasts The yeasts do not produce definite sex organs, such as antheridia or oogonia. They reproduce sexually by the union of two somatic cells or two ascospores, which assume the function of copulating gametangia. Plasmogamy and karyogamy take place and diploid zygote is formed, which eventually changes into an ascus. Ascospores develop in the ascus. Number of ascospores depends on the number of nuclear divisions in the zygotic nucleus, but the usual number is eight. On liberation the ascospores germinate and form new cells by budding or fission.

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Bud

In spite of the above-mentioned general process, the sexual reproduction in yeasts is not so simple. Guilliermond (1940) believed that yeasts show three different types of life-cycle patterns, exemplified by Saccharomyces cerevisiae, Saccharomycodes ludwigii and Schizosaccharomyces octosporus.

C

a

Gametangia

a

D

Bud

a a B

a

a a

a

a

Plasmogamy

H

ap

lo

ph

as

e

(

)

It is a heterothallic speAscospores a a cies. Out of the 4 haploid ascospores liberated from an a a A ascus (Fig. 13.6 A), two carry one mating type (a) and other two carry other mating type (a). Both these types a develop into independent cells of their respective mata E a a ing types (Fig. 13.6 B). a n a n) It has been shown by Hartwell (1974) that when cells (2 e s of both these mating types (a and a) are not in contact Ascospores K ha op pl Young with each other, a sex hormone develops. In response to i D ascus this hormone these elongate and enlarge towards the cells Zygote of opposite mating types and bring about their fusion. The molecular weight of factor (a) is about 1400, as menF tioned by Hartwell. J Meiosis Normally these haploid cells of different matDiploid Ascus mother bud ing types multiply by budding (Fig. 13.6C). The hapcell (2n) loid buds so formed lead independent life and keep on producing new haploid cells for several generations. Under certain conditions, two somatic cells of opposite I mating behave as gametangia and undergo sexual fusion G (Fig. 13.6D). Plasmogamy (Fig. 13.6E) and karyogamy H take place and a diploid zygote is formed (Fig. 13.6F). Zygote The zygotic cells (Fig. 13.6F) are comparatively larger than the haploid cells (Fig. 13.6 B) and are ellipsoidal. Diploid cells The zygote also undergoes budding (Fig. 13.6G) and keeps on forming diploid cells for several generations. Fig. 13.6 A-K, Life-cycle of Saccharomyces cerevisiae These diploid cells (Fig. 13.6H) of S.cerevisiae also lead (based on Guilliermond). independent life like that of haploid cells. Under shortage of water, food and low temperature, the diploid cells start to function as asci (Fig. 13.6I). The diploid nucleus undergoes meiosis. and four haploid daughter nuclei are formed (Fig. 13.6J). Electron microscopic studies of lllingworth et al. (1973) and Beckett et al. (1974) have shown that meiosis is S. cerevisiae is also intranuclear like mitosis. During meiosis the nuclear membrane remains intact, and the four haploid nuclei remain within the original membrane for some time. Such a nuclear division has been named uninuclear by Moens and Rapport (1971). Two of these four nuclei formed after meiosis belong to the mating type (a) and other two to the mating type (a). All these four haploid nuclei accumulate some cytoplasm around themselves and change into ascospores (Fig. 13.6K). These haploid ascospores rupture the ascus wall, come out (Fig. 13.6A ) and develop into fresh haploid cells, of which two belong to mating type (a) and the remaining two to the mating type (a). Such a life-cycle of heterothallic strains of S. cerevisiae indicates the existence of independent haploid and diploid phases of equal importance. Such a fusion of cells or nuclei of opposite mating types is called legitimate copulation. Sometimes, fusion as of the cells of the same mating type results in the formation of a diploid cell, which changes into an ascus. Lindegren (1949) called such a fusion as illegitimate copulation.

Hemiascomycetes

157

According to Kirk et al. (2001), the yeast Saccharomycodes belongs to family Saccharomycodaceae of order Saccharomycetales, class Saccharomycetes of phylum Ascomycota. In this yeast four ascospores, formed in an ascus, do not rupture the ascus wall (Fig. 13.7A). They start to behave as ‘gametangia’. Fusion (plasmogamy and karyogamy) of two ascospores of the opposite mating types (A1 and A2) takes place within the ascus (Fig. 13.7 B). This results in the formation of two diploid zygotic cells within the wall of the ascus (Fig. 13.7 C). Each such diploid cell starts germination within the ascus. A germ tube develops, which ruptures the ascus wall (Fig. 13.7 C). The germ tube becomes multicellular and functions as a diploid sprout mycelium (Fig. 13.7 D). From the cells of this sprout mycelium develop some diploid buds. The diploid buds so formed get detached from the parent sprout mycelium by the formation of a septum at the base and function as diploid sprout cells (Fig. 13.7 E). Under suitable conditions this sprout cell functions as an ascus. Its diploid nucleus undergoes meiosis, and four haploid ascospores are formed within the ascus (Fig. 13.7 A). Out of these four ascospores two belong to mating type (A1) and other two to the mating type (A2). Thus the life-cycle is completed. Thus, in Saccharomycodes ludwigii the entire life-cycle is dominated by diploid stages. The haploid phase is represented only by the ascospores, which also remain within the ascus and soon undergo fusion to form diploid zygote. Kirk et al. (2001)treated Schizosaccharomyces as an yeast belonging to family Schizosaccharomycetaceae, order Schizosaccharomycetales, class Schizosaccharomycetes, phylum Ascomycota and kingdom Fungi. This yeast species is homothallic (Guilliermond, 1931). Its somatic cells (Fig. 13.7 F) are haploid, and keep on dividing by simple transverse division into two daughter cells (Fig. 13.7 G, H). Each haploid somatic cell is a potential gametangium. At the time of sexual reproduction two such cells come together and send a small protuberance towards each other (Fig. 13.7 I). The protuberances of both these cells come in contact with each other and the wall at the point of contact dissolves to form a common passage, called conjugation tube or conjugation canal (Fig. 13.7 J). The nucleus of both these gametangia moves into this tube. Two nuclei fuse in this region of conjugation tube and form a diploid zygotic nucleus (Fig. 13.7 K). The cytoplasm of the two gametangial cells mixes to form a common zygote cell. It now functions as an ascus. The zygotic nucleus divides first meiotically to form four haploid nuclei (Fig.13.7L) and then meiotically to form eight nuclei (Fig. 13.7 M). All these nuclei organize themselves into ascospores (Fig. 13.7N). The ascospores rupture the ascus wall, come out (Fig. 13.7O), and develop into individual uninucleate, haploid somatic cells (Fig. 13.7F). Thus the life-cycle is completed. The above-mentioned details of Schizosaccharomyces octosporus indicate that its life-cycle is dominated by haploid stages. The diploid phase is represented only by the zygote cell, which also soon undergoes meiosis and results first into 4 and then 8 haploid nuclei. Thus, its life-cycle is just opposite to that of Saccharomycodes ludwigii. In the former it is dominated by the haploid stages, whereas in the latter it is dominated by the diploid stages. And, in Saccharomyces cerevisiae both diplophase and haplophase are well-represented and are of equal importance.

13.8

ECONOMIC IMPORTANCE OF YEASTS

Yeasts play several roles in human life, and are of particular importance to the modern civilization. Some of their important economic aspects are mentioned below: 1. They have the ability to ferment sugar solution, specially when the oxygen supply is very poor, and this results in the formation of ethyl alcohol and carbon dioxide: C6H12O6 Æ 2 CH3CH2OH + 2 CO2 + energy (ethyl alcohol)

This process of oxidation of sugar into alcohol and carbon dioxide under anaerobic conditions is called alcoholic fermentation, and is brought about by enzymes like zymase and invertase. Only because of this ability of forming alcohol and carbon dioxide the yeasts are used widely in brewing and baking industries, specially in the prepara-

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Fungi and Allied Microbes

Diploid nucleus (2n)

A2

Ascus

C Germ Karyogamy tube

A1 A2

A1

Bud (2n)

Plasmogamy

B

A1 A1

Sprout mycelium (2n)

D

A2

Ascospores (n)

A2

Sprout cell (2n)

is ios

Me

Ascus A

Meiosis

Diploid nucleus (2n)

E Haploid nuclei

8-nucleate stage

L

Karyogamy

M

K Ascospores (n)

Conjugation tube

J

Ascus

Plasmogamy N

Somatic cells (n) I Gametangia (n)

O F

Ascospores

Daughter cells Division H

Fig. 13.7

G

A-E, Life-cycle of Saccharomycodes ludwigii; F-O, Life-cycle of Schizosaccharomyces octosporus (all after Guilliermond).

Hemiascomycetes

159

tion of beer, natural wines and bread-making. Saccharomyces cerevisiae is employed universally in baking and brewing industries. In the bread-making industry CO2 produced during fermentation is responsible for raising the dough and giving the bread a spongy texture. 2. The ability of alcoholic fermentation of yeasts is also utilized in the preparation of many other industrial products, such as ethyl alcohol, glycerol, ethers, fatty acids, acetic acid and succinic acid. Because of their high value, some industrially superior strains of yeasts have been developed by selection and breeding, e.g. brewer’s yeast, baker’s yeast, distiller’s yeast and wine yeasts. 3. Along with starch, the yeast cells are compressed in the form of special cakes, called yeast cakes. Such cakes are used in home as well as in industries throughout the world. 4. Yeasts have high vitamin contents and also some amount of proteins. Vitamins and proteins make yeasts a valuable food. 5. Some yeasts are used in the treatment of certain skin diseases and intestinal disorders. 6. Some yeasts are used in the manufacture of various syrups and confectionary products. 7. Eremothecium and Ashbya are used in the commercial production of vitamin B2 (riboflavin) according to Gray (1959). Yeasts also have some destructive roles to play for human being: 1. Cheese, tomato products and other similar foods are spoiled by yeasts, because they impart an objectionable yeastyflavour to these products. 2. Some yeasts are parasitic on man causing many diseases, such as cryptococcosis, blastomycosis and torulopsis. A mental disorder (cryptococcosis) is caused by Cryptococcus neoformans. Some yeasts may even cause irritation in vagina. 3. Ashbya, Nematospora, Spermophthora and Eremothecium are parasitic on cotton, destroying the crop.

13.9 13.9.1

TAPHRINALES Distinguishing Characteristics

1. All members are parasitic on higher vascular plants. 2. They produce a definite mycelium in nature, but on artificial media no mycelium is formed. 3. The asci produced by the mycelium lie parallel to one another in a palisade-like layer. 4. The asci are not enclosed by any peridium. 5. Like yeasts their ascospores also multiply by budding. 6. Hyphae usually bear terminal chlamydospores. 7. The ascus is produced from a special, binucleate, ascogenous cell developed from the mycelium. According to Kramer (1973) Taphrinales include only a single family (Taphrinaceae) and a single genus Taphrina. Kirk et al. (2001), however, treated Taphrinales as an order of an independent class Taphrinomycetes of phylum Ascomycota, under kingdom Fungi. They divided Taphrinales into 2 families (Protomycetaceae and Taphrinaceae) comprising 6 genera and 115 species. A brief botanical note on Taphrina is undermentioned.

13.10

TAPHRINA

Taphrina is represented by about 95 species (Kirk et al., 2001), majority of which are parasitic on ferns, Fagales and Rosales. T. deformans is the most common species causing leaf curl of peach (Fig. 13.8 A), almonds and other orchard trees. T. pruni and T. cerasi are other common species. Studies of Sziraki et al. (1975) suggest that leaves infected by T. deformans contain increased cytokinin activity, increased indole-3-acetic acid and tryptophane content.

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Fungi and Allied Microbes

The mycelium is multicellular, and the cells are typically binucleate. The hyphae are intercellular or intracellular. Asexual reproduction takes place by small, oval or Ascus spherical, uninucleate, yeast-like conidia called blasAscospores Infected tospores, which develop by budding of ascospores while Stalk leaf cell they are still within the ascus. The blastospores germinate either by producing a germ tube which develops into mycelium, or also by their budding. Host No specific sex organs develop in Taphrina. In majorcell ity of species, a one-celled thick compact mycelial layer develops between cuticle and epidermis of the host tissue. The cells of this layer are more or less rounded and function as ascogenous cells. At first, they are binucleate. Both A B these nuclei fuse, and this initiates the ascogenous hyphae to elongate. Ascogenous cell divides transversely to form Fig. 13.8 Taphrina deformans. A, Showing symptoms on a short stalk cell and a large cell which develops into an an infected leaf of peach; B, Showing formation of ascus and ascospores. ascus (Fig. 13.8 B). The diploid nucleus of the ascus divides meiotically and then ordinarily to form eight nuclei, which get surrounded by some cytoplasm and change into ascospores. According to Syrop (1975) the ascus of Taphrina is unitunicate, and release of ascospores takes place by simple bursting of ascus tip. Released ascospores produce many blastospores by budding. Blastospores germinate by producing germ tube, which infects the host.

TEST YOUR UNDERSTANDING 1. What are yeasts? Explain in about 100 words. 2. Yeasts belong to: (a) Ascomycetes (b) Basidiomycetes (c) Deuteromycetes (d) all of these 3. How can you culture yeast in the laboratory? 4. Draw a well-labelled diagram of a budding yeast cell as viewed under transmission electron microscope. 5. Give a detailed account of asexual reproduction in Saccharomyces and other yeasts. 6. The ‘budding yeast’ is a common name often given to _______ . 7. All species of Schizosaccharomyces reproduce asexually by _______ and are thus commonly called _______ . 8. Write short notes on (a) Budding, (b) Pseudomycelium. 9. Describe economic importance of yeasts. 10. Write a detailed note on structure and reproduction in Taphrina.

14

C H A P

LOCULOASCOMYCETES

T E R

14.1

WHAT ARE LOCULOASCOMYCETES ?

The ascomycetous members in which the asci are bitunicate, and the ascocarps are ascostromata, are placed under class Loculoascomycetes of subdivision Ascomycotina. The asci are borne in locules within a stroma, and not in a hymenium. Each ascostroma later on becomes either perithecioid, or less commonly an apothecioid pseudothecium (Luttrell,1973). The name ‘Loculoascomycetes’ was first proposed by Luttrell (1955) as a subclass of Ascomycota with fissitunicately discharging asci, producing ascospores which are generally septate and borne in unwalled pseudothecia. The group corresponds to the Ascoloculares of Nannfeldt (1932). Kirk et al. (2001) have mentioned that this class is not accepted in the 9th edition of Dictionary of Fungi.

14.2

HABITAT

These fungi occur as superficial epiphytes, parasites, or hyperparasites of superficial fungi and insects. Many species occur as saprophytes on dung, decaying wood, stem and leaves. Genera such as Buellia and Phaeospora are parasitic on lichens, whereas Lizonia occurs parasitically on mosses. Piedraia occurs on human hairs, whereas genera such as Achorodothis and Cerodothis occur on green leaves or young stems. Many species of Didymella, Pleospora (Fig. 14.1 A-C), Microthelia and Buellia are marine. Some Loculoascomycetes also occur on many crop and ornamental plants, such as Triticum, Zea, Avena, Musa, Hevea and Prunus. Patil and Borse (1985) reported some marine Loculoascomycetes from Maharashtra (India).

14.3

SOMATIC STRUCTURE

The mycelium is well-developed, branched, septate, and resembles other Ascomycotina. The mycelium forms a lichen-like thallus in Cucurbidothis, whereas it forms a brown felt over conifer seedlings in Herpotrichia. According to Luttrell (1973) the mycelium forms a sort of membrane of radiating or parallel hyphae on the leaves or young stems of Venturia and Hormotheca. In a majority of the genera it is superficial.

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14.4

Fungi and Allied Microbes

ASEXUAL REPRODUCTION

Many Loculoascomycetes reproduce asexually by conidia. The genera, that do not produce conidia, reproduce only by ascospores.

14.5

SEXUAL REPRODUCTION

It takes place by a variety of ways such as somatogamy, spermatization, and also by gametangial contact in different genera.

14.5.1

Ascocarp

The ascocarp in these fungi is stromatic, bearing the asci directly in locules within the stroma. Such ascocarps are called ascostromata. The stroma is either prosenchymatous or pseudoparenchymatous. The stromatic tissue surrounds the asci and sterile threads. No special wall surrounds the ascocarp centrum. Within the stroma are present one or more locules, which contain the asci. If only one locule is present in the ascostroma, it becomes very difficult to distinguish it from a perithecium. Some prefer to call these unilocular ascostromata as pseudothecia. Luttrell (1973) distinguished six major categories of ascocarps, based on their size. According to him the size of the ascocarps in Loculoascomycetes varies from small (40-150 mm in Dothideales and many Myriangiales) to very large (1-5 mm in Hysteriales). The ascostromatous ascocarps may be bright coloured, gelatinous, lenticular, apothecioid, elongated or even with laterally compressed beak and slit-like ostiole (Luttrell, 1973). Four major types of the ascocarp structures have been distinguished by Luttrell (1973) among Loculoascomycetes: (i) In Myriangiales, the asci remain scattered individually in unaltered stromal tissue; (ii) in Dothideales, the asci are ovate to short-cylindrical, and remain present in fascicles in small perithecioid locule in ascostromata, which may be unilocular or multilocular; (iii) in Pleosporales, the asci are clavate or long-cylindrical, and occur in broad basal layers in perithecioid locules; and (iv) in Patellariaceae, the ascocarps resemble with apothecia.

14.5.2

Ascus Ascospores

Ascus

The distinguishing character of these fungi is the presence of bitunicate ascus, i.e. the ascus wall consists of two separable layers (Fig. 14.1 B). The outer, thin, inextensible layer is called ectoascus, whereas the inner, thick, extensible layer is called endoascus. The asci are thinwalled, when young. At the tip of each ascus is present a tubular channel or subapical chamber, variously called ‘apical basket’, pore or ‘nasse’ (Chadefaud, 1960). The ascospores are ejected through this pore or channel. At the time of the discharge of the ascospores, the endoascus expands twice or thrice the length of the ectoascus. The endoascus is a multi-layered structure in three Loculoascomycetes investigated by Funk and Shoemaker (1967), and their discovery puts a doubt on the universal presence of bitunicate asci in all Loculoascomycetes, as in Pleospora herbarum (Fig. 14.1 B).

Ruptured outer wall

Pseudoparaphyses Young asci

A

Fig. 14.1

B

C

A–C, Pleospora herbarum. A, Ascus and ascospores; B, Stretched bitunicate ascus showing ruptured outer wall; C, Developing asci and pseudoparaphyses.

163

Loculoascomycetes

14.5.3

Ascospores

Except in a few genera (Luttrell,1973), all Loculoascomycetes contain septate ascospores (Fig. 14.1 A, B). Non-septate ascospores are found in Hyalotheles and Hyphotheca. The ascospores are 1-septate in genera such as Micularia and Calyptra, whereas they contain several septa in genera such as Trichometasphaeria and Pyrenophora (Fig. 14.2 B, C). They contain germ slits or germ pores. The ascospores with gelatinous sheaths are found in Julella and Massaria, whereas those with cilia are found in Microthyrium. Slimy appendages are found on the ascospores in Lophiostoma.

Ectoascus Endoascus Ascospore Ascus Ostiole Paraphyses Ascospores

(A)

Fig. 14.2

(B)

(C)

A, A perithecioid pseudothecium of Mycosphaerella; B, An ascus of Trichometasphaeria turcica; C, An ascus of Pyrenophora bromi.

TEST YOUR UNDERSTANDING 1. 2. 3. 4. 5.

What are Loculoasomycetes? Ainsworth (1973) treated Loculoascomycetes under which subdivision of Eumycota? Who proposed the name Loculoascomycetes? Did Kirk et al. (2001) accepted Loculoascomycetes as a class in the 9th edition of Dictionary of Fungi. Describe in brief the ascocarp, ascus and ascospores of Loculoascomycetes.

15

C H A P

PLECTOMYCETES

T E R

15.1

WHAT ARE PLECTOMYCETES?

Class Plectomycetes includes the Ascomycotina in which the asci are irregularly arranged within and ascocarp. Only because of this character, they have been named Ascocarpic Ascomycetes by Alexopoulos and Mims (1979). Ainsworth (1973) included all such Ascomycotina under Plectomycetes, where the asci are evanescent and remain scattered within the astomous ascocarp, which is typically a cleistothecium, and ascospores are aseptate. According to Fennell (1973), Plectomycetes include the fungi that produce ‘closed ascocarps in which globose evanescent asci are borne at all levels from ascogenous hyphae ramifying irregularly throughout the central tissue of the fruiting bodies’. Kirk et al. (2001) mentioned that Plectomycetes was recognized as a class “in the past”, but during the “last 5 –10 years molecular sequence data (especially of the 18Sr DNA gene) have come to the fore”, and based on the available details they have now not recognized this class as they divided Ascomycota into six classes (see Appendix–3).

15.2 1. 2. 3. 4. 5. 6.

GENERAL CHARACTERISTICS

The mycelium is well-developed, branched and septate. From the mycelium develop conidiophores, which bear large number of conidia in a majority of the members. Many members show degeneration of sex organs, especially of male gametangium. The mycelium forms well-defined fruiting bodies or ascocarps, in which develop asci and ascospores. Asci develop from ascogenous hyphae, which are typically dikaryotic. The asci are typically 8-spored. Ascospores develop through the formation of a vesicle, called ascus vesicle. They are unicellular and without germ pores or germ slits. 7. In a few members the ascocarp is a loose, cottony mass of hyphae surrounding the sexual apparatus, whereas in a majority of members the fruiting bodies remain surrounded by a well-defined wall. Only a few species lack ascocarps (Alexopoulos and Mims, 1979). 8. In a majority of the members the ascocarp is of cleistothecium type. But some also have perithecium, apothecium or ascostroma. 9. Paraphyses are absent in the fruiting bodies.

Plectomycetes

15.3

165

CLASSIFICATION

The classification of Plectomycetes is highly variable in different systems of classification, mainly because it is an unnatural class (Fennell, 1973). Webster (1980) even calls Plectomycetes as “really an assemblage of unrelated fungi”. According to Fennell (1973) main taxonomic criteria for classifying Plectomycetes are ascocarp initials, ascocarp peridium, asci, ascospores and imperfect state of the fungus. Fennell (1973) discussed various taxonomic aspects of Plectomycetes in detail, and described only one order Eurotiales. Alexopoulos and Mims (1979) recognized four subclasses under Ascocarpic Ascomycetes viz., Plectomycetidae, Hymenoascomycetidae, Laboulbeniomycetidae and Loculoascomycetidae. Under subclass Plectomycetidae they recognized five orders (Ascosphaerales, Elaphomycetales, Onygenales, Eurotiales and Microascales). Webster (1980) discussed Erysiphales and Eurotiales under Plectomycetes. Discussion of different taxonomic treatments is beyond the scope of this book. Only Eurotiales are discussed here.

15.4 15.4.1

EUROTIALES General Characteristics

The general characteristics of Eurotiales are: 1. Members are primarily saprobic, but may also be parasitic on plants as well as animals, causing skin diseases, such as dermatomycoses. 2. They occur in soil as well as on wood, dung, textiles, feathers, horns, hairs, etc., and are responsible for the decomposition of many organic materials. 3. Members are thermophilic or thermotolerant, i.e. they may occur in a wide temperature range. 4. Majority of the members are homothallic, and only a few are heterothallic. 5. Usually the ascogonia remain free on the mycelium in the early stages. Later on they get surrounded by the mycelium developing from the archicarp and surrounding hyphae and develop into ascocarp. 6. Ascocarps are small, spherical, generally sessile and nonostiolate, i.e. lack ostiolar mouth opening. They are generally of cleistothecial type. 7. Paraphyses are absent in the ascocarps. 8. The asci are globose, sessile, thin-walled, quickly evanescent and typically contain eight ascospores. 9. The asci are distributed irregularly throughout the centrum of the ascocarp. 10. Asci lack pore or operculum. 11. Ascospores are unicellular, variously ornamented, and do not bear germ pores or germ slits. 12. Cleistothecia rupture either because of the internal pressure created by ascogenous hyphae or because of weathering. Fennell (1973) recognized nine families under Eurotiales, viz. Amorphothecaceae, Gymnoascaceae, Onygenaceae, Monascaceae, Thermoascaceae, Trichomataceae, Eurotiaceae, Cephalothecaceae and Pseudoeurotiaceae. Of these, only Eurotiaceae is considered here. Kirk et al. (2001) included order Eurotiales under subclass Eurotiomycetidae of class Ascomycetes under phylum Ascomycota of kingdom Fungi.

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15.5

Fungi and Allied Microbes

EUROTIACEAE

The ascocarps in a majority of the species are of cleistothecial type. However, in Talaromyces they are soft. The peridial layer of ascocarp is either prosenchymatous, pseudoparenchymatous or very thick and sclerotioid. The peridium is without sutures or line of dehiscence. The ascospores are of bivalve construction. They are hyaline or pale yellow. Conidia develop within a specialized cell, called phialide.

Nomenclature in Eurotiaceae Nomenclature is a big problem in Eurotiaceae, e.g. the perfect state of some Aspergillus species have been named under a separate genus Eurotium. The main problem is that perfect state of many species of Penicillium and Aspergillus have not been discovered so far (but certainly they exist). They should, therefore, be classified under Fungi Imperfecti or Deuteromycotina, specially till their perfect states are discovered. Raper and Fennell (1965) have been of the opinion that only conidial names (Aspergillus and Penicillium) should be used irrespective of the presence or absence of ascocarp. On the contrary, Subramanian (1972) and others have advocated the use of only ‘perfect names’. In spite of the existing controversy of nomenclature, details of Aspergillus and Penicillium are discussed here under Plectomycetes, mainly to avoid confusion to the young students.

15.6

ASPERGILLUS

15.6.1 Systematic Position

According to Ainsworth (1973) Division Subdivision Class Order Family Genus

15.6.2

– – – – – –

Eumycota Ascomycotina Plectomycetes Eurotiales Eurotiaceae Aspergillus

Latest Position of Nomenclature of Aspergillus and Penicillium

Regarding Aspergillus, Kirk et al. (2001) have mentioned it as “anamorphic Eurotium, Neosartorya, Emericella, represented by 185 widespread” species. 19 species of Anamorph Eurotium are placed in family Trichocomaceae. 12 species of Anamorph Aspergillus belong to Neosartorya of family Trichocomaceae. 27 species of Emericella are also Anamorph Aspergillus. For more details, refer Advances in Penicillium and Aspergillus Systematics by Samson and Pitt (1985) and Modern Concepts in Penicillium and Aspergillus Classification by Samson and Pitt (1999).

15.6.3 Occurrence This cosmopolitan fungus is represented by about 200 species (Raper and Fennell, 1965), of which about 30 species have been reported from India. It is so common in the air, that if a slide or petridish with suitable medium is exposed to the air for a few minutes, Aspergillus conidia appear in abundance. Many species occur in soil but their role in soil economy is not exactly known. A. niger is a very common contaminant of foodstuffs. Some species occur on leather and cloth fabrics. Species such as A. flavus, A. fumigatus, A. terreus and A. niger occur parasitically on many animals, including man, and cause the disease aspergillosis.

167

Plectomycetes

15.6.4

Economic Importance

1. Aspergillus niger, A. flavus and many other species spoil almost all foodstuffs that remain exposed for some time. 2. A. flavus produces a violent toxin called aflatoxin. It is a highly toxic substance. Aflatoxin B1 has some carcinogenic effects and may cause cancer of liver in humans and animals (Hesseltine, 1974; Kogbo et al., 1985). 3. Conidia of various Aspergillus species are very common in the air, and hence they contaminate the laboratory cultures very commonly. 4. Many species grow on leather and cloth fabrics even in slight humid conditions, and reduce their commercial value. 5. The disease aspergillosis is caused in humans and animals by several species including A. terreus, A. flavus, A. niger and A. fumigatus. In humans and birds, symptoms of aspergillosis of lungs resemble very much that of lung tuberculosis, and sometimes are so diagnosed by the doctors wrongly. 6. Many plant diseases (crownrot of groundnut and boll rot of cotton) are caused by species of Aspergillus. 7. Many Aspergillus species are used in various industries, specially in the production of citric acid, gluconic acid and many other similar products. 8. Aspergillus species are also used in the commercial production of some enzyme preparations and enzymes. 9. Some antibiotics have also been isolated from the cultures of Aspergillus. 10. Aspergillus is also used for bioassay of soil for trace elements, such as copper.

15.6.5 Laboratory Culture Aspergillus can be cultured in the laboratory by placing a moist piece of bread under a belljar for a few days. Aspergillus appears in the form of greenish-blue, yellow or black patches. A. glaucus-group forms greenish-blue moulds, whereas the A. niger-group forms black moulds.

15.6.6 Somatic Structures The mycelium (Fig. 15.1 A) consists of many branched, septate and interwoven mass of hyphae. Some hyphal branches remain on the surface, whereas others penetrate into the substratum and absorb the food material. Each cell remains surrounded by a thin wall, and contains many nuclei distributed in the granular cytoplasm. Mitochondria, endoplasmic reticulum and riboNuclei

Cytoplasm

Oil globules Cell wall

A Young conidiophore

Sterigmata Vesicle

Foot cell Conidiophore Conidiopore B

Fig. 15.1

C

D

E

Aspergillus. A, A portion of hypha; B–E, Development of conidiophore and vesicle.

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Fungi and Allied Microbes

somes are also present in the cytoplasm. Reserve food material is in the form of oil globules. A simple central pore is present in each septum. This pore permits the flow of nuclei and other cytoplasmic contents between adjacent cells.

15.6.7 Vegetative Reproduction Vegetatively it reproduces by the common method of fragmentation. Any of the detached hyphal part or fragment develops into a fresh mycelium under suitable conditions.

15.6.8

Asexual Reproduction

It takes place by the formation of large number of conidia on conidiophores. At the time of asexual reproduction many erect hyphae are seen developing from the somatic hyphae. These long, aseptate and unbranched (Fig. 15.1 B) hyphae are called conidiophores. The hyphal cell that gives rise to a conidiophore is called the foot cell. A conidiophore terminates into a globular or bulbous head, called vesicle (Fig. 15.1C). The vesicle is multinucleate, and from its entire surface develops a layer of conidiogenous cells called sterigmata (Fig. 15.1. D-E) or phialides. Instead of one, sometimes two layers of sterigmata develop one upon the other (Fig. 15.2 B). In such cases the sterigmata of the former layer are called primary sterigmata and that of the latter layer secondary sterigmata. Each sterigma is a multinucleate, finger-shaped or bottle-shaped structure (Fig. 15.2 A, B). At the tip of the mature sterigmata or phialides develop many small, globose, unicellular and uninucleate or multinucleate bodies called conidia. The wall of the conidia is very rough. A conidium is black, brown, yellow or green coloured body. Conidia develop in basipetal succession (Fig. 15.2 A, B), i.e. youngest at the base and oldest at the top. Conidia are dispersed by air currents and germinate by producing germ tubes. At the time of phialide formation, many thin areas appear in the thick vesicle wall owing to the dissolution of wall material. The vesicle cytoplasm near these thin areas is pushed out synchronously in the form of oval or bottle-shaped outgrowths. These outgrowths represent the phialides or sterigmata. A nucleus enters into each phialide (Fig. 15.1 D) along with other cell organelles. A phialide generally has an attenuated base and a tapering apex, but its shape differs in different species.

Sterigmata

Conidia

Vesicle

Conidiophore

A

Foot cell

Conidia Vesicle Secondary sterigma Primary sterigma

The young phialide is a uniB nucleate structure (Subramanian, 1971) in A. niger (Fig. 15.3 A). But in some other species it is a multinucleate body. Single nucleus of a phialide Fig. 15.2 Aspergillus. A, A mature in A. niger divides mitotically into two (Fig. 15.3 B), and its tip expands in conidiophore with conidia; the form of a spherical knob (Fig. 15.3 C). This knob receives one daughter B, A conidiophore with primary nucleus and functions as the initial of the first-formed spore or conidium. and secondary sterigmata. The conidium expands and because of this the phialide wall ruptures near its tip (Fig. 15.3 D). This ruptured wall survives as a cap around the firstformed conidium. Before this rupturing of the wall, a new layer of wall material develops around the first-formed conidium (Fig. 15.3 D). This layer functions as the outer wall layer of the conidium. Later on, an inner wall layer also develops (Fig. 15.3 G). Another mitotic division takes place in the phialide, dividing its nucleus again into two daughter nuclei (Fig. 15.3 D). Around the upper daughter nucleus develops a wall. Along with some cytoplasm it comes out from the broken tip of the phialide

169

Plectomycetes

Conidium initial

Young phialide

Broken phialide wall New wall

A

B

C

D 1st conidium

Broken phialide wall Isthmus

Outer conidial wall Inner conidial wall

Isthmus

2nd conidium

1st conidium

2nd conidium 3rd conidium Phialide

E

Fig. 15.3

F

G

A–G, Aspergillus niger, showing development of conidia from a phialide (after Subramanian, 1971).

and functions as a second conidium (Fig. 15.3 E). The second conidium and all other future conidia are not surrounded by the remnants of broken wall of the phialide. In the early stages there is a cytoplasmic continuity between the first-and the secondformed conidia through a cylindrical isthmus (Fig. 15.3 E, F). But this continuity of cytoplasm is stopped by the formation of an inner conidial wall layer (Fig. 15.3 G). The isthmus, which remains in between two conidia, is also called connective. Further mitosis of the phialide nucleus (Fig. 15.3 F) results in the formation of a third conidium (Fig. 15.3 G). The process is repeated several times, resulting in the formation of a chain of conidia over a phialide. In one chain the youngest conidium is at the base of the chain, and the oldest at the top (Figs. 15.2 A, B, 15.3 G), i.e. they remain arranged in basipetal succession. Electron microscopic studies of the structure and development of the phialides and conidia of some other Aspergillus species have been made by Oliver (1972, A. nidulans), Hanlin (1976, A. clavatus) and Fletcher (1976, A. terreus).

15.6.9

Sexual Reproduction

The sexual reproduction has been discovered only in a few species. But it does not mean that in other species it is absent. The species where the sexual reproduction has not been discovered have perhaps lost their ability to reproduce sexually (Alexopoulos and Mims, 1979). Loss in the ability to reproduce sexually is evident from the fact that in some species the antheridium is either absent or remains non-functional, whereas in others both male and female sex organs are ill-developed.

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Fungi and Allied Microbes

Perfect states of Aspergillus have been assigned to many different ascomycete genera. Subramanian (1972) assigned the perfect stages of Aspergillus in nine different genera, of which some are mentioned in Table 15.1. Table 15.1 Perfect states of some Aspergillus species (according to Subramanian, 1972) Aspergillus

Perfect state genus

A. athecius A. glaucus A. nidulans A. fumigatus A. heterothallicus A. alliaceus

Edyuillia Eurotium repens Emericella nidulans Sartorya fumigatus Emericella heterothallicus Hemicarpenteles

The sex organs are called antheridia and ascogonia. Most of the species, where sexual reproduction has been observed, are homothallic. Only a few species (A. heterothallicus and A. fischri) are heterothallic. It starts to develop on the mycelium (Fig. 15.4 A) in the form of a septate, loosely coiled, female hyphal branch, called archicarp. Young archicarp is differentiated into a lower multicellular stalk, middle unicellular ascogonium and the terminal unicellular trichogyne (Fig. 15.4 B). The trichogyne is elongated and produced by the ascogonium for the reception of the male gamete. All cells of the archicarp are multinucleate. These loosely coiled female structures (Fig.15.4 B) later on become tightly coiled (Fig. 15.4 C). It first develops in the form of a male branch, either from the same hypha (Fig. 15.4 B), or from the another adjacent hypha of the same mycelium. The male branch is also designated as pollinodium. The male branch comes close to the trichogyne and cuts a unicellular antheridium at its tip (Fig. 15.4 B). The lower part is called stalk. The antheridium as well as all other cells of the male branch are multinucleate. The tip of the antheridium comes in contact with the terminal part of the trichogyne (Fig. 15.4 C). The intervening walls at the point of contact dissolve, and the contents of the antheridium possibly mix with that of the trichogyne. At the time of the development of fruiting body, the haploid nuclei in the ascogonium come to lie in pairs. The unicellular ascogonium divides into binucleate cells. Each binucleate cell produces an outgrowth, called ascogenous hypha. Further details are not clearly known in a majority of the species but asci and ascospores are formed. The two nuclei of each ascogenous hypha divide, making the latter again a multinucleate body (Fig. 15.4 D). Ascogenous hypha now becomes septate, and its tip cell, which bends over (Fig. 15.4 E), is uninucleate. The upper cell gets swollen,

Ascospores Vegetative mycelium

Ascus K

Ascospores

A J Ascogonium

Wall

Trichogyne

Asci

Antheridium

Ascospores Stalk B

I Antheridium Trichogyne Ascocarp

H

C Haploid nuclei Ascogenous hypha

Ascus Diploid zygotic nucleus Ascus

G

Ascus mother cell

Tip cell D

F E

Fig. 15.4

A–K, Sexual cycle of Aspergillus

Plectomycetes

171

contains two compatible nuclei, and functions as ascus mother cell. Out of the two nuclei of ascus mother cell one is male and another is female (Miller, 1984). According to some workers there is no crozier formation in the ascogenous hypha, and its terminal binucleate cell starts to function as an ascus mother cell. The two nuclei in the ascus mother cell fuse and form a diploid zygotic nucleus (Fig.15.4 F). Diploid nucleus of the ascus divides by meiosis into four haploid nuclei, each of which divides mitotically, giving a total of eight haploid nuclei (Fig.15.4 G). Each nucleus gets surrounded by cytoplasm and develops into an ascospore. At the time when asci and ascospores are developing from the ascogenous hyphae, many sterile hyphae grow up from the base of the archicarp. These sterile hyphae enclose the developing asci completely from all sides, and form a pseudoparenchymatous sheath or peridium. This peridium is a wall of multicellular sterile hyphae. The entire structure (asci, ascogenous hyphae and peridium) appears like a hollow ball of nearly a pin-head size, and represents the fruiting body or ascocarp (Fig. 15.4 H). Because of its closed nature it is of cleistothecium type. The mature cleistothecia (Fig.15.4 H, J) are often yellow and globose, and contain a wall of multicellular, closely-packed sterile hyphae. Many globose or pear-shaped asci are present inside. Each ascus contains eight ascospores (Fig. 15.4 J). Soon after the ascospore formation, the wall of the asci breaks down, and the ascospores are released into the cleistothecium. The cleistothecium decays and the ascospores are released. Each ascospore is pully-wheel shaped (Fig. 15.4 K), and attains a diameter of approximately 5 mm. The spore wall is differentiated into an outer thick and sculpturous epispore and inner thin endospore. Each ascospore germinates into a new mycelium (Fig. 15.4 A).

15.7 15.7.1

PENICILLIUM Systematic Position (According to Ainsworth, 1973) Division Subdivision Class Order Family Genus

15.7.2

– – – – – –

Eumycota Ascomycotina Plectomycetes Eurotiales Eurotiaceae Penicillium

Nomenclatural Problem

Kirk et al. (2001) mentioned that Penicillium is “anamorphic Eupenicillium – Talaromyces” represented by 223 widespread species. Eupenicillium is represented by 43 widespread species of Anamorph Penicillium belonging to family Trichocomaceae, while 24 species of Talaromyces also belong to Anamorph Penicillium of the same family. Like Aspergillus, Penicillium is also a form-genus based on conidial morphology. Because of the absence of perfect states in a majority of the species, Penicillium also shows the same taxonomic and nomenclatural problems as shown by Aspergillus. Its perfect state has been assigned many different generic names, such as Talaromyces and Eupenicillium.

15.7.3 Occurrence Commonly called ‘green moulds’ or ‘blue moulds’, Penicillium species occur frequently on Citrus and other fruits, cheese and many other similar foodstuffs. Like those of Aspergillus, its conidia are also present almost everywhere in the air, and therefore Penicillium species are also common biological contaminants of the laboratory cultures. Contamination of the bacterial culture of Staphylococcus by Penicillium notatum in the laboratory of Alexander Fleming in 1928 (see Raper, 1978) actually became the basis of the discovery of the antibiotic, penicillin, the first ‘Wonder-drug’ of the world.

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Fungi and Allied Microbes

Besides Citrus fruits, Penicillia occur commonly on apple, leather and other fabrics, and on all kinds of decaying materials and spoiling foods. Some species cause skin and other diseases of man and animals.

15.7.4 Economic Importance 1. Penicillium notatum (Fleming, 1944) and P. chrysogenum (Raper, 1952, 1978) are used for the production of penicillin throughout the world. 2. The antibiotic griseofulvin, used in skin and nail infection, is prepared from P. griseofulvum. 3. Cheese is fermented by P. camemberti and P. roqueforti. 4. Decaying and rotting of Citrus fruits is done by P. digitatum and P. italicum, whereas apples are decayed by P. expansum. 5. Some species destroy the leather and fabrics, whereas others are associated with some human and animal diseases.

15.7.5 Laboratory Culture If a moist Citrus fruit or cheese piece is covered with a hollow large cup and placed in damp warm place, bluish-green patches of Penicillium appear within a few days.

15.7.6 Somatic Structures

Cell wall

The mycelium is well-branched, and consists of many septate hyphae, which are slender, branched, tubular and generally hyaline (Fig. 15.5 A). The septa have central pore, which allows the cytoplasm and nuclei to flow from cell to cell. Some of the hyphae enter into the substratum to absorb the food material, whereas others remain on surface to produce conidiophores and conidia. Each cell is usually uninucleate, but may also be bi- or multinucleate. The cytoplasm encloses mitochondria, ribosomes, endoplasmic reticulum and other usual inclusions of a fungal cell. Reserve food is present in the form of oil globules.

Cytoplasm Oil globules Septum pore

Nucleus

A Conidia

Conidia

Phialide

Phialide

Metula Conidiophore

Ramus

15.7.7 Vegetative Reproduction B

It takes place by fragmentation. The vegetative mycelium breaks up into two or more fragments, each of which develops into a new mycelium.

Conidia Conidiophore

15.7.8 Asexual Reproduction Conidia developing on conidiophores are the means of asexual reproduction. Electron microscopic studies of Penicillium conidia have been made by Kolesnik (1981, Photoplate 3 A-J) and many others. A conidiophore is an erect, tubular hyphal outgrowth which may develop from any cell of the mycelium, but not from a specialized foot cell as in Aspergillus. The conidiophores of only some species are unbranched as in P. spinulosum (Fig. 15.5 B) and P.thomii. But in a majority of species the conidiophores are branched as in P. expansion (Fig. 15.5 C) and P. digi-

C

Conidia

Phialide Conidiophore E

Fig. 15.5

D G

F

H

Penicillium. A, A portion of hypha; B, Unbranched conidiophore of P. spinulosum; C, Branched conidiophore of P expansum; D-E, Conidiophore and conidia of P. digitatum; F-H, Conidia and their germination.

173

Plectomycetes

tatum. Successive whorls of conidiophore branches terminate into clusters of phialides or sterigmata. If the conidiophore is unbranched, the phialides are borne directly on its tip (Fig. 15.5 B). But in species with branched conidiophores, the ultimate branches, which contain the tufts of phialides, are called metulae (sing. metula). Usually the conidiophores show more branchings, and these lower branches that support the metulae are called rami (sing. ramus) as in P. expansun (Fig. l5.5 C) and P. digitatum (Fig. l5.5 D). A conidiophore with phialides and terminal chains of conidia appears like that of an artist’s brush, and hence is technically called penicillus(L. penicillum, small brush). Only because of its brush-like appearance the generic name Penicillium is given. From the tip of bottle-shaped phialides develop conidia. The conidia (Plate 3 A-J) are globose to ovoid in shape with smooth or rough wall. Usually, they are uninucleate (Fig. 15.5 F) but in some species they are multinucleate. They are green, blue, or yellow-coloured structures, and are produced in enormous quantities to provide the characteristic colour to the colony. Martinez et al. (1982) studied the morphology of the conidia of 211 species or Penicillium, and classified them into six major groups: smooth-walled (7% of the species), delicately roughened (13%), warty (28%), echinate (10%), striate with low irregular ridges (36%) and striate with scarce high ridges and bars (6%). According to Alexopoulos and Mims (1979) the conidia in Penicillium develop exactly in the same manner as in Aspergillus. At the time of the formation of conidia (Fig. 15.5 E), the nucleus of a phialide divides, and one daughter nucleus migrates into the narrow portion at the apex. The apex is then cut off by a cross-wall, and this separated uninucleate cell develops into a conidium. The conidium later on secretes its own spore wall. Electron microscopic studies of Fletcher (1971) in P. clavigerum (Fig. 15.6) suggest that at the time of the conidiumformalion an apical plug of material (Fig. 15.6 A) lines the neck of the phialide. This apical plug remains separate from the phialide wall. The primary wall of the conidium is formed by this apical plug. A centripetal ingrowth of the wall material (Fig. 15.6 B) develops into a septum. Thus septum separates the protoplast of the conidium from that of the phialide (Fig. 15.6 C). The septal pore is closed and the conidial protoplast is separated completely. Inside the primary wall of the conidium develops a new wall layer (Fig. 15.6 D). The wall that separates two conidia functions as a connective (Fig. 15.6 D). The primary wall of each conidium becomes folded, and because of this the conidium appears rough.

Apical plug Connective Conidium

Phialide wall

Surface layer Septal pore Septum

Nucleus A

Fig. 15.6

B

Septum C

New wall

D

A–D, Penicillium clavigerum, showing development of conidia as viewed under electron microscope (after Fletcher, 1971)

The conidia are dispersed by wind. On getting the suitable conditions of moisture and temperature, each conidium swells, and germinates by producing a germ tube. The latter becomes septate and develops into the mycelium.

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Fungi and Allied Microbes

Penicillium

A different terminology has been used to describe the different conidiogenous structures in Penicillium by Samson and Pitt (1985) and Kirk et al. (2001), as is clear from Fig. 15.7.

15.7.9

Conidiogenous cell Cell supporting conidiogenous cell

Sexual Reproduction

Branch cell About sexual reproduction in most of the species of PenicilConidiophore Branch lium, nothing is known. Only a few species are known to reproduce sexually. The sexual or perfect state of Penicillium has been assigned to different genera, such as Talaromyces, Eupenicillium and Carpenteles (Webster, 1980). Alexopoulus and Mims (1979) mentioned that Carpenteles and Eupenicillium are the two names of the same genus, and now the valid name is Eupenicillium. Fig. 15.7 Diagram to describe the different conidiogA few species that show sexual reproduction are Penicilenous structures in Penicillium (after Samson lium vermiculatum (= Talaromyces vermiculatus) and P. stip& Pitt, 1985). itatum (= T. stipitatus). Cleistothecial type of fruiting bodies are formed. According to Miller (1984) the details of formation of cleistothecia, ascus formation and release of ascospores are very similar to that of Aspergillus. Dangeard (1907) studied the sexual reproduction in Penicillium vermiculatum (= Talaromyces vermiculatus), and noted the following details: The mycelium contains uninucleate cells (Fig.15.8 A), from which develops a uninucleate ascogonium. The ascogonium elongates, and its nucleus divides several times to produce as many as 64 nuclei (Fig. 15.8 B). Simultaneously, a uninucleate antheridium also develops and coils spirally around the multinucleate ascogonium (Fig. 15.8 B). The antheridial

Mycelium Ascogonium

G er

Antheridium

io at

in

m A

n

Ascospores

F Sterile hyphae

Peridium

B

Ascospores

Ascogoniu with binucleate cells E

Ka Me ry ios og is am ? y ?

Ascus

C D

Fig. 15.8

A–F, Sexual reproduction in Penicillium vermiculatum (= Talaromyces vermiculatus) (after Dangeard, 1907).

175

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tip touches the ascogonium at one point, and the walls of the two, at the point of contact, dissolve. Side by side the ascogonium gets segmented in the form of many binucleate cells (Fig. 15.8 C). According to Dangeard the antheridial nucleus does not migrate into the ascogonium. It remains very much there in the antheridium, even after the development of ascogenous hyphae. This indicates that antheridia are formed but they remain non-functional. From the binucleate cells of the septate ascogonium develop the ascogenous hyphae. The nuclei in the ascogenous hyphae, therefore, are derived from the original nucleus of the ascogonium. Many sterile hyphae develop and enclose the young asci from all sides. The fruiting body, therefore, is of cleistothecium type. Many asci, each with eight ascospores, are seen in the cleistothecium (Fig. 15.8 E). Dangeard (1907) did not observe the actual processes of karyogamy and meiosis. But two nuclei of each ascogenous hypha (Fig. 15.8 D) might have fused to form a diploid nucleus in the young ascus mother cell, and this zygotic nucleus might have passed through meiosis to form eight ascospores in each ascus (Fig. 15.8 E). The asci are globose or pear-shaped bodies. Their walls soon dissolve, and the ascospores are released in the cleistothecium. As in Aspergillus, the ascospores are pully-wheel shaped and uninucleate bodies. Each ascospore germinates into a fresh mycelium.

15.8

DIFFERENCE BETWEEN TALAROMYCES AND EUPENICILLIUM

Table 15.2 No. 1. 2. 3.

15.9

Talaromyces

Eupenicillium

Fruiting body grows indefinitely and its size keeps on increasing even after the maturation of ascospores. Cleistothecial wall is made up of loosely arranged interwoven hyphae. Asci are produced from croziers in most of the species.

Fruiting body atttains a definite size. Cleistothecial wall is hard and made up of thick-walled pseudoparenchyma. The asci generally develop on short branches of the ascogenous hyphae.

DIFFERENCE BETWEEN ASPERGILLUS AND PENICILLIUM

Table 15.3 No.

Difference between Talaromyces and Eupenicillium

Difference between Aspergillus and Penicillium.

Aspergillus

Penicillium

3.

The conidiophores are unseptate and unbranched. A conidiophore develops from a specialized,thick-walled cell called foot cell. Each conidiophore enlarges into a swollen vesicle at its tip.

4. 5. 6.

Metulae are not present below the phialides. Mature conidia are yellow, green, brown or black in colour. Wall of the cleistothecium is loose.

The conidiophores are septate and branched. Conidiophore develops from any vegetative cell of the mycelium; foot cells are absent. Vesicle is not formed at the tip of conidiophore, and the entire structure appears like that of an artist’s brush. Metulae are present. Usually the mature conidia are green in colour. Wall of the cleistothecium is comparatively thick.

1. 2.

15.10

ROLE OF ASPERGILLUS AND PENICILLIUM IN BIOTECHNOLOGY

After Saccharomyces cerevisiae, two most important organisms used in biotechnology are Aspergillus and Penicillium. Brief consideration of some of the examples the role of these two organisms in biotechnology is undermentioned.

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1. Aspergillus oryzae is used in the degradation of rice starch in the sake production, which “technically a beer rather than a wine” (Webster and Weber, 2007). 2. A. oryzae and A. sojae are also used in production of soya – sauces, in which raw materials used are soyabeans and wheat. 3. Specialized types of cheeses are prepared using Penicillium roqueforti and P.camemberti. Large quantities of extracellular enzymes are used with the help of several species of Penicillium and Aspergillus. Proteases, amylases, lipases and pectinases are used in many industrial processes, including the manufacture of several dairy, bakery, distillery and brewery products. Initially extracted from citrus fruits, citric acid has been produced commercially using Aspergillus niger since 1923, and this fungus still remains world’s most important producer, with a total current annual world production of citric acid about 9 x 106 tons (Webster and Weber, 2007). Besides production of several antibiotics from various species of Aspergillus and Penicillium, some of the widely known ones used throughout the world are (1) penicillin from Penicillium notatum, (2) cephalosporins from Cephalosporium chrysogenum, (3) semisynthetic penicillin G from Penicillium chrysogenum, and (4) griseofulvin (used as a systemic antifungal drug) from P.griseofulvum.

15.11

MYCOTOXINS FROM ASPERGILLUS AND PENICILLIUM

Aspergillus and Penicillium are these days widely used in the production of secondary metabolites, which are highly toxic against many different organisms. These are called mycotoxins. Aflatoxins, ochratoxin-A and patulin are some of the

Cleavage point of penicillin acylases

A O

O H

S O N

O

C

O

O

Griseofulvin

Cl

COOH O

O

OO

H

N H

Penicillin G

Methyl groups

B

COOH O

D

OH

O

O N H

O

Aflatoxin B1

O

O

E

O

Cl Ochratoxin A

O O

O OH Patulin

Fig. 15.9

A–E, Some major secondary metabolites. A, Penicillin G; B, Griseofulvin; C, Aflatoxin B1; D, Ochratoxin A; E, Patulin.

Plectomycetes

177

mycotoxins found in these fungi which are often found as food contaminants and are thus proving major health hazards. Some important metabolites produced by members of Trichocomaceae are depicted in Fig. 15.9 A – E.

TEST YOUR UNDERSTANDING 1. 2. 3. 4. 5.

6. 7. 8. 9. 10. 11. 12. 13.

Write one single character on the basis of which Ainsworth (1973) recognized Plectomycetes. Whether Kirk et al. (2001) recognized the class Plectomycetes? The fruiting body in majority of Plectomycetes is typically a _______ . Write any five general characteristics of Plectomycetes. Asci in the fruiting body of Plectomycetes are: (a) irregularly arranged, (b) evanescent, (c) scattered, (d) all of these. Write a brief note on the economic importance of Aspergillus. How can you culture Aspergillus in the laboratory? Describe in detail the asexual reproduction in Aspergillus. Write a brief nomenclatural note with particular reference to the anamorphic nature of Aspergillus and Penicillium. With the help of a diagram, differentiate between conidiophore, ramus, metula and phialide mentioning at least one sentence about each of them. Write at least three differences between Penicillium and Aspergillus. Describe in brief the role of Aspergillus and Penicillium in biotechnology. What are mycotoxins? Name any two mycotoxins found in Aspergillus and Penicillium.

16

C H A P

LABOULBENIOMYCETES

T E R

16.1

WHAT ARE LABOULBENIOMYCETES?

The fungi that occur parasitically on arthropods, and contain highly reduced thallus, perithecium type of fruiting bodies and inoperculate asci, have been placed under class Laboulbeniomycetes of subdivision Ascomycotina by Ainsworth (1973). Hawker (1966) mentioned that this group of fungi is still of doubtful affinity. Many workers still include them in Pyrenomycetes. Regarding Laboulbeniomycetes, Kirk et al. (2001) mentioned that this belongs to phylum Ascomycota and contains 2 orders (Laboulbeniales and Pyxidiophorales), 4 families, 141 genera and 1869 species. In Laboulbeniales stomata usually present, composed of a basal black haustorium and a dark cellular thallus. Ascomata perithecial, often surrounded by complex appendages, translucent, ovoid and thin–walled. Interascal tissue absent. Asci few, clavate, thin-walled, usually 4-spored. Ascospores hyaline, elongate, 1-septate. These are ectoparasites of Insecta (Kirk et al., 2001). Pyxidiophorales are mainly coprophilous. Stroma (thallus) absent. Ascospores 1–to 3-septate. This order contains 1 family, 2 genera and 16 species (Kirk et al., 2001).

16.2

A BRIEF BACKGROUND

Peyritsch (1873) gave the first authentic record of these fungi. He recognized 5 genera and 12 species and included them under order Laboulbeniales. Roland Thaxter (1896,1908, 1924, 1926, 1931) should rightly be called the “father of Laboulbeniomycetes” because of his thorough study of these fungi from 1890 till his death in 1932. He described 103 genera and 1260 species. Benjamin’s (1971, 1973) work should also be consulted for getting the latest position of these fungi. So far more than 1500 species of these fungi have been described. Benjamin (1973) included all Laboulbeniomycetes under one order, Laboulbeniales. But Kohlmeyer (1973) described 5 species of Spathulospora, all parasitic on marine red algae, and placed them in a second order Spathulosporales. Sugiyama and Tomasz (1985) described 16 species of Laboulbeniomycetes from Malayasia, of which Rickia sakke on Lordithon is new to science.

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Laboulbeniomycetes

16.3

OCCURRENCE

Laboulbeniales are obligate parasites of arthropods. A majority of them occur on true insects, whereas some occur on mites and millipeds. They occur on insects occurring in a variety of habitats, including water, soil, decomposing plants and animal remains and even on the bodies of many living animals. Members of Spathulosporales occur parasitically on red algae. Laboulbeniales are host specific, some attacking individuals of only one sex of an insect species.

16.4

THALLUS

A true mycelium is absent. The ascospores are always bicelled in Laboulbeniales. The lower basal cell gets enlarged and develops into a foot cell. The foot is a sucker-like organ. From the lower surface of the foot cell arises a small and simple haustorium. This haustorium penetrates the integument of the host and reaches up to the epidermis. However, a simple and branched rhizomycelium-type of the structure develops in some genera, such as Microsomyces and Trenomyces. The basal cell divides many times to form a thallus or receptacle. From the thallus develop some appendages.

16.5

SEX ORGANS

A majority of the species are monoecious. However, some species of Laboulbenia are dioecious. Sex organs develop on the appendages present on the thallus. Simple, rod-like branchlets, called spermatia, develop directly on the appendages in species of Zodiomyces and Drepanomyces. But flask-shaped antheridia containing endogenous spermatia are seen in Laboulbenia flagellata (Fig. 16.1 A). In some other genera, such as Haplomyces (Fig.16.1 B), Dichomyces and Monoicomyces (Fig. 16.1C) compound antheridia develop. In such cases many simple antheridia unite to form a compound antheridium. The female sex organ or ascogonium is generally a 3-celled structure, of which the terminal cell elongates to form trichogyne. Its middle cell is called trichophoric cell and the lowermost carpogenic cell (Benjamin, 1973). The trichogyne and the middle trichophoric cell ultimately disappear. From the lowermost carpogenic cell develop 1-8 ascogenous cells, each of which develops into an ascus. Benjamin (1971) observed the conditions in which the spermatia were seen attached with the trichogyne, but details of the plasmogamy, karyogamy and meiosis, etc. are not known. Hill (1977) observed the presence of ‘Woronin bodies’ in the ascogenous cells of Herpomyces.

16.6

A

B

Fig. 16.1

C

A-C. Some Laboulbeniomycetes. A, Laboulbenia flagellata ; B, Haplomyces texanus; C, Monoicomyces.

PERITHECIA, ASCI AND ASCOSPORES

The perithecia are either sessile or shortly-stalked, flask-shaped bodies containing only a small number of asci. Benjamin (1973) described three types of development of perithecia in Laboulbeniomycetes. In general, the perithecium consists of a pair of stalk cells and three basal cells. The perithecia remain united with the receptacle of the thallus.

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Fungi and Allied Microbes

The asci are usually elongate-clavate bodies. Each ascus usually contains four ascospores. The ascospores are hyaline, elongate or spindle-shaped and two-celled in Laboulbeniales. However, in Spathulosporales the ascospores are one-celled (Kohlmeyer, 1973). Both the cells of the ascospores are generally unequal in length. When the spores are discharged from the perithecium the longer basal cell comes out first. Because of the presence of a very small upper cell in Amorphomyces, its spores were first thought to be unicellular.

TEST YOUR UNDERSTANDING 1. What is the position of Laboulbeniomycetes in Ainsworth’s (1973) system on classification? 2. Majority of Laboulbeniomycetes occur parasitically on _______ . 3. Discribe briefly the sex organs, fruiting bodies, asci and ascospores of Laboulbeniomycetes.

17

C H A P

PYRENOMYCETES

T E R

17.1

WHAT ARE PYRENOMYCETES?

Pyrenomycetes is a class of subdivision Ascomycotina (Ainsworth, 1973), and includes the fungi in which ascocarp is usually of perithecium type and the asci are inoperculate with an apical pore or slit. Formerly, only the ascomycetes with perithecium-type of fruiting bodies were included under Pyrenomycetes. But now these fungi include all the ascomycetes where the fruiting bodies are ‘entirely surrounded by a peridial wall and contain unitunicate asci’ arranged primarily in a hymenial layer (Muller and von Arx, 1973). Some Pyrenomycetes also contain cleistothecial-type of fruiting bodies. Kirk et al. (2001) mentioned Pyrenomycetes as a “class of Ascomycota”, which according to them is now “not generally accepted but was reintroduced by Berbee and Taylor (1992) in a restricted sense”. According to them this word (Pyrenomycetes) is used “mostly for fungi with perithecioid ascomata which are ascohymenial in ontogeny and have unitunicate asci often with an apical annulus” (Kirk et al., 2001).They further mentioned that the term “pyrenomycetes” still “has value as colloquial term for all ascomycetes with flask–shaped ascomata”.

17.2

DISTINGUISHING FEATURES

1. The Pyrenomycetes occur on wide variety of habitats such as dung, decaying stems and leaves and soil. Some occur as common plant pathogens, e.g. Claviceps and Nectria. Some (Verrucaria) remain as fungal partner in lichens. 2. The asci are unitunicate, and club-shaped or cylindrical. 3. The asci remain arranged in a hymenial layer. 4. The hymenial layer of asci usually lines the base and the sides of the inner wall of the fruiting body. 5. Usually the asci are persistent, but they may also be evanescent. 6. In a majority of the members the ascocarp is a perithecium. However, some members contain cleistothecial type of fruiting body. 7. The ascocarp remains surrounded by a definite peripheral wall called peridium. 8. Perithecium usually contains an apical opening called ostiole. Rarely, the ostiole is lateral in position. 9. The ostiole remains covered inside by many hypha-like periphyses. 10. The perithecia either occur singly or remain clustered together, on or within a stroma.

182

17.3

Fungi and Allied Microbes

CLASSIFICATION

The Pyrenomycetes have been classified differently by various workers. Formerly,the type of the ascocarp (whether apothecium, perithecium or cleistothecium) was the main criterion of classifying these fungi. But now the points on which main emphasis is being given for classifying Pyrenomycetes, are (i) structure and chemistry of the ascal apex, and (ii) the type of ascocarp centrum. Luttrell (1951), followed by Alexopoulos and Mims (1979), emphasized the importance of ascocarp centrum and recognized four main types among Pyrenomycetes. These are (i) Phyllactinia-type centrum, (ii) Xylaria-type centrum, (iii) Diaporthe-type centrum, and (iv) Nectria-type centrum. Miller and von Arx (1973) discussed the taxonomy of Pyrenomycetes in detail. Their classification is widely accepted these days, and is followed also in this book. An outline suggested by them is mentioned below: Class PYRENOMYCETES Order 1. Erysiphales Families: Erysiphaceae, Perisporiaceae Order 2. Meliolales Family: Meliolaceae Order 3. Coronophorales Family: Coronophoraceae Order 4. Sphaeriales Families: Ophiostomataceae, Melanosporaceae, Sphaeriaceae, Hypocreaceae, Polystigmataceae, Coryneliaceae, Sordariaceae,Diaporthaceae, Halosphaeriaceae, Diatrypaceae, Amphisphaeriaceae, Xylariaceae, Verrucariaceae, Clavicipitaceae and Hypomycetaceae. Only Erysiphales and Sphaeriales are discussed further.

17.4

ERYSIPHALES

17.4.1 Why should Erysiphales be discussed under Pyrenomycetes? Webster (1980) discussed Erysiphales under Plectomycetes. But the presence of scattered asci in the ascocarp has been considered the main character of Plectomycetes, which however is not seen in this order. Asci in Erysiphales form a basal hymenium layer, which is the chief character of Pyrenomycetes. Because of this, Erysiphales have been discussed under Pyrenomycetes by a majority of the workers including Luttrell (1951), Muller and von Arx (1973), Yarwood (1973) and Alexopoulos and Mims (1979). In this book, author also follows the classification of Yarwood (1973). Kirk et al. (2001), however, discussed Erysiphales under subclass Erysiphomycetidae of class Ascomycetes of phylum Ascomycota. Under Erysiphales, they recognized only one family (Erysiphaceae), 13 genera and 494 species.

17.4.2 Classification Yarwood (1973) recognized two families (Erysiphaceae and Perisporiaceae) within Erysiphales. However, Alexopoulos and Mims (1979) placed all Erysiphales in one family, i.e. Erysiphaceae.

Pyrenomycetes

183

17.4.3 General Characteristics (Erysiphales and Erysiphaceae) 1. Members are mainly obligate parasites, occurring on the leaves and stems of a large number of angiosperms. Kirk et al. (2001) described 13 genera and 494 species under Erysiphaceae. 2. They produce enormous number of conidia, which appear as white powdery coating on the surface of the host. Only because of this, the group of plant diseases caused by Erysiphales is commonly called powdery mildews. 3. The mycelium is superficial, well-developed, branched, septate, and contains short, uninucleate cells. Powdery mildews grow luxuriantly in rain-free seasons. 4. From the mycelium develop some one-celled haustorial branches, which are generally globular in shape. 5. During asexual reproduction many unicellular conidiophores develop from young mycelial hyphae. The conidiophores give rise to many conidia or conidiospores in succession. 6. The conidiophores develop singly and at right angle to the host surface. 7. The conidia are uninucleate, oval, thin-walled, and contain numerous vacuoles of water. They are produced in very large number. 8. Sexual reproduction takes place by well-defined antheridia and ascogonia, or by vegetative cells, which perform the same function. 9. Dikaryotization of the ascogonium ultimately results in the formation of a dark-coloured, globose, non-ostiolate fruiting body, called cleistothecium. Yarwood (1973) called them ‘non-ostiolate perithecia’. 10. The cleistothecia remain enveloped by pseudoparenchymatous peridium, made up of 6-10 layers. It helps the fruiting bodies to survive during unfavourable conditions. 11. The asci form a basal hymenium layer inside the fruiting body. 12. The mature cleistothecia bear some characteristic appendages, basically of four different types: (i) mycelioid, resembling the somatic hyphae, e.g. Erysiphe; (ii) Circinoid or hooked with curled tips, e.g. Uncinula; (iii) dichotomously branched tips, e.g. Microsphaera, and (iv) with bulbous base, e.g. Phyllactinia. 13. The cleistothecia split, or explode to release the ascospores. As soon as the ascospores land on the suitable host, they start germinating by producing a short germ tube. Blumer (1967) and Yarwood (1973) recognized only eight genera in Erysiphaceae, viz. Sphaerotheca, Podosphaera, Erysiphe, Microsphaera, Uncinula, Leveillula, Phyllactinia and Acrosporium. Kirk et al. (2001) mentioned that Erysiphaceae comprises 13 genera and 494 species. Only Erysiphe, Phyllactinia and Sphaerotheca are discussed here.

17.4.4

Control of Powdery Mildews

Because of the presence of superficial mycelium the powdery mildews are controlled by sprinkling water on the host. In severe infections sulfur- based fungicides are used. Use of fungicides such as dinocap (dinitrocaprylphenyl crotonate), ethirimol and benomyl has also proved useful in controlling the mildews (Yarwood 1957; Johnston, 1970; Bent, 1978). Use of mildew-resistant varieties has also been advocated (McIntosh, 1978).

17.5 17.5.1

ERYSIPHE Systematic Position (According to Ainsworth, 1973) Subdivision Class Order Family Genus

– – – – –

Ascomycotina Pyrenomycetes Erysiphales Erysiphaceae Erysiphe

184 17.5.2

Fungi and Allied Microbes

Occurrence

It is an obligate, ectoparasitic fungus, which occurs commonly on aerial parts of many angiosperms. Its wide distribution is exemplified by Erysiphe polygoni, which has been reported on 352 different host species (Salmon, 1900). Over 10 species of this genus are known, and a majority of them are cosmopolitan in distribution. Some of the common mildews are grass mildew (E. graminis), mildew of pea (E. polygoni) and mildew of cucurbits (E. cichoracearum). E. aggregata and E. trina are two other common species (Yarwood, 1973). Kirk et al. (2001) treated Erysiphe as an “Anamorph Oidium subgenus Pseudoidium”.

17.5.3

Somatic Parts

The mycelium grows superficially on the host epidermal cells. It is composed of short hyphae, made up of uninucleate cells. From the mycelium develop some unicellular haustorial branches, which penetrate the wall of the host epidermal cells and absorb the food. The haustoria are globular in E. polygoni, E. cichoracearum (Fig. 17.1 A) and a majority of the other species, but they are digitate and develop several tubular processes in E.graminis (Fig.17.1 B). Mckeen et al. (1966) studied the haustorium of E. cichoracearum, whereas Bracker (1968) of E. graminis under electron microscope. According to these workers haustorium contains a tubular neck and a swollen uninucleate body (Fig. 17.1 C). The haustorial body or its lobes remain enclosed in a sheath. In between the sheath and the body of the haustorium is present the extensive matrix. The sheath remains in contact with the tonoplast of the host. The sheath membrane contains many invaginations. In the tubular lobes are present many mitochondria and endoplasmic reticulum. The neck of the haustorium remains surrounded by a thick collar deposited on the host cell wall. Bracker (1968) believed that the sheath surrounding the haustorium is perhaps the invaginated plasmalemma of the host. Only because of this the haustorium does not lie freely in the Mycelium

Haustorium Haustorium Host epidermis B A Hypha

Collar

Channel Host cell wall

Neck Cross wall Body of haustorium Invagination Matrix

Host cytoplasm Host tonoplast

Sheath membrane Nucleus

Fig. 17.1

C

Erysiphe. A, A part of vegetative hypha of E. cichoracearum with haustoria in the host epidermal cells; B, Haustorium of E. graminis; C, Diagrammatic representation of the electron micrograph of haustorium of E. graminis (C, after Bracker).

185

Pyrenomycetes

host cytoplasm. The interchange of the food material between the host and the fungus takes place perhaps through the tubular invaginations of the sheath membrane.

17.5.4

Asexual Reproduction

From the fully established mycelium over the host epidermis develop many short, erect unicellular conidiophores within 2-3 days (Fig. 17.2 A). Usually, each unicellular conidiophore divides into a long stalk cell and a short terminal cell (Fig. 17.2 B, C). The terminal cell cuts off conidia in succession (17.2 D-F). The conidia are dispersed readily by wind. The conidia are hyaline and uninucleate. They are oval or barrel-shaped, and have a water content as high as 70%. Lipid granules and fibrosin bodies are also present. As soon as they land over a suitable host, the conidia start to germinate even at very low relative humidity. The germination starts by putting out one or more germ tubes. In contact with the cuticle of the host the germ tube forms an appresorium. From the appresorium develops a small peg-like hyphal outgrowth, which penetrates the cuticle and epidermal cell of the host, to form a haustorium. The new mycelium so formed becomes capable of producing conidiophores and conidia within a few days.

17.5.5

Sexual Reproduction

Terminal cell Conidiophore Stalk cell A

B

C

Conidia

D

E

F

When the process of conidia formation slows down the sexual reproduction starts, specially in the late summer. Most of the species are homothallic, and only a few are heterothallic (Yarwood, Fig. 17.2 A–F, Erysiphe cichoracearum, showing 1935). E. cichoracearum (Yarwood, 1957) and E. graminis (Hidevelopment of conidia. ura, 1962; Yarwood, 1978) are heterothallic species. The sexual branches develop from the somatic mycelium. At the ends of these branches develop sex organs, which arise near each other in pairs. The sex organs (antheridia and ascogonia) lie either parallel, or more or less twisted around each other. Of the two twisted hyphae (Fig. 17.3 A), the terminal cell of one behaves as an antheridium and that of the another as ascogonium. The walls, at the point of contact of ascogonium and antheridium, dissolve and a pore is formed. Through this pore the antheridial nucleus and some of its cytoplasmic content move towards the ascogonium, and plasmogamy takes place. But the nuclei fuse only at the time of formation of ascus mother cells. In E. aggregata the number of nuclei increases in the ascogonium (Fig. 17.3 B). Septa are formed in the ascogonium (Fig. 17.3 C). In this so formed multicellular ascogonium the penultimate cell contains two or more nuclei. From this penultimate cell develop many ascogenous hyphae (Fig. 17.3 D). The actual process of ascosporogenesis is still not clearly known in most of the species. Some cells of the ascogenous hyphae are binucleate and change into ascus mother cells. Each ascus mother cell increases in size, and its both the nuclei fuse (Fig. 17.3 E) to form a synkaryon. The synkaryon-containing cell is called the ascus. Its diploid nucleus divides meiotically and then mitotically, resulting into eight haploid nuclei, which develop into eight ascospores. The number of ascospores differs from 2 to 8 in different species. The species with less than eight ascospores in an ascus show degeneration of haploid nuclei. Simultaneously, from the stalk of the young developing ascogonium develop many sterile hyphae, which envelope the sexual apparatus from all sides and form a thick sheath or peridium (Fig. 17.3 C-F). The fruiting body is, therefore, cleistoth-

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Fungi and Allied Microbes

Ascogenous hyphae

Ascogonium

Ascogonium

Sterile hyphae A

B C

Peridium

D

Ascospores

Peridium

Ascus

Asci

E

F

Appendages

Cleistothecium

G

Fig. 17.3

A–F, Stages of sexual reproduction in Erysiphe aggregata; G, A cleistothecium of E. polygoni.

ecium type. In the beginning the peridium is only 1-2 cells thick (Fig. 17.3 B) but in mature cleistothecia it becomes 6-10 cells thick (Fig. 17.3 F). In the mature cleistothecia the cells of the outer few layers are thick-walled (Fig. 17.3 F). Some superficial cells of the peridium develop into long, unbranched, hypha-like appendages (Fig. 17.3 G). Mature cleistothecia are dark-brown and globose in E. graminis. For quite some time the mature cleistothecia may remain attached with the host surface. They are detached and dispersed either accidentally or by wind. Throughout the winter season the asci remain enveloped by the thick peridium. Irregular cracking of the upper part of the peridium results in the splitting of cleistothecia. This exposes the asci. The ascospores are forcibly ejected from asci in E. graminis.

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Pyrenomycetes

Falling over a suitable host, each ascospore starts to germinate immediately by producing a germ tube, which soon develops into a young somatic mycelium. However, ascospores of E. graminis may survive even up to 13 years (Moseman and Powers, 1957).

17.6

PHYLLACTINIA

17.6.1 Systematic Position (According to Ainsworth, 1973) Subdivision Class Order Family Genus

– – – – –

Ascomycotina Pyrenomycetes Erysiphales Erysiphaceae Phyllactinia

Cleistothecia

17.6.2 Occurrence Phyllactinia is an obligate hemiendoparasitic fungus, causing powdery mildews of different hosts including Dalbergia sissoo, Cassia fistula, Morus alba and Corylus avellana. In India the most commonly occurring species is Phyllactinia dalbergiae on Dalbergia sissoo. P. guttata occurs on Corylus avellana. On Dalbergia sissoo white powdery mass is present on the lower surface of the leaf (Fig. 17.4). Young cleistothecia are orange to yellow-coloured, round bodies, which become black on maturity. The leaves first become dry, and ultimately they fall on the ground. Kirk et al. (2001) mentioned Phyllactinia as anamorphic Ovulariopsis.

17.6.3

Leaf

Fig. 17.4

Phyllactinia dalbergiae, causing powdery mildew of Dalbergia sissoo.

Somatic Parts

The mycelium is septate, well-branched, superficial and spreads over the host surface (Fig. 17.5 A). It is hemiendophytic in nature. The hypha consists of uninucleate cells. Some of the hyphae may enter through the stomata and penetrate the mesophyll tissue. The haustoria enter inside the mesophyll cells, but they never enter the epidermal cells.

17.6.4 Asexual Reproduction It takes place by conidia developing on multicellular and unbranched conidiophores. The conidia are formed singly and never in chain. The conidiophore (Fig. 17.5 B-D) may be thin-walled (P.suffulta and P.dalbergiae), thick-walled (P. rigida) or even somewhat twisted at the place of its origin (P. subspiralis). The conidia are uninucleate, thin-walled and typically slippershaped or club-shaped. They are disseminated easily by wind, and germinate by producing new hyphal branches.

17.6.5 Sexual Reproduction The sexual reproduction takes places usually in the late summer. The sexual branches develop on different hyphae. Both the branches are uninucleate and more or less similar in the beginning. But soon the female branch becomes curved and stouter (Fig. 17.6 A). The division of the nucleus followed by a septum formation in the male branch result in the formation of a small terminal antheridium and a stalk cell (Fig.17.6 B). Simultaneously, the division of the nucleus and the septum formation in the female branch result in the formation of a large terminal ascogonium and a stalk cell. From the stalk cell of the female branch

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Conidia

Host cell Conidiophores

Hypha

A

Fig. 17.5

B

C

D

A, Phyllactinia corylea, showing mycelium and hypha; B, Conidiophore of P.suffulta; C, Conidiophore of P. rigida; D, Conidiophore of P. subspiralis (all after Smith, 1900). Antheridium Female branch

Male branch Stalk cells

Degenerating nucleus Ascogonium

Sterile hyphae

D

Ascogonium C

A

Sterile hyphae

B

F E

Fig. 17.6

Multinucleate ascogonium

Septate ascogonium G

A–G, Stages of sexual reproduction in Phyllactinia corylea.

start to develop some sterile hyphae at this stage. Sometimes the sterile hyphae also grow from the vegetative hyphae. The nucleus of the antheridial cell degenerates at this stage. No fertilization takes place. The ascogonium becomes binucleate (Fig. 17.6 C), perhaps because of the division of its single nucleus. Both the nuclei of the ascogonium divide (Fig. 17.6 D). Septum formation takes place in such a manner that a short filament of three cells is formed. The middle cell of this filament is binucleate and the other two end-cells are uninucleate (Fig. 17.6 E, F). Two nuclei of the middle cell belong to the different nuclei of the original pair of the ascogonium. The terminal tip cell takes no further part in the ascocarp development. From the basal uninucleate cell develop some hyphae, which form a layer lining the young peridium. The middle binucleate cell elongates and its nuclei divide. At this stage many short ascogenous hyphae develop from the middle cell.

189

Pyrenomycetes

Each ascogenous hypha elongates into a branch. Its tip cell is uninucleate and the penultimate cell is binucleate. From this binucleate cell develops an ascus. Its nucleus fuses to form a synkaryon. The diploid nucleus undergoes meiosis and ordinary mitotic divisions, resulting in the formation of eight haploid nuclei. Of these eight nuclei, six degenerate, and the remaining two develop into ascospores. Simultaneously the vegetative hyphae surround the developing asci all over, and the ascocarp, so formed, is of cleistothecial-type (Fig. 17.6 G). The cleistothecia are globular and orange to yellowcoloured when young, but at maturity they are black. The cleistothecia (Fig. 17.7 A) bear two types of appendages (i) long, unbranched, radiating appendages, each with a bulbous base (Fig. 17.7 B), and (ii) a crown of repeatedly branched appendages, which secrete mucilage (Fig. 17.7 C). The appendages with a bulbous base function as ‘flights’ or vanes (Webster, 1980), and the secretory appendages help in sticking the cleistothecia on the substratum with the mucilage. With the help of bulbous-base appendages the cleistothecia move like a shuttle-cock (Webster, 1979). Each ascus contains two ascospores (Fig. 17.7 D, E). The ascospores are discharged towards the apical side. On being liberated, each ascospore germinates to give rise to a fresh mycelium.

17.7

SPHAEROTHECA

Bulbous appendages

Secretory appendage Fruiting body A

Mucilage

Ascus Ascospores

Secretory appendage B E C

Ascus Ascospores

Mucilage

Fruiting body D

Sphaerotheca is also a member of Erysiphaceae, and causes the mildews of many Asteraceae (S. fuliginea), Fig. 17.7 Phyllactinia corylea. A, A fruiting body with two strawberries and gooseberries (S. mors-uvae), hops (S. types of appendages; B, A bulbous-base aphumuli) and roses (S. pannosa). About 8 species of pendage; C, A branched secretary appendage; Sphaerotheca have been reported from India. D, An opened fruiting body, showing asci; E, An The mycelium consists of white, branched, septate ascus with two ascospores. hyphae with uninucleate cells (Fig. 17.8 A). The asexual reproduction takes place by conidia, which are abstricted in chain at the tip of short unbranched conidiophores (Fig. 17.8 B). The conidia are small, oval uninucleate structures (Fig. 17.8 C) disseminated by the wind. On falling over a suitable host, each conidium may germinate by producing germ tubes (Fig. 17.8 D). Sexual reproduction takes place by uninucleate antheridia and ascogonia, which develop on closely situated hyphae (Fig. 17.8 E). The two sex organs come in contact with each other. The wall at the point of contact dissolves, and the male nucleus enters in the ascogonium (Fig. 17.8 F). The ascogonium soon undergoes transverse division to from a row of 3-5 cells. Only the penultimate cell contains two nuclei, whereas all remaining cells are uninucleate (Fig. 17.8 G). The binucleate penultimate cell changes into an ascus (Fig. 17.8 H). Further details are not clearly known. Sphaerotheca is peculiar in containing only one ascus in the fruiting body. Many sterile hyphae grow around the sex organs and produce the pseudoparenchymatous tissue, which forms the wall of the cleistothecium type of fruiting body (Fig. 17.8 I). The fruiting body contains simple mycelloid appendages, which resemble somatic hyphae (Fig.17.8 I). Electron microscopic studies show that the outer cells of the cleistothecium wall are uninucleate, whereas the cells of the inner layers

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Conidia

C

D Conidiophore

B A

Somatic hypha

Germ tube Antheridium Ascogonium

K Ascospores

E

Ascus Cleistothecium

se

a loph

Hap se pha Diplo Young ascocarp

J

Mei

ase

h ryop Dika

osis

Plasmogamy F

Appendage Zygote

Ascus mother cell

I G Karyogamy

Fig. 17.8

H

A–K, Life cycle of Sphaerotheca castagnei.

are binucleate. The cleistothecium contains only a single ascus with eight ascospores (Fig. 17.8 I). Fibrosin bodies are present in the ascospores as also in conidia. The ascospore germinaes (Fig. 17.8 K) by producing a germ tube and infects the host.

Pyrenomycetes

17.8 17.8.1

191

SPHAERIALES Delimitations and General Characteristics

Muller and von Arx (1973) included the majority of the Pyrenomycetes under Sphaeriales. They have treated Sphaeriales in such a ‘broad sense’ that the majority of the members included by different workers under other orders (Diaporthales, Xylariales,Clavicipitales, Hypocreales and Sphaeriales) of Pyrenomycetes have been included by them under only one order, i.e. Sphaeriales. According to them, the ascomata or fruiting bodies in Sphaeriales are mostly ostiolate and only rarely astomatous, but without an apical layer of gelatinous cells. Some of their general characteristics are mentioned below: 1. Members are mostly saprophytic, but many are also parasitic on higher plants. 2. The mycelium is well-branched, and contains elongated, uninucleate or multinucleate cells. 3. Asexual reproduction takes place by conidia developing on conidiophores. The conidiophores are either enclosed in a fruiting structure, or organized into groups, or show no definite organization at all. 4. Some species produce spermatia or microconidia. These are small, uninucleate, spore-like bodies, which bud-off laterally from some erect, conidiophore-like bodies. 5. Sexual reproduction takes place by spermatia uniting with trichogyne of ascogonia. Many species bear antheridia and ascogonia. But antheridia are generally non-functional. 6. After spermatization, many binucleate ascogenous hyphae develop from the ascogonium. Asci develop from the ascogenous hyphae. The asci are spherical, clavate, fusiform or cylindrical. 7. The fruiting bodies are generally perithecium-type. They are spherical, hemispherical, or flask-shaped, i.e. botuliform. The peridium of perithecia is pseudoparenchymatous. The upper portion of peridium becomes a long neck, terminating into a pore. 8. Along with the asci are present many sterile hairs or paraphyses in the fruiting body. The ostiole of the perithecium is also provided internally by ostiolar hairs or periphyses. 9. The ascospores are unicellular or septate, and hyaline or coloured. Muller and von Arx (1973) included 15 families under Sphaeriales. Only Sordariaceae and Clavicipitaceae are examined here in some details. Kirk et al. (2001) did not recognize order Sphaeriales, and treated it as “Xylariales” in a strict seuse.

17.9

SORDARIACEAE

Perithecia are dark-coloured, and not contained within a stroma. The ostiole of perithecium is lined by periphyses. However, a few genera are astomous, i.e. fruiting bodies are closed or lack ostiole. Ascospores are mostly dark at maturity. They have germ pores or germ slits. The ascospores have either a mucous sheath, or have mucilaginous appendages. Muller and von Arx (1973) included 25 genera in Sordariaceae, of which only Neurospora is briefly discussed here. Kirk et.al. (2001) treated Sordariaceae as a taxon belonging to order Sordariales of subclass Sardariomycetidae and class Ascomycetes. They included 6 genera and 37 species in this family.

192

17.10

Fungi and Allied Microbes

NEUROSPORA

17.10.1 Systematic Position

According to Muller and von Arx (1973) Subdivision Class Order Family Genus

– – – – –

Ascomycotina Pyrenomycetes Sphaeriales Sordariaceae Neurospora

According to Kirk et al. (2001) Kingdom Phylum Class Subclass Order Family

– – – – – –

Fungi Ascomycota Ascomycetes Sordariomycetidae Sordariales Sordariaceae

17.10.2 Neurospora and Haploid Genetics Neurospora is universally known to the mycologists, geneticists and biochemists as an important experimental organism. It is so widely used as an organism for the study of laws of heredity that it is popularly named ‘Drosophila of plant kingdom’. Prior to 1927, the fungus was known only by its conidial stages, belonging to the form-genus Monilia. Its perithecial and other stages were discovered by two American mycologists (C.L.Shear and B.O. Dodge) in 1927. Dodge (1927, 1932, 1935, 1942) published a series of papers on Neurospora, and laid the foundation of a new branch of science, called ‘Haploid Genetics.’ Further researches of many different workers on Neurospora showed the way in which genes control enzymes, and this ultimately gave the way to the discovery of ‘One-gene-one-enzyme-theory’ of G.Beadle and E.Tatum of Stanford University (U.S.A), for which they were awarded Nobel Prize in 1958. They showed that Neurospora would thrive on a basic medium and synthesize all its own amino acids. They produced mutants using X-rays. These mutants could not grow on basic medium because they had lost the ability to synthesize a particular amino acid. This trait, according to Beadle and Tatum, could be inherited in Mendelian fashion. This all ultimately suggested that the amino acid synthesis was because of the presence of one enzyme controlled by a single gene. Neurospora has proved to be a useful material in genetical and biochemical research because of following four reasons, according to Tatum (1946), Beadle (1959) and Webster (1980): 1. Simple nutritional requirements of its wild type strains. 2. Mutation can be induced very easily. 3. It grows and reproduces very rapidly. 4. Analysis of its tetrads is very easy.

17.10.3 Some Aspects of Life History Neurospora is a saprophytic fungus. Kirk et al. (2001) have treated it as anamorph Chrysonilia. Its species (N .sitophila, N.crassa, N.tetrasperma, etc) occur on rotting leaves, leather, bread, burnt ground and charred vegetation. N. crassa is called red mould of bread, whereas N .sitophila is commonly called bakery mould. It is a common contaminant of laboratory cultures of fungi and bacteria. A review on the natural populations of Neurospora has been recently published by Turner et al. (2001). The mycelium is well-branched and multicellular (Fig. 17.9 A). From the aerial hyphal branches develop branched conidiophores (Fig. 17.9 B), which produce large number of pink conidia. These conidia are oval and multinucleate, and because of their large size they are called macroconidia (Fig. 17.9 C). Uninucleate microconidia or spermatia also bud off laterally from erect

193

Pyrenomycetes

branches resembling conidiophores (Fig. 12.8 B¢, C ¢). The cells, from which microconidia develop have been named reduced phialides by Subramanian (1971). These macroconidia and microconidia germinate very easily into new vegetative mycelium. Macroconidia

Spermatia

C

B

C¢ B¢

Conidiophore

Spermatium or macroconidium A

Ascospores Ascus G

Beak

Ascospores

Periphyses

Trichogyne

Ostiole

Ascus F

Neck

Sterile hyphae D envelope Ascogonium

Perithecium

Ascospores E

Fig. 17.9

Neurospora crassa. A–C, Asexual cycle; D–G, Sexual cycle.

Sexual reproduction takes place by multicellular ascogonium and spermatia or conidia. N. crassa and N. sitophila are eight- spored, hermaphroditic and heterothallic (Alexopoulos and Mims, 1979).N. tetrasperma is four-spored and secondarily homothallic species. N. dodgei and N. terricola are homothallic species. The spermatia or conidia act as male cells. Female sex organ is represented by protoperithecium or bulbil (Webster, 1980). In a protoperithecium the vegetative hyphae enclose a multinucleate ascogonium, containing long hyphal branches behaving as trichogyne. The spermatia or conidia are chemically attracted to the trichogyne (Fig. 17.9 D) and unite with it. The walls between the trichogyne and the fusing spermatium or conidium dissolve. This initiates the migration of the nuclei of spermatium or conidium into the trichogyne. Shortly after this develop binucleate ascogenous hyphae, and from these hyphae develop the asci. This shows that heterokaryons in N. crassa are not formed between different mating-type strains, because plasmogamy occurs between a trichogyne of one strain and a fertilizing agent (spermatia or conidia) of the opposite strain. Such a condition is called ‘restricted heterokaryosis’. On the contrary, in N. tetrasperma the heterokaryons are formed between mycelia of opposite mating types. Such a condition is called ‘unrestricted heterokaryosis’. An ascus bearing ascospores is shown in Fig. 17.10.

Ascus Ascospores

Fig. 17.10

An ascus bearing 8 ascospores in Neurospora crassa.

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Fungi and Allied Microbes

The globose envelope of sterile hyphae encloses the maturing ascogonium from all sides, resulting in the formation of young perithecium. The development of mature perithecia follows the typical Ascomycetous pattern. The mature perithecia are pyriform, dark coloured and beaked structures, which remain surrounded by dark pseudoparenchymatous peridium. A long neck opening by an ostiole is also present (Fig. 17.9 E). Ostiole and neck are internally provided by many periphyses. Basal swollen part of the fruiting body contains many asci. Each ascus contains eight ascospores (Fig. 17.9 F), of which four are of one mating type, and the remaining four are of the other mating type. The centrum of a young perithecium contains many paraphyses along with asci. But these paraphyses disappear at maturity. The ascospores (Fig. 17.9 G) are dark brown or black, and contain nerve-like ridges on their outer wall. Because of these nerve-like ridges, the name Neurospora is given to the genus (Hohl and Streit, 1975). When young, the ascospores are uninucleate. But they are binucleate when discharged. The asci probably break down in the perithecium. The ascospores are shot out of the fruiting body. They germinate by producing coarse, septate, rapidly growing mycelium, made up of multinucleate cells. A simplified diagram of T.S. ascospore of Neurospora tetrasperma, as studied under electron microscope by Lowry and Sussman (1966) is shown in Fig. 17.11.

Mitochondria Endospore Vacuole

Fig. 17.11

17.11

Epispore Perispore Surface layer Nucleus

Diagrammatic structure of T.S. ascospore of Neurospora tetrasperma as seen under electron microscope (after Lowry & Sussman, 1966).

CLAVICIPITACEAE

Kirk et al. (2001) treated Clavicipitaceae as a family of Hypocreales, whereas Alexopoulos and Mims (1979) raised them to the rank of an order, viz. Clavicipitales. Muller and von Arx (1973) treated them as a family belonging to Sphaeriales. Some of their important characteristics are mentioned here: (i) a majority of the members are parasitic on Gramineae; (ii) perithecia develop on welldeveloped, fleshy stroma of fungal tissue; (III) each ascus contains a thick apical cap, perforated by a long pore; (iv) the paraphyses develop on the lateral walls of the fruiting body, and are not mixed with asci; and (v) ascospores are long, narrow, thread-like and often fragmented into short segments. Some important genera of Clavicipitaceae are Claviceps, Epichloe, Cordyceps, Barya, Ustilaginoidea and Romanoa. Kirk et.al. (2001) included 31 genera and 157 species in family Clavicipitaceae of order Hypocreales. Only Claviceps is discussed further.

195

Pyrenomycetes

17.12

CLAVICEPS

17.12.1 Systematic Position

According to Ainsworth (1973) Subdivision Class Order Family Genus

– – – – –

Ascomycotina Pyrenomycetes Sphaeriales Clavicipitaceae Claviceps

According to Kirk et al. (2001) Kingdom Phylum Class Subclass Order Family

– – – – – –

Fungi Ascomycota Ascomycetes Sordariomycetidae Hypocreales Clavicipitaceae

17.12.2 Occurrence and Importance Kirk et al. (2001) treated Claviceps as anamorph Sphacelia. Claviceps purpurea occurs parasitically on many species of Poaceae, Gramineae, and causes the ergot disease of grasses and cereals. It is most common on Secale cereale causing ergot of rye. C. microcephala attacks Phragmites and Nardus. Barley and wheat are also attacked by C. claviceps, specially at flowering. The fungus attacks the ovaries of the flowers, which soon turn dark coloured, black, compact masses of fungal tissue, called sclerotia (Fig. 6.6 A-C, 17.12 E). The sclerotium of Claviceps is called ‘ergot’, and hence the disease is called ergot disease. When the grains infected by ergot, are used by men or domestic animals, they cause a serious physiological disease, called ergotism (Caporael, 1976). Sclerotia of Claviceps purpurea contain several toxic alkaloids, including ergotamine, ergometrin and ergonovin. Some of these toxins actually constrict the blood vessels, resulting ultimately into gangrene or loss of limbs. Some ergot alkaloids show direct effect on nervous system. If the sclerotia are eaten by pregnant woman or animals, they may suffer from abortion (Ainsworth and Austwick, 1973). Sclerotia are also used to control haemorrhage during child birth, and to induce labour pains in pregnant women. Sclerotia are mainly composed of lipid. Ergotism may even lead to death. Ergot sclerotia contain lysergic acid, from which the well-known hallucinogen (LSD) is synthesized.

17.12.3 Somatic Parts The mycelium develops within the ovary of the infected flower. It is well-branched, septate and hyaline (Fig. 17.12 A). Cells are either uninucleate or multinucleate. Infection takes place by ascospores, disseminated by the wind. On being settled on the stigma or ovary of the flower they germinate to form mycelium.

17.12.4 Asexual Reproduction It takes place by conidia, which develop on conidiophores. The developing mycelium destroys the tissues of the ovary and replaces them by white, soft, cottony structures of the mycelial mat (Fig. 17.12 B). From the latter develop some short and erect hyphae, called conidiophores. At the tips of the conidiophores develop many minute oval conidia (Fig.17.12 C). The mycelial mat at this stage also secretes some nectar-like, sweet, sticky secretion called ‘honeydew’. The origin of this nectar-like secretion is not known. Many conidia get mixed in this honeydew, which also attracts the insects. The honeydew contains glucose, fructose, sucrose and other sugars (Mower and Hancock, 1975). These insects distribute the conidia to uninfected flowers, where they germinate (Fig. 17.12 D) by producing new mycelial hyphae. The mycelial mat soon becomes very hard and change into a pink, purplish or black pseudoparenchymatous body, called sclerotium (Figs. 6.6 A-C, 17.12 E). Some grains in the rye spikelet are now replaced by these sclerotia. The sclerotia are actually the ergot of commerce. The uninfected ovaries develop into normal grains.

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Fungi and Allied Microbes

Germinating conidium D

C

Conidia

Somatic hypha

Conidia Conidiophores

Cap

A

B Mycelial mat

Ascus M

Germinating ascospore

Sclerotium Rye spikelet E

Ascospores

Stroma Germinating sclerotium F Fertile head

L

Cavities Perithecial wall

G Antheridium

Asci

Ascogonium

K

Ka M ry eio og si am s y

Ascus mother cell Ascogenous hypha J Ascogonium

I Antheridium

Gametangia H

Plasmogamy

Fig. 17.12

Claviceps purpurea. A, Somatic hypha; B, Mycelial mat with conidiophores and conidia; C, Conidia; D, A germinating conidium; E, Spikelet of rye with sclerotium; F, A germinating sclerotium; G, V.S. of a fertile head; H, Gametangia; I, Plasmogamy; J, An ascogenous hypha; K, An ascocarp; L, An ascus and ascospores; M, Germinating ascospore.

Germination of sclerotium starts by falling on the ground during the harvesting of the crop. Either in the same winter or next spring season the sclerotia germinate by producing several stromata (Fig. 17.12 F). The stromata are stalked, mushroom-like, purple or black coloured bodies, each with a spherical or capitate head. They are easily visible to the naked eye. Each stromatal head contains many minute cavities (Fig. 17.12 G). Each cavity remains surrounded by pseudoparenchymatous tissue of the stromata. A multinucleate broad ascogonium, and one or more multinucleate and tubular antheridia are present in each cavity.

197

Pyrenomycetes

17.12.5 Sexual Reproduction Tip portions of one of the antheridia and ascogonium come in contact with each other. The walls at the point of contact dissolve and plasmogamy takes place. Male nuclei migrate into the ascogonium (Fig. 17.12 H, I). From the ascogonium develop the ascogenous hyphae. The asci are formed at the tips of the ascogenous hyphae from the binucleate penultimate cell (Fig. 17.12 J). Simultaneously, the perithecial walls develop around the developing asci within the stromatal heads. The swollen heads of each stromata contain many flask-shaped perithecia (Fig. 17.12 G) in different stages of development. Each perithecium contains many elongated and cylindrical asci, and opens by an ostiole (Fig. 17.12 K). The penultimate cell of each ascogenous hypha elongates to form an ascus. Its both the nuclei fuse to form a synkaryon. It undergoes meiosis and then divides ordinarily to form ascospores. Each ascus contains eight needle-like or thread-like, elongated ascospores (Fig. 17.12 L), lying parallel to one another in the ascus. A conspicuous cap is present at the tip of the ascus. The ascospores are discharged forcibly. They are transported by wind up to the flowering head of the rye plant, where they germinate (Fig. 17.12 M) to produce the fresh mycelium. Some details of the stages of the perithecial stroma, sclerotium, ascus and ascospores are shown in Fig. 17.13.

Ascospores Ascus cap Ascus

A Phialoconidia

B

Fig. 17.13

C

Claviceps purpurea. A, L.S. perithecial stroma; B, T.S. young sclerotium; C, Ascus and ascospores.

TEST YOUR UNDERSTANDING 1. The fungi having perithecium type of ascocarp and inoperculate asci, each with an apical pore or slit belong to class _______ . 2. Write any five general characteristics of Erysiphales. 3. Describe in brief the sexual reproduction in Erysiphe. 4. Phyllactinia is a member of: (a) Uredinales, (b) Ustilaginales, (c) Agaricales, (d) Erysiphales. 5. Each ascus of Phyllactinia usually contains how many ascospores? (a) 2, (b) 4, (c) 6, (d) 8. 6. In Phyllactinia, the fruiting body is of cleistothecium type and contains which type of appendages? 7. Give a brief illustrated account of life-cycle of Sphaerotheca. 8. Neurospora belongs to family _______ of order _______ of Pyrenomycetes. 9. The sclerotium of _______ is called ergot. 10. Name any two toxic alkaloids found in sclerotia of Claviceps purpurea. 11. Draw a graphic life-cycle of Claviceps purpurea. 12. Ergot fungus (Claviceps purpurea) most commonly attacks the ovaries of the flowers of _______ .

18

C H A P

DISCOMYCETES

T E R

18.1

GENERAL CHARACTERISTICS

1. These are the Ascomycetes with apothecium type of fruiting bodies, which are cup-shaped, saucer-shaped or disc-shaped. The shape of the ascocarp provides them the common name ‘Cup-fungi’. 2. The fruiting bodies contain an open hymenium, consisting of both asci and paraphyses. 3. Besides having a cup or disc-shape, the apothecia may also be in the form of bell, sponge, tongue, saddle, wing or even brain. But they all open by a well-developed opening. 4. The colour of the apothecia may be yellow, red, brown, orange or even black. Some are even hyaline. 5. Tuberales, commonly called truffles, have underground fruiting bodies with enclosed hymenium, whereas in all other Discomycetes the fruiting body is epigean or above the ground. 6. Members are either terrestrial, coprophilous (Pezizales), saprophytes or parasites (Helotiales and Phacidiales). Fruiting bodies are common on dung, rotten woods and decaying leaves, fruits, etc. Many occur as fungal components of a large number of lichens. 7. A majority of the members lack conidiophores and conidia. 8. Members show progressive reduction in their sexuality. The antheridia and ascogonia are both functional in Pyronema. In Lachnea stercorea the antheridia are nonfunctional, and in L.cretea the antheridia are totally absent. Along with the total absence of antheridia, Humaria granulata shows the disappearance of trichogyne from the ascogonium. Morchella shows the total absence of both antheridia and ascogonia. 9. An apothecium is made up of hymenium, hypothecium and excipulum. The hymenium forms the hollow part of the cup or disc, and consists of asci and paraphyses. The hypothecium is a thin layer of hyphae present just below the hymenium. The remaining fleshy part of the fruiting body that supports the hymenium and hypothecium is called excipulum, whereas its outer layers represent ectal excipulum. The apothecia of Discomycetes are made up of as many as nine different types of tissues (Korf, 1973). 10. All non-lichen-forming Discomycetes have unitunicate asci. 11. The ascospores in all Discomycetes, except Tuberales, are ejected forcibly through different types of apical openings. Korf (1973) reported ten different types of asci openings in Discomycetes. According to Kirk et al. (2001), the term ‘Discomycetes’ is a “formerly used taxa” for a class of Ascomycota, and is now “not accepted in modern classification”.

Discomycetes

18.2

199

CLASSIFICATION

Korf (1973) classified Discomycetes into following seven orders: (i) Medeolariales, (ii) Cyttariales, (iii) Tuberales, (iv) Pezizales, (v) Phacidiales, (vi) Ostropales, and (vii) Helotiales. Alexopoulos and Mims (1979) divided Discomycetes as under: (A) Epigean Discomycetes (fruiting bodies above the ground) 1. Inoperculate (release of ascospores through an apical, circular perforation) Orders: Ostropales, Phacidiales, Helotiales 2. Operculate (release of ascospores through an operculum, a structure like a cap or lid) Order: Pezizales (B) Hypogean Discomycetes (fruiting bodies underground) Order: Tuberales Only Pezizales and Tuberales are discussed here.

18.3 18.3.1

PEZIZALES General Characteristics

They are found on wood, dung, soil and plant debris. These are the operculate Discomycetes, in which the asci open by a lid or operculum. The asci are arranged in a distinct hymenium along with paraphyses. The apothecia vary in size from less than 1mm to 15 cm in diameter. Some are edible, whereas others are poisonous. Some Pezizales possess exceptionally beautiful fruiting bodies. The asci are generally eight-spored. However, some Pezizales have two or four-spored asci, whereas some members of Theleboleae have more than 7000 spores in each ascus (Korf, 1973). The ascospores are unicellular, hyaline, brown or rarely purple, and smooth or variously sculptured. The ascospores are released from the asci by producing a hissing sound (Buller, 1934).

18.3.2 Classification Korf (1973) divided Pezizales into two suborders and seven families as under: Order Pezizales (A) Suborder Sarcoscyphineae Family: Sarcosmataceae, Sarcoscyphaceae (B) Suborder Pezizineae Family: Ascobolaceae, Pezizaceae, Morchellaceae, Helvellaceae, Pyronemataceae. Kirk et al. (2001) treated Pezizales as an order of subclass Pezizomycetidae, class Ascomycetes, phylum Ascomycota. It contains 15 families, 166 genera and 1125 species (Kirk et al., 2001). Representative members of only Ascobolaceae (Ascobolus), Morchellaceae (Morchella), Pezizaceae (Peziza) and Pyronemataceae (Pyronema) are discussed.

18.4

ASCOBOLACEAE

Young ascospores are thick-walled and hyaline. But at maturity the yellow, orange, purple or red pigments (carotenoids) make the asci and ascospores coloured. The ascospores are uniformly uninucleate.

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Fungi and Allied Microbes

Kirk et al. (2001) included 5 genera and 118 species under family Ascobolaceae.

18.5 18.5.1

ASCOBOLUS Systematic Position

According to Korf (1973) Division Subdivision Class Order Suborder Family Genus

18.5.2

– – – – – – –

Eumycota Ascomycotina Discomycetes Pezizales Pezizineae Ascobolaceae Ascobolus

According to Kirk et al. (2001) Kingdom Phylum Class Subclass Order Family Genus

– – – – – – –

Fungi Ascomycota Ascomycetes Pezizomycetidae Pezizales Ascobolaceae Ascobolus

Brief Life History

It is a coprophilous fungus, growing on old dung of cow (A. furfuraceus and A. immerius) and other herbivorous animals (Fig. 18.1 A). Kirk et al. (2001) treated Ascobolus as anamorph Rhizostilbella. Some species of Ascobolus grow on old bonfire sites (A. carbonarius). The mycelium is composed of many branched, septate hyphae. The hyphal cells are short and multinucleate. Asexual reproduction, by means of spores, is generally absent. However, in A. furfuraceus, chains of arthrospores or oidia develop (Webster, 1980). Sexual reproduction is oogamous. A. immersus and A. carbonarius are heterothallic, whereas A. crenulatus is homothallic. Sexual reproduction is highly variable in different species. In A. carbonarius (Fig. 18.1 B) the antheridium is replaced by a conidium-like body. In A. citrunus the antheridium is completely absent, and the apothecium develops from the female branch without copulating with the male branch, i.e. parthenogamically. Typical sex organs are found in A. magnificus (Fig. 18.1 C, D). Multinucleate antheridium and ascogonium develop from the underlying hyphae. Antheridium is cylindrical and clavate, whereas the ascogonium is globose and contains long trichogyne of 5-8 cells. The trichogyne coils round the ascogonium. Its apical cell touches the antheridial apex. The walls at the point of contact dissolve and a pore is formed. The male nuclei migrate through trichogyne and reach the ascogonium, because the septa of the trichogyne are perforated. In the ascogonium pairs of male and female nuclei develop at this stage. From the fertilized ascogonium develop the ascogenous hyphae. Long, clavate, eight-spored asci, along with paraphyses, develop (Fig. 18.1 F) in the usual way as in other Discomycetes. The asci are phototropic, i.e. grow towards light. From the lower stipe cells of the ascogonium develop many sterile hyphal branches, which surround the ascogonium. Ultimately, a cup-shaped, yellowish apothecium with purple dots develops (Fig. 18.1 E). The details of the development of apothecium in A. furfuraceus have been studied by Wells (1970, 1972) and O’Donnell et al. (1974). The apothecium develops either in ‘angiocarpic’ or ‘gymnocarpic’ fashion. In the angiocarpic or cleistohymenial forms the hymenium is enclosed, whereas in gymnocarpic or gymnohymenial forms the hymenium is exposed (Brummelen, 1967). Oso (1969) has studied the ascus development in Ascobolus (Fig. 18.2 A-F). Young ascus shows formation of membrane bound vesicles from the nucleus, and the ascus wall is lined by plasmalemma (Fig. 18.2 A). Ascospore membrane appears at the tip of the ascus and the vesicles arrange along the periphery of the ascus (Fig 18.2 B). Soon, a peripheral

201

Discomycetes

Apothecia Trichogyne

Antheridium Ascogonium A Ascogonium Male conidium Trichogyne

C

B Female conidium Sheath of sterile hyphae

Trichogyne Antheridium Ascogonium

Mature ascus Operculum

Ascospores

D

Projecting ascus

Paraphysis Ascus

E

Fig. 18.1

Ascus

F

G

Ascobolus. A, Habit of A. furfuraceus on cow dung; B, Sex organs of A. carbonarius; C-D, Sex organs of A. magnificus; E, An apothecium of A. immersus with few projecting asci; F, A few asci with paraphyses; G, A discharged ascus with ascospores.

tube is formed by the ascospore membrane (Fig. 18.2 C) and the diploid nucleus divides. Ascospore membrane invaginates between the haploid nuclei (Fig, 18.2 D). The young ascospores are delimited by ascospore membrane from the epiplasum (Fig. 18.2 E). Two layers of the asospore membrane separate due to the formation of primary spore wall between them (Fig. 18.2 F).

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Fungi and Allied Microbes

Ascus tip

Ascospore membrane Vesicles

Ascus wall Vesicles Plasmalemma Nucleus Vesicle

C

Epiplasm

Primary spore wall

Ascospore membrane

Haploid nuclei Invaginated ascospore membrane

D

Fig. 18.22

B

Young Ascospores

A

E

F

A–F, Development of ascus in Ascobolus (after Oso, 1969).

Mature ascospores are purple coloured, and have a mucilaginous epispore. They are liberated through a lid-like operculum (Fig. 18.1 G). The ascospores in A. immersus are very large (approx. 70 mm × 30 mm). Tip of a ripe ascus showing operculum and only three ascospores and the tip of a discharged ascus are shown in Fig. 18.3 A–B. Each ascospore germinates by germ tube to form new mycelium of Ascobolus.

Operculum

Mature ascus

A Ascospores

18.6

Operculum

MORCHELLACEAE

Apothecia are large, stalked and sponge-like. Pileus is pendant or campanulate, and always brown or buff. Carotenoids are absent in the pileus. Ascospores are generally hyaline. They are multinucleate, and each contains 20-60 nuclei (Korf, 1973). Kirk et al. (2001) included 3 genera and 38 species under Morchellaceae. Only Morchella is discussed here.

Tip of discharged ascus B

Fig. 18.32

Ascobolus immerseu showing tip of mature ascus with three ascospores and operculum (A), and tip of a discharged ascus (B).

203

Discomycetes

18.7 18.7.1

MORCHELLA Systematic Position

According to Korf (1973) Division Subdivision Class Order Suborder Family Genus

18.7.2

– – – – – – –

Eumycota Ascomycotina Discomycetes Pezizales Pezizineae Morchellaceae Morchella

According to Kirk et al. (2001) Kingdom Phylum Class Subclass Order Family Genus

Fungi Ascomycota Ascomycetes Pezizomycetidae Pezizales Morchellaceae Morchella.

Occurrence and Economic Importance

Kirk et al. (2001) treated Morchella as anamorph Costantinella. Morchella species are commonly called “morels’, ‘sponge mushrooms’ or ‘guchhi’. It is a saprophytic fungus, which grows commonly on humus soil, decaying wood, leaves and other similar substances. Although, M. esculenta is the most common morel, the other common species are M. conica (conic morel), M.crassipes (thick-stemmed morel), M. deliciosa (delicious morel) and M.hybrida (hybrid morel). Some workers name Morchella as Mitrophora. Waraitch (1978) reported four species (M.angusticeps, M. esculenta, M. crassipes and M. conica) from India. Morchella is perhaps the most highly-prized edible fungus. These days it is considered a delicious and tasteful dish of the modern dining tables. The edible part is actually the fruiting body (Fig.18.4) or apothecium of the fungus. The dish is prepared with rice and vegetables, or in the form of soups. It is widely grown in India, specially in Punjab and Jammu and Kashmir.

18.7.3

– – – – – – –

Pileus

Stipe Mycelial part

Fig. 18.42

Apothecium of Morchella esculenta.

Somatic Parts

The mycelium is underground, growing a few centimetres deep in the soil, and consists of branched, septate hyphae. Each cell is multinucleate. The hyphal mass develops into a fruiting body or ascocarp (Fig. 18.4) and comes out of the soil after the rains.

18.7.4 Ascocarp Development Young ascocarp develops in the form of a dense knot of hyphal mass, specially when conditions of food and moisture are favourable. Hyphal knots are underground and cup-shaped for some time. They soon come out of the soil and develop into stalked fruiting bodies. Further growth makes the hymenium convex and its asci face towards outer side. Because of the unequal growth of the hymenial surface, the smooth hymenium becomes folded in the form of many ridges and depressions. At this stage the hymenium appears like that of a sponge or honeycomb.

18.7.5 Mature Ascocarp It is a greyish white or yellowish brown structure ranging from 2-24 cm in height. The colour of the ascocarps is due to the pigmented oil drops present in the paraphyses (Hawker, 1966). The ascocarp consists of a stalk or stipe and a cap-like pileus

Fungi and Allied Microbes

(Fig. 18.4). In M.esculenta, the pileus and stipe are nearly of same length, both 6-12 cm in height. But in M. semilibera the stalk is nearly twice in length than that of pileus, and in M. crassipes the pileus is nearly twice in length than that of stalk. The apothecia of M. crassipes are largest (Alexopoulos and Mims, 1979). The stipe is fleshy and hollow. The pileus is smooth, when young, but at maturity the margins of the pileus become folded, and thus many ridges and depressions are seen. The depressions on the pileus actually represent the fertile portions. On the contrary, the ridges represent the sterile portions. The cap-like pileus is also hollow from the centre (Fig. 18.5 A). Because of ridges and depressions the pileus appears like a honeycomb or sponge. Anatomically, the fertile region of the pileus is differentiated into a subhymenium and hymenium (Fig. 18.5 C). Subhymenium forms the boundary of the hollow cavity, and consists of many closely packed interwoven hyphae. The hymenium consists of asci and paraphyses, which are arranged perpendicular to the surface of the depression and form a palisade-like layer. Each ascus is a long cylindrical body, containing eight ascospores. The ascospores are arranged obliquely and uniseriately. They are large, hyaline, oval and multinucleate. In between the asci are present many, elongated, unbranched and multicellular paraphyses. Dehiscence of ascus and dispersal of ascospores take place through an apical pore present at the top of the ascus. The ascospores are thrown out by an internal force. They are then carried by wind to distant places. On falling over a suitable substratum, the ascospore germinates into a fresh mycelium (Fig. 18.6 J-M). The asexual reproduction by means of spores is absent.

Ridge

Pileus

Hymenium

Hollow cavity

Stipe

Groove

Sub-hymenium

A

B

Paraphyses Ascus Ascospores

Hymenium

204

Subhymenium Cavity C

Fig. 18.52

Morchella esculenta. A, L.S. of fruiting body; B,T.S. of fruiting body; C, Section through fertile region of pileus showing hymenium and subhymenium.

18.7.6 Sexual Reproduction Typical sex organs in the form of antheridia and ascogonia are not formed in Morchella. In a majority of the species the apothecia are formed as a result of somatogamy, i.e. copulation and fusion between two somatic cells. But in some species ‘autogamy’ also takes place. Under somatogamy the terminal multinucleate cells of two adjacent somatic hyphae of subhymenium region of pileus come in contact (Fig. 18.6 A). The intervening walls of both the copulating cells dissolve at the point of contact and a pore is formed. The protoplasts of both the cells intermingle with each other, and a fusion cell is formed. All nuclei in the fusion cell disappear except two. These two functional nuclei form a dikaryon (Fig. 18.6 B). Many ascogenous hyphae develop from the fusion cell (Fig. 18.6 C). Its dikaryon divides by conjugate divisions. Each ascogenous hypha receives a pair of nuclei, which are derived from the parent dikaryon. The terminal cell of each septate ascogenous hypha functions as an ascus mother cell. Two nuclei of the ascus mother cell (Fig. 18.6 D) fuse to form a diploid synkaryon (Fig. 18.6 E). The ascus cell elongates, and its diploid nucleus undergoes meiosis, resulting into four, haploid nuclei (Fig. 18.6 F-G). One ordinary mitotic division results in the formation of eight haploid daughter nuclei (Fig.18.6 H), all of which ultimately develop into eight ascospores (Fig. l8.6 I). Dehiscence of ascus, dispersal of ascospores and their germination (Fig. 18.6 J-M) take place as mentioned earlier. The tips of the asci in Morchella are curved (Webster.1980). Because of this the ascospores are projected outwards.

205

Discomycetes

Conjugation

Ascogenous Fusion nucleus Ascus hyphae

Fusion cells

A

B

C

Ascus mother cell

D Ascospores

Ascus

Germ tube

Fusion nucleus

Ascospore

K E

F

G

L

Fig. 18.62

H

I

J

Mycelium M

Morchella esculenta. A–B, Somatogamy; C-D, Formation of ascogenous hyphae; E–J, Development of ascospores; K-M, Germination of ascospore.

Under autogamy, as in M. elata, two nuclei of the same vegetative cell of the subhymenium pair together to form a dikaryon. All the remaining nuclei in this cell disorganize and disappear, and it starts to function as an ascus. Both its nuclei fuse to form a diploid nucleus, which first undergoes meiosis and then mitosis to form eight haploid nuclei. The latter change into ascospores.

18.8

PEZIZACEAE

Chief distinguishing character of Pezizaceae is the presence of uninucleate ascospores (Korf, 1973). The ascospores are hyaline or brown, rarely greenish-yellow. The apothecia are cup-, disc- or lentil-shaped. Kirk et al. (2001) recognized 19 genera and 160 species under Pezizaceae.

18.9 18.9.1

PEZIZA Systematic Position

According to Korf (1973) Division Subdivision Class Order

– – – –

Eumycota Ascomycotina Discomycetes Pezizales

According to Kirk et al. (2001) Kingdom Phylum Class Subclass

– – – –

Fungi Ascomycota Ascomycetes Pezizomycetidae

206

Fungi and Allied Microbes

Suborder Family Genus

– – –

Pezizineae Pezizaceae Penza

– – –

Order Family Genus

Pezizales Pezizaceae Peziza

18.9.2 Nomenclatural Limits Korf (1973) mentioned that some workers place spherical-spored forms of Peziza under Plicaria, whereas the species without oil granules are placed under separate genus Aleuria (Boudier, 1907). Its sparassoid forms are placed by some workers under Daleomyces. LeGal (1941) advocated the use of the name Galactinia instead of Peziza. According to him the name Peziza creates confusion and should be abandoned. Kirk et al. (2001), however, mentioned that species of Peziza now belong to anamorphs Chromelosporium and Oedocephalum. Apothecia

18.9.3 Occurrence Over 100 species of Peziza are known (Korf, 1973), and a majority of them occur as saprophytes during rainy season on dung, decaying or burnt wood, or on richly manured soil. Kirk et al. (2001) mentioned only 84 species of Peziza. Because of its welldefined, cup-shaped apothecia (Fig. 18.7), Peziza is commonly called cup-fungus. P. aurantia (Fig. 18.8 A), commonly called ‘Orange peel Peziza’, grows on bare gravel, paths, lawns or bare soil in woods. P.coccinia (Fig. 18.8 B) grows on decayed branches in woods on mineral rich soil.

Fig. 18.72

Apothecia of Peziza vesiculosus.

Disc

Disc

Substratum

B

A Stipe

Paraphysis

Ascus Excipulum

Ascospores

H menium

Ascus Ascospore Paraphysis Hypothecium

Stipe C

Fig. 18.82

Subhymenium D

A, An apothecium of Peziza aurantia; B, Two apothecia of P. coccinea; C.V.S. of an ascocarp; D, A magnified part of the ascocarp.

207

Discomycetes

18.9.4

Somatic Parts

The mycelium is profusely branched, septate and forms dense network of hyphae. Cells are uninucleate or multinucleate. The perennial mycelium remains hidden in the substratum on which it is growing.

18.9.5 Asexual Reproduction Peziza reproduces asexually by the formation of conidia and chlamydospores. Conidia are thin-walled, and develop exogenously on the tips of the specialized hyphae called conidiophores. Conidia occur in P. fuckeliana, P. ostracoderma, P. repanda and P. vesiculosa. Chlamydospores are thick-walled and intercalary. On germination, each chlamydospore also forms new mycelium.

18.9.6 Sexual Reproduction The sexual reproduction follows almost the same pattern as in Morchella. No definite sex organs (antheridia and ascogonia) are formed in a majority of the species, including P. vesiculosus. The apothecia are formed either by somatogamy (copulation of the terminal cells of two different vegetative hyphae) or by autogamy (fusion of two nuclei of the same vegetative cell). The fusion cell contains the dikaryon. From the fusion cell develop ascogenous hyphae. These hyphae are multicellular, and their cells are binucleate. The terminal cell of each of the ascogenous hypha functions as an ascus mother cell. Two nuclei of the ascus mother cell fuse to form a synkaryon. This diploid nucleus divides reductionally and then ordinarily to form eight haploid nuclei, which get organized into eight ascospores. Intermingled with the asci are present many sterile hyphae, called paraphyses (Fig. 18.8 C, D). The ascospores are uninucleate bodies. Each ascospore germinates into a new mycelium.

18.9.7

Mature Ascocarp

The mature ascocarp is a cup-shaped apothecium (Fig. 18.8 A-C). It contains a short stipe and a cup-shaped disc. The cup is 1-12 cm in diameter in P. aurantia, whereas it is only 2-6 cm in diameter in P. coccinea. Clear cup-shaped discs are seen in P. vesiculosus and P. coccinia, but in P. aurantia the cups are often splitted (Fig. 18.8 A). The apothecia may be whitish-yellow (P. coccinia), orange (P. aurantia), or bright red or grey (P. vesiculosus) in colour. The basal portion of the apothecium is called hypothecium. It is generally thick and fleshy. The outer layer of the hypothecium is called excipulum. The brim of the cup is formed by the hymenium, made up of asci and paraphyses, which are arranged perpendicular to the surface of the hymenium, and are almost parallel to each other. Each ascus contains eight ascospores.

18.10

PYRONEMATACEAE

Apothecia are small, discoid or cupulate and only rarely stalked (Korf, 1973). Ascospores are usually uninucleate, and only rarely binucleate. They are hyaline or brown coloured, and smooth or variously sculptured. Kirk et al. (2001) recognized 68 genera and 462 species of Pyronemataceae. Some details of only Pyronema are given here.

18.11

PYRONEMA According to Korf (1973) Division Subdivision Class Order Suborder

– – – – –

Eumycota Ascomycotina Discomycetes Pezizales Pezizineae

According to Kirk et al. (2001) Kingdom Phylum Class Subclass Order

– – – – –

Fungi Ascomycota Ascomycetes Pezizomycetidae Pezizales

208

Fungi and Allied Microbes

Family – Pyronemataceae Family – Pyronemataceae Genus – Pyronema Genus – Pyronema Pyronema is represented by two species i.e. P. domesticum and P.confluens (= Trichogyne P. omphalodes). It occurs in terrestrial conditions, specially on burnt grounds. Ascogonium It is sometimes observed to grow in the soil of green houses, and also on halfcharred wood and decaying leaves. Mycelium is profusely branched. The hyphae are made up of short cells, each having 6-12 nuclei (Harper, 1990). The hyphae grow superficially on the soil in the form of white cottony layer. Pyronema does not show regular formation of any type of asexual spores. OcAntheridium casionally, there may be the formation of oidia in some species. Sexual reproduction is oogamous, and both the species (P. domesticum and P. omphalodes) are homothallic. Both the sex organs are multinucleate, and develop quite close to one another at the tip of the hyphae belonging to the same mycelium. In P. domesticum the antheridia and ascogonia develop in pairs by Fig. 18.92 Sex organs of the repeated dichotomy of a single hypha. An antheridium develops as a termiPyronema domesticum. nal cell of a 2 to 4 celled male branch. The terminal multinucleate cell becomes club-shaped and functions as an antheridium (Fig. 18.9). Lower cells of the male branch constitute the stalk. The nuclei in the antheridium keep on dividing repeatedly to form hundreds of nuclei. The ascogonium is also a multinucleate body with a short, 2 to 3-celled stalk, and a slender, tubular, curved structure, which represents trichogyne (Fig. 18.9). In the later stages the trichogyne gets separated from the ascogonim by a septum. The trichogyne is unicellular and multinucleate. At the time of plasmogamy the trichogyne grows towards the antheridium and makes a contact with it. The walls at the point of the contact dissolve resulting into the development of a pore. Numerous antheridial nuclei enter into the trichogyne through this pore. Through the central pore of the septum of the trichogyne, the male nuclei enter into the ascogonium. Webster (1980) mentioned that there is no nuclear fusion at this stage. From the ascogonium develop many small multinucleate ascogenous initials, which develop into ascogenous hyphae (Fig. 18.10 B, C). The mature ascogenous hyphae are multicellular and often branched (Fig. 18.10 D) In P. domesticum the tips of the ascogenous hyphae get curved to form croziers. At first the crozier tip is binucleate (Fig.18.10 E). Both the nuclei divide mitotically to form four nuclei (Fig. 18.10 F). Septa formation at this stage results into a uninucleate terminal cell, a binucleate penultimate cell and a uninucleate stalk cell (Fig. 18.10 G). Two nuclei of the binucleate penultimate cell are non-sister nuclei, and this cell develops into an ascus. Its both the nuclei fuse to form a diploid nucleus. It undergoes meiosis, followed by ordinary mitotic division to form four (Fig. 18.10 H-J) and then eight haploid nuclei. Some cytoplasm is gathered round these nuclei and the ultimate result is the formation of eight ascospores (Fig 18.10K). Numerous sterile hyphae develop from cells below the ascogonium, immediately after the contents of the antheridia start to flow into the ascogonium (Fig. 18.10 A-D). These hyphae soon form a loose envelope of interwoven hyphae overarching the sex organs. The entire structure represents the apothecium (Fig. 18.11 A, B). Electron microscopic studies of Reeves (1967) and others show that paraphyses also develop from the ascogenous hyphae (Fig 18.10K). In P. domesticum the apothecia also bear some tapering excipular hairs on the outer side (Fig.18.11B). The ascospores are discharged through an apical operculum (Fig. 18.10 K) of the ascus. Each ascospore germinates to form a new haploid mycelium under suitable conditions.

18.12

TUBERALES

18.12.1 General Characteristics 1. Tuberales are commonly called truffles. They are the most highly esteemed of all edible fleshy fungi. In France, Italy and many other European countries, truffles are regularly collected and sold in the market for delicious table dishes.

209

Discomycetes

Antheridium Antheridium

Trichogyne Ascogonium Ascogenous initial Ascogonium Sterile hyphae

A

B Sterile hyphae Ascogenous hyphae Ascogonium

C Operculum

Paraphysis

Discharged ascus

D Penultimate cell

Binucleate tip Crozier

Ascus

E Ascospores

Ascogenous hypha

Terminal cell Stalk cell F

G

Ascus mother cell

K

Fig. 18.10

H

I

J

A–K, Pyronema confluens showing sex organs, development of ascus and ascospores.

2. They form underground fruiting bodies or ascocarps. Because of this Tuberales are also called ‘Hypogean Discomycetes’. 3. Some species (Tuber aestivum, T. albidum, T. melanosporum ) occur in the form of mycorrhizal association with many trees including Pinus, Quercus, etc. 4. The underground ascocarps contain a special flavour and emit strong smell. Because of this they are dug and eaten by wild animals. Webster (1980) mentioned that because of the strong smell the truffles are sometimes collected by the help of trained dogs or pigs, who can detect them by smelling. 5. The ascocarps remain closed in most of the species. 6. The asci are spherical to clavate, and do not contain lid or operculum. They are arranged in a hymenium or scattered in the tissues. 7. The hymenium of the ascocarps is not open to the outer side.

210

Fungi and Allied Microbes

Paraphyses Asci

Excipular hairs

Apothecium B

A

Fig. 18.11

A, V.S. of a young apothecium of Pyronema confluens; B, An apothecium showing hymenium of P. domesticum.

8. The number of ascospores in an ascus is usually eight. 9. The ascospores are not discharged violently by the asci. They are mainly dispersed by animals. 10. The ascospores are colourless or brown, usually variously sculptured (Plate-4), and contain cyanophillic markings. 11. The ascospores are always unicellular.

18.12.2 Classification Korf (1973) included 31 genera under Tuberales, and divided the order into following four families: 1. Elaphomycetaceae, 2. Terfeziaceae, 3. Geneaceae, 4. Tuberaceae. However, Gilkey (1954) included only three families (Terfeziaceae, Geneaceae and Tuberaceae) under Tuberales. Kirk et al. (2001) treated Tuberales as equivalent to Pezizales and discussed all these members under family Tuberaceae of order Pezizales. They discussed 6 genera and 87 species under Tuberaceae.

18.13

TUBER

18.13.1 Systematic Position

According to Korf (1973) Division Subdivision Class Order Family Genus

– – – – – –

Eumycota Ascomycotina Discomycetes Tuberales Tuberaceae Tuber

According to Kirk et al. (2001) Kingdom Phylum Class Subclass Order Family Genus

– – – – – – –

Fungi Ascomycota Ascomycetes Pezizomycetidae Pezizales Tuberaceae Tuber

Discomycetes

211

Ascocarp Tuber (Photoplate 4) is a large genus, represented by about 63 species which are hypogeous (Kirk et al., 2001). Its ascocarps occur 10-30 cm below the surface of the soil, from where they may be collected by digging. Scrapings of squirrels, rabits and other wild animals are often a useful indication of their possible occurrence. These animals are attracted by the strong odour of the ripe fruiting bodies of Tuber. Many species (T. aestivum, T .albidum, T. brumale, T. melanosporum and T. maculatum) occur in the form of sheathing mycorrhizas around the roots of many trees, inA B cluding Pinus, Quercus, etc. Tuber melanosporum, T. aestivum and T. magnatum are the highly-prized edible species Ascus in many European countries. Ascospores The thallus consists of colourless, underground mycelium made up of well-branched hyphae, consisting of many short, uninucleate cells. Sometimes thick strands or rhizomorphs are formed by the hyphae. The hyphae in the rhizomorphs lie parallel to one another. T. candidum occurs as a true saprophyte, whereas many species occur as mycorAscus rhizal symbionts around the roots of many trees. D Conidia formation, observed in Tuber melanosporum, has not been observed in most of the species of Tuber. C Some details of the sexual reproduction are known only in a few species of Tuber. Ascus development has been studied Fig. 18.12 Tuber candidum. A, A mature ascocarp in surface in T. aestivum and T. brumale. The sexual reproduction is view; B, V.S. of the young ascocarp; C, A portion by somatogamy. Two uninucleate cells fuse and bring about of the fertile region of a mature ascocarp; D, An the binucleate condition. From the binucleate fusion cell deascus with three mature ascospores. velop many ascogenous hyphae. The cells of these hyphae are binucleate. The tip of each ascogenous hypha becomes recurved Ascocarp to form croziers. The penultimate binucleate cell of the ascogenous hypha functions as an young ascus. Its both the nuclei unite and the fusion nucleus divides meiotically and then mitotically to form eight daughter nuclei. Out of these eight nuclei, only 2-5 remain functional and mature into ascospores. The remaining nuclei degenerate. The ascocarps are disc-like, globose or irregular masses of hyphae Ascospores A (Figs. 18.12 A, B, 18.13 A). An ascocarp may reach to 3-8 cm in Ascus diameter. It contains a large basal opening, which leads to a hollow chamber. It has a smooth, verrucose or coarsely tuberculate surface. Anatomically, the outer portion of the ascocarp consists of more or Ascospores less thick-walled cells, which form pseudoparenchymatous cortex or Ascus outer peridium. The central portion is called gleba. The gleba is the fertile part, and consists of narrow partitions, which separate narrow hyphae-filled canals or veins. The veins are of two types, (i) venae externae, which are anastomosing loose-textured veins that open to C B the surface of the fruiting body at one or more points, and (ii) venae internae, also called tramal plates, which are dense-textured, darker Fig. 18.13 A, An ascocarp of Tuber rufum; B, An veins. A palisade-like layer of paraphyses develops over the surface ascus of T. rufum with three asof venae externae. Many asci remain irregularly embedded in the tiscospores; C, An ascus of T. puberulum. sue present in between the veins (Fig. 18.12 C).

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Fungi and Allied Microbes

The asci are globose and generally contain less than eight ascospores (Figs. 18.12 C, D; 18.13 B, C). Often their number is two to four (Fig. 18.13 B, C; Plate 4). The ascospores are unicellular and thick-walled. In T. rufum (Fig. 18.13 B) and T. candidum (Fig. 18.12 C, D) they are ornamented by spines. But in T. puberulum (Fig. 18.13 C) the outer wall of the ascospore bears reticulate foldings. Ascosporogenesis in T. aestivum, T. melanosporum and T. rufum has been studied by Paraguey-LeDuc and Janex-Favre (1977, 1982) and Janex-Favre and Paraguey-LeDuc (1976, 1983). The ascospores are liberated only by the decay of the outer cortex region of the ascocarps. The ascospores are dispersed through animals such as rabbits, squirrils and wood rats. The ascospores germinate into new mycelial hyphae. Details of the ascospores and ascoma ontogeny have been observed by Pegler et al. (1993). Phylogeny of Tuber has been studied by Roux et al. (1999).

TEST YOUR UNDERSTANDING 1. Due to the shape of the ascocarp, the common name “ _______ ” is often given to the members of Discomycetes. 2. Write any five general characteristics of class Discomycetes. 3. In Discomycetes, the members of order Tuberales are commonly called _______ . 4. Ascobolus, Peziza and Morchella are all the members of order _______ of class _______ . 5. Describe in brief the life-history of Ascobolus. 6. In Ascobolus, the ascogonium contains a long trichogyne made up of _______ to _______ cells. 7. ‘Morels’, “Guchhi” and “Sponge mushroom” are all the names of: (a) Peziza, (b) Morchella, (c) Agaricus, (d) none of these. 8. Morchella is: (a) a poisonous fungus (b) highly toxic fungus (c) an edible fungus (d) none of these 9. Draw a labelled diagram of V.S. of an apothecium of Peziza. 10. The name “hypogean Discomycetes” is often given to the members of order _______ of class Discomycetes. 11. Which of the following are considered as highly esteemed edible fleshy fungi? (a) Pezizales (b) Tuberales (c) Ustilaginales (d) Uredinales

19

C H A P T

BASIDIOMYCOTINA (GENERAL ACCOUNT)

E R

19.1

WHAT ARE BASIDIOMYCOTINA ?

Basidiomycotina include those eumycotaceous fungi in which the zygospores are absent, and the perfect-state spores are basidiospores. The basidiospores are the meiospores, and are produced on the outside of a specialized spore-producing body, called basidium. Basidiomycotina include rusts, smuts, mushrooms, jelly fungi, puffballs, shelf fungi, toadstools, bird’s-nest fungi, bracket fungi, stinkhorns, fairy clubs and earth-stars. A majority of the large fleshy fungi belong to Basidiomycotina. The subdivision Basidiomycotina, as treated here, is equivalent to class Basidiomycetes. It is equivalent to Basidiomycota of Kirk et al. (2001).

19.2

GENERAL CHARACTERISTICS

1. The members are terrestrial, and saprophytic or parasitic. Saprophytic Basidiomycetes cause decay of wood, litter, dung, wet leaves and other organic matter. Ustilaginales (smut fungi) and Uredinales (rust fungi) cause serious diseases of cereal crops. Many toadstools form mycorrhizal associations with higher plants, and prove extremely valuable in nature. Armillaria mellea is a serious parasite and destroys many angiospermic herbs as well as woody plants. Kost (1984) reported several Basidiomycetes inhabiting mosses, and called them ‘bryicole’. 2. Basidiomycetes are confined to only living host plants in nature. 3. The mycelium is well-developed, branched and septate. The hyphae penetrate the substratum to absorb the food. The mycelium is of primary, secondary and tertiary types. 4. Sometimes the hyphae develop closely together to form thick strands of mycelium enveloped in a sheath or cortex. These hyphal units or tissues are called rhizomorph (Fig. 6.5). 5. In a majority of Basidiomycetes the clamp connections are formed in secondary mycelium. 6. Dolipore septa are present in most of the genera. 7. Cell wall, in a majority of the members investigated so far consists of microfibrils of chitin and glucans with l Æ 3 linked and l Æ 6 linked b-D glucosyl units (Bartnicki-Garcia, 1973). 8. Basidiomycetes reproduce asexually by conidia, arthrospores, oidia, fragmentation or budding. 9. No specialized sex organs develop in Basidiomycetes. Plasmogamy takes place by somatogamy or spermatization. 10. The characteristic spores are the basidiospores, which develop on a basidium. Usually four basidiospores develop on a basidium. The basidia are either non-septate (holobasidia) or septate (phragmobasidia).

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Fungi and Allied Microbes

11. Each basidiospore is typically uninucleate and germinates into a primary mycelium. 12. In economic importance, Basidiomycetes are harmful as well as useful. Crops worth millions of rupees are destroyed annually by rusts and smuts throughout the world. Several Basidiomycetes attack food and ornamental plants, as well as forest trees. On one hand, mushrooms are used as a delicious food throughout the world. Agaricus bisporus is the most commonly used mushroom. On the other hand one should be very careful while collecting the mushrooms for food, because some species are highly poisonous.

19.3

MYCELIUM AND DIKARYOTIZATION

Three types of mycelium are found: the primary, the secondary and the tertiary mycelium. It develops from the germination of a basidiospore. It consists of uninucleate cells, and therefore also called homokaryon. However, the early stages of the primary mycelium may be multinucleate but soon septa formation divides it into uninucleate cells. Basidia never develop on primary mycelium.

Basidiospores

1

B Oidium

2 Primar y mycelium 3

C The secondary mycelium consists of binucleate cells, and develops by the fusion of two uninucleate 4 cells. In heterothallic species cells fuse when the primary A mycelia of opposite sex grow together in close association. D But in homothallic species the fusion takes place between Basidiospore two hyphae of the single primary mycelium. The process of conversion of primary mycelium into secondary or dikaryBasidium otic mycelium is called ‘dikaryotization’ or ‘diplodization”. Dikaryotization in Basidiomycetes may take place by the fusion of: (i) vegetative cells of two hyphae belonging to the priE mary mycelia of opposite strains (Fig. 19.1 A); (ii) two basidiospores of opposite strains (Fig. 19.1 B); F G (iii) an oidium (or spermatium) and a cell of the primary mycelium of opposite strains (Fig. 19.1 C); (iv) a germinating basidiospore and a haploid cell of a basidium (Fig. 19.1 D); Fig. 19.1 A–F, Different processes of dikaryotization in Basidiomycetes; G, Secondary mycelium (v) two haploid cells of the basidium (Fig. 19.1 E); producing basidia and basidiospores. and (vi) two basidia formed by the germination of smut spores of opposite strains (Fig. 19.1 F). The binucleate cells of the secondary mycelium divide to produce daughter binucleate cells by the simultaneous division of two nuclei. The basidia develop (Fig. 19.1 G) from such binucleate cells of the secondary mycelium.

The secondary mycelium in some higher Basidiomycetes gets organized into complex tissues to develop sporophores or basidiocarps. This stage of the secondary mycelium is called tertiary mycelium.

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Basidiomycotina (General Account)

19.4

CLAMP CONNECTION

Kirk et al. (2001) defined clamp connection as “a hyphal outgrowth which, at cell division, makes a connection between the resulting two cells by fusion with the lower”. Clamp connections are formed in most of the genera of Basidiomycetes. These are formed during the division of the cells of the secondary mycelium. Usually the cell division in the hyphae is restricted to the terminal cells. The following stages come across (Kniep, 1917) during the formation of a clamp connection: 1. At the time of the division of binucleate cell (Fig. 19.2 A) a short lateral outgrowth develops (Fig. 19.2 B) between two nuclei x and y. This outgrowth begins to form a hook-like structure and represents clamp connection. 2. One nucleus (y) migrates into the outgrowth and the other remains within the cell (Fig. 19.2 C). 3. Both the nuclei (x and y) now show a simultaneous division (Fig. 19.2 D). 4. Oblique orientation of one division results in the formation of one daughter nucleus (y) in the clamp connection and the other nucleus (y ¢) in the dividing cell (Fig. 19.2 E). Simultaneously, the orientation of the second division along the long axis of the dividing cell results in the formation of one daughter nucleus (x) near the lower basal end of the cell and the other daughter nucleus (x¢) near the nucleus (y¢) (Fig. 19.2 E). 5. Simultaneously, the clamp bents over and its free end fuses with the cell (Fig. 19.2 E). The wall at the place of fusion dissolves, and the daughter nucleus (y) approaches the daughter nucleus (x) as shown in the Fig. 19.2 F. 6. The clamp is closed by the formation of a septum at the point of its origin (Fig. 19.2 F). A second vertical septum develops just below the level of the outgrowth of the clamp (Fig. 19.2 F). This second septum divides the parent cell into two daughter cells (Fig. 19.2 G), of which the terminal cell contains the daughter nuclei (x¢) and (y¢), whereas the other daughter cell contains the daughter nuclei (x) and (y).

y

x

y

x B

A y

y x

x C

D

y x

x





y

E

x





F

y





G

Fig. 19.2

A–G, Successive stages in the formation of a clamp connection in a hypha (after Kniep, 1915).

216

19.5

Fungi and Allied Microbes

DOLIPORE SEPTUM

Amorphous layer In a majority of the Basidiomycetes the septum is characterized by Ectoplast Lateral wall of lateral wall the presence of a septal pore in the centre. This pore remains surrounded by the barrel-shaped swellings. Such a septum (Fig. 19.3) has been named dolipore septum (Moore and McAlear, 1962). Kirk et al. (2001) defined it as “a septum of a dikaryotic basidiEndoplasmic Pore reticulum omycete hypha which flares out in the middle portion forming a Septal pore barrel-shaped structure with open ends”, as revealed by electron microscopy. Ultrastructural studies of dolipore septum (Fig. 19.3) show the following details: 1. The septum is pierced by a narrow pore, called septal Septal plate pore, which is 0.1-0.2 mm in width. Septal pore cap 2. The septal pore remains surrounded by a barrel-shaped swelling, called septal swelling. Septal swelling 3. On each lateral side of the septal pore is present a septal plate. Fig. 19.3 A dolipore septum of Rhizoctonia solani 4. On either side of the dolipore septum is present a dome(after Bracker and Butler, 1963). shaped membranous structure, called septal pore cap. This cap has been variously named as ‘parenthosome’ (Moore and McAlear, 1962; Webster, 1980), ‘Verschlussband’ (Girbardt, 1961) or simply ‘pore cap’. 5. The septal pore cap is made up of modified endoplasmic reticlum. A summary diagram of the several forms of dolipore/parenthosome septum, as suggested by Hawksworth (1994), is shown in Fig. 19.4.

Perforated parenthosome

P1

P2

Imperforated parenthosome Granule Striated bands

Vesiculate parenthosomes

Without parenthosome P6

Fig. 19.4

P5

P4

P3

Summary diagram of the several forms of the dolipore/parenthosome (d/p) septum in Basidiomycotina. The mushroom-shaped occlusions of the outer part can appear either as a granule (O1) or a striated band (O > sub (2) >); the parenthosomes can be regularly perforate (P1), Imperforate (P2), Vesiculate (P3, P4, P5) or absent (P6).

Basidiomycotina (General Account)

217

In spite of such a complex structure, cytoplasmic continuity between adjacent cells, as well as the flow of the mitochondria and other cell organelles through the septal pore, has been observed in some members. About the function of the dolipore septum, Alexopoulos and Mims (1979) have mentioned that ‘it is probably safe to say that its exact function remains unclear’. Dolipore septa have not been observed clearly in Uredinales and Ustilaginales (Webster, 1980).

19.6

ASEXUAL REPRODUCTION

It takes place either by conidia, arthrospores, oidia, fragmentation or budding. (i) Conidia are produced commonly by smuts, and also often by rusts. They are buded off either from the mycelium or from the basidiospores in smuts. The origin and functions of uredospores in rusts is also conidial in origin. (ii) Arthrospores are the unicellular fragments or sections of many basidiomycetes. An arthrospore neither gets rounded up nor become thick-walled. The arthrospores produced from primary mycelium are uninucleate, whereas those produced from secondary mycelium are binucleate structures. (iii) Oidia (Fig. 6.8 A) develop on specialized, short hyphal branches called oidiophores. They are cut off in succession from the oidiophore tip. Each oidium is a small, hyaline, thin-walled and unicellular fragment of the mycelium. The oidia developing on primary mycelium are uninucleate, whereas those produced on secondary mycelium are binucleate bodies. The oidia are not common in homothallic species. (iv) Fragmentation of the filamentous members, and the budding in some Basidiomycetous yeasts are also not uncommon.

19.7

SEXUAL REPRODUCTION

It takes place by the fusion of the nuclei in a basidium. No specialized sex organs are formed in Basidiomycetes. The sexual process involves processes of plasmogamy, karyogamy and meiosis. The plasmogamy is brought about either by ‘somatogamy’ (i.e. fusion of two somatic hyphae of primary mycelia of opposite strains) or “spermatization” (i.e. fusion of a spermatium with that of a receptive hypha). Basidia develop only on the dikaryotic secondary mycelium. Septate basidia are called phragmobasidia, whereas non-septate basidia are called holobasidia. Karyogamy involves the fusion of two haploid nuclei in the basidium, and results in the formation of diploid zygotic nucleus. The latter undergoes meiosis to give rise to four haploid nuclei, which migrate into the sterigmata present at the apex of the basidium. At the tip of each sterigma develops a basidiospore, which is the characteristic feature of all Basidiomycetes.

19.8 19.8.1

BASIDIUM Structure

A basidium is an organ or a fungal cell, bearing a definite number of basidiospores on its surface. Usually the basidiospores are formed after karyogamy and meiosis, and their typical number on a basidium is four. However, in genera such as Dacrymyces and Calocera, each basidium contains only two basidiospores. The basidia of some members contain more than four basidiospores.

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Fungi and Allied Microbes

According to Talbort (1954) a basidium may be divided into three parts, viz. probasidium, metabasidium and sterigmata. The portion where the nuclear fusion takes place is called ‘probasidium’ whereas that where meiosis occurs is called ‘metabasidium’. Any portion present between the metabasidium and the basidiospore is called ‘sterigma’. Typically the basidium is a club-shaped body (Fig. 19.5 A). But in Calocera and Dacrymyces it is tuning-fork type (Fig. 19.5 B). The sterigmata are very long in Tremella (Fig. 19.5 C), whereas the basidium is transversely divided in Auricularia (Fig. 19.5 D) and longitudinally divided in Exidia(Fig. 19.5 E). In Ustilago avenae the germinating chlamydospore functions as a basidium and bear basidiospores (Fig. 19.5 F), whereas in Puccinia graminis each cell of the teleutospore germinates to produce a segmented basidium, bearing basidiospores (Fig. 19.5 G). Basidiospores Sterigmata Basidiospores

Sterigmata Basidium

A

B

C

D

E F

Fig. 19.5

19.8.2

G

Various types of basidia of some Basidiomycetes. A, Oudemansiella; B, Dacromyces; C, Tremella; D, Auricularia; E, Exidia; F, Ustilago; G, Puccinia.

Kinds of Basidia

Basically only two types of basidia are recognized, viz. halobasidium and phragmobasidium. Single-celled, aseptate basidia are called holobasidia, whereas transversely or longitudinally septate basidia are called phragmobasidia. A phragmobasidium contains a first formed portion called hypobasidium and a later-formed portion called epibasidium. Some types of holobasidia and phragmobasidia are shown in Fig. 19.6.

19.8.3

Development of Holobasidium

The basidia develop in a definite, palisade-like layer of the fruiting body (basidiocarp). This layer is called hymenium. Any terminal cell (Fig. 19.7 A) of a binucleate hypha may develop into the basidium. Usually this terminal cell develops a clamp connection at its base (Fig. 19.7 B-E). The terminal cell becomes elongated and broader, and its two nuclei fuse (Fig. 19.7 E) under the process of karyogamy. The fusion nucleus migrates towards the apex of the basidium (Fig. 19.7 F). The

219

Basidiomycotina (General Account)

Lycoperdales

Tulostomatales

A

Agaricales

B

C

(Apobasidial)

Basidiomycetes F

G

Dacrymycetales Tulasnellales D (Autobasidial)

Teliomycetes H

I

E

Ustomycetes K J

(Phragmobasidial)

Fig. 19.6

Major basidial types as proposed by Kirk et al. (2001). A–E, Holobasidia; F–K, Phragmobasidia (A, Lycoperdales; B, Tulostomatales; C, Agaricales; D, Dacrymycetales; E, Tulasnellales; F,Tremellales; G, Auriculariales; H, Uredinales; I, Septobasidiales; J, Ustilaginales; K, Cryptobasidiales).

zygotic nucleus soon undergoes meiosis to form four haploid nuclei (Fig. 19.7 G, H). Rarely, these four nuclei may also undergo one more mitotic division to form eight nuclei in the young basidium (Maire, 1902). At the top or distal end of the basidium develop four small, slender projections or outgrowths, called sterigmata (Fig. 19.7 H). According to Corner (1948) there exist four elastic areas in the distal end of the basidium, and from these elastic areas develop four sterigmata. The tips of the sterigmata enlarge into basidiospore initials (Fig. 19.7 I). A nucleus from the basidium migrates into each basidiospore initial. The latter cuts off by a cross wall at the base and develops into a basidiospore (Fig. 19.7 J). Actually, after the formation of the basidiospore initials, many smaller vacuoles of the basidium coalesce to form a large vacuole. This large vacuole increases in size and pushes the contents of the basidium into the basidiospore initials, which finally develop and separate in the form of basidiospores (Fig. 19.7 G-I).

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Fungi and Allied Microbes

Basidiospore Sterigma Basidium

Clamp connection

A

Fig. 19.7

19.8.4

B

C

D

E

F

G

H

I

Nine successive stages in the development of a holobasidium (after Kniep, 1928).

Development of Phragmobasidium

Rusts and smuts usually contain phragmobasidia. A phragmobasidium in smuts develops by the germination of thickwalled binucleate spore, produced by the rounding up of the binucleate cell of a dikaryotic mycelium. Two nuclei of such a spore fuse to form a diploid zygotic nucleus. The spore germinates to form a germ tube or epibasidium. At this stage the first-formed portion of the spore is called hypobasidium. Diploid nucleus undergoes meiosis. Four haploid nuclei are formed, which migrate into epibasidium. The latter gets segmented into four uninucleate cells. From each cell of the epibasidium develops a sterigma, at the tip of each of which develops a basidiospore.

19.9 19.9.1

BASIDIOSPORE General Morphology

The basidiospores are typically unicellular and uninucleate haploid structures. But in some genera they contain two nuclei, and in Dacrymycetaceae the basidiospores are septate (Reid, 1974). They may be globose, oval, sausage-shaped, elongated, fusoid or flattened in shape. In colour they vary from colourless, yellow, green, pink, orange, brown, violet, creamish and purple to even black. The wall of the basidiospore may be smooth or variously ornamented. The number of the basidiospores produced by the fruiting bodies is very high. An idea about their very large number may be gathered from the fact that a fruiting body of Agaricus campestris produced 1.8 × 109 spores in 2 days, at an average of 40 million spores per hour (Buller, 1909). The number of basidiospores discharged per day by Ganoderma applanatum is 3 × 1010 (Buller, 1922). The point of the attachment of the basidiospore to the sterigma is called hilum (Fig. 19.8 A). Near the hilum is present a minute, conical projection, called hilar appendix. According to Pegler and Young (1971), the basidiospore wall consists of five layers, viz. ectosporium, perisporium, exosporium, episporium and endosporium. The ectosporium is the outermost layer enveloping the perisporium. The perisporium envelopes the ornamented exosporium. The exosporium is a colourless layer. The episporium is the electron-dense layer and determines the exact form as well as size of the basidiospore. The innermost layer is the endosporium, which is transparent in nature.

221

Basidiomycotina (General Account)

Germ pore Ectosporium Perisporium Exosporium Episporium Endosporium Body of basidiospore

Supra-hilar depression Hilar appendix

Sterigma wall extension Drop Mitochondrion Ectoplasm Parahilar layer Spore wall Discharged

Hilum A Basidiospore

basidiospore Buller’s drop

B

Sterigma Hilum

Sterigma

C

Fig. 19.8

19.9.2

D

E

A, Diagrammatic representation of the electron micrograph of a cut-away view of a basidiospore, showing wall layers; B, Sterigma and basidiospore of Schizophyllum commune as viewed under electron microscope (diagrammatic); C-E, Mechanism of spore discharge (A, modified after Pegler and Young, 1971; B, after Wells, 1965; C-E, after Buller, 1922).

Development and Fine Structure

The apex of the sterigma inflates or extends (Fig. 19.8 B) in the form of basidiospore initial, and for quite some time there exists a cytoplasmic continuity between the two. Young basidiospore remains enveloped by only two membranes called external basidial pellicle and the internal basidial layer. Successive wall layers are deposited on these two layers, and the mature basidiospore wall consists of five layers (ectosporium, perisporium, exosporium, episporium and endosporium), as mentioned earlier. The first layer to develop near the plasmalemma is the episporium. It is the thickest layer. The episporium and endosporium become very thin at one end, and this thin place represents the germ pore. In the later stages the continuity between the spore and sterigma is disrupted by the endosporium. The exosporium is responsible for the formation of spines or folds. The pigment responsible for the colour of the spore is present either in the cytoplasm or in the episporium, or in both.

222

19.10

Fungi and Allied Microbes

DISCHARGE MECHANISM OF BASIDIOSPORES

Many different possible mechanisms of discharge of the basidiospores have been suggested by different workers, but so far there is no general agreement among the mycologists over this aspect. In genera with exposed basidia, the basidiospores are discharged violently. Many workers believe that in the maturing basidiospores a bubble or drop forms at the hilar appendix, and this drop is responsible for the basidiospore discharge. According to Buller (1909, 1922), this drop of liquid (Fig. 19.8 C) keeps on increasing in size (Fig. 19.8 D) until the basidiospore is suddenly shot off the sterigma (Fig. 19.8 E). This drop is called ‘Buller’s drop’. Electron microscopic studies of Wells (1965) confirm the findings of Corner (1948), who believed that this drop of the liquid remains surrounded by an extension of the sterigma wall (Fig. 19.8 B). According to these workers the turgour pressure within the basidium is responsible for the discharge of the basidiospore. Olive (1964) and Ingold and Dann (1968) opined that this drop, instead of being liquid in nature, is actually a gas bubble, possibly of CO2.The bubble explodes and discharges the basidiospores. This mechanism has been named ‘explosive discharge’. According to van Niel et al. (1972) the bubble below the basidiospore contains gas and also possibly liquid, and both (liquid as well as gas) are responsible for the spore discharge. Webster (1980) discussed the various mechanisms of basidiospore discharge proposed by different workers, e.g., ‘jet-propulsion’ mechanism proposed by Brefeld (1877). According to this mechanism the basidiospores are propelled from the sterigmata by tiny jets of liquid. He also mentioned the ‘surface tension’ theory of Buller (1922), according to which the surface tension of the Buller’s drop is responsible for spore projection. According to Gregory (1957) ‘electrostatic repulsion’ is responsible for the projection of spores. The spores, discharged violently, have been termed ballistospores by Derx (1948). According to Webster (1980), most basidiospores are ballistospores. But in Gasteromycetes the spores are not projected violently and called statismospores.

19.11

BULLER PHENOMENON

Usually, a dikaryotic mycelium is formed by the somatogamy between two primary or monokaryotic hyphae. But, dikaryotization may also take place between a monokaryotic mycelium and a dikaryotic mycelium. Such a phenomenon of dikaryotization of a monokaryotic mycelium by a dikaryotic mycelium was first discovered by Buller (1930) and called ‘Buller phenomenon’. Kirk et al. (2001) defined “Buller–phenomenon” as “the dikaryotization, in Basidiomycetes and Ascomycetes, of a homokaryon by a dikaryon”. According to Buller (1930, 193l), production of basidia and basidiospores by a dikaryotized monokaryotic mycelium depends actually on the nature of the dikaryotizing nucleus. Buller (1941) divided this phenomenon into following two categories: A combination in which neither factor in the monokaryotic mycelium is duplicated by the entering nucleus it called legitimate combination. A combination in which either factor in the monokaryotic mycelium is duplicated by the entering nucleus is called illegitimate combination.

19.12

DIFFERENCES FROM ASCOMYCOTINA

Some major differences between Basidiomycotina and Ascomycotina are listed in Table 19.1.

Basidiomycotina (General Account)

Table 19.1

Differences between Basidiomycotina and Ascomycotina

Basidiomycotina 1. Dolipore septa are present in a majority of the members. 2. Dikaryophase is long-lived and independent. 3. Primary mycelium containing the cells with haploid nuclei is short-lived. 4. Sexual apparatus is ill-developed or even lacking. 5. Clamp connections occur commonly in the secondary mycelium. 6. Formation of basidia and basidiospores is the major characteristic of these fungi. 7. Basidiospores are formed exogenously on the basidium. 8. Basidiospores are usually four on a basidium. 9. Fruiting body consists entirely of dikaryotic hyphae, and called basidiocarp.

19.13

223

Ascomycotina 1. Dolipore septa are absent. 2. Dikaryophase is short-lived and dependent. 3. Primary mycelium is dominant and long-lived. 4. Sexual apparatus shows gradual degeneration, i.e. the sex organs are present in lower Ascomycetes whereas absent in higher forms. 5. Instead of clamp connections the crozier formation is common feature; clamp connections are not formed. 6. Formation of asci and ascospores is the chief characteristic. 7. Ascospores are formed endogenously in the ascus. 8. Ascospores an usually eight in an ascus. 9. Fruiting body consists of both monokaryotic as well as dikaryotic hyphae and called ascocarp.

CLASSIFICATION

Ainsworth (1973) divided subdivision Basidiomycotina into the following three classes, and the same classification has been adopted in this book: Basidiocarps are absent and replaced by chlamydospores (smuts) or teleutospores (rusts). These spores are grouped in sori or remain scattered within the host tissue. Members are parasitic on higher plants. Basidiocarps are well-developed and the basidia are organized in a hymenium. The basidiocarps are gymnocarpous or semiangiocarpous. Basidiospores are ballistospores. Members are saprobic and rarely parasitic. Basidiocarps are well-developed and typically angiocarpic. Basidiospores are not ballistospores. Members ate saprobic and only rarely parasitic. All these three classes (Teliomycetes, Hymenomycetes and Gasteromycetes) are discussed in Chapters 20-22. Kirk et al. (2001) divided phylum Basidiomycota into three classes as under : 1. Basidiomycetes 2. Urediniomycetes 3. Ustilaginomycetes It is divided into two subclasses, 16 orders, 112 families, 1037 genera and 20391 species. Two subclasses are Tremellomycetidae (containing 8 orders) and Agaricomycetidae (containing 8 orders). Eight orders of Tremellomycetidae are Auriculariales, Ceratobasidiales, Christianseniales, Cystofilobasidiales, Dacrymycetales, Filobasidiales, Tremellales and Tulasnellales. Eight orders included under Agaricomycetidae are Agaricales, Boletales, Cantharellales, Hymenochaetales, Phallales, Polyporales, Russalales and Thelephorales. It includes 5 orders, 25 families, 195 genera and 8057 species. The orders are Agaricostibales, Atractiellales, Microbotryales, Septobasidiales and Uredinales. It includes 3 subclasses, 10 orders, 31 families, 119 genera and 1464 species. The subclasses alongwith their orders are (i) Entorrhizomycetidae (Entorrhizales), (ii) Exobasidiomycetidae (Doassansiales, Entylomatales, Exobasidiales, Georgefischeriales, Tilletiales and Microstromatales), and (iii) Ustilaginomycetidae (Urocystales, Ustilaginales and Cryptomycocolacales).

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TEST YOUR UNDERSTANDING 1. The eumycotaceous fungi lacking zygospores and possessing basidiospores are placed under subdivision _______ . 2. The basidiospores are: (a) asexual spores, (b) mitospores, (c) meiospores, (d) none of these. 3. Basidiomycotina includes: (a) rusts, shell fungi and smuts, (b) jelly fungi, stinkhorns and puffballs, (c) toadstools, fairy clubs and bracket fungi, (d) all of these. 4. The usual number of basidiospores which develop on a basidium are: (a) two, (b) four, (c) six, (d) eight. 5. What do you mean by clamp connection? Describe successive stages of its formation in a hypha. 6. What is a dolipore septum? Describe its ultrastructure with the help of suitable diagrams. 7. Write only one sentence explaining major difference between a holobasidium and a phragmobasidium. 8. Describe general morphology and fine structure of basidiospore with the help of suitable diagrams. 9. Write short notes on: (a) Buller phenomenon (b) Discharge mechanism of basidiospores 10. Tabulate any four main differences between Ascomycotina and Basidiomycotina. 11. Kirk et al. (2001) divided Basidiomycotina into three classes. These are _______, _______ and _______ . 12. Ainsworth (1973) divided Basidiomycotina into three classes. These are _______, _______ and _______ .

20

C H A P

TELIOMYCETES

T E R

20.1

GENERAL CHARACTERISTICS

1. Members of class Teliomycetes include rusts and smuts, which are parasitic on many vascular plants. Smuts (Ustilaginales) are usually parasitic on angiosperms, whereas rusts (Uredinales) occur parasitically also on pteridophytes and gymnosperms along with angiosperms. 2. The mycelium is well-developed and septate, but the septa lack dolipores or parenthesomes. The septa are of simple type. 3. Members lack basidiocarps. They possess thick-walled, binucleate resting spores called teliospores, and hence the name Teliomycetes is given to the class. 4. Fusion of the two nuclei takes place within the teliospores. 5. From each cell of the teliospore develops a short germ tube, called promycelium. 6. The diploid nucleus moves into promycelium, where meiosis takes place and haploid basidiospores are formed. 7. The number of the basidiospores, developing on the promycelium, are typically four in rusts. But in smuts the number is large and indefinite. 8. Teliospores of Teliomycetes are equivalent to the basidia of other members of subdivision Basidiomycotina. 9. Sterigmata-like outgrowths are present on the promycelium only in rusts. They are absent in smuts.

20.2

CLASSIFICATION

Class Teliomycetes is divided into two orders: 1. Uredinales, which include rusts. 2. Ustilaginales, which include smuts and some basidiomycetous yeasts.

20.3 20.3.1

UREDINALES General Characteristics

1. Because of the reddish-brown colour of some of their spores, these fungi are popularly called rust-fungi or simply rusts.

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2. In nature, all members are obligate parasites of vascular plants, parasitizing a number of green plants belonging to ferns, conifers, dicotyledons and monocotyledons. Rusts of cereals, coffee, beans, asparagus and carnation are known throughout the world. Cereal rusts have been known since Roman times. (The God, Robigus, was believed to be responsible for cereal rusts by Romans. And to satisfy Robigus, Romans used to plan annual festival, Robigalia.) 3. Obligate parasitic nature of rusts has been questioned by many workers, because many rusts have been grown in artificial culture. Puccinia graminis has been grown on Czapek-Dox, yeast agar extract (Williams et al., 1966). Nine rusts could be grown in laboratory till 1973 (Wolf, 1974). 4. Within the host tissue the rusts grow with intercellular mycelium, which rarely bears clamp connections. Clamps develop in the binucleate mycelium of only some species. 5. The mycelium is uninucleate at first, becoming binucleate later on. It penetrates the host cells by haustoria, and absorbs the nutrients. 6. Electron microscopic studies revealed that rust cytoplasm contains mitochondria, ribosomes, endoplasmic reticulum, glycogen particles and lipid granules. 7. Rust septa arc more simple in comparison to that of other Basidiomycetes, and resemble the septa of Ascomycetes. The septum develops in a centripetal fashion, i.e. tapers in thickness towards central pore. The septum consists of two electron-dense layers, which remain separated by a thin electron-transparent layer (Harder, 1976). 8. Rusts are polymorphic because they form more than one type of spores to complete their life-cycle. 9. Basidiocarps arc not formed by rusts. 10. The rusts in which life-cycle is short and completed by only two types of spores (teleutospores and basidiospores) are called ‘microcyclic rusts’. On the contrary, the rusts which form all the five stages of the spores (teleutospore, basidiospore, spermatia or pycniospores, aeciospores and uredospores) in their life-cycle are called macrocyclic rusts (e.g. Puccinia graminis). Rusts, in which uredospores are not formed, are called demicyclic rusts. 11. Roman numerals (0, I, II, III, IV) are customarily assigned to different stages of a macrocyclic rust as under: Stage 0

: Spermogonia (or pycnidia) with spermatia and receptive hyphae. (It is called O-stage because spermatia and spermogonia were earlier thought to be functionless spore-like bodies, and by this time the names of the other stages (aecia, uredia, etc.) were well-established in the literature. Later it was discovered that spermogonial stage is an essential stage in the life-cycle and it precedes aecial stage. Instead of disturbing the designations of the other stages, it was thought by the mycologists to be safest to call spermogonial stage as O-stage).

Stage I

: Aecia with aeciospores

Stage II

: Uredia with uredospores

Stage III : Telia with teleutospores Stage IV : Basidia with basidiospores For different types of sori and spores, Laundon (1973) used the terms pycnia and pycniospores, aecia and aeciospores, uredinia and urediniospores, telia and teliospores, and basidia and basidiospores. 12. Rusts, which complete their life-cycle, on a single host, are called autoecious rusts. On the contrary those, which complete their life-cycle on two different hosts, are called heteroecious rusts. 13. All rusts produce teliospores or teleutospores. Processes of karyogamy and meiosis take place in teleutospores, and therefore a teleutospore is considered to be the perfect stage of rusts.

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Teliomycetes

20.3.2

Classification

Arthur (1934) divided Uredinales in two families: 1. Melampsoraceae, with sessile teliospores 2. Pucciniaceae, with pedicellate teliospores. Savile (1978) divided Uredinales into five families (Pucciniastraceae, Melampsoraceae, Phragmidiaceae, Raveneliaceae and Pucciniaceae), whereas Alexopoulos and Mims (1979) restricted rusts upto three families only (Pucciniaceae, Melampsoraceae and Coleosporiaceae). Kirk et al. (2001) placed Uredinales under class Urediniomycetes of phylum Basidiomycota. They included 14 families, 163 genera and 6927 species in this order. Only family Pucciniaceae and some details of Melampsora of family Melampsoraceae are discussed here.

20.3.3

Pucciniaceae

Presence of pedicellate or stalked teliospores is the chief characteristic feature of the family. The promycelium, formed after the germination of teliospores, is septate. The teliospores are either free or variously united but never present in the form of layers or crusts. Some details of Puccinia, Uromyces, Phragmidium and Ravenelia are given here.

20.4

PUCCINIA

20.4.1 Systematic Position

According to Ainsworth (1973) Division Subdivision Class Order Family Genus

– – – – – –

Eumycota Basidiomycotina Teliomycetes Uredinales Pucciniaceae Puccinia

According to Kirk et al. (2001) Kingdom Phylum Class Order Family Genus

– – – – – –

Fungi Basidiomycotina Urediniomycetes Uredinales Pucciniaceae Puccinia

20.4.2 Occurrence Puccinia occurs as an obligate parasite of many cereals, millets and many other crops of economic importance. More than 700 species have so far been reported from all over the globe, of which over 150 species occur in India. The most common cereal host of Puccinia graminis is wheat, although it also occasionally occurs on barley, oat, rye, etc. Puccinia graminis and many other species are heteroecious, because they complete their life-cycle on two different hosts. Wheat is the primary host of P. graminis, whereas its secondary host is Berberis vulgaris. On the contrary, many others are autoecious rusts because they complete all the stages of their life-cycle on the single host, e.g. Puccinia butleri on Launea and P. chondrillina on Chondrilla juncea (Adams and Line, 1984). Some of the species, and the diseases caused by them, are Puccinia graminis (cereal rust), P. asparagi (asparagus rust), P. antirrhini (snapdragon rust), P. coronata (crown rust of oats), P. malvacearum (hollyhock rust), P. penniseti (Bajra rust), P. sacchari (sugarcane rust), P. sorghii (corn rust) and P. striiformis (yellow rust of wheat). Kakishima et al. (1984) studied the details of life-cycle of Puccinia japonica on Anemone flaccida. Three common rusts of wheat arc black rust (P. graminis tritici), orange rust (P. recondita) and yellow rust (P. striiformis). For comparison between these three rusts, refer Article 20.8, Table 20.1.

228 20.4.3

Fungi and Allied Microbes

Laboratory Culture of Rusts

Obligate parasitic nature of rusts was questioned by the successful culturing of a rust (Gymnosporangium juniperi virginianae) in the laboratory by Hotson and Cutter (1951). Later on, Cutter (1960) cultured Puccinia malvacearum and observed teliospore formation on 3 months old callus tissue. Williams et al. (1966) obtained successfully the first axenic culture of a rust from uredospores of an Australian isolate of Puccinia graminis tritici. The uredospores were incubated by these workers on an agar medium containing Czapek-Dox, 1% yeast extract and 3% sucrose. Within 48-96 hr successful germination of uredospores and their further branching took place. Within 2-4 weeks uredospores and teleutospores were produced. Later on (Williams et al., 1967) these workers successfully reinfected the host when uredospores were placed under the epidermis in contact with mesophyll cells. Because of this study, Puccinia graminis tritici could no longer be considered as an obligate parasite. Bushnell (1976) also cultured successfully P. graminis tritici.

20.4.4

Biological Specialization in Puccinia

Puccinia graminis causes rust of cereals. But there are present a number of different subspecies of P. graminis which attack specific hosts, e.g. P. graminis tritici occurs on wheat, and P. graminis avenae occurs on oats but never on wheat. Similarly, P. graminis hordei occurs on barley, and P. graminis secalis occurs on rye but never on barley, wheat or oat. This phenomenon of attacking only the specific host by the specific pathogen is called ‘biological specialization’. Each of these subspecies contains many physiological races, which again differ in their parasitism of different host varieties, e.g. P. graminis tritici contains more than 300 physiological races (Webster,1980), which are specific in their occurrence on different varieties of hosts. But for all of them the alternate host is the Berberis vulgaris. The details of the life-history of Puccinia graminis tritici are given below.

20.5

PUCCINIA GRAMINIS (LIFE CYCLE)

Puccinia graminis is a macrocyclic, heteroecious rust, of which wheat (Triticum aestivum) is the primary host and the barberry bush (Berberis vulgaris ) is the secondary host. Out of the five stages of the life-cycle, three (urediniospores, teliospores and basidiospores) are produced on wheat plant, whereas the remaining two (pycniospores and aeciospores) are produced on Berberis vulgaris. The life-cycle is, therefore, completed only when both the hosts are present.

20.5.1

Symptoms

Its symptoms on wheat are seen in the form of large, elongated, brown pustules on the stem, leaf sheath and lamina. These are the pustules of uredinia-bearing urediniospores (Fig. 20.1 A). These brown pustules change into black-coloured large pustules of telia bearing teliospores (Fig. 20.1 B). These pustules provide rusty appearance to the infected region. Its symptoms on the barberry bush are first observed on the dorsal, surface of the leaf (Fig. 20.1 C) in the form of yellow spots of pycnial cups which bear pycniospores. The ventral surface of the leaves (Fig. 20.1 D) bears the aecial cups with aeciospores.

20.5.2

Stages on Wheat

The dikaryophase of the life-cycle of P. graminis is confined to its primary host, i.e. wheat. It consists of dikaryotic mycelium, and stages of uredinia and telia (Fig. 20.2 A). It is produced by the germinating aeciospore or urediniospore on wheat. It enters through the stoma and develops in the intercellular spaces of the tissues of the leaves and stem of wheat. Each cell of the dikaryotic mycelium is binucleate (Fig. 20.2 B). From the mycelium are given out some haustoria, which penetrate the host cells. Usually, the haustoria are knob-like, but rarely they may be convoluted or finger-like. Electron microscopic studies (Ehrlich

229

Teliomycetes

Uredinia

Leaf Pycnial cups

Telia

Aecial cups

Stem

A

Fig. 20.1

B

C

D

Puccinia graminis tritici. A, Uredinia on the leaf of wheat; B, Telia on the stem of wheat; C, Pycnia on the upper surface of the leaf of Berberis vulgaris; D, Aecial cups on the lower surface of the leaf of Berberis vulgaris.

and Ehrlich, 1963; Manocha and Shaw, 1966) show that the haustoria are not surrounded by the plasma membrane of the host. Each haustorium is rather surrounded by a distinct capsular wall, either of fungal or host origin. The septa of dikaryotic mycelium have a central pore but no dolipore-parenthesome complex (Ehrlich et al. 1968). Clamp connections are also not formed. The dikaryotic mycelium develops into the reproductive phase, and produces urediniospores (also called uredinospores or uredospore) in uredinia (also called uredium or uredosorus) and teliospores in telia (also called teleutosorus). The dikaryotic mycelium, present subepidermally in the stem, leaf or leaf sheaths of wheat, develops into uredinial cells. These uredinial cells form a palisade-like layer of hyphal tips below the epidermis. The urediniospores are produced from this palisade-like layer of dikaryotic hyphae. The account of the ‘development of uredinia and urediniospores’ mentioned below is based on the ultrastructural studies of Harder (1976): From the sporogenous cells of this palisade-like layer originate some buds which are responsible for the formation of urediniospores. Each bud enlarges in size and divides transversely by a septum into two cells. The lower cell is called a pedicel and develops into the stalk, whereas the upper cell enlarges into urediniospore proper. Many developing urediniospores press against the host epidermis from the inside and finally rupture it. The ruptured epidermis makes the group of the urediniospores exposed (Fig. 20.2 C). Such groups are variously called uredinia, or uredosori, or uredopustules. A mature urediniospore or uredospore is a stalked structure, bearing a swollen round, globose or oval body. Each urediniospore contains two nuclei, and remains surrounded by a thick spiny wall (Fig. 20.2 D). The wall contains four (Webster, 1980) thin areas called germ pores. As many as 50,000 to 400,000 urediniospores or uredospores may be present in a single uredinium(Webster, 1980). The uredinia or uredopustules are seen on the stem and leaves of the wheat in the form of elongate, reddish-brown or blackish, granular pustules, and provide the rusty appearance to the host (Fig. 20.1 A). A crop of highly susceptible grains provides rusty appearance to the entire field. A person passing through such a heavily infected field comes away with clothes covered with a rusty dust (Alexopoulos and Mims, 1979). Because of the rupturing of the host epidermis, the urediniospores in an uredinium remain freely exposed (Fig. 20.2 C). A urediniospore germinates within a few hours after falling upon a suitable host plant, i.e. wheat. It sends out a germ

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Binucleate mycelium

Urediniospore

Stem

Host cell Rusty patches (uredinia)

Ruptured epidermis

B

Leaf Host cells

C

A Germ pore Nuclei

Germinating urediniospore Wheat leaf cell

Germ tube Germinating Appresorium urediniospore Stoma Stoma Vesicle

Germ tube

Host cells

D E

Fig. 20.2

F

Binucleate mycelium

Puccinia graminis tritici. A, Infected leaf and stem of wheat bearing uredinia; B, Binucleate mycelium in wheat plant; C, A uredinium bearing urediniospores; D, A few urediniospores showing germ pores; E, Germination of two urediniospores on wheat leaf; F, Infection of wheat leaf by a germ tube through a stoma.

tube (Fig. 20.2 E) through one or more germ pores. For quite some time, the germ tube grows over the surface of the host epidermis (Fig. 20.2 E). On reaching a stoma (Fig. 20.2 F) the tip of the germ tube develops into a vesicle, called appressorium. The binucleate protoplast of the germ tube migrates into the appressorium, which soon gets separated from the germ tube by a septum (Allen, 1923). On entering through the stomatal slit, the appressorium develops into a vesicle and empties its contents into it. From the binucleate vesicle develops a well-branched mycelium containing binucleate cells. The mycelium keeps on developing only near the point of its enterance in the host, killing the nearby cells. Within 4-5 days this binucleate mycelium again starts to produce urediniospores on the same or nearby wheat plants. The urediniospore stage is also called a ‘repeating stage’ of Puccinia and other rusts because several crops of spores may be produced in one growing season. In the late growing season, instead of the urediniospores the mycelium begins to produce teliospores or teleutospores. Instead of uredinia, the teliospores bearing sori are now called teleutosori (Fig. 20.3). They are dark brown or black couloured (Fig. 20.1 B). In the middle of the season the same sorus usually contains many

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Teliomycetes

urediniospores as well as some teliospores in the different stages of their development. The groups of the binucleate cells which give rise to teliospores are called telia (sing. telium) or teleutosori. The teliospores are stalked, bicelled, spindle-shaped structures, each constricted slightly at the septum (Fig. 20.3). The wall of the teliospores is thick and smooth, and their tip is usually pointed or round. Each cell of the teliospore contains a germ pore. The germ pore is at the apex in the upper cell (Fig. 20.4 A), whereas it is a little below the septum in the lower cell of the teliospore (Fig. 20.4 B). Each cell of the teliospore is binucleate. Because of their thick wall the teliospores may survive even in very unfavourable conditions. At maturity, the two nuclei of each cell of the teliospore fuse to form a diploid nucleus. A teliospore cannot again infect the wheat plant The teliospores undergo a long resting period, and do not ordinarily germinate until the next spring. During this resting period they may be lying on the ground or may remain attached to the dead host plants. Each teliospore germinates and produces the basidiospores, and this stage represents the basidial stage (Fig. 20.4 A, B). Both cells of the teliospore function as hypobasidia. From one or both the hypobasidia develop a long tube called epibasidium (Fig. 20.4 A, B). The diploid nucleus of each cell moves into the epibasidium and divides meiotically to form four haploid nuclei. Both the divisions, constituting the meiosis, are followed by the formation of transverse walls in the epibasidium. Four uninucleate cells are thus formed in each epibasidium. From each of these four cells develop a lateral sterigma and a basidiospore (Fig. 20.4 A, B). Out of the four basidiospores of an epibasidium, two belong to plus strain and the remaining two to the minus strain. Each basidiospore is small, unicellular, uninucleate and haploid structure. According to Buller (1924) the basidiospores discharge explosively, or are discharged with a force into the air. The basidiospores are unable to infect the wheat. They germinate only if they happen to fall upon a barberry bush (Berberis vulgaris), which is the alternate or secondary host. It is because the barberry protoplasm is the only food to which a germinating basidiospore mycelium is able to use in nature (Alexopoulos and Mims, 1979). Fast flowing winds carry these basidiospores from plains to hills, where barberry bushes occur quite frequently. A majority of the basidiospores never reach the barberry bushes and ultimately perish. But the life-cycle is carried on only by those basidiospores that ultimately reach up to barberry bush and germinate.

20.5.3

Teliospore Ruptured epidermis Stalk Host cells

Fig. 20.3

Transverse section of the host through a teleutosorus of Puccinia graminis tritici.

Basidiospores

Sterigmata Epibasidium Hypobasidium

A

B

Fig. 20.4

A–B, Puccinia graminis tritici, showing germinating teliospores.

Stages on Barberry

The haplophase of the life-cycle of P. graminis is confined to its secondary host, i.e. barberry (Berberis vulgaris). It consists of primary or haplomycelium, and stages of pycnidia (or spermogonia) and aecia. The symptoms produced by Puccinia graminis tritici on upper as well as lower surface of leaves of barberry plants are shown in Fig. 20.5 A, B. The haploid basidiospore (of + or – strain) settles on the leaf or young twig of the barberry plant and infects it by producing primary mycelium. Each basidiospore sends out a germ tube, which grows directly

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Fungi and Allied Microbes

Pycnia

Lower surface of Barberry leaf

Aecia Upper surface of Barberry leaf

A

Fig. 20.5

B

A–B. Symptoms produced by Puccinia graminis tritici on barberry leaves. A, On upper surface of leaf showing pycnial pustules; B, On lower surface of leaf showing aecia.

through the outer wall of an epidermal cell. Here, the germ tube develops into a hypha, made up of 4-6 uninucleate cells. From each cell of this hypha develop small hyphal branches, made up of uninucleate cells. These hyphal branches grow through the intercellular spaces of the host cells, and represent primary mycelium, also called monokaryotic mycelium or haplomycelium. The mycelium so formed would be of plus or minus strain. Usually several basidiospores of different strains infect the same leaf and produce mycelia of different strains. Within 3-4 days after infection, dense hyphal mats develop here and there within the host tissue. These palisade-like hyphal mats actually represent the primordia of pycnia or spermogonia. The primary mycelium forms palisade-like hyphal mats, which become visible externally on the barberry leaves in the form of small, yellowish, circular pustules. Each pustule represents a sperOstiole mogonium (or pycnium ) of plus or minus strain. Nectar A mature spermogonium is a flask-shaped body, opening exterReceptive hypha nally by a pore-like mouth opening, called ostiole (Fig. 20.6). The Periphyses cavity of the spermogonium is lined with palisade-like layer of nuSpermatia merous, short, uninucleate tapering cells, which represent spermatiophores. From the tip of each spermatiophore are abstricted many, Spermatiophores small uninucleate spermatia, which keep on accumulating within the spermogonial cavity as well as outside the ostiole on the barberry Spermogonial cavity leaf. Hughes (1970) named these tapering cells as ‘phialides’, whereas they have been named ‘annellophore’ by Rijkenberg and Truter (1974). Near the ostiole also develop some long, sterile, pointed hyphae, which project through and beyond the ostiole. These are Fig. 20.6 Vertical section of a spermogocalled periphyses. Some of the hyphae near the ostiole become more nium of Puccinia graminis. elongated than the periphyses, and also come out through the ostiole. These are called receptive hyphae or flexuous hyphae. Spermatia were earlier thought to be functionless structures in connection with the life-cycle of P. graminis. But, it was Craigie (1927) who discovered that the spermatia function as male cells, and the long receptive hyphae behave as trichogyne. He also reported that spermatia are essential in the formation of dikaryotic aeciospores. It is brought about by a spermatium and a receptive hypha of different strains. All spermatia in a single spermogonium carry the same strains. At the time of the extrusion of spermatia through the ostiole, the spermogonium secretes a droplet of sweet-smelling nectar-like liquid. Groups of spermatia, periphyses and receptive hyphae are all covered by this nectar drop. According to Craigie (1927) some insects are attracted towards this nectar. The insects visit-

233

Teliomycetes

Nectar

ing several spermogonia transfer spermatia from one spermogonium to another. They are thus dispersed from leaf to leaf and from one spermogonium to another spermogonium. Thus, the spermatium of one strain reaches upto the receptive hypha of the other strain. The walls at the point of contact between the spermatium and receptive hypha dissolve, and the spermatium nucleus moves into the receptive hypha. Through the septal pores the spermatium nucleus reaches upto the nucleus of the basal cell of the receptive hypha. Both these nuclei of opposite strains lie side by side in a pair called dikaryon. The cell possessing this dikaryon is now said to be diploidized. According to Buller (1950) the well-developed primary mycelium within the barberry leaf produces a globose hyphal mass (protoaecium) just within the lower epidermis. The protoaecium does not grow any further if there is no spermatization. And if there is spermatization, the spermatial nuclei enter through the receptive hyphae, migrate through the central pores present in the cross walls of the mycelial cells, and finally dikaryotize the basal cells of the protoaecidium. Only in case of such a dikaryotization, the protoaecium develops into an aecium.

Spermogonium Spermatia

Sporophores

Disjunctor Aeciospores Aeciospores

Peridium

B

Aecium

The aecial cups containing the aeciospores are A present on the lower surface (Fig. 20.7 A) of the leaves of Berberis vulgaris. The basal cells of each aecium are dikaryotized and become Fig. 20.7 Puccinia graminis. A, V.S. of a elongated into stalks or sporophores. From each of these dikaryotized long barberry leaf, showing spermogobasal cells are cut off a chain of dikaryotic cells on the side towards the nium on the upper surface and an lower epidermis of the host. All these derivative cells, so formed, cut aecium on the lower surface; B, A off from the basal cells, divide themselves into alternate bands of large chain of aeciospores alternating binucleate cells and small binucleate intercalary cells (Fig. 20.7 B). The with small intercalary sterile cells or disjunctors. small intercalary cells (disjunctor) remain sterile and soon disintegrate. The large binucleate cells mature into aeciospores. The wall of the aecial cup is made up of a sterile protective layer, called peridium. Meiosis A developing aecium pushes and ruptures the host epidermis and Basidiospores Nuclear fusion exposes the aeciospores for dispersal. Each aeciospore is a polyhedral, binucleate structure having an Infection of Teliospores outer thick and smooth exine and an inner thin intine. They are shed Berberis late in the spring. They cannot reinfect the barberry plants. The air Urediniospores currents carry the aeciospores to the primary host, i.e. wheat, on Spermogonium which they germinate under suitable conditions. The young germ or pycnium tube hypha enters through a stoma in the wheat and develops into an Uredinium intercalary dikaryotic mycelium. Within 10-12 days of the infecSpermatia or pycniospores Infection of tion the uredospores start to develop from this dikaryotic mycelium wheat in the tissues of the wheat. The life-cycle (Figs. 20.8 and 20.9) is Spermatization again repeated in the same manner. Aeciospores

20.6

CONTROL MEASURES OF RUSTS

Fig. 20.8

Aecium

Life-cycle of Puccinia graminis

1. The best control measure of rust is the eradication of the alternate host, i.e. Berberis vulgaris, because by such an action the life-cycle of this macrocyclic heteroecious rust will not be completed. But only by eradicating berberry

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Fungi and Allied Microbes

Flexuous hypha Germination by repetition

Basidiospores (wind-dispersed)

Spermatia (insect-dispersed)

Spermogonium (o) Meiosis

Plasmogamy Spermatium Flexuous hypha

Karyogamy Urediniospores (wind-dispersed)

Dikaryotization

Teliospores

Telium (

)

Dikaryotic mycelium (

Uredinium ( )

)

Aecium (

)

Aeciospores (wind-dispersed) Rounding-off

Heterokaryotic phase on cereals

Fig. 20.9

2. 3. 4. 5. 6.

7.

Homokaryotic phase on Berberis

Semidiagrammatic life-cycle of Puccinia graminis.

bushes the problem is not going to be solved so easily because the production of several crops of urediniospores in a season goes on regularly in the wheat itself. Moreover, the urediniospores can also travel for very long distances. Stakman and Harrar (1957) reported the clouds of urediniospores travelling distances of upto 2000 miles between the USA and Canada. Johnson et al. (1967) reported the similar type of long-distance transport of urediniospores from India and Europe. Moreover, urediniospores may also remain viable in the soil for several months. However, barberry eradication will provide positive results because it will lessen the incidence of infection, as well as will reduce the possibility of the formation of new physiological races. According to Webster (1980) there are over 300 known physiological races of Puccinia graminis tritici. Mehta (1929) suggested that rust epidemics can be controlled by growing rust-resistant wheat varieties. ‘Lerma Rojo’, ‘Sonalika,’ ‘Sonora 64’, ‘N P 700’.and ‘N P 800’ are some of the Indian rust-resistant varieties of wheat. Mixed cropping of wheat and barley with suitable crops reduces the amount of secondary infection and spread of rust disease. Crop rotation may also prove useful. Use of nitrogenous fertilizers increase the susceptibility of wheat crop to rusts. Fungicide application to wheat and other cereal crops also controls the rust partly. Use of chemicals, such as zinc sulphate, dithane, sulphur dusting, RH-124 and Plantavax, has given quite encouraging results in rust control. Four sprayings of Parzate liquid along with zinc sulphate at a fortnightly intervals also provide satisfactory results. Timely forecast of rust disease, so that effective measures are taken in advance, can also minimize the chances of the rust epidemics.

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Teliomycetes

20.7

ANNUAL RECURRENCE OF RUST IN INDIA

Puccinia graminis tritici is a macrocyclic heteroecious rust. But in India and many other countries the alternate host, i.e. barberry bushes, do not play any role in the recurrence of rust. Late Professor K.C. Mehta (1929,1933, 1940, 1952) has been the pioneer worker in connection with annual recurrence of rust in India. According to him, only urediniospores are responsible for the initiation and spread of rust disease from year to year. Berberis does not play any active role as an alternate host. The aecial stages present on barberry in India actually belong to Aecidium montanum and not to Puccinia graminis tritici. Moreover, teliospores do not survive a temperature over 26°C. And in the Indian plains the teliospores on wheat are produced in February-March. After March the day temperature in plains is always more than 26°C. Therefore, the urediniospores are the chief source of the rust infection on wheat in India. But, urediniospores also cannot survive the high summer temperature of Indian plains. For that, Mehta investigated and proved that these are those urediniospores that oversummer in the northern hills at higher altitudes of 1300-2500 m, where they survive on self-sown wheat plants and tillers. The conditions of high altitudes and low temperature on hills are congenial for the survival of urediniospores. Hence, they retain their vitality in hills. These surviving urediniospores of hills first infect the wheat crop near or at the foot-hills, where they are carried easily by the air currents. From these infected wheat plants in the foot-hills they are carried by the winter winds to the plains, where they infect the wheat crop in January-February. Mehta, therefore, suggested that the rust severity in the plains can be reduced if there is no wheat cultivation on the hills for some time.

20.8

COMPARISON BETWEEN BLACK, ORANGE AND YELLOW RUSTS

Table 20.1 No. 1. 2. 3. 4. 5.

6.

7.

20.9

Comparison between three rusts

Black rust (Puccinia graminis tritici)

Orange rust (Puccinia recondita = P. triticina)

Yellow rust (Puccinia striiformis = P. glumarum)

Alternate hosts are species of Berberis and Mahonia Appears in plains during February April More severely attacked parts are stems

Alternate hosts are species of Thalictrum and Isopyrum Appears in January

Alternate host is not known

Leaves are most severely attacked

Uredinia are large, elongated and coalescing Urediniospores are oval, 25-30 × 17-20 mm in diameter, and contain 4 germ pores Telia are black and found on all green parts with very low frequency on leaf blades Teliospores are bicelled, 40-60 × 15-20 mm in diameter and dark brown; tip of the upper cell is pointed or rounded.

Uredinia are small, oval or round, and never in long rows Urediniospores are spherical, 16-20 mm in diameter, and wall is echinulate with 7-10 germ pores Telia are very rare. If present, they are distributed mainly on undersurface of leaves Teliospores are bicelled and flattened at the top

Leaves are more severely attacked than stems and leaf sheaths Uredinia are very small, oval, and arrranged in rows or stripes Urediniospores are spherical to ovate, 23-35 × 20-35 mm in diameter, and contain 6-16 germ pores Telia are dull black, and arranged in rows

Appears during December-January

Teliospores are dark brown, flattened at the top, bicelled, 35-63 ×12-24 mrn in diameter.

UROMYCES

This member of Pucciniaceae occur as an obligate parasite of legumes such as pea, gram, bean, etc. Some of its common species include U. appendiculatus (on Phaseolus radiatus), U. cicer-arietini (on Cicer arietinum), U. decoratus (on Cro-

236

Fungi and Allied Microbes

tolaria juncea), U. fabae (on Pisum sativum) and U. trigonellae (on Trigonella foenium-graceum. The urediniospores of Uromyces are spherical, light brown coloured, stalked structures. Each teliospore (Fig. 20.10) is a stalked, ovate, unicellular structure, characteristically bearing a hyaline papilla at the apex. The papilla is hemispherical and very clear.

Hyaline papilla

Teliospore

Stalk

20.10

PHRAGMIDIUM

It is also a member of Pucciniaceae, but Kirk et al. (2001) mentioned Phragmidium of family Phragmidiaceae is now anamorph Physonema, occuring as an autoecious rust on Rosaceae. It occurs as an obligate parasite on members of Rosaceae. Phragmidium mucronatum is an autoecious and macrocyclic rust. Its urediniospores are borne singly on the pedicels of rosaceous hosts. The teliospores (Fig. 20.11) are stalked, multicellular (3-10 celled), robust, cylindrical, and contain horizontal septa. Each cell of the teliospore bears 2-3 germ pores, and its wall is characteristically pigmented. Generally, it is brown coloured. P.violaceum occurs on blackberry, P.rubi-idaei on raspberry and P. mucronatum on rose.

20.11

RAVENELIA

Ravenelia is also an autoecious rust genus of Pucciniaceae. Kirk et al. (2001) treated it as a member of family Raveneliaceae of Urediniales. It occurs as an obligate parasite on members of Leguminosae, Euphorbiaceae and Teliaceae. Its characteristic teliospores (Fig 20.12) are united in the form of radially arranged discoid spore heads. The spore heads are present on the fascicled pedicels, and remain subtended by many hygroscopic cysts. The cysts are present at the tip of fascicled pedicel. The teliospore wall is pigmented. Each teliospore bears one germ pore.

20.12

Teliospores of Uromyces appendiculatus.

Teliospores

Fig. 20.11

Two germinating teliospores of Phragmidium mucronatum. Teliospore Hygroscopic cysts

MELAMPSORACEAE

This family of order Uredinales contains only 1 genus (Melampsora) and 90 species. Spermogonia discoid, with bounding structures. subepidermal or subcuticular; uredinia with pedicellate urediniospores and capitate paraphyses; teliospores unicellular, sessile and form single–layered crusts.

20.13

Fig. 20.10

MELAMPSORA

Fascicle of pedicels

Head of teliospore

Mycelium

Host cells

Melampsora of family Melampsoraceae occurs as an obligate parasite of linseed Fig. 20.12 Teliospores of Ravenelia plant, and hence called “rust of linseed”. The most common species is M. lini, with host tissue. which is an autoecious rust. Because of the infection of Melampsora, bright orange pustules develop on leaves, stem and other parts of the host. Its urediniospores are stalked structures (Fig. 20.13 A). The stalk is 2-3 celled. The urediniospores are binucleate, globose, and contain echinulate wall. Each uredinium contains many capitate and unicellular paraphyses alongwith urediniospores. The

237

Teliomycetes

Capitate paraphyses Urediniospores Ruptured epidermis Mycelium Host cell A Teliospores Epidermis

Mycelium Host cell B

Fig. 20.13

Melampsora lini on linseed plant. A, V.S. through a uredinium bearing many urediniospores; B, Many teliospores arranged in a palisade-like layer.

teliospores develop on the host at the end of the season. The teliospores remain characteristically arranged in a palisadelike manner (Fig. 20.13 B). Each teliospore is unicellular, sessile, cylindrical, long and reddish-brown structure.

20.14

USTILAGINALES

20.14.1 General Characteristics 1. Represented by over 1100 species (Duran, 1973), the Ustilaginales are commonly called ‘smut fungi’. They are so named because they form black dusty masses of spores on or within the tissues of their hosts. These masses of spores resemble soot or smut. 2. They occur parasitically in more than 75 families of angiosperms, of which the most favourable hosts are the members of Poaceae and Cyperaceae. Floral parts of the host are most commonly attacked. 3. The mycelium is intercellular, with no specialized haustoria. But here and there short hyphal branches may enter within the host cells, making the mycelium intracellular. 4. Clamp connections are also seen in the mycelium of many species. 5. No well-organized sex organs are formed in smuts. Plasmogamy takes place either by the fusion of two compatible basidiospores, or two mycelial fragments or two conidia. The ultimate result is the formation of dikaryotic mycelium. 6. The cells of the dikaryotic mycelium are binucleate, which round off to form thick-walled resting spores, variously called smut spores, chlamydospores, teliospores, brand spores, ustospores or even melanospores. But because the name of the class is Teliomycetes, these binucleate spores should better be called only ‘teliospores’.

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Fungi and Allied Microbes

7. Two nuclei of the teliospore later on fuse to make it a mature, uninucleate, diploid structure. These diploid teliospores often have thick dark walls, which are usually ornamented with spines or reticulations, but in some cases the wall is smooth (Plate 6 A-M). 8. Teliospores are usually blackish, brownish or yellowish, and are commonly dispersed by wind. 9. A teliospore germinates into a promycelium. The nucleus moves into the promycelium and divides meiotically to form haploid nuclei, which enter into the basidiospores (= sporidia). 10. No sterigmata are formed. The sporidia develop direcly on the promycelium. 11. Smuts reproduce asexually by the formation of conidia. The conidia develop from uninucleate as well as binucleate mycelium. 12. Budding is also very common in the basidiospores and conidia of smuts.

20.14.2 Classification Ustilaginales are usually divided into two families: 1. Ustilaginaceae, with transversely septate promycelium, having lateral and terminal basidiospores. 2. Tilletiaceae, with aseptate promycelium, having only terminal basidiospores. But author believes that there should also be a third family Graphiolaceae (Sharma, 1967) with only one genus Graphiola. Kirk et al. (2001) placed Ustilaginales under class Ustilaginomycetes. The order contains 2 families (Tilletiaceae and Ustilaginaceae), 40 genera and 672 species (Kirk et al., 2001).

20.15

USTILAGO

20.15.1 Systematic Position

According to Ainsworth (1973) Division Subdivision Class Order Family Genus

– – – – – –

Eumycota Basidiomycotina Teliomycetes Ustilaginales Ustilaginaceae Ustilago

According to Kirk et al. (2001) Kingdom Phylum Class Order Family Genus

– – – – – –

Fungi Basidiomycota Ustilaginomycetes Ustilaginales Ustilaginaceae Ustilago

20.15.2 Occurrence Almost all the so-far known approximately 400 species of Ustilago are parasitic on plants. The most common hosts of Ustilago are the members of Poaceae and Cyperaceae. Crops of great importance to man, such as wheat, oat, sugarcane, barley, maize, rye and pearlmillet (bajra) are the common hosts. Sometimes they are very badly damaged. Some of the Ustilago species and the diseases caused by them are: U. tritici (loose smut of wheat, Fig. 20.14 A, B), U. maydis (corn smut, Fig. 20.14 C; Plate 5 G), U. avenae (loose smut of oat, Fig. 20.14 D), U. nuda or U. hordei (covered smut of barley, Fig. 20.14 E; Plate 5 A, B), U.cynodontis (loose smut of doob grass, Fig. 20.14 F; Plate 5 D), U.scitaminea (whip-smut of sugarcane, Fig. 35.8), U.violacea (anther smut of Caryophyllaceae) and U. hypodytis (stem smut of grasses). Kandua and kangiari are the local names of smut diseases in Uttar Pradesh and Punjab, respectively. Smut spores of some species of Ustilago are shown in Plate 6 A-M (Kakishima, 1981).

Teliomycetes

239

20.15.3 Common Symptoms of Smuts Grains of the inflorescence transform into black powdery mass (Plate 5 A-G). Except the awns and the central spikelet axis, the inflorescences are completely destroyed. Infected plants are reduced in size. Sometimes the grains get hypertrophied as in U. maydis (Fig. 20.14 C). Ultimately the yield of the crop is greatly reduced, sometimes as high as 70%.

20.15.4 Kinds of Smut Diseases Smut diseases are of two types, i.e. loose smut and covered smut. In loose smut the spores are not covered by any membranous structure. They are directly exposed to the air at the time of flowering, and at maturity they are easily disseminated by wind, e.g. loose smuts of wheat, oat and doob-grass. In covered smut the spores remain enclosed in a membranous covering of the grain, i.e. they are not directly exposed to the air. Mature spores are therefore not easily disseminated by wind. They are liberated only by the breaking up of the wall of the grain, e.g. covered smuts of barley and sugarcane. Oat and barley suffer from both loose as well as covered smuts.

20.15.5 Mycelium The mycelium is branched, septate, intercellular, hyaline and usually inconspicuous. As shown by the nuclear behaviour the nucleus passes through following two distinct stages: It is produced by the germination of a basidiospore. It consists of slender, hyaline, septate hyphae, each cell of which contains a single haploid nucleus. Based on the strain of the basidiospore, the primary mycelium is either of plus or of minus strain. The primary mycelium is less extensively developed, and also called haplomycelium or monokaryotic mycelium. It either changes soon into secondary mycelium, or dies. It contains two haploid nuclei in each cell. It consists of septate, extensively branched, intercellular hyphae. This is the mycelium that is usually found within the host. In U. maydis, intracellular hyphae are also common. In some species the haustoria develop from the intercellular hyphae. The secondary mycelium in some species remains scattered throughout the different parts of the host, and is called ‘systemic’. On the contrary, in some species it spreads only near the point of infection, and is called ‘localized’. The secondary mycelium is also called dikaryotic.

20.15.6 Methods of Diploidization The process by which the primary mycelium changes into a secondary mycelium is called diploidization or dikaryotization. In this process two haploid cells of opposite strains copulate to form a binucleate cell. These two nuclei of such cells do not fuse in the vegetative phase and constitute a dikaryon. The binucleate cell so formed develops into a dikaryotic hypha, each cell of which is binucleate. In Ustilago, diploidization may take place by any of the following methods: 1. By somatogamy (hyphal fusion) between the hyphae of the primary mycelia belonging to the conidia of opposite strains (Fig. 20.15A), e.g. U. maydis. 2. By conjugation between the germ tubes of two germinating basidiospores (Fig. 20.15 B), e.g. U. anthearum. 3. By the fusion of a basidiospore of one strain and the germ tube of a basidiospore belonging to another strain. 4. By the fusion of two adjacent haploid cells of the same epibasidium (Fig. 20.15 C, D), e.g. U .hordei. 5. By the fusion of two basidia formed by the germination of teliospores of opposite strains (Fig. 20.15 E), e.g. U. nuda. 6. By the fusion of a basidiospore and one of the cells of a basidium of opposite strains (Fig. 20.15 F), e.g. U. violacea.

240

Fungi and Allied Microbes

Infected grains

Infected ear

A

C

B

Infected inflorescence

Infected grains

Leaf Stem

D

Fig. 20.14

E

F

Symptoms of some smut diseases. A-B, Ustilago tritici, loose smut of wheat; C, U. maydis, maize (corn) smut of Zea mays; D, U. avenae, loose smut of oats (Avena sativa); E, U. nuda, covered smut of barley (Hordeum vulgare); F, U. cynodontis, loose smut of doob grass (Cynodon dactylon).

20.15.7 Reproduction It takes place by the production and germination of ‘teliospores’ into basidiospores. Basic aspects of these stages in some species are discussed. The dikaryotic mycelium in U. maydis (corn smut) grows usually in the intercellular spaces of the host cells. Because of the infection of secondary mycelium the host cells divide and redivide quite actively in the infected region. And this ultimately results in the formation of some gall-like swellings (Fig. 20.16 A). At maturity the growth of the dikaryotic secondary mycelium stops, and its most of the binucleate cells start to function as spore initials. The spore initials are at first enclosed in a gelatinous matrix, but at maturity the matrix disappears. They become globose, enlarged and develop into thick-walled unicellular spores called teliospores (Fig. 20.16 B; Plate 6D).

241

Teliomycetes

Basidiospores

B

+ F –

Conidia

Fig. 20.15

Epibasidium

A

C

D

E

Various methods of dikaryotization in Ustilago. A, Some maize leaf cells (in surface view) showing formation of secondary mycelium by the conjugation of two germinating conidia of U. maydis; B, Conjugation between two germinating basidiospores of U. anthearum; C-D, Fusion of two haploid cells of the same epibasidium in U. hordei; E, Fusion of the basidia of two germinating teliospores of U. nuda; F, Fusion of a basidiospore and a cell of the basidium in U. violacea.

Some prefer to name them ‘chlamydospores’, ‘smut spores’, ‘brand spores’ ‘ustilospores’ or even ‘hypobasidia. The thick wall of each teliospore consists of two layers, of which the outer, usually spiny or reticulate layer is called exine, whereas the inner thin layer is called intine (Fig. 20.16 C). Two nuclei of these thick-walled spores fuse (Fig. 20.16 D) to form a diploid nucleus. Smith (1955) mentioned that these teliospores function as hypobasidia. Each hypertrophied gall (Fig. 20.16 A) contains innumerable hypobasidia or teliospores. These spores are disseminated by first drying and then rupturing of the epidermis of the galls. The mature teliospore is uninucleate and diploid (Fig. 20.16 D). Immediately or after a short resting period, each teliospore germinates. At the time of germination the spore wall cracks and a germ tube comes out in the form of a promycelium (Fig. 20.16 E). The diploid nucleus migrates into the promycelium, divides meiotically and results in the formation of four haploid nuclei (Fig.20.16 F, G). The septum formation in the promycelium transforms it into a septate structure made up of uninucleate cells (Fig. 20.16 H). The nucleus within each cell of the promycelium divides into two daughter nuclei, of which one migrates into a bud that develops at the side of each promycelial cell and the other nucleus remains within the cell (Fig. 20.16 I). The so-formed uninucleate buds function as basidiospores (Fig. 20.16 I). Usually, the nucleus remaining within the cell of the promycelium divides again and results in the budding off of a second basidiospore (Fig. 20.16 I). Genetic studies have confirmed that the diploid nucleus (Fig. 20.16 D-I) of the teliospore divides meiotically, and out of the four cells of the promycelium two cells produce the basidiospores of one sex (+) and the remaining two cells produce the basidiospores of opposite sex (–). Basidiospores are thin-walled, oval or round, haploid, uninucleate and unicellular structures (Fig. 20.16 J). Each basidiospore germinates to produce a monokaryotic mycelium (Fig. 20.16 K.), which is either of (+) strain or of (-) strain. Within the host tissue two hyphae of opposite strains come in contact with each other, fuse and form a dikaryotic cell, which develops into a dikaryotic secondary mycelium (Fig. 20.16 L). Over 200 long fimbriae or hair-like structures develop from the pairing basidiospores in U. maydis and U. violacea. These fimbriae are made up of proteins and they vary in length between 0.5 and 10 mm. On being detached from the promycelium, sometimes basidiospores keep on dividing by budding. These uninucleate buds of the basidiospores are called conidia. The conidia also behave and germinate like basidiospores.

242

Fungi and Allied Microbes

Galls Teliospores

A

B

C Exine Intine

Primary mycelium Nucleus (2x)

– + +

D



Germ tube

L Binucleate cell E Germ tube Promycelium K + – J F Promycelium 1st Basidiospore

+ + – –

2nd basidiospore I

Fig. 20.16

H

G

A–L, Life-cycle of Ustilago maydis on Zea mays.

In this species (Fig. 20.17 A-B) the teliospore germinates into a 3-celled promycelium. It gets detached from the teliospore. Even after being detached the promycelium keeps on producing the basidiospores or sporidia.

243

Teliomycetes

Basidiospore Promycelium Basidiospore Detached promycelium

Promycelium

A

B A

Fig. 20.17

Ustilago violacea. A , Germinating teliospore showing 3-celled promycelium; B, Detached promycelium producing basidiospores.

Fig. 20.18

B

A–C, Various stages of the development of the promycelium from the teliospore of Ustilago nuda.

No sporidia or basidiospores arc produced in U. nuda (Fig. 20.18 A-C), which causes the loose smut of barley and wheat. A septate promycelium, of course, develops by the germination of the teliospore. From the individual uninucleate cells of the promycelium develop the germ tubes, which fuse and bring about dikaryotization. The teliospore in U .longissima (Fig. 20.19 A, B) does not germinate into a promycelium. It germinates only into a short tube, which successively buds off basidiospores. Teliospore germination in Ustilago avenae is shown in Fig. 20.20 A-C.

20.15.8 Control of Smuts

C

Teliospore Basidiospore

A

Fig. 20.19

B

Germination of teliospore and development of basidiospore in Ustilago longissima.

Loose smut of wheat (Fig. 20.14 A, B), caused by Ustilago tritici, is controlled by hot water treatment. Under this treatment the seeds are first soaked in water for 4-5 hr at a temperature of 26-30°C, and then quickly transferred to hot water at 54°C for 10 min. The seeds are finally dried. Solar energy treatment (Luthra and Sattar, 1934) is also effective, under which the seeds are soaked in water for about 4 hr in the forenoon on a bright summer day and then dried in the sun for about 4 hr. Loose smut of wheat is also controlled by anaerobic seed treatment, under which seeds are water soaked for 2-4 hour at a temperature of 16-21oC, kept in the air-tight containers for about 3 days and then finally dried. Use of the fungicides such as Carboxin, Benomyl etc. is also effective. Use of resistant varieties of wheat is the most effective control measure of loose smut. Some smut-resistant varieties are ‘Kalyansona’,’Kalyan 227’. ‘PV 18’, and ‘C 302’. It is (Fig. 20.14 C) caused by Ustilago maydis, and is controlled by breeding of resistant varieties. However, crop rotation and field sanitation (Mehrotra, 1980) also provide satisfactory results.

244

Fungi and Allied Microbes

B

A

Germinating Teliospore

Promycelium (4-celled)

Sporidia

Young dikaryon C

Fusing conjugation tubes Basidiospores

Teliospore

Fig. 20.20

A-C, Teliospore germination in Ustilago avenae. A, Germinating teliospore showing 4-celled promycelium each cell producing basidiospores; B, Germinating teliospore showing fusion of two terminal cells to initiate a dikaryon; C, Fusion of conjugation tubes from two basidiospores to form a dikaryon.

It is (Fig. 35.8 G) caused by Ustilago scitaminea, and controlled by removing the smutted whips from the field, planting unsmutted setts of canes, and disinfecting the setts by Agallol, Aretan or mercuric chloride. Use of resistant varieties is also effective. It is (Fig. 20.14 E) caused by Ustilago hordei or U. nuda, and is controlled by treating the seeds by fungicides, such as Agrosan.

20.16

GRAPHIOLACEAE AND GRAPHIOLA

Graphiola, still an enigma to the systematists, should finally be placed under a separate unigeneric family Graphiolaceae of Ustilaginales (Sharma, 1967, 1974, 1975; Shiam and Sharma, 1975). G. phoenicis, commonly called “false smut”, occurs in the form of cup-shaped fruiting bodies (Fig. 20.21 C, D) on both the surfaces of pinnae, all round the rachis and on the leaf bases of Phoenix sylvestris (Fig. 20.21 A. B). Sharma (1967) reported over 600 fruiting bodies of this fungus on a single pinna of Phoenix sylvestris (Fig. 20.21 A). The mycelium is hyaline, septate, well-branched, intercellular as well as intracellular. Each fruiting body is cup-shaped or crater-like structure and consists of peridium, sporogenous hyphae and bundles of sterile hyphae (Fig. 20.21 C-H). The peridium is black, complex mass of thousands of sterile hyphae, which together form the boundary of the fruiting body. The sporogenous hyphae (Fig. 20.21 C-F) are unbranched, hyaline and erect fertile hyphae, arranged parallel to one another. Each hypha consists of 5-20 basipetally arranged cells, each of which functions as a teliospore. From each teliospore buds off 3-4 sporidia or basidiospores. Millions of sporidia are present in a mature fruiting body. Intermixed with sporogenous hyphae are also present many bundles of sterile hyphae (each of 2-10 cells

245

Teliomycetes

Pinna

Bundles of sterile hyphae

Peridium

Rachis Fruiting B bodies Pseudoparenchymatous mass

C

Spores

Peridium Spores

Bundle of sterile hyphae

Sporogenous hyphae

D

Epidermis Mycelium Sclerenchyma

Spores Sporogenous hypha

A

Fig. 20.21

E

F

H

G

Graphiola phoenicis on Phoenix sylvestris. A-B, Fruiting bodies on pinna and rachis; C, A single fruiting body; D, Vertical section of the leaf of Phoenix sylvestris through a mature fruiting body; E, A few sporogenous hyphae; F, A single sporogenous hypha; G-H, Mature sterile bundles (all after O.P. Sharma, 1967).

thickness) in each fruiting body (Fig. 20.21 C, D, G, H). Each sterile bundle is made up of parallel mycelial threads which come out of the fruiting body. Bundles of sterile hyphae help in dispersal of sporidia. Graphiola has been placed variously among Myxomycetes, Pyrenomycetes, Discomycetes, Uredinales, Ustilaginales and Hyphomycetes during the past 130 years (see Fischer, 1883, 1920, 1922; Killian 1924; Sharma, 1967; Cole, 1983). Oberwinkler et al. (1982) placed it under a separate order Graphiolales. But the author feels that it should be placed in family Graphiolaceae under Ustilaginales. The supporting evidences of its inclusion under Ustilaginales, and that too also under a separate family Graphiolaceae (Sharma, 1967, 1974, 1975, 1987; and Shiam and Sharma,1975), include: 1. Smut-like external appearance, 2. Mode of teliospore germination, 3. Resemblance of bundles of sterile hyphae with the sterile bundles of Farysia javanica, 4. Mode of the attachment of sporidia on sterile bundles similar to that of Farysia javanica, 5. Budding of the sporidia from teliospores as in Ustilago longissima, and 6. Resemblance of diploid teliospores with that of other Ustilaginales. Kirk et al. (2001) also treated Graphiola under a separate family Graphiolaceae but under order Exobasidiales (= Graphiolales) of Ustilaginomycetes.

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TEST YOUR UNDERSTANDING 1. Teliomycetes include: (a) only rusts (b) only smuts (c) both rusts and smuts (d) neither rusts nor smuts. 2. Write any seven general characteristics of order Uredinales. 3. Write one major difference between microcyclic and macrocyclic rusts. 4. What is missing in the series? Spermatia, Aeciospores, _______, Teleutospores, Basidiospores. 5. Puccinia graminis is an autoecious rust or heteroecious rust? 6. Puccinia butleri on Launea is an autoecious or heteroecious rust? 7. The primary host of Puccinia graminis is _______ and its secondary host is _______ . 8. Write in brief the life-history details of Puccina graminis. 9. Make digram of a germinating teleutospore of Puccinia graminis labelling basidiospores, sterigmata, epibasidium and hypobasidium. 10. Draw neat and well-labelled diagrams of following stages of Puccinia graminis: (a) T.S. host through uredosorus (b) T.S. host through teleutosorus (c) V.S. barberry leaf showing a spermogonium and an aecium. 11. Write a note on control measures of rusts. 12. Describe in brief the annual recurrence of rust in India. 13. Write brief scientific notes on: (a) Uromyces (b) Ravenelia (c) Melampsora 14. What is the main difference between loose smut and covered smut? 15. Name any four species of Ustilago along with the botanical names of their hosts. 16. Draw a pictorial life-cycle of Ustilago maydis. 17. Write a scientific note on “Graphiola: an enigma to systematists”. 18. The most common host of Graphiola phoecinis is _______ .

21

C H A P

HYMENOMYCETES

T E R

21.1

WHAT ARE HYMENOMYCETES?

Hymenomycetes is the largest class of subdivision Basidiomycotina, in which basidiocarp is well-developed. Well-known mushrooms, jelly fungi, bracket fungi, toadstools, boletes, fairy clubs, tooth fungi, pore fungi, coral fungi and other similar forms are included under Hymenomycetes. The hymenium of the basidiocarp is fully exposed at maturity and consists of large number of basidia arranged in a palisade-like manner. A majority of the Hymenomycetes are saprobic, and only rarely they are parasitic. Their basidiospores are ballistospores. On the basis of the basidial structure, Hymenomycetes have been divided into the following two subclasses by Ainsworth (1973): 1. Holobasidiomycetidae: The basidium is a holobasidium, which is a single-celled structure, not divided by septa. A basidium typically bears four basidiospores at its apex. 2. Phragmobasidiomycetidae: The basidium is a phragmobasidium, which is a segmented, multicellular structure. Kirk et al. (2001) did not use the term ‘Hymenomycetes’ and treated all these fungi under various orders of Basidiomycetes. They treated “Hymenomycetidae” as synonym to subclass Agaricomycetidae under class Basidiomycetes of phylum Basidiomycota in their proposed system of classification.

21.2

HOLOBASIDIOMYCETIDAE

This subclass includes the holobasidium-containing hymenomycetous Basidiomycetes. The basidia are produced in a welldeveloped hymenium. The hymenium gets exposed during sporulation before the maturation of basidiospores. Members may be saprobes (Dacrymycetales, Aphyllophorales), facultative parasites (Tulasnellales) or even parasites (Exobasidiales) on stems, leaves, or flower buds of many Phanerogams. McNabb and Talbot (1973) divided Holobasidiomycetidae into six orders, viz. Exobasidiales, Brachybasidiales, Dacrymycetales, Tulasnellales, Agaricales and Aphyllophorales, Only Agaricales and Aphyllophorales are considered here.

21.3

AGARICALES

The general characteristics of Agaricales are as follows:

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1. Except a few parasitic species, the majority of Agaricales are saprophytic, growing on dead and decaying wood, leaves and on soil. Many members occur in mycorrhizal associations with forest trees. Mushrooms found on dung, wood and dead leaves are called ‘coprophylous’, ‘lignicolous’ and foliicolous’ respectively. Mushrooms growing on basidiocarps of other mushrooms are called ‘fungicolous’. For detailed treatment of mushroom cultivation, refer Chapter 26. 2. Many Agaricales have a disagreeable taste, but there are many edible species which are specially searched for food purposes. Edible mushrooms are famous throughout the world, not only for their nutritional value but also for their special flavours. 3. Some Agaricales are highly poisonous (Amanita phalloides and A. muscaria), whereas others (Psilocybe) produce hallucination. Some Agaricales also produce antibiotics but none of them have proved to be of clinical use. 4. The mycelium is septate, and the cells are either uninucleate or binucleate. The primary mycelium develops from a homokaryotic basidiospore. It develops into the secondary mycelium. 5. The secondary mycelium consists of binucleate cells, and in many members clamp connections are formed. The secondary mycelium transforms into the complex tissues of tertiary mycelium in the form of mushrooms or fruiting bodies. 6. Mushroom mycelium grows in all directions from a common point and forms an invisible circular colony. At the time of sporulation the fruiting bodies or sporophores develop at the periphery of the colony, and thus form a ring. Such rings of the fruiting bodies are called ‘fairy-rings’, so named because it was thought by the early people that the mushrooms growing in a circle represent the path of the dancing fairies. 7. Mycelium of many Agaricales shows the phenomenon of bioluminescence. The organic matter, on which a bioluminescent fungus (e.g. Omphalotus olearius, Armillariella mellea) is growing, glows in darkness. This glowing phenomenon is also called ‘fox-fire’. 8. In some Agaricales members the hyphae lie parallel to one another, or remain twisted together, to form thick stranded structures, called rhizomorphs (Armillariella mellea, Fig. 6.5). 9. Asexual reproduction is very rare. Only a few species show the production of thin-walled oidia (Coprinus lagopus), chlamydospores (Volvariella volvacea) or conidiospores as in some members of Aphyllophorales. 10. Sexual reproduction takes place by hyphal fusion, and results in the formation of basidia and basidiospores, present together in the form of fruiting body, called basidiocarp (Fig. 21.1 A). 11. The basidia develop in the form of hymenium on gills, or on lamellae. Pileus

Pileus Scales Gills Stipe

Annulus

Young sporophore

Stipe Rhizomorph Volva

A

Fig. 21.1

B

C

Structure of basidiocarps of some Agaricales. A, Coprinus atramentarius; B, Agaricus campestris; C, Diagrammatic representation of a basidiocarp showing volva formed after the rupturing of universal veil.

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249

12. Each basidium is unicellular and bears two, four, or eight spores (Smith, 1973). 13. The gills develop on the under-surface of the fruiting body or sporophore, usually in a radial arrangement. The gills remain exposed from the very beginning in some simple Agaricales. But in higher members the young developing gills remain covered by a partial veil. The enlargement of the pileus ruptures this veil or covering, and finally a ring or annulus (Fig. 21.1 B) is left around the stipe of the fruiting body. 14. Some Agaricales possess a universal veil, which completely encloses the young fruiting bodies, and ruptures only by the elongation of the stipe. Rupturing of this veil leaves a bag-like structure (volva) around the base of the stipe (Fig. 21.1 C). 15. Except a few homothallic species, the majority of the Agaricales are heterothallic.

21.4

EDIBLE AND POISONOUS MUSHROOMS

Mushrooms are both edible (Chang and Haves, 1978) as well as poisonous. The eating of mushrooms is called mycophagy (Gr. mykes, mushrooms, phagein, to eat). Nutritive value of mushrooms lies in their protein and vitamin contents, as well as in their delicious nature and special flavours. Mushroom cultivation has become a good business these days, and according to Hayes and Nair the annual world mushroom production was well over 300,000 metric tonnes in 1975. Agaricus bisporus, Lentinus edodus, Volvariella volvacea, Volvaria sp., Amanita vaginata, A. fulva and Boletus edulis are some edible mushrooms. However, some species of Amanita and Boletus are poisonous. Because of its highly poisonous nature, Amanita phalloides is commonly called ‘death cup’. Amanita verna, Boletus satanus and many species belonging to Galerina, Coprinus, Inocybe and Psilocybe are highly poisonous, and result first into gastrointestinal upset and then even into death, if taken in large quantity. For more details of edible and poisonous mushrooms and mushroom cultivation, refer Chapter 26.

21.5

FAIRY RINGS

More than 60 species of Basidiomycetes have so far been recorded to form some fungal rings called fairy rings. These are of frequent occurrence in grass and grassland and also in woods. Three major types of fairy rings are: 1. Those in which the development of the sporocarps has no effect on the vegetation, e.g. Lepista sordida, Chlorophyllum molybdites. 2. Those in which there is increased growth of the vegetation, e.g Calvatia cyathiformis, Disciseda subterranea. In these members, the basidiomata (or basidia producing organs) are at outer edge of the ring, e.g. Lycoperdon gemmatum. 3. Those in which the vegetation is damaged, sometimes so badly as to have an effect on its value, e.g. Agaricus tabularis, Calocybe gambosa. Usually, outer and inner rings are present in these type of fairy rings. The development of fairy rings starts from a mycelium, the growth of which is at all times on the outer edge because of the band of decaying mycelium and used–up soil within the ring of the active hyphae of the fungus. In Agaricus tabularis the mean growth of a fairy ring is approximately 12 cm in radius every year. In Calvatia cyathiformis it is about 24 cm. Kirk et al. (2001) mentioned that from these measurement details of fairy rings “the ages of rings of these two fungi, 60 and more than 200 m diameter, were thought to be 250 and 420 years; parts of Agaricus tabularis rings were possibly 600 years old”.

21.6

CLASSIFICATION OF AGARICALES

Smith (1973) divided Agaricales into 16 families, whereas Singer (1983) divided them into three suborders (Agaricaneae, Boletineae and Russulineae).

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Kirk et al. (2001) treated Agaricales under subclass Agaricomycetidae of class Basidiomycetes under phylum Basidiomycota. They discussed 26 families, 347 genera and 9387 species under order Agaricales. Only Agaricaceae is considered here in some details.

21.7

AGARICACEAE

1. The stipe and pileus are clearly separated. 2. The cuticle of the pileus is fibrillose or squamulose. 3. Lamellae are free. 4. An annulus is typically present on the stipe. 5. The spore deposits are blackish or dark chocolate-brown. 6. An apical germ pore is absent in spores. Kirk et al. (2001) discussed fungi having basidiocarps with velar structures under family Agaricaceae. According to them, Agaricaceae includes 51 genera and 918 species.

21.8

AGARICUS

21.8.1 Systematic Position

According to Ainsworth (1973) Division Subdivision Class Subclass Order Family Genus

21.8.2

– – – – – – –

Eumycota Basidiomycotina Hymenomycetes Holobasidiomycetidae Agaricales Agaricaceae Agaricus

According to Kirk et al. (2001) Kingdom Phylum Class Subclass Order Family Genus

– – – – – – –

Fungi Basidiomycota Basidiomycetes Agaricomycetidae Agaricales Agaricaceae Agaricus

Occurrence

It is a saprophytic fungus, which was known with the generic name Psalliota in the earlier literature. It occurs in the fields, lawns, wood logs, manure piles, and other similar surroundings, commonly in rainy season. Agaricus campestris is one of the most commonly occurring field mushrooms. It is fairly common in pastures and meadows where horses have been kept. It thrives best on soils with a high organic content. Agaricus campestris, A. brunnescens (=A.bisporus), A.rodmani and larger specimens of many other species are edible and cultivated for commerce. Solan in Himachal Pradesh is the main cultivation centre of this mushroom in India. A. xanthodermus is a poisonous species (Webster, 1980), whereas A.placomyces and A. silvaticus may cause gastrointestinal disturbances in some persons (Alexopoulos and Mims, 1979).

21.8.3

Mycelium

What we usually see in the form of a mushroom (Fig. 21.3) is not actually the whole fungus. It is simply the fruiting body, and its vegetative part is in the form of inconspicuous subterranean mycelium.

251

Hymenomycetes

Secondary mycelium

Hyphal knots C Primary mycelia + B

D

– B

Basidiospores + A

Button stage

– A E

Pileus Basidium

Gill chamber

M

Stipe

Basidiospores

F Gill chamber

Basidium

Pileus Gills

L Sterigma Haploid nuclei Basidium

K

Velum Stipe

Zygotic nucleus

Annulus Pileus

J

Gills Binucleate basidium

I

G

Stipe

Hyphal strands

H

Fig. 21.2

A–M, Life-cycle of Agaricus campestris (A–D, I–M, after Miller).

The primary mycelium in A.campestris is septate, short-lived, and develops from the germination of a basidiospore. The nucleus of the haploid, uninucleate basidiospore undergoes an ordinary division before discharge (Miller, 1984). Mature and discharged basidiospores are therefore binucleate (Fig. 21.2 A). They germinate into multicellular primary mycelia, whose each cell contains many nuclei (Fig. 21.2 B). According to some workers, however, the basidiospores are uninucleate, and germinate into primary mycelia having only uninucleate cells. Fusion of two primary mycelia of opposite strains gives rise to a secondary mycelium (Fig. 21.2 C). It consists of branched septate hyphae, where cells are multinucleate. According to Miller (1984), no clamp connections are formed in A. campestris. The secondary mycelium contains dolipore septa. The hyphae of the secondary mycelium twist together to

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form white hyphal cords, called rhizomorphs. The hyphae actually develop in circular rings (fairy-rings), from the periphery of which arise the fruiting bodies. The secondary mycelium perennates in the substratum or soil for many years. It develops into tertiary mycelium, made up of complex tissues of the fruiting body.

21.8.4

Asexual Reproduction

Except the formation of smooth or verrucose thick-walled chlamydospores by only a few species, the asexual reproduction in Agaricus is absent.

21.8.5

Sexual Reproduction

Definite sex organs are absent. A majority of the species are heterothallic. The fusion between the two somatic hyphae of opposite strains (somatogamy) brings about diploidization and formation of secondary mycelium (Fig. 21.2 C). Diploidization may also be achieved by the fusion between an oidium and a cell of the primary mycelium. From the underground rhizomorphous secondary mycelium (Fig. 21.2 C) develop some white hyphal knots (Fig. 21.2 D) or swellings. These swellings are simply the aggregations of the mycelium. Rhizomorphs are visible at this stage as white hyphal strands. Each hyphal knot consists of pseudoparenchymatous mass of hyphae. This hyphal swelling enlarges, becomes round or ovoid in shape and represents the ‘button-stage’ (Fig. 21.2 E). The slightly mature button stage is differentiated into a stalk region or stipe and a hemispherical upper part, called pileus or cap. Some of the hyphae, at the junction of the stipe and pileus (Fig. 21.2 F), are drawn apart to form a ring-like cavity or chamber, called gill chamber (Fig. 21.2 F). Some thin plates of the tissue develop from the roof of the gill chamber and radiate outward from the stipe. These thin plates are called gills. A membrane, called velum or inner veil, connects the margin of the pileus with the stipe (Fig. 21.2 G). The stipe elongates and the button enlarges in size. In comparison with the lower portion, the growth is more rapid in the upper portion of the button. Such a growth opens the young fruiting body in the form of an umbrella-shaped cup (Fig. 21.2 G, H) and also ruptures the velum or inner veil (Fig. 21.2 H). The rupturing of the velum exposes the gills. At this stage some remnants of the velum remain attached to the stipe in the form of a ring, called annulus (Fig. 21.2 H). Annulus

A mature basidiocarp is an umbrella-shaped structure, having a long, massive stalk or stipe and a broad cap or pileus (Fig. 21.3). The stipe is thick, fleshy and a whitish-pink and hollow structure. On the upper part of the stipe is present a membranous structure called annulus. The upper, convex surface of the umbrella-shaped pileus is white or cream coloured. Numerous pendant gills or lamellae hang down from the underside of the pileus. The gills at maturity are dark brown or purplish black structures. All gills are not of the same length. They may be long, of half-length, and of quarter-length in a mature fruiting body. On both the sides of the gill surface is present a fertile layer of hymenium. The basidia and the basidiospores are produced in the hymenium. The entire fruiting body consists of pseudoparenchymatous mass of hyphae. The stipe is hollow from the centre (Fig. 21.4 A). Its central region or medulla consists of loosely arranged hyphae, whereas the peripheral region is made up of compactly arranged hyphae, forming pseudoparenchymatous tissue. Pileus also shows the differentiation of outer cortex and central medulla. Some of the hyphae of pileus develop into gills. Anatomically, a gill consists of three different regions, viz. ‘trama’, ‘subhymenium’ and ‘hymenium’ (Fig. 21.4 B-D).

Pileus Gills

Stipe

Fig. 21.3

A mature fruiting body of Agaricus campestris.

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Hymenomycetes

Pileus

X

Pileus Gills

Veil

Y Stipe A

Quater length gill Half length gill Full length gill

B Trama

Hymenium Subhymenium Hymenium

Pileus

Hymenium

Stipe C

D

Basidiospores Sterigma

Basidium

Paraphysis

Hymenium

Subhymenium

Trama

E

Fig. 21.4

Agaricus campestris showing anatomy of the basidiocarp. A, V.S. of the basidiocarp; B, V.S. of the pileus along the axis ‘x-y’ in A; C, Cross-section of the pileus; D, Section through a gill; E, T.S. of a lamella, showing basidia at various stages of development.

Trama is the central part of the gill. It consists of many loosely arranged (Fig. 21.4 C, D), interwoven hyphae, which are the extensions of the hyphae of the pileus. The cells of the trama are multinucleate. Subhymenium is the middle region of the gill, situated between trama and hymenium. Its cells are isodiametric (Fig. 21.4 D), and each contains 2-3 nuclei. According to Miller (1984) subhymenial cells are binucleate. Hymenium (Fig. 21.4 C, D) is the outermost fertile region of the gill. It is made up of many club-shaped binucleate cells (Miller, 1984), forming a palisade-like layer. All cells of the hymenial region may develop into fertile cells, called basidia. But in many cases some hymenial cells develop into basidia and the others into sterile paraphyses. Sometimes the sterile cells of the hymenium become more large and protrude beyond the other hymenial cells. These enlarged sterile cells are called ‘cystidia’.

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In A.campestris it follows the typical pattern, as in other Basidiomycotina. Basidia are unicellular and binucleate (Fig. 21.2 I; Miller, 1984). Its two nuclei fuse to form a diploid zygotic nucleus (Fig. 21.2 J), which undergoes meiosis to form four haploid nuclei (Fig. 21.2 K). Segregation of sex strains takes place at this stage. Two of these four haploid nuclei are of plus strain and the remaining two are of minus strain. At the top of the basidium develop four peg-like outgrowths, called sterigmata (Fig. 21.2 K). The tips of these sterigmata swell, and the haploid nuclei migrate into these swellings. These uninucleate swellings at the tip of the sterigmata develop into uninucleate basidiospores (Fig. 21.2 L). The nucleus of each basidiospore undergoes a mitotic division before discharge. Thus each basidiospore becomes binucleate (Fig. 21.2 M). Electron microscopy of the spore walls of Agaricus bisporus and A. campestris (Garcia et al., 1979) revealed that the wall is bilayered structure made up of polymers of chitin (N-acetylglucosamine) and chitosan (glucosamine) associated with proteins, lipids, melanin and a low content of b-glucan. Mature basidiospores are discharged explosively, as in many other Basidiomycetes. At the juncture of the basidiospore and a sterigma develops a small lateral projection called hilum. A few seconds before the spore discharge, a drop of liquid appears at the hilum. This drop increases in size and reaches up to about one-fifth of the size of the spore. The basidiospores are then suddenly shot away from the sterigmata, carrying the drop of liquid with them. A mature basidiocarp of A. campestris may produce as many as 1.8 billion basidiospores each year (Hawker, 1966). On falling over a suitable substratum a discharged basidiospore germinates into a primary mycelium of either plus strain or of minus strain.

21.9

APHYLLOPHORALES (= POLYPORALES)

21.9.1 General Characteristics The general characteristics of Aphyllophorales are mentioned below: 1. Members are parasitic (Stereum hirsutum and Sparassis redicata) as well as saprophytic. The saprophytic species occur on rotting wood and cause considerable damage to the stored timber. 2. The so-called ‘bracket fungi’, ‘pore-fungi’, ‘tooth fungi’ and ‘coral fungi’ are all included under Aphyllophorales. 3. The mycelium is septate and well-branched. Many Aphyllophorales have dolipore septa. 4. Many members show clamp connections. 5. Asexual reproduction is by means of conidia or oidia. The conidia develop on spherical conidiophores. The oidia are thick-walled, spherical spores, which develop directly from the mycelium. 6. Sexual reproduction takes place by the fusion of compatible nuclei in a basidium, followed by the formation of basidiospores. 7. The basidia are unicellular (holobasidia), club-shaped and develop in well-defined hymenia. 8. The hymenium is exposed while the spores are still immature. Such basidiocarps are called gymnocarpous. The hymenium is either unilateral, i.e. develops on one side of the sporophore, or amphigenous, i.e. develops all over the surface of the sporophore. 9. The basidiocarps with porous or lamellate hymenial layer appear papery, woody or leathery. The hymenial layer may also be toothed, warted, ridged, or even smooth. The porous forms are called ‘polypores’, and provide the name ‘Polyporales’ to the order. 10. The basidiocarps of some species may be like funnels, clubs, coral-like or even umbrella-like. They may be stalked or sessile. 11. The basidiospores are forcibly discharged.

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255

12. Differing from the Agaricales, the basidiocarps in Aphyllophorales are never soft. Mature basidiocarps are very hard structures. Also they differ from Agaricales in that their developing hymenophores are not surrounded by any type of veil. 13. The nuclear spindles in the basidia are longitudinally oriented, i.e. ‘stichobasidial. 14. A majority of the Aphyllophorales investigated so far are heterothallic.

21.9.2 Classification This large group includes members with great heterogeneity in their characters, and because of this, different workers have proposed different system of classification. Talbot (1973) divided Aphyllophorales into 23 families. Instead of Aphyllophorales, Kirk et al. (2001) used the term Polyporales and placed this order under subclass Agaricomycetidae of class Basidiomycetes. Under Polyporales, they included 23 families, 298 genera and 2253 species. Some details of only Polyporaceae are considered here.

21.10

POLYPORACEAE

1. Members are lignicolous, terrestrial, humicolous, and sometimes parasitic. 2. The basidiocarps are hard, tough, leathery and usually bracket-shaped, and hence the name “bracket-fungi” is given. 3. The basidia line the inner surface of tubes or pores, and hence the name ‘Polyporaceae’ is given. 4. Fruiting bodies are effused, reflexed, and never Clavarioid. 5. The hyphae are hyaline to brown, and with or without clamps. 6. Spores are hyaline or cream-coloured, and only rarely they are ornamented. 7. Basidia are two-or four-spored, clavate or suburniform. Pegler (1973) described 121 genera under Polyporaceae. Kirk et al. (2001) includes 71 genera and 681 species under Polyporaceae. Only Polyporus is considered here.

21.11

POLYPORUS

21.11.1 Systematic Position

According to Ainsworth (1973)

According to Kirk et al. (2001)

Division – Eumycota Kingdom – Fungi Subdivision – Basidiomycotina Phylum – Basidiomycota Class – Hymenomycetes Class – Basidiomycetes Subclass – Holobasidiomycetidae Subclass – Agaricomycetidae Order – Aphyllophorales Order – Polyporales Family – Polyporaceae Family – Polyporaceae Genus – Polyporus Genus – Polyporus Polyporus is represented by about 50 species (Pegler, 1973), distributed throughout the world. It occurs as a common saprobe or parasite on roots, tree trunks and timber plants such as oak, Dalbergia and Albizzia. Polyporus sulphureus, commonly called sulphur mushroom, causes wood rot of oak and other trees, whereas P. squamosus causes heart rot of Ulmus. P. versicolor is a wood rotter of many woody plants, whereas P. bitulinus causes the heart rot of birch and some other coniferous trees. P. frondosus and P. sulphureus are edible species (Miller, 1972) . Because of their attractive reddish-brown colour, the fruiting bodies of P.lucidus are used as good attractive decorative pieces in the houses.

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Fungi and Allied Microbes

The mycelium is subterranean, and composed of many white, slender, branched and septate hyphae. The primary mycelium, developed by the germination of basidiospore, contains uninucleate cells. Hyphal fusion results in dikaryotization, and the cells of the dikaryotic mycelium are binucleate. The lignified walls of the host wood cells are digested by the enzymes secreted by the mycelial hyphae.

21.11.2

Basidiocarp, Basidia and Basidiospores

The basidiocarps (Fig. 21.5) are thick, hard, dense knots which develop from the dikaryotic mycelium. Young knots soon get differentiated into a short stalk or stipe and rounded cap-like pileus. The basidiocarps in Polyporales consist of three different types of hyphae: (i) generative hyFig. 21.5 Basidiocarp of Polypophae, which are thin-walled, contain dense cytoplasmic contents and derus sulphureus. velop clamp connections; (ii) skeletal hyphae, which are thick-walled and unbranched; and (iii) binding hyphae, which are thick-walled but Basidiospores Pore Basidia branched. In Polyporus adustus the basidiocarps consist of only generative hyphae, and such fruiting bodies are called ‘monomitic’. In P. sulphureus the generative hyphae are combined with the binding hyphae, and such fruiting bodies with generative and binding or skeletal hyphae are called ‘dimitic’. In P. versicolor the basidiocarps are made up of all the three types of hyphae, i.e. generative, binding as well as skeletal. Such fruiting bodies are called ‘trimitic’. Each basidiocarp is whitish or slightly brownish, and consists of context, trama, pores and hymenium. Outer thick-walled hyphae form the context. Trama is made up of loosely arranged, wellbranched, sepatate hyphae. Pores or tubes extend from below the context to the lower surface. Because of the presence of many pores, the name Polyporus is given to the genus. The hymenium consists of layer of basidia lining the pores (Fig. 21.6). Each basidium is a club-shaped body projecting into the cavity of the pore. Four sterigmata develop on each basidium. At the tip of each sterigma develops a uninucleate and oval basidiospore. Large number of basidiospores are discharged in the pores. On germination, each Fig. 21.6 A part of basidiocarp of Polyporus sulphureus in section. basidiospore gives rise to monokaryotic primary mycelium. Macfarlane et al. (1978) reported ‘giant sclerotia’ of Polyporus mylittae. Unusually large sclerotia are alveolate bodies, each consisting of a rind, white strata and translucent material.

Hymenomycetes

TEST YOUR UNDERSTANDING 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

13.

What are Hymenomycetes? Explain in about fifty words. Whether Kirk et al. (2001) recognized Hymenomycetes in 9th edition of their Dictionary of Fungi? Ainsworth (1973) divided Hymenomycetes into two subclasses, viz. Holobasidiomycetidae and _______ . Write any five general characteristics of order Agaricales. Define the terms: (a) Mycophagy, (b) Fox-fire, (c) Coprophilous. Write a note on edible and poisonous mushrooms in about 100 words. Describe briefly “fairy rings” in Basidiomycetes. Write only one sentence on each of the following: Volva, Stipe, Gills, Annulus, Pileus Being highly poisonous, Amanita phalloides is commonly called “_______ cup”. Draw a pictorial life-cycle of Agaricus campestris. Draw T.S. of lamella of Agaricus campestris and explain the terms trama, subhymenium and hymenium. Which of the following belong to order Aphyllophorales (= Polyporales) ? (a) Bracket fungi (b) Pore fungi (c) Tooth fungi and coral fungi (d) all of these Explain briefly the basidiocarp of Polyporus.

257

22

C H A P

GASTEROMYCETES

T E R

22.1

WHAT ARE GASTEROMYCETES?

The Gasteromycetes is a class of subdivision Basidiomycotina (Ainsworth, 1973), which includes the fungi having typical angiocarpous type of basidiocarps and holobasidial type of basidia. The basidiospores of Gasteromycetes are not ballistospores. The fungi commonly called puffballs, stinkhorns, bird’s-nest fungi and earthstars, are all included under Gasteromycetes. Kirk et al. (2001) stated that Gasteromycetes is the “morphological category of Basidiomycota, traditionally based on Homobasidiomycetes that do not actively discharge their spores”. In these fungi, the “basidiospores are not forcibly discharged and sterigmata may be absent. The basidia and basidiospores mature within the basidioma, which typically has a peridium covering a fleshy mycelial tissue or gleba”

22.2

GENERAL CHARACTERISTICS

The general characteristics of Gasteromycetes are undermentioned: 1. Almost all members are saprophytic and occur on dung, rotting wood, soil and other decaying substrata. Many members form mycorrhizal associations, e.g. Scleroderma. Limnoperdon (Escobar et al., 1976) and Nia vibrissa are aquatic members, whereas Rhizopogon forms underground fruiting bodies. 2. Mycelium is well-developed, septate, and may or may not form clamp connections. 3. Dolipore septa are present commonly. 4. All members show the production of holobasidia. 5. Basidiocarp development is angiocarpous, i.e. the basidia, at least for the early part of their development, are enclosed within the fruiting body. 6. A distinct outer wall (peridium) encloses the fruiting bodies. 7. The peridium is either single or many layered, and encloses the fertile portion of the basidiocarp. 8. The fertile portion is called gleba or spore mass. 9. The basidiospores are positioned symmetrically on the sterigmata. Dring (1973) called Gasteromycetous basidiospores as statismospores. 10. The basidiospores are not discharged forcibly from the basidium. 11. The hylar appendages are absent. 12. In a majority of the members the basidia open into cavities within a basidiocarp.

259

Gasteromycetes

22.3

CLASSIFICATION

Dring (1973) divided Gasteromycetes into nine orders (Podaxales, Phallales, Hymenogastrales, Lycoperdales, Gautieriales, Tulostomatales, Nidulariales, Melanogastrales and Sclerodermatales), of which only Lycoperdales is discussed here in some details.

22.4

LYCOPERDALES

1. The mature fruiting bodies are usually sessile and epiterranean. However, young fruiting bodies are first hypoterranean, and later on they come out of the ground. 2. The peridium is bilayered, of which the outer one is called exoperidium and the inner one endoperidium. 3. Mature gleba are powdery and have glebal membranes (Kreisel and Dring, 1967). 4. In some cases pseudocolumella-type of central tuft develops by the union of glebal membranes and capillitium. 5. The basidiospores are globose, warted or spiny, and occasionally reticulate or smooth. The diameter of the basidiospores is well below 10 mm (Dring, 1973). Dring (1973) classified Lycoperdales into 4 families (Arachniaceae, Mesophelliaceae, Geastraceae and Lycoperdaceae), of which only Lycoperdaceae is considered here. Kirk et al. (2001) treated Lycoperdales as equivalent to “Agaricales” (Lycoperdales = Agaricales)

22.5

LYCOPERDACEAE

1. It includes the common puffballs, some of which are edible, e.g. Calvatia gigantea (Miller, 1972). 2. Exoperidium is short lived and uni-layered. 3. Dehiscence of spore sac takes place by apical pore. 4. Gleba contains the capillitium, which is branched. 5. Usually the length of the sterigmata is different. Kirk et al. (2001) treated Lycoperdaceae under order Agaricales and mentioned that this family includes 18 genera and 158 species.

22.6 22.6.1

LYCOPERDON Systematic Position

According to Ainsworth (1973) Division Subdivision Class Order Family Genus

– – – – – –

Eumycota Basidiomycotina Gasteromycetes Lycoperdales Lycoperdaceae Lycoperdon

According to Kirk et al. (2001) Kingdom Phylum Class Order Family Genus

– – – – – –

Fungi Basidiomycota Basidiomycetes Agaricales Lycoperdaceae Lycoperdon

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Lycoperdon is represented by about 50 species (Kirk et al., 2001). It includes some of the common ‘ puffballs’, which occur saprophytically on decaying vegetables, humus soil, decaying wood, etc.

22.6.2

Mycelium

The primary mycelium with uninucleate cells develops from the germination of basidiospore. It develops into secondary mycelium, but not much is known about the mating behaviour in Lycoperdon (Webster, 1980). The cells of the secondary mycelium are binucleate, but no clamp connections are formed. Underground mycelial part is present in the form of rhizomorphs or mycelial cords. The rhizomorphs are made up of an outer cortex of loose hyphae, subcortex of compact hyphae and a central core of parallel hyphae. From the underground rhizomorphs come out the epiterranean fruiting bodies or basidiocarps.

22.6.3

Basidiocarp, Basidia and Basidiospores

A mature basidiocarp is a bell-shaped (Fig. 22.1 A), globose or oval structure of 1-8 cm in diameter. It is made up of a basal sterile portion and an apical fertile portion. In a longitudinal section the fruiting body remains surrounded by a double-layered peridium. Hymenium

Endoperidium

Exoperidium

B

Peridium Gleba (fertile part)

Sterigma Basidiospore Columella

Subgleba

Mycelial cord Capillitium thread A

Fig. 22.1

C

Lycoperdon pyriforme. A, L.S. basidiocarp; B, A part of the peridium and gleba; C, A part of the gleba, showing capillitium thread, basidia and basidiospores.

Gasteromycetes

261

The outer layer (exoperidium) is protective and develops some radial cracks on maturity. The inner layer (endoperidium) is dry, leathery, made up of both thin-walled and thick-walled hyphae, and remains unbroken (Fig. 22.1 B). The peridium opens by one or more pores at the apex. The tissue within the peridium is called gleba. The gleba in Lycoperdon is of ‘lacunose-type’ in which many small cavities, each lined with a hymenium, develop within the gleba. The basal, non-sporal region of the gleba is called subgleba. The subgleba extends into the fertile sporal region in the form of columella. Many glebal cavities are present in the glebal tissue, which is a sponge-like structure. The glebal cavities in the fertile portion of the fruiting body are lined by the hymenium. Two adjacent hymenial chambers remain separated from each other by thick-walled and thin-walled hyphae (Fig. 22.1 C). In the mature fruiting bodies these thick-walled hyphae persist in the form of capillitium threads (Fig. 22.1 C). The hymenium in each glebal cavity consists of round or oval basidia. Each basidium is a binucleate body. Its two nuclei fuse to form a diploid zygotic nucleus, which undergoes meiosis to form four haploid nuclei. Each basidium usually contains four basidiospores arranged symmetrically on the sterigmata. However, some believe that there also develop only two or three sterigmata and basidiospores. The sterigmata of a basidium are of different length. At maturity the glebal tissue dries and cracks, and the dusty mass of the basidiospores is left inside the basidiocarp. The basidiospores are therefore not discharged violently from the sterigmata. The basidiospores are uninucleate structures. They are discharged in the form of small spore clouds from the fruiting bodies by large drops of rain. Each basidiospore germinates to form primary mycelium with short uninucleate cells. Basidiospores usually germinate only after they have been alternately moistened and dried several times.

TEST YOUR UNDERSTANDING 1. Gasteromycetes include the fungi commonly called: (a) Puffballs (b) stinkhorns (c) earthstars and bird’s nest fungi (d) all of these 2. What is gleba? Write only one sentence. 3. Give an illustrated account of basidiocarp, basidia and basidiospores of Lycoperdon.

23

C H A P T

ANAMORPHIC FUNGI (DEUTEROMYCOTINA OR DEUTEROMYCETES)

E R

23.1

ANAMORPHIC FUNGI AND THEIR GENERAL CHARACTERISTICS

Anamorphic fungi are the fungi which have variously been named as Deuteromycotina, Deuteromycetes, Fungi Imperfecti, Asexual Fungi, Conidial Fungi or Mitosporic Fungi (Kirk et al., 2001). Some of their chief characteristics listed by Kirk et al. (2001) are mentioned below: 1. These are the “fungi that are disseminated by propagules not formed from the cells where meiosis has occured.” 2. Majority of the propagules of these fungi are conidia. 3. Several of the anamorphic fungi “are correlated with fungal states that produce spores derived from cells where meiosis has, or is assumed to have occured”. 4. Majority of these fungi are members of Ascomycetes or Basidiomycetes. However, in several cases, “these are still undescribed, unrecognized, unconnected or poorly known”. 5. Sexuality in some of these fungi is appeared to have lost, and its functions are sometimes replaced by mechanisms such as parasexual cycle. 6. Using DNA sequencing makes it now possible to place these remaining fungi with the groups of teleomorphic fungi from which they are or once were derived. Teleomorph represents for sexual or “perfect” form or morph, e.g. that characterized by ascomata or basidiomata. On the other hand, anamorph represents for the asexual or “imperfect” form or morph, e.g. that characterized only by presence or absence of conidia. Subdivision Deuteromycotina, as proposed by Ainsworth (1973), thus includes the fungi in which the ‘perfect stage’ (zygote, ascus or basidium) is either lacking or has not been discovered so far. These are therefore the fungi in which zygotes, or ascospores, or basidiospores, are not formed at any known stage in the development of the fungus. Because of the apparent absence of any perfect stage or sexual phase, these fungi are commonly called ‘imperfect fungi’ or technically ‘Fungi Imperfecti’. By the ‘perfect stage’ we mean the sexual stage. Therefore, the characteristic feature of Deuteromycotina or anamorphic fungi is the absence of sexual reproduction. The members reproduce only by asexual methods, and that too also chiefly by conidia, which develop on conidiophores. Subdivision Deuteromycotina may therefore be said to be a purely artificial and temporary assemblage of fungal species, waiting to be included under either the Mastigomycotina, Zygomycotina, Ascomycotina, or Basidiomycotina, after discovery of their perfect stage. Because of the absence of aseptate mycelia characteristic of Mastigomycotina and

263

Anamorphic Fungi (Deuteromycotina or Deuteromycetes)

Zygomycotina, lack of clamp connections and dolipore septa found in Basidiomycotina, and presence of close similarity between the conidial stages of these members and those of Ascomycotina, Deuteromycotina or anamorphic fungi are thought by many mycologists to belong to Ascomycotina. Sutton (1973) defined Deuteromycotina as ‘an assemblage of fungi typically reproducing by spores which are formed without nuclear fusion, followed by meiosis’. Alexopoulos and Mims (1979), however, opined that Deuteromycotina are the conidial stages of Ascomycotina, or more rarely of Basidiomycotina, whose sexual stages have either not been discovered or do not exist at all. Some other characterisites of these members are given below: 1. Majority of Deuteromycotina are terrestrial. There is, however, a long list of aquatic Fungi Imperfecti (Alatospora, Tricladium, Pyricularia) occurring in both marine as well as freshwater habitats. Most of the members are either saprobes or weak parasites, causing a number of diseases of plants as well as animals. 2. Except the unicellular yeast-like members of Blastomycetes, almost all the remaining anamorphic fungi or Deuteromycotina have a true mycelium, consisting of well-developed, well-branched, septate hyphae. 3. The mycelium is usually intercellular or intracellular, and each of its cell contains many nuclei. 4. The septa of all the species investigated so far resemble largely that of Ascomycotina. A simple central pore is present in each septum. 5. Sexual reproduction is absent. 6. Reproduction takes place chiefly by special asexual spores, called conidia. The conidia are non-motile structures which develop exogenously on the conidiophores, and therefore in this regard Deuteromycotina resemble Ascomycotina. The conidia are hyaline or variously coloured, unicellular or multicellular, and transversely septate or contain both transverse as well as longitudinal septa. They may be oval, elongated, spherical, star-shaped, curved, thread-like, disc-shaped, coiled, and of other shape. 7. The conidia are produced either directly on the conidiophores or in some special types of fruiting bodies such as synnemata, acervuli, sporodochia or pycnidia. These fruiting bodies are pseudoparenchymatous structures within which, or on which, conidia are produced.

23.2

TYPES OF FRUCTIFICATIONS

Following types of fruting bodies or fructifications are formed in anamorphic fungi: 1. A synnema (pl. synnemata) is a fruiting body in which the conidiophores are united at the base and become free at the top. At the top of these conidiophores develop conidia (Fig. 23.1). Usually the conidiophores are branched at the top. 2. In an acervulus (pl. acervuli) many short conidiophores arise from a cushionlike, flat mass of hyphae (Fig. 1.9 A). These conidiophores remain closely packed together, usually subepidermally, and come out as an erumpent bed-like mass of hyphae. The acervuli are neither surrounded by any hyphal wall nor do they possess any definite ostiole. A flat or saucer-shaped fruiting body has been defined as an acervulus by Barron (1968). 3. A sporodochium (pl. sporodochia) is an acervulus-like fruiting body, in which the compact mass of conidiophores develop on a cushion-like mass of hyphae or stroma (Fig. l.9 B). According to Barron (1968) a cushion-shaped fruiting body is generally referred to as a sporodochium. 4. A pycnidium (pl. pycnidia) is a globose or flask-shaped body (Fig. 1.9 C) containing many conidiophores lined on its inner side. It usually contains a mouth-

Conidia

Conidiophores

Fig. 23.1

A synnema of Arthrobotryum.

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Fungi and Allied Microbes

opening or ostiole on a papillate or rostrate apex (Sutton, 1973), but it may also be completely closed. The pycnidia of some Coelomycetes contain many ostioles. 5. In some genera the fruiting bodies start to develop as pycnidia and then open out like acervuli. Kempton (1919) called such fruiting bodies ‘pseudoacervuli’. But Poteniba (1910) used the term ‘pseudopycnidia’ for such pycnidia which only partly enclose the conidia.

23.3

PARASEXUALITY IN ANAMORPHIC FUNGI

Parasexuality is shown by some Deuteromycotina. Under this phenomenon, the processes of plasmogamy, karyogamy and haploidization take place, but not at specified time or specified points in the life-cycle of the fungus. This phenomenon was first reported by Pontecorvo and Raper (1952), and further studied extensively by Pontecorvo (1956,1958) and Davis (1966). Some of the basic aspects of this phenomenon include formation of heterokaryotic mycelium, karyogamy and multiplication of diploid nuclei, occurrence of mitotic crossing over, sorting out of the diploid nuclei, and final haploidization of some diploid nuclei in the mycelium.

23.4

CLASSIFICATION

23.4.1 Criteria for Classification The shape, size, septation, colour and ornamentation of conidia have been the major source for the classification of Deuteromycotina. Along with the morphology of the conidia, their development (thallic and blastic types of development, Kendrick, 1971), morphology and ontogeny of conidiophores, as well as their aggregation in the form of definite fruiting bodies (pycnidia, acervuli, synnemata and sporodochia) have all been considered by the fungal taxonomists while classifying these fungi.

23.4.2 Difficulties in Classification Webster (1980) mentioned the following major difficulties in the classification and nomenclature of Deuteromycotina: 1. Production of several different types of conidial apparatus by some Deuteromycotina poses a problem about the stage that should be used in naming the fungus. 2. Production of similar types of conidia by completely unrelated Fungi Imperfecti. This has actually given rise to the terminology of ‘form-genera’ and ‘form-species’, which actually mean that ‘the names are proposed without knowledge of sexual states of the taxa they represent’ (Sutton, 1973). Alexopoulos and Mims (1979) have gone to the extent of dividing the subdivision Deuteromycotina into a ‘form-class’ Deuteromycetes which includes some ‘form-subclasses’, ‘form-orders’, ‘form-families’, ‘form-genera’ and ‘form-species’. 3. No substantial information is available for providing a natural classification of these fungi (Booth, 1978; Kendrick, 1979).

23.4.3 Some Classifications Ainsworth (1973) divided sub-division Deuteromycotina into following three classes: True mycelium is absent or poorly developed; plant body is yeast-like and shows budding. True mycelium is present; budding cells are absent; mycelium is either sterile or bears spores on sporophores, which are never aggregated in pycnidia or acervuli.

Anamorphic Fungi (Deuteromycotina or Deuteromycetes)

265

True mycelium is present; budding cells are absent; spores are aggregated in pycnidia or acervuli. Alexopoulos and Mims (1979) preferred to name the three form-subclasses of Deuteromycotina as Blastomycetidae, Hyphomycetidae and Coelomycetidae. Webster (1980) discussed Deuteromycotina under three ecological groups, viz. Aquatic Fungi Imperfecti, Predacious Fungi Imperfecti, and Seed-borne Fungi Imperfecti. Sutton (1980) proposed a suprageneric classification of Deuteromycotina (Table 23.1), in which differences in conidiogenesis has been given major importance in distinguishing major taxonomic categories i.e., separation of classes into subclasses, and subclasses into orders. Orders have been grouped into suborders mainly on the basis of the type of fruiting bodies. Table 23.1

Suprageneric classification of Deuteromycotina proposed by Sutton (1980)

Division Deuteromycotina Class 1 Thallodeuteromycetes (Conidiogenesis thallic) Subclass 1. Holothallomycetidae (Conidiogenesis holothallic) Order Thallales Suborders 1. Thallohyphineae; 2. Thallopycnidiineae; 3. Thallopycnothyriineae; 4. Thallostromatineae Subclass 2. Enterothallomycetidae (Conidiogenesis enterothallic) Order Enterothallales. Suborder Enterothallineae Class 2 Blastodeuteromycetes (Conidiogenesis blastic) Subclass 1. Holoblastomycetidae (Conidiogenesis holoblastic) Order Blastales Suborders 1. Blastohyphideae; 2. Blastopycnidiineae; 3. Blastopycnothyriineae; 4. Blastostromatineae Subclass 2. Enteroblastomycetidae (Conidiogenesis enteroblastic) Order: Phialidales (phialidic ontogeny) Suborders 1. Phialohyphineae; 2. Phialopycnidiineae; 3. Phialopycnothyriineae; 4. Phialostromatineae Order Tretales (tretic ontogeny) Suborder Tretohyphineae

Kirk et al. (2001) recognized following three morphological groups of anamorphic fungi, all of which have, however, been named as “classes” in the past: These include mycelial forms which bear conidia on separate hyphae or aggregations of hyphae (e.g. synnema or sporodochia type of conidiomata) but not inside discrete conidiomata or fruiting bodies. These include the mycelial forms which are sterile, but may produce vegetative structures like chlamydospores or sclerotia. These include the forms which produce conidia in pycnidial, acervular, pycnothyrial or stromatic conidiomata or fruiting bodies.

23.5

RECOMMENDATIONS OF ICBN ABOUT NOMENCLATURE OF ANAMORPHIC FUNGI

The International Code of Botanical Nomenclature (2000), as adopted by the International Botanical Congress, recommended following for the nomenclature of anamorphic fungi:

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1. The Code provides for the “use of separate names for the different states of pleomorphic fungi”. 2. The Code, however, rules that the “name of the holomorph (the whole fungus in all its correlated states) is that of the teleomorph”. 3. The Code also recommends “that new names for anamorphs are not introduced when the teleomorphic connection is firmly established and there is no practical need for separate names”.

23.6

DELIMITATION OF TAXONOMIC ENTITY OF ANAMORPHIC FUNGI

Kirk et al. (2001) stated that to delimit or recognize a taxonomic entity for anamorphic fungi, e.g., subdivision Deuteromycotina, while convenient for practical purposes, “is meaningless in terms of natural or phylogenetic classification”. For genera of anamorphic fungi, these workers have “assigned them to the appropriate known level in the teleomorphic hierarchy”. Informally well–known groups of anamorphic genera, e.g. ‘Coelomycetes’ and ‘Hyphomycetes’ are likely to continue to be used but their adoption as formal taxa should be avoided”. For more details on the various approaches to the classification of anamorphic fungi, readers may refer “A Century of Mycology” by Sutton (1996). Only selected genera (Sporobolomyces, Candida, Cryptococcus, Alternaria, Cercospora, Curvularia, Pyricularia, Helminthosporium, Dreschslera, Fusarium, Colletotrichum, Phyllosticta, Phoma and Phomopsis) of anamorphic fungi (Deuteromycotina) along with some major characteristics of Blastomycetes, Hyphomycetes and Coelomycetes are considered here.

23.7

BLASTOMYCETES

According to Ainsworth (1973), Blastomycetes is a class of subdivision Deuteromycotina of division Eumycota, the members of which are characterized by the (i) absence or poorly–developed true mycelium, and (ii) budding cells are with or without pseudomycelium. Like other members of Deuteromycotina, Blastomycetes also have no motile zoospores, and the perfect-state spores like zygospores, ascospores and basidiospores are also absent in these fungi. Alexopoulos and Mims (1979) treated Deuteromycetes as a “form-class” of subdivision Deuteromycotina, and included three “form-subclasses” under this “form-class”. These are Blastomycetidae, Coelomycetidae and Hyphomycetidae. Class Blastomycetes (Ainsworth, 1973) or “form-class” Blastomycetidae (Alexopoulos and Mims, 1979) is actually an arbitrary taxon of asporogenous yeasts or imperfect yeasts. They exist as budding cells or pseudomycelium and do not at all produce ascospores or basidiospores. These include those fungal yeasts, which in a stage of their life cycle occur as single cells and reproduce by budding or fission. Majority of the imperfect yeasts are saprobes occurring on bark, leaves, flowers, wood and exudates of large angiospermic plants and animals. Some of these asporogenous yeasts occur in freshwater and seawater, while other cause plant diseases such a powdery mildews, rusts and smuts. Some imperfect yeasts are pathogenic to animals, including man.

23.8

SPOROBOLOMYCES

Sporobolomyces is actually anamorphic Sporidiales as mentioned by Kirk et al. (2001). It belongs to family Sporobolomycetaceae of order Sporobolomycetales, and contains 7 species (Phaff, 1970). Its species grow saprophytically on old and fallen leaves, ripened fruits, smuts and rust-infected leaves, wood pulp, etc. They are common air contaminants. It produces ballistospores in very large number, sometimes as many as 1 million/m3 in the air, and thus becomes a common

Anamorphic Fungi (Deuteromycotina or Deuteromycetes)

267

respiratory allergen according to Evans (1965). S. salmonicolor is the imperfect stage of Aessosporon salmonicolor of family Tilletiaceae of Ustilaginales of Basidiomycetes (Van der Watt, 1970). Sporobolomyces (Fig. 23.2 A-D) is an yeast-like organism made up of oval, uninucleate cells. The pigments present in these cells impart red to pink colour to the colonies of these cells. A large amount of mucilage is secreted by these cells. Sometimes the cells divide and redivide by budding to form pseudomycelium or true mycelium like structures. Reproduction takes place by budding (Fig.23.2 A-C). In the mature organisms, the cells give rise to sickle-shaped or kidney-shaped spores called ballistospores (Fig. 23.2 D). A ballistospore develops on a conical sterigma. Dispersal of ballistospores takes place by a special mechanism. At the time of dispersal, the ballistospores are shot-off violently for a distance of about 0.1 mm or even more. Muller (1954), Olive (1964) and Ingold (1971) have worked in detail on the mechanism of discharge of ballistospores in Sporobolomyces.

Ballistospore Bud Sterigma A

Fig. 23.2

23.9

Mother cell

B C

D

A–D. Sporobolomyces roseus, cells showing various stages of budding (A–C) and formation of ballistospores (D).

CANDIDA

Candida belongs to order Cryptococcales. Its nomenclature is actually comparable with anamorphic Saccharomycetales (Kirk et al. 2001). It is represented by more than 80 species, mainly differentiated on the basis of their physiological properties (Van Uden and Buckley, 1970). The genus comprises imperfect forms of ascomycetous and basidiomycetous yeasts of various genera according to Kreger-Van Rij (1973). Major diagnostic characteristics of species of Candida include that (i) these are yeast-like organisms, (ii) show mycelial or pseudomycelial growth, and (iii) exhibit multilateral budding. Candida species may easily be isolated from plant and animal materials, soil, water, and animal faeces. Some Candida species cause diseases of animals and man. C. albicans causes candidiasis which is an acute or chronic mycosis. Skin specialists can recognise various types of candidiasis, such as (i) cutaneous candidiasis, (ii) oral candidiasis, (iii) pulmonary candidiasis, (iv) bronchocandidiasis, and even (v) vulvovaginal candidiasis. Several anti-Candida drugs are available in the market but some of these drugs have several side-effects. Some of the anti-candida drugs in current use are (1) amphotericin-B, (2) fluconazole, a triazole compound, (3) allylamine terbenafine, (4) 5-fluorocytosine, a fluoropyrimidine compound, and (5) echinocandin caspofungin. All these are shown in Fig. 23.3 A-E. Candida utilis is used as an important source of proteins, fats and vitamins. According to Fell et al. (1969), the perfect state of several species of Candida belongs of genus Leucosporidium of Ustilaginales. Some believe that C.albicans is an imperfect state of Syringospora of family Tulasnellaceare of Tremellales (Van der Watt, 1969).

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Fungi and Allied Microbes

A

OH

HO

O

HOOC

OH OH

OH OH

H2N

OH

O

HO HO

OH

B N N

N

C N

H2N

Amphotericin B

H OH

F

N

O

N H O 5-Fluorocytosine

H N

OH

N H

N H

O

OO H2N

NH2

D

OH

N

O HN

O OH

HO NH O N O H HO N OH O

N

N

O

OH

E

N

F

HO

Terbinafine

Caspofungin

F Fluconazole

Fig. 23.3

23.10

A–E, Some anti-Candida drugs. A, Amphotericin–B: B, Fluconazole; C, Terbinafine; D, 5-fluorocytosine; E, Caspofungin.

CRYPTOCOCCUS

Cryptococcus is anamorphic Filobasidiella (Fig. 23.4) or Filobasidium (Kirk, et al., 2001). It belongs to order Cryptococcales and contains 17 species according to Phaff and Fell (1970). Its species occur in soil, on decaying plant materials, and also as pathogenic to man and animals. Its cells are capsulated. Cryptococcus is a non-filamentous yeast. From the economic point of view, its cells have the ability to form starch. From the pathogenic point of view, Cryptococcus may cause Cryptococcosis in the form of acute, chronic, pulmonary or meningeal mycosis, which may even infect central nervous system.

a a

Basidiospores Conidia Yeast cells

Conidiophores

Basidia

Meiosis

Bud Budding Plasmogamy

Karyogamy

Dikaryotic mycelium

Fig. 23.4

Life-cycle of Filobasidiella (Cryptococcus) neoformans.

Monokaryotic mycelium (a)

Anamorphic Fungi (Deuteromycotina or Deuteromycetes)

269

Species of Cryptococcus are imperfect states of some species of Ustilaginales. According to Kreger-van Rij (1973), C.albidus is the asexual state of the smut genus Filobasidium floriforme while C.neoformans is the asexual state of Filobasidiella neoformans. Hawksworth et al. (1983), however, reported that Cryptococcus is the asexual state of Sporopachydermia of Endomycetales. Recently, Webster and Weber (2007) discussed Cryptococcos (Filobasidiella) as heterobasidiomycete yeast, the lifecycle of which is depicted in Fig. 23.4

23.11

HYPHOMYCETES

According to Ainsworth (1973), Hyphomycetes include is a class of subdivision Deuteromycotina of division Eumycota of Fungi. Differing from Blastomycetes, members included under Hyphomycetes are characterised by the (i) presence of well-developed, septate and well-branched true mycelium, (ii) absence of budding cells, (iii) presence of either sterile mycelium or bears spores on sporophores, (iv) total absence of aggregation of spores or sporophores in the form of pycnidia or acervuli, and (v) reproduction mainly by dry or slimy conidia. Major characteristics shown by other Deuteromycotina like complete absence of zoospores and perfect state spores (e.g. zygospores, ascospores and basidiospores) are also shown by members included under class Hyphomycetes. According to Kirk et al. (2001) Hyphomycetes are anamorphic fungi including 1800 genera and 9000 species.

23.12

ALTERNARIA

According to Kirk et al. (2001) Alternaria is anamorphic Lewia. Ainsworth (1973) treated it under subdivision Deuteromycotina, class Hyphomycetes, order Moniliales and family Dematiaceae. Ascus Alternaria it is a very large, universally occurring fungus, of which many form-species occur as saprobes on dead and dying parts of the plants in the Ascospores soil. Alternaria is the most common contaminant of the laboratory cultures. Its conidia are very common in house dust as well as in the atmosphere, and are responsible for allergies (Hyde and Williams, 1946), skin diseases and some serious disorders in the human body. Many Alternaria species are parasitic on plants. On members of Solanaceae (e.g. potato) Alternaria shows symptoms of blight and occurs earlier than Phytophthora infestans, which is the causal organism of late-blight of-potato. Only because of this, Alternaria is commonly called ‘early-blight’. Early symptoms are in the form of small, yellowish brown spots on the leaves, which enlarge in size and become round to form concentric rings (Fig. 35.15). Entire lamina, petiole, stem and even tubers are badly damaged in severe infections. Edible part of the tuber turns brown. Fig. 23.5 An ascus of Pleospora Alternaria alternata (=A. tenuis) causes ‘black-point’ disease of wheat, infectoria, a Loculoascomycetous fungus that whereas A. triticina causes ‘leaf-blight’ of wheat. A. brassicae and A. brasrepresents the perfectsicicola (Fig. 23.6 H, J) infect Brassica seeds, whereas A. solani causes early stage of Alternaria. blight of potato and other members of Solanaceae. Some of the other Alternaria species (with their hosts in the parentheses) are A. citri (on roots of Citrus sp.), A. helianthi (on Helianthus annuus) and A. palandue (on Allium cepa). Mycelium consists of light brown, slender, profusely branched, septate hyphae, which are first intercellular, and later on may become intracellular. Each cell is usually multinucleate. According to Knox-Davis (1979) the vegetative cells of A. brassicicola contain 1 to many nuclei. The hyphal tip cells have up to 27 nuclei and older cells even up to 33 nuclei.

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Fungi and Allied Microbes

Conidiophore

A

Bud

B

Germ tubes

C Young conidium

Conidia D

E

Lateral bud J F

I

H

Fig. 23.6

G

A–F, Development of a conidium of Alternaria solani; G, Two conidia in chain of A. brassicae; H, A conidium of A. brassicicola showing lateral budding; I, Branching of the conidia chain in A. brassicae; J, A germinating conidium of A.brassicicola.

Alternaria reproduces only asexually by producing conidia (Plate 7A-E). Its perfect stage belongs to Pleospora infectoria (Fig. 23.5), a Loculoascomycetous fungus (Webster, 1980). Conidia develop at the tips of the conidiophores, which are short, dark-coloured and usually undifferentiated. A conidium develops as an apical bud from the uppermost cell of the conidiophore (Fig. 23.6 A-D). It does not develop by constriction and subsequent enlargement of the terminal cell of the conidiophore. The young conidium first divides by transverse septa (Fig. 23.6 E), which develop by annular ingrowths (Campbell, 1970). In the centre of each septum is present a pore that allows the conduction of cytoplasm in between cells of the conidium. Later on some of the cells divide by longitudinal septa (Fig. 23.6 F). Such conidia with transverse as well as longitudinal septa are called ‘muriform’ or ‘dictyospore’. Usually the tip cell of a conidium also shows budding, and the ultimate result is the formation of chain of conidia (Fig. 23.6 G). Occasionally, a bud may also develop from any lower cell of the conidium (Fig. 23.6 H). Such a bud develops into a conidium, resulting in the branching of the spore chain (Fig. 23.6 I). The further extension of the conidial chain is stopped by the plugging of the pore of the basal conidium. A mature conidium is a multicellular body, having transverse as well as longitudinal septa. It remains surrounded by a two-layered wall, of which the outer layer is pigmented and the inner layer hyaline. According to Knox-Davis (1979) mature conidiophores contain few (0-3) nuclei, whereas conidia contain 1-2 nuclei. Purkayastha et al. (1980) studied the surface ultrastructure (Plate 7 A-E) of 5 pathogenic Alternaria species (A. longissima, A. cassiae, A.tenuissima, A. raphani and A.sonchi).

271

Anamorphic Fungi (Deuteromycotina or Deuteromycetes)

The conidia are disseminated readily by wind. In the presence of moisture and suitable temperature, a conidium germinates by producing five to ten germ tubes (Fig. 23.6 J). Crop rotation is helpful because the disease is soil borne. Fungicidal sprays, preferably with copper fungicides or Zineb, at 15-day interval, provide satisfactory control measures. Azariah et al. (1962) advocated the use of Bordeaux mixture, whereas Mathur et al. (1971) recommended the spray of Brestan 60, Zineb and Dithane M-45.

23.13

CERCOSPORA

Like Alternaria, Cercospora is also a very large genus of form-family Dematiaceae, represented by over 2000 species (Ellis, 1971).Its nomenclature is comparable with anamorphic Mycosphaerella (Kirk et al., 2001). Cercospora causes leaf spot disease of tomato, lettuce, potato, cotton, rice, groundnut, chillies, pigeon pea (arhar), beet, tobacco, and many other crops of economic importance. C. personata causes Tikka-disease of Arachis hypogea, whereas C. gossypina causes leaf spot on Gossypium herbaceum, and C. oryzae causes leaf spot disease of rice. C. apii is a human pathogen, and may cause severe lesions on the face, making it horrible (Emmons et al., 1957). The mycelium is well-developed, branched, and consists of septate, slender, intercellular hyphae. Branched haustoria are present in C. personata. The mycelium is both internal as well as external in C. arachidicola. At the time of the formation of conidia, the hyphae accumulate and become compact to form the globular hyphal mass, called stroma (pl.stromata). The stromata develop subepidermally in the substomatal cavity of the leaf. Conidia develop on septate, dark-coloured conidiophores. A great variation exists in the dimensions of conidia and conidiophores. The conidia are long, slender, narrow, tapering and contain many transverse septa (Fig. 23.7). They develop sympodially on clustered, dark, geniculate (knee-jointed) conidiophores, which usually come out through the stomata of the host leaf. On liberation from the conidiophore, each conidium leaves a small scar at the place of its attachment. The conidia are dispersed effectively by rain splash. In suitable conditions of temperature and moisture, each conidium germinates into a new mycelium.

23.14

Conidia

Conidiophore

Fig. 23.7

Conidiophores and conidia of Cercospora beticola.

CURVULARIA

Curvularia is anamorphic Cochliobolus (Kirk et al., 2001). It is a member of family Dematiaceae of Moniliales. Represented by over 30 species, Curvularia occurs on rice (Benoit and Mathur, 1970) and many other crops, causing leaf spots, blights, grain deformation, grain discolouration and even root rot. Its perfect stages are known in the form of species of Cochliobolus,a member of Loculoascomycetes.

A

Fig. 23.8

B

A-B, Conidiophores and conidia of Curvularia lunata.

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Fungi and Allied Microbes

Conidiophores are erect, macronematous and mononematous. The conidia develop either spirally or in whorls on conidiophores (Fig. 23.8 A). The conidia are usually curved. Usually the third cell from the base of the conidium is largest (Fig. 23.8 B). A protruberant hilum is present at the base of the conidium in some species, such as C. combopogonis. Occasionally the conidiophores develop in stromata.

23.15

PYRICULARIA

Pyricularia is anamorphic Magnaporthe (Kirk et al., 2001). It is yet another member of family Dematiaceae of Moniliales (Ainsworth, 1973). P. oryzae causes serious ‘blast disease’ of rice. The fungus often completely kills the rice seedlings, and sometimes the complete rice plants. Even panicles are sometimes badly damaged affecting the ultimate yield of rice. It also infects the plants such as Eleusine coracana and Setaria italica. The mycelium is well-developed and consists of branched, septate, intercellular or intracellular hyphae. The cells are often multinucleate. The conidiophores are simple, long, slender, septate or aseptate, and usually unbranched (Fig. 23.9). The groups of the conidiophores come out through stomata. The conidia are pale-brown, obpyriform and two-septate (threecelled, Fig. 23.9). Each conidium remains attached with the conidiophore by a papilla-like hilum. The conidia are discharged when the humidity is very high, specially during nights. Probable explosion of hilum discharges the conidia violently. Massarina, a Loculoascomycetous fungus, represents the perfect state of Pyricularia aquatica, whereas the perfect stage of P.grisea is Magnaporthe grisea, a pyrenomycetous fungus.

23.16

Conidia

Conidiophores

Fig. 23.9

Conidiophores and conidia of Pyricularia oryzae.

HELMINTHOSPORIUM

Helminthosporium is anamorphic Splanchnonema (Kirk et al., 2001). According to Ainsworth (1973) it belongs to form-family Dematiaceae of Moniliales. It contains approximately 20 species. Its major distinguishing character is the production of large, transversely septate conidia or porospores, apically as well as laterally, from the determinate conidiophore (Fig. 23.10). The conidiophores are usually brown to dark brown in colour and often fasciculate and erect. Conidia develop laterally often in verticils, through pores beneath the septa, while the conidiophore tip is growing actively and conidiophore growth stops with the formation of a terminal conidium. In this way, the terminal conidium is the youngest and the last conidium developed from the conidiophore. Each conidium is pseudoseptate, bearing a dark-coloured protruding scar at the base. Shoemaker (1959) segregated majority of the Helminthosporium species into two genera (Drechslera and Bipo-

Young conidia

Mature conidia

Conidiophores

Brown scar at the base Developing poroconidia

Fig. 23.10

Conidia and conidiophores of Helminthosporium velutinum.

273

Anamorphic Fungi (Deuteromycotina or Deuteromycetes)

laris). Imperfect state of Helminthosporium is produced in Pseudocochliobolus of order Dothideales of Loculoascomycetes.

23.17

DRECHSLERA

According to Kirk et al. (2001), Drechslera is anamorphic Pyrenophora. Some of the Drechslera species, which germinate only from the end cells, were transferred to a new genus Bipolaris by Shoemaker (1962). Major characteristic of Drechslera (Fig. 23.11) include its sympodially extending conidiophore which gives rise to an acropetal succession of multiseptate and cylindric-shaped conidia (porospores). Conidia germinate from any or all cells of the conidiophore. Conidiophores are indeterminate and extend by sympodial growth in both Drechslera and Bipolaris. On the other hand, production of the apical conidia terminates the growth of the conidiophores in Helminthosporium. All the cells of a conidium are capable of germination in Drechslera, but in Bipolaris conidia germinate only by the two end cells, and hence the name Bipolaris is given to the genus. Perfect states of several species of Drechslera belong to Loculoascomycetes.

23.18

Conidia

Conidiophore

Fig. 23.11

Conidiophore and conidia of Drechslera.

FUSARIUM

Fusarium is anamorphic Gibberella and Nectria (Kirk et al., 2001). It is Vessel the largest genus of Tuberculariaceae (Ainsworth, 1973), and occurs either Hyphal saprophytically or parasitically on many crop plants, fruits and vegetables. pluggings It causes serious wilting of the host plants. The mycelium invades the vascular tissue and finally blocks the xylem vessels (Fig. 23.12). Blocking of xylem vessels adversely affects the translocation of water, leading to wilting of plants (Fig. 23.13 A). Fusarium also produces some toxic secretion in the vessels of the host, which might also be the cause of wilting. Many plants (Fig. 23.13A) are attacked by Fusarium. Some of Fig. 23.12 T.S. infected root of arhar the wilt-causing Fusarium species with the names of their hosts in pa(Cajanus cajan) showing hyphal rentheses, are F. udum (on Cajanus cajan, arhar), F. lycopersicum (on pluggings of xylem vessels by Lycopersicon esculentum, tomato), F.lini (on Linum usitatissimum, flax), the mycelium of Fusarium udum. F.solani (on Solanum tuberosum, potato) and F.orthaceras (on Cicer arietinum, gram). The mycelium consists of branched, septate, often colourless hyphae, which turn brown at maturity. The mycelium produces toxic secretion in the vessels, which ultimately blocks the latter and results into wilting of the host plant. Fusarium reproduces asexually by means of three kinds of asexual spores, viz. macroconidia, microconidia and chlamydospores. Macroconidia are long, multicellular, crescent-shaped or sickle-shaped bodies (Fig. 23.13 C) produced in sporodochia (Fig. 23.13 B). Both the ends of macroconidia are pointed. In some species the macroconidia develop separately, and not in sporodochia. The cells, on which the macroconidia develop, are called phialides. Microconidia are small, usually unicellular but sometimes bi-celled, spherical or oval bodies (Fig. 23.13 D) produced from simple phialides or from branched or unbranched conidiophores. The microconidia are often held in small masses. The microconidia of Fusarium resemble very much those of Cephalosporium, and hence this stage is often referred to as Cephalosporium-stage of the fungus.

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Fungi and Allied Microbes

Macroconidia Microconidia

Intercalary chlamydospores

C

A

macroconidia

D

Terminal chlamydospore E

Phialides

B Mycelium

Fig. 23.13

A, Fusarium udum on Cajanus cajan (arhar), showing wilting; B, A sporodochium with macroconidia; C, Macroconidia; D, Microconidia; E,Chlamydospores.

Chlamydospores (Fig. 23.13 E) are round or oval, thick-walled, terminal or intercalary cells of the old hyphae. They either develop singly or in chains. They get detached and germinate by means of germ tubes, if the conditions are favourable. The chlamydospores are very durable and remain viable for a long time.

23.19

COELOMYCETES

Coelomycetes is a class of Deuteromycotina (Ainsworth, 1973), members of which contain well-developed true mycelium and also show absence of budding cells as in Hyphomycetes. But, conidia and conidiophores in these fungi develop in acervuli or pycnidia which are absent in Hyphomycetes. Conidia are unicellular, deciduous and hyaline or pigmented. Like other members of Deuteromycotina, Coelomycetes also show absence of zoospores and also of perfect state spores like zygospores, ascospores and basidiospores. According to Kirk et al. (2001) the term “Coelomycetes” “merely indicates that conidia are formed within a cavity lined by fungal or fungal/host tissue.” Coelomycetes are parasites and saprobes of terrestrial vascular plants. Some are hyperparasites of other fungi. The pycnidia of these fungi are superficial or immersed, spherical, flattened or discoid bodies and contain a multicellular wall made up of isodiametric cells. The acervuli of Coelomycetes are immersed, and made up of a basal stroma lacking lateral and upper walls (Sutton, 1973). According to Sutton (1980) Coelomycetes “are the imperfect fungi which produce conidia in, or on specialized fructifications called pycnidia, acervuli, stromata and pycnothyria”. Majority of the Coelomycetes are the asexual states of Ascomycotina. However, a few Coelomycetes with clamp connection, have also been described thus showing their relation with Basidiomycetes. Hawksworth et al. (1983) have

Anamorphic Fungi (Deuteromycotina or Deuteromycetes)

275

described about 650 genera and 8000 species of Coelomycetes whereas Kirk et al. (2001) mentioned that Coelomycetes contain 1000 genera and 7000 species. Coelomycetes are found commonly in cultivated and uncultivated soils of various types, leaf litter and other organic debris, in saline as well as freshwaters, as hyperparasites on other fungi, and also as parasites of plants and animals both. On plants, different Coelomycetes cause lesions of stem, leaf and roots, blights, cankers, galls and anthracnoses. They also cause anther hypertrophy and dieback in several woody plants. Conidiogenous cells of various Coelomycetes are unicellular, often aggregated into complex structures or conidiophores and produce conidia. Conidia are produced mostly from phialides and annellides. These are unicellular to multiseptate, exhibit various shapes, provided with cellular or extracellular appendages, and are hyaline or pigmented. For detailed studies, readers may consult monographs on Coelomycetes by Mathur (1979) and Sutton (1980). For modern reassessments of Coelomycetes systematics, refer Coelomycetous Anamorphs with Appendage-bearing Conidia by Nag Raj (1993).

23.20

COLLETOTRICHUM

Kirk et al. (2001) mentioned Colletotrichum as anamorphic Glomerella. According to Ainsworth (1973), Colletotrichum belongs to order Melanconiales of Coelomycetes of subdivision Deuteromycotina. It is represented by 11 species (Sutton, 1973). But Alexopoulos and Mims (1979) mentioned that over 1000 form-species of this genus have been described so far, of which a majority of them appear to be synonyms. According to the estimates of Baxter et al. (1985), Colletotrichum is represented by 21 species. C. coccodes, C.dematium, C. gloeosporioides, C. graminicola, C. falcatum and C. capsici are some common species causing ‘anthracnoses.’ The disease “red rot of sugarcane” caused by Colletotrichum falcatum has been discussed in Chapter 35 (Article No. 35.16). Conidia

Seta

Conidium Setae

A

Conidiophore B

Conidia

Hyphal stroma Germ tubes Conidiophore

C

Fig. 23.14

D

A, An acervulus of Colletotrichum lindemuthianum; B, Acervulus of C. falcatum; C, Conidiophores and conidia of C. graminicola; D, Germinating conidia.

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Fungi and Allied Microbes

Mycelium is endophytic and consists of slender, branched, colourless, septate, intercellular as well as intracellular hyphae. Many oil droplets are present in each cell of the mycelium. At maturity, the hyphae become dark, and closely intertwine with one another to form small stromata under the epidermis. Colletotrichum reproduces only asexually, and that too also only by conidia. The conidia develop on conidiophores in acervulus-type of fruiting bodies (Fig. 23.14 A-C). The acervuli are saucer-shaped, flat, and black velvety structures. Each acervulus consists of a stromatic layer, from whose surface are produced simple hyaline conidiophores. The conidiophores are aseptate and give rise to many unicellular, falcate or sickle-shaped, hyaline conidia (Fig. 23.14 C). Along with the conidia and conidiophores, there are also present many setae in each acervulus (Fig. 23.14 A, B). The setae are long, stiff, pointed, unbranched and multicellular bristle-like structures. Formation of the large number of conidia ruptures the overlying host epidermis. When the conditions are favourable, each conidium germinates by producing one or more germ tubes (Fig. 23.14 D) to form mycelium. Appressoria are formed in Colletotrichum, specially in cultures (Sutton, 1968). The older mycelium sometimes shows the formation of thick-walled, dark brown, spherical or irregular-shaped spores, called chlamydospores. They are either terminal or intercalary in position, and remain viable for a long time. On being detached they also germinate into new mycelial hyphae. According to von Arx (1957) ‘sclerotia’ are also formed in some species of Colletotrichum.

23.21

PHYLLOSTICTA

Phyllosticta is anamorphic Guignardia according to Kirk et al. (2001). It belongs to family Sphaeropsidaceae of Sphaeropsidales (Ainsworth, (1973). It causes leaf spot disease of many plants. P. maydis causes leaf spot disease of corns whereas P. phaseolina causes blight of pulse crops. Species of Phyllosticta have also been reported from apples, Artocarpus and Cycas revoluta. Its mycelium is well-branched and septate. The pycnidiospores are the means of the asexual reproduction, which develop in an urn-shaped pycnidium (Fig. 23.15 A-C). The pycnidiospores are small, unicellular hyaline bodies which develop at the tips of pycnidiophores or conidiophores. The conidiophores are very small structures arising from the wall of the pycnidial cavity. A pycnidium opens with a mouth opening or ostiole. Alexopoulos and Mims (1979) believe that Phyllosticta and Phoma are similar. If the fungus occurs on leaves, it is included under the form-genus Phyllosticta. On the other hand, if it occurs on stem, it is either Phoma (conidia measure under 15 mm) or Macrophoma (conidia measure over 15 mm). However, Sutton (1973) recognizes Phyllosticta and Phoma as separate genera. He did not recognize Macrophoma as a separate genus. Pycnidiospores Pycnidium Ostiole

A

Fig. 23.15

Pycnidiophore

B

C

A–C, Phyllosticta phaseolina showing a mature pycnidium (A), pycnidiophores (B) and pycnidiospores (C).

277

Anamorphic Fungi (Deuteromycotina or Deuteromycetes)

23.22

PHOMA

About 40 species of Phoma have been reported so far. It belongs to family Sphaeropsidaceae of order Sphaeropsidales of Coelomycetes. According to Kirk et al. (2001), Phoma is anamorphic Leptosphaeria and Pleospora. Fruiting body of Phoma is a pycnidium (Fig. 23.16 A), a flask-shaped dark-coloured structure opening usually by Mature a single circular opening or ostiole. The fruitconidia Ostiole ing body remains lined inside by a hymenium, Developing made up of conidiogenous cells. Numerous Conidiogenous Pycnidium conidia cells hyaline, thin-walled, aseptate or occasionally septate conidia develop in the pycnidium. They develop by a process of monopolar repHypha ititive budding of the cells of pycnidial wall. The conidia are of various shapes (Fig. 23.16 Wall B) like cylindrical, ellipsoidal, pyriform, globose or even fusiform. A B A number of Phoma species are pathogenic Fig. 23.16 A-B, Phoma herbarum showing V.S. of a pycnidium (A) on many tropical plants. Blight of vine flowers and some magnified conidiogenous cells and conidia (B). and grapes is caused by P. glomerata, which also causes rot of potato, tomato, citrus fruits and leaf and fruit spot of apple. P.prunicola also causes leaf spot of apple. Damping-off of beet is caused by P.betae. Phoma is also known to be associated with several mycotic diseases of man. For more details of morphology and taxonomy of Phoma, readers may consult Boerema et al. (1973).

23.23

PHOMOPSIS

Phomopsis also belongs to family Sphaeropsidaceae of order Sphaeropsidales. About 100 species of this genus have so far been reported. Its pycnidium type of fruiting body (Fig. 23.17 A) produces two types of conidia i.e., alpha (a)-conidia and beta (b)-conidia (Fig. 23.17 B). Alpha conidia are straight, aseptate, fusiform and usually hyaline while beta conidia are somewhat curved or bent, aseptate and filiform. Alpha conidia are usually biguttulate while beta conidia are usually eguttulate. Some prefer to call b-conidia of Phomopsis as stylospores.

a-conidia

Ostiole

A

Fig. 23.17

b-conidia

Pycnidial wall

Conidiogenous cells

B

A-B. Phomopsis abdita, showing V.S. of a pycnidium (A) and some magnified conidiogenous cells with a-conidia and b-conidia (B).

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Fungi and Allied Microbes

TEST YOUR UNDERSTANDING 1. Anamorphic fungi are also named as: (a) Deuteromycetes or Deuteromycotina (b) Fungi Imperfecti or Asexual Fungi (c) Conidial Fungi or Mitosporic Fungi (d) all of these 2. Write any five major characteristics of Anamorphic fungi. 3. Mention one major difference between (a) Teleomorph, and (b) Anamorph. 4. Differentiate between following types of fructifications? (a) Pycnidium (b) Sporodochium (c) Acervulus (d) Synnema 5. Reproduction in Anamorphic fungi takes place chiefly by _______ . 6. Write a brief note on parasexuality in anamorphic fungi. 7. Ainsworth (1973) divided Deuteromycotina into three classes viz. Blastomycetes, Hyphomycetes and _______ . 8. Write any two major recommendations of International Code of Botanical Nomenclature (2000) regarding nomenclature of anamorphic fungi. 9. Sporobolomyces and Candida belong to: (a) Blastomycetes (b) Hyphomycetes (c) Coelomycetes (d) none of these. 10. Write detailed botanical notes on structure and reproduction of: (a) Alternaria (d) Cercospora (c) Curvularia (d) Pyricularia. 11. How will you differentiate between following: (a) Helminthosporium (b) Drechslera (c) Fusarium. 12. Phytophthora infestans causes late blight of potato. Early blight of potato is caused by _______ . 13. Write a brief scientific note on Coelomycetes in about 100 words. 14. Red rot of sugarcane is caused by _______ . 15. How will you recognise following anamorphic fungi? (a) Phomopsis (b) Phoma.

24

C H A P T

ECONOMIC IMPORTANCE OF FUNGI*

E R

24.1

INTRODUCTION

Hardly a day passes when we all are either not benefited or harmed directly or indirectly by fungi. Fungi affect us directly by destroying our food, fabrics, leather and other similar articles, by causing many common and dangerous diseases of man and animals, and by producing majority of the plant diseases. On the contrary, preparation of bread, wines, beers etc., which involve fermentation, cannot be completed without these tiny microorganisms. Use of fungi in the preparation of many antibiotics (penicillin, griseofulvin) organic acids (oxalic, citric, gluconic acids), vitamins (Vitamin B-complex, riboflavin), hormones (gibberellin), enzymes (taka diastase, digestin) and many other drugs (cortisones, ergometrine) is also widely known throughout the world. Their negative role in causing innumerable plant and human fungal diseases, and their positive role in maintaining the fertility of the soil is certainly beyond anybody’s expectations.

24.2 24.2.1

NEGATIVE ASPECTS OF FUNGI Diseases of Man

Nearly all persons of the world, on one or a number of occasions in their life, become infected by fungi. The most fungal infections are of skin, but other body parts like respiratory tract, lungs, bones, viscera, intestine, liver, kidney, nasal sinuses, corneal tissue of eye, etc., are also sometimes severly affected. Owing to the presence of abundant water, high carbohydrate level and easily available nitrogen compounds in the form of amino acids and proteins in the human body, almost all nutritional requirements of most fungi are fulfilled. And perhaps only because of the fulfilment of their nutritional requirements fungi grow easily on human body. Some species of Rhizopus and Mucor are common fungi infecting lungs, brain (Webster, 1980) and gastric tissues, whereas Neurospora and Fusarium infect corneal tissue of the eye, and Histoplasma infects lungs, spleen, liver, kidney, nervous system and also lymphatic system (Cooke, 1977). Aspergillus is a common infectant of lungs and nasal sinuses. Many fungi are skin pathogens or dermatophytes of man (Ajello, 1977). Pandey et al. (1981) reported that skin infection of penis, caused by the species of Trichophyton and Candida, is more prevalent in langota1-wearing men. (‘Langota’ is a semiocclusive undergarment put on by a large number of Indian men). Wszelaki and Kuzminska (1981), while investigating the diseases of vulva and vagina in little girls, reported mycotic vulvovaginitis in 53% of the girls studied with *

For more details refer Chapters 25-27 (Fungi and Biotechnology, Mushroom Cultivation and, Single-cell Protein).

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vulvovaginal inflammatory processes. In yet another study, Pagani and Libshitz (1981) reported fungal pneumonias in 92 cancer patients, in which different fungi were Candida (50 patients), Aspergillus (35 patients), members of Mucoraceae (4 patients), Cryptococcus (2 patients) and Histoplasma (1 patient). Aspergillosis, whose symptoms closely resemble those of tuberculosis in many men, is caused by Aspergillus niger, A. flavus and A. terreus ((Alexopoulos and Mims, 1979), whereas zygomycosis is caused by many species of Mucor and Rhizopus (Ajello, 1976). Candida albicans is the cause of various diseases in man. Various types of candidiasis of humans are skin candidiasis, bronchocandidiasis, oral candidiasis, pulmonary candidiasis and vulvovaginal candidiasis (Emmons et al., 1977). In a study of 388 female patients of vaginal candidiasis, Martin and Julio (1978) reported that 126 (32.46%) of them were infected with Candida organisms. The Candida-infected patients showed symptoms like leucorrhoea (42%), vaginal pruritus (22%), and low abdominal pain (16%). Besides the above-mentioned human fungal diseases, some other human fungal diseases along with their causal organisms are mentioned in the Table 24.1. Table 24.1 Some human fungal diseases Disease

Causal organisms

Athlete’s foot Blastomycosis Chromomycosis Coccidioidomycosis Cryptococcosis Dermatomycosis Geotrichosis Histoplasmosis Paracoccidioidomycosis Rhinoentomophthoromycosis Ringworm Sporotrichosis

Epidermophyton floccosum Ajellomyces and Emmonsiella Cladosporium carrionii Coccidioides immitis Crytpococcus neoformans Gymnoascus, Actinodendron Geotrichum candidum Ajellomyces and Emmonsiella Paracoccidioides brasiliensis Conidiobolus Gymnoascus, Myxotrichum Sporotrichum schenkii

Many broad-spectrum antibiotic compounds are available for the treatment of bacterial or viral infections but only few non-toxic antifungal compounds are known. Hence, fungal diseases are comparatively difficult to be cured in comparison to the bacterial or viral diseases. One of the successful antifungal compounds is potassium iodide and a well-proven antifungal antibiotic is amphotericin-B (Fig. 23.3 A). This antibiotic works satisfactorily because it rapidly enters the fungal cells, causes them to quickly respire and reduce their internal reserves of carbohydrates. Griseofulvin is also an effective antifungal antibiotic, specially in dermatophyses.

24.2.2 Diseases of Wild Animals and Pests As in man, the fungal diseases are very common in domestic and wild animals also. Monga and Mohapatra (1980) reported number of cases of aspergillosis, candidiasis, phycomycosis, rhinosporidiosis and mycotic abortions in a large number of animals in India. The most prevalent fungi were the species of Trichophyton and Microsporum. Microsporum canis causes common ringworm of dogs and horses, and make them unusable in houses and also unsaleable. Aspergillus fumigatus causes bovine abortion in many animals including birds, ducks and chickens. Mycotic abortions of cattle and aspergillosis in birds are also common fungal diseases of animals.

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Entomophthorales are the parasites on insects and some other animals. Most of the Entomophthora species are insect parasites (Waterhouse, 1975), and Blackwell and Rossi (1986) worked on the fungi growing on termites. They mentioned that termites are unusual among insects in that they are hosts for at least 20 species of obligate ectoparasites, the chief among them are Laboulbeniopsis, Termitaria and Antennopsis.

24.2.3 Diseases of Fishes Many fishes, moluscs and crustaceans, which form a ‘food crop’ of a large of population of the world, are also infected by fungi. Sometimes the fungal infection is so severe that the entire ‘crop’ is finished within a short time. Saprolegniales (Saprolegnia, Achlya) are the common fungal parasites of fishes. In some European countries Ichthyophonus haferi is commonly called a ‘fish-destroying fungus’. The fishes of the domestic aquaria are commonly infected by Saprolegnia ferax and S. parasitica.

24.2.4 Nematode — Destroying Fungi Many members of Deuteromycetes, Chytridiomycetes, Zygomycetes, Oomycetes and Basidiomycetes destroy the nematodes (Alexopoulos and Mims, 1979).

24.2.5 Spoilage of Food and Stored Grains In our daily routine we see that the food articles, if not properly stored, are spoiled by fungi. Exposure of bread and other articles even for a few minutes makes them infected. Common food spoiling fungi are Aspergillus, Rhizopus, Mucor and Penicillium. Some fungi infect the food even at very low temperature. Cladosporium herbaceum grows on meat stored at –6°C, and at –4°C it grows profusely (Cooke, 1977). Therefore, this notorious fungus spoils the food kept in many domestic refrigerators. Some ascomycetous and deuteromycetous fungi contaminate the stored harvested grains with their spores. The infected grains of wheat, maize, etc. may even contain the hyphae of the fungi beneath their outer layers. The hyphae require high relative humidity for their growth. Therefore, if the grains are carefully dried before storage, the hyphae cannot develop within them. The grains should therefore be dried well before their storage.

24.2.6 Poisonous Fungi Some fungi are deadly poisonous, and, if ingested, they may prove even fatal. Amanita phalloides, A. verna and Boletus satanus (Webster, 1980) are highly poisonous. Some other poisonous mushrooms include several species of Galerina, Helvetia, Coprinus, Inocybe and Psilocybe. Great care, therefore, must be taken while purchasing mushrooms for food purposes. A recognized manual should be used for determining the edibility of mushrooms. A slight carelessness may result in a very unpleasant gastrointestinal upset or even death. For more details refer to Chapter 26 (Mushroom Cultivation).

24.2.7 Diseases of Crops Almost all plants, from minute algae to giant forest trees, are attacked and destroyed by fungi. Thousands of crop diseases are known, and many more are yet to be discovered by the mycologists and plant pathologists. The common fungal diseases include smuts, rusts, mildews, blights, rots and wilts. Smuts and rusts are together responsible for destroying many crops worth several hundred crores of rupees in the world. Fungal diseases may result even into a catastrophe if allowed to run their course unchecked. Disastrous effects of Irish Potato Famine of 1845-1849, caused by Phytophthora infestans, are still known to the world. ‘By 1851 approximately one million people had died’ (Cooke, 1977) because of this famine. This is not the only example of fungal catastrophe. People are mostly still unaware of the great damages caused by fungi. A few examples are given below: 1. Tobacco yield reduced to over 60% in north Africa and Middle East in 1962 because of the infection of Peronospora tabacina.

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2. Annual coffee yield in Sri Lanka fell from 42 million kg to less than 3 million kg in 1890 because of the infection of Hemileia vastatrix. 3. Maize was so much damaged by Helminthosporium maydis in 1970 in USA that a large part of the crop was totally lost while in other cases the yields reduced by 50%. 4. Eucalyptus marginata, the famous Australian timber tree, is attacked badly by Phytophthora cinnamomi. This fungus also causes great loss to over 400 species of Australian plants belonging to about 130 genera and 48 families. 5. In England over 5 million elm trees have been destroyed during 1967-77 because of infection of Ceratocystis ulmi. Some other havoc-creating fungal diseases of India include damping-off, potato blight, downy mildews of grapes, ergot of rye, smut of many crops, rust of many cereals, red rot of sugarcane and wilt of cotton.

24.2.8 Toxins and Aflatoxins If the mushrooms like Amanita phalloides (the ‘death cup’) and A. verna (the’destroying angle’) are eaten, the symptoms like diarrhoea and vomitting are seen within 2-3 hr. If the quantity of the ingested fungus is more, it results into liver damage, kidney failure, complete unconsciousness, and even death. This poisonous or fatal quality of these fungi is because of the presence of some compounds known as protoplasmic toxins. 1. About 10 toxins extracted from Amanita phalloides fall broadly in two groups, i.e. phallotoxins and amatoxins. The most extensively studied phallotoxin is phalloidin whereas amatoxin is a-amanitin. Phalloidin attacks plasma membrane of the liver cells, whereas a-amanitin causes lesions in stomach and intestine cells. Both affect kidney and liver in the later stages. Cytochrome-C and thioctic acid are used in the treatment of phalloidin and a-amanitin poisoning. 2. Claviceps purpurea, an ascomycetous fungus, causes the ‘ergot’ disease of rye. The sclerotia of this fungus contain many poisonous alkaloids like ergotamine, ergometrimine, ergocrystinine, ergocristine and ergonovin. The legs, hoofs and tail may become gangrenous, of the animals which feed on these sclerotia. The cows may even abort. Ergot-poisoning in man yields into diarrhoea, abdominal pain and vomitting. It decreases the diameter of terminal blood vessels. Its effect on nerves results into psychotic disturbances. Ergot derivatives have some beneficial aspects also, if properly used. They are used for uterine contraction during childbirth, for reduction of bleeding during labour, and for the treatment of migraine. 3. Some toxins like luteoskyrin, rubratoxin and cyclopiazonic acid are produced from some species of Penicillium, whereas toxins like zearalenone and sporofusarin are produced from Fusarium. 4. A toxin obtained from Entomophthora coronata induce intoxication, damage of blood cells and early death to several insects. 5. Aspergillus flavus and A. niger may infest dried foods and groundnut meal, and produce a carcinogenic toxin called aflatoxin. It is known to induce liver cancer in man and poultry (Kogbo et al., 1985). Researchers have proved that increased evidences of liver cancer might be because of aflatoxins.

24.2.9

Hallucinogenic Drugs

Some Agaricales (Psilocybe mexicana, Amanita muscaria) have some hallucinogenic properties. Because of such property of mushroom, Amanita muscaria was considered as one of their Gods by ancient Indians (Alexopoulos and Mims, 1979). Psilocybe mexicana is one of the well-known hallucinogenic mushroom. Its hallucinogenic effects are because of two compounds, i.e. psilocybin and psilocin. Psilocybin resembles lysergic acid from which the famous psychoactive drug LSD (Lysergic Acid Diethylamide) is derived. Psilocybin and baeocystin have also been extracted from Inocybe. Ingestion of Amanita muscaria results into trembling of limbs within an hour. After a short period of excitation and euphoria the person becomes unconscious. Some early symptoms like perspiration, salivation, nausea, vomiting, diarrhoea etc. are because of two alkaloids, i.e. muscarine and bifotenine, but the central nervous system is affected because of muscimol.

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24.2.10 Wood-Rotting Fungi Many wood-rotting fungi cause a lot of monetary loss. Good timber businessmen are well aware of the dangers of dry rot and wet rot in construction timber. Dry rot is caused by Serpula lacrymans and wet rot by Coniophora cerebella. These fungi break down the wood components and reduce its mechanical strength.

24.2.11 Fungi in Aircraft Fuel Tanks Fungal mycelium has been reported growing in storage tanks and fuel tanks of aircrafts containing kerosene-based fuel. The fungal hyphae may plug the pipes and valves of the aircrafts and might prove dangerous, specially during flight. Amorphotheca resinae is the main organism of the fuel tanks of aircrafts. It is also commonly called ‘kerosene-fungus’. This fungus may also grow luxuriantly on aluminium alloy and may corrode the metal. Because of this fungus aluminium foil may lose up to 27% of their weight within 6 months.

24.3 24.3.1

POSITIVE ASPECTS OF FUNGI Religious Importance of Fungi

The ancient Greeks and Romans attached great religious importance to many fungi, specially mushrooms. Romans thought that the mushroom appearance on earth is a serious warning of Jupiter. The appearance of Amanita muscaria is still considered as a warning of thunder and lightning in many parts of the world (Alexopoulos and Mims, 1979). This fungus also ‘played a powerful part in the Mayan religious life’ of Guatemala (Cooke, 1977). Many Mexican Indians used the mushroom Psilocybe mexicana in their religious rites for centuries. They actually called it ‘sacred mushroom’. It is still believed in some Siberian tribes that persons after eating these mushrooms (A. muscaria and P. mexicana) will receive the messages of God.

24.3.2

Fungi as Research Tools

Fungi are used as a basic material for the study of various fundamental biological processes. As many fungi grow very fast and require a shorter period to complete one generation, they are good research materials for many geneticists. Neurospora has become these days an ideal material for the study of laws of heredity by geneticists. Many biochemical processes are also studied exclusively by using several fungi. Physarum polycephalum, a slime mould, is a good material for the study of DNA synthesis, morphogenesis, mitotic cycle and many other processes. Various aspects of the use of Saccharomyces cerevisiae and other yeasts in modern researches are discussed in Chapter 25 (Fungi and Biotechnology).

24.3.3

Antibiotics from Fungi

Role of fungi in the production of antibiotics is well-known. Antibiotics are the substances produced by some living organisms which injure or kill another living organisms. Alexander Fleming (1944) for the first time extracted the great antibiotic drug penicillin from Penicillium notatum. Later on, Raper (1952) also extracted penicillin from Penicillium chrysogenus. Griseofulvin, one of the best known antibiotics against fungal diseases of skin, is extracted from Penicillium griseofulvum, whereas chaetomin is extracted from Chaetomium cochliodes. Many antibacterial and antifungal antibiotics are produced by Armillariella mellea in culture.

24.3.4 Preparation of Organic Acids Many organic acids are produced commercially by the biochemical activities of many moulds. Aspergillus niger is used in the preparation of citric acid and gluconic acid, whereas Rhizopus stolonifer is used for the manufacture of fumaric acid and lactic acid. Many other species of Rhizopus and Mucor are used for the production of oxalic acid, succinic acid and gallic acid. Jun and Yeeh (1985) isolated ethanol, acetic acid and acetaldehyde from Candida.

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24.3.5 Fungi and Alcoholic Fermentation Alcoholic fermentation with the help of fungi is actually the universal basis of brewing and baking industries. Both these industries are based on the fact that the fermentation of sugar solution by yeast produces ethyl alcohol and CO2. Alcohol is the important product of the brewing industry, whereas in bread-making or baking industry the carbon dioxide is the useful product.

24.3.6 Fungi and Enzymes Many products of high enzymatic activity like taka diastase, digestin and polyzime are produced by Aspergillus flavus. Amylase is produced by Aspergillus niger and A. oryzae. The yeast Saccharomyces cerevisiae is used for the extraction of invertase.

24.3.7 Fungi and Vitamins Saccharomyces cerevisiae and some other yeasts are the good source of vitamin B-complex and riboflavin. Because of their high vitamin content and a satisfactory amount of proteins the yeasts are considered as a valuable food throughout the world.

24.3.8 Fungi and Hormones Gibberellin, a group of plant hormones used to accelerate the growth of many crops, is produced by a fungus Gibberella fujikuroi. Van den Ende and Stegwee (1971) isolated a hormone trisporic acid from Mucor mucedo and Choanephora trispora.

24.3.9 Fungi as Insecticides Many fungi attack a number of insects that are harmful to the crops, and thus play a very important indirect role in our economy. Most of the insect-attacking fungi belong either to Entomophthorales or to Deuteromycetes. About 35-95% of the looper caterpillars infesting soybean are attacked by fungi in the USA, whereas more than 60% of female wheat-bulb flies are killed by fungi in the UK. Many insect pests are being controlled by using fungi like Beauveria bassiana and Metarrhizium anisopliae since the beginning of the 20th century. Coelomyces is an aquatic fungus which attacks mosquito larvae and kills them within a short period, and thus play role in the control of malaria.

24.3.10 Fungi as Food While certain species of mushrooms like Amanita and Boletus are highly poisonous, Amanita vaginata, A. fulva and Boletus edulis are edible (Webster, 1980). Mushroom cultivation for food is well-known these days, because of their fairly large protein content and being a good source of vitamins. Some other edible mushrooms, grown commercially for the purpose, include Agaricus bisporus, Volvaria, Lentinus edodes and Volvariella volvacea. (For more details of mushroom cultivation, refer Chapter 26, Mushroom Cultivation). Saccharomyces cerevisiae is universally used for the production of yeast cake, whereas ‘sufu’, a popular food is produced from species of Mucor and Antimucor. Yeast protein is commonly called ‘single-cell protein’, and its protein content is more than 40%. (For more of its details, refer Chapter 27, Single-cell Protein). Some of the other fungal-based processes are the preparation of leavened bread, cheese manufacture and clarification of fruit juices.

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24.3.11 Latex-Exuding Fungi The laticiferous hyphae of Lactarius contain latex, which exudes if the flesh is broken. Webster (1980) mentioned that if the stipe of Mycena galopus is broken the latex is exuded. Same condition of latex exudation is observed in the bleeding fruiting bodies of Stereum gausapatum.

24.3.12 Fungi and Pigments Matsueda et al. (1978) reported a novel pigment, neocercosporin (I), C29H26O10 (m.p. 237°C), from Cercosporina kikuchii. Its crystals are reddish violet.

24.3.13 Fungi and Luminescence Bioluminescence is the ability of an organism to produce visible light in the dark. Many members of Basidiomycetes such as Armillaria mellea, show bioluminescence. The luminous part of the fungus is either its fruiting body or the non-fruiting mycelium. In some cases the entire fungus body is luminous. In earlier days the luminous fruiting bodies were used as ornamentals, as well as the path makers at night.

24.3.14 Mycorrhizal Association A symbiotic relationship between the hyphae of some soil-borne fungi and roots of the other plants is called mycorrhiza. A majority of the deciduous or evergreen trees have ectomycorrhizas, in which the roots are entirely surrounded by the hyphae of fungal genera like Amanita, Boletus, Phallus, Tricholoma and Scleroderma. These fungi decompose the leaf litter and other forms of soil-organic matter. The ectomycorrhizas enhance the growth of the tree seedlings. This aspect is of particular importance when the plants are growing in nutrient-poor soils. Nutrients like phosphorus, nitrogen, potassium and calcium are easily absorbed by the fungal hyphae, and are then passed to the root tissues, and hence to the rest of the plant. (For details of Mycorrhizae, refer Chapter 30).

24.4 24.4.1

FUTURE EXPECTATIONS FROM FUNGI AND MYCOLOGISTS Favourable Expectations

1. Some new highly useful antibiotics may be obtained from fungi. 2. Commercial production of some useful enzymes, vitamins and hormones will continue to help the mankind. 3. Researches on some psychoactive fungal metabolites may be helpful in the treatment of some kind of mental disturbances. 4. Possibility of the production of large-scale protein-rich food from fungi raises high expectations of mycologists. 5. Role of fungi in disposal of solid and liquid pollutants and their active role in solving the great problem of pollution is also one of the chief expectations. 6. Some new fungal materials for fundamental biological research are also expected to be discovered soon.

24.4.2

Unfavourable Expectations

Though we should not be passimistic in our thinking and approach, fungi might become a major future health hazard for man by producing many new diseases of skin, lung, respiratory tract, intestine, kidney, liver and other body parts. In spite of the best use of many fungicides and disease-resistant crops, many new and old plant diseases may pose a great threat to the mycologists and plant pathologists.

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TEST YOUR UNDERSTANDING 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Write an essay on economic importance of fungi. “Fungi are our friends and foes”. Explain. Name any five diseases of human beings caused by fungi. Why is it difficult to cure fungal diseases? Name any three antifungal drugs. Name any three common food-spoiling fungi. Write a note on toxins and aflatoxins in about 100 words. Write short note on hallucinogenic drugs, obtained from fungi. Give a detailed account of positive aspects of fungi. Describe briefly the role of fungi as human food. What do you mean by mycorrhiza? Agaricus bisporus, Lentinus edodus and Volvariella volvacea are all _______, _______ .

25

C H A P T

FUNGI AND BIOTECHNOLOGY

E R

25.1

BIOTECHNOLOGY AND ITS RELATION WITH FUNGI

“Biotechnology” is the use of organisms or their components in industrial or commercial processes, which can be aided by the techniques of genetic manipulation in developing e.g. novel plants for agriculture or industry. It includes genetic engineering and molecular biology. Carlile et al. (2001) defined biotechnology as “the application of scientific and engineering principles to the processing of materials of biological agents to provide goods and services”. With the advancements in the fields of genetic manipulation fungi are widely used as biological agents in providing goods (e.g. alcoholic beverages, antibiotics, etc.) and services to mankind in different parts of the world. Using biotechnological techniques, fungi have now been variously exploited (i) as food, (ii) in processing various foods, (iii) in brewing industry, (iv) in production of several antibiotics and drugs, (v) in producing enzymes, (vi) vitamins, (vii) plant growth regulators, (viii) agricultural fungicides, insecticides, herbicides and nematicides, (ix) in cultivation of mushrooms, (x) single-cell protein, and also (xi) in gene cloning. Some of the beneficial activities of yeasts and other fungi are listed in Table 25.1. Table 25.1 Some major products of fungal biotechnology S.No

Application

Notes/Major Sources

1. 2. 3. 4. 5. 6. 7.

Agrochemicals Alcoholic beverages Antibiotics Biochemical agents Cheeses Citric acid Enzymes

8. 9. 10. 11. 12. 13. 14. 15.

Fermented foods Heterologous gene expression Industrial alcohol Mushrooms Pharmaceutical drugs Single-cell protein Yeast biomass Vitamins

Fungal fungicides, insecticides, nematicides, herbicides, plant growth regulators. From Saccharomyces cerevisiae Penicillins, Cephalosporins and many others. Various weeds, invertebrate pests, plant pathogenic fungi Mainly blue –vein and also several soft cheeses Mainly by Aspergillus niger Mainly for food processing, e.g. producing high fructose syrups; or in industries, e.g. lipases, proteases, cellulases, etc. From soyabeans, cereals, meat, etc. Mainly using Saccharomyces cerevisiae, also with Aspergillus species Mainly by Saccharomyces cerevisiae Several species, including Agaricus bisporus Secondary metabolites, e.g. controlling blood pressure, cholesterol synthesis,etc. Mycoprotein from Fusarium graminearum Mainly Saccharomyces cerevisiae, for brewing, baking Such as riboflavin.

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FERMENTATION TECHNOLOGY

25.2.1 What is Fermentation Technology? Technology of large-scale culture of microorganisms in fermenters is known as fermentation technology. It also includes recovery of useful products (i) contained within the microbial cells and/or (ii) released into the surrounding medium in the fermenter. [Fermentation is a process of “breaking down of organic molecules, specially by yeast or bacteria, under anaerobic conditions, to produce carbon dioxide and alcohol or lactic acid ”. Alcohol is “any one of a class of organic compounds with one or more hydroxyl groups (– OH), e.g. ethanol (CH3 CH2 OH)”. Lactic acid (CH3 CHOH COOH) is one of the end products of fermentation.] However, in modern fermentation technology the terms “fermentation” and “fermenter” do not necessarily involve anaerobic metabolism or fermentation as it is implied in physiological sense. It is so because majority of the microorganisms used in the fermentation industry these days are aerobes and not anaerobes. Moreover, fermenters, used in fermentation technology these days, also require adequate aeration.

25.2.2

Major Aspects of Fermentation Technology

Four major aspects of fermentation technology include: (i) Research and laboratory studies on the organism used in the technology; (ii) Suitable materials (feedstocks) on which this organism can be grown easily and on large scale; (iii) Fermenters of appropriate design and proper optimum conditions for its operation; and (iv) “Downstream processing” for efficiently recovering the desired product. Some details of all these aspects are discussed below :

25.2.3

Research and Laboratory Studies on the Organism

The most initial consideration for a fermentation process is to find out that a microorganism makes a useful product. Research on this aspect is necessary because (i) the organism may grow poorly, or (ii) the medium used may be expensive, or (iii) yields of the desired product may be very low. To overcome these problems, we may require to improve the performance of the microorganism genetically and by providing it with optimum environment for growth and product formation. When commercial production of fungal metabolites is being done, hundreds of thousands of litres of medium may be there in the large – sized fermenters. Research on fungal metabolism is, however, done only with flasks containing about 100ml of medium. These flasks may be kept static or shaken. In the static flasks the fungi will grow as a mat on the surface. But in the shaken liquid cultures fungi occur as dispersed hyphae submerged in the medium. The important step towards efficient commercial production is to achieve good yields. Commercial production is done only when it has finally been established through researches that (i) the organism is quite efficient in growth, (ii) there is a required amount of product formation, (iii) recovery of the final product is quite satisfactory, and (iv) the entire process is quite economical also. Culture preparation is the next major step in the research and laboratory activities associated with fermentation processes. A fungal strain which gives high yields of a valuable product is a precious asset. The ideal starting point

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for preparing cultures for preservation will be a “master culture”. It is developed from a single uninucleate haploid cell, and is hence genetically uniform. Such cultures have the desired properties. They are stored after they have been grown on agar media in screw-capped vials. Longer survival, usually for over a year, may be obtained by covering the cultures with sterile mineral oil or medicinal paraffin. These days, more sophisticated methods of preserving fungal cultures are used, and probably best amongst them is “liquid nitrogen refrigeration.” Inoculum preparation is the final step of research and laboratory activities associated with fermentation processes. The final inoculum must be of adequate size, i.e, it should have a biomass of about 10% of that to be grown in the fementer. Care should be taken that during the inoculum preparation and its production, there should be no contamination.

25.2.4 Suitable Material (Feedstocks) for Fermentation Processes Two main properties of a suitable material (feedstock) for any fermentation process are that (i) it should contain utilizable sources of carbon, nitrogen and other essential elements in appropriate amounts and ratios, and (ii) it should be cheap. Selection of right feedstock or raw materials for preparation of medium is also an extremely important factor in the success of an industrial fermentation. Majority of the fungi are capable of utilising feedstocks such as sugarcane juice, molasses, hydrolysed starch and unrefined sugar. Cellulose is usually unsuitable as a feedstock, because it is attacked by relatively few fungi and that too only slowly. Some other major carbon sources, other than carbohydrates, are vegetable oils such as soyabean, palm and maize oil. Groundnut meal, beet molasses and whey are good sources of both nitrogen and carbon. If vitamins are needed, yeast extract is their best source. While deciding about suitable material or feedstocks, factors such as (i) transport cost, (ii) storage cost, and (iii) ease of handling, are also important.

25.2.5 Fermenters and Their Operation Fermentations are carried out in fermenters. The size, type and characteristics of fermenters are different in different types of industrial activities and nature of the products, to be fermented. Most common fermenters in various industrial fermentations are stirred tank fermenters. Gas outlet

Stirring shaft Manhole

Head space

4-Times Height

Some basic features of these fermenters are given below: (i) It is a cylindrical vessel (Fig. 25.1) of high grade stainless steel, which is filled with sterile medium. (ii) It is so designed that the medium in the vessel can be inoculated, aerated, stirred, cooled or heated and sampled, without bringing about any type of contamination. (iii) The height of the cylindrical fermenter is one to four times its diameter. (iv) About ¾th part of the vessel is filled with nutrient medium containing suspended microorganisms. (v) The space above the medium is called head space. It contains air which has passed through the medium, due to which it is depleted in oxygen. (vi) The sterile air enters the fermenter through a pipe which terminates in a single aperture or a sparger containing many small apertures. (vii) The sterile air leaves the head space of the fermenter via a gas outlet.

Cylindrical vessel Nutrient medium with suspended microorganisms Baffle

Diameter 1-Time

Rushton impellers

Sparger Harvest line

Fig. 25.1

Sterile air to sparger

Outline diagram of a stirred tank fermenter. (Structures like temperature jacket, cooling coils, steam lines, inlets for inoculation, sampling, additions, etc. are not shown in the diagram).

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(viii) A stirring shaft carries many Rushton impellers, horizontal discs bearing vertical plates. Turbulence is enhanced by baffles. (ix) For cleaning purposes, a manhole is provided in the upper part of the cylindrical vessel. It has a glass window which allows viewing during the fermentation process. (x) At the base of the vessel is present a harvest line through which the culture is harvested. (xi) Stirred tank fermenter also has a temperature jacket, cooling coils, steam lines, inlets for inoculation, sampling and additions during the fermentation process. Both fermenter and medium are sterilized before starting any fermentation process. Sterilization is done by heating upto a temperature of 120 – 150°C. Medium sterilization is usually done within the fermenter, in which steam is passed through the temperature control jacket which surrounds a fermenter. After sterilization, the fermenter is cooled by passing water through the jacket. All components related to the fermenter and medium should also be sterilized. Since most of the industrial fermentations are aerobic, they must all be supplied with air. The air is pumped through a filter and enters the fermenter through a sparger containing many apertures which help in producing many small air bubbles. This air helps in oxygen diffusion into the medium. Mechanical agitation or stirring helps in uniform distribution of dissolved air throughout fermenter. Agitation in the fermenter is done by Rushton impellers (Fig. 25.1) mounted on a stirring shaft. Temperature control jacket, through which cooling water flows, is of great utility for controlling temperature in small fermenters. In larger fermenters, there are cooling coils within the fermenters. Appropriate “sensors” help in detecting departure from optimal conditions (e.g. cell density, acids, pH, etc.) of the medium. In good quality fermenters, various parameters (e.g. growth, nutrient levels, dissolved oxygen, etc.) are automatically corrected by these sensors.

(i) It is a tall fermenter of about 30 meter or more height and used for production of several products including mycoproteins. (ii) Compressed air and ammonia used in this fermenter, are injected into the system (Fig. 25.2A). The compressed air provides oxygen and brings about circulation via air-lift. Ammonia acts as the nitrogen source. (iii) The spent gas contains carbon dioxide and leaves the fermenter through the gas exit at the top. (iv) The nutrient solution is pumped into the fermenter at a constant rate near the lower part of the down corner. The nutrient solution contains glucose, biotin, mineral salts etc. (v) A cooling coil (Fig. 25.2) works as a heat exchanger. It maintains a constant temperature of 30°C. (vi) Continuous harvesting of culture at the same rate is done through a separate tube at lower end of the fermenter. (i) (ii) (iii) (iv)

It is a cylindrical fermenter with a tapering base (Fig. 25.2B) used widely in brewing. No mechanical agitation is done in these fermenters. At the center of this fermenter, yeast cells with associated CO2 bubbles rise to the surface. The liquid in such fermenters cools at the circumference and descends through enhanced specific gravity. This maintains a circulation of cells and nutrients. Circulation occurs usually due to the production of CO2 by the yeast.

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Exit for spent gas Exit for carbon dioxide ‘Riser’ ‘Downcomer’

Entry of ammonia and compressed air

Carbon dioxide sparging

Nutrient solution Continuous harvesting

A

B

Cooling coil Airlift Fermenter

Fig. 25.2

25.2.6

Valve for filling with medium and for harvesting

Cylindroconical Fermenter

An airlift fermenter (A) and a cylindroconical fermenter (B).

Downstream Processing

After the completion of the fermentation process, it contains cells/biomass in a large volume of spent medium. In processes of brewing, the spent medium in itself is the desired product but in single-cell protein this is the biomass. The desired product is, however, usually a minor component of the cells or broth (i.e. liquid nutrient medium). For example, a litre of broth with biomass may contain only 1 gm of a required enzyme or only 5gm of an antibiotic. Such a small amount of the desired product has to be separated from large amount of waste material in the process of downstream processing. The entire process of concentration and purification of the product completes after a series of steps called unit operations. Each unit operation may be quite expensive in terms of equipment, manpower, chemicals, energy, etc. Two commonly used downstream processing equipment are (i) Rotary Vacuum Filter, and (2) Continuous Flow Centrifuge. Some basic aspects of both of these equipment are mentioned below: (i) The medium and suspended organisms are fed into the trough (t) of rotary vacuum filter (Fig. 25.3). (ii) The aqueous phase passes through a filter cloth on the surface (d) of a slowly rotating drum and also through several hollow spokes to the hollow axle (a) of the drum of this equipment. Suction is also applied. (iii) The solid phase (p) is held on the outside of the drum by suction, and is removed with the help of a knife scraper (K). (iv) Washing is done by a sprayer (w). It ensures that the products in the solution are separated from the solids and pass into the drum. (v) In case the aqueous phase is to be used, a filter aid (e.g. diatomite) is added to the medium being fed into the trough (t). Due care should, however, be taken for disposal of diatomite, if used. (vi) In case the solid phase is to be used, then filter aid is not used. (i) It is used if a high degree of microbiological containment is required. (ii) It has numerous stacked conical discs (Fig. 25.4), situated quite close to each other.

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Solidphase (p)

Direction of rotation Stacked conical discs (d)

Hollow space (s) Hollow axle (a)

Washing (W)

Nozzle (n)

Knife scrapper (k) Surface of rotating drum (d) Trough (t) Rotary Vacuum Filter Continuous Flow Centrifuge

Fig. 25.3

A Rotary Vacuum Filter.

Fig. 25.4

A Continuous Flow Centrifuge.

(iii) Liquid with suspended particles is pumped into the rotating centrifuge. It takes a path that takes it to the outer wall and then inwards between the discs as shown by arrows (Æ) in Fig. 25.4. (iv) The discs in this centrifuge are close together and steeply sloped. The particles in these discs move outwards by centrifugal force. They finally reach up to the undersurface of discs. (v) The particles slide along the discs for reaching up to the wall of the centrifuge. (vi) Solids pass out through a fine nozzle (n) of the centrifuge.

25.2.7

Solid Substrate Fermentations

Solid substrate fermentations include several traditional Asian food fermentations. These make several unappealing foods more nutritious, attractive and digestive. These involve microorganisms such as moulds, yeasts or bacteria. Some of these fermented foods include ontjom of Indonesia, cassava or gari of Nigeria and Aug – Kak of China. Aspergillus, a potent producer of extracellular enzymes is prominent in solid substrate fermentations. Solid substrate fermentation are also used to give a range of other products. For example, growing of mushrooms, in which production of compost and composting of organic wastes are examples of solid substrate fermentations. In Japan, these fermentations are now widely used in production of citric acid and other organic acids and industrial enzymes. Some of the merits of solid substrate fermentations include (i) cheap raw materials, (ii) sterilization is usually not required, and (iii) low energy requirements.

25.3

ENZYME TECHNOLOGY

Enzyme is a protein which, in very small quantities, catalyses and controls the natural chemical reaction of metabolism. Enzymes are usually large complex molecules, and most of them are responsible for one or two particular reactions in the cell. Cells contain many thousands of different enzymes. Some enzymes of great potential and immense commercial importance are produced by fungi.

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25.3.1

293

Fungal Enzymes of Commercial Importance

A very large number of fungi produce enzymes that are of considerable industrial importance. These may be grouped into two categories, viz. extracellular fungal enzymes and intracellular fungal enzymes. Many fungi produce extracellular enzymes that enable them to break down polysaccharides and proteins into sugars and amino acids which can be easily assimilated. Some examples of such extracellular fungal enzymes are listed below: 1. Bacterial a – amylase is used to treat starch slurry to produce liquefied starch. 2. Glucoamylase produced from Aspergillus niger is used to treat liquefied starch to produce high glucose syrup which contains 97% glucose, 1.5% maltose and oligosaccharides. High glucose syrups are suitable for jams, confectionery, baking and brewing industries. 3. Fungal a – amylase produced from Aspergillus oryzae is used to treat liquefied starch to produce high maltose syrup, which contains 56% maltose, 4% glucose and 28% maltotriose and other oligosaccharides. 4. A b (1 Æ 3) – gluconase, produced from Aspergillus niger, is used to remove haze from beer by degrading glucans. 5. Fungal pectinase produced from Aspergillus niger or A.oryzae is used to clarify haze in fruit juices and wine. 6. Lactase obtained from the yeast Kluyveromyces and Aspergillus niger is used to convert lactose into glucose and galactose. 7. Cellulases obtained from Trichoderma and some species of Aspergillus are used in food processing and production of lignocellulose, a cheapest and abundant source of organic carbon. 8. Alkaline proteases produced from bacteria are in great demand as detergent additives. 9. An important acid protease produced from fungi (Rhizomucor pusillus, R. miehi) is chymosin. It is used variously in cheese manufacturing. Fungal proteases are also used to remove protein hazes from beer and also biscuit – making. 10. Nuclease obtained from Penicillium citrinum is used to give meaty flavours to several foods in food industry. 11. Lipases obtained from Aspergillus niger are used in flavoring cheese and also in detergents. 12. Phytase obtained from Aspergillus niger is used as an additive to animal feedstuffs. Only few intracellular fungal enzymes are of commercial importance. A few examples are listed below: 1. Glucose oxidase produced from Penicillium chrysogenum and Aspergillus niger is used in bottled fruit juices to remove traces of oxygen. It thus functions as a preservative. It is also used in detection of glucose in urine and blood. 2. Catalase obtained from Aspergillus niger is used to remove H2O2 traces from milk. 3. Alcohol dehydrogenase obtained from Saccharomyces cerevisiae is used in ethanol estimations. 4. Asparaginase obtained from Aspergillus, Penicillium and some bacteria is used in the treatment of leukaemia.

25.4

PRODUCTION TECHNOLOGY OF ALCOHOLIC BEVERAGES

Many yeasts (e.g. Saccharomyces cerevisiae, S.ellipsoideus, S.carlbergensis, etc.) have been utilized for commercial prodution of several alcoholic beverages, wine, cider, perry, bear, etc. In majority of the fermentation processes S.cerevisiae is used. Under aerobic conditions yeasts metabolize sugar into CO2 and water. In the scarcity or complete absence of oxygen and also if the sugar concentration is high, fermentation occurs with the production of ethanol and CO2. This alcoholic fermentation is actually the main basis of the production of large number and huge quantities of alcoholic beverages used by man today in the world.

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On the basis of the procedure involved in the production of alcoholic beverages, they may be grouped into following three categories: 1. Those beverages which are made by fermenting plant juices rich in sugars, e. g. wine, cider, perry, etc. 2. Those beverages which are made from plant materials having substantial amount of starch, which must first be converted into sugars before fermentation starts, e.g. beer. 3. Those beverages in the production of which distillation is required to obtain alcoholic concentrations higher than it can be achieved by fermentation alone, e.g fortified wines, and spirits. In majority of the wine – producing countries there are laws to define wine “as the product of alcoholic fermentation of the juice of fresh grapes” (Vitis vinifera; Vitaceae), and strictly limit the materials which may be added during its production. In the production of some fine white wines, however, a mould (Botrytis cinerea) plays an important role, and it is wided utilized in France and Germany. The yeast, principally used for alcoholic fermentation, is Saccharomyces ellipsoideus which is now regarded as a variety of S.cerevisiae. Cider is produced from apples (Malus pumila; Rosaceae). It is produced almost in the same manner as wine from the grapes. Fermentation is brought about by selected strains of Saccharomyces. Perry is prepared from the juice of perry pears and unripe dessert pears and method of its preparation is similar to that of cider. Basically, beer is an alcoholic beverage made from malted barley (Hordeum vulgare; Poaceae) and flavoured with hops (Humulus lupulus; Cannabinaceae). Saccharification (process of conversion of starch into sugar) process in the production of beer is called malting, and it should be carried out before the start of fermentation. In many countries, including Germany, there exists law for producing beer, according to which the materials used in brewing should only be malted barley, water, hops and yeast. Like beer, several other starch – based alcoholic beverages are produced from other cereal grains such as rice, maize, and sorghum, and also from root crops such as cassava (Manihot esculenta; Euphorbiaceae). Beverages with high alcohol concentrations (e.g. spirits) are made only by distilling the product of a fermentation. A wide range of spirits are prepared from sugary or starchy materials. Among the spirits made from sugary materials are brandy prepared from grape juice, fruit brandies (e.g. applejack from apple juice) or rum prepared from the juice of sugarcane. Among the spirits made from starchy materials are whisky (e.g. Scotch whisky from Scotland). Several cereals are used for their manufacturing, e.g wheat, rye, barely and maize. Gin is prepared from rye (Secale cereale, Poaceae) or maize. Vodka, a Russian whisky, is prepared from non-cereal starchy materials (e.g. potatoes). In the production of whiskies, the alcohol production is due to inoculation with pure strains of Saccharomyces cerevisiae. This yeast is also used for the fermentation phase with gin, vodka and light aroma varieties of rum produced from fresh cane juice. However, most rum is of high aroma varieties, for which raw material is molasses. During production of rum from molasses, fermentation is done with fission yeast i.e Schizosaccharomyces. Sherry and port are two best – known fortified wines. Sherry is made from grape juice, which is first fermented and then neutral grape spirit is added to bring the ethanol concentration to 15%. Port is made from a brandy which checks fermentation, leaving some sugar unfermented. Alcohol concentration in port is kept only upto 17%. Fortified wines require a prolonged period of further maturation, before being used.

25.5

CULTIVATION OF MUSHROOMS AND OTHER MACROFUNGI*

Mushroom is the name for the reproductive structure (or fruiting body) in basidiomycete fungi of the family Agaricaceae. Throughout the world the mushrooms are cultivated and used as highly delicious table delicacies.

* For details refer Chapter 26 (“Mushroom Cultivation”).

Fungi and Biotechnology

25.6

295

SINGLE-CELL PROTEIN

Several yeasts, bacteria, moulds and algae are grown for their use as a source of protein in foods or animal feeds. The microbial product or dried cells of these single-cell organisms is called single-cell protein. The detailed treatment of single-cell protein is given in a separate Chapter 27.

25.7

FUNGI IN FOOD PROCESSING INDUSTRY

A crucial role is played by fungi in processing many of our foods used daily. Fungi also improve texture, digestibility, flavour, appearance and even nutritional value of the several raw materials used in our various food articles. Some of such aspects and articles are discussed here.

25.7.1

Bread

Man has been preparing bread by using cereal seeds as food since times immemorial. Today also we grind cereals to prepare flour, mix it with water to form dough and bake it on heated plates to prepare flat or round bread. But, such breads, get easily contaminated by yeasts. CO2 formation by the yeasts makes them unpleasant and unsuitable for use. They are therefore baked to prepare a loaf. Even in the early 19th century thousands of the tonnes of brewer’s yeasts, were used in bread-making. Such yeasts used for bread making are now almost pure strains of Saccharomyces cerevisiae. Production of baker’s yeast is now carried out in well-aerated fermenters. In smaller bakeries, yeast is compressed, extruded as a strip and cut into yeast cakes, which may be kept at about 5°C for about a week. For bread-making, wheat is (i) milled, (ii) sieved to obtain wheat flour, (iii) mixed with water, yeast and salt to prepare dough, and (iv) added with several other materials, such as sugar, fat, milk, malt, eggs, spices, dried fruit, etc. Amount of yeast to be added in the dough varies from 1 – 6% depending on the type of the bread to be prepared. Salt concentration (1.5 – 2%) usually inhibits some yeast enzymes and helps in giving an optimum dough for bread. Yeast ferments the sugar and produces CO2 which inflates the dough. It also produces ethanol which is driven off during baking. Proper baking finally kills the yeast providing characteristic taste and texture to the bread.

25.7.2

Soyabean Products

Soyabeans (Glycine max) have a high protein content and also yield an oil useful for cooking and many other purposes. Several types of fermentations yield many harmless, nutritious and palatable products from soyabeans. Soy sauce is extensively used in many countries including Japan, China and Indonesia. It is actually a condiment which makes a monotonous diet more appetizing. Some definite strains of Aspergillus are used for fermentations. Aspergillus produces a wide range of hydrolylic enzymes. It also breaks down proteins, polysaccharides, etc. of this substrate during the process of preparation of soy sauce. In the later stages of fermentation, the liquid is drained from the remaining solids. It is then clarified, pasteurized, bottled and marketed. Miso is a fermented soyabean paste prepared in Japan and used widely in preparation of soups and sauces. Two main raw materials used in preparing miso are soyabeans and rice. While preparing miso, rice is (i) soaked, (ii) steamed (iii) inoculated with Aspergillus oryzae, (iv) fermented for about 48 hours, (v) mixed with soaked and steamed soyabeans,(vi) passed through the action of several enzymes, (vii) added with salt and passed through some more stages before being used. Miso is a good source of vitamins and proteins. It contains sufficient quantity of amino acids, like lysine, due to which it is a widely recommended diet for vegetarians. Tempe is a delicious fermented food of Indonesians. It is actually a cake covered in white mould (Rhizopus oligosporous). When soyabeans are used for its preparation, tempe is called tempe kedele.

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Tofu and sufu are highly flavoured foods prepared from soyabeans. In place of animal milk, a vegetable milk is prepared by soaking, grinding and straining soyabeans in China, Japan and many other countries. A curd prepared from soyabean milk is called tofu. In China, tofu is used in place of cheese. Fermentation of tofu yield sufu, a more highly flavoured food. Fungi responsible for fermentation of tofu and production of sufu are Rhizopus chinensis, Mucor racemosus and Actinomucor elegans. Characteristic flavors of tofu and sufu are because of enzymes like proteases.

25.7.3

Cheese, Fermented Milks and Other Fermented Foods

Preparation of several types of cheeses (e.g. surface – ripened cheese, blue – vein cheese), fermented milks (e.g. yoghurt, villi, koumiss, dahi, etc) and other fermented foods (e.g. ontjom, tape ketan, gari, ang-kak, etc) has now developed into a well-developed fermentation industry, which now stands second only to brewing industry in importance. Lactic acid bacteria are the main microorganisms involved in this fermentation industry. They ferment lactose of milk into lactic acid and several other metabolites responsible for characteristic flavour. A definite role in production of cheeses and fermented milks is played also by fungi. Brie and Camembert are the two best known surface – ripened cheeses. Penicillium camembertii, Geotrichum candidum and some acid tolerant yeasts play definite role in preparation of these cheeses. Roquefort, Stilton and Danish-blue are some best-known blue-vein cheeses. Penicillium roquefortii and some bacteria play definite role in blue - vein cheeses. Blue colouration of these cheeses is due to the spores of P.roquefortii. Both proteolytic and tipolytic enzymes are produced by this fungus. In yoghurt, a fermented milk, lactic acid bacteria are responsible for curdling. Villi is a fermented milk of Finland. It has a surface growth of Geotrichum candidum which lowers acidity by metabolizing some of the lactic acid. Koumiss, a Russian fermented milk, contains lactic acid produced by lactic acid bacteria. Kefir, a yet another famous Russian fermented milk is produced by using lactic acid bacteria and yeasts. Dahi is the Indian fermented milk. Yeasts play definite role in its preparation. Ontjom is a nutritious and digestible fermented food of Indonesia. It is prepared from cakes left after groundnuts have been pressed to extract oil. Rhizopus oligosporus or Neurospora intermedia are used in its preparation. Tape ketan is yet another nutritious and much-liked Indonesian fermented food. An yeast (Endomycopsis burtonii) and a fungus (Amylomyces rouxii) play definite role in preparation of tape-ketan. Gari, a Nigerian fermented food, is prepared from roots of Cassava (Manihot esculenta; Euphorbiaceae). Fermentation in the preparation of Gari occurs due to Geotrichum candidum, a fungus and Corynebacterium manihot, a bacterium. Aug – Kak is a fermented food produced from rice in China. Monascus purpureus, an Ascomycetous fungus plays an important role in preparation of ang-kak.

25.8

PRODUCTION OF PRIMARY METABOLITES BY FUNGI

The metabolites that have to be produced for the occurrence of growth are known as primary metabolites. For the production of any special primary metabolite in the metabolism process, the biotechnologist has to select from a wide range of fungi and bacteria. Commercial production of primary metabolites is done mainly by bacteria because they have higher growth rates than fungi. In some cases, however, yeasts and moulds, are also utilized for production of primary metabolites such as industrial alcohol and citric acid.

25.8.1

Ethanol

Industrial alcohol or ethanol is an important primary metabolite of commercial importance. It is also a major feedstock in the chemical industry. Formerly, major part of industrial ethanol was produced by fermentation. In several developing countries production of ethanol by fermentation is still promoted and about 20% of the world output of industrial alcohol

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is still done by fermentation. Fermentations for the production of ethanol or industrial alcohol are generally carried out with Saccharomyces cerevisiae. In some cases, however, the fission yeast (Schizosaccharomyces pombe) is also used. Some major substrates are sugarcane juice, molasses and starchy materials like potatoes, cassava and surplus grains. The starch has, however to be converted into sugars before alcoholic fermentation can proceed, and this is done either with fungal amylases or acid hydrolysis. In some systems the yeast is recycled and used to generate more ethanol.

25.8.2

Citric Acid

Most of the aerobic organisms produce citric acid because it is a component of tricarboxylic acid (TCA) cycle. Commercial production of citric acid from Aspergillus niger began about a century ago in 1923 and it still remains the main microorganism for the production of this acid. In the recent years, however, the production of citric acid in submerged culture has also been carried out using Candida sp.

25.8.3

Some Other Primary Metabolites Produced by Microorganisms

Production of some other primary metabolites is also done by microorganisms including bacteria and fungi. Representatives of most classes of primary metabolites have been produced commercially by moulds and yeasts. A few such examples include production of (i) nucleotides and amino acids by bacteria, (ii) proteins and single-cell proteins by 6 CH2OH O

5

1 H

H 4 3

6 CH2OH O

O

5 H

H

2

Kojic acid OH

H

4

COOH

1

OH OH 3

H 2

HCOH

OH

HOCH OH

H

HCOH

Glucose HCOH CH2OH Gluconic acid

HO

COOH

COOH

COOH

CH2

CH2

CH2

C

COOH

COOH

CH2

CH

COOH

COOH

Citric acid

Fig. 25.5

C

Cis-aconitic acid

Some economically important organic acids produced by fungi.

C

COOH

CO2

Itaconic acid

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moulds as well as bacteria, and (iii) many extracellular polysaccharides by bacteria and fungi. Some moulds and yeasts accumulate large amounts of fats. Fusarium and Candida are used for the accumulation of fats. Glycerol is obtained from the anaerobic metabolism of Saccharomyces cerevisiae. Besides citric acid, several other organic acids (Fig. 25.5) are also produced using moulds, such as Rhizopus. Itaconic acid is produced by Aspergillus terreus. Gluconic acid and kojic acid are also produced by several species of Aspergillus. Several vitamins are also purified by yeast extract.

25.9

PRODUCTION OF SECONDARY METABOLITES BY FUNGI

Several secondary metabolites of exceptional biological activities and importance are now provided to us by fungi. These are the metabolites which are not essential for vegetative growth in pure culture. Most fungal secondary metabolites show an enormous variety of structures and biosynthetic origins. The production of any one of them tends to be limited to one or a few organisms, e.g. cyclosporin - A is produced by many strains of three species of Tolypocladium, but

A

OCH3

O

OCH3

B

CO2H H OAc

HO

O O

CH3O

CH3

Cl

H

Griseofulvin HO H Fusidic Acid

OH C

HO

CH3

D O

OCH3 H3COOC O

O

Strobilurin

CO2H O H

E CO

CHO HO Sordarin

Fig. 25.6

OH

A–E, Some secondary metabolites produced by fungi.

H

COOH

Gibberellin GA3

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also by strains of species of Fusarium, Neocosmospora and a few more genera. Secondary metabolites are produced in families of closely related molecules. Some examples of the fungal secondary metabolites are griseofulvin, fusidic acid, sordarin, strobilurin-A and gibberellin GA3 as illustrated in Fig. 25.6A-E. A widely exploited secondary metabolite is Cyclosporin-A, which is a cyclic peptide. The largest enzyme protein known is cyclosporin synthetase from Tolypocladium niveum. It has a molecular weight of 1689243 with eleven amino acid – activating domains and forty catalytic functions involved in its biosynthesis. Screening of fungal isolates for the production of secondary metabolites has proved to be of great profit, and it has shown exceptional development and advancement of medicine and agriculture. Some such examples to prove this are discussed below:

25.9.1

Role of Secondary Metabolites in Medicine

Penicillin amidase Substances produced by some microorganisms which injure or kill another microorganisms are called H H CH3 S R C NH antibiotics. Such an action is due to the production of second1 C6 C5 2C ary metabolites by one organism (e.g. Penicillium) that interfere O CH3 with the metabolism of the other organism. These secondary 7 4 3 metabolites are known as antibiotics. These are produced by C N C some Ascomycetes and related mitosporic fungi and also by COOH O many soil bacteria, particularly of Actinomycetes (e.g. StrepH tomyces) or spore forming soil bacteria (e.g. Bacillus). For exb-lactamase ample, the antibiotic penicillin-G, obtained from Penicillium Penicillin Molecule notatum, if administered by injection, could combat potentially OCH3 lethal infection by Staphylococcus and other bacteria. Similar R groups to penicillin-G, several thousand antibiotics have now been isoCH2 lated till date. Majority of the antibiotics discovered so far are too toxic to humans, and only about 50% have been used clinically. OCH3 Methicillin Penicillin-G, penicillin-V, methicillin and ampicillin are Penicillin G some of the penicillins (Fig. 25.7) which differ in side chain. Methicillin and ampicillin are semi – synthetic antibiotics. OCH2 Cephalosporins have been produced from a strain of CeCH phalosporium acremonium and inhibit the growth of both NH Gram positive and Gram negative bacteria. Cephalosporin-P Ampicillin (active against Gram positive bacteria), Cephalosporin-N (ac- Penicillin V tive against both Gram positive and Gram negative bacteria), Fig. 25.7 Penicillin molecule and various penicillins cephalosporin-C (a relatively weak antibiotic) and cephalexin which differ in side chain (Penicillin-G, (a fourth generation semi – synthetic cephalosporin) are now penicillin-V, methicillin and ampicillin. widely used globally. Cephalosporins are also now biosynthesized by the actinomycete, Streptomyces clavuligerus. Some other antibiotics of clinical significance are griseofulvin (from Penicillium griseofulvum), fusidic acid (from Fusidium coccinium), fumagillin (from Aspergillus sp.) and sordarin, an antifungal antibiotic obtained from Sordaria aroneosa.

Most important group of the fungal compounds used in cholesterol – lowering drugs are mevinic acids. Mevastatin obtained from Penicillium citrinum and lovastatin from Monascus ruber are examples of some of such fungal secondary metabolites used in cholesterol-lowering drugs. These all are small organic acids with an acidic side group.

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Fungi and Allied Microbes

In the fields of medicine and surgery, a global revolution has now been observed in the ability of the scientists/doctors to transplant organs, such as heart, lung, kidney, pancreas, liver, skin, bone marrow etc. Transplantation of these organs have been made possible only because of the discovery of Cyclosporin-A (Fig. 25.8). It is a potent immunosuppressive drug, and a secondary metabolite of an isolate of a fungus (Tolypocladium inflatum). It acts on the T-lymphocytes of the immune system of the person who is to be operated for organ transplantation . Cyclosporin – A is also used in treatment of resistant forms of eczema and psoriasis. It is actually a very complex cyclic peptide with many of the constituent amino acids methylates (Fig. 25.8). MeBmt

CYCLOSPORIN-A

H

CH3 C MeLeu CH3

MeVal

CH3 CH

CH3

CH3

CH3 CH

CH2

Abu

C H

CH2

OH

CH

CH3 CH

Ser

CH3 CH3

CH2

CH3

CH3 — N — CH — CO — N — CH — C — N — CH — CO — N — CH — C — N — CH2 10

MeLeu CH3

CO

CH — CH2 — CH CH3

1

11

3

2

O

H

O

CO

H

O

H

N — CH3

9

CH3 — N 8

5

6

7

4

CO — CH — N — CO — CH — N — C — CH — N — C — CH — N — CO — CH CH3

H

CH3

O

CH2

CH3 CH3

CH CH3 D-Ala

Fig. 25.8

Ala

CH

CH3

MeLeu

CH2 CH3

CH CH3

Val

CH3

MeLeu

Cyclosporin – A, a cyclic peptide. (D-Ala, D-alaline; Meleu, methyl leucine; Val, Valine; MeVal, methyl valine; MeBent, butenyl-methyl-L-threonine).

Ergot are the sclerotia produced by a fungus (Claviceps purpurea). Ovaries of the cereal rye (Secale cereale) are infected by this fungus, and a sclerotium (ergot) instead of a grain develops. The contaminated grains are highly toxic. The alkaloids in the ergot are nitrogen – containing basic compounds. Lysergic acid and its amide are produced by Claviceps paspali. One synthetic derivative, lysergic acid diethylamide (LSD) is a globally well-known hallucinogen. A medicinally important derivative of lysergic acid is ergotamine, obtained from C.purpurea. Some other ergot alkaloids of medical importance are ergometrine and ergotamine. Ergometrine and its derivatives act upon uterus and given during childbirth, to facilitate delivery. They are given even after childbirth if there is excessive bleeding.

Fungi and Biotechnology

25.9.2

301

Role of Secondary Metabolites in Agriculture

Strobilurins is a class of antifungal antibiotics, identified as secondary metabolites of mushrooms. Introduction of strobilurins has brought significant advancements in the field of agricultural fungicides. Mucidin, described in 1960 from ‘porcelain fungus’ (Oudemansiella mucida) and Strobilurin-A from yet another small-sized mushroom (Strobilurus tenacellus) described in 1980 are the famous strobilurins known. Since 1990, some synthetic strobilurins have also been introduced in the market . These broad – spectrum systemic agricultural fungicides have a much longer life in the field, and upto 2005 these have captured a wide market in several countries, specially for their specific use for cereal fungicides. Gibberellins are group of plant hormones, discovered first as secondary metabolites of Gibberella fujikuroi, a rice pathogen. These cause increased growth of rice plants, which soon fall over and rot. Over two dozen gibberellins have so far been produced from G.fujikuroi. Most of them are diterpenoids with 19 or 20 carbon atoms (Fig. 25.6E), biosynthesized from mevalonic acid as are carotenoids and sterols. In industries, gibberellins are produced by fermentation of improved strains of G.fujikuroi. Gibberellins are widely used to control development and ripening of apples and to improve fruit size in seedless grapes and citrus fruits.

25.10

ROLE OF BIOTECHNOLOGY IN SELECTION AND MUTATION OF FUNGAL STRAINS

Any fermentation technology is accepted in the modern society if it provides us a suitable microorganism, the performance of which can be improved by genetic modification for the benefit of the mankind. Fungal biotechnology is now widely known for its role in selection and genetic improvement of fungal strains.

25.10.1 Selection of Fungal Strains Biotechnology helps us to select suitable fungal strains for the desired purpose from amongst the large number of strains of large number of fungal species in culture collections. For example, recently, during the screening for insulin-like activity, involving over 50,000 mixtures of synthetic and natural products, a metabolite was discovered from an Ascomycetous fungus (Pseudomassaria sp.) which shows insulin-like effects in some types of diabetes. Fungal biotechnology techniques also provide us the guidelines of screening for strains with improved performance. These techniques help us in increasing the yield of the metabolite treatments. They should also show absence of toxic constituents. Fungal biotechnology techniques also provide us the guidelines of screening for microorganisms giving new products. For example, while isolating new strains of Claviceps, some new ergot alkaloids have been discovered by the biotechnologists. Similarly, the starch – utilizing Fusarium, that is used to produce mycoprotein, was isolated from a starch - rich effluent. In a similar fashion, the inhibition of chitin synthase, an essential requirement for cell wall growth in fungi, has been used as a test for the detection of new antifungal antibiotics. Our knowledge of physiology, biochemistry and fungal biotechnology has helped us in devising tests for detection of (i) antitumour agents, (ii) antiviral agents, (iii) hormones, (iv) immunosuppressants and many other substances which are pharmacologically active.

25.10.2 Fungal Strain Selection and Mutation After selecting a proper strain, the biotechnology can greatly increase the rate of mutation by the use of mutagens. Mutagen is a substance which causes mutation. Artificial mutagenesis is actually the basis of most programming of strain improvement. While using in fermentations, a fungal strain should be genetically stable. It should, therefore, not be an aneuploid (a heterokaryon or a heteroplasmon), because all aneuploids are genetically unstable.

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A series of mutagenic agents are generally used in a strain improvement programme. Some commonly used mutagenic agents include X-rays, ultraviolet radiation, dimethylsuphonate and nitrosoguanidine. While using mutagenic agents proper precautions and technical expertise are needed because most mutagens are carcinogens.

25.11

ROLE OF BIOTECHNOLOGY IN GENETIC RECOMBINATION AND GENE CLONING

25.11.1 Genetic Recombination The process by which offspring can gain combinations of genes (a length of DNA in a chromosome) different from the combinations in either of their parents is called recombination of genes or genetic recombination. In fermentation industry, the strain improvement is generally done by taking a single promising strain and then carrying out the mutation programme and strain selection. When two strains with different features bring about genetic recombination between them, the recombinants with desirable features of both the original strains may be selected. Such genetic recombination of two strains with different features, can be carried out by two processes known as sexual hybridization and parasexual hybridization. In sexual hybridization in Agaricus bisporus, homokaryons of opposite mating types can be obtained, and mated to yield new dikaryotic strains that give fruiting bodies. Genetics and life cycle of Saccharomyces cerevisiae using sexual hybridization have been studied in detail. Its entire genome (the genetic material on the sets of chromosomes in a cell) has been sequenced and functional role for all of these genes are now being worked out in detail. In several fungi of industrial importance (e.g. many species of Aspergillus and Penicillium) sexual cycle is absent. They are able to undergo a parasexual cycle, in which the genetic system allows limited recombination as a result of doubling of the chromosomes in a nucleus, followed by crossing-over and a gradual return to the haploid state by progressive chromosome loss”. The parasexual cycle has proved useful in bringing about genetic recombination between closely related industrial strains, e.g. recombination between strains of Penicillium chrysogenum with high yields of penicillin and some ancestral strains with lower yields but higher growth and sporulation.

25.11.2 Gene Cloning A length of DNA in a chromosome is known as gene. Clone may be defined as “ a set of cells, organisms or microorganisms all derived from a single progenitor by asexual means”. All members of a clone have exactly the same genome or genetic material. In several developed and developing countries, currently a great deal of research is being done on the application of gene cloning in fungi. It is now expected that gene cloning in fungi will soon bring a revolution in the genetic improvement of industrial strains.

25.12

GENE CLONING AND FUTURE OF FUNGAL BIOTECHNOLOGY

Due to the development and discovery of several new techniques in the field of genetics and biotechnology during past 40 years, a new and powerful technology has now developed. This new technology is variously termed as genetic engineering, genetic manipulation, recombinant DNA technology or gene cloning. Almost a revolution has now come in our knowledge of living organisms. Due to these new techniques, following can now be achieved: (i) DNA can be extracted from a donor-microbe, plant or animal and can be treated in many different ways. (ii) A desired portion of donor – DNA can now be introduced into the desired organism (host).

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(iii) In the host organism, this desired portion of host DNA may be replicated, or it may also be expressed into RNA and may then be translated into protein. Large amount of protein and peptide hormones have now been produced using this technique with the host bacterium Escherichia coli. (iv) Techniques have now been developed using yeasts and moulds as hosts for gene cloning. (v) Some fungi can now be used for producing heterologous proteins e.g. yeasts (Kluyveromyces lactis and Hansenula polymorpha). (vi) Success has now been achieved in the production of mammalian proteins by fungi. The first microbe used for commercial production of mammalian proteins was Escherichia coli. This bacterium was also, of course, the first host used for gene cloning. (vii) Human insulin, human growth hormone and interferon (the antiviral factor ) have also been produced using Escherichia coli by these new techniques. (viii) In mammalian cells a peptide can now be modified in several ways following its synthesis. (ix) In place of Escherichia coli, the researches are now aimed at exploring ways of using other organisms as hosts for gene cloning. The success has now been achieved with the yeast (Saccharomyces cerevisiae). This yeast may be of great help because it neither produces toxins nor infects humans. (x) Efforts are now focusing using another yeast (Kluyveromyces lactis) for all the above-mentioned aspects of gene cloning. (xi) For mammalian gene expression, several species of Aspergillus are also now used. The utility and efficiency of this fungus in fermentation technology are well -known. (xii) Scientists have now been able to obtain DNA sequences that code for mammalian proteins. (xiii) If Escherichia coli is being used for the production of mammalian protein, it has to be provided with a DNA sequence corresponding to that of processed mRNA. This type of DNA sequence is known as complimentary DNA or cDNA. (xiv) Methods have now been developed for dealing with the cells that contain more than one type of mRNA. (xv) Techniques have now been developed in which the enzyme reverse transcriptase can be used to make a single –stranded DNA copy of the mRNA, and then a DNA polymerase to obtain double-stranded cDNA. (xvi) Biotechnologists have now been successful in the introduction of DNA into the fungal cells. For gene cloning, the most primary requirement is the availability of a vector, which is a DNA molecule that will survive and replicate in the selected host. Usually, a vector is a circular DNA molecule that may be opened with a restriction enzyme, an endonuclease. This enzyme recognizes a specific nucleotide sequence and cuts the molecule at the desired point and at no other point. (xvii) Most extensively used yeast as a host in the gene cloning is Saccharomyces cerevisiae. (xviii) Biotechnologists have now been successful in employing a polylinker cloning site, particularly in S.cerevisiae. The entire polylinker can now be removed with a single restriction enzyme. It can also be replaced with inserted DNA. (xix) Yeast artificial chromosomes (YACs) have now been developed. They resemble chromosomes in that they are linear with a centromere and at each end a telomere, a sequence that checks attacks on chromosome ends by exonucleases. YACs are ideal for cloning long DNA sequences. (xx) Besides S.cerevisiae vectors have now also been developed for many other fungal hosts, e.g. Kluyveromyces lactis. (xxi) Some success has also now been achieved in the expression of heterologous DNA in fungi. On the basis of the above-mentioned successes, we are soon to get many more required important practical applications of gene cloning in fungal biotechnology.

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TEST YOUR UNDERSTANDING 1. For commercial production of enzymes, the most important fungi are species of: (a) Neurospora (b) Agaricus (c) Saccharomyces (d) Aspergillus 2. Most wine is made commercially with: (a) a culture of Saccharomyces cevevisiae (b) the natural fungal flora of grapes (c) both (a) and (b) (d) an inoculum from the previous fermentation 3. Make a list of at least ten major products of fungal biotechnology. 4. Write an essay on “Fungi and Biotechnology”. 5. What is fermentation technology? Give brief account of some of its major aspects. 6. What are fermenters? Describe some major types of fermenters and their operation. 7. Make diagrammatic diagrams of airlift fermenter and cylindroconical fermenter. 8. What do you mean by enzyme technology? Give an account of some major extracellular and intracellular fungal enzymes. 9. Explain in brief the production technology of some common alcoholic beverages. 10. Technically, “wine” is the product of alcoholic fermentation of the juice of fresh _______, ( _______ ) . 11. “Cider” is produced from _______ . 12. “Sherry”, a fortified wine is made from the juice of _______ . 13. Give an account of “fungi in food processing industry” with particular reference to bread, cheese and fermented milks. 14. Describe the production of secondary metabolites by fungi. 15. Write brief notes on: (a) Cholesterol-lowering drugs (b) Cyclosporin-A (c) Strobilurins. 16. Describe briefly the role of biotechnology in (a)genetic recombination, (b) gene cloning. 17. Discuss some major aspects involving “gene cloning and future of fungal biotechnology”.

26

C H A P T

MUSHROOM CULTIVATION

E R

26.1

MUSHROOMS AND MYCOPHAGY

Mushrooms are the fleshy fungi of Basidiomycotina. They develop basidia within a fruiting body called basidocarp (Fig. 26.1). The basidiocarps develop from an extensive mycelium, often buried in some substratum. The characteristic basidiospores develop at the tip of basidia in the basidiocarp. The basidia remain arranged along the gills of the basidiocarp or fruiting body. Today, hundreds of mushrooms are well-known table delicacies in many parts of the world, and this has all resulted in the development of a separate science of mycophagy (Gr. mykes, mushrooms or fungi; phagein, to eat) which means “eating of mushrooms” or “use of fungi as food”. They are now considered excellent table delicacies, and there has been an increasing global demand for choice mushrooms during last few decades. Because of their increasing demand as food, several growers in India and several other countries have taken up mushroom cultivation on a commercial basis.

26.2

Remains of universal veil Pileus or cap Gills

Annulus or ring

Stipe or stalk

FOOD VALUE OF MUSHROOMS

Mushrooms are not only delicious but nutritious too. They have a high percentage of proteins, vitamins and minerals. They are now considered a good supplementary protein-rich food on the global level, mainly because their protein content is higher than that of several vegetables and pulses including soybeans. In general, 1 kg of fresh edible mushroom contains 50-80 gm of protein, 2-10 gm of fats, 10-20 gm of carbohydrates, 5-10 gm of minerals, 50-150 mg of vitamins and the rest remains the water. Protein content of fresh mushroom (5-8 percent) is much higher than that or cauliflower (2.7%), cabbage (1.5%), carrot (1.1%), banana (1.1%), and

Volva or cup Underground mycelium

Soil

Fig. 26.1

Generalized diagram of a basidiocarp or fruiting body of a mushroom.

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fresh apple (0.3%). Several enzymes reported from edible mushrooms are amylase, maltase, protease, catalase, glycogenase, triosinase, polyphenolase and dehydropeptidase. Edible mushrooms are excellent source of vitamins of B-complex, and they also contain vitamin A, vitamin C, vitamin D and vitamin K. Mushrooms are highly useful in treating anemia because they contain large quantity of folic acid and vitamin B12. They are also considered as an ideal diet for diabetics because of their low caloric value (18-20 k cal./100 gm) and little fat and starch. Some well-known mushroom dishes include mushroom soups, curries, stews in combination with fried rice, pulses and vegetables, mushroom sandwiches, sauce, egg omlette, pakodas, noodles and pickles.

26.3

EDIBLE AND POISONOUS MUSHROOMS

Mushrooms are edible as well as poisonous. And there is no single test of knowing whether a mushroom is edible or poisonous. Therefore, it is advised that unless a wild mushroom is identified by a specialist, the only safe procedure is to consider all wild mushrooms as inedible. Majority of the 200 available species of Agaricus are edible except a few which are poisonous (e.g. A. silvaticus and A. placomyces). Field mushroom or “Khumb” (Agaricus campestris) is the most common wild edible mushroom. Agaricus bisporus is the most common cultivated mushroom of commerce. It is, therefore, commonly called “cultivated mushroom” or “white button mushroom”, and is being eaten all over the world. Some other edible mushrooms cultivated for commercial purposes are paddy straw mushroom (Volvariella volvacea), oyster mushroom (Pleurotus spp.), shiitake (Lentinus edodes), Auricularia sp., Flammulina velutips, Tremella sp., Hypsizygus marmoreus, Pholiota nameko, Grifola frondosa and matsutake (Tricholoma matsutake). Several species of Coprinus, Tricholoma, Volvaria, Amanita (A. fulva and A. vaginata) and Boletus (B. edulis) are also edible. Some of the poisonous mushrooms are Amanita phalloides (death cup), A. verna, A. muscaria, Boletus satanus and several species of Coprinus, Galerina, Inocybe and Psilocybe. Some of these mushrooms are so highly poisonous that they first result into gastrointestinal upset and then into dizziness, unconsciousness and even into death, if taken in large quantity.

26.4

COMMERCIAL CULTIVATION OF MUSHROOMS

Agaricus bisporus, Volvariella volvacea and two species of oyster mushroom (Pleurotus flabellatus and P. sajorcaju) are cultivated on commercial basis in India. At present, there is a well established National Centre for Mushroom Research and Training (NCMRT) at Solan (Himachal Pradesh), with its three sub-centres located at Bangalore, New Delhi and Ludhiana. All these centres are under the control of Indian Council of Agricultural Research (ICAR), New Delhi. Several universities, botanical gardens, horticultural institutes and private organizations in the country are also producing mushrooms on a large scale. These days, it is produced in purpose built mushroom-houses, with an annual output of over several million tonnes. Some other Indian institutes engaged in mushroom cultivation and to supply its technical know-how are Rashtriya Chemicals and Fertilizers, Mumbai; Lalbagh Botanical Garden, Bangaluru and Indian Institute of Horticultural Research, Bangaluru. Over 100 countries in the world are now producing more than 5 million tonnes of mushrooms annually. Agaricus (56%), Lentinus (14%), Volvariella (8%) and Pleurotus (7%) are the four largest cultivated mushroom genera. U.S.A., China, France, Netherlands, U.K., Taiwan and India are some of the largest mushroom producers in the world.

26.5

CULTIVATION OF WHITE BUTTON MUSHROOM

Following steps are followed for cultivation of white button mushroom or cultivated mushroom, i.e., Agaricus bisporus on commercial basis:

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The spawn is a pure culture of mycelium of the mushroom grown on a special growth medium. This is produced by the experts in the laboratory under highly controlled conditions. The spawn can also be produced by culturing a piece of tissue of fresh basidiocarp of the mushroom on the base medium. The base medium usually contains chalk, water and powdered grains (e.g. wheat, sorghum, pearl millet or rye) in the bottles. The compost is a substrate on which the spawn grows and ultimately the mushrooms develop. Ususlly the horse or cow manure is used for the compost production. Horse dung, collected from stables, in which abundant wheat straw has been used, is the best material for compost. For compost production, 28 lb of gypsum is added to every ton of fresh horse or cow manure. The mixture is turned after regular intervals until it is not completely decomposed. 14 lb of superphosphate per ton is added in the mixture at the time of last turning. Thus-formed mixture is allowed to undergo natural fermentation for about 2 weeks with the help of fungi, bacteria and other organisms. In this process the compost is filled in large wooden trays arranged in tiers for 7-10 days or more at a temperature of about 55°C. Instead of wooden trays, the compost may be placed on the ground or floor of the mushroom house (any “kuchha” or “pucca” house) or in specially prepared sheds. 6-9 inch thick layer of compost is usually filled in wooden trays. With the help of a wooden board, compress the compost of the trays, leaving 1 cm clear space on the top of each tray, Light is not required for mushroom cultivation. Spawning means “inoculation of compost beds” or planting of mushroom mycelium in the compost.” Chunks of spawn, growing on a suitable substrate, are cut into small squares and temperature of about 80°F. (At this stage, maintenance of proper environmental conditions is essential. Otherwise it is usually seen that several undesired weed fungi develop in the compost). Thus, spawned beds are covered with newspaper sheets, and the water is sprinkled every other day or daily on the newspaper sheets. This will provide the desired humidity and moisture to the growing mycelium. Within 10 – 15 days, a thick growth of white cottony mycelium covers the top surface of compost in the beds. Casing means covering the compost with a thin layer of soil or soil-like casing material. It is usually done after 10-15 days of spawning when the compost beds are covered by white cottony mycelium. The casing material usually consists of top soil or sub-soil, sand, loam, gravel, ash, litter or alkaline peat. Newspaper sheets are removed and the beds are covered with a 2-2.5 cm thick layer of sterilized casing material. The beds covered with casing soil are sprayed with fine nozzle of a sprayer with definite intervals. This will maintain a desired relative humidity between 70 – 80 per cent. After a period of about 3 to 8 weeks, the mushrooms start appearing in the form of small white button-like growths. The sporophores are soon produced at their top. Harvesting of mushroom is the final stage. The crop is harvested with a knife daily or even twice in a day. The right stage to harvest a mushroom is when its cap is still in the tight position with its stalk. A bed may go on producing mushrooms for about 3-4 months. If the liquid manure is applied twice a week, the mushroom production is stimulated and may be maintained for 6-8 months. Mushrooms may be stored at 4°C in a refrigerator but only for a few days.

26.6

MUSHROOM GROWING IN LABORATORY

In this section, we will study different methods of growing mushrooms in laboratory:

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The growth medium is prepared in the following proportion: Dextrose ................................................................................15.0 gm Brewer’s yeast .......................................................................1.0 gm Casein hydrolysate ................................................................2.5 gm CaCO3 ...................................................................................3.0 gm 1MKH2 PO4 (136 g/l H2O) ....................................................10.0 ml Prepare the stock solution (a), trace-element solution (b) and final Hoagland’s solution (c) as under: (a) Stock Solution Prepare each of the following in 100 ml H2O: 1. Ca (NO3)2 . 4H2O .............................................11.8 gm 2. KNO3 ...............................................................5.0 gm 3. MgSO4 . 7H2O ..................................................1.4 gm 4. KH2PO4 ............................................................1.4 gm 5. Iron chelate ......................................................1.0 gm (b) Trace-element Solution 1. ZnSO4 . 7H2O ...................................................2.2 gm/100 ml H2O (Use 10 ml)

2. CuSO4 . 5H2O ...................................................0.8 gm/100 ml H2O (Use 10 ml)

3. Na2MoO4 . 2H2O ..............................................2.5 gm/100 ml H2O (Use 1 ml)

Final Trace – element Solution 10 ml each of Nos. (1) and (2) and 1 ml of No. (3) MnC12 . 4H2O ..........................................................1.8 gm H3BO3 ......................................................................2.8 gm Water ........................................................................979 ml (c) Final Hoagland’s Solution To 940 ml H2O add 10 ml each of the five stock solutions mentioned above in (a) and also add in it 10 ml of the final trace element solution. 1. Take 500 ml of final Hoagland’s solutions (B) and add in it 500 ml H2O. 2. Add enough of this diluted Hoagland’s solution in the growth medium (A) to make 1 litre of final medium. 3. Take a large-sized beaker and prepare a growth chamber by putting in it 600 ml of vermiculite of perlite. Add 450 ml of the final medium prepared in step 2. 4. The beaker is covered first with a paper towel and then with an aluminium foil sheet (Fig. 26.2A). Make the two sheets tight with a string around the top of the beaker. 5. Sterilize the covered growth container in an autoclave for 20 minutes at 15 1b pressure. 6. Cool the growth chamber completely and transfer the portions of the spawn of mushroom about 1.5 to 2.5 cm deep into the growth medium with a transfer loop. 7. Again cover the growth chamber with paper towel and aluminium foil sheet, and keep it in dark for about 3 weeks at 20°C. Within 3 to 4 weeks, white cottony growth of the fungal mycelium spreads throughout the medium (Fig. 26.2B).

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Aluminium foil sheet

Small fruiting body Soil layer White cottony mycelium Paper towel Growth medium with vermiculite Beaker

A

Fruiting bodies of mushroom

C

Fig. 26.2

B

D

A method of growing mushrooms in the laboratory.

8. About 2.5 cm thick layer of moist, fine-textured, unsterilized loam is added on the top of the growth chamber. This is called casing. This induces the formation of small fruiting bodies of the mushroom in the top layer (Fig. 26.2B). 9. Cover the growth chamber, keep it back under same environmental conditions and wait for about a week. Sprinkle the water on loam on alternate days to keep it moist. Small white fruiting bodies of the mushroom begin to appear (Fig. 26.2C) within 7-10 days. 10. The white buttons soon enlarge and open (Fig. 26.2D). Harvesting is done usually at button stage of the mushroom.

26.7

CULTIVATION OF SHIITAKE (LENTINUS ELODES)

In Japan and several other countries, shiitake is cultivated on a large scale. In 1999, a total world production of shiitake was 1564000 tones while Agaricus bisporus was produced 1456000 tonnes (Chang, 1999). Lentinus elodes grows in nature on dead wood of oaks, chestnuts, etc. It is cultivated in young oak trunks with a diameter of 5-15 cm. Trunks are felled in late autumn when they have high concentration of sugar. Trunks are cut into pieces or logs of 1 metre lengths and stored for about two months. About two dozen holes are drilled into these logs to receive the pure culture inoculum of Lentinus elodes. These holes are sealed with hot wax to check evaporation or infection. These inoculated logs are now laid on racks on woodland. Mycelium starts developing after about one year. The logs are now transferred in late autumn to well-sheltered, cool, humid places and kept upright against bamboo fences. In the spring season a good crop of high

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quality mushroom is obtained. An inferior quality of crop is obtained in the following autumn. These mushrooms may be sold fresh, canned or dried. Shiitake has a stronger taste in comparison to common cultivated mushroom (Agaricus bisporus). While cultivated in greenhouses, shiitake is cultivated on a mixture of sawdust and rice bran.

26.8

PADDY STRAW MUSHROOM

Volvariella volvacea is a very delicate mushroom used as a table delicacy in many parts of the world. It is commonly called paddy straw mushroom, or straw mushroom. It is now grown commercially in several countries including China, India, Myanmar, Indonesia, Philippines, Malayasia and Nigeria. The general process of its cultivation is almost the same as that of cultivated mushroom described above under Article 26.5. The traditional method of cultivating V. volvacea involves the use of 20-80 cm wide beds under partial shade or in open field. The paddy straw is the most commonly used substrate. It is soaked in water by complete immersion in large tanks for about 24 hours. Preferably the straw bundles are sterilized using an autoclave. The straw is now folded and arranged into stack on a soil base. The stack is then set up in several layers, and each layer is spawned with pure culture of the mushroom grown on sterilized straw at points about 20 cm apart. The procedure of spawning is repeated with each layer, decreasing gradually in width. Usually the beds are watered after 5 days. Within 12 to 15 days after spawning, the mushrooms start appearing in the bed. Under controlled conditions of temperature, humidity and ventilation, this mushroom is now cultivated indoor in plastic houses. In the indoor cultivation method, the pasteurized compost is used and the mushroom beds are cased with a clayey loam layer of about 2 cm thickness. In India, the paddy straw mushroom is cultivated on a large scale at several stations, including Indian Agricultural Research Institute, New Delhi; National Botanical Research Institute, Lucknow; Regional Research Laboratory, Jammu and; Agricultural Research Institute, Coimbatore.

26.9

OYSTER MUSHROOM

The oyster mushroom (Pleurotus ostreatus, P. flabellatus and P. sajorcaju) is edible and has been cultivated successfully in India on a commercial basis. It proliferates well on wheat straw, sugarcane bagasse, groundnut pod shells and paper waste. The mushroom can be easily cultivated in polybags in empty balconies and sunshades in multistoried buildings. In the recently improved polybag technique of cultivation of oyster and other mushrooms, polybags (30x45 cm size) and nylon nets are used. Chopped paddy straw (750 gm) is soaked in water for about 24 hours. The excess water is drained off soon. The substrate is preferably sterilized in pressure cooker for about 10 minutes and then dried in shade for about an hour. About three-week-old spawn is added to the substrate and mixed thoroughly. This mixture is packed in polybags having several holes in their middle portion. The mouth of the bag is tied with nylon thread, and the bags are kept in shade in a dry place at a temperature of about 30 – 35°C. Bags are cut open very gently after about ten days. During this period the fungus spreads and the pulp becomes compact resembling a ball. It is now transferred to a nylon net and hung on a hook in the balcony. Water is sprinkled twice or thrice daily. Within 3-4 days, small, whitish-brown bodies of the mushroom appear. Insecticides (e.g. gammexane) may be sprayed to protect mushrooms from flies, etc. An yield of about 250 gm of mushroom may be obtained daily from each nylon net.

26.10

COMMERCIAL PRODUCTION OF SOME OTHER MACROFUNGI

Some highly prized and expensive edible macrofungi are also now produced commercially. These include some Basidiomycetes (e.g. Catharellus, Cibarius and Boletus) and Ascomycetes e.g. morel (Morchella esculenta) and truffle (Tuber melanosporum).

Mushroom Cultivation

26.11

311

MUSHROOM PARASITES

Some of the common mushroom parasites, known for causing diseases and sometimes great loss in yield, are Cladobotryum dendroides, Fusarium solani, Myceliophthora lutea, Mycogone perniciosa and Verticillium lamellicola.

26.12

MUSHROOM DISHES

Mushroom dishes are famous in many countries of the world, and a large number of delicious mushroom recipes are now available. They are famous due to their highly delicious taste, food value and special flavour. These dishes also have quick-cooking properties. Mushroom soups, curries, and stews in combination with fried rice, pulses and vegetables are highly-liked dishes. Some of the other famous preparations are mushroom sandwiches, sauce, ketchup, egg omlette, pakodas, pickles and noodles. Delicious dishes are also prepared by cooking mushrooms with other vegetables such as potato, tomato, cauliflower, peas and beans.

TEST YOUR UNDERSTANDING 1. 2. 3. 4. 5. 6.

7. 8. 9. 10. 11. 12.

Define mycophagy. Mushrooms are the fleshy fungi of _______ . Mushrooms have high percentage of vitamins, minerals and _______ . Mushrooms are considered as an ideal food for _______ . Most common cultivated mushroom of commerce is _______ _______ . Which of the following is edible mushroom? (a) Volvariella volvacea (b) Lentinus edodus (c) Tricholoma matsutake (d) All of these Amanita phalloides, A. verna, A.muscaria and Boletus satanus are all _______ mushrooms. Give an account of various steps followed for the cultivation of white button mushroom on commercial basis. Explain following terms used in the process of mushroom cultivation: (a) Spawn (b) Compost (c) Casing (d) Harvesting Enlist various steps used for growing mushroom in laboratory. Write a note on cultivation of Shiitake. Write botanical names of following mushrooms: Oyster mushroom, Paddy straw mushroom, Shiitake.

27

C H A P T

SINGLE-CELL PROTEIN

E R

27.1

WHAT IS SINGLE-CELL PROTEIN?

The microbial product or the dried cells of certain yeasts, bacteria, moulds, higher fungi and algae grown in mass culture for its use as a source of protein in foods or animal feeds is called Single-Cell Protein or SCP. According to Roth (1982), “the microbial fermentation results in quick growth of selected microorganisms which are rich in protein and the dried biomass of the end product of these organisms is referred to as single-cell protein or SCP. Single-cell protein is thus the “protein-rich material from cultured algae, fungi (including yeasts) or bacteria, used (potentially) for food or as animal feed”. The principal organisms that have been used for SCP production in the world are certain yeasts and methylotrophic bacteria (e.g. Methylophilus methylotrophyus). Production of SCP requires no farms and may be done in the laboratories or large factories by means of natural microorganisms. Inspite of the fact that a very large amount of protein may be obtained form the microorganisms, and the protein deficiency problem may be solved easily, the microbial protein is not yet directly consumed by human beings. It is because of their high nucleic acid content. SCP is being used for supplementing the feed ration of animals such as hens, pigs, calves and cows. For human beings, they are used as sources of dietary protein because some specific amino acids are also produced form them. We use wheat, rice, corn or pea in our daily diet. But wheat protein is low in lysine, rice protein in lysine and threonine, corn protein in lysine and tryptophan and pea protein in amino acid methionine. The addition of a particular deficient amino acid to our diet is, therefore, essential. The specific amino acids, produced from the microorganisms, may be used for this purpose.

27.2

WHY DO WE NEED TO PRODUCE SCP?

We need to produce SCP because of following main reasons: 1. A very large section of world’s population is poorly nourished, and the dietary component that is usually in short supply is protein. 2. Plants, being largely carbohydrates, have a relatively low protein content. 3. Some essential amino acids (e.g. methionine and tryptophan) are very low in plant protein. 4. Meat, possessing high-grade protein, is too expensive for much of the world’s population. 5. Both plants and animals are easily affected by climate, and unseasonable weather, droughts and floods also drastically reduce their yield.

313

Single-cell Protein

On the other hand, many microorganisms (e.g. fungi, bacteria, algae as the source of a single-cell protein are useful because of the following: 1. They are able to use cheap sources of nitrogen (e.g. ammonium salts and nitrates). 2. They are able to use abundant carbon sources such as starch, natural gas and petroleum hydrocarbons. 3. The resulting biomass produced by SCP-producing microorganisms has a high protein content. 4. Growth rates of SCP-producing microorganisms are very high. Because of this quality, a compact fermentation plant can produce as much protein as a large area of agricultural land in any climate. Because of these qualities, massive industrial research and development programmes have been initiated globally to aim at the large scale production of single-cell protein for human consumption.

27.3

MICROORGANISMS USED FOR SCP-PRODUCTION

Hundreds of microorganisms are currently exploited for production of single-cell protein in the world. These include over four dozen yeasts, about a dozen bacteria and many filamentous fungi and algae. All these microbes are very rich in protein. Principal organisms utilized for SCP-production are Candida, Saccharomyces, Rhodotorula, Rhodopseudomonas, Methylophilus, Methanomonas, Cellulomonas, Thermoactinomyces, Hydrogenomonas, Spirulina, Chlorella, Scenedesmus, Paecilomyces, Chaetomium, Cephalosporium, Fusarium and Penicillium. In India, National Botanical Research Institute, Lucknow and Central Food Technological Research Institute, Mysore started work on mass cultivation and utilization of Spirulina platensis as a source of SCP. This alga contains 60% crude protein and is quite rich in lysine, tryptophan and vitamin B12.

27.4

COMPOSITION OF SINGLE-CELL PROTEINS

Basically, single-cell proteins are composed of proteins, fats, carbohydrates, water and ash ingredients including potassium and phosphorus. Their composition, however, depends mainly on the organism and the substrate on which they grow. Generalized composition of some representative single-cell proteins is given in Table 27.1. Table 27.1 Generalized composition of some representative single-cell protein Organisms

1. 2. 3. 4.

27.5

Bacteria Yeasts Fungi (filamentous) Algae

Composition, wt.% True protein

Lysine

Methionine

Fats

50-80 45-65 30-40 40-50

5.8 7.4 6.5 4.6

2.2 1.8 1.5 1.4

8 9 5 5

ADVANTAGES AND DISADVANTAGES OF USING MICROORGANISMS FOR ANIMAL OR HUMAN CONSUMPTION

There are both advantages and disadvantages of using microorganisms for animal or human consumption, and specially for their utility as single-cell proteins. Bacteria have a rapid growth rate and are usually very high in their protein content (50-80 per cent; see Table 27.1). But they have many disadvantages: (i) Bacterial cells have very small size and low density, due to which it becomes very difficult to harvest them from the fermented medium, and the entire process becomes very costly; (ii) their high nucleic acid content may prove detrimental to human beings because it increases the uric acid

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Fungi and Allied Microbes

level in the blood, causing ultimately uric acid poisoning. The advantages of using yeasts include their (i) comparatively larger size than that of bacteria, which makes them easy to harvest; (ii) lower nucleic acid content; (iii) high lysine content; (iv) ability to grow at acid pH; and (v) familiarity and acceptability by the general public because of the long history of their use in traditional fermentation processes. The disadvantages of using yeasts in comparison to bacteria include their (i) lower growth rate; (ii) lower protein content (45-65 per cent) and; (iii) lower methionine content (Table 27.1). The advantages of using filamentous fungi include their easy cultivation but their disadvantages include their lower growth rate, lower protein content, and least familiarity and acceptability by the general public. Main disadvantage of using algae lies in their cellulosic cell wall which is not digested by human beings.

27.6

MYCOPROTEIN

Several fungi, including yeasts and moulds, produce single-cell protein called mycoprotein. Fusarium graminearum is used on large scale production of SCP for human consumption in U.K. Quorn mycoprotein produced from this fungus has a high protein content with a satisfactory amino acid composition. Its lipid content is lower than in meat, and the animal sterol cholesterol is absent. Although, a detailed technology is involved, “the production of Quorn mycoprotein is a major success story for fungal biotechnology” according to Carlile et al. (2001). Several yeasts are good source of protein and vitamins and can be easily grown on a variety of waste materials. Various species of several genera (Saccharomyces, Candida, Torulopsis) are grown as source of SCP. Huge production of Candida yeast protein from hydrolysed peat is done in Russia each year. Dried yeast is sold as a nutritional supplement in many health stores throughout the world.

27.7

SINGLE-CELL PROTEIN FROM CYANOBACTERIA

Some Cyanobacteria (e.g. Spirulina) are used these days for producing single-cell protein. Spirulina has been grown for centuries in alkaline lakes of Africa, Mexico and Peru. Its cells are harvested, sun-dried, washed to remove sand and made into several articles, like cakes, for human consumption. Dried Spirulina has about 65% protein, and is now proving a very valuable food in many developing countries.

27.8

SINGLE-CELL PROTEIN FROM ALGAE

Chlorella and Scenedesmus are excellent examples of algae which have been cultured in several countries of Asia, Europe and central America as a source of single-cell protein and thus as a major food source. SCP-production from algae is advantageous because these organisms utilize solar energy and thus reduce the amount of fuel resources required to produce single-cell protein.

27.9

SINGLE-CELL PROTEIN FROM ORGANIC WASTES

Large amounts of organic wastes are produced in the industries based on agriculture and forestry produce. Such wastes are now converted into single-cell protein in many countries, including India. Candida utilis (food yeast) can be grown in the industries where these wastes are rich in sucrose or glucose. Molasses is used for this purpose in many countries including India, Cuba and Brazil. In U.K. the “food yeast” is grown on a small scale with confectionery waste. Whey is a waste product obtained from cheese manufacturing. It is used for SCP production by a yeast (Kluyveromyces fragilis) in many countries including USA and France. This yeast has the ability to utilize lactose of the whey. Starch-rich wastes are used for SCP production in Sweden using Candida utilis. In these processes preliminary hydrolysis of starch into sugar is carried out by yet another yeast, Endomycopsis fibuliger. A small amount of SCP is produced from cellulose wastes using

Single-cell Protein

315

an Ascomycetous fungus Chaetomium. However, if not processed properly, toxic phenolic compounds, may be formed during the production of SCP from cellulose wastes.

27.10

SCP FROM PETROLEUM HYDROCARBONS AND OTHER RELATED SUBSTRATES

Crude oil possesses n-paraffins, unbranched hydrocarbon molecules, which is used by some yeasts. These yeasts are used for the production of SCP with n-paraffins as the source of carbon. One raw material is gas oil, a crude petroleum fraction with a boiling temperature of 300-380ºC and 10-25% n-paraffins. One oil company produced 16000 tonnes of SCP per year from this gas oil. Another oil company used purified n-paraffins and produced about 4000 tonnes of SCP per year. Some aerobic microorganisms have the ability to use natural gas which mainly consists of methane. Mixtures of methane and air are explosive and hence dangerous. Biotechnological techniques have now been developed for conversion of dangerous methane into much safer methanol, which can be used by some yeasts and bacteria. Later on, a process was developed for the bacterial production of SCP form methanol. Since crude oil contains carcinogenic materials, production of SCP form n-paraffins and gas oil should be done with utmost care involving latest available biotechnological techniques. Several moulds (e.g. species of Fusarium) produce mycotoxins and should be used for SCP-production with great care making trials with several toxicological tests.

TEST YOUR UNDERSTANDING 1. 2. 3. 4. 5.

What is a single-cell protein (SCP)? Why do we need SCP? Make a list of at least ten microorganisms used for SCP-production. Write a detailed note on single-cell protein obtained from algae including Cyanobacteria. What are mycoproteins? Discuss them briefly Which of the following is/are used these days for production of single-cell protein? (a) Chlorella (b) Spirulina (c) Scenedesmus (d) all of these 6. Write a scientific note on “single-cell protein from organic wastes”.

28

C H A P T

HETEROTHALLISM IN FUNGI

E R

28.1

WHAT IS HETEROTHALLISM?

A condition shown by heterothallic species is called heterothallism. What are then heterothallic species? Alexopoulos and Mims (1979) mentioned that there are two versions about a heterothallic species. According to one version, a species consisting of self-incompatible or self-sterile individuals, which for sexual reproduction requires the union of two compatible thalli (mating types), is called heterothallic. According to another version, a species, in which the sexes are segregated in separate thalli, and therefore two different thalli are required for sexual reproduction, is called heterothallic. Thus it is a condition where the thalli of a single species are morphologically similar but physiologically different. Physiologically the mycelia are unisexual, but there is no apparent distinction between the male and female mycelia. They differ only in their sexual behaviour. Such a condition of sexual incompatibility in fungi was first observed in Mucorales by an American geneticist, A.F. Blakeslee in 1904. The term “heterothallism” was first used by Blakeslee (1904) for describing a condition of sexual reproduction in some species of Mucorales. Blakeslee (1904) observed that “conjugation is possible only through the interaction of two different thalli”. He divided various species of Mucorales into two major categories as homothallic and heterothallic, and these categories “correspond respectively to monoecious and dioecious forms among the higher plants”. Regarding heterothallic species, Blakeslee (1904) opined that “The condition is essentially similar to that in dioecious plants and animals” and finally concluded “that the formation of zygospores is a sexual process; that the mycelium of a homothallic species is bisexual, while the mycelium of a heterothallic species is unisexual, and further, that in the (+) and (–) series the heterothallic groups are represented in two sexes”. In this way, Blakeslee believed that plus (+) and minus (–) strains of the heterothallic Mucorales differ in sex, and so the term “heterothallism is equivalent to dioecism in a haploid organism”. In general, the term “heterothallism” may be defined as “The condition in which there are two (or more) mating types with sexual reproduction only successful between individuals of a different type”. An “individual plant or a clone, incapable of self fertilization” is called self-incompatible.

28.2

HETEROTHALLISM IN MUCORALES

According to Blakeslee (1904) a heterothallic species in Mucorales contains two physiologically and sexually different strains or races. Such races, when grown apart, produce only asexual bodies or sporangia (Fig. 28.1 A, B). But, when they are allowed to come in contact, they fuse and from zygospores (Fig. 28.1 C) along the line of their union. Blakeslee named these different strains as + (plus) and – (minus).

317

Heterothallism in Fungi

Zygosporangium

Zygospore Sporangia

+

Sporangiophores +

+

Zygospores

– Promycelium

Spore

A +



B – Spore



E – Spore

D –

+ C Spore

Spore

No zygospores A

Fig. 28.1

B

C

D

Phenomenon of heterothallism. A, Plate with only + strain producing sporangia and no zygospore; B, Plate with only – strain producing sporangia and no zygospore; C, Plate with + and – strains producing zygospores; D, Zygospore formation at the junction of mycelia produced from spores carrying different strains.

Of course, morphologically + and – strains are indistinguishable, but often the + strain shows more vigorous growth than – strain, and the gametangia of the former (+ strain) are larger than that of the latter (– strain). Some workers are, however, of the opinion that this difference in gametangial size is not because of any difference of sex but because of available nutrients. Blakeslee also noted that + strain generally grows better on maltose than the – strain. Blakeslee (1904) proved also experimentally this phenomenon of heterothallism in Mucorales. He noticed clearly that conjugation took place only in between the hyphae of two different strains. The zygospores were formed only along the line of the union of + and – hyphae. Figure 28.1 D shows a petri-dish containing a suitable sterilized medium inoculated with the spores A, B, C, D and E of known strains. A and C spores belong to +strain, whereas B, D and E belong to – strains. The zygospore formation takes place only at the union of mycelia produced along AB, BC, CD, AE and AD, because in all these cases they belong to two different strains. No zygospores are formed along DE and AC, because along these lines the mycelial strands belong to one and the same strain, i.e. – and – along DE, and + and + along AC. Blakeslee (1904) also showed that opposite mating types of different species of Mucorales might also sometimes interact with one another to from zygophores, but in such cases plasmogamy and zygospores are rarely formed. Burgeff (1924) demonstrated experimentally that for the initiation of the sexual reproduction in Mucorales, a diffusible substance is responsible. Burgeff’s discovery demonstrated that the sexual reproduction mechanism is a hormonal reaction. Observations of Gooday (1973), Sutter (1977) and Van den Ende (1978) also showed the existence of hormonal sexual mechanism in Mucorales. In a majority of the mucoraceous fungi investigated so far the hormone responsible for the sexual mechanism is trisporic acid. According to Gooday (1973) the trisporic acid in Mucorales is not produced in detectable amounts in + and – strains when grown separately. But when both these strains (+ and –) are grown and mated in the same culture dish, trisporic acid is produced, and it induces the phenomenon of zygotropism, i.e.formation of zygophores ant their fusion resulting into the ultimate formation of zygospores, as shown below: (+) Mycelium

Trisporate synthesis

(–) Mycelium

Trisporate synthesis

(+) Zygophores Zygotropism (–) Zygophores

Zygospores

Researches of Werkman and Van den Ende (1974) suggested that each + and – molecule produces precursor molecules, which are converted by compatible strains into trisporic acid. According to Van den Ende and Stegwee (1971) normally a mixture of trisporic acids A, B and C is found in cultures, and for zygophore formation mainly trisporic acids B and C are responsible.

318

28.3

Fungi and Allied Microbes

HETEROTHALLISM IN SOME OTHER LOWER FUNGI According to Gallindo and Gallegly (1960) P. infestans is heterothallic. Two types of mycelia (A and B) are formed by this species, and both these mycelial types produce both the sex organs i.e. antheridia as well as oogania. They are, however, self-sterile and cross-fertile. In species occurring in Mexico, the oospores are common and both the mating types of mycelium occur in plenty. In species occurring in other parts of the world, however, oospores are rare, and only one mating type of the mycelium occurs. Both these species of Achlya show the occurrence of heterothallism. In both these species Raper (1955, 1957) discovered a definite role of special hormonal mechanism in sexual reproduction.

28.4

28.4.1

HORMONAL BASIS OF SEX AND HETEROTHALLISM IN LOWER FUNGI Oomycetes

As mentioned above, Achlya bisexualis and A. ambisexualis exhibit a complicated type of heterothallism (Raper, 1939, 1951, 1955, 1957). When thalli of these species are grown all alone, there is no formation of sex organs (gametangia). However, if two kinds of thalli are grown in close contact, they show the formation of gametangia. Following four different kinds of thalli are produced in these species according to Raper. (i) Pure male thalli; (ii) Predominantly male thalli having a hidden capacity to produce oogonia; (iii) Pure female thalli; and (iv) Predominantly female thalli having a hidden capacity to produce antheridia. When any of these above-mentioned kinds of the thalli is grown in contact with any of the other thalli, they start producing sex organs. This shows that heterothallism does exist, and this type of heterothallism is called gynandromictic. Raper (1955, 1957) has also shown that development of sex organs in Achlya bisexualis and A.ambisexualis is governed by a set of hormones. It is so because when grown all alone neither antheridia nor oogonia are formed. These hormones have been referred as sex hormones, and have been discussed in detail in Chapter 29. It is thus clear that heterothallism as well as formation of sex in these fungi is governed by the secretion of sex hormones.

28.4.2

Zygomycetes

Blakeslee discovered heterothallism in Mucorales in 1904 and after about two decades, Burgeff (1924) demonstrated that a diffusible substance is responsible for the initiation of sexual reproduction in Mucorales of Zygomycetes. Fungi thus show a hormonal sexual mechanism, at least in some Mucorales (Burgeff, 1924). Several workers then started working on this aspect, and noted the nature of these hormones involved in sexual mechanism (Gooday, 1974; Bu’lock, 1976; Van den Ende, 1978).

28.5

HETEROTHALLISM IN ASCOMYCETES

In comparison to Lower Fungi, much more accuracy or exactness is shown by members of Ascomycetes in the regulation of their mating systems. Many of the Ascomycetes are homothallic or homomictic while many others are heterothallic

Heterothallism in Fungi

319

or dimictic with a well-defined genetic control of their mating systems. Sordaria macrospora and S. fimicola are the two most studied ascomycetous fungi showing homothallism. In heterothallic Ascomycetes, compatibility is determined by a pair of genes A1 and A2. These genes are segregated at meiosis just before the formation of ascospores. Only because of this, out of eight ascospores of an ascus four contain gene A1 and the remaining four ascospores contain gene A2. On germination, each of these ascospores will give rise to such a mycelium whose nuclei will carry only one gene, i.e. either A1 or A2. Antheridia (or spermatia) and ascogonia are formed on this mycelium. Both these sex organs (male and female) will carry the same gene (factor), i.e. either A1 or A2, and therefore will remain unable to mate among themselves. For the sexual fusion, therefore, two thalli of different genetic make-up must be brought together, so that an A1 ascogonium may come in contact with an A2 antheridium, and vice versa. In this type of heterothallism the mating individuals consist of two groups that differ in their genetic make-up for the compatibility factor, and hence it is called bipolar heterothallism. Some Ascomycetes show tetrapolar heterothallism. Instead of two, four mating types of mycelia are contained in these fungi, and the compatibility in them is controlled by two pairs of factors, i.e. A1 A2 and B1 B2. These two pairs of factors are located on different chromosomes. Only those thalli are compatible whose nuclei contain opposite genes of both Mendelian pairs A1 A2 and B1 B2. The species like Neurospora crassa and N. sitophila are hermaphroditic, heterothallic and octosporous. In both these species, the incompatibility is controlled by a pair of alleles A and a which segregate at meiosis immediately before ascospore formation. Due to this, four ascospores in each ascus carry gene A and the remaining four ascospores carry gene a. Each ascospore germinates and produces mycelium in which all the nuclei carry only one factor i.e. either A or a. Both the sex organs formed on this mycelium carry the same factor and thus will be self sterile. But, if two compatible strains are grown together in the same culture tube for a few days, male gamete of one strain A can be transferred to the trichogynes of the ascogonia of opposite strain a. Soon, plasmogamy between the trichogyne of the ascogonium and the male gametes results in the formation of dikaryons (A+a). Fusion between two nuclei results in the formation of a diploid nucleus (Aa). This further gives rise to eight ascospores in the ascus due to ascosporogenesis. These 8 ascospores are of two mating types (A and a). Compatibility at this stage is governed by a pair of alleles A/a at the same locus, and such a one-locus, two-allele type of compatibility has been named as bipolar heterothallism by Whitehouse (1949). Such a bipolar heterothallism has been reported in many other Ascomycetes by different workers, like Sordaria brevicolis by Olive and Fantini (1961), S. heterothallis by Fields and Maniotis (1963) and several species of Aspergillus by Raper and Fennell (1965).

28.6

HETEROTHALLISM IN BASIDIOMYCETES

Heterothallism in Basidiomycetes is of common occurrence. Only about 10 per cent of Basidiomycetes are homothallic, and show primary homothallism as in Coprinus sterquilinus, or secondary homothallism as in C. ephemerus form bisporus. In approximately 25 per cent of Basidiomycetes homokaryon compatibility is controlled by a single gene, designated as A. Many alleles of this gene exist in the population in such Basidiomycetes. Homokaryons with different A alleles (e.g. A1 + A2; A1 + A3) are compatible, and in them the fusion of the hyphae results in the formation of a dikaryon, whereas with the same A alleles these fungi are incompatible. Compatibility control of this type is called bibolar. It is so because as a result of meiosis half the basidiospores from a single fruiting body contain only A allele while the remaining half the other allele. Due to the involvement of only a single factor (or single gene), the genetic basis for the bipolar compatibility is called unifactorial, as in most of the rusts and smuts. Instead of a single factor (or gene) a more complex compatibility control is seen in some Basidiomycetes by the existence of two factors (or two genes), called A and B with two alleles at each locus, of which both have many alleles, e.g. A1, A2, A3, A4....An. Due to the involvement of two separate factors, the genetic basis is called bifactorial. In such cases, the compatibility occurs only when alleles of both A and B are different, i.e. A1, B1 and A2, B2. If one or other allele is common, the hyphal fusion, of course, may occur, but there is no formation of a dikaryon. A and B factors are

320

Fungi and Allied Microbes

located in different chromosomes and four combinations of A and B alleles (e.g. A1 B1, A1 B2, A2 B1 and A2 B2) are derived in basidiospores from a single basidium in equal frequency. Two gene systems are therefore present. The species having the two gene systems are called tetrapolar, as in Hymenomycetes and Gasteromycetes. More than 50 per cent of Hymenomycetous and Gasteromycetous fungi are tetrapolar according to Whitehouse (1949). In Psathyrella coprobia, the compatibility has been octopolar because it is determined by three factors A, B and C, and its genetic basis is called trifactorial (Jurand and Kemp, 1973).

TEST YOUR UNDERSTANDING 1. 2. 3. 4.

Explain heterothallism in about 100 words. Who was the first to observe heterothallism in Mucorales? How did Blakeslee proved the phenomenon of heterothallism experimentally? Who was the first to demonstrate experimentally that the sexual reproduction mechanism in Mucorales is a hormonal reaction? 5. Explain in brief the hormonal basis of sex and heterothallism in lower fungi. 6. Discuss bipolar heterothallism and tetrapolar heterothallism in Ascomycetes. 7. What do you mean by bipolar compatibility? Explain this phenomenon in terms of heterothallism in Basidiomycetes.

29

C H A P T

SEX HORMONES AND PHEROMONES IN FUNGI

E R

29.1

HORMONES, SEX HORMONES AND PHEROMONES

A substance which, in very small amounts, controls growth and development is called hormone. Hormones are actually chemical messengers which are usually produced in one organ and transported to another part of the plant, where they have their effects. Usually, a hormone is an organic substance produced in minute quantity in one part of an organism and transported to other parts where it exerts profound effect. Sex hormone is a “diffusible substance playing a specific role in sexual reproduction of the organism that produces it” (Van den Ende, 1978). Several reviews on the sex hormones in fungi have been published by mycologists, including Machlis (1966), Gooday (1974) and Bu’Lock (1976). Pheromone is a term which was earlier used for “a chemical substance secreted by an animal, which influences the behaviour of other animals of the same species”. Pheromones have now been detected in several other organisms, including algae and fungi. Carlile et al.(2001), however, opined recently that “Animal physiologists use the term hormone for a factor concerned with chemical coordination within an individual, and pheromone for a factor that is emitted and produces effects in other individuals in the same species. The distinction is an artificial one in the fungi since it is probable that the same factors are involved in the coordination of sexual activity both in self-sterile species , in which interaction is between different sites in the same colony”. In fungi, however, the general use has settled on “hormone for the lower fungi, and pheromone for Ascomycetes and Basidiomycetes” (Carlile et al., 2001). According to these workers, therefore, the diffusible factors involved in the regulation of sexual processes in fungi have been termed as hormones and pheromones.

29.2

SOME EARLIER STUDIES ON SEX HORMONES AND PHEROMONES IN FUNGI

The involvement of hormones in sexual reproduction in fungi was first proposed by de Bary (1881) for Achlya bisexualis and A. ambisexualis, and this is now an established fact. Kauffman (1908) then concluded that certain “inorganic salts caused the synthesis of hormones which induced antheridial hyphae” in Saprolegnia hypogyna. In the later years, Couch (1926) provided evidence of the hormonal regulation of sexual reproduction in Dictyuchus of Saprolegniaceae. Raper (1954) studied in detail and established the definite involvement of hormonal regulation of sexual reproduction in Achlya bisexualis and A. ambisexualis of Saprolegniaceae. The terms erotactin (for substance, which attracts motile gametes), erotropin (for substance that induces chemotactic growth of sexual structures), and erogen (for substance which controls the induction and differentiation of sexual structures) have been proposed by Machlis (1972), but these have not been accepted by many of the later workers.

322

29.3

Fungi and Allied Microbes

WHAT HAS FINALLY BEEN ESTABLISHED WITH SEX HORMONES IN FUNGI?

Researches of various recent workers have finally established that there exists a definite involvement of sex hormones in sexual processes of several groups of fungi. However, only some of these hormones have so far been identified and chemically analyzed and characterised.

29.4

SEX HORMONES ISOLATED FROM LOWER FUNGI

The sex hormones isolated from lower fungi (including Chytridiomycota, Oomycota and Zygomycota) are isoprenoids (the sterols named antheridiol and oogoniol) from Achlya; the sesquiterpene (named sirenin) from Allomyces of Chytridiomycota, and trisporic acid and its precursors from Mucor and Blakeslea of Zygomycetes. (i) Antheridiol (Fig. 29.1 A) is a sterol (C29 H42 O5), the site and specificity of which are female cells of Achlya sp. (ii) Oogoniol (Fig. 29.1B) is also a sterol, the molecular structure which is C33H54O6, and site and specificity of its synthesis are male cells of Achlya sp.

A

B OH HO

O O

OH HO

O Antheridiol (C29H42O5)

Fig. 29.1

O

RO

Oogoniol (C33H54O6)

Sex hormones of Achlya. A, Antheridiol (C29H42O5; MW470), a sterol produced by female strain; B, Oogoniol (C33H54O6; MW546), a sterol which induces oogonial initials in female strain.

(iii) Sirenin (Fig. 29.2) is a sesquiterpene with its molecular structure being C15 H24 O2. The site and specificity of its synthesis are female gametes of Allomyces. Response of its bioassay include chemotaxis of male gametes. (iv) Parisin’s molecular structure is not yet established. The site and specificity of its synthesis are male gametes of Allomyces. Response of bioassay include chemotaxis of its female gametes. (v) Trisporic acid (Fig. 29.3) is a hormone, the molecular structure of which is C18H26O4. Site and specificity of its synthesis are (+)/(–) cells of species of Mucorales. Various characters of these isoprenoid hormones of fungi are also given in Table 29.1.

CH2OH

CH2OH Sirenin (C15H24O2)

Fig. 29.2

Sirenin, an attractant released by female gametangia and gametes of Allomyces.

323

Sex Hormones and Pheromones in Fungi

Table 29.1 Isoprenoid hormones of fungi (Adapted from Gooday, G.W., 1999) S.No.

Hormone

Molecular Structure; Molecular Weight

Site and specificity of synthesis

Response of bioassay

1.

Antheridiol Oogoniol

Female cells of Achlya species, Constitutive Male cells of Achlya species, induced

Antheridia by males

2.

Sterol (C29H42O5; MW470) Sterol (C35H54O6; MW546)

3.

Sirenin

Female gametes of Allomyces

Chemotaxis of male gametes

4. 5.

Parisin Trisporic acid

Sesquiterpene (C15H24O2; MW236) Not worked out Apocarotenoid* (C18H26O4; MW306)

Male gametes of Allomyces (+) / (-) cells of species of Mucorales, in collaboration

Chemotaxis of female gametes Zygophores by (+) and (-) strains.

Oogonia by females

*Apocarotenoid is a degradation product of Carotene (C40H56, an orange carotenoid pigment), via retinal (C20H28O).

29.4.1

Common Properties of Hormones Isolated From Lower Fungi

The above-mentioned groups of hormones, although structurally very different, have some properties common in all. Some of such common properties are mentioned as follows: (i) These hormones are all specific, affecting only fungi of the same species or closely related species. (ii) These are produced in very low concentrations. Blakeslea trispora, however, produces trisporic acids in high concentrations. (iii) All these hormones are active in very low concentrations. (iv) These are unstable, being metabolized by the cells which respond them.

O

COOH OH Trisporic Acid (C18H26O4)

Fig. 29.3

Trisporic acid C (C18H26O4; MW306), the major zygophore inducing hormone in Mucor mucedo.

What is Most Important About the Studies of Sex Hormones? As is clear from the above-mentioned properties, sex hormones and pheromones are produced in very low concentrations. Due to this, very sensitive bioassays (quantitative determination techniques or methods) have had to be developed for their quantitative determination. Very sophisticated and minutely managed techniques are needed for their studies.

29.5

29.5.1

SOME EXTENSIVELY STUDIED SEX HORMONES OF LOWER FUNGI Sirenin

Machlis (1958, 1966) and his co-workers observed that in Allomyces of Blastocladiales of Chytridiomycota, male gametes are attracted to female gametangia due to the production of an “attractant”. This attractant or the chemical factor involved in this attraction process has been named as sirenin by Machlis (1958). In later studies, Carlile and Machlis (1965) showed the removal and inactivation of sirenin by male gametes. Nutting et al. (1968) established the structure of sirenin (C15H24O2) which is a bicyclic sesquiterpene (Fig 29.2) with a molecular weight of 236. Sirenin in Allomyces is produced by female gametangia and female gametes and released in the surrounding water. Under the influence of sire-

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nin, male gametes start collecting around the female gametangia. Due to this, the female gametes are faced with several male gametes immediately after their release. Both male and female gametes undergo pairing which results in the formation of motile diploid zygotes. The process which limits interspecific mating is, however, still not clear. A chemical, parisin (Table 29.1), has also been shown to be released by male gametes involved in the chemical attraction of female gametes. Its chemical characteristics (e.g. molecular structure, probable precursor, optimal yield, sensitivity, etc.) could not be ascertained so far.

29.5.2

Antheridiol and Oogoniol

Raper, in his pioneering work published in a series of papers during 1939 and 1952, proved the occurrence of sex hormones in Achlya. In his classical work on A. ambisexualis and A.bisexualis, Raper found that vegetative female filament in these species secreted hormone A which induce antheridia on the male filament. Raper observed that this hormone A is a mixture of four hormones, two produced by male hyphae (A1 and A3) and two secreted by female hyphae (A2 and A). Some of these hormones increased the hormonal activity of the vegetative female and others decreased this activity. The male hyphae then produced another hormone called hormone B. It induced the development of oogonia on the female filament. Thus developed oogonial initial released another hormone called hormone C. It induced the antheridial growth towards the oogonia. Ultimately, the antheridia secreted hormone D. This hormone has the quality of controlling cleavage of the oogonium into oospores. McMorris and Barksdale (1969) investigated the preparation of a compound from Achlya ambisexualis that have activities like that of hormone A and named it antheridiol. The structure of antheridiol (C29H42O5), as shown in Fig. 29.1 A, was proposed by Arsenault et al. (1968). It is a colourless crystal. The molecular weight of antheridiol is 470. Edwards et al. (1969) also synthesized antheridiol. Four responses induced by antheridiol include (i) induction of antheridial hyphae on male plants, (ii) stimulation of male hyphae to produce hormone B, (iii) chemotropic stimulation of antheridial hyphae, and (iv) delimitation of antheridia in male hyphae. McMorris et al. (1975) isolated three compounds from Achlya heterosexualis showing hormone B activity. These are steroids and induce oogonial initials in female strains. They have been named as oogoniols (Fig. 29.1B), as oogoniol-1, oogoniol-2 and oogoniol-3. Instead of the presence of four hormones in Achlya ambisexualis, collectively known as A-complex (Raper, 1952), Van den Ende (1976) opined that hormonal regulation of sexual reproduction in this species of Achlya involves only two hormones, i.e. antheridiol and oogoniol.

29.5.3 Trisporic Acid Trisporic acids are the progametangia-inducing substances in some fungi. In Mucorales (e.g. Rhizopus nigricans and Mucor hiemalis), the phenomenon of sexual incompatibility was discovered by Blakeslee (1904). Since two incompatible strains in these fungi could not be distinguished morphologically, Blakeslee named them as + and -. Burgeff (1924) demonstrated the presence of sex hormones in Mucor mucedo after about two decades of the discovery of heterothallism by Blakeslee (1904). Burgeff opined that a diffusible substance is responsible for the initiation of sexual reproduction in the investigated Mucorales. In Mucor hiemalis, Burgeff (1924) observed that zygophore-inducing substances could be transferred through air. Such zygophore-inducing substances were also reported in both mating types of Mucor mucedo by Banbury (1954). These substances were isolated and purified by Plempel (1963) of his co-workers. Caglioti et al. (1967) and some other workers identified two compounds which enhanced the production of carotenoids in Choaneophora trispora (= Blakeslea trispora). These compounds were named as trisporic acids B and C (Fig. 29.3) because trisporic acid A was biologically inactive. Later workers confirmed that trisporic acids were actively involved in the production of zygophores in Mucor mucedo. Three kinds of trisporic acids normally reported from mated cultures of some Mucorales are Trisporic acid A, B and C. According to Van den Ende and Stegwee (1971) trisporic acid A shows only 1-2 per cent of the activity while trisporic acid B shows 15 per cent hormone activity, and trisporic acid C shows as high as 80 per cent activity. Low hormonal activity of trisporic acid A is perhaps because it lacks the functional group in the side chain. The empirical formula of trisporic acid is C18H26O4 and its molecular weight is 306. Austin et al. (1969) also isolated trisporic acid (progametangia-inducing factors) from culture media of Blakeslea trispora and Mucor mucedo.

325

Sex Hormones and Pheromones in Fungi

The substances responsible for induction of progametangia were named as gamone by Plempel (1963), who also proposed a scheme of operation of sex hormones in Mucor mucedo, as outlined in Fig. 29.4. Cainelli et al. (1967) and Van den Ende et al. (1970) suggested the existence of one sexual factor in (+) mycelium of Mucor mucedo. They named this factor as trisporone. Gooday (1973) suggested the existence of more or less equal involvement of both (+ as well as-) mating types in the production of trisporic acids. According to Gooday (1999), structurally the trisporic acid (C8H26O4) is a apocarotenoid, which is a degradation product of carotene (C40H56) via retinal (C20H28O), and its sensitivity is 10-8 .

29.6

(–) Mating type

(+) Mating type

(–) Progamone

(+) Progamone

(–) Gamone

(+) Gamone

(+) Zygophores

(–) Zygophores

Zygotrophic hormone

Fig. 29.4

Zygospore

Zygotrophic hormone

Scheme of operation of sex hormones in Mucor mucedo (after Plempel, 1963).

SEX PHEROMONES IN HIGHER FUNGI

Regarding the terms ‘hormones’ and ‘pheromones’, Carlile et al. (2001) may be quoted that general use “has settled on ‘hormone’ for lower fungi, and ‘pheromone’ for Ascomycetes and Basidiomycetes”. According to these workers, several sex pheromone systems have now been characterized in higher fungi i.e. Ascomycetes and Basidiomycetes. All these systems are peptides (e.g. a-factor of Saccharomyces cerevisiae) or more usually lipopeptides, “such as a-factor of S.cerevisiae and tremerogens A-10 and A-13 of Tremella mesenterica” (Fig. 29.5). NH2 — Trp — His — Trp — Leu — Gln — Leu — Lys — Pro — Gly — Gln — Pro — Met — Tyr — COOCH a -factor of Saccharomyces cerevisiae

S NH2 — Tyr — IIe — IIe — Lys — Gly — Val — Phe — Trp — Asp — Pro — Ala — Cys — COOCH3 a-factor of S. cerevisiae CH2OH

S NH 2 — Glu — His — Asp — Pro — Ser — Ala — Pro — Gly — Asn — Gly — Tyr — Cys — COOCH3 2

Tremerogen A-10 of Tremella mesenterica

S NH 2 — Glu — Gly — Gly — Gly — Asn — Arg — Gly — Asp — Pro — Ser — Gly — Val — Cys — COOH 2

Tremerogen A-13 of T. mesenterica

Fig. 29.5

Sex pheromones of Ascomycetes (a-factor and a-factor of yeast Saccharomyces cerevisiae) and Basidiomycetes (tremerogen A-10 and tremerogen A-13 of Tremella mesenterica).

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The a-factor of Saccharomyces cerevisiae (Fig. 29.5) is actually a tridecapeptide. It is so named because it is produced by cells of mating type a of S.cerevisiae. Two unlinked genes (MFa1 and MFa2) are responsible for its formation. The a-factors of two other species (Saccharomyces exiguus and S.kluyveri) are closely related to that of S.cerevisiae with four and five amino acid replacements, respectively. The a-factor of Saccharomyces cerevisiae (Fig. 29.5) is so named because it is produced by mating type a of this yeast. It occurs in two forms, one having the amino acid leucine (Leu) instead of valine (Val). Due to the methylation of the carboxyl group and farnesylation of the sulphur (S) atom of its cysteine, the molecule of a-factor of S.cerevisiae is highly hydrophobic. According to Carlile et al. (2001) “the effects of a-factor are similar to those of a-factor, a are exercised a-cells”. An a-factor has also been discovered by Hartwell (1974) and Bu’Lock (1976). Apparently, this factor has a similar effect on a-cells. The tremerogens (Tremerogen A-10 and Tremerogen A-13; Fig. 29.5) are the sex pheromones of genus Tremella mesenterica of Basidiomycetes. In tremerogens; (i) ‘trem’ refers to genus Tremella; (ii)’erogen’ refers to sexual activity; (iii) A and a refer to the mating types of the producer cell of Tremella; and (iv) numerals (10 and 13) refer to the used strains. Pheromones similar those of Tremella mesenterica are found in some other Basidiomycetes e.g. Ustilago naydis. Upto 2005, only a few of the lipopeptide pheromones could be characterized directly i.e. by sophisticated and painstaking methods of purifications and chemical analysis. Many have, however, been discovered indirectly by cloning genes involved in mating.

29.6.1

P-Factor and M-Factor From Ascomycetous Yeasts

Sex pheromones have also been isolated from some other Ascomycetous yeasts, e.g. Schizosaccharomyces pombe, in which mating type Plus (P) cells produce a peptide pheromone, P-factor, and mating type Minus (M) cells produce farnesylated and methylated lipopeptide, M-factor.

29.6.2

Sex Pheromones From Mycelial Ascomycetes

In mycelial Ascomycetes, usually a single mating type produces both male and female structures. Isolation of pheromones is more difficult in these fungi. Several such fungi have, however, shown some evidence for the occurrence of sex hormones. A few such examples are listed below: 1. In Neurospora crassa of Sordariaceae, microconidia of each mating type produce a factor which attracts trichogynes of the other mating type. Isolation of these factors, however, could not be made possilbe. 2. In Bombardia lunata (Lasiosphaeriaceae of Sordariales) trichogynes are attracted chemotropically to differentiate male spermatia or vegetative mycelium of opposite strain. 3. Bistis (1957) reported the existence of a multihormonal mechanism controlling and regulating sexual reproduction in Ascobolus stercorarius. 4. Wolf and Mirocha (1973) demonstrated the presence of a hormonal system controlling perithecia formation in Gibberella zeae. 5. In Cryphonectria parasitica of Valsaceae, sex pheromones have been characterized. The pheromones Mf2/1 and Mf2/2 have been characterized in Mat-2 mating type strain, while Mf1/1 in Mat-1 mating type strain.

29.6.3 Why do Basidiomycetes have Great Chances of Outbreeding? The great chances of outbreeding among several Basidiomycetes are due to the presence of a very large number of mating types. Recent studies have shown that tetrapolar mating systems of Schizophyllum commune (Schizophyllaceae) give rise at more than 20,000 mating types and of Coprinus cinereus (Coprinaceae) give rise at more than 12,000 mating types. This gives an outbreeding potential of as high as 98%. The pheromone receptor proteins of Schizophyllum commune show homology with the pheromone receptors of Saccharomyces cerevisiae and Ustilago maydis. They resemble to some extent with a-factor of S.cerevisiae.

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Sex Hormones and Pheromones in Fungi

TEST YOUR UNDERSTANDING 1. Discuss briefly the terms “hormones”, “sex hormones” and “pheromones”. 2. Pheromones are now known to occur in: (a) animals (b) algae (c) fungi 3. Give an account of sex hormones isolated from lower fungi. 4. Which of the following is/are sex hormones? (a) Antheridiol and Oogoniol (b) Sirenin and Parisin (c) Trisporic acid (d) All of these. 5. Both antheridiol and oogoniol are sterols while sirenin is a _______ . 6. Write some common properties of hormones isolated from lower fungi. 7. How do the sex hormones operate in Mucor mucedo? 8. Give a brief account of pheromones in Ascomycetes and Basidiomycetes.

(d) all of these

30

C H A P

MYCORRHIZAE*

T E R

30.1

WHAT ARE MYCORRHIZAE?

The roots of many plants enter into a mutualistic symbiotic association with fungi. The existence of such a relationship was first noted in 1885 by A.B. Frank, a German botanist, who coined the term “mycorrhiza” (Gr. mykes = fungus + rhiza = root). Earlier, the mycorrhizae were believed to be infrequent and abnormal structures. But today we know that mycorrhizal associations are very common in natural and agricultural ecosystems. According to Smith and Read (2008), more than 80% of land plants, liverworts, ferns, woody gymnosperms and angiosperms show mycorrhizal associations. They have been in existence since Devonian period. In fact, some primitive plant orders, like Magnoliales, have an obligate dependence on mycorrhizae.

30.2

NATURE OF MYCORRHIZAL RELATIONSHIP

The mycorrhizal relationships represent mutualistic relationships. The fungi which normally participate in the mycorrhizae do not have the ability to utilize complex polysaccharides, normally available in the soil. By getting associated with the roots, these fungi are able to obtain an abundant supply of simple carbohydrates (like glucose) from the plant. On the other hand, the hyphal network of mycorrhizal fungi is specialized for nutrient and water uptake from soil so that the plants are able to obtain enhanced supply of nutrients and waters in addition to other benefits. This leads to improved plant fitness. Probably this is the reason why mycorrhizal plants normally occupy the closed plant communities where competition for soil nutrients has to be faced. On the other hand, non – mycorrhizal plants tend to occupy open habitats where availability of soil nutrients is not a major problem.

30.3

TYPES OF MYCORRHIZAE

There are six major types of mycorrhizae, which can be categorised as under: (A) Ectomycorrhizae(or Ectotrophic mycorrhizae) (B) Endomycorrhizae (includes 3 major types viz. Arbuscular mycorrhizae or AM, Ericoid mycorrhizae, and Orchidoid mycorrhizae). (C) Ectendomycorrhizae (includes 2 major types viz. Arbutoid mycorrhizae and Monotropoid mycorrhizae) * Contributed by M.U. Charaya, Professor of Botany, C.C.S. University, Meerut-250004

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Mycorrhizae

Some of these mycorrhizae are depicted in Fig. 30.1. (A) Ectotrophic mycorrhiza Sheath of fungai hyphae Hartig net (B) Vesicular arbuscular mycorrhiza (VAM) Arbuscules Vesicle

Coiled hyphae

Germinating spore

(C) Endotrophic mycorrhiza

Fig. 30.1

30.3.1

Three types of mycorrhizae shown diagrammatically in transverse section of root. A, Ectotrophic mycorrhiza; B, Vesicular-arbuscular mycorrhiza; C, Endotrophic mycorrhiza of an orchid.

Ectomycorrhizae

The ectomycorrhizae are characterized by the presence of an external pseudoparenchymatous sheath, called mantle, on the terminal nutrient absorbing rootlets. The sheath may be more than 40 mm thick, and may constitute up to 40% of the dry weight of combined (root + fungus) structure. Beneath the sheath, the fungal hyphae penetrate the intercellular spaces of epidermis and cortex to form an intercellular network of hyphae called Hartig net. Though the hyphae of the Hartig net are in close contact with the root cells in the region, there is no penetration of host cells. The ectomycorrhizae occur in only about 3% of plant species most of which are coniferous and broad – leaved trees in temperate and boreal environments (pines, spruce, fir, oak, beech, birch, eucalyptus etc.). More than 5000 species of fungi énter into mycorrhizal associations. These include ascomycetes (such as truffles) are basidiomycetes (like Amanita, Boletus, Cenococcum, Cortinarius and Leccaria). Ectomycorhizal fungi do not exhibit high degree of host specificity so that mycorrhizae belonging to several different fungi can be found on the root system of a single tree. As a result of ectomycorrhizal infection, the rootlets of the plant branch dichotomously repeatedly so as to form a cluster. These mycorrhizae in most roots are believed to be due to auxin and other fungal metabolites produced by the fungal partner. The formation of root hairs is suppressed and their function is taken over by fungal hyphae which radiate into the soil from the mantel. As a result, root’s absorptive surface is greatly affected. All the increased mineral nutrients that enter the plant are chanelled through the fungus. Nitrogen-containing compounds, calcium and phosphate ions first get absorbed/accumulated in the fungal sheath, and are then transferred to the plant root. The ectomycorrhizal roots take up ions like phosphate and potassium at rates much higher than do the non-mycorrhizal plants.

30.3.2

Endomycorrhizae

In case of endomycorrhizae, the fungal partner grows mainly inside the roots, penetrating the outer cortical cells of the plant root. Only a small portion of the fungal component lies externaly as a loose mass of hyphae in soil. Three major

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types of endomycorrhizae are: (a) arbuscular mycorrhizae (AM); (b) ericoid mycorrhizae; and (c) orchidoid mycorrhizae. Also known as Vesicular Arbuscular Mycorrhizae (VAM), these are the most common type of mycorrhizae found worldwide. About 70-90% of land plant species form AM associations. These include angiosperms, gymnosperms, pteridophytes and bryophytes. Most of the major crop plants like maize, wheat, potatoes, beans, tomatoes, tea, coffee, sugarcane, cotton etc. form AM. Fungi belonging to seven genera i.e., Glomus, Acantospora, Gigaspora, Entrophospora, Archaeospora, Paraglomus and Scutellospora enter into AM associations. By and large, the AM fungi are obligate biotrophs and it has not been possible to grow them in culture/ without the host plant. Of course, their spores can germinate in the absence of host plants. These depend upon a living plant partner to complete their life cycle and for producing next generation of spores. Hildebrandt et al. (2006), however, have reported that it has been possible to grow Glomus intraradices in the absence of host, and it could produce spores when co-cultured with the bacterium Paenibacillus validus. An interesting feature of arbuscular mycorrhizal association is that individual fungal strains do not show much specificity with respect to colonisation of plants – a strain that enters into mycorrhizal association with tomato roots may grow on roots of many other plants. Similarly, the roots of single plant can be colonised by many different AM fungal species. As compared with the ectotrophic mycorrhizae, the AM fungi neither form any mantle nor cause any visible changes in the roots. These are visible only when root tissues are appropriately stained. A network of intercellular as well as intracellular hyphae is seen. The hyphae, upon penetrating cortical cells, form very fine structures showing tree-like branching. These structures are called arbuscles (Latin ‘arbusculum’ = bush/little tree). These are thought to be the major sites of the nutrient exchange between the fungal and plant partners. Apparently, these look similar to the haustoria of biotrophic parasites. But, arbuscles do not have a neck, and have a very limited life (of about 14 days only). The arbuscles are surrounded by a plant-derived membrane called periarbuscular membrane (PAM). The interface between the fungal plasma membrane and PAM is called periarbuscular space (PAS). PAS contains fungal and plant cell wall material. Some (but not all) AM fungi form spherical or oval, lipid – rich storage structures called vesicles in the root cells. Earlier, it was believed that all the AM fungi produce vesicles and were, therefore, called vesicular arbusular (VAM) fungi. From the colonised roots, the AM fungi grow out into the soil forming a ramifying set of hyphae called extraradical hyphae, which produce the spores. It is a spore or an extraradical hypha, which on coming in contact with appropriate root colonises it leading to AM development. Our knowledge regarding development of AM has increased substantially during last few years. Parnishe (2008) has given an excellent coordinated account of the events leading to the development of AM, which has been summarised as under and also illustrated in Fig. 30.2. Spore Striga seedling

Mutual recognition ‘presymbiotic phase’ Hyphopodium

Strigolactone Myc factor

Formation of Fungal PPA penetration

Plant root

Nucleus Epidermis

Calcium spiking

Outer cortex

Inner cortex Endodermis Vascular cylinder

Fig. 30.2

Steps in arbuscular mycorrhiza (AM) development

Arbuscule

Mycorrhizae

331

(i) The plant roots exude lactone compounds called strigolactones. The strigolactones induce the germination of spores of AM, stimulate hyphal growth as well as hyphal branching. (ii) The AM fungi produce some signalling molecules called Myc factors, which induce calcium oscillations in root epidermal cells and also activate plant symbiosis-related genes. (iii) The AM fungi approach epidermis and form hyphopodia (special types of appressoria that develop from mature hyphae, not from germ tubes) on the epidermal cells. (iv) 4-5 hours after the formation of hyphopodium, the epidermal cell produces a prepenetration apparatus (PPA). PPA is a thick cytoplasmic bridge across the vacuole of the host cell. It contains microtubules and microfilaments which, together with dense endoplasmic reticulum cisternae, forms a hollow tube within the PPA. The nucleus of the plant cell keeps moving ahead of the developing PPA, perhaps guiding its growth direction in the cell. The PPA ultimately reaches the other end (towards cortex) of the epidermal cell-thus a ‘transcellular tunnel’ is formed. (v) After the transcellular tunnel formed by PPA is complete, the fungal hypha (from hyphopodium) penetrates the host epidermal cell and, through the channel passes through the cell towards the cortex. (vi) The fungal hypha now leaves the epidermal cell and enters the apoplast. In the apoplast, it branches. The branches grow laterally along the root axis, obviously come in contact with the cortical cells, and induce the development of PPA – like structures in cortical cells. (vii) The fungal hyphae now enter the cortical cells and branch to form arbuscles. Arbuscular mycorrhizal associations results in increased uptake of phosphate, zinc, sulphate and ammonium from soil. AM fungi can also acquire nitrogen from organic materials and make it available to the plant. In return, AM fungi obtain carbohydrates from plant. According to some estimates, upto 20% of the photosynthates produced by terrestrial plants are consumed by AM fungi. Many members of three families-Ericaceae, Epacridaceae and Empetraceae – belonging to the order Ericales enter into this distinctive type of mycorrhizal association. Of course, some members of the Ericaceae (Arbutus, Arctostaphylos) have another type of mycorrhizae. The ericoid roots, also known as ‘hairy roots’, are quite delicate and lack root hairs; their epidermis is ephemeral-the epidermis being lost as the roots grow older so that outer layer of cortex become suberized and thickened to form the outer surface of the root. The cortex itself is only two layered. The diameter of ericoid roots is less than 10 m m. The fungal partners are mostly basidiomycetes but some are ascomycetes. These include Hymenoscyphus ericae (earlier known as Pezizella ericae) which forms association with many ericoid plants. Four species of mitosporic genus Oidiodendron (O.cerialis, O.matus, O. rhodogenum and O. griseum) and some sterile, so far unidentifiable fungi also form ericoid mycorrhizal associations. A number of species form ericoid mycorrhizae with different species of ericaceous plants in culture. The species of Clavaria too have been reported to have mycorrhizal association with Rhododendron. The fungal hyphae penetrate the epidermal and cortical cells and ramify within each cell to form a coil or knot of filaments occupying much of the volume within the cells. The stele is not invaded. The nutrient exchange occurs between the hyphal coils and cell cytoplasm. One of the most important advantage of ERM is that the partner fungi provide its host with nitrogen in natural soils. The ericaceous plants typically occur in cool, acidic sites where the rate of mineralisation of nitrogen by saprobic fungi is very low. ERM fungi make N and P from organic sources accessible to the plant. Thes fungi secrete the enzyme proteinase and phosphodiesterase which can function efficiently at low pH (pH 3.0 to 5.5) releasing amino acids and P from the soil organic matter. Production of indole acetic acid and siderophores by ERM fungi leading to better plant growth and iron uptake by ericaceous plants as a result of mycorrhizal associations. Thus, mycorrhizal fungi play a major role in supplying minerals to ericaceous plants in such nutrient-deficient soils. Hence, it is not surprising that all members of Ericales are strongly mycorrhizal. The orchids form an unusual type of mycorrhiza where plant appears to be parasitic on the fungus. The seeds of orchids are very small with minimal amount of food reserves, and they geminate only when infected by endomycorrhizal fungi. Majority of fungal strains involved belong to the genus Rhizoctonia,

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rest of them to Armillaria, Fomes, Marasmius and occasionally to Corticium. These fungi grow on soil organic matter, degrading cellulose and other structural polymers. The seeds and adult plants are colonised by the fungi at almost equal frequency. Thin-walled hyphae enter the seed through suspensor and rhizoidal cells while in adult plants, the fungi enter the roots through root hairs and\or velamen cells. After passing through the surface layers, the fungal hyphae penetrate cortical cells. These form coils, called peletons, within the cells of outer cortex. However, these coils are short-lived. The host cells secrete certain enzymes to break down the peletons and utilise the nutrients brought by the fungus as well as break-down products (tolypophagy). Alternatively, the hyphal tips burst and the cytoplasm so released forms free fungal bodies called ptyosomes, which are digested later on (ptyophagy). In both cases, the orchids gain material through fungal digestion. New coils take the place of digested/autolysed coils. In this way, the fungus continues to provide the nutrients (including carbon) to all orchids through the life of those orchids which cannot carry out photosynthesis and during the first years of life of photosynthetic orchids until these plants develop chlorophyll. Consequently, the orchids are undoubtedly mycoheterotrophic being actually parastic on the fungus. In addition to C and other nutrients, the partner fungus provides certain vitamins like niacin, thiamine and nicotinic acid etc. to the host cells. The orchid seeds also provide p-aminobenzoic acid, a constituent of folic acid, to the fungal partner. Not only this, when the photosynthetic orchids develop chlorophyll, the carbon diffusion gradient is reversed i.e. orchids start supplying food to the fungus, at least partly. Symbiosis, in such cases, consists of two phases: (a) a protocorm phase, in which orchid receives carbohydrates from the fungus; and (b) an autotrophic phase, when orchid ‘pays off’ carbohydrates. The term ‘phase separation symbiosis’ is used for such an interaction.

30.3.3

Ectendomycorrhizae

In certain cases, mycorrhizae share characteristics of both ecto- and endo-mycorrhizae. The fungal partner forms mantle on the root surface and Hartignet (like ectomycorrhizae) as coils as haustoria or hyphal coils in the cortical cells (like endomycorrhizae). These are of two types: arbutoid and monotropoid. era Arbutus, Arctostaphylos, Arctous (of family Ericaceae) and Pyrola (of family Pyrolaceae). Fungal health (mantle) is well – developed, and Hartig net is also present. The cortical cells are extensively penetrated by the intercellular hyphae. After penetrating the cortical cells, these hyphae form hyphal coils (peletons) inside there in and the cells appear to be filled with the coils. The fungi entering into this type of association are basidiomycetes and include species of Amanita and Boletus. These mycorrhizae are limited to achlorophyllous plants from a small sub-family Monotropoidae (Ericales). The best studied example of monotropoid mycorrhiza is Monotropa indica (known as Indian pipe). It is a non – chlorophyllous plant that grows on the forest floor under Fagus, Pinus, Quercus, Salix and other trees. Earlier, Monotropa was believed to be a root parasite of these trees. However, later on it became clear that Monotropa is in contact with these trees through a mycorrhizal fungus (e.g., Boletus) which forms mycorrhizal association with the tree (e.g. pine) as well as with Monotropa. Thus, the mycorrhizal fungus forms a sort of bridge between the tree (s) and Monotropa. The nutrients from the roots of chlorophyllous host (e.g. pine) enter the mycorrhizal sheath and through the mycelial cords reach the roots of Monotropa. Labelled 14CO2 provided to pine tree moves down to the roots of pine as labelled sucrose, but after entering the mycorrhizal sheath it is detected as labelled sugar alcohol or trehalose, but after being translocated through mycelial cords it is found as labelled sugar in Monotropa. This clearly demonstrates that Monotropa receives sugars from green plants through the mycorrhizal association, not by direct parasitic association. Similarly, labelled 31P-phosphate, when injected into the phloem of the tree, was translocated to Monotropa through the mycorrhizal fungus. Thus, Monotropa seems to have a complex associaton – a three tier association involving (i) a chlorophyllous partner (e.g. pine), a non – chlorophyllous partner (e.g. Monotropa) and a mycorrhizal fungus bridge. The monotropoid mycorrhizae form develop a mantle (around the roots of Monotropa) as well as Harting net in the root cortex, and infection pegs (hyphal pegs) in the cortical cells.

Mycorrhizae

30.4

333

MYCORRHIZOSPHERE AND “MYCORRHIZATION-HELPER BACTERIA” (MHB)

It is being gradually realised that the fungal and plant partners are not the only components of mycorrhizal associations. Bacteria, whether tightly or loosely associated, with mycorrhizal fungi represent a ‘third’ component of mycorrhizae. Daponnois and Garboye (1991) observed that a strain of Pseudomonas fluorescens stimulated ectomycorrhizal formation significantly. Garboye (1994) coined the term “Mycorrhiza Helper Bacteria” (MHB) for bacteria involved in the establishment and /or functions of mycorrhizae. Physical contact between fungi and bacteria, as also the release of active diffusible molecules by them, appear to be important for the establishment of both plant-fungus and mycorrhiza – bacteria networks. In fact, as the external hyphal network of mycorrhizae radiates into the soil, a mycorrhizosphere is formed as a result of flow of carbon from the plant into the mycorrhizal hyphal network, and then into the surrounding soil where bacterial communities are influenced by mycorrhizal fungi. A number of bacteria associated with mycorrhizal fungi colonise the extraradical hyphae. Not only this, some bacteria live inside the hyphae of mycorrhizae fungi as endosymbionts. Though the exact role of these bacteria is not precisely known, one possible role which is ascribed to them is that these assist in the synthesis of essential amino acids. Arthrobacter, Bacillus, Pseudomonas etc. are associated with extra-radical hyphae. Some of the endosymbiotic bacteria of mycorrhizal fungi belonging to Gigasporaceae have now been identified as true bacteria related to Burkhoederia. However, since these are not culturable, these are given the name “Candidatus Glomeribacter gigasporum”. The interactions between mycorrhizal fungi and bacteria exert a beneficial effect of fungi on bacterial development and vice versa. Thus, the mycorrhizae (especially AM) represent a tri-partite association (involving three partners – the fungi, the bacteria and the plants) instead of bipartite one.

30.5

SIGNIFICANCE OF MYCORRHIZAE

Different types of mycorrhizal associations have different roles to play. In general, the benefits that accrue to plants forming mycorrhizae are as under: 1. In the case of orchids and Monotropa, the fungal partner supplies the plants with carbon either throughout the life or at least until the plants develop chlorophyll and become self-sufficient for carbon. However, in most other cases, it is the fungus which depends upon the plant for most of or all of its carbohydrate requirements. 2. Improved mineral nutrients availability is considered to be a major benefit of mycorrhizal association to the plant. Through their extensive hyphal network, the mycorrhizal fungi, not only influence the physiochemical properties of the soil, but also contribute to the release of phosphate from relatively lesser soluble inorganic complexes. In warmer climates especially, phosphorus is growth-limiting nutrient; and AM are dominant. The AM plants absorb P through two pathways (i) direct (through epidermis and root hair); (ii) AM pathway, in which P is absorbed by extraradical mycelium, and then transferred to plant cortical cells. It has been shown conclusively that in many cases, most of the P enters the plant via AM pathway; direct pathway makes a negligible or reduced contribution (Li et al., 2006). Improved phosphorus uptake is considered to be a main benefit of AM symbiosis. In the more acidic soils of moorlands and in woodlands where the rates of litter decomposition are relatively slower, the mycorrhizal fungi obtain nitrogen from organic sources in the soil by means of the protease enzyme. The nitrogen so-obtained is released in C-free (probably as ammonium) to the plant. Thus, in some situations, mycorrhizae contribute significantly to N-uptake of plants. 3. Mycorrhizal plants have been found to be more tolerant to drought stress. It is believed that roots shrink under high transpiration conditions, creating air gaps between the root and soil leading to a large drop in water potential adjacent to the roots. The hyphae of mycorrhizal fungi bind roots to the soil and probably maintain water continuity. The water absorbed by the fungal hyphae is delivered to the cortical cells where it joins the water taken up by the roots directly.

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4. Mycorrhizae provide protection to roots from plant pathogens by: (i) acting as a mechanical barrier. Fungal sheath/mantle drastically reduces the chances of contact of pathogen with the root; (ii) improving the nutritional status of the plant, thereby increasing its tolerance to pathogen attack; (iii) competing with pathogens for infection sites as well for ecological niches to be occupied by the pathogens; (iv) producing antibiotics, for example Leucopaxillus produces diatretyne nitrile (a polyacetylenic antibiotic) which strongly inhibits the growth of Phytophthora cinnamomi; (v) inducing changes in root exudates which may alter interactions between soil microbial communities that may become detrimental for root pathogens. 5. Mycorrhizal fungi also protect plants from the toxic effects of pollutants (including acid rain, SO2 toxic heavy metals). The toxic metals get accumulated and immobilised in the mycorrhizal sheath. 6. Mycorrhizal hyphae connect plant roots of the same or different species, and serve as conduits for distribution of photosynthates between plants. Some plants are able to exist in a given habitat only because they are ‘nursed’ by photosynthetic plants through mycorrhizal mycelium. Thus, a greater variety of plants are able to colonise and flourish a given habitat leading to greater species richness.

TEST YOUR UNDERSTANDING 1. What are mycorrhizae? 2. In which of these types of mycorrhizal symbiosis, the carbon compounds are transferred mainly from fungi to plants? (a) Ectomycorrhiza (b) Orchid mycorrhiza (c) VAM (d) Ericaceous mycorrhiza. 3. Structures formed by symbiotic fungi inside plant cells are: (a) appressoria (b) Rhizomorphs (c) haustoria (d) Hartig nets. 4. Give a detailed account of types of mycorrhizae. 5. Differentiate between: Arbuscular mycorrhizae, Ericoid mycorrhizae and Orchidoid mycorrhizae. 6. Describe briefly Vesicular Arbuscular Mycorrhizae (VAM). 7. Write a note on significance of mycorrhizae.

31

C H A P

LICHENS

T E R

31.1

WHAT IS A LICHEN?

Lichen is such an association of an alga and a fungus in which two organisms remain so closely associated with each other that they appear to be a single plant. The definition of the ‘lichen’ which was the winner in a poll amongst the members of the ‘International Association for Lichenology’’ held in 1981 is: ‘A lichen is an association of a fungus and a photosynthetic symbiont, resulting in a stable thallus of specific “structure”. However, in 1983 edition of the Dictionary of the Fungi, a lichen has been defined as ‘a stable self-supporting association of a mycobiont and a photobiont’. The mycobiont is the fungal partner, whereas the photobiont is the photosynthetic partner in a lichen association. According to the majority of the lichenologists a symbiotic relationship exists between the two partners of the association. The fungus parasitizes the algal cells, as well as also lives saprobically on the algal cells, which die because of either parasitism or other causes. On the contrary algal cells are protected from high light intensity. Water and some nutrients are also being made available to the alga by the fungus. Kirk et al. (2001) defined lichens as “essentially a stable self-supporting association of a fungus (mycobiont) and an alga or cyanobacterium (photobiont).” It is actually an “ecologically obligate stable mutualism between an exhabitant fungal partner and an inhabitant population of extracellularly located unicellular or filamentous algal or cyanobacterial cells.”

31.2

COMPONENTS OF LICHENS

A lichen consists of two components, an alga (photobiont) and a fungus (mycobiont). The mycobiont is usually a member of Ascomycotina, less commonly of Basidiomycotina, and only rarely of Deuteromycotina. The photobiont is usually a member of Myxophyceae (blue-green algae), and less commonly of Chlorophyceae (green-algae). In most of the earlier literature, however, instead of ‘photobiont’ the word ‘phycobiont’ (Gr. phykos, alga; bios, life) is used, which indicates its algal nature. But the recent trend is to treat blue-green algae as Cyanobacteria and not as algae. Based on this trend Hawksworth and Hill (1984) divided the photobionts into ‘phycobionts’ (Green algae) and ‘Cyanobionts’(Cyanobacteria). A majority of the ascomycetous lichens belong to either Discomycetes or Pyrenomycetes. Some also belong to Loculoascomycetes. But none belongs to Mastigomycotina, Zygomycotina, Hemiascomycetes, Plectomycetes and Laboulbeniomycetes.

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Hawksworth and Hill (1984) mentioned that out of a total number of about 13,500 lichenized fungi, about 13,250 species (98%)belong to Ascomycotina. According to these workers 46% of all species in the Ascomycotina are lichenforming species. All species belonging to Graphidales, Gyalectales, Peltigerales, Pertusariales and Teloschistales of Ascomycotina form lichens. Galloway (1992) estimated that a total of about 17000 to 20000 species of lichens are probably existing in the world. Karlile et al. (2001) mentioned that about 14000 species of lichens are known. Some of the lichen-forming genera of Basidiomycotina are Dictyonema, Omphalina and Multiclavula. Only 55 species of lichen-forming Deuteromycotina or Anamorphic fungi have so far been described (Hawksworth and Hill 1984), of which Blarneya hibernica is very common. According to Hawksworth and Hill (1984) only 37 genera have so far been identified as lichen photobionts, mostly belonging to blue greens (Cyanophyta) or green algae (Chlorophyta). Common lichen-forming blue – green algae are Anabaena, Calothrix, Chroococcus, Gloeocapsa, Nostoc, Scytonema and Stigonema. Common Chlorophyta are Cephaleuros, Chlorella, Phycopeltis, Trebouxia and Trentepohlia.

31.3

A BRIEF HISTORY

Theophrastus was the first person who introduced the word ‘Lichen’ (lie, ken) into Greek literature in about 300 BC. He used the word primarily to describe the outgrowths from the bark of olive trees. P.A. Micheli (1729), an Italian botanist, described about 300 species of lichens in his Nova Plantarum Genera. Hawksworth and Hill (1984) mentioned the Erik Acharius (1757 – 1819), a Swedish doctor, must be credited as the founder of the systematic study of lichens. The works of the Acharius are discussed in Methodus Lichenum, Lichenographia Universalis and Synopsis Methodica Lichenum. Among the recent workers the name of V. Ahmadjian comes on top. The author of this book feels that because of the Ahmadjian’s detailed study on lichens (Ahmadjian, 1960, 1962, 1965, 1967, 1970, 1980, 1982; Ahmadjian and Hale, 1973; Ahmadjian and Jacob, 1983, etc.), he should be named as the ‘Father of Modern Lichenology’.

31.4

OCCURRENCE

Lichens, are found growing in a wide variety of situations from the Arctic to Antarctic and all regions in between. Some lichens are able to live where there is no other vegetation, and thus prove important colonizers of bare rocks. They may grow on leaves, bark of trees, soil, bare rocks, and many other similar situations. On one hand lichens grow commonly on exposed rocks in the desert, as well as near volcanoes, whereas on the other some lichens grow luxuriantly on frozen substrata in polar regions (Cladonia rangifera). A number of species occur only on the seashore. Peltigera canina (Fig. 31.1 A) and Xanthora parietina grow commonly on walls, rocks, soil, and also amongst grass in woods, on lawns and sand-dunes. Parmelia physodes (=Hypogymnia physodes) is common on twigs (Fig. 31.1B), branches, wood, rocks and walls forming large, well-branched masses. Usnea subfloridana (Fig. 31.1C) grows commonly on trees, specially in hilly regions, whereas Cladonia coccifera (Fig. 31.1D) and C.floerkeana (Fig. 31.1E) are common on moorland and in hilly regions. Cora pavonia (Fig. 31.1F), a basidiolichen, occurs on bare soil and on trees. Based on their place of occurrence, lichens may fall in following groups: Lichens developing on bark of trees, e.g. species of Parmelia, Alectoria, Usnea, Graphis, etc. Lichens developing directly on wood, e.g. Calicicum, Chaenotheca, Cyphelium, etc. Lichens developing on rocky substrata, e.g. Verrucaria, Porina, Dermatocarpon, Xanthora, etc. Lichens growing on the ground, e.g. Cladonia floerkeana, Lecidea granulosa, Collema tenax, etc. Lichens developing on siliceous rocky shores of sea, e.g. Verrucaria mucosa, Caloplacentum marinae, Caloplaca marina etc.

337

Lichens

A

B

C

D E

Fig. 31.1

F

Thalli of some lichens. A, Peltigera canina; B, Parmelia physodes; C, Usnea subfloridana; D, Cladonia coccifera; E, C. floerkeana; F, Cora pavonia.

Lichens developing on hard siliceous rocks in freshwater, e.g. Hymenelia lacustris, Epheba lanata, etc. Many man-made substrata, such as leather, silk, wool, hairs, bone, glass fibre, timber, walls, paints, sculptures, asbestos-cement, worked-iron, glass etc., may also be colonized by lichens (Hawksworth and Hill, 1984). Singh (1981) reported 145 microlichen taxa belonging to 43 genera from Manipur (India).

31.5

CLASSIFICATION

Under the rules of the International Code of Botanical Nomenclature, no taxonomic significance is attached to the algal component in a lichen. The lichen classification is based solely on the fungal partner. It is because the thalli and fruiting bodies of the lichens are largely fungal in structure. According to Miller (1984) lichens are assigned subdivision status in true fungi (Eumycophyta), and are divided into two classes: 1. Class Ascolichens: Fungal partner is an Ascomycotina. 2. Class Basidiolichens: Fungal partner is a Basidiomycotina. However, Poelt (1973), Henssen and Jahns (1974) and Alexopoulos and Mims (1979) divided lichens into three groups as under: 1. Basidiolichens: Fungal partner is a Basidiomycotina. 2. Deuterolichens: Sterile lichens that do not produce spores. 3. Ascolichens: Fungal partner is an Ascomycotina.

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(a) Hymenoascolichens with unitunicate asci 1. Order Caliciales 2. Order Lecanorales (i) Suborder Peltigerineae (ii) Suborder Lecanorineae (iii) Suborder Cladonineae 3. Order Graphidiales (b) Loculoascolichens with bitunicate asci in apothecia 1. Order Arthoniales (c) Loculoascolichens with bitunicate asci in pseudothecia 1. Order Dothideales 2. Order Verrucariales 3. Order Pyrenulales

31.6

LICHEN THALLUS (MORPHOLOGY AND ANATOMY)

The association of the mycobionts and the photobionts in the lichens results in the formation of a thallus-type plant body. In general, the lichen thallus is irregular, variously coloured, and shows several morphological types. Formerly, only three basic types (crustose, foliose and fruticose) of lichen thalli were recognized. But, on the basis of their detailed studies, Hawksworth and Hill (1984) described the following categories and subcategories in the morphology of the lichen thallus: This is the simplest type of thallus organization, in which the fungal hyphae envelope either single or small cluster of algal cells. A distinct fungal layer does not envelope the algal cells all over. The so-formed simple lichen thallus grows superficially over the substratum, provides a powdery appearance, and is called leprose, e.g. Lepraria incana (Fig. 31.2). In these lichens (Buellia, Strigula, Dimerella, Graphis) the thallus is very closely adhered to the substratum, and provides a crust-like appearance (Fig. 31.3). It is very difficult to separate them from the substratum. The photobiont (algal) cells are covered by a distinct layer of fungal tissue (cortex). In crustose lichens the outer surface is smooth, and may be continuous or dissected by wandering cracks. In some genera, such as Rhizocarpon, the outer surface contains many polygonal structures, called areolae. When the areolae grow out to form coralloid tufted cushions, they are called suffruticose. Some variations of the crustose types are mentioned below:

Fig. 31.2

Fig. 31.3

A leprose lichen thallus of Lepraria incana. Rock

Lichen

Caloplaca thallincola, a crustose lichen.

When the outer surface is radially striate and contains slightly raised marginal tissues, the lichen is called placodioid or placoid, e.g. Lecanora and Caloplaca. When the outer surface contains overlapping scale-like squamules, the crustose-lichen is called squamulose, e.g. Psora.

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Lichens

Internally, a crustose lichen is usually differentiated into cortex, algal zone and medulla (Fig. 31.4A ). The cortex and medulla are made up of fungal hyphae. Some hyphae of the medulla may form pointed rhizoids, which enter into the substratum. The thallus is flat, leaf-like, well-branched, lobed, and attached to the substratum with the help of rhizoid – like rhizines. The external appearance is like that of crinkled and twisted leaves, e.g. Parmelia (Fig. 31.1B), Physcia, Collema, Peltigera (Fig. 31.1A ) etc. Internally, along with the presence of a separate upper cortex on the upper side followed by an algal layer and a thick medulla, foliose lichens also have a well – organized lower cortex. From some cells of this lower cortex develop rhizines or some other type of attachment organs. The regions of the upper cortex, medulla and lower cortex are formed by the fungal hyphae. Foliose lichens may fall into any of the following categories (Hawksworth and Hill, 1984): The algae in some lichens arc distributed more or less evenly throughout the thallus, as in Collema (Fig. 31.4B). Such lichens are called homoiomerous. The algal cells in majority of the foliose lichens form a distinct layer (algal zone) within the thallus (Fig. 31.4C). Such lichens are called heteromerous e.g. Parmelia, Physcia, etc.

Cortex Algal cells

Upper cortex

Hypha

Algal zone

Medulla

Rhizoid

Substratum

A

Algal cells

Medulla Hyphae

Lower cortex

Rhizines

B C

Fig. 31.4

Internal organization of lichens. A, Crustose lichen; B, Foliose (homoiomerous) Iichen; C, Foliose (heteromerous) lichen.

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Fungi and Allied Microbes

These are well-branched, generally erect or pendulous structures, which provide shruby appearance, e.g. Usnea (Fig. 31.1C), Cladonia (Fig.31.1D,E), Letharia, Bryoria, etc. Internally, the fruticose lichens have a heteromerous structure, which is not dorsiventral but develops around a vertical axis. The erect or pendulous nature of these lichens is maintained either because of the presence of a thickened outer cortex or by a specialized central elastic axis, called chondroid axis, as in Usnea. In all the above-mentioned lichen types (leprose, crustose, foliose and fruticose) the fungus has the main role in the formation of the structure of the lichen thallus. But in some lichen genera (Coenogonium, Ephebe, Racodium, Cystocoleus) the algal partner is filamentous, well-developed, and remains ensheathed or covered by only a few fungal hyphae. The lichen thalli so formed have the dominance of algal partner, and have been named filamentous by Hawksworth and Hill (1984).

31.7

INTERACTION BETWEEN PHYCOBIONT AND MYCOBIONT

Ahmadjian and Jacobs (1981) and Honegger (1986) have traced the details of interactions between phycobiont cells and mycobiont hyphae of a lichen thallus in Cladonia cristatella (Fig. 31.5 A-D). In the early stages of this lichen a single algal cell of phycobiont (Trebouxia) comes in contact with the mycobiont hyphae (Fig. 31.5A), and soon the algal cell gets ensheathed by the mycobiont (Fig. 31.5B). Tubular intracellular haustoria are formed by the mycobiont (Fig. 31.5C), and it has been observed that the same phycobiont cell may be repeatedly penetrated by the mycoboint hyphae. In some stratified lichens, intraparietal haustoria may be formed by the mycobiont as shown in Fig. 31.5 D. Exchange of nutrients takes place apoplastically. The walls of both phycobiont and mycobiont are surrounded by a common hydrophobin sheath, which is produced by mycobiont.

Phycobiont cell

Hyphae ensheathing algal cell

Intraparietal haustorium of mycobiont

Mycobiont

Tubular intracellular haustorium

Hydrophobin sheath produced by mycobiont Mycobiont hyphae Chloroplast A

Fig. 31.5

31.8

B

C

Photobiont

D

A–D. Interaction between phycobiont cells and mycobiont hyphae of the lichen thallus in Cladonia cristatella. (A-B, after Ahmadjian and Jacobs, 1981; C – D, after Honegger, 1986).

TISSUE TYPES IN LICHENS

Not much is known about the tissue types in leprose, crustose and filamentous lichens. However, in foliose and fruticose lichens the fungal tissues are of taxonomic significance. According to Hawksworth and Hill (1984) the cortex in foliose and fruticose lichens consists of following two main type: The cells are oriented randomly and provide a cellular appearance. The elongate fungal hyphae are oriented in a particular direction.

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Lichens

However, in some lichens (Parmelia), the cortex is overlined by a thin layer of polysaccharide, called ‘epicortex’ (Hale, 1973). Loosely interwoven hyphae, constituting the medulla, have been termed ‘chalaroplectenchyma’ by Hawksworth and Hill (1984).

31.9

ATTACHMENT ORGANS IN LICHENS

Lichens remain attached to the substratum by following means: In the absence of lower cortex, some fungal hyphae from the medulla (Fig. 31.4A) penetrate the substratum, e.g. a majority of the leprose and crustose lichens. These are complex, tough, irregularly branched, thick strands, e.g.Buellia pulchella (Fig. 31.6 A). In Psora decipiens (Fig. 31.6 B) the fungal hyphae form delicate, reticulately branched netlike structures, called hyphal nets. Simple, unbranched or branched attachment organs of foliose lichens are called rhizines (Fig. 31.6 C).

Rhizinose strands

Hyphal net A

Fig. 31.6

Rhizines B

C

Attachment organs of some lichens. A, Rhizinose strands of Buellia pulchella; B, Hyphal net of Psora decipiens; C, Rhizines of Physconia pulverulacea.

A thick, black, spongy, algal-free tissue on the lower surface of genera, such as Anzia, is called hypothallus. It is the basal, black, algal-free, persistent region of some lichens, such as Usnea and Letharia. These are the short, apical, penetrating branches of some large pendulous lichens which loose their attachment with the point of their origin, as in Alectoria sarmentosa.

31.10

PROPAGULES ASSOCIATED WITH LICHEN THALLUS

Along with the tissues already mentioned (such as upper cortex, algal layer, medulla, lower cortex, etc), some other vegetative structures (propagules) may also be associated with a lichen thallus. Some of these structures are breathing pores,

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cyphellae, pseudocyphellae, cephalodia, isidia, soredia, lateral spinules, blastidia, phyllidium, schizidium, goniocyst and hormocyst (Figs. 31.7, 31.8). These are the areas in the cortex where loosely interwoven (Smith, 1955) hyphae are present, e.g. Oropogon. Just below a breathing pore the tissue is more or less medullary in nature. Sometimes, breathing pores are elevated in the form of a cone-like structure. In foliose lichens they develop only in the upper cortex. Breathing pores are supposed to function in gaseous exchange. These are the neat circular depressions present only on the lower surface of certain lichens, such as Stricta (Fig. 31.7 A). The depressed region of a cyphella remains lined by somewhat specialized rounded cells. The cyphellae play some role in facilitating gaseous exchange (Hawksworth and Hill, 1984). In genera such as Alectoria, Bryoria, Coelocaulon and Pseudocyphellaria, loose hyphal medullary tissue comes to the surface of the lichen thallus in the form of discrete patches, called pseudocyphellae (Fig. 31.7 B). These are also present on the lower surface. But, in the region of pseudocyphellae the lower cortex is absent. Pseudocyphellae

Algal cells

Fungal hyphae

Lower cortex

Cyphella A Rhizine

Blue-green algal filaments Cephalodium Algal cells

B Isidia Algal cells

Upper cortex E

Green algal cells C D

Fig. 31.7

Some lichen propagules. A,Cyphella of Stricta; B, Pseudocyphellae of Alectoria nigricans; C, Cephalodium of Solorina crocea; D, V.S. of thallus of Parmelia conspersa, showing two isidia; E, A soredium of Parmelia (A, after Schneider; B, D, after Ahmadjian; C, after Hawksworth).

Some of the lichens have two phycobionts, of which one is a blue-green alga and the another is a green alga. Such lichens, having three-membered symbiosis (2 algae + 1 fungus), are called ‘diphycophilous lichens’. In such lichens the blue-green alga is segregated in special external or internal swellings (Fig. 31.7C) called ‘ cephalodia’ . In diphycophylous lichens the main thallus is formed by the green alga. According to Hawksworth and Hill (1984) cephalodia are known to occur in 520 species of lichens, belonging to 21 genera of 8 families. Some of the cephalodiaproducing lichens are Lobaria amplissima, L.pulmonaria and Solorina crocea. An isidium is a small and corticated outgrowth present on the upper surface of lichen thallus. It is made up of both fungal hyphae and algal cells (Fig. 31.7D). A soredium is a small but non-corticated bud-like outgrowth present on the upper surface of the lichen thallus. It is made up of only a few algal cells, enclosed by only a few fungal hyphae (Fig. 31.7E).

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31.11

VEGETATIVE REPRODUCTION

31.11.1 Common Methods In many foliose and fruticose lichens the thallus breaks into small fragments, each of which develops into new thallus under favourable conditions. Fragmentation is a major method of reproduction in fruticose lichens, such as Cladonia uncialis, C.stellaris and Bryoria capillaris. Small, corticated outgrowths, made up of both fungal hyphae and algal cells, and situated on the upper surface of the lichen thallus, are called isidia (Fig. 31.7D). The isidia are smooth, coral-, papilla-, scale-, or petal-like outgrowths (Hawksworth and Hill, 1984). They are constricted basally, and are easily broken away from the thallus. A detached isidium develops into a new lichen thallus. Isidia are common in genera such as Parmelia (Fig. 31.7D), Pseudoevernia, Bryoria and Usnea. As mentioned earlier, the soredia are small, non-corticated, bud-like, powdery masses or outgrowths, made up of only a few algal cells surrounded by only a few fungal hyphae (Fig. 31.7E). They develop either over the entire upper surface of the lichen thallus, or in special pustule-like areas, called ‘soralia’ . Each soredium may develop into a new lichen thallus, provided satisfactory conditions of its growth are available. It is the most common method of vegetative reproduction in many lichens, including Parmelia and Bryoria.

31.11.2 Some Rare Methods According to Hawksworth and Hill (1984), lichens may also reproduce rarely by any of the following methods of vegetative reproduction: These are abstricted, leaf- or scale-like, dorsiventral portions (Fig. 31.8A) of the entire thallus of some foliose lichens, e.g. Peltigera praetextata. These are the yeast-like segmented propagules (Fig. 31.8 B) of some lichens e.g. Physcia opuntiella. These are the splitted, scale-like segments of some lichens (Parmelia taylorensis) made up of the upper layers of the thallus (Fig. 31.8C). When an algal cell and its derivatives remain wrapped in fungal hyphae in the form of an unsorallium-like structure, it is called a goniocyst (Fig. 31.8 D). These are formed in goniocystangia. When algal filaments and fungal hyphae grow together in a chain-like manner (Fig. 31.8E) and break into clumps, these are called hormocysts, e.g. many species of Lempholema.

Blastidia

Schizidium

Goniocysts

C

D

Hormocysts

Phyllidium

A

Fig. 31.8

B

E

Diagrammatic representation of vegetative propagules of some lichens.A, Phyllidium;B, Blastidium;C, Schizidium; D, Goniocyst; E, Hormocyst.

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31.12

Fungi and Allied Microbes

ASEXUAL SPORES

31.12.1 Conidia The conidia of different shape and size develop in special multihyphal structures in many lichens, such as Anthonia (Fig. 31.9 B), Lecanactis, Peltigera (Fig. 31.9C), Roccella (Fig. 31.9D), Cladonia (Fig. 31.9E), Lobaria (Fig. 31.9F) and Xanthoria (Fig. 31.9G). These conidia-containing multihyphal structures are termed ‘conidiomata’ by Vobis and Hawksworth (1981). The conidiomata occur in many ascolichens. In the thallus the conidiomata always remain immersed in flask-shaped bodies, called pycnidia. A majority of the Deuterolichens reproduce only by producing conidia. Upper cortex

Conidia Pycnidium Conidiophores

Algal cells

B

D

C

Conidia Conidia

E

Medulla A F

Fig. 31.9

G

(A) V.S. of a pycnidium of Physcia; B-G, Conidia and conidiophores of Arthonia (B ), Peltigera (C ), Roccella (D), Cladonia (E ), Lobaria (F ) and Xanthoria (G).

A pycnidium opens by a mouth-opening or ostiole (Fig. 31.9A). From their inner lining wall develop some hyphae, called conidiophores. The conidia are produced either terminally, laterally or in an intercalary manner. The conidia so formed may be cylindrical, filiform or sickle-shaped. The conidia are colourless and germinate by producing a germinating hypha. Such developing hyphae produce a lichen if they come in contact with an appropriate phycobiont. Honegger (1984) showed that in Cladonia furcata the conidia are attracted by the trichogyne and bring about fertilization. They have therefore a sexual role also to perform in some lichens.

31.12.2 Oidia According to Smith (1921) the hyphae of certain lichens break up into small bodies, called oidia. The oidia may germinate into hyphae.

Lichens

31.13

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SEXUAL REPRODUCTION OF MYCOBIONT

31.13.1 In Ascolichens It develops from certain hyphae situated deep in the algal layer. The ascogonia are multicellular, and their lower portion is usually coiled (Fig. 31.10A). Usually the cells of the ascogonia are uninucleate. The upper portion of the multicellular ascogonium usually projects above the level of the cortex. Such projected portion represents trichogyne. The conidia produced in pycnidia might also function as male cells (Honegger, 1984). Such conidia with a sexual function are called ‘spermatia’ (Hawksworth and Hill,1984). At the time of ‘fertilization’, many spermatia are lodged against the sticky tips of the trichogyne. Lichen thalli having many ascogonia but no spermatia do not show the production of ascocarps. This is also an indication that spermatia function as male ga