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ENTOMOPATHOGENIC BACTERIA: FROM LABORATORY TO FIELD APPLICATION

Entomopathogenic Bacteria: from Laboratory to Field

Application

Edited by

Jean-Fran<;ois Charles Annelle Del6cluse

and

Christina Nielsen-Le Roux Institut Pasteur, Paris, France

SPRINGER-SCIENCE+BUSINESS MEDIA, B.Y.

A C.I.P. Catalogue record for this book is available from the Library of Congress.

ISBN 978-90-481-5542-2 ISBN 978-94-017-1429-7 (eBook) DOI 10.1007/978-94-017-1429-7

Printed an acid-free pa per

AII Rights Reserved © 2000 Springer Science+Business Media Dordrecht

Originally published by Kluwer Academic Publishers in 2000 Softcover reprint of the hardcover 1 st edition 2000

N o part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical,

including photocopying, recording or by any information storage and retrieval system, without written permission from the copyright owner.

Contents

Contributors Xlll

Preface XIX

SECTION 1: THE ENTOMOPATHOGENIC BACTERIA 1

1.1 Biodiversity of the entomopathogenic, endospore-forming bacteria (FG Priest) 1

1. Introduction 2 2. The genus Bacillus - a brief history 3 3. The genus Bacillus becomes several genera 4 4. Bacillus sensu stricto (rRNA group 1 sensu Ash et al., 1991) 7 5. Round-spore-forming bacilli (rRNA group 2 sensu Ash et ai.,

1991) 10 6. Paenibacillus (rRNA group 3 sensu Ash et al., 1991) 12 7. Brevibacillus (rRNA group 4 sensu Ash et ai., 1991) 16 8. Concluding remarks 16

1.2 Natural occurrence and dispersal of Bacillus thuringiensis in the environment (PH Damgaard) 23

l. Occurrence in soil 23 2. Occurrence on foliage 24 3. Occurrence in specific insects habitats 26 4. Occurrence in foods 28 S. Clinical infections 29 6. Epizootiology of B. thuringiensis 30 7. Factors governing B. thuringiensis-caused epizootics 32 8. Conclusions 34

v

VI Contents

1.3 Virulence of Bacillus thuringiensis (BM Hansen & S Salamitou) 41

1. Introduction 42 2. Taxonomy and relations to B. cereus 42 3. Non-insect pathogenesis of B. thuringiensis and B. cereus 43 4. Virulence of B. thuringiensis and B. cereus spores 44 5. Virulence factors of B. thuringiensis and B. cereus 46 6. Expression of virulence factors 52 7. B. thuringiensis in the environment 53 8. Consequences for application of B. thuringiensis 54

SECTION 2: TOXINS AND GENES 65

2.1 The diversity of Bacillus thuringiensis B-endotoxins (N Crickmore) 65

1. Toxin nomenclature 65 2. Sequence comparisons 70 3. Related sequences 77 4. Summary 78

2.2 Insecticidal proteins produced by bacteria pathogenic to agricultural pests (T Yamamoto & DH Dean) 81

1. Description of B. thuringiensis 81 2. Extra-cellular insecticidal proteins 83 3. Characterisation of the crystal protein 83 4. Three domain structure of the crystal protein 85 5. Domain I function 86 6. Receptor binding sites in domain II 90 7. Domain III function 93 8. Cry2a family proteins 94 9. Future for the bacterial insecticidal proteins 96

2.3 Vector-active toxins: structure and diversity (A Delecluse, V Juarez-Perez & C Berry) 101

1. Introduction 1 0 1 2. Dipteran active bacteria 102 3. Mosquitocidal toxins 105 4. Summary and conclusions 114

2.4 Toxin and virulence gene expression in Bacillus thuringiensis (D Lereclus & H Agaisse) 127

1. Expression of the insecticidal toxin genes 128 2. Virulence gene expression 137 3. Conclusion 139

Contents

2.5 Genetic and genomic contexts of toxin genes (M-L Rosso, J Mahillon & A Delecluse)

1. Introduction 2. The entomopathogenic bacteria genome 3. Genomic location of toxins 4. Virulence gene mobility and transfer 5. Concluding remarks

SECTION 3: MODE OF ACTION AND RESISTANCE

3.1 Pathogenesis of Bacillus thuringiensis toxins

Vll

143

143 144 149 151 156

167

(P LUthy & MG Wolfersberger) 167

1. Introduction 167 2. From the bacterial inclusion to the active polypeptide 169 3. Reaction of insect larvae to the 8-endotoxin 171 4. The gut epithelium as the target tissue 172 5. In vitro studies 175 6. On the origin of the 8-endotoxin: a hypothesis 177

3.2 Investigations of Bacillus thuringiensis Cryl toxin receptor structure and function (SF Garczynski & MJ Adang) 181

1. Cryl toxin binding to brush border membrane vesicles 181 2. Cryl receptor detection using toxin overlays 184 3. Identification of Cryl receptor proteins 185 4. Characteristics of Cryla binding molecules in M sexta BBMV 186 5. Cryla toxin-induced pores 189 6. Functional Cryl receptors 190 7. Concluding remarks 191

3.3 Membrane permeabilisation by Bacillus thuringiensis toxins: protein insertion and pore formation (J-L Schwartz & R Laprade) 199

1. Introduction 199 2. Toxicity at the molecular level 200 3. Approaches and techniques 203 4. Microenvironment of target cells 206 5. Role of the receptor 206 6. Conformational changes and pore structure 207 7. Postbinding events, resistance 211

V111 Contents

3.4 Insect resistance to Bacillus thuringiensis insecticidal crystal proteins (J Van Rie & J Ferre) 219

1. Introduction 219 2. Biochemical basis of resistance 220 3. Genetics of resistance 227 4. Conclusions 232

3.5 Mode of action of Bacillus sphaericus on mosquito larvae: incidence on resistance (J-F Charles, MH Silva-Filha & C Nielsen-LeRoux) 237

1. Introduction 237 2. Cytological and physiological effects 238 3. Binding of the binary toxin to a specific receptor 240 4. The toxin receptor in Culex pipiens 243 5. Toxin receptor interaction in B. sphaericus-resistant colonies

of C. pipiens 246 6. Toxin structure and in vivo/in vitro activity 248 7. Conclusions/perspectives 249

SECTION 4: SAFETY AND ECOTOXICOLOGY OF

ENTOMOPATHOGENIC BACTERIA 253 (LA Lacey & JP Siegel) 253

1. Introduction 253 2. Direct effects of Bacillus entomopathogens on invertebrate

non target organisms 254 3. Indirect effects of Bt on nontarget invertebrates 257 4. Effects of Bacillus entomopathogens on vertebrates 258 5. Indirect effects of Bacillus entomopathogens on vertebrates 263 6. Long term impact of Bacillus pathogens used as microbial pest

control agents 264 7. Conclusion 266

SECTION 5: STANDARDISATION, PRODUCTION AND

REGISTRA TION

5.1 Is Bacillus thuringiensis standardisation still possible?

275

(0 Skovmand, I Thiery & G Benzon) 275

1. Introduction 275 2. History of Bt standardisation 277 3. Standard procedures 278 4. In vitro assays 285 5. Future of bioassays 286 6. Suggestion for new type of standard 287

Contents IX

5.2 Industrial fermentation and formulation of entomopathogenic bacteria (TL Couch) 297

1. Introduction 297 2. Culture selection 298 3. Laboratory techniques for culture maintenance 299 4. Fermentation inoculum preparation 299 5. Fermentation medium selection 30 1 6. Fermentation process 302 7. Recovery of entomopathogenic bacteria 303 8. Formulation 304 9. Quality control requirements 313 10. Conclusion 314

5.3 Rural production of Bacillus thuringiensis by solid state fermentation (E Aranda, A Lorence & M del Refugio Trejo) 317

1. Introduction 3 1 7 2. Strategies for insect control 3 18 3. Solid state fermentation (SSF) 320 4. Concluding remarks 329

5.4 Registration of biopesticides (GN Libman & SC MacIntosh) 333

1. Introduction 333 2. What are biopesticides? 334 3. Registration of products containing Bacillus thuringiensis

toxins as the active ingredient 336

SECTION 6: FIELD ApPLICATION AND RESISTANCE

MANAGEMENT 355

6.1 Bacillus thuringiensis application in agriculture (A Navon) 355

1. Introduction 355 2. Considerations of Bt uses in the field 3 56 3. Combinations of Bt with other means of pest management 362 4. Future prospects 365

6.2 Application of Bacillus thuringiensis in forestry (K van Frankenhuyzen) 371

1. Introduction 371 2. Field development 372 3. The biological interface: reducing the efficacy bottleneck 377

x Contents

6.3 Bacterial control of vector-mosquitoes and black flies (N Becker) 383

1. Introduction 384 2. Mosquitoes 387 3. Blackflies 394 4. Future prospects 396

6.4 Resistance management for agricultural pests (RT Roush) 399

1. Introduction 399 2. Factors that influence selection 401 3. Myths about management of resistance 405 4. Promising tactics for resistance management for bacteria and

sprays 408 5. Resistance monitoring 412 6. Implementation 413 7. Conclusions 414

6.5 Management of resistance to bacterial vector control (L Regis & C Nielsen-LeRoux) 419

1. Introduction 419 2. Case histories of B. sphaericus resistance in mosquito

populations 420 3. Mechanisms and genetics of resistance in terms of stability and

reversibility 422 4. Factors influencing the rate of development of resistance in

the field 425 5. Cross-resistance and toxin receptor interaction 427 6. Strategy for the management of resistance to B. sphaericus 429 7. Conclusions and perspectives 433

SECTION 7: BIOTECHNOLOGY AND RISK ASSESSMENT

7.1 Biotechnological improvement of Bacillus thuringiensis for agricultural control of insect pests: benefits and

441

ecological implications (V Sanchis) 441

1. Introduction 441 2. Improvement of Bt strains 443 3. Expression of cry genes in plants 449 4. Ecological risks associated with the use of transgenic Bt crops 450 5. Future challenges and prospects 452 6. Summary and conclusions 455

Contents Xl

7.2 Genetic engineering of bacterial insecticides for improved efficacy against medically important Diptera (B Federici, H-W Park, DK Bideshi & B Ge) 461

1. Introduction 461 2. Properties of mosquitocidal bacteria 463 3. Factors for enhancing endotoxin synthesis 466 4. Improvement of mosquitocidal bacteria 474 5. Summary and conclusions 479

7.3 Bacillus thuringiensis : risk assessment (A Klier) 485

1. Introduction 485 2. Taxonomy of Bacillus thuringiensis and its occurrence in the

environment 3. Risk assessment 4. Conclusions

Index

485 492 499

505

Contributors

Michael J. Adang Affiliation Departments of Entomology, and Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 3060, USA

e-mail: [email protected]

Herve Agaisse Biochimie Microbienne, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France, and Station de Lutte Biologique, INRA, La Miniere, 78285 Guyancourt Cedex, France

e-mail: agaisse@pasteurfr

Eduardo Aranda Centro de Investigaci6n en Biotecnologia (CEIB), Universidad Aut6noma del Estado de Morelos (UAEM). Av. Universidad 1001, Col. Chamilpa. c.P. 62210, Cuernavaca, Morelos, Mexico


Norbert Becker German Mosquito Control Association, Ludwigstrasse 99, 67165 Waldsee, Germany


Gary Benzon BenzonResearch, 208 Burt HouseRoad, Carlisle, PA 17013, USA


Colin Berry Cardiff School of Biosciences, cardiff University, Museum Avenue P.G. Box 911, Cardiff CFlO 3 US, Wales, UK


Xlll

XIV Contributors

Dennis K. Bideshi Department of Entomology and Interdepartemental Graduate Program in Genetics, University of California-Riverside, Riverside, California 92521, USA


Jean-Fran.;ois Charles Bacteries et Champignons Entomopathogenes, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France

e-mail: jcharles@pasteurfr

Terry L. Couch Beker Microbial Products, 9464 NW lith st., Plantation, FL 33322, USA


Neil Crickmore School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG E Sussex, UK


Per H. Damgaard The Royal Veterinary and Agricultural Thorvaldsensvej 40, 1870 Frederiksberg C,


Donald H. Dean

University, Denmark

Department of Ecology,

Department of Biochemistry, The Ohio State University, Columbus, OH 43210, USA e-mail: dean. 1 [email protected]

Armelle Delecluse Bacteries et Champignons Entomopathogenes, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France

e-mail: armdel@pasteurfr

Brian A. Federici Department of Entomology and Interdepartemental Graduate Program in Genetics, University of Cahfornia-Riverside, Riverside, California 92521, USA


Juan Ferre Department of Genetics, Faculty of Biology, Universitat de Valencia, 46100 Burjassot, Spain


Stephen F. Garczynski Affiliation Departments of Entomology, and Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 3060, USA


Baoxue Ge Department of Entomology and Interdepartemental Graduate Program in Genetics, University of California-Riverside, Riverside, California 92521, USA


Contributors xv

Bjarne M. Hansen Department of Marine Ecology and Microbiology, National Environmental Research Institute, Frederiksborgvej 399, DK-4000 Roskilde, Denmark


Victor Juarez-Perez Bacteries et Champignons Entomopathogenes, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France

e-mail: vicjua@pasteurfr

Andre Klier Biochimie Microbienne, URA 1300 CNRS, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France

e-mail: aklier@pasteurfr

Lawrence A. Lacey Fruit and Vegetable Research Unit, Yakima Agricultural Research Laboratory, USDA-ARS, 5230 Konnowac Pass Road, Wapato, WA 98951, USA

e-mail: l/[email protected]

Raynald Laprade Groupe de recherche en transport membranaire, Universite de Montreal, Montreal, Quebec, Canada


Didier Lereclus Biochimie Microbienne, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France, and Station de Lutte Biologique, INRA, La Miniere, 78285 Guyancourt Cedex, France

e-mail: lereclus@pasteurfr

Gary N. Libman Ecogen Inc. Company, 39 Sage Hill Drive, Placitas, NM 87043, USA


Argelia Lorence Centro de Investigaci6n en Biotecnologia (CEIB), Universidad Aut6noma del Estado de Morelos (UAEM). Av. Universidad 1001, Col. Chamilpa. c.P. 62210, Cuernavaca, Morelos, Mexico


Peter Liithy Institute of Microbiology, Swiss Federal Institute of Technology, 8092 Zurich, Switzerland


Susan C. Macintosh AgrEvo USA Company, 7200 Hickman Road, Suite 202, Des Moines, fA 50322, USA


Jacques Mahillon Unite de Genetique, Universite Catholique de Louvain, Place Croix du Sud, G 1348 Louvain-la-Neuve, Belgium


XVI Contributors

Amos Navon Department of Entomology, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, Israel


Christina Nielsen-LeRoux Bacteries et Champignons Entomopathogenes, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France

e-mail: cnielsen@pasteurfr

Hyun-Woo Park Department of Entomology and Interdepartemental Graduate Program in Genetics, University of California-Riverside, Riverside, California 92521, USA


Fergus G. Priest Department of Biological Sciences, Heriot-Watt University, Edinburgh EH14 4AS, Scotland, UK


Ma. del Refugio Trejo Centro de Investigaci6n en Biotecnologia (CE1B), Universidad Aut6noma del Estado de Morelos (UAEM). Av. Universidad 1001, Col. Chamilpa. c.P. 62210, Cuernavaca, Morelos, Mexico


Leda Regis Centro de Pesquisas Aggeu Magalhiies-FiOCRUZ, Av. Moraes Rego sin 50670-420 Recife PE, Brazil


Richard T. Roush Centre for Weed Management Systems, Waite Institute, University of Adelaide, South Australia, Australia


Marie-Laure Rosso Bacteries et Champignons Entomopathogenes, 1nstitut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France

Sylvie Salamitou Biochimie Microbienne, Institut Pasteur, 25 Rue du Dr Roux, 75724 Paris Cedex 15, France

e-mail: mitou@pasteurfr

Vincent Sanchis Biochimie Microbienne, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France and Station de Recherches de Lutte Biologique, INRA, La Miniere, 78285 Guyancourt Cedex, France

e-mail: vsanchis@pasteurfr

Contributors XVll

Jean-Louis Schwartz Biotechnology Research Institute, National Research Council of Canada, Montreal, and Groupe de recherche en transport membranaire, Universite de Montreal, Montreal, Quebec, Canada


Joel P. Siegel Horticultural Crops Research Laboratory, USDA-ARS, 2021 S. Peach Ave., Fresno, CA 93727, USA


Ole Skovmand Intelligent Insect Control, 80 rue Paul Ramart, Montpellier 34070,France


Maria Helena Silva-Filha Centro de Pesquisas Aggeu Magalhaes-FIOCRUZ, Av. Moraes Rego sin 50670-420 Recife PE, Brazil


Isabelle Thiery Bacteries et Champignons Entomopathogenes, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France


Kees van Frankenhuyzen Canadian Forest Service, Great Lakes Forestry Centre, P.D. Box 490, Sault Ste. Marie, Ontario P6A 5M, Canada


Jeroen Van Rie Aventis CropScience N. V, J. Plateaustraat 22, 9000 Gent, Belgium


Michael G. Wolfersberger Biology Department, Temple University, Philadelphia, PA 19122, USA


Takashi Yamamoto Maxygen, Inc., 515 Galveston Drive, Redwood City, CA 94063, USA


Preface

The discovery of a bacterium with specific insecticidal activity was made with Bacillus thuringiensis (Bt) in 1911 and the first attempts to use Bt in insect control were reported as early as the 1930's. Today Bt is the most successful commercial microbial insecticide, comprising about 90% of the biopesticide market. Its application for the protection of crops and forests and for the prevention of human diseases is occurring world-wide and is replacing chemical insecticides in some areas. This is due to both its specific activity with less damage to the environment and also to insect resistance to chemicals. The development of Bt has more recently led to the industrial development of another bacterium, Bacillus sphaericus (Bsp) as a biopesticide, with restricted activity against some Dipteran larvae. Apart from Bt and Bsp, other insecticidal bacteria have been identified, but none has yet been industrially developed.

The main interest in biopesticides as opposed to chemical pesticides is their high specificity and thus less damage to non-target fauna and flora. The insecticidal activity of Bt and Bsp is due to the presence of parasporal protein inclusion bodies, also called crystals, produced during sporulation. These inclusions are composed of one or several specific crystal protoxins (Cry, Cyt and Bin toxins) which act like a stomach poison. Upon ingestion by insect larvae, the inclusions are solubilised in the insect midgut, and protoxins proteolysed into active toxins which interact with specific midgut receptors. Pores and/or channels are formed, creating osmotic imbalance, cell lysis, and larval death.

In this book, we have attempted to bring together all recent studies regarding both fundamental and more applied research aspects related to entomopathogenic bacteria, in order to facilitate their development and

XIX

xx Preface

further success. The last book covering such various topics was published in 1993 by Entwistle et al. (Bacillus thuringiensis, An Environmental Biopesticide: Theory and Practice). Since that time, there have been substantial research advances that are included here. The 26 different chapters written by the leading researchers in the field comprehensively update the entire subject.

• Section 1: The entomopathogenic bacteria

The first three chapters provide a wide overview of the entomopathogenic bacteria, including their classification using molecular tools, their occurrence in different environments, and, for Bt strains, the nature of virulence factors they produce. This latter point has recently gained importance, as Bt seems to produce similar virulence factors as those found in B. cereus. That could have implications for human pathogenesis.

• Section 2: Toxins and genes

This section includes five chapters discussing the large diversity of cry toxins genes and the presence of important insecticidal polypeptides which do not enter the Cry nomenclature (Vip, Bin, Mtx, Cyt ... ). For some of the Cry toxins, the tridimensional structure has been determined. It seems to fit a common Cry toxin model of three domains, where domain II is variable and responsible for insect target specificity and receptor interactions. Considerable efforts have been made to elucidate the mechanisms of regulation of the cry and virulence genes. Both transcriptional and post-transcriptional controls were found to regulate their expression. Their location (plasmid versus chromosome) and association with mobile elements (insertion sequences and transposons) is also presented in relation to their dispersion among the bacteria.

• Section 3: Mode of action and resistance

The chapters of this section deal with the mode of action of the insecticidal toxins and implications for resistance development. In several insects, an aminopeptidase serves as the membrane receptor for the Bt Cry toxins, but a cadherine-like protein is also found in one insect species, and membrane lipid interactions are important. On the other hand, the receptor for B. sphaericus crystal toxin has been identified as an a-glycosidase. The resistance mechanism, in most cases, is due to the loss of a functional receptor, but proteinase modifications might also be involved.

Preface xxi

• Section 4: Safety and ecotoxicology

This unique chapter is specifically devoted to the safety and environmental impact of entomopathogenic bacteria, an emergmg concern.

• Section 5: Standardisation, production and registration

This part deals in four chapters with the production of entomopathogenic bacteria, either at large industrial sites or on a rural scale, the latter considering the use of native strains and local wastes to reduce the cost. The registration procedures for use of all organisms expressing Bt toxins (either wild-type and recombinant Bt strains or transgenic plants) are also clearly defined, and the use of standard procedures for insecticidal potency evaluation is discussed.

• Section 6: Field application and resistance management

These chapters are dedicated to the field application of entomopathogenic bacteria, to control agricultural and forestry pests (Lepidopteran, Coleopteran larvae) as well as vectors of tropical diseases (Dipteran). The impact of large and/or frequent application of Bt on the development of insecticidal resistance as well as resistance/susceptibility management are also well documented for both agriculture and health. The advantages and performances of these bacteria in IPM programs are also debated.

• Section 7: Biotechnology and risk assessment

Finally, this last part considers all the advantages and drawbacks of biopesticide development and suggests methods to increase the use of entomopathogenic bacteria through improvement of these bacteria by way of genetic engineering or construction of transgenic plants. Already much has been done and interesting recombinants have been obtained. The last chapter of this section evaluates the benefits for agriculture and the environment of both native and recombinant bacteria and addresses the question of possible adverse environmental impact to both fauna and flora.

Research in all areas of entomopathogenic bacteria has been investigated in recent years. However, lots of work still has to be done to improve the successful use of bacteria in insect control. In fact, the biopesticide market is expected to reach at least 10% of the whole pesticide market in the next few years. However, it is important to state that the appropriate measurement of this increase should be based on the amount of treated areas rather than on an economical base. There is a need for users to become aware of the benefits obtained from using biopesticides,

XXll Preface

especially in IPM programs. It is in forestry where Bt has best demonstrated its efficiency and competitivity, mainly because 1) chemicals were not allowed in these sensitive ecosystems and 2) the methods and equipments were better developed. The lessons learn in forestry should be extended to agriculture and human health and wherever biopesticides can replace chemicals.

The challenge is great and there is still a need for expanded research and cooperation among a variety of disciplines; entomologists, microbiologists, biochemists, ecologists, pest control executors and authorities need to interact. Our hope is that this book will provide the basic information to stimulate more research in the area of biopesticides and that it will be a useful tool for all, when considering bacteria for both fundamental and applied needs of insect control. We would like to take the opportunity to thank all authors, and hope that their contributions will enhance the interest of the larger public in entomopathogenic bacteria.

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