BIOASSAYS OF ENTOMOPATHOGENIC MICROBES AND NEMATODES Bioassays of Entomopathogenic Microbes and Nematodes Edited by A. Navon and K.R.S. Ascher Department of Entomology Agricultural Research Organization Bet Dagan Israel CABI Publishing CABI Publishing is a division of CAB International CABI Publishing CABI Publishing CAB International 10 E 40th Street Wallingford Suite 3203 Oxon OX10 8DE New York, NY 10016 UK USA Tel: +44 (0)1491 832111 Tel: +1 212 481 7018 Fax: +44 (0)1491 833508 Fax: +1 212 686 7993 Email: [email protected] Email: [email protected] Web site: http://www.cabi.org © CAB International 2000. 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. Library of Congress Cataloging-in-Publication Data Bioassays of entomopathogenic microbes and nematodes / edited by A. Navon and K.R.S. Ascher. p. ; cm. Includes bibliographical references and index. ISBN 0-85199-422-9 (alk. paper) 1. Nematoda as biological pest control agents--Research--Technique. 2. Microbial pesticides--Research--Technique. 3. Biological assay. I. Navon, Amos. II. Ascher, K. R. S. [DNLM: 1. Nematoda--parasitology. 2. Biological assay--methods. 3. Parasites--parasitology. 4. Pest control, Biological--methods. QX 203 B615 2000] SB933.334.B56 2000 632′.96--dc21 99–046507 ISBN 0 85199 422 9 Typeset by Columns Design Ltd, Reading. Printed and bound in the UK by Biddles Ltd, Guildford and King’s Lynn. Contents Contributors vii Foreword ix 1 Bioassays of Bacillus thuringiensis 1 1A. Bioassays of Bacillus thuringiensis Products Used Against Agricultural Pests 1 A. Navon 1B. Bioassays of Genetically Engineered Bacillus thuringiensis Plant Products 25 S.R. Sims 1C. Bioassays of Bacillus thuringiensis subsp. israelensis 41 O. Skovmand and N. Becker 1D. Production of Bacillus thuringiensis Insecticides for Experimental Uses 49 S. Braun 2 Bioassays of Replicating Bacteria Against Soil-dwelling Insect Pests 73 T.A. Jackson and D.J. Saville 3 Bioassays of Entomopathogenic Viruses 95 K.A. Jones 4 Bioassays of Entomogenous Fungi 141 T.M. Butt and M.S. Goettel 5 Bioassays of Microsporidia 197 J.V. Maddox, W.M. Brooks and L.F. Solter v vi Contents 6 Bioassays of Entomopathogenic Nematodes 229 I. Glazer and E.E. Lewis 7 Statistical and Computational Analysis of Bioassay Data 249 R. Marcus and D.M. Eaves 8 Legislation Affecting the Collection, Use and Safe Handling of Entomopathogenic Microbes and Nematodes 295 D. Smith Index 315 Contributors K.R.S. Ascher, Department of Entomology, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel N. Becker, German Mosquito Control Association, (KABS), Ludwigstrasse 99, D-67165, Waldsee, Germany S. Braun, The Biotechnology Laboratory, Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel W.M. Brooks, Department of Entomology, 2315 Gardner Hall, North Carolina State University, Raleigh, NC 27695, USA T.M. Butt, School of Biological Sciences, University of Wales, Singleton Park, Swansea, SA2 8PP, UK D.M. Eaves, Department of Mathematics and Statistics, Simon Fraser University, Burnaby, British Columbia, V5A 1S6, Canada I. Glazer, Department of Nematology, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel M.S. Goettel, Lethbridge Research Centre, Agriculture and Agri-Food Canada, PO Box 3000, Alberta, T1J 4B1, Canada T.A. Jackson, AgResearch, PO Box 60, Lincoln, New Zealand K.A. Jones, Natural Resources Institute, University of Greenwich, Central Avenue, Chatham Maritime, Chatham, Kent ME4 4TB, UK E.E. Lewis, Department of Entomology, Virginia Tech, Price Hall, Blacksburg, VA 24061, USA J.V. Maddox, Center for Economic Entomology, Illinois Natural History Survey and Illinois Agricultural Experiment Station, 607 Peabody Drive, Champaign, IL 61820, USA R. Marcus, Department of Statistics, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel vii viii Contributors A. Navon, Department of Entomology, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel D.J. Saville, AgResearch, PO Box 60, Lincoln, New Zealand S.R. Sims, Whitmire Micro-Gen, 3568 Tree Court Industrial Blvd, St Louis, MO 63122, USA O. Skovmand, Intelligent Insect Control, 80 Rue Pauline Ramart, F-34070, Montpellier, France D. Smith, CABI Bioscience UK Centre (Egham), Bakeham Lane, Egham, Surrey TW20 9TY, UK L.F. Solter, Center for Economic Entomology, Illinois Natural History Survey and Illinois Agricultural Experiment Station, 607 Peabody Drive, Champaign, IL 61820, USA Foreword Since World War II, pest control methods have been dominated by the use of synthetic chemical insecticides. These insecticides have been very suc- cessful in the marketplace and have contributed greatly to the production of food and fibre, as well as to the control of many diseases of medical and veterinary importance transmitted by insects, especially mosquitoes and blackflies. Despite these benefits, the use of chemical insecticides has not been without problems. Among the most important of these are the devel- opment of high levels of resistance in target pest and vector populations, high mortality rates in non-target beneficial insect, mite and spider popula- tions, and contamination of the environment, particularly water supplies, with chemicals that the public perceives as being harmful. Due to these problems, scientists in academia, government and industry have placed a great deal of effort over the past few decades in developing more environ- mentally compatible pest control products and methods. The best of these include safer chemical insecticides, microbial insecticides based on pathogens that cause disease in insects, parasitic nematodes, and during the last decade, insect-resistant transgenic plants based primarily on insecticidal proteins produced by Bacillus thuringiensis. Products based on these new technologies are increasingly being accepted in the marketplace, and legis- lated into use, either by the banning of certain chemical insecticides or by legal restrictions placed on their use by environmental protection agencies around the world. The need for new products and pest control technologies will continue to accelerate due not only to demands for safer pest control technologies, but to the continuing increase in the world’s population. Many of the new technologies noted above offer excellent promise for the development of safe and effective pest control methods. However, each ix x Foreword of the various new types of agents being developed requires special exper- tise and methods for accurate determination of efficacy through bioassays. Though many methods have been published in the scientific literature, these are typically found in a variety of disparate journals and technical manuals. In the current text, Dr A. Navon and Dr K.R.S. Ascher have enlisted the assistance of experts on all of the new and developing pest control tech- nologies based on viruses, microorganisms and nematodes, to bring together the most pertinent methods in a single volume. The volume begins with a series of subchapters on B. thuringiensis, the most successful of the microbial insecticides. These subchapters cover a wide range of topics ranging from how to isolate and grow new bacterial strains, assess their efficacy and evaluate the potential of wild-type and recombinant bacterial strains and proteins for commercial development. Methods are provided for assessing the activity of strains, proteins and for- mulations against numerous insect pests of crops, forests, and the larvae of pest and vector mosquitoes and blackflies. Perhaps the most important advance of the last decade emerging from the field of microbial control is the development of insect-resistant transgenic crops based on the insectici- dal crystal proteins of B. thuringiensis (Bt). Millions of acres of Bt-transgenic cotton and maize are already being planted in the USA. Yet methods for assessing the efficacy of these are not widely known or available. The cur- rent text fulfils this need by providing bioassay methods for different plant tissues including leaves, roots and pollen. Whereas most of the focus on bacterial insecticides has been on B. thuringiensis, other bacteria continue to show potential for control of soil-dwelling pests. To treat this area, a chapter has been included on the recent development of a species of Serratia to control soil-dwelling beetle grubs. Though Bt has been the most successful commercial microbial insecti- cide, insect-pathogenic viruses, fungi, microsporidia and nematodes have been used or are being considered for use against many crop pests, espe- cially against pests not controlled easily by bacteria. These insects include locusts, sucking insects such as whiteflies and aphids, termites, ants, and even lepidopterous pests insensitive to Bt. Viruses must be eaten to be effective, whereas the fungi typically invade the insect target through the cuticle. Both of these pathogen types can cause acute diseases. Microsporidia, which are commonly transmitted horizontally by feeding or transovarially on or within eggs, cause chronic diseases. The nematodes are minute parasitic worms which, unlike most pathogens, can actually seek out their host, invade the body through various orifices (the mouth, anus, or spiracles), and cause death within