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This page intentionally left blank S.E. Albrechtsen Danish Seed Health Centre for Developing Countries Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark formerly Danish Government Institute of Seed Pathology for Developing Countries CABI Publishing CABI Publishing is a division of CAB International CABI Publishing CABI Publishing CAB International 875 Massachusetts Avenue Wallingford 7th Floor Oxfordshire OX10 8DE Cambridge, MA 02139 UK USA Tel: +44 (0)1491 832111 Tel: +1 617 395 4056 Fax: +44 (0)1491 833508 Fax: +1 617 354 6875 E-mail: [email protected] E-mail: [email protected] Website: www.cabi-publishing.org S.E. Albrechtsen 2006. All rights reserved. No part of this publication may be reproduced in any form or by any means, electronically, mechanically, by photocopying, recording or otherwise, without the prior permission of the copyright owners. A catalogue record for this book is available from the British Library, London, UK. ISBN 0 85199 016 9 Library of Congress Cataloging-in-Publication Data Albrechtsen, S.E. (Sven Erik) Testing methods for seed-transmitted viruses: principles and protocols / S.E. Albrechtsen. p. cm. Includes bibliographical references and index. ISBN 0-85199-016-9 (alk. paper) 1. Seeds--Testing. 2. Seed pathology. 3. Virus diseases of plants. I. Title. SB117.A35 2005 632′.8--dc22 2005003343 Typeset by AMA DataSet Ltd, UK. Printed and bound in the UK by Biddles Ltd, King’s Lynn. Contents Preface ix Acknowledgements xi PART I 1 Introduction 1 1.1. Seed-transmitted Viruses and Viroids 1 1.2. The Development of the Science 2 1.2.1. Seed-health testing for viruses 3 1.3. Economic Importance of Seed-transmitted Viruses 4 1.4. Testing of Seeds for Viruses: Why? 6 1.4.1. Seed certification 7 1.4.2. Quarantine 8 References 9 2 Seed Transmission of Viruses 13 2.1. Location of Inoculum in the Seed 13 2.2. Plant-to-seed and Seed-to-plant Transmission 14 2.2.1. Plant-to-seed transmission 14 2.2.2. Seed-to-plant transmission 20 2.2.3. Factors affecting seed transmission 21 References 23 3 Ecology, Epidemiology and Control 27 3.1. Ecology and Epidemiology 27 3.1.1. Viruses and virus–host interaction 27 3.1.2. Vector transmission 29 3.1.3. Environment and cultural practices 32 3.2. Control Strategies 34 3.2.1. Control measures at farm level 34 v vi Contents 3.2.2. Control measures at research and technology level 38 References 43 PART II 4 Biological Assays 47 4.1. Symptomatology 48 4.1.1. Indicator plant symptoms 50 4.2. Facilities and Equipment for Biological Assays 51 4.2.1. Growth facilities 51 4.2.2. Plant culture materials and equipment 56 4.3. Growing-on Tests 57 4.3.1. Growing-on test, standard protocol 59 4.4. Infectivity Assays 60 4.4.1. Production of indicator plants 60 4.4.2. Inoculation techniques and standard protocol 62 4.4.3. Seed as inoculum, protocols 67 4.4.4. Plants and seedlings as inoculum, protocol 75 4.4.5. Differential hosts 78 4.5. Maintenance of Isolates 78 4.5.1. Long-term storage of dehydrated samples, protocol 79 References 81 5 Serological Testing Methods 84 5.1. Antigens and Antibodies 84 5.1.1. Polyclonal antibodies 87 5.1.2. Monoclonal antibodies 88 5.2. Enzyme-linked Immunosorbent Assay (ELISA) 90 5.2.1. General principles of ELISA 91 5.2.2. General components and steps of ELISA 93 5.2.3. Double-antibody sandwich ELISA (DAS-ELISA), protocols 103 5.2.4. Indirect antigen-first (AgF) ELISA, protocol 111 5.2.5. Triple-antibody sandwich ELISA (TAS-ELISA), protocol 115 5.2.6. Penicillinase-ELISA, protocol 118 5.2.7. Other variants of ELISA 120 5.2.8. Remarks on ELISA systems, including protocol for plate reuse 122 5.2.9. Recording and interpretation 128 5.3. Dot Immunobinding Assay (DIBA) 129 5.3.1. Principles of DIBA 129 5.3.2. General components and steps of DIBA 130 5.3.3. Indirect AgF-DIBA, protocol 135 5.4. Tissue Blotting Immunoassay (TBIA) 140 5.4.1. Tissue blotting immunoassay (TBIA), protocol 142 5.5. Other Serological Test Methods 145 References 147 Contents vii 6 Nucleic Acid-based Testing Methods 153 6.1. Nucleic Acid Hybridization 155 6.1.1. General principles and components of nucleic acid spot hybridization (NASH) assays 156 6.1.2. Non-isotopic nucleic acid spot hybridization, protocol 163 6.2. Enzymatic Nucleic Acid Amplification 171 6.2.1. Principle of the polymerase chain reaction (PCR) 173 6.2.2. General components of the reverse-transcription polymerase chain reaction (RT-PCR) assay 174 6.2.3. Protocols for RT-PCR assays 183 6.2.4. Analysis of PCR products and protocol 203 6.2.5. Detection of virus species and strains 208 6.2.6. Variants of molecular amplification assays 211 6.2.7. Remarks on molecular amplification 216 References 219 7 Epilogue 226 7.1. Other Detection Techniques 226 7.1.1. Electron microscopy 226 7.1.2. Return electrophoresis for viroid detection 227 7.2. Organization and Interpretation of Seed-health Assays 228 7.2.1. Sampling and test methodology 229 7.2.2. Group testing of seeds 231 7.3. Tolerance Levels of Infection, and Pathogens in Germplasm 234 7.4. Standardization and Cost of Tests 235 7.4.1. Comparative testing 236 7.4.2. Cost of tests 238 References 238 Appendix 1. List of Seed-transmitted Viruses and Viroids 241 Appendix 2. Reagents, Solutions and Buffers 247 Appendix 3. Suppliers of Laboratory Equipment and Materials 257 Index 259 This page intentionally left blank Preface The yield losses and reduced crop quality caused by plant virus diseases have gained increasing recognition in recent years. The use of virus-resistant cultivars appears to be one of the best control strategies, but is not always possible or effective. Outbreaks of new diseases due to new variants of viruses and viroids continue to occur. Several important plant viruses are, in addition to their sap and vector transmission, also transmitted through seed. Seed transmission of plant pathogens plays an important role for the early outbreak of crop diseases and for the survival of inoculum from one crop season to the next. And seeds are instrumental in an effective worldwide spread of a range of diseases through international exchange of seed. One of the key principles of seed-transmissible viruses is that their elimination by seed treatment is virtually impossible. In the tropical and subtropical zones, where a great variety of virus-susceptible crop species are grown and virus vector pressure continues year-round, effective management of viral diseases is particularly important. And effective management of seed-transmitted viruses depends upon the use of healthy planting material, through seed quality control and by plant quarantine monitoring and testing. Tests of seed for viral infection are crucial for providing adequate supplies of virus-free seeds or seed with very low infection rates to avoid intra-national and international dispersal, leading to potentially high losses. Until 1970–1980, large-scale testing of sowing material for viruses was impracticable, but a number of suitable techniques have since been (and are being) developed. The primary aim of this book is to provide principles and protocols for a number of well-established routine detection methods for viruses and viroids in seeds of, primarily, field and vegetable crops, methods that should also be appli- cable for the detection of many other viruses and viroids. In the introductory Part I, an attempt is made to provide a ‘mini-review’ of important aspects of seed-transmitted viruses, especially their economic importance, location in seeds, seed transmission and strategies for their control. ix x Preface Chapters 4, 5 and 6 in Part II describe principles and stepwise protocols for the detection methods, and also contain information on method optimization and variations, and discuss plusses and drawbacks of different methods. Further included are practical notes, interpretation of data, notes on pitfalls and trouble shooting. The chapters contain both techniques manageable in modestly equipped laboratories and those requiring more sophisticated facilities. The last chapter provides philosophies and outlines for setting up seed-health testing for viruses, including seed sampling, group testing, statistical analyses of test results, tolerance levels and standardization of seed-health assays. A list of seed-transmitted viruses and viroids can be found in Appendix 1. A few recipes for reagents and buffers appear in the protocols, but most of them are placed in Appendix 2, and suppliers of equipment and materials are indi- cated in Appendix 3. Some manufacturers and sources are mentioned in the text but this does not infer that other comparable makes and brands may not be equally satisfactory for use. The author’s experience of virus seed pathology was obtained during 30 years of appointment, the final 12 years as Associate Professor, at the Danish Government Institute of Seed Pathology for Developing Countries (DGISP), an institute funded by the Danish Development Assistance (DANIDA). In 2004 the institute was changed to a centre, the Danish Seed Health Centre for Developing Countries (DSHC) under the Royal Veterinary and Agricultural University, Copenhagen, Denmark, and DANIDA. The book is based on methods and materials used in teaching and training students in seed virology courses held regularly in Denmark as well as in the developing countries, for which the author was responsible during the last 16 years of his appointment. The materials used are updated on the basis of consultations of the latest and most relevant litera- ture. The book is primarily technical, and thus such topics as, for example, sys- tematic descriptions of individual viruses are not included; for details on individual viruses or for further information on plant virology, the reader is referred to other sources (books and websites) specified in the text.

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