Microbial plant pathogens : detection and management in seeds and propagules 9781119195771, 1119195772, 9781119195788, 1119195780, 9781119195801

Table of contents :
Content: Preface xv Acknowledgement xvii Volume 1 Pathogen Detection and Identification 1 1 Introduction 3 1.1 Concepts and Implications of Pathogen Infection of Seeds and Propagules 3 1.2 Economic Importance of Seed ] and Propagule ]Borne Microbial Pathogens 4 1.3 Nature of Seed ] and Propagule ]Borne Microbial Pathogens 6 1.4 Development of Crop Disease Management Systems 8 References 9 2 Detection and Identification of Fungal Pathogens 12 2.1 Detection and Differentiation of Fungal Pathogens in Seeds 12 2.2 Detection and Differentiation of Fungal Pathogens in Propagules 86 2.3 Appendix 104 References 112 3 Biology of Fungal Pathogens 134 3.1 Biological Characteristics 135 3.2 Physiological Characteristics of Fungal Pathogens 144 3.3 Genotypic Characteristics of Fungal Pathogens 147 3.4 Influence of Storage Conditions 165 3.5 Appendix 166 References 166 4 Process of Infection by Fungal Pathogens 174 4.1 Invasion Paths of Seedborne Fungal Pathogens 174 4.2 Invasion Paths of Propagule ]Borne Fungal Pathogens 207 References 210 5 Detection and Identification of Bacterial and Phytoplasmal Pathogens 220 5.1 Detection and Identification of Bacterial Pathogens 220 5.2 Detection of Bacterial Pathogens in Propagules 273 5.3 Detection of Phytoplasmal Pathogens 326 5.4 Appendix 343 References 352 6 Biology and Infection Process of Bacterial and Phytoplasmal Pathogens 375 6.1 Biology of Bacterial Pathogens 375 6.2 Disease Cycles of Seedborne Bacterial Pathogens 377 6.3 Disease Cycles of Propagule ]Borne Bacterial Pathogens 409 6.4 Biology of Phytoplasmal Pathogens 429 6.5 Disease Cycles of Phytoplasmal Pathogens 431 6.6 Appendix 437 References 437 7 Detection and Identification of Viruses and Viroids 457 7.1 Detection of Viruses in Seeds 457 7.2 Detection of Viruses in Propagules 493 7.3 Detection of Viroids in Seeds 572 7.4 Detection of Viroids in Propagules 577 7.5 Appendix 590 References 594 8 Biology and Infection Process of Viruses and Viroids 619 8.1 Characteristics of Plant Viruses 619 8.2 Biological Properties of Viruses 620 8.3 Infection Process of Plant Viruses 632 8.4 Characteristics of Viroids 646 8.5 Infection Process of Viroids 651 References 656 Index 669 Volume 2 Epidemiology and Management of Crop Diseases 1 9 Epidemiology of Seed ] and Propagule ]Borne Diseases 3 9.1 Epidemiology of Fungal Diseases 4 9.2 Epidemiology of Bacterial Diseases 27 9.3 Epidemioloy of Virus Diseases 37 References 42 10 Crop Disease Management: Exclusion of Pathogens 52 10.1 Health Status of Seeds and Propagules 52 10.2 Plant Quarantines for Preventing Entry of Microbial Pathogens 63 10.3 Production of Disease ]Free Seeds and Propagules 72 10.4 Appendix 89 References 91 11 Crop Disease Management: Reduction of Pathogen Inoculum 100 11.1 Reduction of Pathogen Inoculum by Cultural Practices 100 11.2 Reduction of Pathogen Inoculum by Physical Techniques 123 11.3 Reduction of Pathogen Inoculum by Chemical Techniques 132 References 133 12 Crop Disease Management: Enhancement of Genetic Resistance of Crop Plants 142 12.1 Types of Disease Resistance 142 12.2 Identfication of Sources of Resistance to Crop Diseases 145 12.3 Improvement of Disease Resistance Through Biotechnological Approaches 188 References 205 13 Crop Disease Management: Biological Management Strategies 224 13.1 Evaluation of Biotic Agents for Biological Control Potential 225 13.2 Evaluation of Abiotic Agents for Biological Control Potential 262 13.3 Methods of Application of Formulated Products of Biological Control Agents 283 13.4 Integration of Biological Control with Other Management Practices 289 References 290 14 Crop Disease Management: Chemical Application 306 14.1 Application of Fungicides 307 14.2 Application of Chemicals Against Bacterial Diseases 341 14.3 Application of Chemicals Against Virus Diseases 348 References 351 15 Crop Disease Management: Integration of Strategies 361 15.1 Development of Integrated Disease Management Systems 361 15.2 Management of Fungal Diseases 364 15.3 Management of Bacterial Diseases 369 15.4 Management of Virus Diseases 373 References 377 Index 383

Citation preview

Microbial Plant Pathogens

Microbial Plant Pathogens Detection and Management in Seeds and Propagules

Volume 1

P. Narayanasamy

Former Professor and Head Department of Plant Pathology Tamil Nadu Agricultural University Coimbatore, India

This edition first published 2017 © 2017 John Wiley & Sons, Ltd. Registered Office John Wiley & Sons, Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial Offices The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley‐blackwell. The right of the author to be identified as the author of this work has been asserted in accordance with the UK Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. Limit of Liability/Disclaimer of Warranty: While the publisher and author(s) have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. It is sold on the understanding that the publisher is not engaged in rendering professional services and neither the publisher nor the author shall be liable for damages arising herefrom. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Library of Congress Cataloging‐in‐Publication Data Names: Narayanasamy, P., 1937– author. Title: Microbial plant pathogens : detection and management in seeds and propagules / P. Narayanasamy. Description: Hoboken : John Wiley & Sons, 2017. | Includes bibliographical references and index. Identifiers: LCCN 2016036177 (print) | LCCN 2016037345 (ebook) | ISBN 9781119195771 (cloth) | ISBN 9781119195788 (pdf ) | ISBN 9781119195795 (epub) Subjects: LCSH: Seed-borne phytopathogens. | Seed-borne plant diseases. Classification: LCC SB732.8 .N27 2017 (print) | LCC SB732.8 (ebook) | DDC 632/.3–dc23 LC record available at https://lccn.loc.gov/2016036177 A catalogue record for this book is available from the British Library. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Set in 10/12pt Warnock by SPi Global, Pondicherry, India 10 9 8 7 6 5 4 3 2 1

Dedicated to the memory of my parents for their love and affection

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Contents Preface  xv Acknowledgement  xvii Volume 1 

Pathogen Detection and Identification  1

1 Introduction  3

1.1 ­Concepts and Implications of Pathogen Infection of Seeds and Propagules  3 1.2 ­Economic Importance of Seed‐ and Propagule‐Borne Microbial Pathogens  4 1.3 ­Nature of Seed‐ and Propagule‐Borne Microbial Pathogens  6 1.4 ­Development of Crop Disease Management Systems  8 References  9

Detection and Identification of Fungal Pathogens  12 2.1 ­Detection and Differentiation of Fungal Pathogens in Seeds  12 2.1.1 Conventional/Isolation‐Dependent Methods  13 2.1.1.1 Dry seed examination  14 2.1.1.2 Histological and Cytological Methods  15 2.1.1.3 Seed Washing Test  16 2.1.1.4 Blotter Test  16 2.1.1.5 Direct Plating  18 2.1.2 Isolation‐Independent Methods  19 2.1.2.1 Grow‐Out/Seedling Symptom Test  19 2.1.2.2 Physical Methods  20 2.1.2.3 Chemical Methods  22 2.1.3 Immunoassays  24 2.1.4 Nucleic Acid‐Based Techniques  28 2.1.4.1 Hybridization‐Based Nucleic Acid Techniques  28 2.1.4.2 PCR‐Based Techniques  29 2.1.4.3 Combination of PCR‐Based Assays with Other Techniques  59 2.1.4.4 Reverse‐Transcription (RT) Polymerase Chain Reaction  61 2.1.4.5 Random Amplified Polymorphic DNA Technique  61 2.1.4.6 Amplified Fragment Length Polymorphism  67 2.1.4.7 Restriction Fragment Length Polymorphism  70 2.1.4.8 DNA Sequence Analysis  75

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2.1.4.9 Phylogenetic Analysis  79 2.1.4.10 DNA Array Technology  80 2.1.4.11 Matrix‐Assisted Laser Desorption Ionization Time‐of‐Flight Mass Spectrometry  81 2.1.4.12 Biochemical Methods  81 2.1.4.13 Assessment of Variations in Biological Characteristics of Fungal Pathogens  84 2.2 ­Detection and Differentiation of Fungal Pathogens in Propagules  86 2.2.1 Conventional/Isolation‐Dependent Methods  87 2.2.2 Isolation‐Independent Methods  88 2.2.2.1 Immunoassays 88 2.2.2.2 Nucleic Acid‐Based Techniques  90 2.2.2.3 Loop‐Mediated Isothermal Amplification  101 2.2.3 Techniques Based on Biological Characteristics of Fungal Pathogens  102 2.2.3.1 Vegetative Compatibility Groups  102 2.3 ­Appendix  104 2.3.1 Alkaline Blotter Method  104 2.3.2 Detection of Teliospores of Tilletia indica in Seed by Acid Electrolyzed Water   104 2.3.2.1 Preparation of Acid Electrolyzed Water and Sodium Hypochlorite Solutions  104 2.3.2.2 Assessment of Effectiveness of AEW and NaOCl  104 2.3.3 Detection of Sporisorium reilianum and Ustilago maydis by Dot‐Blot Hybridization  105 2.3.3.1 Isolation of Smut Pathogens  105 2.3.3.2 Nucleic Acid Hybridization  105 2.3.4 Detection of Phoma ligulicola in Pyrethrum Seed by Polymerase Chain Reaction  106 2.3.4.1 Isolation of the Pathogen  106 2.3.4.2 Extraction of Pathogen DNA  106 2.3.4.3 PCR Amplification  106 2.3.5 Detection of Plasmopara halstedii in Sunflower Seed by Optimized Duplex Real‐Time PCR Assay  106 2.3.5.1 Preparation of P. halstedii‐Infected Seed Materials  106 2.3.5.2 Optimized Duplex qPCR Assay  107 2.3.6 Detection of Colletotrichum lindemuthianum in Common Bean Seed by PCR Assay  107 2.3.6.1 Extraction of Pathogen DNA  107 2.3.6.2 Seed Powder Preparation  107 2.3.6.3 PCR Amplification  107 2.3.7 Detection of Alternaria brassicae by Conventional and Real‐Time Pcr Assays in Radish Seeds  108 2.3.7.1 Seed Sample Preparation  108 2.3.7.2 PCR‐Based Assays  108 2.3.8 Assessment of Genetic Diversity of Fusarium fujikuroi by UP‐PCR Analyses  109 2.3.8.1 Extraction of DNA from Pathogen Mycelium  109 2.3.8.2 UP‐PCR Amplification  109

Contents

2.3.9

Detection and Quantification of Veriticillium dahliae in Spinach Seed by Real‐Time PCR Assay  110 2.3.9.1 Extraction of Pathogen DNA from Infected Spinach Seed  110 2.3.9.2 Quantitative PCR Assay  110 2.3.10 Assessment of Variation Among Isolates of Fusarium graminearum Using Random Amplified Polymorphic DNA (RAPD)  111 2.3.11 Differentiation of Tilletia spp. by PCR‐RFLP Technique  111 2.3.11.1 Extraction of Pathogen DNA  111 2.3.11.2 PCR Amplification  111 2.3.11.3 DNA Sequencing and Analysis  112 References  112 Biology of Fungal Pathogens  134 3.1 ­Biological Characteristics  135 3.1.1 Disease cycles of Fungal Pathogens  135 3.1.1.1 Reproduction of Fungal Pathogens  135 3.1.1.2 Survival and Dispersal of Fungal Pathogens  136 3.1.2 Host range of Fungal Pathogens  143 3.2 Physiological Characteristics of Fungal Pathogens  144 3.2.1 Variations in Virulence of Fungal Pathogens  144 3.3 Genotypic Characteristics of Fungal Pathogens  147 3.3.1 Pathogenicity of Fungal Pathogens  147 3.3.2 Production of Mycotoxins by Fungal Pathogens  149 3.3.3 Production of Enzymes and Other Compounds by Fungal Pathogens  157 3.3.4 Sensitivity of Fungal Pathogens to Fungicides  160 3.4 Influence of Storage Conditions  165 3.5 Appendix  166 3.5.1 Isolation of Fusarium oxysporum f.sp. vasinfectum from cotton seed  166 3.5.1.1 Fusarium selective liquid medium  166 3.5.1.2 Isolation of the pathogen  166 References  166

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4 Process of Infection by Fungal Pathogens  174 4.1 Invasion Paths of Seedborne Fungal Pathogens  174 4.1.1 Direct Infection of Floral Parts  174 4.1.2 Infection Through Inoculum in Soil/Plant Residues  190 4.1.3 Infection Through Inoculum Present in Seed‐Bearing Organs  197 4.1.4 Role of Enzymes and Toxins in Pathogenesis  201 4.2 Invasion Paths of Propagule‐Borne Fungal Pathogens  207 References  210 5

5.1 5.1.1 5.1.2 5.1.2.1 5.1.2.2 5.1.3

Detection and Identification of Bacterial and Phytoplasmal Pathogens  220

Detection and Identification of Bacterial Pathogens  220 Detection of Bacterial Pathogens in Seed  220 Conventional/Isolation‐Dependent Methods  222 Isolation of Bacterial Pathogens  222 Bacteriophage Typing  225 Isolation‐Independent Methods  226

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5.1.3.1 Seedling grow‐Out assay  226 5.1.3.2 Biochemical Methods  227 5.1.3.3 Physical Techniques  228 5.1.4 Immunoassays  229 5.1.4.1 Agglutination‐/diffusion‐Based Tests  230 5.1.4.2 Immunofluorescence Microscopy  231 5.1.4.3 Enzyme‐Linked immunosorbent Assay  232 5.1.4.4 Immunomagnetic Separation of Bacterial Pathogens  237 5.1.4.5 Immunolabeling and Electron Microscopy  238 5.1.4.6 Flow Cytometry  240 5.1.5 Nucleic acid‐Based Assays  242 5.1.5.1 Nucleic acid Hybridization Methods  242 5.1.5.2 Restriction Fragment Length Polymorphism  243 5.1.5.3 PCR‐Based Assay  244 5.1.5.4 Real‐Time Polymerase Chain Reaction Assay  254 5.1.5.5 Integration of PCR with Other Detection Methods  261 5.1.5.6 Assessment of Genetic Diversity of Bacterial Pathogens  270 5.2 Detection of Bacterial Pathogens in Propagules  273 5.2.1 Conventional/biological Methods  273 5.2.2 Phage Typing  275 5.2.3 Microscopy  276 5.2.4 Physical Techniques  276 5.2.5 Immunoassays  278 5.2.6 Nucleic Acid‐Based Techniques  282 5.2.6.1 Nucleic Acid Hybridization Methods  282 5.2.6.2 PCR‐Based Methods  283 5.2.6.3 Loop‐Mediated Isothermal Amplification Technique  318 5.2.6.4 Cycleave Isothermal and Chimeric Primer‐Initiated Amplification of Nucleic Acids  321 5.2.6.5 DNA Array Technology  322 5.2.6.6 Nucleic Acid‐Based Amplification Assay  325 5.3 Detection of Phytoplasmal Pathogens  326 5.3.1 Immunoassays  328 5.3.2 Nucleic Acid‐Based Techniques  330 5.3.2.1 Hybridization Techniques  330 5.3.2.2 Polymerase Chain Reaction Techniques  330 5.3.2.3 Loop‐Mediated Isothermal Amplification Assay  343 5.4 Appendix  343 5.4.1 General/Semiselective Media for Isolation of Bacterial Pathogens from Seed  343 5.4.2 Stem‐Printing Technique for Evaluation of Seed Transmission of Pantoea stewartii ssp. stewartii in Corn Seedlings  344 5.4.3 Detection of Acidovorax avenae ssp. citrulli (Aac) by MAb‐Captured‐Sandwich ELISA (MC‐sELISA) in Seed Extracts  344 5.4.3.1 Detection of the Pathogen in Culture  344 5.4.3.2 Detection of the Pathogen in Artificially and Naturally Infested Seeds  345 5.4.4 Detection of Clavibacter michiganensis subsp. michiganensis (Cmm) in Tomato Seeds by Immunomagnetic Separation (IMS) Plating Technique  345

Contents

5.4.4.1 5.4.4.2 5.4.5 5.4.5.1 5.4.5.2 5.4.6

Coating of Magnetic Beads  345 Standardization of Immunoseparation Technique  345 Detection of Xanthomonas campestris pv. carotae by PCR Assay  346 Extraction of Pathogen DNA from Seeds  346 Polymerase Chain Reaction (PCR) Assay  346 Detection of Burkholderia glumae in Rice Seeds by Real‐Time PCR Assay  346 5.4.6.1 Preparation of Seed Wash  346 5.4.6.2 Real‐Time PCR Assay  347 5.4.7 Detection and Quantification of Burkholderia spp. in Rice Seeds by Real‐Time PCR Assay  347 5.4.7.1 Isolation of DNA for Real‐Time PCR Assay  347 5.4.7.2 Real‐Time PCR Assay  347 5.4.7.3 Preparation of Standard Curve  348 5.4.8 Detection of Pseudomonas syringae pv. phaseolicola in Bean Seed Washings by Membrane Bio‐pcr Assay  348 5.4.9 Detection of Acidovorax avenae subsp. citrulli in the Seeds of Watermelon by IMS‐Real‐Time PCR Assay  348 5.4.9.1 Immunomagnetic Separation (IMS)  348 5.4.9.2 Real‐Time PCR Assay  349 5.4.10 Detection of Acidovorax avenae subsp. citrulli (Aac) by Direct PCR and Immunocapture (IC‐) PCR Assays in Watermelon Seeds  349 5.4.10.1 Selection of Antibodies and Primers Specific to Aac 349 5.4.10.2 Detection of Aac by Standard PCR Format  349 5.4.10.3 Detection of Aac by IC‐PCR Assay  349 5.4.11 Detection of “Candidatus Liberibacter solanacearum,” Causative Agent of Potato Zebra Chip Disease by PCR Assay  350 5.4.11.1 Extraction of DNA from Plant Tissues  350 5.4.11.2 PCR Amplification  350 5.4.12 Detection of Erwinia carotovora subsp. atroseptica (Eca) by Loop‐Mediated Isothermal Amplification (LAMP) Assay in Potato  350 5.4.12.1 Preparation of Genomic DNA of the Pathogen  350 5.4.12.2 Preparation of Primers  351 5.4.12.3 LAMP Assay  351 5.4.13 Detection of Flavescence Dorée Phytoplasma in Grapevine by Reverse‐Transcription PCR Assay  351 5.4.13.1 Extraction of Leaf Sap  351 References  352 6

6.1 6.1.1 6.2 6.2.1 6.2.2 6.2.3 6.2.4 6.2.5

Biology and Infection Process of Bacterial and Phytoplasmal Pathogens  375

Biology of Bacterial Pathogens  375 General Characteristics of Bacterial Pathogens  375 Disease Cycles of Seedborne Bacterial Pathogens  377 Xanthomonas spp.  377 Pseudomonas spp.  391 Burkholderia spp.  395 Acidovorax spp.  397 Clavibacter spp.  401

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6.2.6 Curtobacterium spp.  406 6.2.7 Pantoea spp.  407 6.2.8 Xylella sp.  408 6.3 Disease Cycles of Propagule‐Borne Bacterial Pathogens  409 6.3.1 Erwinia spp.  410 6.3.2 Clavibacter spp.  414 6.3.3 Ralstonia sp.  414 6.3.4 Xanthomonas spp.  417 6.3.5 Candidatus Liberibacter  421 6.3.6 Streptomyces spp.  426 6.4 Biology of Phytoplasmal Pathogens  429 6.4.1 General Characteristics of Phytoplasmal Pathogens  430 6.5 Disease Cycles of Phytoplasmal Pathogens  431 6.5.1 Apple Proliferation Phytoplasma  434 6.5.2 Lime Witches’ Broom Phytoplasma  435 6.5.3 Flavescence Dorée (FD) Phytoplasma  436 6.5.4 Palm Lethal Yellowing (LY) Phytoplasma  436 6.6 Appendix  437 6.6.1 Stab‐Inoculation of Xanthomonas campestris pv. vitians on Lettuce Plants  437 6.6.1.1 Isolation of the Pathogen  437 6.6.1.2 Determination of Pathogen Location in Lettuce Stems  437 ­References  437 7 Detection and Identification of Viruses and Viroids  457 7.1 Detection of Viruses in Seeds  457 7.1.1 Plant Virus Taxonomy  458 7.1.2 Biological Methods  458 7.1.2.1 Symptoms Induced by Viruses  458 7.1.2.2 Modes of Transmission of Viruses  460 7.1.3 Immunoassays  463 7.1.3.1 Enzyme‐Linked Immunosorbent Assay  464 7.1.3.2 Dot‐Immunobinding Assay  476 7.1.3.3 Petridish‐Agar Dot‐Immunomagnetic Assay  477 7.1.3.4 Fluorescent Antibody Technique  477 7.1.3.5 Microscopy‐Based Techniques  478 7.1.4 Nucleic Acid‐Based Techniques  480 7.1.4.1 Nucleic Acid Hybridization  480 7.1.4.2 Polymerase Chain Reaction Assays  483 7.1.4.3 Combination of PCR Assay with Other Diagnostic Methods  491 7.2 Detection of Viruses in Propagules  493 7.2.1 Bioindexing Methods  494 7.2.2 Immunoassays  495 7.2.2.1 Enzyme‐Linked Immunosorbent Assays  499 7.2.2.2 Tissue‐Blot Immunoassay  509 7.2.2.3 Immunoblot Assay  512 7.2.2.4 Dot‐Immunobinding Assay  514

Contents

7.2.2.5 In Situ Immunoassay  515 7.2.2.6 Immunosorbent Electron Microscopy  516 7.2.3 Nucleic Acid‐Based Techniques  517 7.2.3.1 Nucleic Acid Hybridization  518 7.2.3.2 Polymerase Chain Reaction Techniques  521 7.2.3.3 Combination of RT‐PCR with Other Diagnostic Assays  550 7.2.4 Real‐Time PCR Assay  556 7.2.5 Molecular Beacon  562 7.2.6 Single‐Strand Conformation Polymorphism Analysis  564 7.2.7 Reverse‐Transcription Loop‐Mediated Isothermal Amplification  565 7.2.8 DNA Array Technology  568 7.2.9 Small‐RNA Deep‐Sequencing Analysis  570 7.3 Detection of Viroids in Seeds  572 7.3.1 Classification of Viroids  572 7.3.2 Biological Methods  573 7.3.3 Physico‐Chemical Methods  574 7.3.4 Nucleic Acid‐Based Techniques  575 7.4 Detection of Viroids in Propagules  577 7.4.1 Nucleic Acid‐Based Techniques  577 7.4.1.1 Nucleic Acid Hybridization  578 7.4.1.2 Reverse‐Transcription (RT‐) PCR Techniques  581 7.4.1.3 Reverse‐Transcription Loop‐Mediated Isothermal Amplification  588 7.4.1.4 Single‐Strand Conformation Polymorphism Analysis  590 7.5 Appendix  590 7.5.1 Detection of Citrus Tristeza Virus by Improved Direct Tissue‐Blot Immunoassay (I‐DTBIA) in Citrus Plants  590 7.5.1.1 Preparation of Antibodies  590 7.5.1.2 Improved DTBIA Procedure  591 7.5.2 Detection of Tomato Spotted Wilt Virus (TSWV) in Ranunculus Tubers by Tissue‐Blot Immunoassay  591 7.5.2.1 Preparation of Plant Tissue Samples  591 7.5.2.2 Tissue‐Blot Immunoassay (TBIA)  591 7.5.3 Detection of Citrus Tristeza Virus (CTV) by Hybridization Assays  592 7.5.3.1 Preparation of Nucleic Acid Extracts  592 7.5.3.2 Hybridization 592 7.5.4 Detection of Plum Pox Virus (PPV) by Real‐Time RT‐PCR and Its Variants  592 7.5.4.1 Preparation of Crude Extracts of Plant Tissues  592 7.5.4.2 Real‐Time RT‐PCR and Dilution, Spot, Tissue Print, and Squash Variants  593 7.5.5 Detection of Potato Spindle Tuber Viroid by Reverse‐Transcription Loop‐Mediated Isothermal Amplification (LAMP)  593 7.5.5.1 Extraction of Total Nucleic Acid from Potato Leaf, Tuber, and Tomato Leaf Tissues  593 7.5.5.2 RT‐LAMP Reaction  594 References  594

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8

Biology and Infection Process of Viruses and Viroids  619

8.1 Characteristics of Plant Viruses  619 8.2 Biological Properties of Viruses  620 8.2.1 Types of Symptoms Induced by Viruses  620 8.2.2 Host Range of Viruses  623 8.2.3 Genetic Variability of Viruses  623 8.2.4 Modes of Transmission of Viruses  627 8.2.5 Cross‐Protection of Viruses  628 8.3 Infection Process of Plant Viruses  632 8.3.1 Virus Replication  632 8.3.2 Movement of Plant Viruses  634 8.3.2.1 Cell‐to‐Cell Movement  634 8.3.2.2 Long‐Distance Movement of Viruses  637 8.3.3 Colonization of Plant Tissues by Viruses  640 8.4 Characteristics of Viroids  646 8.4.1 Biological Properties of Viroids  647 8.4.1.1 Types of Symptoms  647 8.4.1.2 Diagnostic Hosts and Host Range  647 8.4.1.3 Modes of Viroid Transmission  649 8.5 Infection Process of Viroids  651 References  656 Index  669 Volume 2 

Epidemiology and Management of Crop Diseases  1

  9

Epidemiology of Seed‐ and Propagule‐Borne Diseases  3

10

Crop Disease Management: Exclusion of Pathogens  52

11

Crop Disease Management: Reduction of Pathogen Inoculum  100

12

Crop Disease Management: Enhancement of Genetic Resistance of Crop Plants  142

13

Crop Disease Management: Biological Management Strategies  224

14

Crop Disease Management: Chemical Application  306

15

Crop Disease Management: Integration of Strategies  361

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Preface Agricultural and horticultural crops are raised by using seeds or propagules whose health status has to be ensured for healthy and high‐quality produce to satisfy the needs of human and animal populations. From time immemorial, seeds and propagules were obtained from selected plants growing in the wild environment, saved to grow plants of subsequent generations. In doing so, the improvement of quality parameters such as appearance, color, aroma, and taste was achieved by selecting plants with desired characteristics. However, due importance was not allocated to select plants with resistance to diseases caused by microbial plant pathogens, resulting in progressive increase in the susceptibility of plants to diseases and phenomenal crop losses which were considered responsible for dreadful famines and untold human suffering. Several diseases transmitted through seeds/propagules have been found to be highly destructive, with the potential to ruin the economy of certain countries. Such critical conditions were ­primarily ascribed to the failure to select disease‐free seeds and propagules for future generations of crops. Early detection and precise identification of the pathogen(s) present in seeds/­ propagules which are involved in a disease(s) occurring at a geographical location constitute the basic strategy for development of effective disease management systems suitable for various agroecosystems. Studies of pathogen biology, the infection process, and epidemiology of crop diseases have highlighted the weak links in the life cycles of microbial pathogens in order to disrupt pathogen development and the progress of disease under field conditions. The principles of crop disease management are essentially based on exclusion, eradication, and immunization and various disease management strategies emerge from these principles. The need to produce disease‐free seeds and propagules to restrict the introduction of pathogens into fields/new locations and subsequent disease spread has been clearly indicated by different investigations. The role of quarantines and certification programs in excluding the introduction of new diseases into a country where the pathogen may be absent or less important has been well ­realized. The effectiveness of adoption of simple cultural practices in restricting disease incidence and further spread has been indicated in some pathosystems. The development of crop cultivars with built‐in resistance to diseases, the most economical disease management strategy, has been achieved through traditional breeding methods or ­biotechnological approach and has been shown to be instrumental in keeping many diseases under check. Employing biological control agents is advantageous, since this strategy has been demonstrated to be effective not only in restricting the disease incidence and spread but also in preserving the ecological environments. The application of

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chemicals, although more effective, must be restricted because of the accumulation of chemical residues in grains and other food materials as well as significant environmental pollution. The integration of compatible disease management strategies has been shown to provide additive effects, resulting in enhancement of effectiveness of the disease management systems; greater efforts have therefore been made to explore the possibility of developing integrated disease management systems for several ­economically important diseases that need to be tackled more effectively. This book includes information distilled from over 2500 citations following an extensive literature search, with the aim of providing a comprehensive knowledge of various aspects of microbial plant pathogens transmitted via seeds and propagules, particularly pathogen detection and management of diseases caused by them. Graduate students, researchers, and teachers of Plant Pathology, Plant Protection, Microbiology, Plant Breeding and Genetics, Agriculture and Horticulture, and especially certification and quarantine personnel, will find the information presented in this book useful. Protocols appended at the end of relevant chapters form a unique feature of this book and may assist researchers in fine‐tuning their projects. P. Narayanasamy Coimbatore, August 2016

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Acknowledgement The author gratefully expresses his sincere appreciation to his alma mater for being an unlimited source of inspiration in reaching his present position of being able to share his experience and thoughts with those who have contributed to the advancement of science in general and plant pathology in particular. He heartily thanks the staff and students of the Department of Plant Pathology, Tamil Nadu Agricultural University for their assistance in one way or another in improving the usefulness of this book to the audience. The efforts of the author to provide useful information on the selected subject matter in this book have become fruitful thanks to the enormous loving support of his wife Mrs N. Rajakumari whom he thanks profusely. With immense pleasure, the author conveys his appreciation to his family members Mr N. Kumar Perumal, Mrs Nirmala Suresh, Mr T. R. Suresh and Mr S.Varun Karthik for encouraging and uplifting his ­spirits with their affectionate, amiable attitude and deeds. Various copyright holders/publishers and authors of research papers have kindly granted permission to use the figures published in different journals, and have been credited at the appropriate pages in this book.

Microbial Plant Pathogens

Microbial Plant Pathogens Detection and Management in Seeds and Propagules

Volume 2

P. Narayanasamy

Former Professor and Head Department of Plant Pathology Tamil Nadu Agricultural University Coimbatore, India

This edition first published 2017 © 2017 John Wiley & Sons, Ltd. Registered Office John Wiley & Sons, Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial Offices The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley‐blackwell. The right of the author to be identified as the author of this work has been asserted in accordance with the UK Copyright, Designs and Patents Act 1988. All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. Limit of Liability/Disclaimer of Warranty: While the publisher and author(s) have used their best efforts in preparing this book, they make no representations or warranties with respect to the accuracy or completeness of the contents of this book and specifically disclaim any implied warranties of merchantability or fitness for a particular purpose. It is sold on the understanding that the publisher is not engaged in rendering professional services and neither the publisher nor the author shall be liable for damages arising herefrom. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Library of Congress Cataloging‐in‐Publication Data Names: Narayanasamy, P., 1937– author. Title: Microbial plant pathogens : detection and management in seeds and propagules / P. Narayanasamy. Description: Hoboken : John Wiley & Sons, 2017. | Includes bibliographical references and index. Identifiers: LCCN 2016036177 (print) | LCCN 2016037345 (ebook) | ISBN 9781119195771 (cloth) | ISBN 9781119195788 (pdf ) | ISBN 9781119195795 (epub) Subjects: LCSH: Seed-borne phytopathogens. | Seed-borne plant diseases. Classification: LCC SB732.8 .N27 2017 (print) | LCC SB732.8 (ebook) | DDC 632/.3–dc23 LC record available at https://lccn.loc.gov/2016036177 A catalogue record for this book is available from the British Library. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Set in 10/12pt Warnock by SPi Global, Pondicherry, India 10 9 8 7 6 5 4 3 2 1

Dedicated to the memory of my parents for their love and affection

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Contents Preface  xi Acknowledgement  xiii Volume 1 

Pathogen Detection and Identification  1

1 Introduction  3 2

Detection and Identification of Fungal Pathogens  12

3

Biology of Fungal Pathogens  134

4

Process of Infection by Fungal Pathogens  174

5

Detection and Identification of Bacterial and Phytoplasmal Pathogens  220

6

Biology and Infection Process of Bacterial and Phytoplasmal Pathogens  375

7

Detection and Identification of Viruses and Viroids  457

8

Biology and Infection Process of Viruses and Viroids  619 Volume 2 

9

9.1 9.1.1 9.1.1.1 9.1.1.2 9.1.1.3 9.1.2 9.2 9.2.1 9.2.1.1 9.2.1.2 9.2.1.3 9.2.2 9.3 9.3.1 9.3.2 9.3.3

Epidemiology and Management of Crop Diseases  1

Epidemiology of Seed‐ and Propagule‐Borne Diseases  3

Epidemiology of Fungal Diseases  4 Dynamics of Host–Fungal‐Pathogen Interactions  4 Pathogen Factors  5 Host Factors  13 Environmental Factors  16 Molecular Ecology and Epidemiology of Fungal Diseases  24 Epidemiology of Bacterial Diseases  27 Dynamics of Host–Bacterial‐Pathogen Interactions  27 Pathogen Factors  27 Host Factors  32 Environmental Factors  34 Molecular Ecology and Epidemiology of Bacterial Diseases  35 Epidemioloy of Virus Diseases  37 Dynamics of Host–Virus Interactions  37 Quantitative Epidemiology  38 Molecular Ecology and Epidemiology of Virus Diseases  39

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Contents

9.3.3.1 Molecular Biology of Virus Infection  40 9.3.3.2 Molecular Biology of Virus Transmission  41 References  42 10 Crop Disease Management: Exclusion of Pathogens  52 10.1 Health Status of Seeds and Propagules  52 10.1.1 Certification of Seeds and Propagules  53 10.1.1.1 Application of Diagnostic Techniques  53 10.2 Plant Quarantines for Preventing Entry of Microbial Pathogens  63 10.2.1 Principles of Plant Quarantines  63 10.2.2 Functions of Regional Plant Quarantine Organizations  64 10.2.3 Impact of Introduced Pathogens  65 10.2.3.1 Potato Late Blight Disease  65 10.2.3.2 Citrus Canker Disease  65 10.2.3.3 Panama (Fusarium) Wilt Disease of Banana  66 10.2.3.4 Banana Bunchy Top Disease  66 10.2.3.5 Strawberry Anthracnose Disease  67 10.2.3.6 Potato Virus Diseases  67 10.2.4 Assessment of Efficiency of Quarantines  68 10.3 Production of Disease‐Free Seeds and Propagules  72 10.3.1 Production of Virus‐Free Plants  72 10.3.1.1 Meristem‐Tip Culture  72 10.3.1.2 Chemotherapy 80 10.3.1.3 Electrotherapy 81 10.3.1.4 Cryotherapy 81 10.3.2 Production of Viroid‐Free Plants  83 10.3.3 Production of Bacterial and Phytoplasmal Pathogen‐Free Plants  84 10.4 Appendix  89 10.4.1 Elimination of Sweet Potato Little Leaf Phytoplasma from Sweet Potato by Cryotherapy of Shoot Tips  89 10.4.1.1 Preparation of Plant Materials  89 10.4.1.2 Cryotherapy of In‐Vitro‐Grown Shoot Tips  91 References  91 11 Crop Disease Management: Reduction of Pathogen Inoculum  100 11.1 Reduction of Pathogen Inoculum by Cultural Practices  100 11.1.1 Elimination of Infected Plants and Debris  100 11.1.2 Effects of Tillage Practices  102 11.1.3 Effects of Sowing Time and Planting Density  104 11.1.4 Effects of Irrigation Practices  106 11.1.5 Effects of Crop Nutrition  108 11.1.5.1 Use of Organic Matter  110 11.1.5.2 Use of Inorganic Fertilizers  111 11.1.6 Effects of Other Crops  115 11.1.6.1 Crop Sequence  115 11.1.6.2 Monoculture 121 11.1.6.3 Multiple Cropping  121 11.2 Reduction of Pathogen Inoculum by Physical Techniques  123

Contents

11.2.1 Treatment of Seeds and Propagules  123 11.2.1.1 Heat Treatments  124 11.2.1.2 Forced‐Air Treatment  128 11.2.1.3 Radiation and Microwave Treatments  128 11.2.1.4 Use of Inorganic Mulches  129 11.3 Reduction of Pathogen Inoculum by Chemical Techniques  132 11.3.1 Treatment of Seeds  132 11.3.1.1 Chemical Application  132 11.3.1.2 Fermentation 133 11.3.2 Treatment of Soils  133 References  133 12

12.1 12.1.1 12.1.2 12.2 12.2.1 12.2.1.1 12.2.1.2 12.2.1.3 12.2.1.4 12.2.1.5 12.3

Crop Disease Management: Enhancement of Genetic Resistance of Crop Plants  142

Types of Disease Resistance  142 Vertical and Horizontal Resistances  143 Durable Resistance  144 Identfication of Sources of Resistance to Crop Diseases  145 Screening for Disease Resistance  145 Assessment of Resistance by Visual Examination  145 Assessment of Resistance by Quantification of Pathogen Biomass  167 Molecular Basis of Resistance to Fungal Diseases  170 Molecular Basis of Resistance to Bacterial Diseases  179 Molecular Basis of Resistance to Virus Diseases  184 Improvement of Disease Resistance Through Biotechnological Approaches  188 12.3.1 Tissue and Cell Culture Techniques  189 12.3.1.1 Somaclonal Variation  189 12.3.1.2 In Vitro Selection for Disease Resistance  190 12.3.1.3 Induction of Mutation Using Chemicals  192 12.3.2 Transgenic Resistance to Crop Diseases  192 12.3.2.1 Transgenic Resistance to Virus Diseases  193 12.3.2.2 Transgenic Resistance to Fungal Diseases  197 12.3.2.3 Transgenic Resistance to Bacterial Diseases  203 References  205 13

13.1 13.1.1 13.1.2 13.1.3 13.1.3.1 13.1.4 13.1.4.1 13.1.4.2 13.1.4.3 13.1.5 13.1.6

Crop Disease Management: Biological Management Strategies  224 Evaluation of Biotic Agents for Biological Control Potential  225 Fungal Biological Control Agents  225 Bacterial Biological Control Agents  227 Viral Biological Control Agents  234 Mild Strains as Biological Control Agents  237 Mechanisms of Action of Biological Control Activities of Biotic Agents  244 Mechanisms of Action of Fungal Biological Control Agents  244 Mechanisms of Action of Bacterial Biological Control Agents  249 Mechanisms of Action of Mild Strains of Viruses  258 Improvement of Biological Control Potential of Biotic Agents  260 Transformation Crop Plants with Genes of Biological Control Agents  261

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13.2 13.2.1 13.2.2 13.2.3 13.2.4 13.2.5 13.3

Evaluation of Abiotic Agents for Biological Control Potential  262 Effects of Addition of Organic Matter  262 Effects of Products from Plant Sources  265 Effects of Products from Animal Sources  269 Effects of Organic Compounds  271 Effects of Inorganic Compounds  280 Methods of Application of Formulated Products of Biological Control Agents  283 13.3.1 Treatment of Seeds  283 13.3.2 Treatment of Propagules/Transplants  286 13.3.3 Treatment of Soils  288 13.3.4 Treatment of Foliage of Plants  289 13.4 Integration of Biological Control with Other Management Practices  289 13.4.1 Integration with Fertilizer Application  289 13.4.2 Integration with Fungicide Application  290 References  290 Crop Disease Management: Chemical Application  306 14.1 Application of Fungicides  307 14.1.1 Seed/Propagule Treatment  307 14.1.2 Soil Treatment  316 14.1.3 Treatment of Aerial Plant Parts  317 14.1.4 Development of Resistance in Fungal Pathogens to Fungicides  324 14.2 Application of Chemicals Against Bacterial Diseases  341 14.3 Application of Chemicals Against Virus Diseases  348 14.3.1 Application of Antiviral Chemicals  348 14.3.2 Chemicals Applied Against Vectors of Viruses  349 References  351

14

Crop Disease Management: Integration of Strategies  361 15.1 Development of Integrated Disease Management Systems  361 15.1.1 Selection of Strategies for Integration  362 15.2 Management of Fungal Diseases  364 15.2.1 Fusarium Head Blight Disease  364 15.2.2 Rice Blast Disease  365 15.2.3 Rice Sheath Blight Disease  366 15.2.4 Late Blight Diseases of Tomato and Potato  367 15.3 Management of Bacterial Diseases  369 15.3.1 Rice Bacterial Leaf Blight Disease  369 15.3.2 Tomato Bacterial Canker Disease  371 15.3.3 Potato Scab Diseases  371 15.3.4 Citrus Canker Disease  372 15.4 Management of Virus Diseases  373 15.4.1 Management of Seedborne Virus Diseases  374 15.4.2 Management of Propagule‐Borne Virus Diseases  375 References  377

15

Index  383

xi

Preface Agricultural and horticultural crops are raised by using seeds or propagules whose health status has to be ensured for healthy and high‐quality produce to satisfy the needs of human and animal populations. From time immemorial, seeds and propagules were obtained from selected plants growing in the wild environment, saved to grow plants of subsequent generations. In doing so, the improvement of quality parameters such as appearance, color, aroma, and taste was achieved by selecting plants with desired characteristics. However, due importance was not allocated to select plants with resistance to diseases caused by microbial plant pathogens, resulting in progressive increase in the susceptibility of plants to diseases and phenomenal crop losses which were considered responsible for dreadful famines and untold human suffering. Several diseases transmitted through seeds/propagules have been found to be highly destructive, with the potential to ruin the economy of certain countries. Such critical conditions were ­primarily ascribed to the failure to select disease‐free seeds and propagules for future generations of crops. Early detection and precise identification of the pathogen(s) present in seeds/­ propagules which are involved in a disease(s) occurring at a geographical location constitute the basic strategy for development of effective disease management systems suitable for various agroecosystems. Studies of pathogen biology, the infection process, and epidemiology of crop diseases have highlighted the weak links in the life cycles of microbial pathogens in order to disrupt pathogen development and the progress of disease under field conditions. The principles of crop disease management are essentially based on exclusion, eradication, and immunization and various disease management strategies emerge from these principles. The need to produce disease‐free seeds and propagules to restrict the introduction of pathogens into fields/new locations and subsequent disease spread has been clearly indicated by different investigations. The role of quarantines and certification programs in excluding the introduction of new diseases into a country where the pathogen may be absent or less important has been well ­realized. The effectiveness of adoption of simple cultural practices in restricting disease incidence and further spread has been indicated in some pathosystems. The development of crop cultivars with built‐in resistance to diseases, the most economical disease management strategy, has been achieved through traditional breeding methods or ­biotechnological approach and has been shown to be instrumental in keeping many diseases under check. Employing biological control agents is advantageous, since this strategy has been demonstrated to be effective not only in restricting the disease incidence and spread but also in preserving the ecological environments. The application of

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  Preface

chemicals, although more effective, must be restricted because of the accumulation of chemical residues in grains and other food materials as well as significant environmental pollution. The integration of compatible disease management strategies has been shown to provide additive effects, resulting in enhancement of effectiveness of the disease management systems; greater efforts have therefore been made to explore the possibility of developing integrated disease management systems for several ­economically important diseases that need to be tackled more effectively. This book includes information distilled from over 2500 citations following an extensive literature search, with the aim of providing a comprehensive knowledge of various aspects of microbial plant pathogens transmitted via seeds and propagules, particularly pathogen detection and management of diseases caused by them. Graduate students, researchers, and teachers of Plant Pathology, Plant Protection, Microbiology, Plant Breeding and Genetics, Agriculture and Horticulture, and especially certification and quarantine personnel, will find the information presented in this book useful. Protocols appended at the end of relevant chapters form a unique feature of this book and may assist researchers in fine‐tuning their projects. P. Narayanasamy Coimbatore, August 2016

xiii

Acknowledgement The author gratefully expresses his sincere appreciation to his alma mater for being an unlimited source of inspiration in reaching his present position of being able to share his experience and thoughts with those who have contributed to the advancement of science in general and plant pathology in particular. He heartily thanks the staff and students of the Department of Plant Pathology, Tamil Nadu Agricultural University for their assistance in one way or another in improving the usefulness of this book to the audience. The efforts of the author to provide useful information on the selected subject matter in this book have become fruitful thanks to the enormous loving support of his wife Mrs N. Rajakumari whom he thanks profusely. With immense pleasure, the author conveys his appreciation to his family members Mr N. Kumar Perumal, Mrs Nirmala Suresh, Mr T. R. Suresh and Mr S.Varun Karthik for encouraging and uplifting his ­spirits with their affectionate, amiable attitude and deeds. Various copyright holders/publishers and authors of research papers have kindly granted permission to use the figures published in different journals, and have been credited at the appropriate pages in this book.

1

Volume 1 Pathogen Detection and Identification

3

1 Introduction Plant diseases are caused by biotic and abiotic stresses. The microbial plant pathogens – oomycetes (fungus‐like), fungi, bacteria, phytoplasmas, viruses, and viroids – form a major group of biotic stresses causing a variety of diseases in various crops grown in different agroecosystems, whereas abiotic stresses are due to adverse environmental conditions which may have an influence both on the plants and the pathogens. The ­environmental conditions have an important role in the buildup of inoculum and in predisposing the plants to infection by the pathogens to a different extent, depending on the geographical locations where the infected seeds/propagules are to be planted.

1.1 ­Concepts and Implications of Pathogen Infection of Seeds and Propagules Use of clean seed and propagules (vegetatively propagated planting materials) is emphasized as the basic step for the commencement of implementation of strategies of crop disease management systems. Seedborne pathogens are not confined to the spermosphere alone and they may also be present in or on the surface of other organs or storage tissues of the plant. Seed pathology is considered a branch of plant pathology, devoted to the detection of microorganisms associated with seeds. That seeds are carriers of microbial plant pathogens, resulting in the infection of developing seedlings, has been known for a long time. The Danish Government Institute of Seed Pathology (DGISP) for developing countries was established in 1967 at Copenhagen by the concerted efforts of Dr Paul Neergaard, who is considered as the Father of Seed Pathology. The term “seed pathology” was introduced by him during investigations of seedborne pathogens and the diseases caused by them. As the Chairman of the Plant Disease Committee (PDC) of the International Seed Testing Association (ISTA), Dr Neergaard has contributed immensely to the standardization of methods for detection of ­seedborne fungi. He authored the book Seed Pathology, which contains voluminous information on the various aspects of seed pathology and is the main reference book for all plant pathologists. Seed pathology has developed into an important discipline within plant pathology. The propagules are the “seed” for the asexually propagated crops, and the importance of propagules carrying pathogens in the incidence and spread of crop ­diseases has been emphasized by several investigations.

Microbial Plant Pathogens: Detection and Management in Seeds and Propagules, First Edition. P. Narayanasamy. © 2017 John Wiley & Sons, Ltd. Published 2017 by John Wiley & Sons, Ltd.

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Microbial Plant Pathogens

The role of seed‐ and propagule‐borne inoculum in crop disease epidemiology and the indispensability of using disease‐free seeds and propagules for profitable crop cultivation are well realized. Several seed‐ and propagule‐borne pathogens have been shown to be disseminated and perpetuated through various other means. Comprehensive studies are therefore essential to elucidate the host–pathogen interactions, leading to the incidence and spread of diseases caused by seed‐ and propagule‐borne pathogens and consequent substantial losses in quantity and quality of the produce induced by them. Some seedborne pathogens may become soilborne when the infected seeds are planted, and the pathogen present in the soil can infect the plants systemically and infect different tissues en route to the seeds as in the case of Rhizoctonia solani causing rice sheath blight (Narayanasamy 2002). Fusarium graminearum, incitant of the Fusarium head blight (FHB) disease of wheat, develops on the infected plant debris left on the soil after harvest. The inoculum from the infested tissues reaches spikes at the time of anthesis through wind currents (Inch and Gilbert 2003). Verticillium dahliae is  seedborne in spinach. The possibility of spinach seeds becoming soilborne and ­infecting lettuce plants was explored under microplot conditions. Verticillium wilt developed on lettuce following two or three plantings of Verticillium‐infested spinach seeds/plant tissues. Polymerase chain reaction (PCR) assay was employed to detect and confirm the identity of V. dahliae in infected lettuce plants obtained from the microplots. In addition, transmission of a green fluorescent protein (GFP) ‐tagged mutant strain of V. dahliae from spinach to lettuce was demonstrated in greenhouse experiments. The results provided clear evidence that the transmission of V. dahliae introduced through infected spinach seed could be the source of inoculum for ­ Verticillium wilt disease in lettuce (Short et al. 2015). Seeds and propagules infected by microbial pathogens form the primary sources of inoculum from which they are disseminated by other modes of transmission such as soil, water, or wind. As the environmental conditions have significant effects on the pathogen and the crops, incorporation of epidemiological concepts and the effects of various disease management strategies have to be considered as components of seed pathology. As the propagules are the seed for the vegetatively propagated crops, the propagule‐borne pathogens have to be managed by applying different strategies as in the case of seedborne pathogens. It is logical to widen the scope of research and applications pertaining to the microbial pathogens associated with the seeds and propagules, and to develop management systems for restricting the incidence and spread of the diseases caused by them.

1.2 ­Economic Importance of Seed‐ and Propagule‐Borne Microbial Pathogens Microbial pathogens have been demonstrated to be the causes of various crop diseases of great historical and economic importance. A major catastrophe due to potato late  blight disease caused by Phytophthora infestans occurred during the 1840s, ­resulting in the infamous Irish potato famine. The acute food shortage was primarily responsible for  the migration of about 1.5 million people from Ireland to other ­countries (Large 1940). A similar magnitude of human suffering due to another fungal pathogen, Helminthosporium oryzae, ruined the rice crops extensively in India. It has

Introduction

been e­ stimated that about 2 million persons perished due to starvation, because of the exorbitant cost of rice which was beyond the reach of the majority of Indians during that decade (Padmanabhan 1973). These two pathogens, transmitted through infected potato tubers and rice grains respectively, revealed the economic importance and social relevance of the pathogens which are either seed‐ or propagule‐borne. Fusarium head blight (FHB) disease infecting wheat and barley is caused by different Fusarium spp. and still remains an enigma for all involved in cereal production. FHB disease is considered as a re‐emerging disease of devastating impact on cereal grain production. A series of severe FHB epidemics that occurred in the USA and Canada during 1991–1996 led to unparalleled economic and sociological impact in the Northern Great Plains region. The combined direct and secondary economic losses between 1993 and 2001 were estimated to cost US $7.7 billion in nine states of USA (Nganje et  al. 2004; McMullen et  al. 2012). In addition, the fungal pathogens involved in FHB disease ­produce different kinds of mycotoxins capable of causing ailments in human beings and animals if contaminated grains and feed are consumed. The quantitative and ­qualitative losses are very difficult to estimate precisely. Rice bacterial leaf blight (BLB) disease caused by Xanthomonas oryzae pv. oryzae is  widespread in all SE Asian countries. In Japan, rice crops in 300,000–400,000 ha were infected by BLB disease with yield losses ranging over 20–30% (Ou 1972). The estimated yield losses due to BLB disease in tropical Asia may vary over 2–74% depending on the location, season, weather, cultivar, and stage of crop growth at the time of infection. Yield loss induced at the kresek phase of the disease was found to be greater in India (Srivastava and Kapoor 1982; Reddy 1989). The bacterial pathogen Xanthomonas axonopodis pv. citri causing citrus canker disease was considered to have been introduced into the USA from Asian countries through infected budwood materials. Quarantine laws were imposed as soon as the first incidence of citrus canker was observed, and the massive eradication of 20 million citrus trees at a cost of US $20 million effectively checked the spread of the disease (Schumann 1991). Tomato ­bacterial canker disease caused by Clavibacter michiganensis subsp. michiganensis is considered to be one of the most destructive bacterial diseases in tomatoes with the potential to cause heavy yield losses. In Ontario, Canada yield losses of up to 84% in commercial fields were recorded (Poysha 1993). In Michigan State, USA tomato bacterial canker disease accounted for as much as US $300,000 in a single year for individual processing tomato growers (Hausbeck et al. 2000). Because of the potentially destructive nature of the pathogen, the Good Seed and Plant Practices Organization functioning in the Netherlands and France directed disease management strategies for preventing infection of tomato seeds and avoiding the use of infected seedlings (de León et al. 2011; Sen et al. 2015). Huanglongbing (HLB), also referred to as greening of citrus caused by Candidatus Liberibacter asiaticus, has been known to affect citrus production in East Asia for more than a century (Bové 2006); the disease is now found in threatening proportions in the USA and other countries. Tree decline, premature fruit drop and formation of small misshapened fruits lead to drastic production losses, incurring an estimated loss of US $3.63 billion in revenue in Florida alone (Hodges and Spreen 2013; Wang and Trivedi 2013). The HLB disease can be spread through infected nursery stock and the Asian citrus psyllid Diaphorina citri, indicating the need for directing management strategies toward production of certified ­pathogen‐ free nursery stock, eradication of infected trees, and reduction of vector populations through chemical application (Gergerich et al. 2015).

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Microbial Plant Pathogens

The socioeconomic consequences of seedborne viruses such as Bean common mosaic virus (BCMV) and Lettuce mosaic virus (LMV) have been revealed by several investigations (Stewart and Reddick 1917; Zink et al. 1956; Grogan 1983). The number of seedborne viruses reported increased from 47 in 1969 (Bennett 1969), to 85 in 1974 (Phatak 1974) to 119 in 1981 (Mandahar 1981), and the number of viruses reported to be transmitted via seeds is increasing steadily with time. In addition, viruses are also known to be readily transmitted through propagules. Infected plants may remain asymptomatic because of long latent (incubation) periods required for symptom expression in the mother plants, especially in perennial fruit trees if infection occurs late in the growing season. Planting materials prepared from such plants carry the virus(es) and symptoms of infection may be expressed when they are planted later. These plants may establish poorly, exhibit decline, and the produce from such plants may be of poor quality; infection by viruses leads to both quantitative and qualitative losses (Narayanasamy 2011). Plum pox virus (PPV) causes one of the most devastating virus diseases (sharka) infecting stone fruits in all countries. Sharka disease, once limited to Europe for most of the twentieth century, has spread to Africa, South America, North America, and Asia (OEPP/EPPO 2004). Long‐distance spread occurs primarily through grafting for producing propagation materials, and aphids may have an important role for local dispersal in some locations. Transmission of PPV via pollen also has been observed (Isac et al. 1998). The incidence of PPV reached such alarming proportions that the destruction of 648 ha of stone fruit orchards in Pennsylvania was necessary during the decade from when the disease incidence was first observed in 1999. The eradication cost was estimated at US $53 million (Welliver 2012). On the other hand, the cost associated with sharka disease management in Europe was estimated to exceed 10 billion Euros prior to 2006 (Cambra et al. 2006). Citrus tristeza virus (CTV), existing in the form of many strains, induces different kinds of symptoms depending on the virulence of the CTV strain, cultivar, and the scion/rootstock combinations. CTV spreads primarily through infected plants and budwood; secondary spread is through different aphid species, Toxoptera citricida being most efficient in transmitting the virus from infected to healthy plants. Citrus decline due to CTV has taken a heavy toll in several countries and the virus killed more than 100 million trees propagated widely on sour orange rootstock during the last 80 years in South America, the USA, Spain, and Israel (Moreno et al. 2008). When two or more CTV strains infect citrus plants simultaneously complex symptoms appear, making it difficult to establish the identity of the strain(s). The complexity increases further because of continuous recombinations occurring in nature, resulting in the appearance of new or more virulent strains (Mukhopadhyay 2011). Severe epidemics occurred in locations where the vector T. citricida was active and was found in large populations in Brazil and Venezuela (Michaud 1998). The economic impact of CTV on the citrus industry could be lessened considerably by strengthening quarantine and certification programs and using certified virus‐tested budwood (Gergerich et al. 2015).

1.3 ­Nature of Seed‐ and Propagule‐Borne Microbial Pathogens Microbial pathogens infecting plants may be carried in and/or on the seeds and propagules in an active or dormant stage. These planting materials may or may not exhibit visually recognizable symptoms of pathogen infection. Under favorable environments,

Introduction

the fungal pathogens may multiply and infect the emerging seedlings or become ­systemic and develop in the apical tissues of the plants without expressing symptoms of infection, as in the case of cereal smut diseases. Detection of the presence of the pathogens based on the symptoms alone will not be sufficient to establish the identity of the pathogens present in seeds and propagules. Several techniques – conventional methods, immunoassays, and molecular techniques – have been applied for the detection, identification, differentiation, and quantification of fungal pathogens rapidly and precisely (Chapter 2). The genetic diversity to determine the variability of isolates/strains in pathogenicity, survival ability, production of toxins, and sensitivity to environments and fungicides has to be assessed to develop effective systems of management of diseases caused by the fungal pathogens (Chapter  3). Fungal pathogens follow different paths of invasion to reach the seed tissues or storage organs. Monitoring the movement of pathogens from seed‐to‐plant and plant‐to‐seed is essential to break the vulnerable link in the life cycle of the fungal pathogens so that the progress of disease development may be arrested, resulting in the failure of infection/disease development (Chapter 4). Bacterial pathogens with simpler structural characteristics compared to fungal ­pathogens require detection techniques based on their biochemical, physiological, immunological, and molecular properties for their detection, identification, differentiation, and quantification. Nucleic acid‐based techniques have been shown to be more sensitive and reliable and provide precise results rapidly. The phytoplasmal pathogens defying all efforts to culture them in cell‐free media have to be examined in planta only. Most of them are unable to become seedborne because they induce sterility in plants to different extents. Infection of vegetative propagules is the most important method of the transmission of phytoplasmas. They are also transmitted by natural biological ­vectors. Various diagnostic techniques have therefore been evaluated for their efficacy in detecting and differentiating different phytoplasmal pathogens present in the propagules of plants selected for propagation (Chapter 5). Biological properties of and the process of infection by bacterial pathogens are discussed, with a view to focusing ­attention on the vulnerable stages in the population buildup and formation of infection foci of bacterial pathogens to facilitate the application of effective disease management strategies. The sensitivity of bacterial pathogens to chemicals and adverse environmental conditions indicates the possibility of selecting suitable disease management ­strategies for suppressing the development of the diseases caused by these pathogens (Chapter 6). Plant viruses and viroids have the simplest structure with no physiological activity of their own; they are therefore dependent on the synthetic machinery of the host plants for their replication. Both viruses and viroids are seed‐ and/or propagule‐borne in several plant species while the viruses have, in addition, specific biological vectors for their dissemination to different healthy plants and in locations. On the other hand, viroids do not appear to have any proven vector for their dispersal. Various techniques based on the biological, immunological, and nucleic acid properties of viruses have been applied for the detection, identification, and differentiation of viruses and their strains with varying degrees of efficiency, depending on the sensitivity of the techniques employed. Nucleic acid‐based techniques are preferred because of their high levels of sensitivity, rapidity, and reliability. Viroids with single‐stranded RNA alone as their genome are detected and identified more frequently by employing nucleic acid‐based techniques as compared to biological tests which require a longer time and more labor and space (Chapter 7). The process of infection of plants by viruses has been intensively studied using electron microscopy, immunoassays, and nucleic acid‐based methods.

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Microbial Plant Pathogens

The movement of viruses from the point of inoculation to other tissues systemically and finally to seed tissues has been monitored. The investigations have indicated the obstacles to be overcome to target the viruses by using antiviral chemicals which cannot interact with the viruses without affecting plant tissues (Chapter 8).

1.4 ­Development of Crop Disease Management Systems The management of crop diseases caused by seed‐ and propagule‐borne microbial pathogens depends largely on the understanding of the effects of various factors – for example, susceptibility/resistance levels of crop cultivars, virulence of the pathogen species/strains, and the prevailing environmental conditions during crop seasons – on the incidence and severity of diseases caused by the pathogens. These factors favoring incidence and severity of diseases leading to epidemics are discussed in Chapter 9. All research efforts to understand the host–pathogen interactions have been made with the ultimate goal of containing the development and spread of crop diseases under various agroecosystems. Disease management strategies are based on a three‐pronged thrust on the pathogens which are endowed with various armories to deactivate the defense mechanisms of the host plant species. The major principles of disease management systems are exclusion, eradication, and immunization. The disease management strategies based on the principle of exclusion include the establishment of certification and quarantine programs and the use of disease‐free seeds and propagules to prevent the introduction of pathogens from other countries or areas where the pathogen may not be present or not important (Chapter 10). Various cultural practices and physical and chemical techniques have been shown to reduce the pathogen inoculum, resulting in the reduction in the rate of pathogen population buildup and consequent reduction in incidence and severity of crop diseases (Chapter  11). The selection of crop genotypes for high yield potential has led to increased levels of susceptibility of cultivars to several disease(s). The genetic potential of the cultivars to tolerate/resist infection by microbial pathogens could be enhanced by adopting traditional breeding procedures. However, in the absence of dependable sources of resistance among wild relatives of the crop species, biotechnological methods have extended a helping hand to enhance the levels of resistance of cultivars by introgressing gene(s) from diverse sources of plants and animals; such methods are not possible through conventional breeding procedures (Chapter 12). Crop disease management strategies aimed to adversely affect the pathogen development directly involve the use of biotic and abiotic biological control agents and/or chemicals. Fungi, bacteria, and viruses have been demonstrated to inhibit the pathogen development to varying degrees. The biocontrol potential of the biotic agents differed widely with species/strains which suppress the pathogen development through different mechanisms of action. As can be expected of biological materials, the effectiveness of most biological control agents varies widely because of the competition with other microorganisms as well as the pathogens for nutrients and space. In general, mixtures of strains/species of biological control agents have been found to be more effective than an individual species/strain. Abiotic agents such as manures, organic compounds of plant and animal origin, and inducers of resistance to crop diseases have been shown to have potential for reducing the incidence and spread of crop diseases. Use of biotic and abiotic biological control agents for crop disease management is an ecofriendly strategy

Introduction

with no adverse effect on nontarget organisms, and is also compatible with existing cultural practices applied in different geographic locations. However, commercially available formulations are very few compared with the number of chemicals available in the market. The enhancement of resistance of crop cultivars using inducers/activators of resistance of crop cultivars against microbial pathogens appears to be a productive approach for containing crop diseases (Chapter 13). Disease management using fungicides, bactericides, antibiotics, and antiviral chemicals is reported to be more effective than other disease management tactics which may be difficult to apply because of crop‐imposed limitations or a lack of cost‐effectiveness or impractical nature. Several kinds of chemicals have been used for seed/propagule treatment, soil application, and foliar spray, and different levels of effectiveness of chemical application have been demonstrated. The development of resistance to c­ hemicals in fungal and bacterial pathogens has been observed as these pathogens are able to produce new strains that can tolerate the dosage of chemicals recommended for application, necessitating the replacement or withdrawal of such chemicals to which the pathogens have become less sensitive/resistant. In addition, the presence of residues of chemicals in treated grains, vegetables, and fruits and the environmental pollution remain strong impediments for the continued use of chemicals against plant pathogens. A constant search is underway for alternative strategies leading to a reduction in the quantum of chemicals applied or to replacement with other biological materials (Chapter 14). Various strategies of crop disease management may be able to individually offer ­protection to plants, but often below the desired levels. Some efforts have been made to integrate strategies that are compatible with each other (Chapter 15), so that the combined application of two or more tactics may be more effective than when applied individually. It will be worthwhile intensifying such investigations in order to assess the advantages of integrating as many strategies as possible to derive more benefits to the growers and to provide the consumers with healthy food materials in future. No single management tactic is known to be effective under the varied ecological conditions in which various crops are cultivated. Choosing suitable strategies and integrating them into crop production systems is therefore essential. As management decisions are ­primarily based on economic consideration (benefit: cost ratio), some technologies have not been implemented due to a lack of cost‐effectiveness, despite having been shown to be effective in suppressing disease development. The impact of adopting effective management systems on social systems and human welfare is discussed to indicate their sustainability over several years. This book contains the latest information gathered from an extensive literature search that will assist researchers, teachers in departments of Plant Pathology, Microbiology, Molecular Biology and Biotechnology, Plant Physiology, and Plant Breeding and Genetics, as well as the personnel of Certification and Plant Quarantine programs. The protocols presented at the end of certain chapters as appendices will provide a useful start to investigations.

­References Bennett CW (1969) Seed transmission of plant viruses. Advances in Virus Research 14: 221. Bové JM (2006) Huanglongbing: A destructive newly emerging, century old disease of citrus. Journal of Plant Pathology 88: 7–37.

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Cambra M, Capote N, Myrta A, Llacer G (2006) Plum pox virus and estimated costs associated with sharka disease. OEPP/EPPO Bulletin 36: 202–204. de León L, Siverio F, Lopez MM, Rodriguez A (2011) Clavibacter michiganensis subsp. michiganensis, a seedborne tomato pathogen: Healthy seeds are still the goal. Plant Disease 95: 1328–1339. Gergerich RC, Kamenidou S, Fuchs M, Welliver RA, Gettys S, Martin RR, Vidalakis G, Osterbauer NK, Golino DA, Tzanetakis IE, Eastwell K (2015) Safeguarding fruit crops in the age of agricultural globalization. Plant Disease 99: 176–187. Grogan RG (1983) Lettuce mosaic virus control by use of virus indexed seed. Seed Science and Technology 11: 1043–1049. Hausbeck MK, Bell J, Medina‐Mora C, Podolsky R, Fulbright DW (2000) Effect of bactericides on population sizes and spread of C. michiganensis subsp. michiganensis on tomatoes in the greenhouse and on disease development and crop yield in the field. Phytopathology 90: 38–44. Hodges A, Spreen T (2013) Economic impacts of citrus greening (HLB) in Florida, 2006/7‐2010/11. University of Florida, IFAS Ext Bull #FE903. Inch SA, Gilbert J (2003) Survival of Gibberella zeae in Fusarium‐damaged wheat kernels. Plant Disease 87: 282–287. Isac M, Preda S, Marcu M (1998) Aphid species–vectors of Plum pox virus. Acta Virologica 42: 233–234. Large EC (1940) Advance of the Fungi. Dover Publications, New York. Mandahar CL (1981) Virus transmission through seed and pollen. In: Maramorosch K, Harris KF (eds) Plant Diseases and Vectors. Academic Press, New York, pp. 241. McMullen M, Bergstrom G, De Wolf E, Dill‐Macky R, Hershman D, Shaner G, van Sanford D (2012) A unified effort to fight an enemy of wheat and barley: Fusarium head blight. Plant Disease 96: 1712–1728. Michaud J (1998) A review of the literature in Toxoptera citricida (Kirkaldy) (Homoptera: Aphididae). Florida Entomologist 81: 37–61. Moreno P, Ambrós S, Albiach‐Marti M, Guerri J, Pena L (2008) Citrus tristeza virus: A pathogen that changed the course of the citrus industry. Molecular Plant Pathology 9: 251–268. Mukhopadhyay S (2011) Plant Virus, Vector, Epidemiology and Management. CRC Press, Taylor & Francis Group, Boca Raton, New York. Narayanasamy P (2002) Microbial Plant Pathogens and Crop Disease Management. Science Publishers, Enfield, USA. Narayanasamy P (2011) Microbial Plant Pathogens–Detection and Disease Diagnosis, Vol 1–3. Springer Science + Business Media BV, Heidelberg, Germany. Nganje WE, Kaitibe S, Wilson WW, Leistritz FL, Bangsund DA (2004) Economic impacts of Fusarium head blight in wheat and barley: 1993–2001. AgriBusiness Applied Economic Report No. 464. Available at: http://ageconsearch.umn.edu/handle/23515 (accessed July 2016). OEPP/EPPO (2004) EPPO Standards: Diagnostic protocols for regulated pests. Plum pox virus. OEPP/EPPO Bulletin 34: 247–256. Ou SH (1972) Rice Diseases. Commonwealth Mycological Institute, Kew, England. Padmanabhan SY (1973) The Great Bengal Famine. Annual Review of Phytopathology 11: 11–26. Phatak HC (1974) Seed‐borne plant viruses–identification and diagnosis in seed health testing. Seed Science and Technology 2: 3.

Introduction

Poysha V (1993) Evaluation of tomato breeding lines resistant to bacterial canker. Canadian Journal of Plant Pathology 15: 301–304. Reddy APK (1989) Bacterial blight: Crop loss assessment and disease management. In: Proceedings of International Workshop on Bacterial Blight of Rice, International Rice Research Institute, Philippines, pp. 79–88. Schumann GL (1991) Plant Diseases–Their Biology and Social Impact. The American Phytopathological Society, St Paul, MN, USA. Sen Y, van der Wolf J, Visser RGF, van Heusden S (2015) Bacterial canker of tomato: current knowledge of detection, management, resistance and interactions. Plant Disease 99: 4–13. Short DPG, Gurung S, Koike ST, Klosterman SJ, Subbarao KV (2015) Frequency of Verticillium species in commercial spinach fields and transmission of V. dahliae from spinach to subsequent lettuce crops. Phytopathology 105: 80–90. Srivastava MP, Kapoor RT (1982) Yield loss due to BLB. International Rice Research Newsletter 7(3): 7. Stewart VB, Reddick D (1917) Bean mosaic. Phytopathology 7: 61. Wang N, Trivedi P (2013) Citrus huanglongbing: A newly relevant disease presents unprecedented challenges. Phytopathology 103: 652–665. Welliver R (2012) Plum pox virus case study: The eradication road is paved in gold. Phytopathology 102: S4.154. Zink FW, Grogan RG, Welch JE (1956) The effect of the percentage of seed transmission upon subsequent spread of Lettuce mosaic virus. Phytopathology 46: 662.

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2 Detection and Identification of Fungal Pathogens Microbial plant pathogens  –  oomycetes, fungi, bacteria, viruses, and viroids  –  infect true seeds and propagules such as tubers, corms, setts, and cuttings. These become the primary sources of inoculum that may introduce the disease(s) into a new location or field where the incidence of the disease(s) may be limited or absent, and also carry the pathogens to the subsequent generations of crop plants. In order to prevent the establishment of new pathogens, early detection and precise identification of pathogens in seeds and propagules is an important component of integrated disease management systems. As specific restrictions are imposed by different governments to protect domestic crop production from pathogens likely to be introduced through imported seeds and ­propagules, sensitive detection and precise identification of the fungal pathogens is essential. Both conventional isolation‐based methods and modern isolation‐­independent techniques have been applied with varying degrees of effectiveness and reliability. Quarantine and certification programs have been established in most countries around the world to examine different kinds of plants and plant materials for freedom from ­diseases and pests affecting the crop(s) concerned. Conventional methods employed for examining seeds and propagules include visual inspection for disease symptoms, microscopic examination, and culturing the fungal pathogens in appropriate culture media. Blotter test, grow‐ out test, and direct plating on agar media are applied to assess the extent of seed health. Bait tests have been found to be useful for the detection of fungal pathogens in the roots of susceptible crop plants. Isolation‐­independent immunoassays and nucleic acid (NA‐) based procedures provide more precise, sensitive, reliable, and rapid results. Further, the results of these techniques are not influenced by the environmental conditions to which the test materials are exposed prior to testing (Narayanasamy 2001, 2011).

2.1 ­Detection and Differentiation of Fungal Pathogens in Seeds Fungi associated with seeds may be pathogenic or they may be saprophytes developing on seed surface competing with the pathogens for space and nutrients. The saprophytic fungi may produce toxic compounds that may cause seed rots, resulting in reduction in germination and gappiness in the field. Fungi that infect seeds also reduce the seed ­ germination and induce disease symptoms in roots and also in aerial plant organs. Various methods have been followed to detect and identify the fungal ­pathogens in seeds of different crops. Microbial Plant Pathogens: Detection and Management in Seeds and Propagules, First Edition. P. Narayanasamy. © 2017 John Wiley & Sons, Ltd. Published 2017 by John Wiley & Sons, Ltd.

Detection and Identification of Fungal Pathogens

2.1.1  Conventional/Isolation‐Dependent Methods

The Handbook on Seed Health Testing containing Worksheets has been published by the International Seed Testing Association (ISTA). The Worksheets have specific ISTA number with description of methods for detecting seedborne fungi, bacteria and viruses (ISTA 1994). In addition, the International Seed Health Initiative (ISHI) coordinates the production and supply of healthy seeds of vegetable crops. ISHI provides status reports which focus the efforts on the importance of different pathogens transmitted through seeds to the cooperating agencies (Meijerink 1997). The morphological characteristics are basically considered for the identification of the seedborne fungal pathogens which are classified into different phyla, class, families, genera, and species. The morphologic (phenotypic) characteristics of the asexual and sexual spores and spore‐bearing structures form the basis for classical taxonomy of oomycetes (fungus‐ like) and fungal pathogens for assigning them into various groups from kingdom down to species level (Agrios 2005) as detailed below. Fungus‐like microorganisms (oomycetes) Kingdom: Protozoa Phylum: Myxomycota: producing a plasmodium or plasmodium‐like structure Phylum: Plasmodiophoromycota (Plasmodiophoromycetes): endoparasitic slime molds Order: Plasmodiophorales: plasmodia produced within cells of roots and stems of infected plants; obligately parasitic in nature Genus: Plasmodiophora  –  P. brassicae, causing clubroot of cruciferous plants; Spongospora – S. subterranea, causing potato powdery scab disease Kingdom: Chromista (Stramenopiles) Phylum: Oomycota with biflagellate zoospores Class: Oomycetes: zoospores produced in sporangia Order: Saprolegniales Genus: Aphanomyces – A. euteiches, causing pea root rot disease Order: Peronosporales Family: Pythiaceae Genus: Phytophthora – P. infestans, causing potato late blight disease Family: Peronosporaceae Genus: Plasmopara  –  P. halstedii, causing sunflower downy mildew disease; Sclerospora  –  S. graminicola, causing pearl millet downy mildew disease; Peronosclerospora – S. sorghi, causing sorghum downy mildew disease True fungi Kingdom: Fungi, producing achlorophyllous mycelium with cell walls containing glucans and chitin Phylum: Chytridiomycota, producing zoospores with single posterior flagellum Class: Chytridiomycetes, producing aseptate mycelium Genus: Synchytrium – S. endobioticum, causing potato wart disease Phylum: Ascomycota, producing sexual spores (ascospores) generally in groups of eight within an ascus enclosed in specialized structures known as ascocarps and asexual spores in pycnidia or acervuli Class: Pyrenomycetes: filamentous ascomycetes Order: Hypocreales

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Genus: Gibberella – G. fujikuroi, causing rice bakanae disease; Claviceps – C. purpurea, causing ergot diseases of grain crops; C. sorghi, causing sorghum ergot disease Class: Loculoascomycetes Order: Diaporthales Genus: Diaporthe – D. phaseolorum, causing soybean pod and stem rot disease; Magnaporthe – M. grisea, causing rice blast disease Order: Xylariales Genus: Eutypa – E. lata, causing grapevine canker disease Order: Dothidiales Genus: Mycosphaerella  –  M. graminicola, causing cereal leaf spot diseases; Pyrenophora  –  P. teres, causing barley net blotch disease; Leptosphaeria  – L. maculans, causing cabbage black leg disease Class: Discomycetes Order: Helotiales Genus: Sclerotinia – S. sclerotiorum, causing canola stem rot disease Phylum: Basidimycota, producing sexual spores known as basidiospores externally on a club‐like one‐ or four‐celled spore‐bearing structure called basidium Order: Ustilaginales Genus: Ustilago – U. maydis, causing corn smut disease; U. tritici, causing wheat loose smut disease; Sporisorium  –  S. sorghi, causing sorghum kernel smut ­disease; Tilletia  –  T. caries, causing wheat bunt disease; T. indica, causing wheat Karnal bunt disease Order: Ceratobasidiales Genus: Thanatephorus  –  T. cucumeris (anamorph‐ Rhizoctonia solani), causing rice sheath blight disease Class: Deuteromycetes or Mitosporic fungi existing only in the asexual stage in their life cycle; conidia are formed on conidiophores separately or in specialized structures such as sporidia, synnemata, pycnidia, or acervuli Genus: Alternaria spp., Bipolaris spp. Drechslera spp., Fusarium spp., Septoria spp., and Verticillium spp. infecting seeds of several crops resulting in seed rot, seedling blight, anthracnose, head blight and scab diseases 2.1.1.1 Dry seed examination

During harvest, seeds may get admixtures of fungal structures such as ergot sclerotia, smutted kernels, discolored and shriveled seeds, and free insects. A dry examination is carried out using a stereoscope or an illuminated swinging‐arm desk magnifier of × 2 magnification. Usually samples of 400 or more seeds are subjected to examination under stereoscopic microscope for detection of fungal fructification such as pycnidia, acervuli, and smut sori. Seeds have to be incubated under high‐humidity conditions to induce the fungal pathogens to sporulate, making it easier to detect their presence in the seeds (Narayanasamy 2002). Incidence and contamination of Tilletia species in conventionally produced, nonprocessed wheat seeds were assessed. Analysis of 151 samples of basic, certified, and commercial seed lots showed that 129 samples were contaminated with Tilletia spp. The teliospores had prominent gelatinous sheath together with conspicuous deep reticulation. The contamination level of basic and certified seed was about 1 teliospore per 10 seeds, whereas 4 of 16 commercial seed samples were contaminated with more than 900 teliospores per seed, higher than the permissible contamination level (Župunski et al. 2012). Infestation level of Alternaria brassicola in commercial batches of

Detection and Identification of Fungal Pathogens

cabbage seeds produced between 1984 and 2001 in Japan was investigated. A total of 100 seeds from each lot were incubated separately on agar at 25°C. A. brassicola was most frequently isolated (up to 94%) depending on seed lot. The pathogen was isolated even from seeds refrigerated for 17 years. A. alternata was also isolated, although less frequently (