n P-1

IpRC - The Biopesticide Bacillus thuringiensis and its Applications in Developing Countries

Edited by

H. S. SALAMA, Ph. D., D. Sc. National Research Centre ( NRC), Cairo, Egypt

O. N. MORRIS, Ph. D. Agriculture Canada Research Station, Manitoba, Canada E. RACHED, Ph. D. International Development Research Centre ( IDRC), Canada

Proceedings of an International Workshop organised by NRC-Cairo, Agriculture Canada and IDRC ( 4 - 6 November,1991 )

Cairo 1993 Published by : National Research Centre, Cairo, Egypt and International Development Research Centre (IDRC), Ottawa, Canada.

The views expressed in this publication are those of the authors. Mention of a proprietary name does not constitute endorsement of the product, and is given only for information.

Layout : A. A. Abdel-Maguid, NRC, Cairo.

Printed in Egypt by : AL-AHRAM Commercial Press, Qualiub. Egypt. Legal Deposit No.: 7706/1993 - ISBN : 977-5041-31-7 This volume presents the contributions of the international workshop "Bacillus thuringiensis and its Applications in Developing Countries", that was held in Cairo during the period 4 - 6 November, 1991.

Honorary President Dr. Mahmoud Hafez

Steering Committee : - Dr. H.S. Salama NRC, Cairo. - Dr. M.S. Foda NRC, Cairo. - Dr. Oswald Morris AGRIC. Canada. - Dr. Eglal Rached IDRC, Cairo. - Dr. Ken McKay IDRC, Ottawa.

Organizing Committee - Dr. F. Zaki NRC, Cairo. - Dr. S. Salem NRC, Cairo. - Dr. Magda A. Amer NRC, Cairo. - Mr. Atef Abdel-Razek NRC, Cairo. - Mr. Atef Abdel-Rahman NRC, Cairo.

The generous contributions made by the following organisations are gratefully acknowledged : - International Development Research Centre (IDRC), Canada. - Food and Agriculture Organization of the United Nations, (FAO), Italy. - UNESCO Regional Office for Science and Technology for the Arab States, Egypt. - Canadian International Development Agency (CIDA), Canada. - Sandoz Ltd, Switzerland. - Abbott Labs. North Chicago, U.S.A. CONTENTS

PW Preface ...... I Introductory Remarks ...... 3 List of Contributors ...... 9

Part I : BaciUus thuringiensis Utilization in Developing

Countries :

- Development of isolates of Bacillus thuringiensis and similar aerobic microbes for use in developing countries.

H. T. Dulnrage ...... 15 - Simple techniques for the application of Bacillus thuringiensis in field crops suitable for developing countries. A. Jones ...... 43

- Use of Bacillus thuringiensis in Italy : Current status. E. Pasqualini ...... 53 - Potential of Bacillus thuringiensis in integrated pest management for developing countries.

S. Barbosa ...... 59 - Development of a new ELISA method to measure the concentra- tion of delta-endotoxin produced by Bacillus thuringiensis. A. Margiarkis, J. Absir and D. to DAM ...... 73 - Persistence of Bacillus thuringiensis in the tropical environment. O.- N. Morris ...... 93 - Enhancement of Bacillus thuringiensis for field application. H. S. Sakaw ...... 105 - The genetics and molecular biology of Bacillus dwringiensis. A. M. M. Ali ...... 117 Page Part II. Production and Utilization Constraints of Bacillus thuringiensis in Developing Countries - Novel simple production and formulation techniques for Bacillus thuringiensis in Thailand.

S. Pantuwatana, W. Panbangred and A. Bhumiratana ...... 123 - Development of a high production process for the production of bioinsecticides by Bacillus thuringiensis. M. Rodriguez, E. Razo, J. Villafana, E. de Urquijo, and M. de La Torre ...... 137

- Local production of Bacillus thuringiensis in Egypt : Advantages and constraints.

M.S. Foda, H.S. Salama and M. Fadel...... 149 - Applications of Bacillus thuringiensis preparations against the diamondback moth, Plutella xylostella (L.), in Taiwan. Roger F. Hou and Tao-mei Chou ...... 167 - Constraints on the use of Bacillus thuringiensis in the Philippines. L.E. Padua ...... 179 - Identification and purification of different exotoxins from nine strains of Bacillus thuringiensis. B.A. Afef, L. Ferid and B. Omrane ...... 189 - Application of biotechnology in pest control using Bacillus thuringiensis formulations. A. Merdan ...... 197 - Utilization of Bacillus thuringiensis for crop protection in Egypt. Emphasizing constraints.

F. N. Zaki ...... 205 - Farmers acceptability of the microbial control application in Egypt. S.A. Salem ...... 211 - Metabolic characteristics of Bacillus thuringiensis during submerged fermentation. Me Tianjian, Ma Tianliang, Xie Xinzhu and Yang Zhiwen ...... 213

11 Page - Study on optimization of dissolved oxygen to raise spore count of Bacillus thuringiensis. Me Tianjian, Ma Tianliang, Me Xinzhu and Ding Qiumei ...... 221 - Production and Utilization of Bacillus thuringiensis for crop protec- tion in Brazil. L O. Moraes ...... 227 - Optimization of process parameters for an economic production of biocide, Bacillus thuringiensis active against lepidopteran agri- cultural pests by the use of continuous culture studies. R. Sachidanandham, N. Rajendran, E. Sivamani, K. Jayaraman, K. Jenny, R. Laforce and A. Fiechter ...... 233 - Bioassay of Bacillus thuringiensis. A. Sharaby ...... 253 - Bacillus thuringiensis and environmental safety. M. Matter ...... 257

Part III. Commercialization of Bacillus thuringiensis : - Commercialization of Bacillus thuringiensis and other bacterial insecticides. R. A. Daoust ...... 267

Development of Bacillus thuringiensis insecticides in Ciba-Geigy as exemplified with CGA 237'218. K. Bernhard ...... 283

Canadian policy and regulations for the adoption of naturally occurring and genetically modified Bacillus thuringiensis. J. E. HoUebone ...... 303 Commercialization and utilization of Bacillus thuringiensis for crop protection in China. Xie Tianjian, Huang Bingao, Zhong Liansen and Wu Girin ...... 311

All Page - Investigations with Dipel ES and Dipel ES-Chemical insecticide mixtures for control of Lepidoptera pests in cotton in the United States and Australia. R. A. Asco ...... 317

Conclusions and Recommendations ...... 331

IV Preface

In many countries of the world, agricultural plant protection relies heavily on pesticides. While providing effective control, chemical pesticides have produced major well known problems : health hazards to humans and animals, destruction of natural biotic control agents and increased resistance of major insects species and steady increase in dosages required to control them. In spite of a more than 10 fold increase insecticide use since 1940, crop losses due to insects have nearly doubled in the same period. This situation accelerates the movement towards more sound control methods of which the microbial control proved to be the most efficient. The most promising biological agent to date to satisfy this goal is the bacterium Bacillus thuringiensis (B. t. ). This organism proved to be a highly successful weapon for fighting some agricultural pests and some vectors of diseases but its use is still limited in developing countries except China where it is widely produced and used. It is non toxic to people, most other non target insects and the environment. It can be targeted to specific pests. The safety ofB. t. is associated with a much lower development cost, in large part due to the reduced expenses of obtaining registration.

Plans have been made to organise "The International workshop on Bacillus thuringiensis (B.t.) and its application as it relates to developing countries "at the National Research Centre (NRC), Cairo, Egypt, from November 4 - 6, 1991.

This workshop was organized jointly by the National Research Centre, the International Development Research Centre, IDRC, Ottawa and Agriculture Canada, Winnipeg, Manitoba.

The objectives of the workshop were as follows :

1. Identifying the constraints facing increased utilization of B. t. in developing countries. 2. Discussing recent research on specific topics as they relate to developing countries. 3. Identifying needs and priorities for the increased utilization of B. t. in developing countries. This volume provides a comprehensive coverage of the present and potential use of Bacillus thuringiensis in developing countries. It aims to collate the existing information and research activities in B. t. research in one source to present important basic and applied advances and above all to provide a stimulating forum for the discussion of news ideas and observations on the microbial control agent Bacillus thuringiensis. The contributors are well known in their fields. Each has prepared a thorough treatment of his data together with his personal interpretations and conclusions. Although many comments were given following each presentation, they have not been included in this review. We wish to pay special tribute to all authors for their excellent contributions.

We gratefully thank Prof. Dr. M.S. Foda for his constructive contribution and the members of the organising committee for their excellent cooperation. We also thank all our sponsors who through their generous contributions made it possible to hold this workshop in such a pleasant setting. The editors thank Prof. Dr. Mahmoud Hafez, Honorary President of the Workshop ; the representative of H.E. Deputy Prime Minister and Minister for Agriculture and Land Reclamation, the representative of H.E. State Minister of Scientific Research and Dr. H. Dulmage for their welcoming remark's at the workshop. Thanks and gratitude to the colleagues who assisted in the preparation of this volume, Dr. M. Ragaei, Mr. A. Abdel-Rahman and Mr. A. A. Abdel-Maguid.

Editors

2 Introductory Remarks

Mahmoud Hafez, Honorary President it is my honour and privilege to welcome you to this workshop organised by the National Research Centre of Egypt in collaboration with Agriculture Canada and International Development Research Centre.

It is indeed a memorable scientific event to have this distinguished gathering of leading scientists from different countries who all came to participate in the various activities of this workshop and to exchange views and ideas about the biopesticide Bacillus thuringiensis and its applications in developing countries. Insect pathogens as microbial control agents are gaining increasing support at both the national and international levels against insect vectors of diseases by the WHO and against agricultural pests by FAO and the International Organization for Pest Control (IOBC). The status of insect control by microbial pathogens is encouraging and promising and a great successes against mosquitoes and other pests with Bacillus thuringiensis new strains has been attained during the last few years.

Pests of different nature are to a great extent responsible for the considerable losses of world production in agriculture. According to FAO recent reports, estimates of crop losses caused by pests and diseases amount to about 30070 preharvest and about 10% post-harvest. We can easily imagine how tremendous is this loss which takes place concurrently with an explosive increase of world population that is expected to reach 7 billions by the turn of the century. Unfortunately, this unhappy picture is most pronounced in developing countries and Egypt is no exception. This is associated with extensive use of chemical pesticides. Intensive research programmes will have to be embarked upon to explore new avenues and develop non conventional methods of pest control and to advocate and strongly adhere to a sound programme of pest management. Currently, research is being undertaken on the use of the biological control agent Bacillus thuringiensis against agricultural pests at the National Research Centre, Cairo ; other research institutions and Egyptian Universities.

Finally, I would like to express my sincere thanks to Professor Dr. Youssef Waly, Deputy Prime Minister and Minister of Agriculture and Land Reclamation, and his representative, Professor Dr. Adel-Ezz State Minister for Scientific Research and his representative for their support and encouragement. Deep

- 3 - appreciation is also expressed to Prof. Dr. H. S. Salama, President of the National Research Centre for organising this workshop.

I hope that our guests from other countries will have a pleasant stay in Egypt which is memorable and scientifically productive.

4 H. S. Salama, National Research Centre, Cairo

It is my pleasure and privelage to welcome you all, this morning at the opening session of the International Workshop on "The use of Bacillus thuringiensis for insect control and its applications as it relates to developing countries". It is indeed a very important scientific occasion to have this gathering of distinguished scientists and leading experts from Egypt and other countries and who came to participate in this workshop. The National Research Centre (NRC) of Cairo is pleased to organise this workshop in cooperation with Agriculture Canada, and Canada International Development Research Centre and in this concern, I should applaud the existing scientific cooperation between these agencies that dated back to 6 years.

The workshop today, deals with a high priority subject. As you know, crop losses from various pests are estimated by around 35070 of potential production of all crops. An estimate by UNIDO places the total loss from agricultural pests about 90 billion $ per year the world over. The present situation with hazards and indiscriminate use of chemical pesticides accelerates the movement towards the possible reliance on more safe biological control methods. The most promising biological control agent to date to satisfy this goal is the bacterium Bacillus thuringiensis which proved to be a highly successful weapon for fighting some agricultural pests and it offers many advantages over chemical insecticides.

I feel confident that this workshop will be productive and hopefully lead to strong and fruitful cooperation in this area between various countries and to develop 'means and avenues that would facilitate the utilization of Science and Technology output for the human welfare in this fast growing and complex world.

Finally, I would like to express sincere thanks to members of steering committee and organising committee for the efforts they have made in the preparation for this workshop. I wish to deeply thank all our invited speekers for the contributions they are going to present to the workshop. Thanks for the Canadian International Development Agency CIDA, UNESCO, FAO, Abbotts and Sandoz for sponsoring this workshop.

May God bless all of you and bless our efforts for the welfare of human being. Oswald Morris, Agriculture Canada

I welcome you to this first International Workshop on the constraints encountered by Developing Countries in the use of Bacillus thuringiensis (B. t.) an alternative to chemical pesticides for pest control. I have had for many years now a special interest in promoting B. t. use in these countries, so this conference presents a good opportunity to pursue this interest. You are no doubt wondering how this meeting actually came about. About 10 years ago, I was invited as Canadian representative to an international workshop on B. t. to which Prof. Salama, President of NRC, was also invited as delegate from Egypt. This workshop was organized by Dr. Howard Dulmage of the United States Department of Agriculture and funded by the Rochefeller Foundation, New York. The meeting place was the Rochefeller Foundation Study Centre, situated on the shores of Lake Como in Northern Italy. In the course of my discussion on the status of B. t. use in Developing Countries with Prof. Salama, I suggested that Canada might be interested in supporting research and development designed to reduce the use of chemical pesticides and increase the use of biological methods in crop protection in these countries. Pursuant to these discussions, the International Development Research Centre (Ottawa) in 1985 funded a cooperative research program between myself at Agriculture Canada and Dr. Salama at NRC (Cairo). The cooperative work that ensued was beneficial to both sides and now we are both approaching the point of evaluating our work for commercialization. This is a good example of successful scientific cooperation between Developed and Developing Countries. One of the provisions of the IDRC agreements with the researchers was that a workshop be held towards the end of the cooperative project with selected Developing Countries to review the status of B. t. in these countries and to evaluate the constraints to its use. This workshop, then is the culmination of many years of scientific cooperation by Canada and Egypt and I welcome you all to what I hope will be a productive meeting. Eglal Rached, IDRC

It is a great pleasure to welcome you, on behalf of IDRC, to this very important gathering, where highly dedicated and respected B.t. workers from both developed and developing countries will meet to discuss potentials and constraints related to the utilization of B.t. in developing countries.

The topic of microbial control of insect pests is of crucial interest to developing countries. The overuse or misuse of chemical pesticides and their negative impacts on soil and water quality, human health, wildlife and the ecological balance within agro-ecosystems are increasingly becoming cause for concern underlining the need for development of alternative pest control methods.

IDRC, a Canadian Crown Corporation dedicated to support development oriented research projects, has for a long time been concerned with issues of environmental sustainability, farmer's welfare and improved agricultural productivity. We have supported several projects related to B. t. both in developing countries and in Canada, several of which will be described in this forum. One of these, initiated in 1985 is the very fruitfull collaboration on B. t. between Agriculture Canada in Winnipeg and the National Research Centre of Egypt, which is hosting the present conference.

The utilization of Bacillus thuringiensis biocides started in the 1960's and initially one of the countries where it was put into widespread use was in China. Though China remains a world leader in using B.t., its utilization has not spread widely to other developing countries. Worldwide some 2.3 tons of commercial B. t. products are used each year to control insect pests of agricultural crops, forest trees, ornamentals, etc. but of this only a small proportion is used in developing countries.

The question that this workshop wishes to address is why, and what can be done to improve the impact of B. t. products. The program of this workshop has been drafted in an effort to reflect the broad range of constraints to greater use

of B. t. in developing countries :

Some are scientific and technical, such as for example those relating to the need to optimize strains, medium and formulations to increase effectiveness of B. t. products against specific pests and under specific agro-ecological conditions of individual countries. These issues, as might be expected in the early stages of

7 research, have received most attention in many projects including those supported by IDRC.

However with success in these areas it is vital to consider downstream issues, those that lie beyond biological efficacy. There is first, the need to evaluate the economics of production.

Are the increases in efficacy that we find when adding adjuvants and sunscreens for example worth the additional costs in production ? Then, what happens to the product once it comes out of the production plant : It must compete in the market with chemical pesticides, whose production or importation is often subsidized. Our efforts in reducing costs of producing the B. t. may not be sufficient to make it a commercial success if government policies do not allow, it to compete fairly with other pesticides. This area of micro and macro-economic analysis is not one in which many of us feel comfortable, but it may be crucial in pointing out the conditions under which B. t. can have an appreciable impact.

Another key question is that of farmer acceptability. To what extent, for example, is the less rapid mortality of pest larvae with B. t. compared to chemical pesticides constraining the adoption of B. t. that we see as an advantage in reducing the disruption of natural biological control, may be a problem from the point of view of the farmer confronted by a pest complex. It is a mistake to put off consideration of such issues until field testing of a finished product. They should and must influence decisions on what to produce, how many strains, what compromise needs to be made in the choice between a strain that has higher efficacy against one pest versus another with lower efficacy against many pests.

We hope that the discussions during the conference will help to identify common problems that are most important in constraining the wider use of B. t. in developing countries and around which it may be possible to define priorities for future research and cooperation.

I would like to extend IDRC's thanks to NRC, Dr. Salama and his colleagues, for the excellent organization of this workshop. Thanks are also due to Dr. Morris, of Agriculture Canada, who has played an important role in getting the workshop off the ground. Finally, I would like to acknowledge the invaluable support of CIDA and FAO who have contributed funds, encouragement and commitment to this workshop.

- 8 - List of Contributors

Afef, Ben Ali Laboratoire de Biochemie, Faculte des Sciences de Tunis, Tunis. Ali, A.M.M. Genetic Engineering and Biotechnology Division, National Research Centre, Cairo, Egypt. Barbosa, S. FAO/AGP, via delle Terme di Caracalla 00100, Rome, Italy.

Bernhard, K. Ciba - Geigy Ltd. R-1093. 4.41 CH 4002, Basel, Switzerland. Bhumiratana, A. Department of Biochemistry, Faculty of Science, Mahidol University, Rama VI Road, Bangkok, 10400, Thailand.

Bingao, A. Hubei Academy of Agriculture Science, Wuhan, P.R.C. China. to Bokkel, D. Department of Chemical and Biochemical Engineering University of Western Ontario, London. Ontario, N6A 5139, Canada. Chou, Tao-mei Department of Entomology, National Chung Hsing University, Taichung, Taiwan 402, ROC, China. Daoust, R. A. Ecogen Inc., 2005 Cabot Boulevard West, Langhome, Pensylvania, 19047 - 1810, U.S.A. Dulmage, H.T. HD - Associates, P.O. Box 4113, Brownsville, Texas, 78521, U.S.A. Fadel, M. Microbial Chemistry Department, National Research Centre, Cairo, Egypt. Ferid, L. Laboratoire De Biochemie, Faculte des Sciences de Tunis, Tunis.

Fiechter, A. '-lstitut fur Biotechnologie, ETH. Zurich, Switzerland.

- 9 - Foda, M.S. Microbial Chemistry Department, National Research Centre, Dokki, Cairo, Egypt. Fusco, R.A. 4083 Rosewall Court, Harrisburg, PA, 17112, U.S.A. Gixin, W. Hubei Academy of Agriculture Science, Wuhen, P.R.C. (China). Hollebone, J.E. Director of Issues, Planning and Priorities Division, Agriculture Canada, Pesticide Directorate, Food Production and Inspection Branch, Ottawa, Ontario, K1AOC6, Canada. Hou, R.F. Department of Entomology, National Chung Hsing University, Taichung, Taiwan 402, Republic of China. Jayaraman, K. Director for Biotechnology, Anna University, Madras, 600 025 - India. Jenny, K. Institut fur biotechnologie, ETH, Zurich, Switzerland. Jones, K. A. Natural Resources Institute, Central Avenue, Chatham, Maritime. Chathan, Kent ME4, 4TB, UK. Kosir, J. Department of Chemical and Biochemical Engineering, Faculty of Engineering Science, University of Western Ontario, London, Ontario, N6A 5139, Canada. Laforce, R. Institut fur biotechnologie, ETH, Zurich, Switzerland.

Liansen, Z. Hubei Academy of Agriculture Science, Wuhan P.R.C., (China). Loevinsohn Michael Consultant in Applied Ecology, Butane, Rwanda. Margaritis, A. Department of Chemical and Biochemical Engineering, Faculty of Engineering Science, University of Western Ontario, London, Ontario, N6A 5139, Canada.

- 10 - Matter, M. Pests and Plant Protection Department, National Research Centre, Dokki, Cairo, Egypt.

Merdan, A. Faculty of Science, Ain Shams University, Cairo, Egypt. Moraes, I.O. Food Engineering and Technology Dept., UNESP, C.P.-136-CEP 15055 S-J., Rio Preto - SP - Brazil. Morris, O.N. Agriculture Canada, Research Station, 195 Dafoe Road, Winnipeg, Manitoba, R3T 2M9 Canada. Omrane, B. Laboratoire de Biochemie, Faculty des Sciences, de Tunis, Tunis. Padua, L.E. National Institutes of Biotechnology and Applied Microbiology (Biotech.), University of the Philippines at Los Banos College, Laguna, Philippines. Panbangred, W. Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand. Pantuwatana, S. Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand. Pasqualini, E. Institute of Entomology, University of Bologna, Via Filippo Re, 6, 40126, Bologna, Italy. Qiumei, D. Academy of Agriculture Science, Wuhan, P.R.C. (China). Rajendra, N. Centre for Biotechnology, Anna University, Madras, India. Razo, E. Department of Biotechnology, Cinvestav, P.O. Box 14-740, Mexico City, 07000, Mexico. Rodriguez, M.

Department of Biotechnology, Cinvestav, P.O. Box 14-740 , Mexico City, 07000, Mexico. Sachidanandham, R. Centre for Biotechnology, Anna University, Madras, India. Salama, H.S. National Research Centre, Tahrir Street, Dokki, Cairo, Egypt. Salem, S.A. Pests and Plant Protection Department. National Research Centre, Dokki, Cairo, Egypt. Sharaby, A. Pest and Plant Protection Department. National Research Centre, Dokki, Cairo, Egypt. Sivamani, E. Centre for Biotechnology, Anna University, Madras, India. Tainjian, X. Hubei Academy of Agricultural Sciences, Plant Protection Institute, Wuhan, People's Republic of China. Tianliang, M. East China Chemical Technology College, China. de la Torre, M. Department of Biotechnology, Cinvestav, P.O. Box 14-740, 07000, Mexico City, Mexico. de Urquijo, E. Department of Biotechnology. Cinvestav, P.O. Box 14-740, Mexico City, Mexico. Villafana, J. Department of Biotechnology, Cinvestav, P.O. Box 14-740, Mexico City, Mexico.

Xinthu, X. East China Chemical Technology College. P.R.C. (China). Zaki, F.N.

Pests and Plant Protection Department, National Research Centre ; Dokki, Cairo, Egypt. Zhiwen, Y. Hubei Academy of Agriculture Science, Wuhan, P.R.C. (China).

- 12 - . Part I

Bacillus thuringiensis Utilization in Developing Countries

Development of Isolates of Bacillus thuringiensis and Similar Aerobic Microbes for Use in Developing Countries

H. T. Dulmage H.D. Association, P.O.Box 4113, Brownsville, Texas, 78520

ABSTRACT Progress has been made in developing better isolates of Bacillus thuringiensis (B. t.) for control of Trichoplusia ni and Heliothis virescens. The initial HD-I formulations were about 100 times as potent as were the commercial products of its day. Since then, yields of the B. t. toxins have been increased a 100fold in fermenters. Unfortunately, the massive increase in yields reflects how weak the original formulations were. So even with the increase, these new formulations were not potent enough or economical enough to be used widely in the control offorestinsect pests. The addition of support from formulation chemists and application engineers has resulted in products and procedures that have made the HD-1 endotoxin effectively economically in the forest. Formulations of insect pathogens can be improved markedly. It takes time, but, with the proper effort, (and it is worth repeating) formulations of insect pathogens can be improved. This is an important concept, but one that is frequently forgotten. While it would seem obvious to me that we are only in the early stages of knowledge about these entomopathogens or their toxin that we can make marked improvements in activity, yield, and formulation and application technique, there is a strong tendency by many scientists and by the administrators who prepare our budgets to believe that what we have now is the best that can be found. Too many of our evaluations of entomopathogens are restricted to judgements based on present knowleege and do not allow for possible improvements in strains or techniques. There are better isolates to be found, if we search for them. There are better fermentation and recovery procedures, if we pursue a logical course in fermentation research. There are better ways of using

- 15 - H. T. Dulmage

and applying these pathogens and toxins, if we seek and develop close cooperation between entomologists, microbiologists, chemists, and

engineers to , find them.

1. PRINCIPLES OF FERMENTATION

I.A. Introduction

Over the past few years, the successful use of isolates of Bacillus thuringiensis (B.t.) to control many agricultural and vector insect pests, has stimulated world-wide interest in the design of research programs that aimed at finding even more potent fermentations. Two isolates of this genus are highly active against insects of great economic importance ; (B.t.) subsp. kurstaki, active vs. lepidopterous insects and B. t. subsp. israelensis, active vs. mosquitoes and black flies). Progress has been slowed by an incomplete understanding of just what B. t. is ; what it can or can't do, and why. This led me to believe that the most useful contribution that I could make to this workshop would be to review basic fermentations principles and to show how these principles apply to all fermentations, whether they be bacterial, fungal, or actinomycete. I will review what fermentation have done for us in the past, what we can hope from fermentations in the future, and then discuss how the production of B. t. relates to the studies in this workshop.

Much of this discussion will be devoted to examples of B. t. fermentations. To simplify the analyses of the fermentations, most of the fermentations were carried out in a single medium, 13-4b. (Table 1)

I.B. History of Fermentation

In the early days of fermentation research, any insecticidal activity observed in a microbial fermentation was widely presumed to be the result of a straight-forward competition for nutrients, with the slower growing microbe losing the battle. Unfortunately, infection plays little or no role in most microbial- insecticidal activities and a failure to recognize this delayed many important discoveries. For example, scientists had, for many years, reported that when the fungus, Penicillium notatum, (a common contaminant on agar plates), was grown in a mixed culture with a gram-positive bacterium, it could kill the bacterium by

- 16 - B.I. in developing countries

Table (1) Fermentation Medium B-4b.

Proflo 20.0 g/liter Peptone 2.0 Dextrose 15.0 Yeast Extract 2.0 CaCO3 1.0 MgSO4.7H2O 0.3 FeSO4.7H2O 0.02 ZnSO47H2O 0.02 Distilled H2O to 1000 ml out-competing the bacterium for the available nutrients. This belief was wrong : P. notatum was active because it produced a toxic chemical - penicillin. Failure to recognize the difference between toxin activity (antibiotic) production and infectivity delayed the use of this highly effective therapeutic agent for as much as 40-50 years. As a matter of interest, recognizing and proving that the activity of this and similar fungi was due to the production of an antibiotic rather than an infection earned Fleming and his co-workers the Nobel Prize.

Fleming's work was shortly followed by the discovery of streptomycin by Waksman. Streptomycin, produced by Streptomyces griseus, was the first known agent that could actually cure tuberculosis. Waksman was also awarded the Nobel Prize for his discovery. This opened the door to the possibility that many biologically active agents could be produced in fermentation. Since then, several hundred different microbes have been shown to produce a wide range of biological agents. Of major significance was the observation that the agents produced were complex chemicals, mostly unrelated to each other and that many types of microbes - fungi, bacteria and actinomycete - have been sources of valuable biologically active chemicals whose molecular structures were so complex that it would be too expensive to synthesize them, they could only be produced by fermentations. Fortunately these products can usually be produced economically.

This is not the end of the story. Once it had been shown that fermentation products could have useful biological activity, laboratories around the world have

- 17 - H. T. Dulmage been examining fermentation beers for other unique products, - and the results have been spectacular. Fermentation products are now used to control many types of pathogenic microbes. They are useful in the synthesis of steroids. They play an important role in several cancer chemotherapy regimes (actinomycin-D). And perhaps their most unexpected use is in the prevention of tissue rejection after organ transplants (cyclosporin). They are being used to control many vector insects around the world, and, germane to this discussion, many isolates of bacilli produce insecticidal agents useful in agriculture for the control of insect pests (HD-1 and H-14).

I.C. Submerged Fermentations

I.C-1. Principles

B. t. fermentations have a need for high levels of carbon, nitrogen, and oxygen (a) Aerobic microbes such as B.t. have a heavy demand for air in the early stages of fermentation. It is almost impossible to saturate actively growing B.t.'s with air, in the log phase in 14 L fermenters, even at an aeration rate of 2.0 v/m.

(b) The high levels of carbohydrates needed in B. t. fermentation are usually supplied by dextrose or starch. However, carbohydrates can produce large amounts of organic acids that can drop the pH of the fermentation beer to 5.2 - 5.4, a pH at which the bacilli cannot grow and the fermentation stops. (Neutralizing such beers usually allows the microbe to resume growing).

(c) High levels of nitrogen, (from waste proteins, hydrolysates, or corn steep) also stimulate growth, and, in this case, release organic bases.

Thus the balance of nitrogen and oxygen can have a profound influence on the pH during the fermentations. Used properly, the pH of the fermentation (ranging from pH 5.4 to pH 8.4) can be controlled through the correct selection of nutrients, balancing acids from the carbohydrates and bases from the proteins. Finally, a proper balance of mineral salts is also required. A typical medium is shown in Table(t)

- 18 - B.t. in developing countries

LC-2. Philosophical Differences between Bioassays of Chemical and Biological Insecticides

Scientists familiar with the assay of insecticides enter a new world when they turn to assaying microbial insecticides. In the case of a chemical insecticide, the assayer already knows the quantity and purity of the insecticide that he is testing. The assay is not an accessory to the production process, it needs only be confirmatory. In the case of a microbial agent such as B. t. the assay must monitor what sort all stages of the production process : the quality of the fermentation, of losses of activity occur during the recovery of the active material from the fermenter beer, the quality of the product recovered, and the characteristics of the final formulation. All this must be known accurately, and this knowledge can be achieved only through bioassay. The bioassay is not peripheral or accessory accuracy that we to the B. t. process : it is central to it. Thus the reliability and must demand from the assay of a microbial insecticide must be much greater than that demanded from the assay of a chemical (Dulmage, 1990).

The recovery processes for a chemical insecticide are usually carried out in a high concentration of product whereas the recovery processes, of a microbial insecticide, such as HD-1, usually start at a very low concentration of the product (10 mcg harvested beer/liter or less), a much more difficult process.

II. BACILLUS THURINGIENSIS II.A. Discovery and Classification of B.t.

B. t. was first observed by Ishiwata, who found it in a colony of sick or dying silkworms, in Japan, in 1901. Although Ishiwata's description was good enough to let us recognize that he had isolated a B. t. , his nomenclature was improper, and it was left to Berliner in 1911 to name his isolate, "Bacillus thuringiensis ", after the Province of Thuringia in Germany where he had found it.

II.B. Flagellar Serology

B. t. is apparently widely distributed in the soil and is frequently found there, but no one has ever, so far as I know, isolated a B. t. from a feral insect except in the vicinity of laboratories where B. t. had been grown in the past.

- 19 - H. T. Dulmage

Much of the early work with B. t. isolates was confused and marked by disagreement among scientists as to how to sort the various B. t. 's found in different laboratories. Then, in 1958, Bonnefoi and de Barjac proposed that classification of B. t. isolates be based on the antigenic reactions of the flagellae associated with young, actively motile, cells of the Bacillus.

Nomenclature under the Bonnefoi and de Barjac system is straight-forward. Using this system, the various isolates can be divided into groups, with each group having its own antigenic characteristics. Based on these characteristics, cultures are either assigned an "H-number" (also called serotype or subspecies) with an "H-number" assigned in consecutive order or placed in a group of other isolates. There is an obvious correlation between "H-types" and insecticidal activity. It is difficult to visualize a relationship between the flagellar antigen of a vegetative cell of B. t. and the insecticidal toxin that the cell produces when it sporulates, but it exists, although the correlation, as we shall see later in this paper, is not complete. Serotyping is a relatively simple technique and easy to use, and is the accepted method for classifying the various isolates of B.t.

The discoverer of a new isolate has the privilege of naming his discovery. Three important isolates of more than thirty five known isolates are :

H-3a,3b kurstaki HD-1 (Dulmage, 1967) H-7 aizawai (Aizawai, 1962) H-14 israelensis (Goldberg, 1980)

II.C. Discussion of the Role of the Spore in B.t. Fermentations

The spore is at best only a secondary factor in killing insects. The insecticidal activity of B. t. isolates and formulations are associated with a crystalline, proteinaceous toxins produced by the B. t. cell and called the delta-endotoxin. However, as we now know, spores and crystals are not produced in parallel and do not correlate with each other or with the toxic activity. This was not known for many years and our predecessors' in research on B. t. , in all good faith, relied upon.the spore count to evaluate their studies. Indeed the regulatory agencies in the U.S. required that commercial formulations of B.t. offered for sale in the U.S.

- 20 B.t. in developing countries be standardized by spore count and that the U.S. label contain a statement expressing potency as spores of B. t. per gram of formulation - an essentially meaningless exercise. As we shall discuss in the next section, the only way to standardize or evaluate B. t. formulations is through bioassay.

However, the spore count does give a reasonably good estimate of the growth of the Bacillus in the original formulation. Unfortunately, a reliance on spore count to measure the potency of a preparation is wrong for two reasons, aside from the inaccurracies faced in any spore count (a) there is no way to differentiate subspecies by spore count, and (b) or to learn anything about the attenuation or virulence of an isolate through a spore count. Spore counts, however, can be useful in following the growth of B. t. in a fermentation or in fractionating samples during a recovery study.

Tables 2-A and 2-13 illustrate the problems that arise in the use of spore counts. Table 2-A compares the spore count with the potencies in International Units (IU) for of 8 isolates of B. t. grown in identical media. The spore counts determined

Table (2-A)

Interrelationship between spore counts and activity of different isolates of Bacillus thuringiensis.

Activity vs. Culture Serovar. Spore count H. virescens kIU/ 109 Spores No. x 109/G (kIU/G)

HD-2 H-1 12 1,410 120 HD-83 H-3A 6 Inactive 0 HD-1 H-3A, 313 17 15,400 910 HD-263 MA, 3B 11 54,600 4,210 HD-244 H-3A, 313 13 70,600 5,400 HD-305 H-5A, 513 16 943 59 HD-519 H-14 63 Inactive' 0 HD-563 H-14 62 Inactive 0

- 21 - H. T. Dulmage

6 of these isolates varied over a narrow range (6 - 7 x 106 viable spores/gm) and did not correlate with each other. Similarly, the potencies measured in bioassays vs. H. virescens ranged from zero to 70,600 IU/mg with no indication of any correlation between spores and crystals. Two isolates (HD-244 and HD-263) produced formulations much more potent than the rest and were saved for further work. The table is admittedly biased by the inclusion of HD-519 and HD-563 with high spore counts, since both cultures are sterotype H-14 (subspecies israelensis). H-14 isolates produce a toxin with lower molecular weight, different toxic activities, and an unique delta-endotoxin with little or no lepidopterous and high mosquito activity. There is some doubt in my mind whether H-14 should be classified as a new species.

Table (2-B)

Correlation between spore counts and activity of the same culture grown under different media.

Activity vs. Culture Medium Spore count H. virescens kIU/109 Spores No. X 109/ML (kIU/ML)

HD-263 A 1.2 5,800 4,830 B 1.7 12,100 7,120 C 2.4 3,830 1,600

Table (2-13) compares one isolate of B. t. (HD-263) grown in 3 different media. Here too, there is no correlation between the potencies of the products and the spore count.

* N.B. The following discussions are derived from a series of workshops conducted in the country and year indicated : New Zealand (1977) ; Italy

(1979 ; ; 1980) Japan (1980) ; USA (1980) ; Israel (1988) ; Brazil (1988 ; 1990). In addition, a series of unpublished workshops were conducted in

Egypt (1980) ; China (1982 ; 1988) ; England (1985) ; and The Philippines (1987) ; were also used as source material for this report.

- 22 - B.t. in developing countries

H.D. Characteristics of Bacillus thuringiensis (B.t.) in size, shape and B. t. closely resembles B. cereus, a common soil organism, behavior, with two notable and very important exceptions.

II.13-1. Cell Type At the same time that the cells of B. t. sporulate, parasporal bodies, usually crystalline in shape, appear in the cells. This is so characteristic of this group of The Bacilli that they are commonly referred to as the "Crystalliferous bacteria". parasporal bodies are loosely referred to as the "crystals", the "crystalline toxins", or, as we shall discuss shortly, as the "delta-endotoxins". The presence or absence of the crystal should not be used as a sole identifying character to distinguish B. t. from B. cereus particularly during fermentation studies. This risk of over-looking an acrystalliferous B. t. is avoided. Thus, it may not always be accurate, but the preliminary classification of cultures meeting the criteria for B. cereus is essentially a straight-forward one. If parasporal bodies are seen the Bacillus is called B. t. If no parasporal bodies are seen, the Bacillus is called B. cereus.

ILD-2. Crystals

Many, if not all, of the crystals produced by B. t. are toxic to one or more species of insects. At one time, it was believed that all of these crystalline toxins were active against insects, but more recently, several isolates have been reported that resemble B. t. in containing a crystal, but which have no known insecticidal activity. Nevertheless, some of the toxicity of B. t. is closely associated with the crystal. These crystalline toxins, as a group, have been named the "delta-endotoxins" according to a system proposed by Heimpel (1966). This is a generic name, and not a specific one, because there are many delta-endotoxins produced by different isolates of B. t.

II.D-3. Delta-endotoxins

It is very important to realize that there is not just one B.t. or one B. t. delta-endotoxin. The species, B. t. , is made up of groups of microbes, each of which has its own characteristics. First, the species can be divided into subspecies or serotypes according to the serological reactions of the thread-like flagellae

- 23 - H. T. Dulmage

described earlier. These subspecies are all given distinguishing names : Thus B. t. subsp. kurstaki ; subsp. galleriae ; subsp. aizawai ; subsp. morrisoni ; subsp. tenebrionis ; subsp. israelensis ; and subsp. sun diego ; to name a few of the more familiar ones. There are more than 35 known subspecies of B. t. In addition to the divisions that can be made by the use of flagellar antigens, Krywienczyk and Angus (1967) and Krywienczyk et al. (1978) proposed a serological classification of the crystals present in B. t. cells. These are frequently appended to the flagellar classification. It is interesting that neither the flagellar nor the crystal serology pinpoints all the delta-endotoxin activity of the various isolates of B. t. This leads us to the key aspect of the B. t. isolates : What makes the species so fascinating and important is that the toxins produced by different isolates of B. t. can differ in their toxicities towards the same insect species or differ in their "spectra of insecticidal activities". Why this is so, and what the interrelationships are between the different delta-endotoxins, are poorly understood. They have similarities. They are all high molecular weight proteins. They all must be eaten and digested to be effective. And there must be some relationship between the serology of the subspecies producing the toxin and the spectra of activities of the delta-endotoxins that they produce, but the correlations are not complete.

II.E. LC50 and the International Unit II.E -1. Introduction As discussed in the previous paragraphs, many, if not all, biologically active fermentation products have complex molecules that are difficult to measure and purify chemically. However, with proper care and techniques, highly accurate bioassays can be developed for many microbial formulations. Bioassays have been and still are used for many such widely diverse commercial products as vitamin B12, erythromycin, and penicillin, as well as many experimental entomocidal fermentation. Dulmage et al., (1973a, 1973b, 1976, 1985) ; de Barjac, (1979, 1984). Replication of these assays is, of course, essential. Using 3 to 4 replications, one can hold the error in an assay to less than 20 percent over a 7-day period. Good, even for a chemical assay of a microbial product.

- 24 - B.t. in developing countries

II.E-2. Mechanics of B.I. Bioassays A bioassay compares the interaction between a test insect and the toxins in a standard. The most dramatic response of an insect to a microbial insecticide and the one that is easiest to observe is death ; and the most accurate expression of the killing power of a B. t. sample is the LC50 : (the theoretical concentration that will kill 50% of the insects). This concentration is determined by exposing groups of larvae to different concentrations of samples in their diet, incubating them for a standardized period of time, recording the per cent kill at each concentration, and then using regression analysis to determine the LC500f each sample.

It is important that the assay achieve the accuracy reported by Dulmage et al. (1981), de Barjac and Larget-Thiery (1983), de Barjac (1984). The secret of an accurate bioassay lies in the use of a standard. Most B. t. 's are evaluated by the LC50. The LC50 for any given sample will vary from day to day, no matter how carefully the test insects are raised, or how uniform the insects are in the colony (Tables 3-A and 3-B). This variation is minimized by assaying a standard formulation along with the test samples using the same population of insects in each day's bioassays, and then comparing the LC50's of the standard vs. the LC50's of the test samples (Dulmage et al., 1981). At the present time, three standards have been adopted by the scientific community. The standards are listed

below ; the HD-1 standards are used in assays against lepidopterous insects and the IPS standard is used against mosquitoes and blackflies.

Name of Standard Potency

HD-1-S-1971 18,000 IU/mg* HD-1-S-1981 16,000 IU/mg** IPS-82 15,000 ITU/mg***

* U.S.D.A. Official Standard ** Proposed U.S.D.A. Replacement Standard *** World Health Organization (WHO) Standard

- 25 - H. T. Dulmage

Table (3-A)

Calculations of potencies of dry powders of delta-endotoxins of B. t.

1. Basic Formula :

LC50 Standard x Potency of Standard (IU/mg) = Potency of Test LC50 Test Sample Sample (IU/mg)

II. When HD-1-S-1971 is used as the standard, the equation becomes LC50 HD-1-S-1971 x 18,000 IU/mg = Potency of Test Sample (IU/mg) LC50 Test Sample

Table (3-B) Hypothetical calculations of Tn/Hv ratios containing one of two delta-endotoxins with different spectra of activity.

Powder A P owder B P owder C P owder D

Assays against Trichopluisa ni LC50 standard, (pg/ml) 11.2 11.2 11.2 11,2 LC50 sample, (pg/ml) 20.0 4.0 4.0 16.0 Potency of sample, (IU/mg) 10,000 50,000 50,000 12,500 Assays against Heliothis virescens LC50 standard, (pg/ml) 2.8 2.8 2.8 2.8 LC50 sample, ()ig/ml) 5.0 1.0 2.0 2.0 Potency of sample, (IU/mg) 10,000 50,000 25,000 25,000 Tn/Hv ratios 1.0 1.0 2.0 0.5

Source : Adopted from Dulmage, 1979, Activity ratios are discussed at length by Dulmage and Cooperators, 1981. Note : Powder A and Powder B are homologous to themselves and to the standard, and Powder C and Powder D are neither homologous to the standard nor to each other. Potency vs. T ni (IU/mg) aTn/Hv ratio Potency vs. H. virescens (IU/mg) - 26 - B.t. in developing countries

II.E.-3. Evaluation of Assays and their Reproducibility

Table (4) summarizes the criteria for evaluating the accuracy of individual assays as follows - Dulmage (1983) :

Table (4)

1. There must be < 10% dead in the control larvae. This is absolute. 2. Dilutions must be selected so that at least five concentrations of each sample and seven concentrations of the standard are valid. with no more than 90% or no less than 10% larval mortality. 3. Slopes of the regression curves must be reasonable, similar to those obtained previously against the test insect, and, within the errors of the day's samples, parallel to each other. 4. If computer analyses are available, the 95% confidence limits around the LC50 should be such that the maximum limit/minimum limit is < 2.0.

5. Similarity, the 95076 confidence limits around the IU's should be determined, and the maximum limit/minimum limit should be < 2.0. 6. Assays should be replicated on three separate days. The IUs determined on each of those days should be averaged, and the standard deviation should be determined and should be < 0.20. These data should be used to determine the coefficient of variation (CV) between the replicated assays (standard deviation/average).

These criteria allow us to "trouble-shoot" the various stages within a bioassay. Experience has taught us that the CVs between reliable assays will be < 0.20 (possibly < 0.15 for assay with A. aegypti). CVs higher than this indicate that something is wrong with the sample being tested, with the insect colony being used, or with the techniques of the assayers.

- 27 - H. T. Dulmage

II.F. Spectra of Activity

Table (5) shows a partial spectra for the delta-endotoxins produced by typical B. t. isolates. The table shows that the spectra of activities of the three isolates of subsp. kurstaki (HD-1, HD-73, and HD-263) differ from each other, even though they have the same flagellar serology. Note that HD-1 and HD-73 differ from each other in crystal serology, as might be expected from the difference in their spectra of activities. However, HD-1 and HD-263 have different spectra of activities even though they appear to have the same crystal serology.

The difference can be striking. It is interesting to compare HD-73 with HD-83, a typical isolate of subsp. alesti : HD-73, along with other isolates of subsp. kurstaki, is active vs. T. ni and H. virescens, while HD-73 has no activity vs. B. mori. In contrast, isolates of subsp. alesti have no activity vs. T. ni or H. virescens, but are highly active vs. B. _mori and H. cunea. HD-1.82 differs from all of them in activity against H. cunea but not against B. mori.

An important and distinctive difference can be observed when the toxins produced by isolates of most subspecies of B. t. are compared with the toxins produced by isolates of subsp. israelensis, such as HD-567. The delta-endotoxins active vs. lepidopterous insects have little or no activity vs. mosquitoes, even though they have high activity vs. lepidoptera. Isolates of subsp. israelensis, in contrast, have little or no activity vs. lepidopterous insects, but have high activity vs. mosquitoes and aquatic blackflies.

The differences that I have just discussed show that some of the toxicity of these toxins are independent of subspecies. The situation becomes still more complicated when the spectra of activities of the B. t. toxins are broadened by including a wider range of insect species in determining the spectra. I will not go into this in detail here, but suggest that reading the chapter by Dulmage and Cooperators (1981) will show how the spectra of activities of these delta-endotoxins increase in complexity as the number of species tested increases.

For many years, it was believed that B.t.-endotoxins were active only vs. lepidopterous insects except for the occasional presence of weak activity vs. a few mosquito species. The commercial development of B.t. formulations paralleled this belief, and the use of formulations of these lepidopterous activities has been

- 28 - Table (5) Comparaison of the activities of 9 isolates of Bacillus thuringiensis against 4 Lepidopterous and 5 mosquito Species.

Potency (IU/mg)

Culture Subspecies Crystal type T. ni H. virescens H. cunea B. mori Mosquito Type Activity

HD-59 thuringiensis thu 7,960 1,440 13,900 1,300 Inactive HD-83 alesti ale Inactive Inactive 93,200 27,500 Inactive

HD-1 kurstaki k-1 39,800 15,400 47,200 50,500 Inactive HD-263 kurstaki k-1 39,600 54,600 18,100 18,400 Inactive HD-73 kurstaki k-73 29,300 34,500 15,400 Inactive Inactive

HD-168 galleriae G-1 16,300 10,100 86,700 6,200 Inactive HD-29 galleriae G-9 6,7616 341 Inactive 7,400 Inactive

HD-135 aizawai aiz 18,000 1,460 134,100 75,900 Inactive

HD-567 israelensis isr Inactive Inactive Inactive Inactive High act. H. T. Duimage increased slowly over the last 15 years. For example, the endotoxin derived from fermentations of subsp. kurstaki (HD-1) is being used in the control of pests of field crops, such as the cabbage looper, Trichoplusia ni and the European corn borer, Ostrinia nubilalis ; stored crops pests such as the almond moth, Ephestia cautella ; tobacco insects, such as the tobacco budworm, Heliothis virescens ; and forest insects, such as the gypsy moth, Porthetria &spar and the spruce budworm, Choristoneura fumiferana. The commercial use of B. t. to control lepidopterous pests has not been restricted to formulations of subsp. kurstaki. An isolate of subsp. aizawai is being used for the control of the wax moth Galleria mellonella and a strain of subsp. san diego for the control of army worms, Spodoptera spp.

A major screening program was designed by Dulmage et al. (1967) searching for more potent isolates of B. t. This lead to the discovery of the HD-1 isolate which was 10 to 30x more virulent than previous isolates. A second major search by Goldberg and Margalit (1977) seeking pathogens for the control of mosquitoes led to the isolate of a new subspecies of B. t. (subsp. israelensis) with high activity against mosquitoes A aquatic blackflies. The earlier observations that there were a few isolates of B.t. (primarily members of the subsp. of HD-1-k-1) with weak activity vs. mosquito was considered only of academic interest. The dimension was broadened still further when a toxin produced by a new species of Bacillus sphaericus was found to be very active against mosquitoes. Subsequent field trials showed H-14 was effective at economic levels.

The discovery of subsp. israelensis has served as catalyst for similar searches for other isolates of B. t. that might produce delta-endotoxin active against other orders of insects. Such searches have been successful, but slow. After a two or three year search, Krieg et al. (1983), discovered a new subspecies of B.t. subsp. tenebrionis which produces a delta-endotoxin active against many species of Coleoptera, particularly against the colorado potato beetle, Leptinotarsa decemlineata. Another new subspecies of B. t. , subsp. san diego has been found by scientists working for Mycogen. This subspecies, is also active vs. Coleoptera.

New isolates are still being found : Padua et al. (1984) have reported the discovery of a new isolate of B. t. subsp. morrisoni with high activity against mosquitoes.

- 30 - B.t. in developing countries

ILG. Activity Ratios II.G-1 Introduction As discussed previously, the bioassay measures an activity, not a finite substance that can be taken out and weighed. The IU's determined in a B. t. bioassay reflect the comparative toxicity of a test sample to a standard formulation against a designated insect species. Thus the IU's found in an assay depend, not only on the amount and kind of endotoxin in the powder, but also on the insects used in the assay. The IU's determined in assays of a powder against insect species will not be the same unless the toxin being assayed is identical to the standard. This leads to several corollaries :

1 - If the standard and the test material contain the same delta-endotoxin, then the IU's determined in bioassays conducted against different insect species will be the same. (In such a case, we are merely comparing the toxin with itself). 2 - If the IU's determined in assays against two different insect species are identical, this is evidence, but not conclusive evidence, that the test sample is homologous to the standard.

3 - If the IU's determined in assays against two different insect species are not identical, this is evidence that the test sample is not homologous to the standard. 4 - The similarity (or dissimilarities) of B. t. powders can be quantitated by a simple mathematical ratio. This ratio is called the "activity ratio".

5 - Activity ratio alone cannot distinguish between different types of B. t. IU's measure the quantity of B. t, endotoxin in a powder or beer, using the LC50 as a measuring tool. The coefficient of variation indicates the type of B. t. endotoxin present using activity ratio as a measuring tool.

II.G-2. Calculation of Activity Ratio Activity ratio can be used to compare any two susceptible insect species. To avoid confusion, it is necessary to have a convenient way of designating what two insect species were used in calculating the ratio. To do this, I have used the first letter of the generic name and the first letter of the species name to indicate the

insect species and the place of the insect in the fraction. Thus : T.ni = Tn and

- 31 - H. T. Dulmage

H. virescens = Hv, and the activity ratio would be identified as the Tn/Hv ratio. Table (3-B) shows the calculation of an activity ratio based on assays against T.ni and H. virescens.

II.G-3. Using CVs to Sort Activity Ratios The technique of using coefficient of variation (CV) can be used to evaluate the activity ratio. Table (6) illustrates the value of the activity ratio. The table shows the potencies of powders of subspecies kurstaki (HD-1) measured in assays versus T. ni and H. virescens. The powders have been produced in several different media and also differ from each other in potency. However, the CVs of the various powders averaged 0.192, well within the experimental error, indicating that the toxins present in each powder were the same despite differences in the quantities of the powders produced or the media in which it was produced.

In actual practice, as a result of several years of using the technique, we have widened the acceptable CV of the ratio to < 0.33. Thus, if the average CV of a series of assays is > 0.33, the assay is not considered valid.

The most valuable contribution of coefficient of variation is their aid in sorting activity ratio in a similar manner to the sorts of the potencies of B. t. powders. If the CV's are < 0.33, we assume that they belong in the same set. If the CV's are > 0.33, then we assume that some of the powders in the group belong in different sets. The assays of the individual powders can be shuttled one at a time between groups until a distribution is found that will fit as many powders as possible into sets. This is not a statistically elegant procedure, but it has worked to help sort out the different activities quickly and easily and has proven to give very reproducible results.

II.G-4. Parameters in B.t. Fermentations.

B. t. fermentations are generally quite easy to conduct, with no special requirement for growth or crystal formation. Not all parameters have been studied in same detail, but the following data gives a picture of a rather broad range. a) Temperature of Incubation As shown in Table (7), growth and yields are about the same at

- 32 - B.I. in developing countries

temperatures between 26'C to WC. At 37°C, microscopic examination of the cells in the fermenter showed the presence of long strands of cells and much lower yields. There seems to be no advantage to growing the B.t. cultures at higher temperature (34°C). Because there is some risk in growing them at higher temperatures, for production purposes, we now grow all of our experimental fermentations at 30°C.

Table (6) Influence of medium on the production of delta-endotoxin by Bacillus thuringiensis strain HD-1 in 14-liters fermenters.

Medium Ingredients Yield (kIU/ml)b (g/liter)a

Experiment Cottonseed Glucose Corn Trichoplusia Heliothis Tn/Hv No. flour steep ni virescens ratio`

20 15 - 414 298 1.39 30 40 25 834 365 2.28 30 20 25 893 472 1.89 20 15 10 711 278 2.56 30 40 10 797 381 2.09 30 40 10* 515 246 2.09 30 40 50* 1730 - -

- 'All media, except those designated by *media contained yeast extract, 2.0 : peptone, 2.0 ; MgSO47H2O, 0.3 ; FeSO4, 7H20, 0.02 ; ZnSO410.02 ; CaCO3, 1.0. - bMeasured against T. ni and:H. virescens, using HD-1-S-1971 as a referenced standard, HD-1-S-1971 contains 18,000 IU/mg ; kIU = international units X 103. - cIU/ml measured against T. ni divided by IU/ml measured against H. virescens. Reproducibility of Tn/Hv ratio : n = 6 ; Avg. = 2.050 ; SD = 0.394 ; CV = 0.192. -33- H. T. Dulmage

Table (7) Effect of temperature of incubation on growth and delta-endotoxin production by Bacillus thuringiensis strain HD-263 in 14-liters fermenters.

Temperature of incubation Peak yield obtained Spore count Yield (°C) at indicated hour (x 107) (kIU/mla)

37 39 - 42 80 283 34 18 - 24 170 1,150 30 34- 39 220 1,500 30 39 240 1,450 26 39 - 42 200 1,630

aMeasured against Heliothis virescens ; kIU = International Units x 103

b) Aeration Aeration is important to B. t. fermentations. Studies by Dulmage et al. (unpub. data) and Foda et al. (1985) indicated that it was very difficult to grow and maintain B. t. during the first 10 hours in submerged fermentation due to the high demand for oxygen.

c) Stability of Activity Ratio Random selected powders of 5 isolates of B. t. produced under a wide variety of conditions were bioassayed against H. virescens. Table (8) shows that while there was considerable range in yields between the different serotypes, the coefficient of variation of all these powders were constant. The CV's of the different powders were extremely close with a range of 0.23 to 0.32. From a practical standpoint, a company producing B. t. must be able to rely on the reproducability of the toxin from run to run, or the overhead cost will be exorbitant.

- 34 - B.I. in developing countries

Table (8) Reproducibility of activity ratios of formulations from fermentation of five isolates of Bacillus thuringiensis in 14 liters fermenters. Number of Culture formulations Average Coefficient testeda Tn/Hv Ration' of variation

HD-1 60 2.40 0.25 HD-129 59 5.63 0.32 HD-241 26 0.63 0.24 HD-244 26 1.54 0.23 HD-263 136 0.44 0.29 aIncludes formulations from different media or fermentation conditions and from different times of harvest. 'International unit (IU)/ml measured against Trichoplusia ni divided by IU/ml measured against Heliothis virescens IU determined against HD-1-S-1971 as a standard. HD-1-S-1971 contains 18,000 IU/mg.

III. RECOVERY The recovery procedure for laboratory scale fermentation (up to 1-2L) is shown in Table (9). The recovery used for shake flasks fermentation is a batch procedure with the initial centrification in centrifuge cups. It is important to add the acetone slowly and particularly to allow it to stand 30 minutes before filtering in suction flasks using Whatman # 2 filter paper. At this stage and thereafter the filtration is rapid and with no problems except from inadequate washing with the acetone.

Different problems arise in spray drying for pilot plant scale Table (10). We use a continuous flow sharples centrifuge although we sometimes have problems with two many solids which force us to shut down and restart. This represent a mechanical problem (not a recovery problem). The inlet and outlet temperatures should be adhered to. At this stage, we add any wetting agent or dispersant to the slurry that we want in the final formulation.

Both procedures can be easily followed with little loss of activity.

- 35 - H. T. Dulmage

Table (9) Recovery process for spore-crystal complex of Bacillus thuringiensis : Flow sheet for laboratory-scale recovery.

Whole beer -pH 8.4 - 8.7 Adjust to pH 7.0 with HCI

Supernate Centrifuge (discard) Residue Suspend in 1/10 - 1/20 vol. 4-6% lactose (vol. based on original beer) Add slowly while stirring 4-5% vol. acetone Stir 30 minutes Let stand 10 30 minutes

Filtrate Filter with suction (discard) Residue Stir with small-volume acetone

Filtrate Filter with suction (discard) Residue Stir with small-volume acetone

Filtrate Filter with suction (discard) Residue Dry overnight B.t. in developing countries

Table (10) Recovery process for spore-crystal complex of Bacillus thuringiensis : Flow sheet for pilot plant and production-scale recovery.

Spray Drying

Whole beer pH 8.4 - 9.0

Centrifuge (tubular bowl continuous flow centrifuge)

Residue (weigh cream)

Prepare slurry based on weight of cream Add 100/'o Lactose 1% wetting agent 1% dispersant

Mix thoroughly

Adjust total solids to approximately 20%

Spray dry (centrifugal atomization feed)

Inlet temperature : 125 - 1500C

Outlet temperature : 75 - 1000C

Formulation

- 37 - H. T. Dulmage

IV. EXTRAPOLATION FROM LABORATORY TO FIELD TRIALS Bacterial and viral insect pathogens all act as stomach poisons. They must be eaten and digested to be effective. This must be remembered in developing practical programs for the use of these pathogens. Not every insect is readily susceptible to an insect pathogen. The habits of the insect may be such that contact between the insect and the insect pathogens may be difficult to achieve. For example, when a formulation of HD-1 in the laboratory is compared with a formulation of Pectinophtora gossypiella or T. ni, it is much more active against P. gossypiella then T. ni. In the field, the reverse occurs and the formulation of HD-1 does not control the P. gossypiella but does control the T. ni on cotton. The reason may be simple : The larvae of T. ni spend their hours feeding on the surface of the crop's leaves, an area effectively covered with HD-1 by our application system. On the other hand, the eggs of P. gossypiella are laid close to the newly developing terminals and squares of the cotton plant. The newly hatched larvae immediately then bore into these terminals and squares, thereby escaping with little more than a brief feeding on the B. t.

Ideally, the target insect used in a laboratory screening program should be the same as the insect that one wishes to control in the field. Sometimes this is impossible because the laboratory supply of target insects available is limited either by the habits of the insect or by the capabilities of the laboratory. In such a case, it may be necessary to screen against a substitute insect. This is risky, however, because the spectra of activities of the different pathogens or formulations can differ so much, as was discussed earlier in this talk and in the chapter by Dulmage and Cooperators. Members of the same genus do not necessarily respond to different delta-endotoxins alike. For example, the responses of insects of Spodoptera littoralis and Spodoptera exigua to the different B. t. 's do not correspond. Likewise, the responses of H. virescens and Heliothis zea differ, both in the laboratory and in the field. Thus, if a substitute insect must be used, the program should be monitored as closely as possible by tests against the true target (Dulmage 1981).

V. DESIGNING A SCREENING PROCEDURE

The term, "Screening Program", probably derives from the process used by builders in separating sand into fine, medium, and coarse grades. The builder

- 38 - B.t. in developing countries cannot sieve sand to separate all grades at the same time. He must run consecutive sievings to accomplish the desired separations. So is it in the insect pathogen screening program. We must design a stepwise program that will select and verify the active cultures that we are seeking. The goal is to devise a program that will detect the few active isolates out of a large number of isolates being screened.

To begin with, we must define our criteria. In a screening program like ours, where the cultures will be judged by their ability to kill a target insect, the most convenient criterion is percent kill. It should be remembered that the test we are running in this type screen is a biological test which is inherently quite variable. There is no rigid rule that will allow us to state what percent kill indicates activity. The decision must be an arbitrary one, based on the experience of the research worker. A reasonable tool for judging a selected criterion is the "cut-off" percentage (i.e., the percentage kill that will be accepted as indicating possible activity) is that no more than 15 - 20% of the cultures tested will pass this chosen criterion. This will detect most active cultures, but will allow some inactive isolates to be selected. The screening procedure should be kept as simple as possible, with all B. t.s used in the screen grown in 100 ml of media. Isolates, that pass this first screening, are reisolated and retested under the same conditions as the primary sort. The isolates that pass the secondary screening are retested and plated on nutrient agar plates and 5 to 10 colonies of B. t. are isolated as stock culture. The direction of further studies and procedures will vary depending on the first two screenings.

SUMMARY

Progress has been made in developing better isolates of B. t. for control of T. ni and H. virescens. The initial HD-1 formulations were about 100 times as potent as were the commercial products of its day. Since then, yields of the B. t. toxins have been increased a 100-fold in fermenters. Unfortunately, the massive increase in yields reflects how weak the original formulations were. So even with the increase, these new formulations were not potent enough or economical enough to be used widely in the control of forest insect pests. The addition of support from formulation chemists and application engineers has resulted in products and procedures that have made the HD-1 endotoxin effectively economically in the forest.

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Formulations of insect pathogens can be improved markedly. It takes time, but, with the proper effort, (and it is worth repeating) formulations of insect pathogens can be improved. This is an important concept, but one that is frequently forgotten. While it would seem obvious to me that we are only in the early stages of knowledge about these entomopathogens or their toxin that we can make marked improvements in activity, yield, and formulation and application techniques, there is a strong tendency by many scientists and by the administrators who prepare our budgets to believe that what we have now is the best that can be found. Too many of our evaluations of entomopathogens are restricted to judgements based on present knowledge and do not allow for possible improvements in strains or techniques. There are better isolates to be found, if we search for them. There are better fermentation and recovery procedures, if we pursue a logical course in fermentation research. There are better ways of using and applying these pathogens and toxins, if we seek and develope close cooperation between entomologists, microbiologists, chemists, and engineers to find them.

REFERENCES Barjac, H., de and Larget-Thiery, I. (1979). Proposal for the adoption of a standardized bioassay method for the evaluation of insecticidal formulations derived from serotype H-14 of Bacillus thuringiensis. WHO/VBC/79., 744. Barjac, H., de and Larget-Thiery, I. (1984). Characteristics of IPS 82 as standard for biological assay of Bacillus thuringiensis H-14 preparation. WHO/VBC 84. 892, 10 pp. Bonnfoi, A. et de Barjac, H. (1963). Classification des souches du Bacillus thuringiensis par la determination de 1'antigene flagellaire. Entomophaga, 8, 223-229. Dulmage, H.T. (1973a). Assay and standardization of microbial insecticides. Ann. N.Y. Acad. Sci. 217, 187 - 199. Dulmage, H.T. (1973b). Bacillus thuringiensis. U.S. assay standard. Report on the adoption of a primary U.S. reference standard for assay of formulations containing the delta-endotoxin of Bacillus thuringiensis. Bull. Entomol. Soc. Am. 19. (4), 200 - 202.

Dulmage, H.T. ; Martinez, A. J. and Pena, T. (1976). Bioassay of Bacillus thuringiensis (Berliner) delta-endotoxin using the tobacco budworm. U.S. Dept. Agric. Tech. Bull. 1528, 16 pp.

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Dulmage, H.T. (1979). In "Genetics in Relations to Insect Management" M.A. Hoy and T.I. McKelvey. eds. pp. 116 - 127. Working papers. The Rockefellows Foundation 1979. Dulmage, H. T. and Cooperators, (1981). Insecticidal activity of isolates of Bacillus thuringiensis and their potential for pest control. In Microbial Control of Pests and Plant Diseases, 1970 - 1980, ed. H. D. Burges, 191 - 220. London Academic Press. Dulmage, H. T. ; McLaughlin, R. E. ; Lacey, L. A. ; Couch, T. L. ; Ails, R. T. and Rose, R. I. (1985). A proposed U.S. standard bioassay for the potency assessment of Bacillus thuringiensis subsp. israelensis H-14. Bull. Entomol. Soc. Am., 31, 31 - 34. Dulmage, H. T. ; Correa, J. A. and Morales, G. M. (1989). Problems and progress in the development of microbial insect control agents, Israel. Journal of Entomology. Dulmage, H.T. (1989). Production and use of Bacillus thuringiensis. Perspective from 1989. Mem. Inst. Oswaldo Cruz, Rio de Janeiro, Vol. 84, Supl. 113 - 122. Dulmage, H.T. ; Gallegos-Morales, G. and Correa, J. A. (1990). Potential for improved formulations of Bacillus thuringiensis subsp israelensis through standardization and fermentation development. In : Bacterial Control of of Mosquitoes and Blackflies : Biochemistry, Genetics, and Application Bacillus thuringiensis subsp israelensis and Bacillus sphaericus. H. de Barjac and D. Sutherland, eds. 110 - 133. Foda, M.S. ; Salama, H. S. ; and Salem, M. (1985). Factors affecting the growth physiology of Bacillus thuringiensis. Appl. Microbiol. Biotechnol. Bioeng. 22, 50- 52. Goldberg, L. J. and Margalit, J. (1977). A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaenia unguiculata, Culex univitattus, Aedes aegypti and Culex pipiens. Mosq. News, 37 (3), 355 - 358. Heimpel, A. (1967). A taxonomic key proposed for the species of crystalliferous bacteria. J. Invertebr. Pathol, 9, 364 - 375. Krieg, A. (1983). Insect pest control in plant protection by Bacillus thuringiensis preparations and their influence on the environment. II., Bekampfung Von Anz. Schadlingskd. Pflanzenschutz. Umweltschutz V. 56 (3), 41 - 52. Krywienczyk, J. and Angus, T. A. (1967). A serological comparison of several crystalliferous insect pathogens. J. Invertebr. Pathol., 9, 126 - 128. Krywienczyk, J. ; Dulmage, H. T. and Fast, P.C. (1979). Occurrence of two serologically distinct groups within Bacillus thuringiensis serotype 3ab var. kurstaki. J. Invertebr. Pathol., 31, 372 - 375.

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Padua, L.E. ; Ohba, M. and Aizawa, K. (1984). Isolation of Bacillus thuringiensis strain serotype 8a 8b highly and selectively toxic against larvae. J. Invertebr. Pathol., 44 (1), 12 - 17. Simple Techniques for the Application of Bacillus thuringiensis in Field Crops Suitable for Developing Countries

K. A. Jones Natural Resources Institute, Central Avenue, Chatham Maritime, Chatham, Kent, ME4 4TB, UK.

ABSTRACT

Recent years have seen rapid developments in the use of Bacillus thuringiensis (B. t.) for insect pest control, notably the isolation and selection of strains that are effective against a broader range on insect pests and improvements in production technology. This, and concerns about the use of chemical insecticides, has led to an increase in the commercial use of B. t. for pest control. With the development of genetically manipulated B. t. strains and products resulting in more effective control, the future of B. t. as an insecticide seems assured. Economic control with B. t. still requires an effective means of delivery of the product to the target insects. With the exception of developments with transgenic plants, this is an area that has, in comparison to strain development, been neglected. B. t. needs to be ingested by the host insect. This requires either delivery to the regions of a plant where the target insects are located or the target insect needs to be attracted to where the B. t. is delivered. The latter case is particularly important where the insect has cryptic habits e. g. bollworms. Application methods mainly rely on the use of spray technology, but the use of solid baits may also be used. This paper will discuss the various options for application of B. t. with particular reference to the use of technology appropriate for developing countries.

INTRODUCTION The potential of Bacillus thuringiensis (B.t.) for control of insect pests was really demonstrated by the pioneering work of Steinhaus in the early 1950's,

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although the pathogenicity of the bacterium to insects was known prior to this time. Indeed the bacterium was isolated as long ago as 1901 and the type species described by Berliner in 1911. However, Steinhaus was the first worker to demonstrate potential for commercial exploitation (Heimpel and Angus, 1963). Since this time interest in the use of B. t. has steadily increased and as long ago as 1971, B. t. was registered for use against 23 insects in the USA on some 20 agricultural crops (Falcon, 1971).

In recent years, there has been a massive upsurge in interest in the potential of B. t. This has been a result of several factors. This includes the growing awareness of problems with conventional insecticides, including toxic residues, especially in the environment ; the development of resistance and the decreasing opportunities for discovering new groups of chemical insecticides. Also in the last five to ten years, there have been great steps forward in the development of B. t. as a pesticide. These include the isolation and seclection of new strains that are effective to a wider range of insect pests e.g. B.t. tenebrionis for control of Coleoptera (Krieg et al. 1983) ; developments in production technology reducing costs ; and improved formulations to increase persistance at the target and control. And, of course, developments in genetic manipulation including insertion of toxin genes into transgenic plants (Anon, 1991) and other bacteria (Gelernter, 1990). Also, through conjugation, development of strains active against both Lepidoptera and Coleoptera (Federici, 1990).

At present B. t. accounts for perhaps 80-90% of the total world microbial insecticide market of about $ 100 million (Carlton, Gawron-Burke and Johnson, 1990). In developing countries, the potential of B. t. has not been ignored and many research groups have been looking at the potential for control, and the developments mentioned above can only increase the possible uses. However, with all new control technologies the B. t. product must still reach the insect for effective control. With the exception of transgenic plants, this means that the B.t. must be applied to the crop in some way. Generally, this is an area in the development of insect control with B.t. that has been ignored.

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APPLICATION REQUIREMENTS

B. t. works essentially as a stomach poison and therefore needs to be ingested by the target insect. Any application technique must therefore deliver the B. t. to the region of the crop where the insect feeds. This is often not an easy task. Take the Egyptian cotton leafworm, Spodoptera littoralis, as an example. S. littoralis is the main early season pest of cotton in Egypt and at present is controlled by the hand-collection of egg masses, supplemented by the use of chemical insecticides (Hosny, 1980). The insect is primarily a leaf-feeder (although the later instars will attack the fruiting bodies) and therefore presents a good target for control with microbial pesticides, including B. t. The targets for control are the first three instars as these cause less damage- cumulatively they account for less than 2% of the total damage caused by the larval stage (Goodyear, 1978) and they are more susceptible to microbial agents. These stages of the insect are mainly located toward the top of the plant on the undersurface of the leaves(Abul-Nasr, et al., 1966, Khalifa, et al., 1982) where they eat only on the underside ; it is here therefore that the spray must be applied ; spray landing elsewhere on the plant could mostly be regarded as wasted.

Similary, larvae of the diamondback moth, Plutella xylostella, a major pest of cruciferous plants world-wide, are also normally found on the undersurface of the leaves, the young instars borrowing into leaves whilst older instars primarily eat only the lower epidermis (Hill, 1983).

The cryptic nature of many insects present severe problems of application. In Central America, Heliothis spp. is a serious pest of maize where it feeds within the leaf whorl. Heliothis and Helicoverpa spp. are also major pests of cotton where, after hatching from the eggs, they move rapidly to feed within the fruiting bodies with only minor feeding on the leaves (Pearson, 1958). In this case good distribution of spray is required to enable the insect to encounter and ingest the B. t. before

it enters the buds or bolls ; once inside these, the insect can be regarded as inaccessible for control by application of B. t.

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OPTIONS FOR APPLICATION

By far, the most widely employed method of delivering pesticides to the target is through spray application. Within this field there has been a lot of research over several years, directed of course toward the application of chemical insecticides. B. t. can be regarded as a particulate stomach poison and thus much of the technology for application of conventional insecticides can be employed for B. t. Indeed it should be said that all pesticides require controlled and accurate applicalion to the target site of the insect. Stomach poisons presenting the further requirement that they must be applied to the feeding site of the insect.

I will limit my discussion to ground application. Aerial application can provide adequate coverage of tree crops and has been used world-wide for the application of insecticides to large areas of field crops. However, with field crops, aerial application often does not provide good underleaf cover or reach enclosed areas of the plant, one of the requirements for effective control with microbial pesticides. More importantly, aerial application is not an option available to the resource poor farmer in most developing countries. For this same reason the use of tractor mounted sprayers will also not be considered.

By far, the most widely used method of ground application is the use of knapsack sprayers. There are many different designs of these but the general principle is the same. The spray is delivered via a lance and nozzle to the crop. Application volumes are usually 100 litres/ha or greater. The lance can direct the spray onto the crop, but in many situations underleaf cover is poor. However, using these sprayers it is possible for an operator to direct the spray at fruiting bodies or in the leaf whorl of a maize plant.

Other relatively high volume sprayers include the motorised hose sprayer used in Egypt. Typically this delivers 400-600 liters/ha. At these volumes plants are sprayed to run off and much of the active ingredient drips off to the soil. Unless the spray is carefully directed only a small percentage will reach the undersurface of the leaves.

In the last twenty years there have been a number of developments in application technology to improve depostition of spray on the target ; to reduce

- 46 - Application of B.t. in Developing Countries. run-off and, with chemical insecticides, resulting environmental pollution ; and through reducing spray volumes, to make spray application physically easier for the operatlor. This has been achieved through the use of controlled droplet application (CDA). The use of spinning disc ultra-low volume sprayers has been recommended for insect control in many crops, including cotton (Mowlan, 1973). Undoubtedly, spray cover with these sprayers can be very good on the upper leaf surface, especially at the top of the plant. However, our experience has been that coverage on the lower leaf surface can be very poor, also penetration of the spray into some crops such as berseem (clover) is also poor. Improvements in the underleaf cover of spinning disc and other CDA sprayers have been achieved with the development of electrostatic spray technology- notably the Electrodyn by ICI (Coffee, 1979) and the APE 80 experimental sprayer by Rothamsted Experimental Station in the UK (Arnold and Pye 1981). These undoubtedly give improved unerleaf cover (Mathews, 1982, Jones, 1990), but the Electrodyn is not widely used in developing countries, and APE 80 is not even commercially available. Farmers that already have a knapsack sprayer will continue to use that sprayer, especially as they know than these sprayers are adaptable and robust.

Thus, we must look toward improving the coverage of the more conventional sprayers. In Egypt we have compared knapsack sprayers, both lever-operated and motorised, with fan-assisted spinning disc sprayers. Trials on cotton have demonstrated that all of these can be adapted to give good underleaf cover (Topper, 1984, Jones, 1990). Thus, the mounting of an fan-assisted sprayer on a single wheeled bogie was required to provide adequate underleaf cover. The motorised knapsack sprayers needed to be held low in the crop and the lance of the knapsack sprayer needed to be adapted to give upward pointing nozzles that could be held low in the canopy. This last option was by far the most suitable, primarily because knapsac sprayers are readily available and can be used for application on several different crops and, as mentioned above, are robust. In cotton, the addition of a cotton tailboom - these having been developed in the 1960's in Central Africa-improved overall coverage even further (Topper, 1984). Trials in Egypt by the Plant Protection Research Institute, in collaboration with NRI, during 1990 have also shown that nozzles with smaller orifices can result in improved underleaf cover, as well as reduced spray volumes. However, a compromise has to be reached

- 47 - K. A. Jones here as nozzles with very small orifices are easily blocked by particulate matter prfsent in water used for spraying.

The choice of a sprayer will depend on crop type as well as availability. In Thailand, the cotton variety grown is taller than that grown in Egypt (up to 2m compared to about 1.5m). This means that good coverage of leaves (upper and lower surface) and fruiting bodies at the top of the canopy is much easier - this being where the main insect target H. armigera lays its eggs - and is possible without the need to employ upward facing nozzles and the widely used motorised knapsack sprayer was shown to give very good results in trials carried out by the Department of Agriculture, in collaboration with NRI, during 1991.

In Malaysia, control of P. xylostella on vegetables requires application to the undersurface of leaves to a crop that is only 0.5 m high. Here, knapsack lances with one or more upward directed nozzles are manufactured locally to satisfy this requirement (Mamat pers. comm.).

It is impossible to consider fully spray application without some consideration of formulation which influences spray distribution, droplet size and spray retention. With particulate material, it will also influence the distribution of the particles- in this case the B. t. - within the drop. With microbial agents, feeding stimulants are often included in the spray such as the commercially available COAX which is based on cotton seed flour (Young and Yearian 1986). These can encourage feeding by the target insect, for example a neonate Heliothis larva may be encouraged to eat before it reaches the cotton boll. Other feeding stimulants have also been identified, and these can be based on simple, locally available products. For example, grassmeal was demonstrated in the field to act as a feeding stimulant for S. littoralis (Jones, 1990).

In some instances alternatives to spray application may be considered. Thus, in berseem, a crop that is difficult to get good spray cover and penetration, the use of a solid bait could be considered. We have tested a grassmeal-based bait in Lucerne fields in Crete and demonstrated that late instar S. littoralis larvae are attracted to it (Campion et a1.,1980).

To control Heliothis in maize whorls, an effective option that could be considered is to mix a dry preparation of B. t. with an inert powder carrier, such

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where as clay. Scoops of this powder can then be dropped into a maize whorl it falls to the region where the insect is feeding.

Finally, it should also be said that a thorough understanding of the target pest's behaviour on the crop will also give an insight into which pests can be effectively controlled with B.t. For example, the pink bollworm, Pectinophora the gossypiella, lays it's eggs on or near cotton buds and squares and on hatching feeding insect immediately moves into the fruiting body with little or no external (Pearson, 1958). This presents and extremely difficult target for control by B.t. and therefore it is preferable to employ other techniques of control which avoid the spray encounter/ingestion problem, such as synthetic pheromones (Campion microbial and Jones 1991). I think this last point is extremely important, too often agents have been directed toward inappropriate targets with resulting poor control. This result will only delay the wider scale use of B. t. and should therefore be avoided.

CONCLUSION

To summarise, knowledge of insect behaviour is required to determine the or requirements of application. Application equipment must then be chosen the adapted in accordance with the crop and the behaviour of the pest to which insect application is made. It is then possible to apply the spray to areas where the is located. This is the key to successful control and may also have additional benefits. For example, B. t. on the undersurface of a leaf will persist considerably to longer than on the upper surface of the leaves. This approach is not exclusive chemical B. t. and many years research has been done on the application of pesticides. This knowledge should be called upon and, if appropriate for developing countries, utilised.

REFERENCES

Ecological studies of Abul-Nasr, S. ; Moussa, M.A. and Naguib, M.A. (1966). the cotton leafworm, Prodenia litura (Fab). I- The egg masses in cotton fields. Bull. Ent. Soc. Egypt, Econ. Ser., 57, 353-360. and Anon. (1991). Gene-engineered cotton in field tests, Ag. Biotech. News Information, 3 (1), 7-8.

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Arnold, A.J. and Pye, B.J. (1981). Electrostatic spraying of crops with the APE80. Proceedings 1981 British Crop Protection Conference, Pests and Diseases, 661-666. Campion, D.G.; Ellis, P.E.; Hunter-Jones, P.; Mckinley, D. J.; McVeigh, L.J.; Murlis, J.; Paton, E.M.; Brimacombe, L.; Bettany, B.W.; Cavanagh, G.G. and Jordon, J. (1980). Pheromones and viruses in the control of the Egyptian cotton leafworm, Spodoptera littoralis (Boisd.) and trials with pheromones of the olive moth, Prays oleae (Bern,) in Crete, report for 1977. COPR unpublished report Natural Resources Institute, Chatham, Kent, UK. Campion, D.G. and Jones, K.A. (1991). Pheromones and microbial insecticides for the control of cotton pests. Proceedings, 501h Plenary Meeting of the International Cotton Advisory Committee, Antalya, Turkey, September 1991. Carlton, B. C.; Gawron-Burke, C. and Johnson, T. B. (1990). Exploiting the genetic diversity of Bacillus thuringiensis for the creation of new bioinsecticides. Proceedings Vth International Colloquium on Invertebrate Pathology and Microbial Control, Adelaide, Australia 20-24 August 1990, 18-22. Coffee, R.A. (1979). Electrodynamic energy - a new approach to pesticide application. Proceedings 1979 British Crop Protection Conference, Pests and Diseases, 777-789. Falcon, L.A. (1971). Use of bacteria for microbial control. In H. D. Burges & N. W. Hussey (eds) Microbial Control of Insects and Mites, 67-95. Academic Press, London. Federici, B.A. (1990). Bright horizons for invertebrate pathology. Proceedings Vth International Colloquium on Invertebrate Pathology and Microbial Control, Adelaide, Australia 20-24 August 1990, 5 - 9. Gelernter, W.D. (1990). MUPTM bioinsecticide : a bioengineered, bioencapsulated product for control of lepidopteran larvae. Proceedings Vth International lepidopteran Colloquium on Invertebrate Pathology and Microbial Control, Adelaide, Australia 20-24 August 1990, 14. Goodyear, R. (1978). Identification of armyworm, cutworm, budworm and looper caterpillar pests. A. G. Bulletin 2, Department of Agriculture, New South Wales, Australia. Heimplel, A.M. and Angus, T.A. (1963). Diseases caused by certain sporeforming bacteria. In E. A. Steinhaus (ed.) Insect Pathology : An Advanced Treatise. 2, 21-73 Academic Press, London. Hill, D.S. (1983). Agricultural Insect Pests of the Tropics and their control, 2nd edition. Cambridge University Press. Hosny, M.M. (1980). The control of cotton pests in Egypt. Outlook in Agriculture 10, 204-205.

- 50 - Application of B.t. in Developing Countries.

Jones, K.A. (1990). Control of Spodoptera littoralis in Crete and Egypt with NPV. In J. E. Casida (ed) Pesticides and Alternatives : Innovative Chemical and Biological Approaches to Pest Control, 131-142. Elsevier, Amsterdam. Khalifa, A.; Iss-Hak, R. R. and Foda, M. E. (1982). Vertical and horizontal distribution of the Egyptian cotton leafworm masses in cotton fields in Egypt. Research Bulletin No. 1749, Faculty of Agriculture, Ain Shams University, Cairo, Egypt. Krieg, A.; Huger, A. M.; Langenbruch, G. A. and Schnetter, W. (1983). Bacillus thuringiensis var. tenebrionis, a new pathotype effective against larvae of Coleoptera. J. Appl. Entom., 96, 500-508. Mathews, G.A. (1982). Prospects of better deposition of microbial pesticides using electrostatic sprayers. Proceedings IIlyd International Colloquium on Invertebrate Pathology, Brighton UK, September, 1982, 55-59. Mowlan. M.D. (1973). Spraying cotton with ULV hand machines SPAN, 16 127-128. Pearson, E.O. (1958). The insect pests of cotton in tropical Africa. CAB, London. Topper, C.P. (1984). Report on the research and development of nuclear polyhedrosis virus of Spodoptera littoralis (Boisd.), 1979-1981, 2, ODA, London. Young IIi, S.Y. and Yearian, W.C. (1986). Formulation and application of baculoviruses. In R. R. Granados and B. A. Federici (eds.) The Biology of Baculoviruses vol. II, Practical Application for Insect Control., 157-179.

Use of Bacillus thuringiensis in Italy Current Status

E. Pasqualini Institute of Entomology "Guido Grandi" University of Bologna Via Filippo Re, 6-40126-Bologna, Italy

ABSTRACT

Two Bacillus thuringiensis (B. t.) based preparations are currently registerd in Italy. These include B. t. subs. kurstaki (B. t. k.) and subs. israelensis (B. t. i. ), and have been available in Italy since early 1987 They are used for the control of several species of Lepidoptera and Diptera, respectively. B. t. k. is employed against several species of leaf-rollers which infest especially apple and pear, as well as other lepidoptera which attack vineyard, cabbage, radish and maize. B. t. k. has been classified as having low toxicity ; no indications as to maximum permissible toxic residues in food is given for this product, which features a decay period of three days. Current consumption of B. t. k. is 35 to 40 tonnes, as follows : 35-40% on fruit trees, 55-60% on vineyards and 5-8% on several grass crops. The amount of the product, sold at an average price of 30,000 Italian lire (about 23 US $) per kilogram to the farmers, are estimated to be around 800,000 US $, accounting for approximately 0.1 % of the total Italian pesticide market. B. t. i. on the other is employed for controlling several species of mosquitoes. Rather than for plant protection and disease control, the product is employed to reduce insect nuisance, especially to farmers and tourists. It is also used against several species of livestock parasites, such as Simulidae. Estimated overall consumption of B. t. i. in Italy is around 20,000 kgs., employed almost exculusively for projects funded by control and local government. B. t. subs. tenebrionis has not yet been registered in Italy, but trials have been carried out with fully satisfactory results, especially against Colorado potato beetle.

The use of Bacillus thuringiensis (B. t.) has been permitted in Italy since early 1987. Two B. t. - based preparations are currently registered, namely B. t. subs. kurstaki (B. t. k.) and B. t. subs. israelensis (B. t. i. ), which are respectively used against

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several species of Lepidoptera and Diptera. Both before and after its registration, several trials have been carried out, and additional tests are currently still being conducted on B.t.k.. In particular, field trials have been performed in order to asses the preparation's efficacy as compared to that of traditional products, such as azinphos-methyl and quinalphos. A number of trials have also been carried out on vegetable and fruit tree crops in order to determine optimum dosage as well as optimum timing. These studies have essentially been carried out by University institutes and other research centers as part of general programmes aimed at developing integrated or biological control strategies to minimize the use of chemical products.

-B. t. k. is currently registered as a product for the control of several species of harmful Lepidoptera known to infest apple and pear orchards, as well as vineyards, cabbage, radish, maize and forestry (Table 1). It is also recommended for the protection of other crops such as strawberry, pepper, tomato, etc., although official registration for use on these crops are still pending. In recent years B. t. k. has been extensively used for controlling infestation caused by Hyfantria cunea larvae (Lep., Arctiidae), which attack a wide number of fruit crops and ornamental plants. Many researches are carried out to verify the efficacy against some species of Lepidoptera on kiwi, cork trees, etc.

Current consumption of B.t.k. in Italy (Data of 1991 from chemical companies) is around 35 to 40 tonnes employed as follows : 35 to 4007o on fruits orchards ; 55 to 60016 on vineyards and 5 to 8016 on vegetable crops. (Table 2). The possible amount of the product used on forestry is not known. but is presumed to be very low. Most of B. t. k. consumption, around 80%, occurs in the northern regions of the country and Sardinia.

Several B.t.k. preparations are currently available in Italy, including Thuricide HP, Bactospeine, Bactucide, Dipel, Biobit, Forey, etc. Overall sales of the product, sold at an average price of 23 US $ per kilogram to the farmers, are estimated to be about 800,000 US $, accounting for approximately 0.1 % of the total Italian pesticide market.

- 54 - Use of B.t. in Italy

Table (1) Crops in which B. t. k. is registered to use, main target harmful species and a.i. doses (16,000 U.I.) g/hl.

Crops Main Harmfull Species g/h

Cabbage Pieris brassicae 30-75 Radish Pieris rapae 30-75 Maize Ostrinia nubilalis 100-150

Apple Pandemis cerasana 100-150 and Pandemis heparana 100-150 Pear Archips podanus 100-150 Argyrotaenia pulchellana 100-150 Adoxophyes reticulana 100-150

Vineyard Lobesia botrana 100-150 Eupoecilia ambiguella 100-150

Forestry Thaumetopoea pytiocampa 50-100 Thaumetopoea processionaria 50-100 Lymantria dispar 50-100

Table (2) Some data on B.t.k. use in Italy (1991).

Fruit orchards 35-40%

Total consumption Vineyards 55-60% 35-40 ton. Other crops 5-10%

- 55 - E. Pasqualini

The B. t. k. has been classified amongst pesticides with the lowest degree of toxicity, so that no maximum permissible toxic residue level in food are given. The products features a decay period, i.e. the period of time between the last treatment and harvest, is three days. The product is selective for the beneficial arthropods and no cases of resistance have been reported to-date in our country (Table 3).

Table (3) Main peculiarities of B.t.k. registration in Italy.

1- Lowest class of toxicity

B. t. k. in law 2- No maximum permissive toxic residue level in food is given

3- Decay period : 3 days

The main reasons for its actually limited use should be : the scarce efficacy at low temperatures, the irregularity of the results, the easy removal by rain, the need to add, in some cases, the sugar to improve the performance, the difficulty to determine the timing, the brief period of action (about one week). The only one reason, however, which prevails over all others in limiting its use is probably the cost factor, which is currently still too high as compared to that of traditional chemical insecticides.

B. t. i. - based preparations are used for controlling insects for different reasons. With regard to mosquitoes, it is employed for the purpose of reducing insects nuisance to farmers and tourists, while it is also used for protecting livestock, especially against Simulidae. Several B. t. i. - based products are currently registered including Vectobac, Teknar, Bactis, Turbac, Bactimos, etc. Overall consumption of preparations with 1500 U.T.I. is estimated to be around 20.000 kg (data 1991). The use of these products is almost exclusively funded by Government agencies and is practically restricted to north-eastern Italy and Sardinia.

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B. t. - based product field trials are particulary widespread and intense. The trials are aimed to optimize the use of already registered products for the purpose to improve their performance. They include, for example, the better timing of treatment, the doses as well as the enhancement of product efficacy and period of action. Trials are also currently carried out on new preparations with a longer persistance. Other researches are carried out to verify the activity of other B. t. - based preparations, like B. t. subs. tenebrionis to control Coleoptera.

In conclusion, it can safely be said that B. t. based product represent an effective solution to control several harmful insects. A more widespread use of these products is to be expected because the good results so far obtained, the possiblity to improve the efficacy, the ever greater need for pesticides with lower impact on environment and the high selectivity. However, the increase in the use depends on reduction of their cost to the farmers.

Potential of Bacillus thuringiensis in Integrated Pest Management for Developing Countries

S. Barbosa Integrated Pest Management Specialist, Food and Agriculture Organization of the United Nations, Via dell Terme di Caracalla, 00100 Rome, Italy.

ABSTRACT

For several decades since its discovery, formulations of Bacillus thuringiensis Berliner (B. t.) have been seen as the ideal means of controlling lepidoteran pests in agriculture because of the many attributes that differentiate this microbial insecticide from the synthetic chemical formulations. No toxicity to mammals, environmental friendliness, apparent immunity to the pesticide resistance phenomenon (no longer true), good integration with other pest control methods and the possibility of being mass produced at farm level at low cost, all made B. t. the much-needed tool for IPM programmes in developing countries. Unfortunately, however, apart from a few very good examples of its use in forestry and crop protection, the worldwide utilization of B. t. did not come up to expectations. Probably the most important factors that prevented B. t. from being more widely accepted were : high cost, inconsistency of results in different environmental conditions and, ironically, too much specificity. Presently, the possibility of using B. t. and other pathogens for insect pest control has gained a new momentum as a result of growing environmental and health scrutiny to which

synthetic chemicals are subject and the many prospects promised by . genetic engineering and bio-technology. However, to become widely accepted as a tool in IPM programmes, B. t. will have to be competitive with other means :,f controlling the same group of pests in terms of effectiveness, cost and availability of the right formulations where they are most needed. INTRODUCTION

Agriculture has changed quite markedly over the last forty years as a consequence of the use of synthetic chemicals to control pests on different crops,

- 59 - S. Barbosa triggered by the discovery of DDT in 1939, which insecticidal activities were only discovered a few years later. From 1943 to 1985, the world consumption of pesticides jumped from less than 30 thousand metric tons to 3 million metric tons in 1985 (Prokopy, 1988).

We should not deny that chemical control has made a significant contribution to the rapid and constant increase of agricultural production around the world but we must realize that it also contributed to a very rapid decrease of the plant health situation (Brader, 1991).

As a consequence of the over-dependence on chemical control, modern agriculture became extremely vulnerable with a host of undesirable side effects to human health and the environment. An estimated 25 million agricultural workers in developing countries are poisoned every year by pesticides (Jeyaratnam, 1990). On the other hand, the effect of pesticides on the environment, although with well documented cases, is still lacking a global assessment. Among the pesticides, insecticides stand out as the most important group when human health and the environment are considered. From the so-called "dirty dozen" list of pesticides (Pan, 1991), ten are insecticides.

Two other very important side effects of the use of pesticides are also more predominant within the insecticides. These are secondary pest outbreaks/pest resurgence, due to the elimination of naturally occurring parasites and predators, and the selection of pests that are resistant to pesticides. Georghiou and Lagunes (1991) reported that from the first detection of the resistance phenomenon in 1914, to 1989, some 504 species of arthropods have shown resistance to different groups of insecticides/acaricides, 283 of which are agricultural pests.

According to Wilcox et al. (1986), more than $3 billion are spent worldwide each year to control insect pests. Ironically, although the use of pesticides worldwide is always increasing, yield losses caused by pests have not decreased since World War 11. Even in developed countries, the plant health condition has deteriorated. In the U.S., for instance, the annual crop losses due to insects have increased from 7% in the 1940's to 13% in the 1980's (Holden, 1989).

Insect pests of the order Lepidoptera constitute the most important group of all the agricultural pests and most of them were pests created by the overuse

- 60 - B.t. in Integrated Pest Management of pesticides. The Lepidopterans also have the leadership of the number of species with insecticide resistance among the agricultural arthropodal pests with 26076 of the total (Georghiou and Lagunes, 1991). Fortunately enough, Bacillus thuringiensis exists and has shown from its discovery a marked efficiency against this group of agricultural pests. Although the recent discovery of the resistance phenomenon (McGaughey, in three lepidopteran species : Indian mealmoth, Plodia interpunctella, the 1985), the almond moth, Cadra cautella (Mcgaughey and Beeman, 1988), and diamondback moth, Plutella xylostella (Tabashnik et al., 1990) may have brought in some disappointment to the promoters of B. t., it still remains the best option several IPM programmes where lepidopteran pests are important components, of the pest complex. As a matter of fact, sales of B. t. have doubled since 1987 and conservative estimates have put the 1995 market at between $300 and $500 million (Anon, 1991).

INTERGRATED PEST MANAGEMENT (IPM)

The FAO Panel of Experts on IPM (or IPC as its was originally called) defined integrated pest control as a pest management system that in the context of the associated environment and the population of the pest species utilizes all suitable techniques and methods in as compatible a manner as possible and manitains the pest populations at levels below those causing economic injury (FAO, 1967). Today, almost 25 years since FAO's official definition of IPM was adopted, the number of successful IPM programmes is still very reduced. Although much awareness has been created for the need to develop alternative non-chemical ways of controlling pests in agricultrue, what we see is an escalation of the use of pesticides in developing countries. Developed countries are enacting and reinforcing stringent legislation to drastically reduce the use of pesticides in their agriculture, mainly because of environmental concern. On the other hand, third world countries in the struggle to develop their agriculturally based and much indebted economies do not have the protection of the environment as their first priority and, thereby, use any pesticides they can get hold of. This situation has evolved in so many cases of disaster in agricultural developing programmes that adopted the green revolution model of high-input agriculture.

Fortunately enough there are a few good examples of successful IPM

- 61 - S. Barbosa

programmes whereby it was possible to reduce the overall amount of pesticides previously utilized and at the same time maintain and sustain high yields. Rice IPM in Asia and soybean IPM in South America stand as good examples to be emulated by other crops and regions where present agricultural production scheme are dependent on chemicals for pest control. These two programmes clearly demonstrated that IPM is decisively the best approach to guarantee crop health at low cost and with many dividends in terms of the sustainability of the farming activity, better health for the farming community and preservation of the environment.

For IPM to be practised by farmers, it needs to be made easy for them. Of course it needs to be based on sound ecological and technical principles but for almost any pest problem there is enough information to permit the starting of a solid IPM programme. It will evolve further as new information from research is validated at field level and becomes a new tool for the IPM practitioners.

Most successful IPM programmes were initiated as an escape from the so-called pesticide treadmill, when the chemical option had reached its limits with all the consequences already mentioned. Only with the partial or total removal of the pesticide pressure from the agroecosystem, natural enemies of insect pests were able to come back and somehow re-establish a balance that existed before pesticide overuse. Probably, the best example of this phenomenon comes from paddy agroecosystems in Asia, where the brown plant hopper, Nilaparvata lugens, became the most serious problem in rice intensification programmes due to the overuse of insecticides. It was demonstrated that spiders maintain brown plant hoppers under check and that the application of insecticides were worthless against the hoppers but were very efficient in killing the spiders (Kenmore, 1980). Ever since, all the outbreaks of this insect in Asia have been traced back to the use of a broad spectrum insecticide. So many other cases exist of insects that historically have no economic importance but that become key pests due to the elimination of their natural enemies.

It is now well known that successful IPM programmes invariably have three principles : in common 1) Grow a healthy crop ; 2) Conserve and enhance the beneficial organisms that naturally occur in the agroecosystems and 3) Use only selective pesticides when an action threshold has been reached.

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Of course, there are situations where insecticides are an important component of IPM programmes. This is the case of cotton growing where varieties are very susceptible to insects and where the whole system is so dependent on chemical pest control. In these circumstances, supervised chemical control based on thresholds may become an important step forward in the development of an IPM programme. Simple general recommendations like destruction of plant residues, establishment of uniform dates of planting, delaying as much as possible the first insecticide application and use of selective insecticides on a need-basis may eventually constitute the backbone of a long-lasting IPM programme if rightly and thoroughly implemented.

CONSTRAINTS TO IPM IMPLEMENTATION

There is much speculation on the reasons why there are not more successful IPM programmes in developing countries considering the economic, social and environmental benefits this approach promised when it was defined one-quarter of a century ago. These reasons could be grouped into three categories economical, social and institutional. made available to farmers at low cost, 1 - Economic reasons : Pesticides are thereby farmers do not save money by sparing on the number of pesticide applications. There are so many forms of direct and indirect subsidies to pesticides, pesticide donations and other mechanisms that eliminate any incentive to adopt IPM. To make things even worse, pesticides are promoted most strongly by government and private enterprises whereby everyone makes a profit. On the other hand, the promotion of IPM is left with individuals of good will but without resources for the implementation of programmes. The farmers do not even hear about IPM but are flooded with massive propaganda on pesticides. 2 - Social reasons : Farmers in developing countries are not organized and do not constitute a political power. They are not used to demand government actions that would bring progress to rural areas and to a betterment of the living conditions. Government officers responsible for agricultural services, extension and research are underpaid and in a constant situtation of low morale, lacking the minimum condition to bring about any change in agricultural technology to

- 63 - S. Barbosa their clientele. By contrast, pesticide salesmen are well paid and have all the incentives to sell more and more pesticides.

3 - Institutional reasons : Agricultural universities in developing countries lack the basic conditions for the preparation of future professionals. Agricultural research and extension are under-staffed and under-funded to carry out their formidable task of changing a subsistance agriculture, into a market agriculture. There are conflicts of interest when a tochnology like IPM, that would certainly reduce pesticide sales, is to be implemented. At policy level, there is no a favourable environment for the development of IPM programmes.

In spite of all these constraints, the side effects of the over use of pesticides on human health and the environment have created an awareness even in the most under-developed countries for the need to develop integrated pest management programmes. As mentioned before, there is so much information on alternative ways of controlling pests in agriculture that at any point in time we could initiate IPM programmes for different crop commodities in different countries. What is necessary is to have farmers as co-participants from the beginning and demonstrate- by-doing to them that IPM is more profitable than unilateral control and that it can be made very simple.

THE ROLE OF Bacillus thuringiensis IN IPM PROGRAMMES IN DEVELOPING COUNTRIES

It has already been mentioned in this paper that larvae of insects of the order Lepidoptera constitute the most important group of agricultural pests. They also have shown to be extremely vulnerable to the phenomena of resistance to insecticides, pest resurgence and secondary pest outbreaks.Table (1) shows a list of important lepidopteran pests to be examined as potential targets for B. t. within the framework of IPM systems. If we try to single out today the most important pests of agriculture worldwide, we most certainly come out with the Heliothis/Helicoverpa complex and the diamondback moth (DBM), Plutella xylostella. The first is truly a complex as it embodies two genera and some four very important species of polyphagous insects whose host range covers some of the most important crops : cotton, pulses, maize, etc. On the other hand, P. xylostella is an oligophagous species with hosts limited to some crucifers.

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(a) The HeliothislHelicoverpa complex : Georghiou and Lagunes (1991) reported 118 cases of insecticide resistance in five species of the world covering six groups of insecticides : DDT, cyclodienes, organophosphates, carbamates, synthetic pyrethroids and miscellaneous which represent 20% of all the reported cases within the order Lepiodoptera. Most of the recorded cases of resistance within the complex come from cotton where species of HeliothislHelicoverpa are commonly called bollworms. It is exactly from cotton growing too that most cases of pest resurgence and secondary pest outbreaks are reported, a direct consequence of pesticide overuse.

Throughout the world, the overuse of pesticides in cotton has made the bollowrms very important from two points of view. Firstly, by making key pests out of them when they were secondary pest. This is the case of cotton growing in the U.S. and Central America where the overuse of broad spectrum insecticides (toxaphene-DDT, parathion, etc.) early in the season to control the boll weevil, Anthonomus grandis, released the bollworm by killing its naturally occurring parasites and predators. (Adkission, 1972). The same situation happened in the Sudan in the late 1970's (Eveleens, 1983) and is happening today throughout Asia. Secondly, by creating key pests out of secondary pests after much insecticide is used to control the now key bollworm pests, just a recycling process. Well known today throughout cotton growing areas of the world are the very difficult to control whiteflies, Bemisia tabacci, which previously were not of much economic importance but became key pests after the over-use of broad spectrum insecticides including the short-lived synthetic pyrethroids.

Cotton is well known as the champion among crops when insecticide use is taken as a comparative criterion. In some developing countries, with their economies bases on cotton production, the situtation is even more serious. Pakistan, for instance, dedicates over 8001o of all pesticides used in their agriculture to cotton. A biological insecticide against the bollworm complex does not only seem to be the long-term solution for this pest but would also alleviate many other problems caused by the over use of broad spetrum insecticides.

Unfortunately, B. t. formulations have not proven efficient against this group of insect pests although in laboratory tests a high percentage of mortality occurs.

- 65 - S. Barbosa

For years now it is accepted that young larvae only spend a very short time feeding on plant terminals before they bury themselves into the fruiting bodies, consequently chances that they eat crystals and spores of sprayed preparations are very minimal. Unless we solve this problem through better formulations, establishing correct timing for B. t. applications or even developing a more virulent B. t. for this pest, chances of using B. t. effectively for cotton protection are not very good. Even after we have resolved all the above constraints and others, farmers will only use B.t. if it is proven to be more effective and at least as cheap as other insecticides.

(b) The diamondback moth, Plutella xylostella : Georghiou and Lagunes (1991) reported 110 cases of insecticide resistance in this species throughout the world covering all the six groups of insecticides as for the bollworm complex referred to earlier in this paper. Of much importance for the future of B. t. in the control of this pest were the findings of Tabashnik et al. (1990) who reported from Hawaii, for the first time, populations of DBM with high levels of resistance against commercial formulations of B.t. kurstaki. With this finding, it became clear that even biological pesticides have to be used intelligently and with moderation if we want to prevent/manage cases of resistance, thereby preserving much needed pest control agents.

DBM is a consmopolitan pest and the development of resistance against pesticides has forced growers to increase dosages and number of applications. This syndrome only exacerbates the development of further resistance and the occurrence of unacceptable levels of pesticide residues in cabbages, broccoli, cauliflower and other crucifers (Lim, 1990).

The use of B. t. formulations to control DBM larvae has produced good results in most places where it has been tried and still constitutes one of the good examples of the use of a microbial pesticide to control an agricultural pest. It controls the pest, does not off-set the natural enemies of the pest, does not pose any risk to field labourers, does not leave residues on product and causes no damage to the environment. Unfortunately, it is more expensive than most chemical insecticides and the recent report of resistance in the DBM may just pre-empt a movement to increase dosages and frequency of applications which will make it evey more expensive and will foster the development of resistance.

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B. t. efficacy, specificity and human and environmental safety associated with its application for plant protection are so extraordinary that if methods for its wise depleyment are not devised promptly, we will risk losing it because of resistance and economic reasons. There are very good prospects for the develpment of IPM programmes for crucifers with DBM as the key pest. The enhancement of natural enemies of the pest and curative treatments with B.t. seems to be the best combination of methods to solve this worldwide problem. Again, in this use, B. t. has advantages over chemical insecticides as it integrates well with the use of parasitoids in IPM programmes.

(c) Other lepidopteran pests. As already mentioned in this paper, there are many other species of the Lepidoptera that are potential candidates for B. t. In China, for instance, local formulations of B. t. are regularly used against the leaf folder, Cnaphalocris medinalis, when the field population levels reach the action threshold. During the first crop of 1991, in the Jinan township of the Hubei Province, only 5007o of the fields reached the action threshold for the leaf folder and B. t. was the only insecticide used (Prof. Zeng Zhaohui, personal communication). In Brazil, commercial formulations of B. t. have shown outstanding results against the cotton leafworm, Alabama argillacea, but the need to spray other insecticides against pests for which B. t. was not recommended prevented the adoption of B. t. by farmers.

Table (1) shows most of the key pests of very important crops in developing countries but much R & D work is necessary before B. t. becomes a regular tool in plant protection for the majority of the conditions under which these crops are grown. S. Barbosa

Table (1) Lepidopteran Agricultural Pests of International Importance as Potential Targets for B. t.

Number of Reported Common Name Scientific Name Cases of Insecticide Resistance *

CABBAGE (and other Crucifers)

Diamondback moth Plutella xylostella 110 Imported cabbageworm Pieris rapae 16 Cabbage looper Trichoplusia ni 46 Lesser armyworm Spodoptera exigua 14 Cabbage webworm Hellual undalis Latin American cabbageworm Ascia monuste orseis Cabbage moth Crocidolomia binotalis COTTON American bollworm Helicoverpa armigera 0 Cotton bollworm Helicoverpa zea 35 Tobacco budworm Heliothis virescens 52 Spotted bollworm Earias vittella Spiny bollworm Earias insulana 4 Pink bollworm Pectinophora gossypiella 4 Red bollworm Diparopsis castanea Cotton leafworm Alabama argillacea 9 Cotton leafworm Spodoptera littoralis 33 Lesser armyworm S. exigua 14 Black armyworm S. frugiperda 14 Fall armyworm S. litura 9 Black cutworm Agrotis ipsilon 6 Common cutworm A. segetum Cotton leaf perforator Bucculatrix thurberiella 20

FRUITS Codling moth (apples) Cydia pomonella 39 Grape moth Lobesia botrana 1 Oriental fruit moth Grapholita molesta 3

GRAIN LEGUMES Velvetbean caterpillar Anticarsia gemmatalis Soybean looper Pseudoplusia includens 6 American bollworm Helicoverpa armigera 30 Bean pod borer (Mung moth) Maruca testulalis

* From Georghiou and Lagunes, 1991 - 68 - B.t. in Integrated Pest Management

Table (1) Continued.

Number of Reported Common Name Scientific Name Cases of Insecticide Resistance *

JUTE AND KENAF Jute semilooper Anomis sabulifera Jute hairy caterpillar Diacrisia obliqua indigo caterpillar Spodoptera exigua

MAIZE African armyworm Spodotera exempta Black armyworm S. frugiperda 14 European cornborer Ostrinia nubilalis Asiatic cornborer 0. furnacalis 2 Spotted stalk borer Chilo partellus Maize stalk borer Busseola fusca Corn armyworm Helicoverpa zea 35 American bollworm H. armigera 30 Pink stalk borer Sesamia calamistis

RICE

Rice stem borer Chilo suppressalis 13 Rice leaf folder Cnaphalocrocis medinalis 1 small rice borer Scirpophaga gilviberbis White rice borer S. nivella Yellow rice borer Tryporiza incertulas

TOMATOES (and other Solanceae) Tomato fruitworm Helieoverpa zea 35 American bollworm H. armigera 30 Lesser armyworm Spodoptera exigua 14 Tomato pinworm Keiferia lyeopersicella ` Tomato leafminer Scrobipalpula absoluta 1 Brinjal borer Leueinodes orbonalis South American fruitworm Neoleucinodes elegantalis Potato tubermoth Phithorimaea operculella 11

* From Georghiou and Lagunes, 1991.

- 69 - S. Barbosa

CONCLUSIONS

Since World War II, we have seen an increase in the use of insecticides for crop protection and a decrease in the plant health status throughout the world with many undesirable consequences to human health, the environment and the sustainability of agriculture. We seem to have understood the limitations of chemical insecticides and we tried to develop alternative tools (microbial insecticides) and approaches (IPM) that would solve the problems brought by insecticides and would still sustain agricultural production. There are positive signs on the horizon but the battle is far from ending and the victory not yet very certain. IPM seems to be the way but it needs to be implemented at field level for various crops and for all the regions. Few examples are good but not enough to liberate modern agriculture from its dependence on the chemical control of pests.

B. t. is available now for several decades but only recently is it raising serious interest, mainly because of the limitations of chemical insecticides. Biotechology and genetic engineering advances may just be what was necessary to make B.t. broader in its effectiveness and more virulent to agricultural insect pests. The detection of cases of resistance against B.t. adds a word of caution to those who thought that by incorporating simple B. t. genes into plants would make them «resistant» forever to pest attack. It also shows that B. t. is more than just another tool for IPM as it has many attributes not possessed by chemical insecticides.

Unfortunately, B.t. was developed as other pesticides, having the profit of the manufacturer as the most important objective. Unless we do something to have B. t. formulations produced at village level, thereby making it available to farmers at low cost where the formulations are needed, I do not see a very bright future for presently available B. t. formulations in IPM programmes for developing countries where lepidopteran species are key pests.

REFERENCES

Adkisson, P.L. (1972). The integrated control of the insect pests of cotton. Proc. Tall Timbers Conf. on Ecological Animal Control by Habitat Management, Tall Timbers Res. Sta. (Tallahasse, FL) 4, 175-188. Anon. (1991). Pesticide News. 12 The Pesticides Trust & Pesticides News. London, p. 16.

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Brader, L. (1991). Global Trends and Constraints of Integrated Pest Management. Paper presented at the XII International Plant Protection Congress. Rio de Janeiro. Brazil. 114 August 1991. Eveleens, K.G. (1983). Cotton insect control in the Sudan Ghezira : analysis of a crisis. Crop Protection. 2(3), 273-287. FAO (1967). Report of the First Session of the FAO Panel of Experts on Integrated Pest Control. Rome. 19 pp. Georghiou, G.P. and Lagunes, A.T. (1991). The Occurrence of Resistance to Pesticides in Arthropods. FAO. AGPP/MISC/ 91-1. 318 pp. Holden, C. (1989). Research News : Entomologists Wane as Insect Wax. Science,

246, 754-757. - Jeyaratnam, J. (1990). Acute pesticide poisoning : A major problem. World Health Stat. Ort. 43, 139-144. Kenmore, P.E. (1980). Ecology and outbreaks of a tropical insect pest of the green revolution : The rice brown planthopper, Nilapavata lugens (Stal). Ph.D. Thesis, University of California, Berkeley (USA). Lim, G.S. (1990). Overview of vegetable IPM in Asia. FAO Plant Protection Bulletin 38 (2), 73-87. McGaughey, W.R. (1985). Insect resistance to the biological insecticide Bacillus thuringiensis. Science,229 , 193-195. McGaughey, W.R. and Beeman, R.W. (1988). Resistance to Bacillus thuringiensis in colonies of Indianmeal moth and Almond moth (Lepidoptera : Pyralidae). J. Econ. Entomol. 81, 28-33. Prokopy, R.J. (1988). Pest management in perspective. In Innovations in Pest Management. Sturbridge, Mass., March 7-8, p. 7-11. Tabashnik, B.E. ; Cushing, N.L. ; Finson N. and Johnson, M.V. (1990). Field development of resistance to Bacillus thuringiensis in the diamondback moth (Lepidoptera : Plutellidae). J. Econ. Entomol. 83, 1671-1676. Wilcox, D.R. ; Shivakumar, A.G. ; Melin, B.E. ; Miller, M.F. ; Benson, T.A. ; Shopp, C.W. ; Casuto, D. ; Gundling, G.J. ; Bolling, T.J. ; Spear B.B. and Fox, J.L. (1986). Genetic Engineering of Bioinsecticides. In M. Inouye & R. Sarma (eds.), Protein Engineering : Applications in Science, Medicine and Industry. Academic, Orlando, Fla, 395-413.

Development of a New ELISA Method to Measure the Concentration of Produced by Bacillus thuringiensis

A. Margaritis , J. Kosir and D. to Bokkel Department of Chemical and Biochemical Engineering, Faculty of Engineering Science, University of Western Ontario, London, Ontario, Canada, N6A 5B9

ABSTRACT

This paper describes the development of a new Enzyme Linked Immunosorbent Assay (EL/SA) method to meausre the concentration of the bioinsecticidal protein delta-endotoxin produced by Bacillus thuringiensis. (B. t.). First, purified polyclonal antibodies from rabbit antiserum were used to measure delta-endotoxin concentration during the course of B. t. HD-1, kurstaki fermentation. Ba1b1c mice were immunized with delta-endotoxin and used subsequently to develop a new hybridoma cell line which produces monoclonal antibodies (MAB) against delta-endotoxin. Results will be presented on the kinetics of hybridoma cell growth, MAB production and their use with the new ELISA method to meausre the concentration of delta-endotoxin in B. t. fermentation samples.

INTRODUCTION

During the course of early work on Bacillus thuringiensis (B. t.) fermentations in our laboratory we recognized the need to test for delta-endotoxin presence on a routine basis. Up till this time the standard method for determining delta-endotoxin presence in a B.t. fermentation has been a bioassay conducted on the final fermentation product. No measurements of delta-endotoxin concentration during fermentation are available in the scientific literature. Only microscopic observations are reported on the presence of the parasporal body i.e. the delta-endotoxin crystal. - 73 - A. Margaritis et at.

The bioassay is not convenient to use for routine testing because it requires the rearing of insects and larvae which is time consuming and expensive. The bioassay suffers from inaccuracies and lack of reproducibility and can only be used on a comparative basis with a large degree of statisitical analysis to ensure the absence of major errors. The results are reported as LD50 values, i.e. the dosage required for 50% mortality of the insect larvae.

We needed a simpler method to use on a routine basis. Rather than subject each and every sample to LD50 testing we chose to develop our own ELISA based technique for measuring delta-endotoxin. The use of an ELISA based method for measurement of delta-endotoxin concentrations in a fermentation broth of B. t. as a function of real fermentation time has not been reported in the literature to date. This test could also have a significant impact on the quality control of delta-endotoxin production and could form the basis of a new standard test for B. t. delta-endotoxin presence.

The ELISA test does not replace toxicity testing for specific organism suceptibility, but it does allow rapid testing of small sample volumes on a routine basis. Since the ELISA test is very sensitive, with detection in the order of a few nanograms of delta-endotoxin per ml of fermentation broth, the early stages of delta-endotoxin production can now be detected during fermentation. Once the more rapid delta-endotoxin biosensor, which is being developed by our research group (Margaritis et al., 1991a), becomes available, on-line monitoring of delta-endotoxin will be feasible. This will allow monitoring the effect of fermentation feeding strategies on delta-endotoxin production more readily.

Two approaches to ELISA development were pursued. The first involved the production of rabbit polyclonal antibodies to solubilized delta-endotoxin of B.t. HD-I (Kosir, 1989). These antibodies were used to test fermentation samples for delta-endotoxin concentration. The second approach was the development of a new hybridoma cell line producing a specific monoclonal antibody aganist delta-endotoxin.

We will also show an analysis of B. t. fermentation samples provided by Dr. 0. Morris from Agriculture Canada and N.R.C., Egypt and own purified delta-endotoxin crystal preparation.

- 74 - Elisa Method to Measure B.t. Endotoxin

DEVELOPMENT OF NEW HYBRIDOMA CELL LIME FOR MONOCLONAL ANTIBODY PRODUCTION

Several BALB/c mice were immunized with alkaline soulbilized delta-endotoxin crystals purified from a B.t. kurstaki HD-1 fermentation. The spleens of these mice were fused with a SP2 mouse tumor cell line using Polyethylene Glycol (PEG) and Hypoxanthine Aminopterine Thymidine (HAT) selection medium by the method of de st. Groth and Scheidegger (1980) with modifications. Figure (1) shows the general scheme of the hybridoma cell fusion.

Figure (2) outlines the detailed steps of the cell fusion and hybridoma cell line selection process. The details of this are available elsewhere, (Margaritis et al., 1991b) and sub-cloning of the fused cells was done and selection for a strain producing monoclonal antibodies specificly against delta-endotoxin was made as described in the above reference. Once a suitable isolate was found it was subcultured in Dulbecco's Modified Eagle Medium (DMEM) tissue culture medium and sample aliquotes were preserved frozen in liquid nitrogen.

Development of Hybridoma Cell System

Myeloma Spleen Cell Hybridoma SP2

Figure 1. Fusion of myeloma cell line with immunized spleen cell to create a monoclonal antibody producing hybridoma cell line.

- 75 - A. Margaritis et al.

Immune mouse

Spleen

Normal antibody-forming cells 0000000000

Plasmacytoma cells

Polyethylene glycol

Unfused I Plasmacytoma cells Heterokaryons Unfused spleen cells f i Q Q Q Q 0000

I HAT medium

Hybrids Die in HAT medium Die in culture 0 00 O

Clone by limiting dilution

Q ® 0000 Clone 1 Clone 2 Clone 3 Clone 4

Figure 2. Detailed schematic diagram of the cell fusion protocol and hybridoma cell line selection process.

- 76 - Elisa Method to Measure B.t. Endotoxin

PRODUCTION OF MONOCLONAL ANTIBODY AGAINST delta-ENDOTOXIN

Two approaches to antibody production were adopted. The first was batch fermentation in a low shear bioreactor system, and the second was semi-batch antibody production in an immobilized cell membrane bioreactor system. In both bioreactor systems, the new hybridoma cell line was used.

The low shear bioreactor system was used to study the effects of Fetal Bovine Serum (FBS) concentration on antibody production. The fermentation was conducted in the bioreactor system shown in Figure (3). The temperature and pH were controlled to 37°C and pH 7.25, respectively. The low shear floating impeller was driven at 90 rpm and gently mixed the bioreactor contents. Surface aeration was provided using a gas mixture containing 5% CO2 and 95% air. This bioreactor was incoulated with a 10% inoculum containing 106 cells/ml. The concentrations of antibody produced were monitored with our new ELISA method. Glucose, lactate, glutamine and ammonia were determined enzymatically with diagnostic kits from Sigma Co., and total and viable cell counts were determined microscopically with a hemocytometer and the trypan blue dye exclusion test. Dissolved Oxygen was measured with a polarographic D.O. probe.

Figure (4) shows the viable cell concentration over course of the fermentation time in the low shear bioreactor. It should be noted that the viable cell concentration was greater for the 1.2507o FBS medium. This medium formulation was based on a serum free medium base called Opti-MEM (Gibco). The 2.501o and 5.007o FBS supplemented media were based on a standard DMEM formulation. Using 1.25076 FBS with DMEM medium, resulted in poor cell viablity and slow cell growth. The change in medium base formulation allowed much lower FBS supplementation to be used with minimal loss of antibody production. Figure (5) shows that the 1.2501o FBS Opti-MEM medium produces antibody more quickly and at a level only slightly lower than the 5.001o FBS DMEM medium production. This allows for minimization of FBS supplementation without undue loss of monoclonal antibody production.

- 77 - A. Margaritis et aL

pH CONTROLLER AIR FILTER A T-

95% AIR 6% C02 EXHAUST GAS ACID/ BASE PUMP CONDENSER

AIR FILTER

pH PROBE-

DO PROBE

SAMPLING PUMP

MEDIUM/ HERMOSENSOR INOCULUM-(;-7

FLOATING MAGNETIC STIRRER 1J

HEATER BASE WITH

ELECTROMAGNETIC

DRIVE ASSEMBLY

Figure 3. Schematic diagram of the low-shear bioreactor system.

- 78 - 14

(o) 1.25076 FBS in Opti-MEM. 2.5% FBS in DMEM. 10 M 5.0% FBS in DMEM.

20 40 60 80 100 120 140 160 180 200 220

Time (hrs.)

Figure 4. Viable hybridoma cell concentration kinetic data from the low-shear bioreactor system. (o) 1.25% FBS in Opti-MEM. 2.5% FBS in DMEM. (p) 5.0% FBS in DMEM. v v v

I 000

I

20 40 60 80 100 120 140 160 180 200 220

Time (hrs.)

Figure 5. Monoclonal antibody production kinetic data from the low-shear bioreactor system.. Elisa Method to Measure B.t. Endotoxin

The immobilized membrane bioreactor system is shown in Figure (6), which has the following essential components : the immobilized cell module, the gas exchange tubing, medium recycle pump and the medium reservoir. The media used in this system were buffered to pH 7.4, the temperature controlled at 37°C in a 6% C02 atmosphere incubator. The membrane module shown schematicly in Figure (7) was loaded with 4.0 x 107 cells initially. The cells were trapped between two membranes which allow the passage of nutrients and the removal of wastes and products including the monoclonal antibody produced by the cell line. Total internal volume of the cell compartments was 12.5 ml, and the distance between the two membranes in each compartment was 800 pm. Monoclonal antibody concentrations were monitored as well as glucose, lactate, glutamine, and ammonia levels. The nutrient media bottle was changed every 3 to 4 or 6 to 7 days depending on the medium formulation. The DMEM media base was used till day 40 after which the Opti-MEM formulation was used. Figure (8) shows the levels of antibody production during the various cycles of the system. Overall, 130 mg of monoclonal antibody against delta-endotoxin was produced over an 83 day period. AIR VENT - +-)`/ SAMPLE PORT

It

MEDIA BOTTLE

SILICONE OAS EXCHANGE TUBING

IMMOBILIZED MONOCLONAL HYBRIDOMA ANTIBODIES CELL MODULE PUMP r_- SUPERNATANT

l

TRAY

Figure 6. Schematic diagram of hybridoma cell immobilization membrane bioreactor system. - 81 - A. Margaritis et al.

MEDIA MEDIA INLET OUTLET

lam- i IMMOBILIZED MICROPOROUS CELL - i COMPARTMENT MEMBRANES

Figure 7. Details of the cell immobilization membrane.

16

14

12

10

8

6

4

2

1 I 1 20 40 60 80 Time (hrs.)

Figure 8. Monoclonal antibody production kinetic data from the immobilized cell membrane bioreactor system.

- 82 - Elisa Method to Measure B.t. Endotoxin

Further details and discussion of the production of this monoclonal antibody are available elsewhere. (Margaritis et al., 1991b).

DEVELOPMENT OF A NEW ELISA METHOD FOR delta-ENDOTOXIN MEASUREMENT

Schematic diagrams of the structure of an antibody are shown in Figure (9). Monomeric antibody molecules consist of two heavy and two light chains, both have variable and constant regions. The space filling model of immunoglobulin G (IgG) shows the branched nature of this complex immunogenic protein. The activity of antibodies from one organism against those of another is the key to the development of the indirect antibody ELISA. In our case a commercially available goat anti-mouse IgG antibody conjugated with biotin forms the basis for a colourimetric reaction system, which is correlated with antibody concentration or antigen concentration depending on which form of the test is used.

The ELISA test for antibody concentration measurement is outlined in Figure

(10) as follows : First a polystyrene microtitre plate is coated with highly purified and alkaline solubilized delta-endotoxin crystals at a concentration of 50 pg delta-endotoxin/ well. It is incubated for 18 hours at 5°C, then washed phosphate buffer three times. The plate is then blocked with 10/4 gelatin in buffer to eliminate non-specific binding of the antibody to the plate. After a 1 hour incubation and three more washes the plate is loaded with serial dilutions of the monoclonal antibody samples and standards and incubated 4-6 hours at room temperature. Three more washes with 0.01 M phosphate buffer pH 7.2 are done. The biotin labelled goat antimouse IgG was added to the wells and incubated overnight at 4°C. The wells were washed again and alkaline phosphatase conjugated strepavidin was added to the wells and incubated at 37°C for two hours. After washing the wells again, the substrate para-nigrophenyl phosphate (1 mg/ml) in 9.7% diethanolamine buffer pH 9.8 was added to the wells and incubated for 15 minutes at room temperature. The reaction was stopped by the addition of 50 pl 3M NaOH to each well. Note that all solutions were added at 100 pl per well. The colour change in the wells was measured using an ELISA plate reader (Titertek Multiscan PLLUS) at 405 nm.

- 83 - A. Margaritis et al.

0

0

(a) The monomeric antibody molecule consists of two heavy, (H) chains and two light (L) chains. Both have variable (V) and constant. (C) regions. (b) Molecular model of IgG.

Figure 9. Diagramatic representations of the structure of IgG.

- 84 - Elisa Method to Measure B.t. Endotoxin

Plating the Wells with Antigen delta-Endotoxin (A) nA AA AAA M

WASH, BLOCK, WASH

Monoclonal Antibody (M-IgG)(,&) from Samples (Dilutions) and Standards (Dilutions)

Biotin-Sp Conjugated Gam-IgG (r )

Alkaline Phosphatase Conjugated Streptavidin (E )

0 Cl Q 0#a a a p-Nitrophenyl Phosphate in Subtrate Buffer pH 9.8

Stop reaction with 3M NaOH,read OD @ 405 nm

Figure 10. Schematic diagram of the indirect antibody ELISA method.

- 85 - A. Margaritis et al.

For the measurement of delta-endotoxin concentration an inhibition ELISA was used as shown in Figure (11). Fermentation samples analysed by this technique were always pretreated by French Pressing at 20,000 psi to release all delta-endotoxin from the cells. These samples were then solubilized with 0.02M NaOH for 16 hours at room temperature. Sample dilutions and standrd solutions of solubilized delta-endotoxin were each mixed with a known antibody concentration and allowed to bind. These samples and standards were then added to a microwell plate as before. Remaining unbound antibody could then bind to the solubilized delta-endotoxin on the plate. The rest of the test proceeded as described above. The antibody concentration measured on the plate is inversely proportional to the delta-endotoxin concentration in the samples. Using pure delta-endotoxin crystal protein standards a standard curve could be developed as shown in Figure (12). Thus colour development could give delta-endotoxin concentration directly.

Plate coated with antigen.

I Wash

1 Add sample containing antigen mixed with antibody

1 Wash

1 Add enzyme - antibody conjugate.

1 Wash 0 0 0 1 Add enzyme substrate. 0 0

I Read absorbance at 405 nm.

Figure 11. Schematic diagram of the inhibition ELISA method.

- 86 - Elisa Method to Measure B.t. Endotoxin

1.0

tn Qor 0.5

0 10 10 10 104 [Crystal protein] (ng/ml)

Figure 12. A standard curve for the inhibition ELISA generated using pure crystal protein dissolved in alkali. The linear portion of the semi-log plot gives the working range of the assay, in this case, 200 to 1200 ng/ml.

MEASUREMENT OF delta-ENDOTOXIN IN FERMENTATIONS OF B.t.

To demonstrate the use of the ELISA method with polyclonal antibodies for measuring delta-endotoxin production, a number of fermentation runs were studied. A typical result for a I Liter fermentation for B.t. kurstaki HD-1 delta-endotoxin production is shown in Figure (13). This shows maximum biomass production in 5 to 6 hours with the onset of delta-endotoxin production commencing at 6 hours and mostly completed by 12 hours. This is well before significant cell lysis has occurred and the majority of the cells are still intact at this time. Complete cell lysis takes another 15 hours more. This could have interesting consequences for industrial production and possibly toxin stability. Glucose usage is mostly complete in 3 hours and corresponds to the minimum pH level. This is due to the production of organic acids during glucose metabolism. (Rowe, 1990) As cell lysis proceeds medium pH becomes more alkaline.

- 87 - I 00 00

4200

' '0 10 20 30 40 Time (hrs.)

Figure 13. Batch fermentation of Bacillus thuringiensis kurstaki HD-1 in 1L Bellco stirred tank reactors. Biomass and delta-endotoxin crystal protein profiles are shown. Elisa Method to Measure B.t. Endotoxin

At the request of Dr. O. Morris from Agricultrue Canada, several fermen- tation samples from the Egyptian NRC were analysed as well as some fermentation samples of Dr. Morris's own strains using our ELISA test with our new monoclonal antibodies. We compared these to the international standard as well as our own purified delta-endotoxin crystals using the ELISA method. These data are shown in Table (1). We showed the presence of antibody activity against all the Egyptian NRC B. t. aizawai strains tested but at levels lower than the international standard B. t. kurstaki HD-1 to which our monoclonal antibodies had been developed. Dr. Morris's B.t. aizawai strain HD-1 showed very good activity as measured by the ELISA method. Our own pure crystal preparation gave about 3 times the activity in I.U. of the International Standard preparation. The cross reactivity of antibodies to delta-endotoxin of various strains of B.t. has been noted before by Wie et al., (1982).

In addition to our ELISA results shown in Table (1), we examined the samples using Scanning Electron Microscopy. Results show the International Standard

preparation of B. t. kurstaki HD-1 at a magnification of 798 times ; with an apparent absence of visible delta-endotoxin crystals, though some are possibly present inside the spheroids, with respect to the Egyptian NRC sample of B. t. aizawai HD-134 ; again no crystals are apparent but many small particles less than 0.5 pm were visible

as well as 1 to 4 pm collapsed hollow spheres. The Egyptian B. t. aizawai HD-282 sample shows many irregularly shaped particles 1-2 um, 2-4 um and less than 0.5 um in diameter. Dr. Morris' HD-133 B.t. aizawai strain shows many irregularly shaped crystals while the unidentified strain coded MB 2.6/60.1 shows the presence of crystals of two apparent shapes, some irregular tending to be spherical and some rhomboid. This is compared to the material 99076 pure after two Renografin-76 density gradient centrifugation runs (Sharpe et al., 1975), prepared in our laboratory where only delta-endotoxin crystals are present.

CONCLUSIONS

We have been able to produce a monoclonal antibody producing hybridoma cell line to B. t. kurstaki HD-1 delta-endotoxin. The kinetics of antibody production

- 89 - Table (1) ELISA analysis of delta-endotoxin B.t. samples using monoclonal antibody.

delta-Endotoxin Ave. Sample Activity Activity Sample No. Origin (IU/MG) (IU/MG) Comments

HD-1-S-1980 International Standard Sample 16,000 16,000 Used as Standard B. thuringiensis (B. t. ) HD-1, kurstaki HD-134 NRC Egyptian Sample, 12,350 12,657 B.t. aizawai Strain 12,310 13,310

HD-282 NRC Egyptian Sample, 11,570 11,680 I B. t. aizawai Strain 11,420 12,050 HD-133 Dr. O. Morris 36,680 35,985 Dr. Morris request. B.t. aizawai Strain 35,290

MB 2.6/60.1 Dr. O. Morris 13,920 15,117 New strain of B. t. isolated by Dr. New strain not 16,180 O. Morris, not identified yet, Dr. Identified yet 15,250 Morris request. AM-Crystals Dr. A. Margaritis 46,110 48,430 Purified crystals of delta- purified crystals of 51,740 endotoxin from B.t. HD-1 delta-endotoxin. B.t. HD-1 var. 47,440 var. kurstaki prepared in the lab. kurstaki. of Dr. A. Margaritis. Used to compare with other B. t. samples from Egypt and O.Morris

Note : All Elisa analysed were done in triplicate with DE NOVO sample preparation and ANA. Dr. A. Margaritis/mm, July 12, 1991. Eiisa Method to Measure B.t. Endotoxin by this cell line has been studied in a low shear batch bioreactor system and in an immobilized cell membrane bioreactor antibody production system. We have shown the ELISA method with polyclonal and monoclonal antibodies can be used for the measurement of delta-endotoxin concentrations in B. t. kurstaki fermentations. We have also noted the cross-reaction of our monoclonal antibodies with the delta-endotoxin from B. t. aizawai and MB2.6/60. I strains obtained from NRC Egypt and Dr. Morris.

AKNOWLEDGEMENTS

The work on the development of the ELISA test using polyclonal antibodies from rabbit antiserum against delta-endotoxin was supported by IDRC, Ottawa, Canada, through a research grant awarded to Dr. A. Margaritis. The work on the development of monoclonal antibodies against delta-endotoxin from a new hybridoma cell line was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada, through a Strategic Biotechnology Grant on Biosensors awarded to Dr. A. Margaritis.

REFERENCES

De St. Groth, S.F. and Scheidegger, D. (1980). J. Immunol. Methods 35, 1-121. Margaritis, A.; Morgan, T.; Jiang, L. and Peterson, N.O. (1991a). Development of a novel optical fiber biosensor to measure delta-endotoxin in Bacillus thuringiensis fermentation, (Work in progress). Margaritis, A.; Pham, T. ; Nichols, S. ; to Bokkel, D. and Strejan, G.H. (1991b). Kinetics of growth of a new hybridoma cell system and production of monoclonal antibodies against the bioinsecticidal protein delta-endotoxin. Presented at Canadian Chemical Engineering Conference, Oct. 9, 1991, Vancouver, B.C., Canada. Kosir, J. (1989). Development of an enzyme-linked immunosorbant assay (ELISA) for measurement of the bioinsecticide delta-endotoxin in Bacillus thuringiensis fermentations, M.E.Sc. Thesis, Univ. Western Ontario, London, Ontario, Canada. Rowe, G. (1990). Central metabolism of Bacillus thuringiensis during growth and sporulation, Ph.D. Thesis, Univ. Western Ontario, London, Ontario, Canada.

- 91 - A. Margaritis et al.

Sharpe, E.S.; Nickerson, K.W.; Bulla, L.A. Jr. and Arosnson, J.N. (1975). Separation of spores and parasporal crystals of Bacillus thuringiensis in gradients of certain X-ray contrasting agents. Appl. Microbiol., 30, 1052.. Wie, S.I. ; Andrews, R.E. ; Hammock, B.D. ; Faust, R.M. and Bulla, L.A. (1982). Enzyme-linked immunosorbent assays for detection and quantitation of the entomocidal parasporal crystalline protein of Bacillus thuringiensis subspp. kurstaki and israelensis. Appl. Environ. Microbiol. 43, 891-894. Persistance of Bacillus thuringiensis in the Tropical Environment

O. N. Morris Agriculture Canada Research Station 195 Dafoe Road, Winnipeg Manitoba, Canada, R3T 2M9

ABSTRACT

The rapid inactivation of entomopathogens, such as Bacillus thuringiensis (B. t.) by sunlight limits their residual activity and effectiveness in the field. It can be especially limiting if the target insect spends much of its feeding time in sites that are sheltered from applied bioinsecticides and if the pathogen is transmitted to subsequent generations by contaminatedfoliage. The germicidal wave lenghts which reach the earth's surface are primarily between 290 nm and 380 nm and constitute only 7% of the total incident sunlight at ground level. Dissolved parasporal crystals which are mainly responsible for B. t. activity absorb radiation at 273 nm, very little of which reaches the earth surface. Notwithstanding the relatively small amount of germicidal radiation reaching the earth, the scientific literature has many examples of rapid inactivation of B. t. when exposed to direct sunlight. This phenomenon can be expected to be most severe in tropical regions of the earth where solar radiation and atmospheric temperature are most intense. Nearly all the available scientific literature on the solar inactivation of microbial agents were generated in temperate regions. In this paper relevant information will be reviewed regarding B. t. var. kurstaki inactivation and some attempts to slow down the rate of inactivation by various methods. The paper will conclude with some suggestions for protecting B. t. from rapid solar inactivation in tropical environments.

INTRODUCTION

The rapid inactivation of entompathogens by sunlight has been observed universally. Sunlight limits their residual activity and effectiveness in the field.

- 93 - O. N. Morris

It can be especially limiting if the target insect spends much of its feeding time in sheltered sites or if the pathogen is transmitted to subsequent generations by contaminated foliage, such as in cytoplasmic polyhedrosis virus.

The germicidal solar wavelengths which reach the earths surface are primarily between 290 nm and 390 nm and constitute about 7% of the total incident sunlight at ground level. Bacterial spores are known to absorb both ultraviolet radiation (330 nm) and visible light (near 400 nm). Dissolved crystals of Bacillus thuringiensis (B.t.) absorb 273 nm but very little of this wavelength 2% ) reaches the earth's surface.

Although the proportion of incident germicidal solar radiation is small, it appears to be quite effective in inactivating B.t. in nature, thus limiting its effectiveness for insect control. This would be particularly so in the tropical environment where the harshest environmental conditions would be encountered by the microorganisms. It should be noted that temperature, atmospheric humidity and leaf surface exudates can also cause microbial inactivation either alone or in combination with solar radiation.

In my presentation, I want to look at some theories on the mode of action of photo inactivation of B. t. , the persistence of B. t. in temperate and tropical environments and methods of extending the residual activity of B. t. in the agricultural and forest environments.

Figure (1) is a schematic representation of a B.t. showing spore and crystal endotoxin and electron micrographs of the delta endotoxin.

Figure (2) shows declines in both spore viability and insecticidal potency as exposure time to solar ultraviolet radiaton increases but spore viability declined significantly more rapidly than did insecticidal activity (Morris, 1983). Spores lost 50% activity after about 26 cal/cmz of sunlight compared with about 60 cal/cm` for endotoxin crystals. The data suggest that if spores contributed to insecticidal activity they did so only during initial sunlight exposure.

Figure (3) illustrates the spectral reflectance of B. t, spores in polychromatic light using Carey model spectrophotometer. This trace of reflectance of a pure suspension of B. t. spores on Millipore filter membrane using polychromatic

- 94 - Persistance of B.t. illumination and monochromatic viewing shows peak absorbance between 280 and 320 nm with decreasing absorbance between 320 and 500 run. The non-uniformity of the curve above 320 nm was probably due to uneven distribution of the spores on the filter paper.

Figure (4) shows spectral absorbance peaks of pure spores in suspension. There is significant absorbance between 250 nm and 400 run, peaking at 270 nm. This measurement was taken on a Beckman ACTA CIII spectrophotometer using a scanning range of 250 to 700 nm.

Chr om y in Cytoplasm

%

Figure 1. A. A schematic diagram of Bacillus thuringiensis cell showing spore and parasporal crystal endotoxin. B. Electron micrograph of spores and crystal. C. Highly magnified parasporal crystal.

- 95 - INSECTICIDAL POTENCY `. Y=66.6 - 0.307x r 2 =0.90

VIABLE SPORES Y=1-(1 -e -0.09168x )6.105 r 2 -0.977

25 50 75 100 125 150

UV RADIATION (295-385 nm) in CAL/CM 2

Figure 2. Decay curves of spores and spore/crystal toxicity of Bacillus thuringiensis var. kurstaki following exposure to sunlight in Ottawa, Canada.

280 300 350 400 450 500

100 %

SPORES

Figure 3. Spectral reflectance of Bacillus thuringiensis spores in polychromatic light. - 96 - Persistance of B.t.

Figure 4. Spectral absorbance peaks of spores of B. thuringiensis.

From my own work, then, I am lead to believe that the main germicidal wavelengths for B.t. are at the lower end of the near ultraviolet, viz . 280 nm to

340 nm . However, Griego and Spence (1978) presented good evidence that solar inactivation of B. t. is due in part to wavelengths near 400 nm which spores readily absorb.

MODE OF ACTION OF ULTRAVIOLET RADIATION ON B.t.

There is general agreement, then, that solar radiation inactivates B. t. spores and crystals and a question that immediately comes to mind is the mechanism by which this comes about. There are several theories but no general agreement.

Firstly, if the germicidal wavelengths are indeed between 280 and 340 nm, this would support the fact that nucleic acids and proteins are known to be denatured by ultraviolet light below 340 nm (Jagger, 1967). If the exact mechanism by which the toxic protein is denatured is determined, then a rational basis for

- 97 - O. N. Morris engineering an improved toxin could be developed. Dr. H. Kaplan, National Research Council, Ottawa, considers it very unusual for proteins to be affected by sunlight. He has discovered, however, an impurity in the crystal protein. When sunlight reaches it, this impurity energizes oxygen which, in turn, attacks the protein rendering it inactive. Dr. Kaplan reports that within 8 hrs. B. t. protoxin loses half its toxicity, partly because sunlight radiation of B. t. crystal renders it very insoluble.

Pozsgay et al. (1987) gave good evidence that the amino acid tryptophan was destroyed in sunlight-mediated crystal inactivation. Kapuscinski and Mitchell (1981) proposed that solar radiation damaged the catalase system in the bacteria Escherichia coll. This injury was repaired or overcome by adding catalase or pyruvate to the culture medium. The most recent study by Kaplan et al. (personal communication) found the 300-350 nm solar spectrum to be largely responsible for crystal damage and loss of toxicity and that the absorption of chromosphores (light absorbing molecules) by the crystals exposed to the fermented liquor made the crystals photosensitive. This is a particularly interesting idea since it suggests that manipulating the culture medium might be used to render B. t. less sensitive to solar radiation. Indeed C.L. Sanders, National Research Council, Ottawa, suggested to me in 1974 that B. t. spores that are well fed will be more resistant to radiation than those that are not well fed.

PERSISTANCE OF B.t. IN THE ENVIRONMENT

The short persistence of B. t. on crops following exposure to direct sunlight have been reported repeatedly. Residual activity on crops appears to be related to more than just ultraviolet radiation. Morris and Moore (1975), for example, reported that B. t. applied to spruce trees lost 50% of their insecticidal activity- in eight days in shade, compared with 50% loss in 2 days in sunlight. The residual activity of B.t. on crops, is illustrated in Table (1) and Fig. (5).

The curves here indicate a significant reduction in the spore viability (Morris, 1977) after one day of weathering under Canadian conditions. Corresponding bioassay data on the same trees indicated a high level of biological activity persisting for up to 20 days due to crystal activity. Under subtropical conditions in the USA, the insecticidal activity of B. t. on cotton plants was between 30 and 48 hr, (Beegle et al., 1981). The half life under Egyptian conditions on castor plants was between

- 98 - Persistance of B.t.

Table (1) Estimated half-life of selected entomopathogens for natural sunlight (North America) .

Pathogen Half-life (Hrs.)

Nosema trichoplusiae 8 Heliothis NPV 24 Trichoplusia NPV 48 Pieriis GV 42 Bacillus thuringiensis spores 24 crystals 72 Metarrhizum anisopliae 8 Nomuraea rileyi 60 Nosema necatrix 1

0 50

40

WN 20 z JO 0 10

O 0 CHECK --- z 0 F a - 0 1 5 10 15 DAYS 20 2 0.7 2.2 4.1 8.4 14.1 KCAL/CM WEATHERING INTERVAL

Figure 5. Degradation curves of spores of Bacillus thuringiensis var. kurstaki aerially applied to white spruce tress in Ontario, Canada.

- 99 - O. N. Moms

19 and 40 hr. as reported by Ragaei (1990). Hamed and Hassanein (1986) in Egypt also reported that after 6 days of exposure to sunlight, B. t. -treated cotton foliage killed 7% of Spodoptera littoralis compared with 27% on treated cotton held in the shade.

EXTENDING THE RESIDUAL LIFE OF B.t.

Several methods have been attempted in recent times for extending the residual life of B. t. exposed to solar ultraviolet radiation. In 1983, Morris recommended that any UV absorbers for B. t. should have high absorbance capability in the range of 330-400 nm wavelengths. Table (2) summarizes the more recent attempts. Morris (1983) used the water soluble dyes viz. DS 49 (Sodium 2, 21-dihydroxy-4-41 dimethoxy-5-sulfobenzophenone) and Erio Acid Red XB100 (sodium salt of formyl-m-benzenedisulphonic acid). This mixture with absorbance peaks at 284, 330 and 564 nm increased residual activity of B. t. on white spruce trees 2.9 fold. A 2% solution of Congo Red increased residual activity on castor plants 3.3 fold in Egypt (Ragaei 1990). Dunkle and Shasha (1989) reported that starch-encapsulated B. t. , with 1% Congo Red added, retained at least 50% of their original activity after 12 days of exposure to sunlight (50% original activity without sunlight screen is about 2 days). Folic acid and p-amino benzoic acid as screens were somewhat less effective. Cohen et al. (1990) reported photoprotection of B. t. by absorption of cationic chromosphores such as Acriflavin and Rhodamine B. After 12 hrs of exposure to UV radiation, mortaliy with Acriflavin was 78% compared with 5% without Acriflavin. This approach involves a specifically intimate alignment between an organic pesticide and a selected organic chromophore. Such spatial arrangement facilitates transfer of energy or electrons between the excited molecule and the chromosphore. In this way, a probable photochemical reaction is fully or partially prevented. Mycogens's M-Cap technique involves encapsulating the delta endotoxin within killed and fixed cells of Pseudomonas fluorescens, a bacteria which colonizes corn roots. I believe this genetically engineered method of increasing residual activity is now patented in the USA and the product is ready for sale. Data in Table (3) show that Orzan, Congo Red and Molasses can protect B. t from rapid solar inactivation following application to castor plants. In Table (4), it appears

- 100 - Persistance of B.t. that Congo Red was not effective in protecting B. t. on canola plants in terms of mortality of Bertha armyworm. However, the sunlight screen was apparently effective in reducting the feeding activity of the Bertha armyworm larvae.

Table (2) Techniques of extending residual activity of B.t. exposed to UV radiation .

Bioassay Technique Substrate Country Reference

1016 DS49 + 101o EAR Balsam Fir Canada Morris, 1983 2016 Congo Red Castor Egypt Ragaei, 1990 Starch-encapsulated Starch granules USA Dunkle, & 1989 + 101o Congo Red Shasha, Acriflavin Artificial diet Israel Cohen et al., M-CAP (Mycogen) ? USA 1990 Mycogen, 1986

Table (3) Relative efficiency of some UV absorbers for B. t. under Egyptian environmental conditions .

Relative Efficiency Artificial Sunlight UV Absorber UV source castor plants

B. t. alone exposed 1.0 1.0 B. t. + 10% Orzan 2.2 5.6 B. t. + 25% Molasses 2.4 3.7 B.t. + 2% Congo Red 2.1 3.3

- 101 - O. N. Morris

Table (4) Mortality and feeding activity of M. configurata feeding on Canola plants treated with B. t. plus sunlight screen (Congo Red) .

Treatment Mortality (%) Mean Wt. Gain of Surviviors (MG ± SD)

B. t. without Congo Red -Shade 93 32 ± 19 B. t. without Congo Red-Sun 22 22 ± 15 B. t. with Congo Red-Sun 22 0+ 0

In conclusion, I suggest that in the developing tropical environments, the relatively simple method of adding the water soluble dye Congo red and 25076 Molasses, is an effective and practical way of extending the activity of B. t. applied to crops. It is probably inadvisable to use this dye on a large scale at this time, however, since the environmental acceptability has not yet been fully explored.

REFERENCES

Beegle, C.C. ; Dulmlage, H.T. ; Wolfersberger, H.D. and Martinez, E. (1981). Persistance of Bacillus thuringiensis Berliner insecticidal activity on cotton foliage. Env. Entomol. 10, 400-401. Cohen, E. ; Rozen, H. ; Joseph, T. ; Braun, S. and Marguiles, L. (1990). Photoprotection of Bacillus thuringiensis kurstaki from ultraviolet radiation. J. Inverteber. Pathol. 57, 343-351. Dunkle, R.L. and Shasha, B.S. (1989). Response of starch-encapsulated Bacillus thuringiensis containing ultraviolet screens to sunlight. Environ. Entomol. 18, 1036-1041. Griego, V.M. and Spence. K.D. (1978). Inactivation of Bacillus thuringiensis spores by ultraviolet and visible light. Appl. Environ. Microbiol. 35, 906-910.

- 102 - Persistance of B.t.

Jagger, J. (1967). Introduction to research in ultraviolet photobiology. Prentice Hall, N.J. 146 pp. Hamed, A.R. and Hassanein. F.A. (1986). Persistance and virulence of Bacillus thuringiensis under sunny shady conditions. Bull. Ent. Soc. Egypt. Econ. Series, 14, 73-77. Kapuscinski, R.B. and Mitchell, R. (1981). Solar radiation induces sublethal injury in Escherichia coli in sea water. Appl. Environ. Microbiol. 43, 670-674. Morris, O.N. (1977). Long term study of the effectiveness of aerial application of Bacillus thuringiensis - acephate combinations against the spruce budworm Choristoneura fumiferana (Lepidoptera, Tortricidae). Can. Ent. 109, 1239-1248. Morris, O.N. (1983). Protection of Bacillus thuringiensis from inactivation by sunlight. Can. Ent. 115, 1215-1227. Morris, O.N. and Moore, A. (1975). Studies on the protection of insect pathogens from sunlight inactivation. II. Preliminary field trials. Rep. Chem. Control Res. Inst. CC-X-113. 34, pp. 8. of sunlight Pozsgay, M. ; Fast, P. ; Kaplan,H. and Carey, P.R. (1987). The effect on the protein crystals from Bacillus thuringiensis var. kurstaki HD-1 and.NRD 50, 246-253. 12 : A Raman spectroscopic study. J. Invertebr. Pathol. ; T. and Carey. P.R. Puztai, M. ; Fast, M. ; Eringorten, L. ; Kaplan, H. Lessard, (1991). The mechanism of sunlight - mediated inactivation of Bacillus thuringiensis crystals. Biochem. J. 273, 43-48. Ragaei, M. (1990). Studies on the effect of Bacillus thuringiensis Berliner on the greasy cutworm Agrotis ypsilon (Rott). Ph.D. thesis, Univ. of Cairo, 266 pp.

Enhancement of Bacillus thuringiensis for Field Application

H. S. Salama National Research Centre Tahrir Street, Dokki, Cairo, Egypt.

ABSTRACT

Bacillus thuringiensis (B. t.) has a very short effective residual life. The persistance of spores of B. t. is progressively correlated with the time of exposure in the field. Various approaches have been adopted to overcome this problem. Plant feeding stimulants, host plants extracts, leafpowders or oils proved to serve as adjuvants to increase the potency of B. t. against various lepidopterous insect species. Biochemical approaches have been also developed with great success. These approaches were based on the incorporation of some selected non toxic chemical compounds, with different modes of action with the endotoxin fed to the larvae and thus resulting in the potentiation. Among the compounds tested some inorganic salts, protein solubilizing agents, amino acids and amides, showed a remarkable potentiation to the endotoxin activity against the target insects. the significance of these findings with respect to the toxicity enhancing effect of some of the tested compounds lies in the fact that most of them are low priced, non toxic to humans and animals which add to their feasibilities in application. These results would undoubtedly be of interest to both research work and industry.

INTRODUCTION

The incorporation of adjuvants with microbial insecticides to achieve high efficacy was practiced by several authors either to extend the spectrum of activity or to overcome the short persistance of these insecticides in the field. Salama and his collaborators developed simple biochemical means for enhancing the potency of Bacillus thuringiensis (B. t.) against Spodoptera littoralis and Agrotis ypsilon.

This presentation gives an account of the results of investigations that have been carried out in Egypt in this concern.

- 105 - H. S. Salama

PERSISTANCE OF B.t. IN THE FIELD It is known that B. t. has a very short effective residual life. The pathogen is not mobile and cannot escape the unfavourable conditions. In cotton fields in Egypt, it has been found that the persistance of spores of B. t. showed an obvious reduction after one day of weathering and decay in its viability was progressively correlated with the time of exposure in the field. The spores half-life of various formulations of B.t.strains varied between 75-256 hours and was not correlated with the temperature attained on the surface of cotton leaves exposed to sun. Ultraviolet radiation seems to be the dominat factor affecting the spores viablibity (Salama et al., 1983).

Naturally, if we succeed to prolong the B. t. persistence in the field, the optimum potential effect can be obtained.

In this concern, investigations have been made in our laboratories to isolate mutants that are resistant to physical factors namely, UV-resistant and high temperature resistant. Several mutants were obtained and their potencies were assessed vs. the target insect. Further work is still in progress and is being carried out by a group of researchers in genetic engineering and biotechnology. Rapid progress can be expeted in this area in the immediate future. The reason for giving this attention through genetic engineering research is that the toxin produced by B. t. is determined by a single gene, thus, greatly simplifying the transfer of toxin production to other biological organisms.

Significant steps have been made to develop strains of B. t. with a modified and more commercially acceptable host range. The development of conjugation techniques for the transfer of toxin-coding plasmids between B. t. isolates may lead to the production of strains which have broader potency ranges. Very recently, transgenic plants have been produced in U.S.A. which constitutively express the B. t. toxin gene. Such development and its progress, we have to be cautious about it although it offers the most exciting possibilities for future crop protection strategies and may provide the greatest commercial opportunity for the use of pest control products based on microbial pathogens.

Parallel to the gentic engineering research, various approaches have been developed to overcome the effects produced due to short persistance of B. t. in the field.

- 106 - Enhancement of B.t. for Field Application

POTENTIATION OF B.t. THROUGH FEEDING STIMULANTS, BAITS AND ADJUVANTS

In the first approach, investigations have been made to find eliciting materials of some of our target insects and to evaluate their role in increasing the potency of B. t. expecting that these materials will make it more acceptable and thus result in increased ingestion. Extract of some host plants, their volatile and nonvolatile fractions showed to be active in potentiating the effect of B. t. when combined with it (Salama et al., 1985a). (Tables 1 and 2).

Also, coax, molasses, soybean flour, cottonseed flour, sucrose and its combination with cotton leaf powder or its extract can be used as adjuvants (Table 3) to increase the potency of B.t. (Hafez et al., 1987).

Based on these findings, El-Nockrashy et al. (1984) carried out some studies aiming to develop bait formulations for the control of S. littoralis with B.t. These formulations when added to the pathogen are expected to make it more acceptable to the target insect and thus result in increased ingestion. Different formulations containing various proportions of ingredients obtained from cottonseed or soybean were tested for their effectiveness with B. t. vs. the target insect and Coax was used as a reference material. Formulations containing cottonseed kernels extracted with 70016 ethanol and then acetone-hexane-water led to a better acceptability of the meal as compared to the known commercial adjuvant "Coax". Increase in the gossypol content decreased the efficiency of the formulations as observed with those containing cottonseed kernels extracted only with hexane. Changes in the total amount of cottonseed oil in the formulation had no effect on the activity, while the addition of fresh ethanol extract of cottonseed kernels increased the effectiveness. Formulations containing soybean flour were highly efficient.

Some field tests were conducted by Salama et al. (1985c) to determine the activity of B. t. formulation when applied to cotton plants (Gossypium barbadense var. Giza 69) against S. littoralis. Based on the results obtained with the role of feeding stimulants to the larvae of S. littoralis, some of these stimulants and others were assessed with regard to their effect in increasing the effectiveness of the formulation through increasing pathogen ingestion. In these experiments, the activity of vars. entomocidus, subtoxicus, galleriae, kurstaki and Dipel were screened at different concentrations singly or in combination with some feeding stimulants - 107 - Table (1) Effectiveness of host plant extracts on the efficacy (potency) of B. t. var. entomocidus vs. Spodoptera littoralis. Water extracts Petroleum ether extracts Plant extract incorp- orated into larval Slope Variance LC50 Potency Slope Variance LC50 Potency diet with B.t. ( pg/ml) (IU/mg) ( pg/ml) (IU/mg)

1st series of tests Untreated 2.31 0.0066 131.7 77889 1.40 0.0022 138.9 73857 (without extract) Cotton 2.08 0.0027 121.6 84359 1.48 0.0041 36.5 281842 (Gossypium barbadense) Clover 1.89 0.0031 152.7 67195 2.30 0.0037 89.4 114743 (Trifoium alexandrinum) 0 Sweet potato 2.09 0.0041 121.8 84241 2.40 0.0023 69.2 148237 00 (Ipomae batatas) Castor oil 2.59 0.0021 121.6 84345 2.40 0.0034 92.4 111018 (Ricinus communis) Cabbage 0.88 0.0250 123.81 28853 1.52 0.0027 50.5 203130 (Brassica oleraceae var. captitata) Lettuce 1.73 0.0029 133.0 77147 2.40 0.0025 115.1 89123 (Lactuca sativa) 2nd series of tests Untreated 1.20 0.0220 223.2 140710 (without extract) Jew's mallow 1.33 0.6530 199.2 157647 1.12 0.0510 112.5 279140 (Corchorus olitorius) Enhancement of B.t. for Field Application as compared to Coax. Results obtained so far revealed that cotton leaves extract increased the effectiveness of the pathogen. Non of the stimulants used had any effect on prolonging the half life of the spores of the preparation, which showed a great variation and with a maximum of 116 hours.

Bioassay of the oils of soybean, cottonseed and corn (as natural hosts of Heliothis armigera), indicate the superiority of soybean oil to other oils in improving the effectiveness of B. t. against H. armigera. Chick peas flour, soybean flour, corn flour, and cottonseed flour were also effective and these were followed by wheat, rice and barley flours.

Table (2) Effect of different concentrations of petroleum ether extract of cotton leaves on the efficacy (potency) of B. t. var. entomocidus vs. Spodoptera littoralis.

% conc. of extract in the diet Slope Variance LC50 Potency with B. t. Pg/ml IU/mg

1 0.97 0.0809 17.3 241034

0.1 1.64 0.0091 51.1 81602

0.01 0.64 0.0358 91.7 45473 0.001 1.06 0.0036 126.2 33042 Untreated 0.92 0.0190 113.0 36902 (without cotton extract)

POTENTIATION OF B.t. THROUGH CHEMICAL ADDITIVES

In a second approach, some biochemical means have been developed to overcome the short persistance of B.t. and at the same time to extend the activity spectrum of delta-endotoxin of B.t. var. kurstaki HD-1. Until the late 1960's, industrial production of B. t. was depending on the serotype 1, B. t. thuringiensis as the standard. Dulmage (1973) reported the discovery of HD-1 strain B.t. kurstaki which was 16X more potent than previous strains being used in commercial production. This was also adopted as a primary U.S. standard reference for assay of formulations containing endotoxins of B.t. In spite of the success achieved in

- 109 - H. S. Salama

Table (3) Effect of different feeding stimulants on the efficacy (potency) of B.t. var. entomocidus vs. Spodoptera littoralis. (Boisduvalle,1883) (Lepidoptera : Noctuidae).

Additives incorporated into Slope Variance LC50 Potency the diet with B. t. µg/ml IU/ml

Untreated (without additives) 1.32 0.015 200.3 30734 20% Coax 0.94 0.005 152.3 40420 5% Coax 0.95 0.005 98.1 62752 10076 Coax 1.01 0.003 75.4 81645 Sucrose 2% 2.01 0.078 110.0 51055 Cottonseed flour 3% + sucrose 1% 1.35 0.041 176.6 31801 Cottonseed flour 3% + sucrose 2% 2.01 0.220 182.6 30756 Cottonseed flour 5% + sucrose 1% 1.13 0.002 158.4 35455 Cottonseed flour 5% + sucrose 2% 1.02 0.005 112.1 50098

Untreated (without additives) 1.46 0.006 185.7 62032 1 % molasses 1.105 0.012 170.1 67709 2% molasses 1.55 0.005 162.5 70884 5% molasses 1.16 0.021 105.1 109568

Untreated (without additives) 2.3 0.002 121.9 91565 Cotton leaves powder 5% 1.4 0.021 123.2 90592 Cotton leaves powder 5% + sucrose 2% 1.5 0.001 93.4 119467 Petroleum ether extract of cotton leaves

(0.1070) 0.7 0.021 88.3 126336 Petroleum ether extract of cotton leaves 0.1% + 2% sucrose 2.1 0.027 64.9 171958 Water extract of cotton leaves 5% 2.1 0.002 121.6 91791 Water extract of cotton leaves 5% + sucrose 207o 0.9 0.003 89.1 125231 5% soybean flour 0.7 0.018 86.9 128489

- 110 - Enhancement of B.I. for Field Application combating a number of lepidopterous insects using this formulation, others are little susceptible to it.

Selected groups of chemical compounds were tested in this respect. In selecting these compounds, certain criteria were considered, for instance, the compound must be non-toxic to man or animal, possess no harmful effect on plants, biodegradable and commonly available at low price. The chemicals tested included representatives of inorganic salts, amino acids, lipid emulsifying agents, protein solubilizing agents, aromatic acids (Salama et al., 1984, 1985b, 1986, 1989).

The results obtained (Tables 4 and 5) indicate that calcium salts, such as calcium carbonate and calcium oxide, drastically enhanced the potency of B.t. against S. littoralis and A. ypsilon. This may be attributed to the fact that the addition of such salts will change pH of the gut, being more alkaline and thus enhancing the endotoxin breakdown and release of toxic fragments. Zinc sulphate also showed a remarkable effect in enhancing B. t. potency. The mode of action of this salt may be correlated to its effect on the proteolytic enzymes present in the insect midgut. The divalent cations are generally known either as activators or co-factors for many proteolytic enzymes.

With amino acids (Tables 6 and 7) acetamide, L-valine, L-arginine and tryptophan enhanced the potency of B. t. against the target insects. Among the protein solubilizing agents, disodium B-glycerophosphate and sodium thioglycollate caused 2-3 fold increase in the potency of B. t. vs. S. littoralis. The mode of action of this group may be correlated to their effect in reducing the disulphide linkage in the protein molecules of B. t. and thus increasing the endotoxin solubility in the insect gut (Table 8).

These promising results have been extended to include other strains of B.t. in the hope to fill the gaps now present with respect to spectra of activity, host ranges and biological specificities.

These approaches should contribute significantly to the feasibility of biological control means by indirectly lowering the production costs through their drasitc effects on their potency vs. the target insect species. The possible application in the commercial production of bacterial insecticides through incorporation of these chemical at a post-harvest stage will be of great value and most economic.

- 111 - H. S. Salams

Table (4) Effect of inorganic salts on the potency of B. t. var. entomocidus against S. littoralis.

Chemical incorporated LC50 Slope Variance Potency tested with B.t. ( pg/ml) (IU/mg)

Control (B. t. ) 333.0 0.17 0.422 182583 Calcium oxide (0.05%) 43.6 1.20 0.004 1394445 Calcium hydroxide (0.01%) 164.2 0.01 1.016 370280 Calcium acetate (1%) 37.7 1.14 0.010 1612732 Calcium carbonate (0.25%) 55.0 1.02 0.042 1105455 Calcium sulfate (1%) 64.0 0.87 0.025 9500000

Control (B. t.) 360 1.30 0.018 168889 Zinc sulfate (0.05%) 15 1.22 0.009 4053333 Zinc sulfite (0.05%) 130 1.28 0.008 467692 Copper sulfate (0.05%) 111 0.91 0.039 547748 Copper oxide (0.05%) 143 1.06 0.008 425175 Sodium sulfite (0.25%) 275 0.40 0.380 221091 Enhancement of B.t. for Field Application

Table (5) Effect of inorganic salts on the potency of B. t. var. galleriae HD-234 vs. A. ypsilon.

LC50 Slope Variance Potency Chemical additive tested (IU/mg) ( Ng/ml)

Control (B. t. ) 661494 (endotoxin only) 138.16 1.81 0.0047 Calcium carbonate (0.101o) 29.65 3.83 0.00097 3082361 Calcium hydroxide (0.1010) 29.91 2.67 0.0018 3055567 Calcium nitrate (101o) 39.48 1.65 0.0047 2314894 Calcium acetate (0.0501o) 37.11 1.98 0.0039 2462732 Calcium sulfate (0.05010) 77.31 1.06 0.0104 869046 Calcium oxide (0.05(1o) 29.06 1.98 0.0044 186286

Zinc sulfate (0.1%) 18.73 0.86 0.0157 4879445 Zinc sulfite (0.1010) 40.15 1.79 0.0099 2276264

Copper oxide (0.0501o) 13.80 1.09 0.0099 6622609 Copper carbonate (0.0501o) 9.45 0.91 0.0123 9671111 Copper phosphate (0.05010) 27.75 0.92 0.0120 3293405 Potassium carbonate (1010) 7.59 0.976 0.0131 12041106

Table (6) Effect of nitrogenous compounds on the potency of B. t. var. entomocidus against S. littoralis.

Chemical incorporated LC50 Slope - Variance Potency tested with B. t. ( Ng /ml) (IU/mg)

Control (B. t.) 360 1.30 0.018 168889

L-Tryptophane (0.5010) 28 1.73 0.013 2171428 L-Arginine (0.1010) 100 1.67 0.012 608000 Acetamide (1010) 15 2.08 0.005 4053333

- 113 - H. S. Salama

Table (7) Effect of amino acids and amides on the potency of B. t. var. galleriae HD-234 vs. A. ypsilon.

LC50 Slope Variance Amino acid tested Potency ( hg/nil) (IU/mg)

None (endotoxin only) 240.70 1.421 0.0040 241961 (DL) Alanine 52.831 0.787 0.0174 1102383 (DL) Serine 33.941 0.983 0.0121 1715919 (DL) Valine 33.030 0.875 0.0129 1763246 (DL) Tryptophane 14.891 1.322 0.0078 3911087 (L) Proline 7.658 1.435 0.0042 7605119 (DL) Ornithine 11.053 2.702 0.0019 4872417 (L) Arginine 6.017 2.263 0.0028 9679242 (DL) Asparagine 9.236 1.252 0.0076 6305760 (DL) Glutamine 8.299 1.042 0.0001 7017713 (DL) Aspartic acid 50.365 1.437 0.0058 1156359 (L) Glutamic acid 76.412 1.041 0.0021 762184

Table (8) Effect protein of solubilizing agents on the potency of B.t. var. entomocidus against S. littoralis.

Chemical incorporated LC50 Slope Variance Potency with B. t. ( Ng/ml) (IU/mg)

Control (B. t.) 333.0 0.17 0.422 182583 Sodium thioglycollate (1%) 20.3 2.70 0.0048 2995074 Urea (0.5070) 121 2.40 0.003 502479 Disodium-B-glycerophosphate

(0.0501o) 56 1.02 0.032 1085714 Dipotassium hydrogen phosphate (1076) 59 0.11 0.022 1030508

- 114 - Enhancement of B.t. for Field Application

EFFECT OF PHOTOPROTECTANTS ON THE ACTIVITY OF B.t.

In a third approach, photoprotectants were evaluated. In a series of field experiments, thirteen compounds were assessed with respect to their effect as protectants of B. t. var. entomocidus vs. S. littoralis. These were as follows : - Egg albumen - Brewer's yeast - Egg albumen + Brewer's yeast - Yeast extract - Starch - Cellulose - Crude cottonseed oil - Cellulose + crude cottonseed oil - Aluminum oxide - "Shade" Commercial product of Sandoz - " Shade " + polyvinyl alcohol - Charcoal - Peptonized milk The data obtained show that the insecticidal activity of B. t. in the control decreased gradually from 92076 (when using freshly sprayed leaves) to reach 43% when using leaves three days after its treatment. Most of the protectants tested, however, showed a protective effect which varied according to the tested material. Thus, it appears that the half life period of B. t. was prolonged to 8.97 days after using peptonized milk at 5% concentration compared to 3.28 in the control. The materials that exhibited protection differed both chemically and physically.

Brewer's yeast 5% and egg albumen 5% singly or combined with Brewer's yeast 5% were efficient as protectants of B. t. against inactivation by UV radiation. The commercial product "Shade" did not increase the half life of B. t. and its combination with polyvinyl alcohol 0.5% did not increase the half life of B.t. H. S. Salama

REFERENCES

Dulmage, H.T. (1973). Bacillus thuringiensis U.S. Assay Standard. Report on the adoption of a primary U.S. reference standard for assay of formultations containing the delta-endotoxin of Bacillus thuringiensis Bull. Entomol. Soc. Amer., 19, 200-202. Hafez, H. ; Salama, H.S. ; About-Ela, R. and Ragaei, M. (1987). Evaluation of adjuvants for use with Bacillus thuringiensis. vs. Heliothis armigera (Hubn). Z. ang. Ent., 103, 313-319. El-Nockrashy, A. ; Salama, H.S. and Taha, F. (1984). Development of bait formulations for control of Spodoptera liltoralis with Bacillus thuringiensis. Z. ang. Ent., 101, 381-389. Salama, H.S. ; Foda, M.S. ; Zaki, F.N. and Khalafallah, M. (1983). Persistance of Bacillus thuringiensis Berliner spores in cotton cultivation. Z. ang. Ent., 95, 321-326. Salama, H.S. ; Foda, M.S. and Sharaby, A. (1984). Novel biochemical avenus for enhancing Bacillus thuringiensis endotoxin potency against Spodoptera littoralis (Lep., Noctuidae). Entomophaga, 29 (2), 171-178. Salama, H.S. ; Foda, M.S. and Sharaby, A. (1985a). Role of feeding stimulants in increasing the potency of Bacillus thuringiensis vs. Spodoptera littoralis. Entomol. Gener., 10 (2), 111-119. Salama, H.S. ; Foda, M.S. and Sharaby, A. (1985b). Potential of some chemicals to increase the effectiveness of Bacillus thuringiensis against Spodoptora littoralis. Z. ang. Ent., 100 425-433. Salama, H.S. ; Foda, M.S. and Sharaby, A. (1986). Possible extension of the activity spectrum of Bacillus thuringiensis through chemical additives. Z. ang. Ent., 101, 304-313. Salama, H.S. ; Foda, M.S. and Sharaby, A. (1989). Potentiation of Bacillus thuringiensis endotoxin against the greasy cutworm Agrotis ypsilon. J. Appl. Ent., 108, 372-380. Salama, H.S. ; Foda, M.S. and Sharaby, A. (1985c). Application of Bacillus thuringiensis and its potency for the control of Spodoptora littoralis in Egypt. Z. ang. Ent. 99 (4), 425-431. Van Mellaert, H. ; Van Rie, J. ; Hofmann,Christina and Reynaerts, A. (1988). Insecticidal crystal proteins from Bacillus thuringiensis : Mode of Action and Expression in Transgenic Plants. Proc. of Conference, Biotech. biological Pesticides and Novel Plant-Pest Resistance for Insect Pest Management, Ithaca, NY, USA, 82-87.

- 116 - The Genetics and Molecular Biology of Bacillus thuringiensis

A.M.M. Ali Genetic Engineering & Biotech. Div., National Research Centre, Dokki, Cairo, Egypt.

ABSTRACT

developing countries Genetic engineering techniques suitable for the strains of Bacillus were attempted in order to develop more efficient high temperature and thuringiensis. These are : B. t. resistance to genes linked to ultraviolet, determination of antibiotic-resistant gene in a genetically endotoxin genes in B. t., cloning of B. t. endotoxin of polyhedrosis stable Azotobacter chrococcum transfomwnt, isolation polyhedrosis virus, like virus from B. t. using B. t. as a host for endotoxin gene to developing a novel method for cloning genomic engineered Azotobacter chrococcum, utilization of genetically gene, in vitro Azotobacter chrococcum strain acquiring B. t. endotoxin fungi, as a new tendency in biological control to some phytopathogenic on B. t. types, effect of inserting different DNA from different sources determination of distribution of B. t. in some Egyptian Governorates, protein crystals B. t. types according to amino acid analysis of their techniques. and construction of B. t. types through genetic engineering

(B.t.) has focused Genetics and Molecular Biology of Bacillus thuringiensis essentially all strains of primarily on the complex plasmid systems harbored by plasmids and the insecticidal this organism, and on the relationship between the toxins produced by the various strains. in the many different The first point of emphasis is the ubiquity of plasmids in both number and size natural isolates of the bacterium and their variability fourteen or more plasmids distribution. Different strains may harbor from two to state of continual change. of different sizes. The plasmids of B. t. are in a dynamic of approaches Evidence from several laboratories, obtained by a combination and southern blotting including plasmid curing and alteration, plasmid transfer,

- 117 - A.M.M. Ali analysis with cloned toxin genes, has led to the general conclusion that the insecticidal toxin genes in different strains are usually located on one or more plasmids and not on the chromosome. For some unknown reason, the plasmids carrying the delta-endotoxin genes are rather large ranging from 44 M daltons to 130 M daltons in size, while the smallest toxin plasmid is the 29-M dalton plasmid of HD-1 (Minnich and Aronson, 1984).

The toxin plasmids may integrate into the chromosome while retaining expression of the toxin gene, and can then be excised from the chromosome to generate new toxin plasmids of novel sizes (Carlton and Gonzalez, 1985 a and b). Strains having integrated toxin plasmids might acquire other toxin plasmids via the natural plasmid transfer process.

Understanding of the genetics of B.t. toxin production advanced considerably during the early 1980's. Toxin genes have been localized on specific plasmids, transferred from one strain to another, and cloned out of several strains. The sequencing of the cloned toxin genes has begun, and the way is now open to other genetic manipulations of toxin genes, whether cloned or in the natural state. The numerous B. t. plasmids, with their toxin and transmissibility genes, their capacity for recombination, and possible transposons and insertion sequences should prove a fruitful field for both basic and applied investigations in the years to come.

The utilization of B. t. in biological control of pests has shown some difficulties in the developing countries and these can be solved through genetic engineering techniques. Some research programmes have been carried out aiming to 1. Obtain high efficient strains for pest control. 2. Obtain stable strains without loss or reduction of their efficiency by UV, temperature or other agents. 3. Produce various types of B.t. specific for the different groups of pests. 4. Reduce the production costs. 5. Overcome insects resistance to endotoxin. 6. Develop microbial systems cheap for determining B. t. efficiencies against different insect species. 7. Develop biological control for pathogenic fungi and bacteria.

- 118 - Genetics of B.t.

In the first article, B. t. strains resistant to high temperature and UV had been selected as single colonies after exposing to high degrees of temperature and/or UV and also through introducing foreign genes from Bacillus sp. which tolerate 50°C and which had been isolated from the soil of Aswan Governorate, Egypt. Unfortunately, reduction in efficiency for pest control had been obtained in these strains which were attributed either to the loss of plasmids carrying endotoxin gene or to some regulatory genetic defect caused by foreign genes.

The second article was by isolating kanamycin resistant strains (resist 100 mg kanamycin/ 100 ml medium) which proved that this gene is linked to endotoxin gene and located in the same plasmid. The growing of these strains in medium supplemented with kanamycin will enrich plasmid contents in the cells and consequently increase the efficiency of endotoxin production.

The B. t. endotoxin gene was cloned to Azotobacter chrococcum strain. Transformants that acquired this gene in their chromosome were isolated. These transformants have stable efficiencies as the gene located in the chromosome and not in plasmids which can be exposed to curing and loss. Moreover, these transformants were able to fix nitrogen and can be used for pest control and fertilization.

it was possible to use B. t. strains as hosts for polyhedrosis virus. This increased their efficiencies.

It was also possible to isolate polyhedrosis like virus from some B. t. strains. The infection of these virus to B. t. increase their efficiencies by isolating lysogenic strains.

As it had been found that these viruses can be lost from the cells through lysis induction by UV or any other agent, a novel method had been done by introducing viral DNA only to cells. It was found that this will not only stop any lysis induction but also acquire the bacterial cells resistance to accept any other virus or foreign DNA.

Fhages carrying endotoxin genes were isolated from B. t. The viral DNA carrying endotoxin genes was used to transform Azotobacter chrococcum. This method help to introduce genes in chromosomes. The transformants were stable

- 119 - A.M.M. Ali and highly efficient in pest control. Moreover, these were found to be able to control biologically some phytopathogenic fungi, e.g., some species of Sclerotium, Rhizoctonia, Fusarium, Aspergillus, Alternaria and Penicillium. This method can also be used to determine the efficiency of B.t.

It was able to change B. t. types through introducing foreign genes from different sources, e.g. Streptomyces thermovirdis, Linen, Cardamom and Fenugreek.

Cheap production for B. t. strains can be made through fermentation of cellulotic plant by-products with specific microorganisms and growing B. t. strains in the fermenting products after autoclaving. In many cases the growth of B. t. was successful.

REFERENCES Carlton, B.C. and Gonzalez, J.M. (1985a). The genetics and molecular biology of Bacillus thuringiensis. In : Molecular Biology of the Bacilli. London Academic Press, 211-289. Carlton, B.C. and Gonzalez, J.M. (1985b). Plasmids and delta-endotoxin production in different subspecies of Bacillus thuringiensis. In. Molecular biology of microbial differentiation. (Eds. by J.A. Hoch and P. Setlow), A. Soc. for Microbiology, Washington, D.C., 252 - 264. Minnich, S.A. and Aronson, A.I. (1984). Regulation of protein synthesis in Bacillus thuringiensis. J. Bacteriol., 158, 447-454. Part II

Production and Utilization Constraints of Bacillus thuringiensis in Developing Countries

Novel Simple Production and Formulation Techniques for Bacillus thuringiensis in Thailand

S. Pantuwatanal , W. Panbangred2 and A. Bhumiratana2 Department of Microbiologyi and Department of Biotechnology2 Faculty of Science, Mahidol University Rama VI Road, Bangkok 10400, Thailand

ABSTRACT

Currently, microbial pesticides especially Bacillus thuringiensis (B. t.) have the greatest potential in intelligently designed and carefully applied pest management programs. However, expanded use of these pesticides will depend heavily on the balance between prodution costs and ecological considerations. Broad range chemical pesticides disrupt ecosystem and affect natural balances in insect populations. One important thing is that the more narrow host range of microorganisms makes them less attractive to industry from a profit perspective. One factor, high cost of shipping from the developed countries, that lead to the development of novel simple production and formulation techniques for B. t. in Thailand. Attempts have been made to find cheap raw materials available in country to use in production and appropriate formulations that are suitable for the application against insect pests have been investigated. Details are discussed.

INTRODUCTION

In recent years, attempts have been made to alternative measures to control insect pests and insect vectors of human diseases. These efforts are primarily due to the development of resistance to chemical insecticides in insect populations, mammalian toxicity, and environmental considerations. Research and development in the area of microbial agents for controlling of insects especially mosquito vectors is rapidly increasing since the discovery of Bacillus thuringiensis subsp. israelensis (B. t. i.) in 1976. However, several strains of Bacillus thuringiensis have been used against insect pests for quite sometime. It has been documented in 1983 that there

- 123 - S. Pantuwatans et at.

are four companies in North America and one in France which are already producing commercial quantities of B. t. i. (Service, 1983).

It has been demonstrated that different strains of subspecies of B. t. have different spectra of insecticidal activity (Dulmage et al., 1990). They, also, produce different activity against the type of insect species that they kill, i.e., subspecies kurstaki (HD-1) is active against many lepidopterous pests, has only very weak activity against mosquitoes, and has no activity against aquatic black flies whereas subspecies israelensis is very active against mosquitoes and blackflies, but has little or no activity against lepidopterous insects (Barjac, 1978a, 1978b, 1978c). The larvicidal activity of these organisms is associated with crystalline parasporal bodies which is called as delta endotoxins. This crystalline parasporal bodies appear in the cells at the time the Bacillus sporulates. The toxins are all high molecular weight protein and their appearance are look alike except their molecular weight and the type of insects they kill. It has been demonstrated that a single isolate may produce more than one toxin, and the spectra of these toxins may differ widely (Dulmage and Cooperators, 1981). In some cases, the physical characteristics of the protein crystal also differ, as in the small, bipyramidal crystal (P2 toxin) of B.t. subsp. kurstaki (HD-1) or B.t.k. described by Yamamoto and McLaughlin (1981).

Since Thailand is located in monsoon region and there are many species of mosquito breeding in Thailand and several species serve as important vectors for certain mosquito borne disease, i.e. dengue hemorrhagic fever, viral encephalitides (Japanese encephalitis virus), filariasis and malaria. It has been demonstrated that many species of these mosquitoes develop resistance to chemical insecticides (Brown, 1986). At present, many concerns on environmental problems created by long residual activity and toxic activity against nontarget organisms in addition to the development of resistance to a number of chemical insecticides have led to the consideration of finding other alternative measures to control mosquito vectors.

B. t. i. and B. sphaericus have been recommended by World Health Organization to be used as an alternative measure for the control of insect vectors. There are several factories in U.S.A. and Europe which produce these two microbial agents commercially for mosquito control. The importataion cost of these microbial insecticides are still too expensive for developing countries such as Thailand due

- 124 - Production of B.t. in Thailand to long distance of transportation. Thus, research and development of local facilities for the production of these microbial agents may be appropriated. It has been demonstrated that these two species can be easily grown in a simple medium but the proper operation is needed according to several factors.

ADVANTAGES OF LOCAL PRODUCTION

There are a number of advantages in promoting development of local production facilities for microbial insecticides in developing countries.

First, the most important advantage of local production concerns stability of the product. One of the drawbacks in using microbial insecticides has been the instability of the microbial agents and the variation in the toxicity of the formulations. This instability is most likely the result of the lengthy shipping periods accompanied by long and variable storage temperatures before the product reached the consumer. It has been demonstrated that these factors have led to the unpopularity of B.t. - based microbial insecticides as the choice for controlling lepidopteran pests. Thus, these similar disadvantages might be encountered in the mosquito control program if the microbial insecticides were to be produced overseas and subsequently imported for use in developing countries. In order to avoid these problems, local production should be encouraged.

The second advantage of local production concerns appropriate formulations. The success in using microbial insecticides to control mosquito larvae relied mainly on appropriate formulations, since individual species of mosquito have different breeding sites and habitats, i.e. Anopheles minimus is found mainly in slow running stream water, Anopheles dirus is commonly found in old gem pits. The application of microbial insecticides in a wide variety of habitats requires appropriate method of application and formulation of B. t. i. and B. sphaericus. However, examining the many formulations of B. t. i. and B. sphaericus available at present, one finds that all have been developed by companies in industrialized nations, with, perhaps, very limited knowledge and few tests appropriate to the actual field condition in tropical countries where the product is most needed. Local production would certainly be highly beneficial in providing material for conducting appropriate field studies and for developing formulations suitable for local environmental conditions. Due to the very different environmental conditions between mosquito

- 125 - - S. Pantuwatana et at.

species and between tropical countries and temperate industrialized countries, there probably will be no single formulation that will be effective for all field conditions.

GROWTH AND CULTIVATING CONDITIONS

In general, bacteria in genus Bacillus do not require a complex growth medium composition, and they are quite easy to be cultivated. Selective media (Yousten, Fretz and Jelley, 1985 ; Roberts and White, 1985) as well as routine media for optimal growth, sporulation and toxin formulation in conventional laboratory conditions, have been worked out (Kalfon et al., 1983). It has been shown that spurting with ordinary air is sufficient for cultivation of Bacillus and that there is no advantage in increasing aeration or even in continuing the expense of aeration after forespore development has been completed (Yousten, Wallis, and Singer, 1984; Yousten, Madehekar, and Wallis, 1984).

There are some evidences that quite drastic modifications in the fermentation can be made without changing the toxin produced. When cells of B. t. subspecies alesti, aizawai, and kurstaki are treated with mutagens to derive asporogenous mutants that would still produce delta - endotoxin (Nishiitsutsuji- Uwo, Wakisaka and Eda, 1975; Wakisaka et al., 1982). The toxins produced are the same as the parent isolates. The apparent reproducibility of the B. t. fermentations is very encouraging and necessary if B. t. fermentations are going to be practical.

However, the relationship between the presence of inclusion bodies and toxicity of B. sphaericus toward mosquito larvae is still somewhat different from those of B. t. species. It has been shown that toxicity of B. sphaericus strain 2297 is increased in correlation with the formation of crystalline inclusions (Kalfon et al., 1984). But certain strains of B. sphaericus may possess toxicity against mosquito larvae in the absence of inclusion bodies. Good growth of B. sphaericus can be obtained by using continuous production techniques, and it has been shown to produce at least 15 to 25 % prespore levels. The final product using this preparation has shown excellent efficacy in operative field spraying (Hertlein et al., 1981).

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POTENTIAL FOR IMPROVEMENT PRODUCTION OF B.t. AND B. sphaericus TOXIN

Selection of Nutrients As stated above, the general requirements of Bacillus for nutrients can vary from species to species. In developing countries such as Thailand, if the fermentation is to be economic, consideration to be made is the cost of nutrients. The cost of nutrient must be cheap. Therefore, it is best to use local available nutrients, either inexpensive raw materials or waste products. It has been documented that several ingredients have been proposed or used for the production of B. t. (Dulmage, Correa and Gallegos-Morales, 1990) as summarized in Table 1.

Table (1) Examples of local inexpensive media ingredients available in developing countries*.

Liquids Coconut milk (waste product) Crude sugar, e.g. jaggery Whey (waste product) Molasses Corn steep liquid Inorganic Nitrogen (NH4)2SO4 Materials of Plant Origin Legumes and other seeds, soya beans, cotton seed meal etc. Cereals, corn, wheat flour, wheat bran etc. Carbohydrate, dextrin, maltose, sucrose, glucose Plant extracts, potato tubers, sweet potato roots, etc. Cassava, yams, sweet potatoes Yeast powder, fodder yeast Materials of Animal (non mammalian) origin Fishmeal Materials of Mammalian origin

_, Blood, chicken slaughter house residue *(From Dulmage, Correa and Gallegos-Morales, 1990) - 127 - S. Pantuwatana et at.

For B. sphaericus, most of the major effort in local production at present is also confined to the search for locally available raw materials for fermentation, and a number of reports have indicated the success in this endeavour. Some examples of raw materials have been documented and summarized (Bhumiratana, 1990) as shown in Table (2).

Table (2)

Examples of raw materials for use in formulating fermentation media for local production of B. sphaericus and B. t. i.

Raw Materials Comment ; Reference

Agricultural Products : Bambara bean, cowpea, groundnut These materials are extracted in cake, soya bean boiling water and added to basal medium containing dried cow blood and mineral salts; Obeta and Okafor, 1983. Ground corn, cottonseed meal, Vandekar and Dulmage, 1983. cassava, yams

Animal products : Blood, beer bones, chicken parts, Vandekar and Dulmage, 1983 animal dung Hertlein et al., 1981

Industrial byproducts : Byproduct from monosodium Dharmsthiti, Pantuwatana, and glutamate factory Bhumiratana, 1985

Other : Coconut milk, corn-steep liquor, Vandekar and Dulmage, 1982 molasses, whey

Most of the studies cited were aimed at obtaining microbial growth similar to or higher than that attained by routine laboratory media. The raw material used included agricultural products. It should be noted that by-products with high sugar content, such as molasses, are not suitable for B. sphaericus cuture, since

- 128 - Production of B.t. in Thailand this Bacillus does not use sugars. In all reports, high cell yields could be demonstrated. However, the preparation of some media required extensive pretreatments such as extraction and/or hydrolysis. In contrast, the hydrolysate- media formulation introduced by Dharmsthiti et al., (1985) possessed several advantages : (1) the media were very easy to prepare and required no pretreatment for any of the constituents ; (2) the media supported high growth and toxicity of B. sphaericus ; and (3) the cost of the major raw material for fermentation was extremely low since it was a by-product from an existing fermentation industry.

The use of media formulated from industrial by-products may result in nutrient fluctuations depending on the quality of the raw material from factory lots. However, this problem can be easily overcome by determining the amount of L-tyrosine in each batch of factory material (called HDL) and making an appropriate dilution. The testing interval for L-tyrosine need not be frequent since each batch of HDL is produced in large quantity and can be stored for very extended periods. Thus, small variations in various factory lots should not create undue difficulties should fermentation process be scaled up.

FERMENTATION

The fermentations of the different strains of B.t. and B. sphaericus regardless of subspecies, have some general characteristics in common. As mentioned above, they have similar requirements for nutrients, however, the individual strains are unique entities, and a particular medium that may support good growth or toxin production by one strain may be less satisfactory for another (Dulmage, 1970,

1971 ; Dulmage and de Barjac, 1973 ; Salama et al., 1983a, 1983b ; Salama, Foda, and Dulmage, 1983 ; and Smith, 1982). Based on the present available data concerning raw materials for production of B. t. and B. sphaericus, it can be concluded that abundant, suitable alternatives to laboratory media can be located readily. Thus, the availability of cheap raw material should not be a major obstacle in the attempt to stimulate local production of the bacterium.

The impetus for promoting a local production capability is not only to make the product available in a relatively short time, but also to reduce the transportation costs, to increase the shelf life, and to reduce the cost so that the locally produced material will be able to compete in both quality and price with an imported product.

- 129 - S. Pantuwatana et al.

Since the cost of the large size fermenter is very high for developing countries, the facility that is currently available in the laboratory is 200 liter fermenter. Thus, the study on the possibility of developing a highly efficient fermentation systems for local production of B. t. i. and B. sphaericus has been carried out in order to produce higher cell yield than the conventional submerged fermentation system. The earlier experiments on the production of B. t. i. had employed the use of "fed batch" and "dialysis" fermentation systems. One problem which is easily to be encountered is the contamination of the systems particularly in the dialysis systems. This problem in contamination will further elevate when attempts were made in scaling-up the production capacity. Therefore, experiments were set up in attempting to use cell recycle with membrane attachment system in production of B. t. i. and B. sphaericus. The system was done by growing bacteria in the conventional fermenter. After 24 h. fermentation period, 4 liters of the cultures were processed through milliopore membrane (pore size 0.45 micron) filter unit. The filtrate was discarded, whereas the cells were recycled back into the fermenter. Subsequently, four liters of freshly sterile medium of the same composition was added into the fermenter to obtain the same original volume of 6 liters. The cycles of cell-recycling were repeated in the same manner after 24, 48, 72, 96 and 120 hours of fermentation periods. Data as indicated in Table 3 showed that after running the systems for 5 cycles or 120 hr period, the numbers of vegetative cells and spores were found to increase to 1 x 1013 cells/ml and 5.9 x 1011 spores/ml, respectively. If one used this data to calculate the productivity based on the amount of cells and/or spore yields per day, it could be clearly shown that the productivity using cell recycle system is much higher than the conventional batch-type submerged fermentation svstem.

Similar results were obtained when B. sphaericus was used (Table 4). However, the yield was lower than those of B. t. i. This is probably due to the difference in size and metabolic pathway between these two organisms. Moreover, B. sphaericus is more active motile in the broth than B. t. i. The numbers of vegetative cells and spores increased in parallel to larvicidal activity against Gr. quinquefasciatus larvae.

Both of hydrolysate by-product obtained from monosodium glutamate and L-lysine factories were used successfully for the production of B. t. i. and B. sphaericus after minor modification of their compositions. The modification was done by adjusting the pH to 7.5 with 0.05 % K2HPO4. The medium developed

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Table (3) Production of B. t. i. using cell recycle system.

Cycle Time Volume Concentration Viable cell Spore count (No.) (h) (liter) factor count (cfu/ml) (cfu/ml)

1 24 6 none - -

24 2 3 x 2.8 x 1011 2.3 x 109 2 48 6 none - 1.6 x 1010 48 2 3x - 7.5x1010 3 72 6 none - - 72 2 3x 7.0x1011 4.9x1010 4 96 6 none - - 96 2 3x 2.1x1012 7.0x109

5 120 6 none - - 120 2 3 x 1.0 x 1013 5.9 x 1011

Table (4) Production of B. sphaericus using cell recycle system.

Cycle Time Volume Concentration Viable cell LC50 vs. (No.) (h) (liter) factor count (cfu/ml) cfu/ml Cx-quin*

1 24 6 none 2.9 x 107 1.2 x 103 24 2 3 x 7.0 x 107 4.2 x 103 2 48 6 none 4.0 x 101 2.8 x 104

48 2 3 x 7.5 x 109 1.2 x 104

3 72 6 none 7.0 x 107 1.9 x 103

72 2 3 x 1.0 x 108 3.9 x 102 4 96 6 none 7.8 x 108 1.6 x 103 96 2 3 x 1.2 x 109 1.6 x 102

5 120 6 none 9.4 x 108 1.7 x 103

120 2 3 x 2.3 x 109 4.4 x 103

*cfu = colony forming unit; Cx. quin = Culex _quinquefasciatus 131 - S. Pantuwatana et al.

for cultivating B. t. i. was composed of 4% hydrolyzed liquor by-product from a monosodium glutamate factory whereas 7% hydrolyzed liquid by-product was used for cultivating B. sphaericus. In the case of B. sphaericus, yeast extract was added in the amount to make a final concentration of 0.05%. The use of cell recycle system has proven to be superior than the "fed batch" or "dialysis" systems. The superiority of cell recycle system relied on the fact that the cell yield as well as the productivity of the system were quite high, there was no problem with contamination after many repeated cycles, and the system was relatively easy to control. Preliminary experiments at 50-liter fermenter also indicated that the performance obtained by using cell-recycle system was also as effective as at the 5-liter fermenter. It appeared that the use of cell recycle system can be applicable to local production of B.t.i. and B. sphaericus at industrial level. Further development is needed to be done to determine the optimal conditions.

FORMULATION

Since different species of target organisms have different behavior and habitats, appropriate formulations are needed to suit their characteristics. Several formulations have been developed and evaluated against Anopheles dirus, Culex quinquefasciatus and Culex spp. both in the laboratory and field conditions. It was demonstrated that the floating briquets formulation gave a good effective levels at the water's surface for a period of 21 days or more. The granular form gave a good activity in dense vegetation area because it could spread and penetrate very well on the water's surface. However, the residual activity released from the granular form on the water surface lasted for 6 days in the field conditions due to the rate of sinking to the bottom of water. The carrier used in these two formulations was ground coconut husk and ground corncob which had been completely dried. The binding agent for making briquet was the rubber glue (latex).

The carrier and glue were mixed in the ratio of 3 : 2 and pressed under the pressure of 5,000 lb/surface area using a hydraulic press. After the briquets were dried, they were soaked in the broth suspenion of bacterial culture obtained from fermenter and then dried at room temperature conditions. Further studies are planed to carry out in order to find the optimal conditions to produce highly effective products.

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STANDARDIZATION

To date, it is very difficult to express the quantity of delta-endotoxin in a formulation in terms of weight of toxin since there is no direct chemical assay for the delta-endotoxin. The purification of delta-endotoxin free from spores is also very difficult, thus, the measurement of the potency of the material containing the toxin and express activity in terms of an insecticidal unit derived by comparing the activity of the sample with that of a standard formulation. Potencies are then expressed in International Units (IU) in the case of formulations of subsp. kurstaki or in International Toxicity Units (ITU) in the case of formulations of subsp. israelensis (H-14). The uniformity of production should be taken into consideration.

REFERENCES Barjac, H. de. (1987a). A new candidate for biological control of mosquitoes Bacillus thuringiensis var. israelensis. Entomophaga, 23, 209-219. Barjac, H. de. (1978b). Toxicity of Bacillus thuringiensis var. israelensis for larvae of Aedes aegypti and Anopheles stephensi. C.R. Acad, Sci. (Paris), 286 D, 1175- 1178. Barjac, H. de. (1978c). Une nouvelle variete de Bacillus thuringiensis tris toxique pour les moustiques : Bacillus thuringiensis var. israelensis serotype, 14. C.R. Acad. Sci. (Paris). 286 D, 797-800. Bhumiratana, A. (1990). Local production of Bacillus sphaericus. Ch. 17 in "Bacterial Control of Mosquitoes and Black flies : Biochemistry, Genetics and Applications of Bacillus thuringiensis israelensis"ed. by H. de Barjac and D.J. Sutherland. Rutger University Press, New Brunswick., 272-283. Brown, A. W. A. (1986). Insecticide resistance in mosquitoes : a pragmatic review. J. Am. Mosq. Control Assoc., 2, 123-140. Dharmsthiti, S.C. ; Pantuwatana, S. and Bhumiratana, A. (1985). Production of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus 1593 on media using a by-product from a monosodium glutamate factory. J. Invertebr. Pathol., 46, 231-238. Dulmage, H. T. (1970). Production of the spore-endotoxin complex by variants of Bacillus thuringiensis in two fermentation media. J. Invertebr. Pathol., 16, 385- 389. Dulmage, H. T. (1971). Production of delta-endotoxin by eighteen isolates of Bacillus thuringiensis, serotype 3, in 3 fermentation media. J. Invertebr. Pathol., 18, 353- 358.

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Dulmage, H.T. and Barjac, H., de. (1973). HD-187 : A new isolate of Bacillus thuringiensis that produces high yields of delta-endotoxin. J. Invertebr. Pathol., 22, 273-277. Dulmage, H. T. et al. (1981). Insecticidal activity of isolates of Bacillus thuringiensis and their potential for pest control. In "Microbial Control of pests and plant diseases, 1970-1980." ed. H. D. Burges, Academic Press : London., 191-220. Dulmage, H.T. ; Correa, J.A. and Gallegos Morales, G. (1990). Potential for improved formulations of Bacillus thuringiensis through standardization and fermentation Development. Ch. 8 in "Bacterial Control of Mosquitoes and Black flies : Biochemistry, Genetics and Applications of Bacillus thuringiensis israelensis" ed. by H. de Barjac and D.J. Sutherland. Rutger University Press, New Brunswick., 110-113. Hertlein, B. C. ; Hornby, J. ; Levy, R. and Miller, T. W., Jr. (1981). Prospects of spore-forming bacteria for vector control with special emphasis on their local production potential. Dev. Industr. Microbiol., 22, 53-60. Kalfon, A. ; Larget-Thiery, I. ; Charles, J. F. and Barjac, H., de (1983). Growth, sporulation and larvicidal activity of Bacillus sphaericus. Eur. J. Appl. Microbiol. Biotechnol., 18, 168-173. Kalfon, A. ; Charles, J. F. ; Bourgouin, C. and Barjac, H., de. (1984). Sporulation of Bacillus sphaericus 2297 : An electron microscope study of crystal- like inclusion biogenesis and toxicity to mosquito larvae. J. Gen. Microbiol., 130, 893-900. Massie, J. ; Roberts, G. and White, P. J. (1985). Selective isolation of Bacillus sphaericus from the soil by use of acetate as the only major source of carbon. Appl. Environ. Microbiol., 49, 1478-1481. Niishiitsutsuji-Uwo, J. ; Wakisaka, K. and Eda, M. (1975). Sporeless mutants of Bacillus thuringiensis. J. Invertebr. Pathol., 25, 355-361. Salama, H.S. ; Foda, M.S. ; F1-Sharaby, A. and Selim, M.H. (1983a). A novel approach for whey recycling in production of bacterial insecticides. Entomophaga, 28, 151-160. Salama, H.S. ; Foda, M.S. ; Selim, M.H. and El-Sharaby, A.M. (1983b). Utilization of fodder yeast and agro-industrial by-products in production of spores and bilolgically-active endotoxins from Bacillus thuringiensis. Zbl. Microbiol., 138, 553-563. Salama, H.S. ; Foda, M.S. and Dulmage, H.T. (1983). Novel fermentation media for production of delta-endotoxins from Bacillus thuringiensis. J. Invertebr. Pathol., 41, 8-19. Service, M. W. (1983). Biological Control of Mosquitoes-has it future ? Mosquito News. 43 : 113-120.

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Smith, R.A. (1982). Effect of strain and medium variation on mosquito toxin production by Bacillus thuringiensis var. israelensis. Can. J. Microbiol., 28, 1089-1092. Wakisaka, Y. ; Masaki, E. ; Koizumi, K. ; Nishimoto, Y. ; Endo, Y. ; Nishimura, M.S. and Nishiitsutsuji-Uwo, J. (1982). Asporogenous Bacillus thuringiensis mutant producing high yields of delta-endotoxin. Appl. Environ. Microbiol., 43, 1498-1500. Wakisaka, Y. ; Masahi, E. and Nishimoto, Y. (1982). Formation of crystalline delta-endotoxin or poly-B-hydroxybutyric acid granules by asporogenous mutants of Bacillus thuringiensis. Appl. Environ. Microbiol., 43, 1473-1480. Yamamoto, T. and Mclaughlin, R. E. (1981). Isolation of protein from the parasporal crystal of Bacillus thuringiensis var. kurstaki toxic to the mosquito larvae, Aedes taeniorhynchus. Biochem. Biophys. Res. Commun., 103, 414-421. Yousten, A.A. ; Wallis, D.A. and Singer, S. 1984. Effect of oxygen on growth, sporulation, and mosquito larval toxin formation by Bacillus sphaericus 1593. Curr. Microbiol., 11, 175-178. Yousten, A. A. ; Madehekar, N. and Wallis, D. (1984). Fermentation condition affecting growth sporulation, and mosquito larval toxin formation by Bacillus sphaericus. Dev. Industr. Microbiol., 25, 757-762. Yousten, A.A. ; Fretz, S.B. and Jelley, S.A. (1985). Selective medium for mosquito pathogenic strains of Bacillus sphaericus. Appl. Environ. Microbiol., 49, 1532-1533.

Development of a High Productivity Process for the Production of Bioinsecticides by Bacillus thuringiensis

M. Rodriguez, E. Razo, J. Villafana, E. Urquijo and M. de la Torre* Department of Biotechnology. Cinvestan. P.O. Box 14-740. Mexico City 07000. Mexico

ABSTRACT

These studies have been devoted to optimize culture medium and fermentation conditions to increase yield and productivity of Bacillus thuringiensis (B. t.) production and to establish the criteria for scale up. We use continuous culture techniques to obtain kinetic and yield parameters of B. t. kurstaki (HD- 73). The fed batch procedure applied produces 2.8 x 1010 spores/ml in 23.5 ,hours of fermentation with a spore : crystal ratio 1 : 1. Oxygen Transfer Rate (OTR) seems to be an adequate criterion for scale up while centrifugation orfiltration are technically feasible for product recovery.

INTRODUCTION

There are two main constrains for widely use of Bacillus thringiensis (B.t.)

in developing countries : manipulation and price. First one requires trained people, knowledge of pest biology and well defined strategies for an integrated insect control program, while the second implies research on process to reduce production cost of B. t. However, B. t. use will increase in near future due to ecology pressure, free trade agreements and insect pests resistance to chemical insecticides.

In terms of commercial scale production of B. t. insecticides, submerged fermentation technology is developed, although publicly available information since the initial patent disclosure, is scant. On the other hand, relatively little effort has been devoted to refinement of substrates and fermentation conditions for

* To whom all correspondence should be addressed.

- 137 - M. Rodriguez et al. increased activity yield in addition to some few studies of process analysis (Rowe and Margaritis, 1987).

Therefore we decided to study substrates and fermentation conditions to increase productivity and activity as well as down stream processing schemes and scale up criteria.

MATERIALS AND METHODS

Microorganism and Culture Conditions.

B. t. var. kurstaki strain HD-73 was obtained from Cotton Research Labs. Brownsville, Texas. The strain was maintained on nutrient agar slants. The culture medium (batch and continuous culture) was prepared as described by de Urquijo (1987). For fed batch experiments a 5 fold concentrated medium was used. The inoculum for the fermentors was grown in Erlenmeyer flask and was incubated for 7 hours at 30°C. A 2% (v/v) inoculum ratio was used. Fermentations were conduced at 30°C and pH 7.0 and ended when more than 90% of the cells had sporulated.

The Fermenters

For laboratory test a 14 L New Brunswick Scientific Co. model CFS-314 was used. The pilot plant fermenter was a 80.1 cm diameter, stirred reactor with HL/D = 1.87 and working volume of 700 L.

Fed Batch It was done in the 14 L fermenter with an initial flow of 1 L/h and a flow rate change of I L/h2.

Oxygen Transfer Rate and KLa

The balance method described by Murkopadhyay and Ghose (1976) was used.

Centrifugation Test A Westfalia separator centrifuge model SA 1-02-175 with an equivalent area of 1382 m2 was used.

- 138 - Production of B.t. in Mexico

Filtration Test An EIMCO leaf filter test with a filtration surface of 0.1 ft2 was utilized. Analytical Methods Bacteria biomass was estimated by counting the total number of cells and multiplying it by the cell unit weight (2.5 x 10-12 g/cell). Glucose was analyzed by DNS method (Miller, 1959). Spores were counted in a Neubauer chamber. Biomass yield was based on glucose consumption.

RESULTS AND DISCUSSION

Studies were first conducted on the optimization of culture medium and culture conditions. Main goal was to use cheap protein source (de Urquijo 1987). It was possible to reach a cell concentration of 13.4 g/L with 9007o sporulation and a total fermentation time of 25 hours. A crystal : spore rate I : 1 was found and the product (remain solids of broth, cell debris, spores, crystals and cells) had a toxicity of CL50 = 166 pg/cm2 against Manduca sexta (Razo, 1991). Continuous Culture

. In order to get kinetic and yield parameters for B. t. growing on glucose, continuous culture experiments were realized. An X-D diagram showed that B.t. has different metabolic states for dilution rates (D) of 0.1 to 0.59 h-1, at D 0.2 h-1 there were cells, sporulating cells and free spores (Rodriguez, 1991). Similar results were previously reported for Bacillus subtilis (Dawes and Mandelstan, 1969, Dawes and Thorley, 1970).

In order to get kinetics data for vegetative cells growing on glucose a D interval of 0.25 to 0.45 h- I was selected (Figure 1). The kinetic parameters obtained are shown in Table (1). The biomass yield data reported in literature are very different and even an author reports very wide intervals, there are values higher and lower than 0.5. Extremely high values indicate that B. t. is using another carbon source in addition to glucose while lower values suggest the accumulation of some metabolic products. Rowe (1990) found that B. t. accumulates piruvate, butirate, lactate, acetate and poly beta hydroxy butirate under different culture conditions, even more, it is able to metabolize them as well as amino acids. Therefore it might be possible that very different Yx/S and specific growth rates values correspond to the utilization of different substrates and the accumulation of metabolic products. However most of the kinetic studies of B. t. do not take into account accumulation of metabolic products or consumption of different carbon sources.

- 139 - M. Rodriguez et al.

In our studies we found that Yx/, for glucose varies in function of the dilution rate but for the chosen interval it is constant and near to 0.5. True oxygen yield (Y02) was 0.73 g cell/g oxygen and maintenance energy was 0.065 g glucose/g cell-hour.

Residual glucose ( g/L) 14

12

10

8

6

4

2

0.20 0.23 0.23 0.29 0.32 0.35 0.38 0.41 0.44 0.47 Dilution rate (L/hr. )

Yxis ( g B.t. /g Glucose )

120

100

80

60

40 0.2 20

0.20 0.23 0.23 0.29 0.32 0.35 0.38 0.41 0.44 0.47 Dilution rate (L/hr. )

Figure 1. X-D Diagram for Bacillus thuringiensis

- 140 - Production of B.t. in Mexico

Table (1) Kinetic parameters of Bacillus thuringiensis

Variety yx/s Amax ks (1/h) (mg/ml)

kurstaki 1990 0.56-1.98 0.6-1.1 - Anderson kurstaki 0.30-0.52 0.47-0.77 - Arcas 1987 Berliner - 0.29-0.83 - Goldberg 1980 thuringiensis - 0.70-1.90 1.8.2.0 Holmberg 1980 kurstaki 0.60-0.67 0.48-0.51 - Rowe 1990 kurstaki 0.32-0.41 0.60-0.78 - this work akurstaki b0.43 1.21 0.48 this work a continuous culture - b true yield for glucose

Fed Batch Culture The kinetic parameters for B.t. growing on glucose were used for fed batch simulations in order to find out the best feeding conditions (flow rate and nutrients concentration) to get a maximal biomass productivity according to our fermentation system. A linear flow was chosen and experimental results are shown in Figure (2) During batch culture, glucose was not consumed and it was accumulated during feeding stage, therefore we assume that the bacteria metabolized another substrate, may be amino acids and peptides. Exponential growth was followed by linear growth and during this last stage, glucose was metabolized, however it was not exhausted at the end of growth phase. Therefore, the sporulation stage was elongated and earlier studies indicated that the remaining glucose has this effect (de Urquijo 1987). Other fed batch experiments were done taking off glucose from feeding, the results are summarized in Table (2). In all cases the fermentation was ended when more than 90% of cells had sporulated. In fed batch culture without glucose feeding, 2.7 x 1010 spores/ml were obtained in 23.5 hr., these results are similar to those reported by Arcas et al. (1987). The toxicity of the product against Manduca sexta was alike to that of batch product.

- 141 - M. Rodriguez et al.

Biomass ( g

Fed Batch 300 batch

250 i 200

150 -4., Feed

100 M Yx/s

50 0.5

0 5 10 15 20 25 30 35 Time (hr.)

Glucose ( g )

1200

1000

800 600 f 400

T 200 I '

0 5 10 15 20 25 30 35 Time (hr.)

Figure 2. Fed batch culture of Bacillus thuringiensis.

- 142 - Production of B.t. in Mexico

Table (2) Comparison between batch and fed batch cultures of B. t.

Biomass Biomass Time (g/L) productivity (h) (g/L-h)

Batch 13.4 0.53 25 Fed batch simulation 49.8 1.99 25 Fed batch with 27.7 0.72 38.5 glucose feeding Fed batch without 23.5 0.92 25.5 glucose feeding

Scale Up

In order to use kLa or oxygen transfer rate (OTR) as scale up criterion, the effect of kLa on spore production in batch culture was studied. Results show a linear correlation (Figure 3). Earlier reports established that oxygen was important for growth and sporulation of B. t. (Holmberg et al., 1980 and Anderson, 1990).

Batch culture was scaled up from 14 L fermenter to 1100 L fermenter at constant oxygen transfer rate (Table 3). Spores concentration and fermentation time were similar, nevertheless yxj, and maximal specific growth rate were different as well as sporulation stage interval. The last one was shorter for the largest fermenter. Holmberg et al. (1980) also found a reduction in specific growth rate of B. t. when they scaled up from 8 L to 1000 L fermenter. Experiments have been carried out to find out the reasons of this behavior.

Recovery Different recovery systems were studied in order to recover spores and crystals together with cell debris, cells and remainent solids. First, the effect of pH, temperature and flocculent agents on the critical sedimentation rate of the solids of fermented broth was investigated. Under all

- 143 - M. Rodriguez et al.

Xs x 109

5

4

3

2 50 100 150 200 250 300 350 KLa (L/hr.) Figure 3. Relationship between spore production and kLa for Bacillus thuringiensis.

Table (3) Scale up of B.t. fermentation at constant OTR.

14 L 1100 L

OTR (g/L-h) 1.75 1.73 Xe (spores/ml) 4.8 x 109 5.1 x 109 xx/s 0.36 0.41 Pv (spores/L-h) 1.9 x 101, 2.2 x 1011 u (L/h) 0.78 0.67 Time (h) 25.5 23

- 144 - Production of B.t. in Mexico conditions tested, the critical sedimentation rate was very low and the best value correspond to pH 4.0. At this pH, the critical sedimentation rate was 1.9 x 10-8 m/s compared to 7 x 10-8 m/s for pH 7.0 (Villafana, 1990). This effect was previously reported by Cords and Fisher (1966). Therefore, for recovery purposes broth was acidified till pH 4.0, this process did not modify the toxicity of the product (Villafana, 1990). Consecutively several runs were performed on the Westfalia Separator Centrifuge (Figure 4). The centrifuge performance deteriorated when the sludge holding space was full. That was evidenced by solid losses on the overflow stream and consequently the solid recovery efficiency decreased. On the other hand, short time discharge intervals reduced the solids content of the concentrated stream. Thus, the selection of the solids time discharge interval is a compromise between good clarification and high solids content in the separated sludge. The highest concentration was 7.2-7.507o (DW) with a recovery efficiency of 99% of spores. Zamola et al., (1981) reported solid contents of 9-15% dry weight when recovering B. t. by centrifugation. Although these concentrations are higher than ours, their losses (about 12%) are higher too. This may be attributed to differences in solids characteristics (packed volume).

Spore recovery (%)

100 [ o--so-SL3--a*-X --X

80

60

40

® Flow 13 L /hr. 20 X Flow 18 L /hr. D Flow 30 L /hr.

0 2 4 6 8 10 12 14 16 Discharge frequency(L/min.)

Figure 4. Influence of flow rate on spore recovery efficiency for Bacillus thuringiensis. - 145 - M. Rodriguez et at.

Filtration

The broth filtration rate was very slow bue to the fast filter surface blocking by the solids cake. Therefore, a continuous rotatory drum filter was simulated by using a leaf test filter with a polyester sheet and a precoat of decalite 4127. The best results were achieved with a 84 seconds cycle (immersion 31 seconds and 44 seconds for drying). M Under these conditions, filtration rate was 187.2 L/h - Z with a spore recovery of 99.5°10. This filtration rate is similar to those reported for Streptomyces fradie and Bacillus subtilis (Villafana, 1990).

CONCLUSIONS

Because of very complex metabolism of B. t. any kinetic study must take into account the metabolites production and consumption, as well as, all compounds that can be used as carbon and energy sources by the bacteria.

Fed batch culture is a good alternative for production of bioinsecticides by B. t. but one must be very careful because spore productivity could fall down and therefore toxin productivity, if glucose is not consumed as predicted. Models for B. t. fermentation should take into account the different metabolic states of the bacteria according to the culture conditions.

Oxygen transfer rate or kLa might be a criterion for scaling up B. t. fermentation, but there are some other factors that should considered.

Either centrifugation or filtration can be used for B. t. crystal and spores recovery when some insoluble proteins remained in the broth if pH is adjusted to 4.

ACKNOWLEDGEMENTS

This work was partially supported by IDRC under Contract B. t. U W O/NICARAGUA/MEXICO.

REFERENCES

Anderson, T. B. (1990). Effects of carbon nitrogen ratio and oxygen on the growth kinetics of Bacillus thuringiensis and yield bioinsecticidal crystal protein. M.Sc. thesis, University of Western Ontario, Canada.

- 146 - Production of B.t. in Mexico

Areas, J. ; Yantorno, O. and Ertola, R. (1987). Effect of high concentration of nutrients on Bacillus thuringiensis cultures. Biotechnol. Lett., 9 (2), 105-110. Cords, H. and Fisher, R.A. (1966). Stable concentrate bacterial insecticide suspensions. USA patent 3 2 71 243. Dawes, I.W. and Mandelstam, J. (1969). Biochemistry of sporulation of Bacillus subtilis. Continuous culture studies. In : Malek, 1. ; Beyan, K. ; Fenel, Z. ; Munk, V. ; Ricica, J. and Smrkova, H. (eds.) Continuous Cultivation of Microorganisms. Acad. Press, 157-162. Dawes, I.W. ; Kay, D. and Mandelstam, J. (1970). Sporulation in Bacillus subtilis. The theoretical and experimental studies in continuous culture systems. J. Gen. Microbiol., 62, 49-66. Holmberg, A. ; Sievanen, R. and Carlberg, G. (1980). Fermentation of Bacillus thuringiensis for exotoxin production : Process analysis study. Biotechnol. Bioeng., 22, 1707-1724. Miller, G.L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugars. Anal. Chem. 31, 426 - 428. Rowe, G.E. and Margaritis, A. (1987). Bioprocess development in the production of bioinsecticides by Bacillus thuringiensis. Crit. Rev. Biotechnol., 6 (1), 87-127. Mukhopadhyay, S.N. and Ghose, T.K. (1976). A simple dynamic method of kLa determination in laboratory fermenter. J. Ferment. Technol., 54, 405-419. Razo, E. (1991). Estudios para el escalamiento de un proceso por Tote de nivel de laboratorio a planta pilot para la produccion de Bacillus thuringiensis. M.Sc. thesis. Unam., Mexico. de Urquijo, E. (1987). Produccion de Bacillus thuringiensis para el control de ciertas plagas agricolas y medicas de importancia en Mexico. M.Sc. thesis. Cinvestav. Mexico. Villafana, J. (1990). Evaluacion de alternativas para la recuperaci6n de los productos entomopatogenos de Bacillus thuringiensis. M.Sc. thesis, Cinvestav. Mexico. Zamola, B. ; Valles P. ; Melig, Miecoli P. and Kajfez, F. (1981). Use of the centrifugal separation based on Bacillus thuringiensis. Biotechnol. Bioeng., 23, 1079-1086.

Local Production of Bacillus thuringiensis in Egypt : Advantages and Constraints

M. S. Foda, H. S. Salama, and M. Fadel Microbial Chemistry and Plant Protection Departments, National Research Centre, Cairo, Egypt.

ABSTRACT

Pilot studies on Bacillus thuringiensis (B. t.) production were carried out using a mobile submerged fermentation unit located in a sugar factory near Cairo. Fermentation media based on locally available agro-industrial byproducts were successfully used for production of B. t. vars. galleriae HD-234 and HD-282 that were active in field studies against Spodoptera littoralis infesting cotton and soyabean crops. Problems encountered in endotoxin precipitation and drying are discussed in the light of some developed feasible solutions.

INTRODUCTION

Although the commercial products and formulations based on Bacillus thuringiensis as a biological control agent are now available in the market, yet they are too costly for the use in most developing countries. The high cost of the biological control agents is due mainly to production being located in the developed countries such as Europe and North America where production costs are higher and also due to the expenses paid in transportation to the operational sites. Thus, the production of such B.t. based formulations in the countries where the insect pests are endemic, should significantly reduce the cost of pest control. It will also help to develop local fermentation industries in the developing countries.

In Egypt where applied B. t. research has been going for more than 14 years and where several insect pests of field crops, oil-seed crops, and vegetables have become endemic, it was necessary to take the decision of trying to establish a pilot production line of B. t. formulations based upon the locally available media ingredients and with the assistance of a local industrial company.

- 149 - M.S. Foda el al.

Thus, to achieve this goal, a survey study was conducted as to select the most suitable location for a pilot scale production line. In such a location, it is assumed that the available facilities would permit the implementation of the preliminary fermentation studies on a suitable scale. The result of the survey study has indicated that the most suitable location for this goal is, thus far, the Sugar and Distillary Company located in Hawamdia, a suburb of Giza City and lies about 20 km south to the National Research Centre of Egypt.

The following factors were decisive in the selection of the sugar and Distillary Company as the suitable site for establishing and implementing a pilot scale production line for B. t. formulations.

a) The location is near to the NRC, thus, facilitates the direct contact and supervision on the fermentation processes. b) The company possesses a large mobile fermentation unit (up to 5 m3 capacity) equipped with necessary accessories required for carrying out the fermentation process, centrifugation and harvest and drying of the product. The unit, though designed for yeast production, constituted in our opinion a prototype of a fermentation line that could be adjusted and modified to fit our purpose of B. t. production. c) The Sugar and Distillary Company is known to produce fodder yeast and molasses that could be used as cheap local byproducts for making of the fermentation media.

BRIEF DESCRIPTION OF THE INDIVIDUAL COMPONENTS OF THE. MOBILE FERMENTATION UNIT

(A) Paddle-wheel Fermenter

Technical data :

- Length 2.15 m - Width 1.40 m - Height 2.12 m - Total volume 5400 liter - Useful volume 1000 liter - Pressure atmospheric

- 150 - Production of B.t. in Egypt

- Type of agitator perforated paddle-wheel - Speed 29.5 r.p.m. - Agitator drive geared motor I1 kw The paddle-wheel fermentor is a rectangular vessel, the bottom of which is adapted to the agitating system so as to ensure that the entire quantity of fermentation medium is stirred by the agitator and that no dead space remains. A horizontally supported paddle wheel serves as stirring element. Three sets of 9 perforated paddles are arranged on the shaft and are offset by 13°C. This agitating system ensures thorough mixing of the liquid and gas. The droplet portion in the gas space of the reactor allows the organism grown to be well supplied with oxygen. The air is laterally fed in the central fermentation space at a slight positive pressure (0.1 bar). The fermentor is cooled by an external cooling cycle. Part of the culture medium is supplied into a laterally arranged pump receiver by means of the paddle wheel. From this receiver, it is returned to the upper fermentor chamber by a centrifugal pump after having passed a plate-type heat exchanger. Due to the fact that this is the only receiver where a certain minimum liquid level is available at all times, it is here where the temperature and pH measurements are performed. In the remaining reactor part, the liquid is dispersed by the paddle-wheel in the form of droplets.

(B) Fedder Water Chiller

The air-cooled water chiller is of a special mobile design for out door use. It serves for dissipating the process heat released during fermentation. Water is used as a coolant. The refrigeration capacity is 120.000 K.J./hr at a water inlet temperature of 14°C and a chilled water outlet temperature of 8°C. The water chiller consists of four main components namely, the compressor, condenser, evaporator and the control box.

(C) Compressed Air Set

The compressed-air set serves for the generation of oil-free compressed air for pneumatic temperature control of the fermenter and for the fluidized-air dryer. The set is designed for outdoor location. It consists of the following essential items : which - Non-lubricated compressor : it is a two-stage reciprocating compressor generates compressed air of 8-10 bar.

- 151 - M.S. Fods et al.

- Low-temperature dryer : where the air is cooled to 10°C above the compressor suction temperature and dewatered.

- Compressed-air reservoir : it has a volume of 350 liter and is designed for a final pressure of Pe = 11 bar. The final pressure downstream is further reduced to Pe = 6 bar.

- Switch box : which contains the entire control system as well as the contactors.

(D) Lateral Channel Blower

The lateral channel blower generates oil free compressed air which serves for aeration of the fermentor. The air is withdrawn via a fine filter, which separates any impurities larger than 5 pm, and via a cooler and forced via a sterile filter into the fermente r. The lateral channel blower is provided by a protective safety valve. At 20°C, the discharge rate is 150 m3/hr without back-pressure and 72 m3/hr at a back- pressure of 0.2 bar.

(E) Decanter Centrifuge

The decanter consists of a horizontal conical rotor which is provided with an internal screw conveyor for continuous discharge of the solids. The screw conveyor moves in the same direction as the rotor with a slower speed. The drum speed is 3390 r.p.m. The screw conveyor speed is 2924 r.p.m. The speed of the drum and conveyor is adjustable by several V-belt palleys. (F) Fluidized-bed Spray Granulator

The spray granulator serves for the drying and granulation of the yeast paste (possibly B. t. to be tried) with solids concentration of 20-25%. It can be operated both batchwise and continuously. The spray granulator consists of a cylindrical filter housing with conical product container. The product container is fed with some granulated product. Heated air is blown through the product from below, which causes the product to be fluidized. The liquid product is sprayed through a nozzle onto this fluidized bed. The water evaporates while granulates form in the steadily growing fluidized bed. These granulates which are reduced to the optimum size by means of a disintegrator, can be discharged via a discharge device (continuous operation). The air required for fluidization and evaporation of water is withdrawn from the outside via a fan cleaned in the coarse dust filter and heated in the air heater. The air is then discharged into the atmosphere via an outlet filter. - 152 - Production of B.t. in Egypt

The technical data of the fluidized-bed spray granulator are as follows - Nominal capacity 20 kg water evaporation/hr - Drying temerature : 130°C (adjustable) - Heating capacity 54 kw - Air rate 0.3 m3/s.

(G) Continuous-flow Heater

The continuous flow heater serves to supply the fermentor with warm water of 40-55°C. At the start of a production process, the fermentation temperature can be set via the continuous-flow heater. The continuous-flow heater is automatically switched on and off upon the use of a special valve.

(H) Tanks Associated with the Mobile Fermentation Unit

A number of calibrated six tanks, and of different sizes are incorporated within the mobile fermentation unit. These are : capacity 1000 liters 1 - Dissolving tank for nutrient medium 2 - Substrate receiver tank capacity 100 liters liters 3 - Acid receiver tank capacity 50 4 - Substrate metering vessel capacity 5 liters 5 - Harvest tank capacity 400 liters 6 - Thermolysis tank capacity 100 liters (I) Pumps in the Mobile Fermentation Unit The various media and ingredients are conveyed by means of centrifugal and positive-dispalcement pumps. A total of six pumps are associated with the mobile fermentation unit. Those are one nutrient medium, one molasses pump, one cooling water circulating pump and three yeast (product) suspension pumps with different discharge rates.

(J) Measuring Instruments and Control Means

The measuring instruments and control means associated with the mobile fermentation unit are as follows

- Temperature control loop : The heat obtained during the fermentation process

- 153 - M.S. Foda et al.

is dissipitated by the temperature control loop. The control loop consists of the electric and pneumatic instrumentation and the external cooling cycle which comprises plate-type heat exchanger and the chilled water system.

- Temperature indicator : It is a resistance thermometer that indicates the actual temperature inside the fermenter.

- Temperature controller : It serves to indicate temperature measured, to compare it with adjustable control variable (set point is the desired fermentation temperature) and to control temperature as required.

- pH measurement : Measuring of the pH value serves to set the desired pH for the nutrient solution and to monitor the pH in the fermentor. The two measuring chains consists of : pH electrode and a transmitter. The pH electrode has a measuring range of pH 2-12. It is protected by a metal cage. The pH value is indicated on the transmitter which also serves for the calibration of the pH electrode. The transmitter is protected by a thermatically sealed housing, so that it can be used outside. The electrode can be adjusted by means of a magnetic tool even when the housing is in closed state.

- Carbon dioxide analyzer : It continuously measures the CO2 concentration in the fermente r outlet air. The pump located in the analyzer withdraws 50 liters outlet air per hour which is analyzed according to the infrared photometric method. The measuring range covers 0-10% by volume COQ.

(K) Energy of the Mobile Fermentation Unit

The unit is supplied with electric power by a Diesel set. The set consists of Diesel engine, three phase synchronous generator and switch gear. If an adequate external electric power is available, the Diesel set can be shut down and the required power is fed (380/220 V, 50 Hz, 110 Km) from external source.

PILOT SCALE PRODUCTION OF B.t. USING THE MOBILE FERMENTATION UNIT

New approaches have been adopted for the develepment of practical media for delta-endotoxin production by B. t. strains (Salama et al., 1982, 1983 a, b). In these approaches cheap ingredients to including agro-industrial byproducts and fodder yeast were used. - 154 - Production of B.t. in Egypt

A series of pilot scale fermentation experiments for production of B. t. endotoxin were carried out over a period of eight months using the forementioned mobile fermentation unit. The production experiments were preceeded by some engineering modifications of the mobile unit as to fit the B. t. fermentation purpose since it was originally designed for yeast production. Such modifications included an increase in the centrifugation efficiency of the decanter centrifuge and also a major change in the dryer design and function in order to be capable of spray drying the B. t. endotoxin preparation at moderate temperature.

(A) Production of B.t. var. galleriae HD-234 on Conventional Fodder Yeast Medium

In previous studies, we have selected a strain of B. t. var. galleriae namely, HD-234 as one of the potent strains against our target insects namely, Spodoptera littoralis and Agrotis ypsilon. A suitable inoculum size (1.5%) was used to inoculate 500 to 1000 liters of the routine fodder yeast medium which has the following composition

Ingredient Amount (gram/liter) Dry fodder yeast 20.0 Yeast extract 2.0 Glucose 2.0 Peptone 2.0 K2HP04 4.3 Mg SO4. 7H2O 0.4

The fermentation medium was placed in the paddle-wheel fermenter of the mobile unit. Strong aeration was effected at a rate of 85 m/hr and the fermentation process was allowed to proceed for 90 hr at 30°C. The changes in pH and density of culture were periodically recorded.

Table (1) shows the recorded data for pH and culture density change during the course of fermentation. The recorded data show a gradual decrease of pH value of the culture from 7.2 to the minimum value of pH 6.0 after 13 hr. of fermentation. The pH, then, started to rise as a result of the assimilation of the acetic acid produced during the phase of fast growth. The pH rise was steady until reaching a maximum value of pH 7.8 by the completion of 30 hr of fermentation.

- 155 - M.S. Foda et al.

The pH was, hereafter, stable at the indicated value until the end of fermentation. With respect to the culture density, a slight drop was noted during the first 15 hr. followed by a small increase until the end of the fermentation process.

Yield and potency of endotoxin : Due to the low speed of the centrifuge, the yield of dry endotoxin was low. However, the potency of the endotoxin as evaluated was high giving 92% kill when tested against second instar larvae of Spodoptera littoralis. Table (1) Changes in pH and density of the culture monitored with time during fermentation process (Batch No. 1) for B.t. var. gallierae HD-234 in the mobile fermentation unit. Aeration rate 85 ml/hr. Total culture volume 250 liters. The growth medium was the routine fodder yeast medium.

Fermentation Temperature Culture densit y p H (hours) oC

0 1.11 7.2 29 4 1.12 7.2 28 5 1.13 7.1 28 7 1.13 7.0 28 8 1.13 6.8 28 9 1.13 6.6 29 10 1.12 6.3 29 11 1.05 6.0 28 12 1.07 6.0 28 13 1.07 6.0 28 14 1.08 6.2 29 15 1.08 6.4 30 16 1.10 7.0 28 17 1.10 7.2 28 20 1.09 7.2 28

22 1.01 7.4 28

27 1.10 7.5 28

- 156 - Production of B.t. in Egypt

Table (1) Continued.

Fermentation Temperature Culture density pH dC (hours)

28 1.10 7.6 27 29 1.09 7.7 26

31 1.09 7.8 25 48 1.10 7.8 27 60 1.10 7.8 30 64 1.11 7.8 30 72 1.11 7.8 30 79 1.12 7.8 28 84 1.13 7.8 29 85 1.13 7.8 28 88 1.13 7.8 27 90 1.13 7.8 28

(B) Effect of Omission of Glucose and Yeast Extract on the Fermentation Process

The fodder yeast medium was modified to lower the production costs of the local product. Thus both yeast extract and glucose ingredients were omitted from the composition of the fermentation medium and the modified medium (FMI) had the following composition : Ingredient gram/liter

Dry fodder yeast 50.0 K,HP04 0.2 Mg S04. 71-120 0.1

The total fermentation volume was rased in this batch to 1 m3 in the paddle- wheel fermenter. After cooling, the pH of the medium was adjusted to 7.5 with sodium hydroxide when cooled to 50°C. The aeration level and inoculum size was that used in the previous batches. Both pH values and culture density were

- 157 - M.S. Foda et al. periodically monitored. Results are shown in Table (2). The pH of the culture dropped to 7.0 within eight hours of fermentation. It started to rise steadily, hereafter, racking pH value of 8.0 after two days. However, the pH continued to rise reaching 9.0 and become stable at this alkaline side till the end of, the fermentation period. Table (2) Effect of omission of free carbon source on culture pH and density in the FMI modification of fodder yeast medium (Batch No. 5). Organism used : B.t. var.

galleriae HD-234. Initial pH was adjusted to 7.4. Total culture volume 1 m'. Fermentation Temperature Culture densit y p H (hours) 0C

0 1.10 7.4 32

2 1.16 7.2 31 3 1.16 7.0 30 7 1.26 6.9 30

8 1.26 7.0 31 9 1.26 7.1 32 11 1.26 7.3 32 12 1.26 7.4 32 13 1.26 7.6 30 20 1.24 7.8 30 32 1.20 7.8 32 50 1.18 8.0 31 51 1.18 8.2 32 52 1.18 8.4 32 53 1.18 8.8 30 54 1.18 4.0 30 55 1.18 9.2 32 63 1.18 9.0 32 66 1.18 8.8 32

This modified medium (FMI) gave relatively high yield of endotoxin that reached more than 20 kg/m3. However, the potency was significantly reduced as compared with the previous batches. This is probably due to the cessation of growth at an early stage of fermentation due to the fast rise of pH of the culture. - 158 - Production of B.t. in Egypt

(C) Incorporation and Optimization of Molasses and Yeast Concentrations in the Fermentation Medium

The possibility of the use of molasses as the major carbon source for B.t. was investigated using different concentrations. In other experiments the concentration of the dry yeast was varied as to optimize the levels of both components. The main goal here, was to arrive to an appropriate medium composition that would be suitable for production of active endotoxin preparation in suitable amounts. The results of this series of batch experiments indicated that the following concentrations of various ingredients of the medium are most suitable for the routine production of endotoxins of B. t. fermentation

Ingredient gram/liter Dry fodder yeast 40.0 Molasses (5501o sugar) 15.0 KzHP04 1.0 Mg S04.71-120 0.2

This composition of the medium was repeatedly employed and gave consistently good results.

Tables (3) and (4) summarise the changes of pH and culture density during the course of fermentation using the above mentioned medium with B.t. var. galleriae HD-234 and B. t. var. kurstaki HD-341, respectively.

The recorded data show the typical behaviour of B. t. strains in this medium where an initial drop in the pH values takes places in the early phases of growth. This is due to the formation of appreciable amounts of acetic acid from sugars present in the molasses.

The exhaustion of sugar from the medium is followed by the assimilation of the accumulated acetic acid in the medium and the formation of basic products that results in a second change in the pH of the growth medium towards the alkaline side. As the pH values continue to rise, sporulation and crystal formation take place simultaneously followed by sporangium lysis and release of spores and endotoxins toward the end of the fermentation period. It is of interest to note that both tested strains gave the same pattern indicating that such a behaviour is an inherent character in the B. t. species. - 159 - M.S. Foda et al.

Table (3) Typical pH and culture density change during endotoxin production of B.t. var. galleriae HD-234 using modified fodder yeast molasses medium (FM4) in the mobile fermentation unit. Total culture volume 1 M3. (Batch No. 14).

Fermentation Temperature Culture densit y p H (hours) 0C

0 1.17 6.9 28 2 1.17 6.7 28

5 1.17 6.5 28 7 1.16 6.2 28 8 1.16 6.1 28 10 1.15 6.2 28

11 1.15 6.3 27

12 1.15 6.4 28 13 1.14 6.5 28 14 1.14 6.6 23 15 1.14 6.7 27 17 1.14 6.7 27 18 1.13 7.0 28 20 1.13 7.2 22 25 1.12 7.4 28

35 1.10 7.5 28

39 1.10 7.6 28

41 1.09 7.7 28

42 1.09 7.8 28 43 1.08 7.9 28 44 1.08 8.0 28

47 1.08 8.2 28

51 1.08 8.3 28 55 1.08 8.4 28 65 1.08 8.5 28

- 160 - Production of B.t. in Egypt

Table (4) Typical pH and culture density changes during endotoxin production of B. t. var. kurstaki HD-341 using modified fodder yeast molasses medium (FM4) in the mobile fermentation unit. Total culture volume 1 M3. (Batch No. 13).

Temperature Fermentation Culture density pH (hours) 0C

0 1.24 6.7 34 4 1.21 6.6 34

5 1.19 6.5 33 7 1.18 6.2 33

10 1.15 6.1 30

15 1.13 6.0 29 19 1.13 5.9 30 20 1.13 6.1 29 21 1.12 6.2 29 22 1.12 6.3 29

23 1.11 6.4 31 24 1.11 6.5 32 25 1.11 6.6 32 26 1.11 6.7 33 27 1.10 6.9 35 28 1.10 7.2 36 29 1.10 7.5 36 30 1.11 7.7 36

31 1.11 7.8 36 32 1.11 7.8 36 33 1.11 8.0 35 55 1.10 8.2 32 58 1.08 8.3 30

59 1.08 8.3 29 60 1.08 8.3 30 62 1.08 8.3 32

- 161 - M.S. Foda et al.

Table (5) summarizes some of the data concerning the potency of endotoxin preparations obtained from B. t. var. galleriae HD-234 using FM4 medium in the mobile fermentation uint. The results are compared to that obtained using Dipel 2X, a commercial product, and tested against the second instar larvae of S. littoralis. The obtained results indicate clearly that the potency of the local B. t. product is comparable to that of the Dipe1 2X in spite of the limitation imposed by the use of the mobile fermentation unit that has limited capabilities regarding the adjustment of fermentation conditions..

Table (5) Effect of concentration of some locally produced endotoxins preparation against larvae of Spodoptera littoralis.

Larval percent kill at concentration (pg/ml) Endotoxin prepartion pH adjustment

batch No. of supernatant 500 250 125

Dipel 2X (commercial - 67 - - control) 16 4.5 78 53 16 16 7.0 33 33 13 17 7.0 68 30 32 18 7.0 48 16 0 19 4.5 55 65 30 19 7.0 58 40 10 20 7.0 63 67 37 21 7.0 60 60 36

FORMULATION OF THE LOCAL B.t. ENDOTOXIN

The biological activity of B.t. depends on two characters which are equally important namely, the endotoxin potency and the additives added to enhance the efficiency, potency and stability. The last factor fulfillment is known as B. t. formulation.

- 162 Production of B.t. in Egypt

In the process of formulation, the conditions of application as well as the properties of B. t. endotoxins are taken into consideration. For example, it is well established that B. t. endotoxin is very sensitive to UV-irradiation; thus, addition of UV-protectant is a significant component of the formulation. Regarding diluents and carriers, local mixture of minerals of particle size less than 44 mu may be selected. In some formulations, molasses in the wettable powders are included. However, this adjuvant is recommended to be tank-mix.

DESIRED PROPERTIES OF THE B.t. FORMULATION

In a series of studies, the B. t. was prepared as water-dispersible powder taking

into consideration the following factors :

I - Flowability, wettability, dispersility and suspensibility to promote stable suspensions with a consequent over plant distribution. 2 - Addition of UV-protectant. 3 - Carriers and adjuvants free of moisture. 4 - Deactivation of the acidic active sites on the carrier to a pka higher than 3.3, to ensure shelf-life stability.

5 - Since foaming is undesirable, adjuvants with antifoaming activity were included.

The following are the basic ingredients reported as percentage of the water- dispensible powder locally devised for B. t. formulation:

Ingredient % of WD powder

B. t. 75.00 Wetting agent 3.00 Dispersant 2.00 UV-protectant 2.00 Oil 0.25 Deactivator 0.25 Antifoaming agent 0.50 Carrier, 300 mesh 17.00 Total 100.00

- 163 - M.S. Foda et al.

Table (6) lists the activity of some locally made formulations from B.t. produced in the mobile fermentation unit. The results are compared to that of Dipel 2X. The data indicate that formulation (D) is the best under the prevailing conditions.

Table (6) Determination of the activity of various locally formulated endotoxin preparations of B. t. var. kurstaki HD-341 against second instar larvae of Spodoptera littoralis.

B. t. formulation used Percent kill after 7 days at 500 pg/ml concentration

Dipel 2X (control) 67 B. t. endotoxin (As such) 63 Local formulation (A) 30 Local formulation (B) 50 Local formulation (C) 25 Local formulation (D) 70

Table (7) shows the values of LC50 determined against second instar larvae of S. littoralis for some B. t. formulations as compared to that of the commercial product Dipel 2X. The results indicate that the local formulations are at least as active as the expensive commercial product Dipel 2X. In future studies on formulations, the shelf life of the preparation will be investigated and developed to ensure that the product will be stable at least for 12 months under ambient storage conditions. Production of B.t. in Egypt

Table (7) Determination of LC50 of some endotoxin preparations produced in the mobile fermentation unit before and after formulation. The values were determined against second instar larvae of Spodoptera linoralis.

Condition of LC50 Compound used B. t. variety preparation pg/ml

Dipel 2X (control) - As such 686

Local production batch (17) HD-341 var. As such 340 kurstaki After 630 formulation

Local production-mixture HD-234 var. As such 580 of four batches galleriae After HD-341 var. formulation 563 kurstaki

REFERENCES for whey Salama, H.S. ; Foda, M.S. and El-Sharaby, A. (1982). A novel approach recycling in production of bacterial insecticides. Entomophaga 28, 151 - 160. yeast Salama, H.S. ; Foda, M.S. and EI-Sharaby, A. (1983a). Utilization of fodder and agro-industrial by products in production of spores and biologically active endotoxins from Bacillus thuringiensis. Zbl. Mikrobiol. 38, 553 - 563. Novel Salama, H. S. ; Foda, M. S. ; Dulmage, H. and El-Sharaby, A. (1983b). fermentation media for production of delta-endotoxins from Bacillus thuringiensis. J. Invertebr. Pathol. 41, 8 - 19.

Applications of Bacillus thuringiensis Preparations against the Diamondback Moth, Plutella xylostella (L.), in Taiwan

Roger F. Hou and Tao-mei Chou Department of Entomology, National Chung Hsing University Taichung, Taiwan 402, ROC

ABSTRACT

Applications ofBacillus thuringiensis (B. t.) preparations for killing the diamondback moth (DBM), Plutella xylostella (L.), on crucifers was studied both in laboratory and field conditions. The DBM strains collected from different localities on Taiwan were found all susceptible to B. t. indicating that the B. t. preparations are effective in killing local DBM populations on this island. Efficacy of B. t. preparations was not affected by temperatures ranging from 20 ° to 35 °C and water pH 4 -10 which are important factors while spraying B. t. in the fields. Addition of sinigrin or cations (Ca++ and K+) to B.t. preparations might enhance its effectiveness to DBM. Applications of B. t. at sublethal doses could deter pupal weight, pupal duration and adult emergence of DBM. This sublethal dose effect is thus significant in practical pest control. Field applications of B. t. preparations for controlling DBM on cruciferous vegetables were found to be more effective than conventional chemical insecticides. The B. t. based products have currently become major control agents for practical DBM control on crucifers in winter in Taiwan.

INTRODUCTION The total vegetable acreage is currently over 200,000 hectare on Taiwan which is an island with the total area of ca. 36,000 sq. km. Many species of insect pests occur all the year round on this island because of its location in tropical and subtropical zones; however, the diamondback moth (DBM), Plutella xylostella (L.), has been regarded to be the most serious pest attacking crucifers since it has more

- 167 - Roger F. Hou and Tao-mei Chou

than 20 generations in a year (Tao and Lee, 1981). Resistance of DBM to various groups of chemical insecticides was variable but serious in Taiwan as well as in other Asian areas as compiled by Cheng (1988). Therefore, alternative control measures are important and required for effective control of this cruciferous pest.

The insecticidal bacterium, Bacillus thuringiensis (B.t.), has been introduced into Taiwan since 1958, and the B. t. based products were first registered for control of insects in 1967 (Hou, 1989). Survey on application priority of insecticides by local farmers revealed that the B. t. products ranked the 6th among 18 different types of insecticides being surveyed (Li, 1984). At present the B.t. products are mostly used for control of vegetable insects in Taiwan; therefore they are apparently widely accepted by vegetable growers for field applications. Unfortunately, information on various phases of applications of B. t. preparations is meager although this microbial agent has been introduced into this island for more than 30 years. This report presents research work on usage of B.t. preparations for controlling DBM on cruciferous vegetables.

MATERIALS AND METHODS

Insects and B.t. Products

Various strains of P. xylostella were collected from six localities distributed at different parts of Taiwan, viz., Ban-chiao (BC), Chang-hua (CH), Lu-chu (LC), Ta-li (TL), Tai-tung (TT), and Tou-cheng (TC). The colonies were reared on cabbage leaves in a growth chamber at 25°C. The 3rd instar larvae were used for various bioassays.

Three B.t. based products were adopted for various tests, i.e., Bactospeine (France Biochem), Dipel (Abbott) and Thuricide (Sandoz), all being 3% WP with 16,000 IU/mg.

Assay of Susceptibility of DBM Strains to B.t.

The 3rd instar larvae of different strains were fed on the cabbage leaves sprayed with various B. t. preparations with dilutions of 1 x 103 to 5 x 106 fold. The larval mortalities caused by B. t. were recorded for 3 days after spraying. Values of LC50, LC95 were calculated by probit analysis.

- 168 - Application of B.I. in Taiwan

Pathogenicity and Spore Viability of B.t. as Affected by pH of Diluting Water and Incubating Temperature

The B. t. preparations were diluted with water at pH 2, 4, 7, 8, 9, and 10, and were then sprayed on cabbage plants. The 3rd instar larvae of DBM were allowed to feed on the treated plants, and their mortalities were recorded for 3 days after spraying. These diluted preparations were also cultured on the plates containing the BTV selective medium (Akiba and Katoh, 1986) at 35°C, and the B. t. colonies in each plate were counted 3 days after culturing. In another tests, the B.t. preparations were diluted 1,000 fold and were then combined with adjuvants, viz., Sandovit (Sandoz), 2,500X and Agral 90 (ICI), 5,000X, respectively. The mixtures were sprayed on cabbage plants and were incubated at 20°, 25°, 30° or 35°C for 1, 2, 4, 7 or 14 days. After incubation, the treated leaves were punched out giving several discs with 6 mm in diameter. The disc test was carried out by washing the leaf discs with sterile distilled water and the washed solutions were cultured with the BTV selective medium at 35°C. The B. t. colonies were counted 3 days after culturing.

Assays of B.t. Efficacy by Adding Sinigrin, and Cations

Sinigrin (Sigma) was added to B.t. preparations at 0.001, 0.01, 0.1 and 1.0 ppm. Discs (1 cm, diam.) of artificial diet without leaf powder (Hou and Hsiao, 1986) were dipped into the mixtures and were fed to the starved 3rd instar larvae. The treated larvae were incubated at 25°C for 3 days, and their mortalities were recorded. For cation tests, various salts, viz., Na2CO3, K2CO3. CaCl2 and MgC121 were added to the B. t. preparations at 0.5%. The bioassay using diet discs as mentioned above was carried out. The bioassays were carried out by spraying on cabbage and cauliflower plants. The larval mortalities were recorded from 1 through 26 days after spraying.

Assay of Effect of B.t. Sublethal Dose on DBM

A series of dosages of B. t. preparations were made up from 3.2 to 320 IU/ml which are either below or above LC50. Each dose was assayed by spraying on cabbage plants. The larval mortality, pupation, pupal weight and adult emergence of assayed DBM were observed and recorded after feeding on the treated cabbage leaves.

- 169 - Roger F. Hou and Tao-mei Chou

Field Tests of B.t. Preparations Against DBM

1. Experimental design :

A cauliflower field of 0.18 ha. at Chang-hua county was selected for trial. The whole field was divided into 36 plots and different treatments were arranged according to the randomized complete block design. Each treatment had four replicates, and those plots sprayed with water served as controls.

2. Field tests :

The field tests were carried out in winter season from November to January. The. B. t. products were diluted by 1,000 fold and adjuvants were added to the diluted solutions. The first spray was done at the time of 3-4 leaves on each plant, the forthcoming 4 sprays were carried out every 7 days. Permethrin and cartap were also applied for comparison. The number of larvae was counted from 20 plants in each plot on one day before and 3 days after spraying. The leaf areas of damaged plants were also measured for evaluating the control efficiency.

RESULTS Pathogenicity of B.t. to Different Strains of DBM

Table (1) shows that DBM strains collected from different localities of Taiwan were all susceptible to the B. t. preparation, the differences in LC50, LC75 and LC95 being no more than 3 fold among various strains while the Tai-tung strain was slightly but not significat;tly poorer than others. The B. t. based products have been applied to control vegetable insects for over two decades; however, 6 representative strains from different parts of Taiwan did not show appreciable differences in pathogenicity to DBM, indicating that the B. t. products still maintain their effectiveness for controlling this pest on Taiwan.

Factors Affecting B.t. Effectiveness Against DBM

1. pH value of diluting water :

In practical application, farmers do not pay attention to the pH value of diluting water available near the vegetable fields. It is important to investigate whether efficacy of B.t. preparations to DBM is affected by different pH values

- 170 - Application of B.t. in Taiwan of water. Table (2) demonstrates the same level of larval mortality at the pH value ranging from 2 to 10 although no viable cell count was obtained from pH 2 dilution. Therefore, farmers do not have to care about the pH value of water with which they usually use for making up B. t. preparations in the field.

Table (1) Comparison of pathogenicity of Bacillus thuringiensis on various strains of Plutella xylostella collected from different localities of Taiwan.

Strain') B. t. conc. (IU/ml)

LC50 Ratio LC75 Ratio LC90 Ratio Slope

BC 98.2 1.0 437.3 1.0 3,757.4 1.0 1.04 CH 132.9 1.4 730.6 1.7 8,507.8 2.3 0.91 LC 134.3 1.4 1,040.9 2.4 9,881.7 2.6 0.76 TC 196.3 2.0 811.8 1.9 6,274.4 1.7 1.09 TL 91.0 0.9 609.9 1.4 9,446.4 2.5 0.82 TT 284.3 2.9 1,309.2 3.0 11,812.0 3.1 0.62

1) BC (Ban-chiao), CH (Chang-hua), LC (Lu-chu), TC (Tou-cheng), TL (Ta-li), TT (Tai-tung)

Table (2) Effects of pH on virulence and spore viability of B. t. to DBM.

pH Mortality (%) Viable cells (colonies/mL)

2 95.7 ± 4 0.000 4 95.8 ± 6 3,030 6 100.0±0 1,346 7 97.6±3 1,710 8 100.0±0 2,083 9 100.0± 0 1,277 10 97.7 ± 0 3,780

- 171 - Roger F. Hou and Tao-mei Chou

2. Effect of Temperature on Spore Viability of B.t :

The plate assays of B.t. viability on culture medium showed variable results; however, B. t. could still grow at high temperatures if mixed with adjuvants (Table 3). The B.t. colonies were detectable at 20° to 35°C after applying for 14 days. It is thus suggested that temperature seems not to be seriously harmful to spore viability after spraying on the plants. -

Table (3) Persistance of B. t.. at various temperatures.

No. colonies/mm2

Treatment Day intervals after application Temp.

(°C) 0 1 2 4 7 14

B. t. only 223.2 20 55.7 152.8 4.6 0.0 4.5 25 77.8 23.1 0.3 4.4 3.7 30 156.2 6.1 4.7 4.7 1.7 35 2.9 14.7 0.0 1.5 2.8 B. t. + Sandovit 78.2 20 13.9 127.3 0.0 0.7 4.4 25 36.8 126.8 0.0 3.7 0.1 30 2.1 5.1 2.8 0.4 0.2 35 9.4 216.3 0.0 15.1 0.3 B. t. + Agral 90 60.5 20 15.9 121.0 0.0 5.1 1.2 25 13.6 8.1 1.6 6.0 1.6 30 10.9 0.6 2.6 0.6 1.5 35 9.8 11.8 0.0 2.3 2.2

3. Effect of Sinigrin on B.t. Effectiveness Against DBM : Enhancement of B.t. effectiveness against DBM was predominant when sinigrin was added to low concentrations of B.t. preparations, e.g., 3.2 IU/ml; however, increase in larval mortality was also observed in 16 and 32 IU/ml. The best effect of sinigrin was obtained from 0.01 ppm in 160 IU/ml ; the optimal

- 172 - Application of B.t. in Taiwan dose of sinigrin was 0.01 ppm based on assays with different B. t. concentrations (Table 4).

Table (4) Effect of sinigrin on virulence of B. t. to DBM.

Larval mortality (%)

B. t. conc. Sinigrin conc. (ppm)

(IU/ml) 0 0.001 0.01 0.1 1.0 79.6 3.2 12.5 23.5 44.8 50.8 16.0 21.1 44.0 54.3 52.8 51.8 32.0 48.2 63.3 72.0 54.2 66.7 160.0 60.0 72.0 82.9 77.0 78.4

4. Enhancement of B.t. pathogenicity by cations :

Table (5) shows that addition of different salts to the B. t. prepartions may enhance pathogenicity to DBM, especially CaC12 increased 6 times based on LC50, and even up to 21 times in term of LC75. Other salts may also promote B. t. pathogenicity to a certain degree. Thus, cations are effective in enhancing B.t. effectiveness against DBM.

Table (5) Effect of cations on pathogenicity of B.t. to DBM.

B. t. conc. (IU/ml)

Salt (0.5%) LC50 LC75 LC95 Slope

B.t. only 209.3 2,382.8 79,158.0 0.64 CaC12 33.5 105.4 548.7 1.36 MgC12 85.6 385.7 3,373.9 1.03 Na2Co3 89.1 338.8 2,319.3 1.16 K2Co3 175.3 254.4 435.0 4.17

- 173 - Roger F. Hou and Tao-mei Chou

Effect of Sublethal Dose of B.t. on DBM.

The sublethal doses of B.t. may cause lowering of pupation rate, pupal weight and adult emergence, and prolong the pupal duration of DBM (Table 6). In our assays, 32 IU/ml or lower was regarded as sublethal dose, the DBM pupation and adult emergence at this concentration were around 60% compared with over 90% in control group although its larval mortality was only 28%. The application of sublethal dose is thus effective in suppressing insect development, and is significant in practical control of pests.

Table (6) Effect of B. t. sublethal dose on larval mortality and development of DBM.

Dose No. Larval Pupation Pupal Pupal Adult larvae mortality wt. duration emergence (IU/ml) (%) (%) (mg) (days) (%)

0.0 81 1.10 97.50 5.05 3.68 92.40 3.2 131 16.11 87.50 4.85 3.64 75.98 16.0 122 14.34 83.54 4.64 3.64 73.58 32.0 148 28.05 63.24 4.70 4.70 60.98 160.0 128 61.18 42.82 3.73 4.13 19.73 320.0 119 69.64 39.90 3.64 3.95 9.78

Field Tests of B.t. Preparations Against DBM on Cauliflower

Table (7) indicates that 3 different B. t. preparations were slightly better than cartap and permethrin in term of the number of larvae on leaves, especially when added Agral 90 to the preparations.

Evaluation of cauliflower leaf areas damaged by DBM has definitely shown superiority of B. t. preparations to cartap and permethrin. Obviously, the B. t. preparations are more effective in killing DBM than chemical insecticides under field conditions in Taiwan.

- 174 - Application of B.t. in Taiwan

Table (7) The number of DBM larvae on cauliflower after spraying various insecticides, Chang-hua, 1987.

No. of larvae')

Treatment Dilution Date Sprayed (103) Nov. 27 Dec.8 Dec. 19 Dec. 29 Avg2)

Cartap lx 44.3a 10.8bc 54.3ab 28.8bc 34.5ab Permethrin 2x 25.3abc 18.9ab 46 Oabc 42.0ab 33 3abc Dipel lx 21.3abc g gbc 35.8bcd 25 8cd 23.lbcd Bactospeine lx 20.0abc 6.8c 33.5bcd 22.8cd 20.8bcd Thuricide lx 12.Obc 10.8bc 18.0cd 19.8cd 15.1cd Thuricide + lx & 8.Oc 6.8c 12.5cd 12.3d 9.9d Agral90 5x Thuricide + 0.8x & 21.2abc g gbc 22.5bcd 20 3cd 18 3bcd Agra190 5x Thuricide + 0.5x & 21.8abc 6.8c 8.9d 12.3d 12.4d Agral90 5x Check 38.0ab 22.0a 73.8a 59.3a 48.3a

1. Means followed by the same letter are not significantly different at 5%.

2. No. of larvae per 10 plants.

DISCUSSION

Resistance of DBM to B. t. preparations has recently been reported in Hawaii (Tabashnik et al., 1990). B. t. has been applied to control vegetable pests for over two decades in Taiwan; however, we collected six strains of DBM from different parts of this island and did not find appreciable differences in susceptibility of this species to B. t. preparations, indicating that the local populations have not yet developed tolerance to B. t. The B. t. products are still effective in killing DBM; in particular, this insect was found to be resistant to many chemical insecticides including those being recommended for control use (Cheng, 1988). Our field tests further showed that the B. t. preparations were superior to chemicals. The B. t.

- 175 - Roger F. Hou and Tao-mei Chou products can thus be regarded to be effective alternatives for practical applications in vegetable fields in Taiwan.

Physical factors are usually important for effectiveness of biocontrol agents and insect development; however, the B. t. preparations were not affected by pH value of diluting water and temperatures ranging from 20°C to 35°C while applying to the crucifers although it was indicated that temperature at 18° to 20°C was most favorable for B. t. applications in the field (Falcon, 1971). The B. t. preparations could be persistent on leaves up to 14 days at 35°C in our assays. These characteristics make B. t. products more applicable and acceptable by. farmers for practical control of DBM on crucifers.

Sinigrin is extractable from cruciferous leaves, being a mustard oil glycoside, and is a feeding stimulant to cabbage butterfly and DBM (Bech and Reeve, 1976). The bait formulation containing cotton seed extracts which could be phagostimulative may enhance effectiveness of B. t. against Spodoptera linoralis (El-Nockrashy et al., 1986). It is then reasonable to believe that sinigrin could also enhance B. t. efficacy against DBM when added to B. t. preparations. It was observed that DBM larvae ingested more diets in the presence of sinigrin in them (data not shown). Addition of sinigrin to B. t. preparations was found to be more effective at lower B. t. concentrations, being possibly due to sustaining the feeding activity of treated larvae at low concentrations so that the enhancement became evident. On the contrary, the larvae lost appetite soon after ingesting high concentrations of B. t. therefore, the enhancement by adding phagostimulants is not detectable under this condition.

Salama et al. (1984) reported that B. t. endotoxin potency against S. littoralis can be strengthened by adding 0.5% salts to the artificial diets, especially CaC12. Similarly, we found that both neutral CaC12 and alkaline K2CO3 could enhance B. t. efficacy against DBM, indicating that ionic strength rather than pH value is virtually effective in promoting B. t. potency.

Effect of B. t. sublethal doses on insect development and reproduction was observed in the spruce budworm, Choristoneura fumiferana and some noctuids (Ignoffo and Gregory, 1972; Alford and Holmes, 1986). Our results revealed that pupating rate, pupal weight and adult emergence of DBM treated with sublethal

- 176 - Application of B.t. in Taiwan

was longer than the controls. doses of B. t. were lower and the pupal duration B. t. for field applications. Hence, sublethal effect may assist the suitability of that B.t. product showed Although Kirsch and Schmutterer (1988) reported ascribed to wide and frequent low efficacy to DBM on cabbage, being possibly in cabbage growing areas in the use of B. t. mixing with chemical insecticides to chemicals by assessing Philippines. Our field tests showed superiority of B.t. Chang (1972) indicated that B.t. larval numbers and leaf areas on cauliflowers. in winter rather than in preparations were more effective for controlling DBM were undertaken in winter, summer or fall in northern Taiwan. Since our tests is usually not abundant in summer B. t. was thus potent for killing DBM. This pest ;sland. Therefore, farmers while crucifers are not widely grown in plain on this DBM during summer. The do not apply B.t. preparations for practical control of DBM in this season might yearly interruption of B. t. application for controlling and currently the B. t. restrain this insect of developing resistance to B. t. products, for practical DBM control on based products have become major control agents crucifers in winter on this island.

ACKNOWLEDGMENTS Agriculture, ROC, and This study was financially supported by Council of Government, ROC. Department of Agriculture and Forestry, Taiwan Provincial

REFERENCES thuringiensis. V. Akiba, Y. and Katoh, K. (1986). Microbial ecology of Bacillus Jpn. Appl. Ent. Selection medium for Bacillus thuringiensis vegetative cells. Zool., 21 , 210-215. (In Japanese). of carbaryl, aminocarb, Alford, A. R. and Holmes, J. A. (1986). Sublethal effects and fecundity of fenitrothion, and Bacillus thuringiensis on the development 79 , 31-34. the spruce budworm (Lep., Tortricidae). J. Econ. Entomol., : nutrition and Beck, S. D. and Reeve, J. C. (1976). Insect-plant interaction Interaction metabolism. In "Recent Advances in Phytochemistry Biochemical Plenum Press, N.Y. and between Plants and Insects" (J. W. Wallace, ed.). London. 10, 41-92. and other insecticides. Chang, L. C. (1972). Vegetable insect control by Thuricide Taiwan Agric. Quart., 8 , 164-169. (In Chinese).

- 177 - Roger F. Hou and Tao-mei Chou

Cheng, E. Y. (1988). Problems of control of insecticide-resistant Plutella xylostella. Pestic. Sci., 23, 177-188. El-Nockrashy, A. ; S. Salama, H. S. and Taha, F. (1986). Influence of bait formulations on the effectiveness of Bacillus thuringiensis against Spodoptera littoralis (Boisd.) (Lep., Noctuidae). J. Appl. Entomol., 101 , 381-389. Falcon, L. A. (1971). Use of bacteria for microbial control. In "Microbial Control of Insects and Mites" (H. D. Burges and N. W. Hussey, eds.). Academic Press, London and N.Y., 67-95. Hou, R. F. (1989). Control of the diamondback moth, Plutella xylostella, using entomopathogens. Chinese J. Entomol., Special Publication No. 4, 46-52. (in Chinese). Hou, R. F. and Hsiao, M. L. (1986). An improved diet for rearing the diamondback moth, Plutella xylostella, and its requirements for fatty acids. Chinese J. Entomol., 6, 31-37. Ignoffo, C. M. and Gregory, B. (1972). Effects of Bacillus thuringiensis -exotoxin on larval maturation, adult longevity, fecundity, and egg viability in several species of Lepidoptera. Environ. Entomol., 1, 269-272. Kirsch, K. and Schmutterer, H. (1988). Low efficacy of a Bacillus thuringiensis (Berl.) formulation in controlling the diamondback moth, Plutella xylostella (L.), in the Philippines. J. Appl. Ent., 105 , 249-255. Li, G. C. (1984). Residue problems associated with insecticide use on vegetables and their preventions. Proc. Symp. Insect Control Vegetables in Taiwan. Dept. of Agriculture and Forestry, Taiwan Provincial Government. 116-133. (In Chinese). Salama, H. ; S. Foda, M. S. and Sharaby, A. (1984). Novel biochemical avenues for enhancing Bacillus thuringiensis endotoxin potency against Spodoptera littoralis (Lep.: Noctuidae). Entomophaga, 29, 171-178. Tabashnik, B. E. ; Cushing, N. L. ; Finson, N. and Johnson, M. W. (1990). Field development of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera : Plutellidae). J. Econ. Entomol., 83 , 1671-1676. Tao, C. C. and Lee, H. S. (1981). Population fluctuation of cruciferous insect pests and their control in Taiwan. Proc. Symp. Production and Insect Control of Cruciferous Vegetable in Taiwan. (C. N. Chen and W. Y. Su, eds.). 16-31. (In Chinese). Constraints on the Use of Bacillus thuringiensis in the Philippines

L. E. Padua National Institutes of Biotechnology and Applied Microbiology (BIOTECH), University of the Philippines at Los Banos College, Laguna, 4031 Philippines

ABSTRACT

Bacillus thuringiensis (B. t.) is the most potential microorganism for control of insects ofeconomic and medical importance in the country. Research and development of this bacterium was conducted at the Microbial Insecticides Laboratory, National Institutes of Biotechnology and Applied Microbiology (BIOTECH), University of the Philippines at Los Banos, College, Laguna, Philippines. At the national research institution in biotechology, the research and development objectives aimed towards utilization of locally available materials, self-reliance and self-sufficiency for sustainable agriculture. Survey and isolation of B. t. from soil samples of dead and living insects around the country are regular activities of the Microbial Insecticides Laboratory. Isolated B. t. strains were kept and used in screening against priority pests of the country. The screening of the isolated B. t. was conducted against mosquito larvae, slug caterpillars of coconut, important lepidopterous pests of rice and vegetable crops and stored grain pests of corn and paddy rice. A local isolate of this bacterium has been developed for the control of corn borer, Ostrinia f irnacalis (Guenee) and mosquito larvae. However, some isolates of this organism were found also effective against some plant bacterial and fungal pathogens of selected agricultural crops. Farmers have accepted the use ofB. t. for controling insects. Their only complaint is that it is expensive and sometimes is not effective. Another constraint on the use of this bacterium is the problem related to the use of sophisticated equipment for mass production. Only a small quantity can be used for large scale field trials of the product. Equipment necessary for mass production and formulation are also expensive. Even if the feasibility study on the commercial production is viable, another problem faced by our local enterpreneurs is the big capital investment.

- 179 - L. E. Padua

INTRODUCTION

Bacillus thuringiensis (B.t.) is the most promising agent for control of insects of economic and medical importance. Investigations on the potential of this bacterium have greatly contributed to its development as a microbial control agent not only against Lepidoptera (Heimpel, 1967; Dulmage and Cooperators, 1981; Faust and Bulla, 1982), but also Diptera (Goldberg and Margalit 1977; de Barjac 1978; Undeen and Nagel, 1978; Padua et al., 1980; Padua et al., 1984). Recently, a new subspecies of B. t. with high toxicity against coleopteran insects was reported from Germany and the United States (Krieg et al., 1983; Hermstadt et al., 1986).

The worldwide distribution of this bacterium has been reported by many workers. In the Philippines, the first attempt to isolate and identify this bacterium was in 1982 (Padua et al., 1982), although Gabriel (1968, 1970) reported the introduction and subsequent use of the commercial preparations of B. t. subsp. thuringiensis, serotype H-1. The discovery of new B.t. isolates having novel insecticidal value and use will add again to the importance of this bacterium for microbial control.

This paper summarizes the research done and some constraints on the use of B. t. in the Philippines. Quantitative toxicity tests of this crystalliferous spore-forming bacterium were carried out against locally important insect pests of corn, crucifers, rice and coconut, some stored grain pests of corn and paddy rice. Screening of this bacterium against some bacterial and fungal pathogens of selected agricultural crops had been also conducted.

B.t. AGAINST INSECTS AND DISEASES OF ECONOMIC IMPORTANCE

Corn, crucifers, rice and coconut are major crops of the Philippines and insect pests and diseases are among the factors that significantly reduce their yields. The control of these pests and diseases relies almost entirely on chemical insecticides. However, the use of chemical insecticides has been found to be effective only to a certain extent. Furthermore, insect populations and plant pathogens have developed resistance to a number of these chemicals due to extremely high and indiscriminate use. This leads to ecological imbalance through the destruction of non-target organisms like predators and parasites of pests and the accumulation

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the development of toxic residues in the environment. Such a situation necessitates of alternative or supplementary methods for pest control.

EVALUATION OF B.t. AGAINST ASIAN CORN BORER

The systematic screening of B. t. against the Asian corn borer, Ostrinia in toxicity funracalis (Guenee), offers the first evidence of considerable variation isolates of the local and imported isolates (Padua et al., 1987a). Based on the 265 PG-02 were used in the screening against third instar larvae, LEP-20, HD-231 and mg/l of diet, the most toxic, each having an LC50 of 85.92, 93.17, and 141.01 was noted as respectively. With the local isolates, LEP-20 and PG-02, mortality consume less early as two days after treatment. Infected larvae were found to treated diet compared to the control.

1. B.t. Against the Diamondback Moth

The diamondback moth, Plutella xylostella (Linnaeus), is a key pest of crucifers that in the Philippines. Control is characterized by heavy insecticide application At a lower is perpetuated by a vicious cycle of rapid development of resistance. LEP-13, dilution of one loopful of the isolate in 100 ml of distilled water, LEP-21, hours after PG-02, HD-54, HD-61 and HD-100 gave 60-88% mortality 24 isolate, treatment against third instar larvae (Padua et al., 1987a). PG-02, a local toxic. was found to be toxic against Plutella and Ostrinia while HD-1 was less

Susceptibility of Some Important Lepidopterous Pests of Rice to B.t. It has been demonstrated that out of the 300 isolates of B. t. several have more hairy than 50% toxicity against leaffolder, Cnaphalocrocis medinalis; green caterpillar, Rivula atimeta; green semilooper, Naranga aenescens; rice caseworm, stem Nymphula depunctalis; yellow stemborer, Scirpophaga incertulas; and striped to B. t. borer, Chilo suppressalis (IRRI Highlights, 1986). The larval susceptibility were isolates varies. However, the rice stemborers, S. incertulas and C. suppressalis, found to be resistant compared to other insect pests of rice.

Among the 287 isolates used in screening against the green semilooper, N. et aenescens, LEP-38, LEP-54, LEP-57 and LEP-59 were the most toxic (Padua the two al., 1987b). The toxicity of the four isolates was comparable to those of isolates taken from the commercial product (I and II) available in the Philippines.

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LEP-59 and LEP-54 were significantly more toxic than I and II, with LC50 values of 2.6 and 3.8 mg/L respectively (Table 1).

Table (1) The LC50 values of the selected Bacillus thuringiensis isolates against third instar larvae of rice green semilooper, Naranga aenescens Moore.

Isolate LC50 (mg/L)* Fiducial Limit (mg/L)

I 4.7a 2.5 - 8.9 II 7.9a 2.7 - 47.5 - LEP 38 16.9a 6.5 - 54.7 - LEP 54 3.8a 1.5 - 9.4 - LEP 57 7.3a 3.9 - 12.3

LEP - 59 2.6a 1.3 - 4.7

* Any values in a group having the same letter are not significantly different at 95% level as indicated by the overlapping of the fiducial limits.

2. B.t. Against Stored Grain Pests

One hundred fifty-four isolates were tested against Rhizopertha dominica, Sitophilus zeamais and Tribolium castaneum. Thirty isolates gave 50-80% mortality against R. dominica. When these isolates were used in the second screening, six isolates gave 50-60% mortality. Most of the screened isolates gave 0-25% mortality only. No isolates were found toxic against S. zeamais and T. castaneum. However, when the adults from the treated medium were examined after two months, there were isolates that gave a population of 50-100% below the control and 50-100% above the control. The low and high population incidence observed in some samples treated by some isolates must be an after-effect of B.t. on the adults of the target pests.

3. Biological Efficacy of B.t. Against Plant Pathogens

Initial screening of B. t. against Xanthomonas campestris pv. oryzicola, X. c. pv. oryzae, Erwinia earotovora, Pseudomonas solanacearum, Pythium debaryanum and

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Sclerotium rolfsii was conducted in the laboratory. Out of 137 B.t. isolates used in the screening 7,5, 7 and 22 showed positive reaction against the four bacterial pathogens, X. c. pv. oryzicola, X. c. pv. oryzae, E. carotovora and P. solanacearum, respectively. For the two fungal plant pathogens, 10 isolates exhibited positive reaction against P. debaryanum and none was observed against S. rolfsii.

There were isolates of B. t. that showed positive reaction against 2 or 3 bacterial plant pathogens. A suppressive action was observed with B.t. when introduced into the mycelial growth of P. debaryanum.

4. B.t. Toxicity Against the Slug Caterpillar of Coconut.

Studies on the pathogenicity of B. t. isolates in the laboratory against fourth instar larvae of the coconut slug caterpillar, Setora sp., have generated initial information (Pacumbaba and Padua, 1987). Among the 150 isolates tested, HD-595, C-27, HD-52, AO-97, and HD-72 exhibited high insecticidal activity. HD-595 and C-27 were the most toxic, with LC50 values of 62.52 and 79.41 mg/L respectively. The two isolates were even more toxic than the pure cells taken from the commercial product.

Generally, the symptoms exhibited by infected insects include sluggishness, cessation of feeding, discoloration, moribundity and death. Dead larvae turned dark brown to black and became shriveled, which are typical characteristics of an insect infected by this bacterium.

5. B.t. Against Insects of Medical Importance The initial biological control of mosquito larvae in the Philippines was done by using indigenous fish species like Gambusia a}finis and Poecilia reticulata. The need for improved biological control agents led to initiation of the search for new methods. In the search for a safe and effective mosquito larvicide, the bacterium PG-14, B.t. subsp. morrisoni (serotype H 8a : 8b) is highly and selectively toxic and is being developed.

PG-14 was isolated from a soil sample taken from a canal in Cebu City, the Philippines (Padua et al., 1982). This isolate produces a spherical or irregular parasporal crystal, highly toxic to mosquito larvae but not to the silkworm, Bombyx mori, and adults of a daphnid. It is also negative for 13-exotoxin. All this is in

- 183 - L. E. Padua contrast to the type strain (Padua et al., 1984). This isolate, being the first discovered from the tropics, is serologically different from B. t. subsp. israelensis, serotype H-14.

Toxicity of PG-14.

PG-14 was found to be highly toxic to several mosquito species in the Philippines. The degree of toxicity is about equal to that reported for subsp. israelensis (Padua et al., 1984; Ibarra and Federici, 1986; Lacey et al., 1988) in some species of mosquito larvae.

The purified parasporal crystal of PG-14 is very similar in toxicity to that of subsp. israelensis, having LC50 values of 2.97 and 3.36 pg/ml, respectively (Ibarra and Federici, 1986). In assays of spore-crystal complex against A. aegypti and Culex molestus, PG-14 was as toxic as subsp. isaelensis, having LC50 values of 0.044 and 0.018 and 0.052 and 0.033 mg/L respectively (Padua et al., 1984).

Lacey et al. (1988) reported the susceptibilities of eight species of mosquito larvae from temperate countries. The range of susceptibilities was similar to but not identical with, the susceptibilities to the toxin of subsp. israelensis. The disparity between LC50 values of PG-14 for Anopheles quadrimaculatus and A. albimanus, principal vector of malaria in Mexico and Central America, was greater than that reported in the toxicity studies with subsp. israelensis and the isolate 73-E-10-2, subsp. darmstadiensis (serotype H-10) (Padua et al., 1980; Lacey and Singer, 1982; Lacey and Oldacre, 1983). The activity of PG-14 against A. albimanus is comparable to its activity against culicines that were tested, an incident not normally observed in subsp. israelensis. This observation warranted further investigation, particularly under field conditions.

In a Philippine field trial, using a local formulation of PG-14 applied against A.. flavirostris, a vector of malaria in the country, about 90076 reduction in the total larval population was observed (Padua, 1988).

CONSTRAINTS ON THE USE OF B.t.

Gabriel (1968, 1970) reported the introduction and subsequent use of the commercial preparations of B. t. particularly against the diamondback moth,

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Plutella xylostella, one of the most important pests of crucifers in the country. Since then the farmers have accepted the use of this bacterium against the diamond- back moth. The first commercial product introduced in the country was subsp. thuringiensis (Serotype H-1) and later subsp. kurstaki (Serotype H 3a : 3b). The use of the commercial product was not very extensive because of the cost and availability. The cost of the product is not stable and not always readily available, being an imported material. Effectiveness of the product also varies. In our laboratory examination, colony count varies, which might be an explanation for variability in toxicity. Another consideration might be the duration of shipping the product from the U.S.A. to the country. In our follow-up of how the imported product proceeds from its place of origin to the local company, it took almost six weeks to reach the port of entry. After this, it takes another month to almost six weeks before the local company could take the product. Hence, the total time from the U.S.A. a temperate country, to the company in the tropics, lasts two to three months. By the second or third month from the day of shipment from the U.S.A., most of the cells must have been dead already and the protein or toxic crystals, already degraded.

Biotech, as the national research and development institution in biotechnology in the country, aiming towards utilization of locally available materials, self-reliance and self-deficiency, focuses the work on microbial insecticides to answer the constraints as gathered from the farmers. After our development of an isolate of B.t. for mosquito larvae (PG-14, subsp. nwrrisoni, Serotype H 8a : 8b) and corn borer (LEP-20, subsp. kurstaki, Serotype H 3a : 3b), the problem is the need of sophisticated equipment for mass production. Fermentors, centrifuge and spray drier are the most needed to have a product. They are very expensive, more so, if the local market demand is targeted.

The feasibility study of the two developed products of B. t. for commercial production in the country presented the viability of the product. The Return on Investment (ROI) is 221.91 %, Internal Rate of Return is 150.70% with a Payback Period of 11.86 months. This project entails a capital Investment of 14,898,032.00 S U.S. 'This big. capital investment presents another problem to our local enterpreneurs.

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CONCLUDING REMARKS

This overview on the work done and constraints presented on the use of B. t. in Philippines is typical of a developing country. The progress in the use of this organism for microbial control for developing countries is hard to predict. We, as researches have done our part. By then, who is really the one responsible to make the dream a reality? What else is to be done?

ACKNOWLEDGMENTS

This research has been financed in part by a grant from UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, International Foundation for Science (IFS), US-Israel CDR Grant, Department of Science and Technology (DOST), National Research Council of the Philippines (NRCP), Philippine Rice Research Institute (PhilRice), UPLB Basic Research and the National Institutes of Biotechnology and Applied Microbiology (BIOTECH), University of the Philippines at Los Banos, Laguna, Philippines.

REFERENCES de Barjac, H. (1978). Un nouvelle vari&e de Bacillus thuringiensis tres toxique pour les moustiques : Bacillus thuringiensis subsp. israelensis serotype 14. C.R. Acad. Sci. Paris Ser. D, 286, 797-800. Dulmage, H.D. and Cooperators. (1981). Insecticidal activity of the isolates of Bacillus thuringiensis and their potential use in pest control, 193-211. In : H.D. Burges (ed.), Microbial Control of Pest and Plant Diseases, 1970-1980. Academic Press, London. Faust, R.M. and Bulla, L.A. Jr. (1982). Bacteria and their toxins as insecticides, pp. 75-208. In : E. Kurstak (ed.), Microbial and Viral Insecticides. Dekker, N.Y. Gabriel, B.P. (1968). Entomogenous microorganisms in the Philippines : new and past records. Philipp. Entomol., 1, 97-130. Gabriel, B.P. (1970). Additional record on the microbiota of Philippine insects. Philipp. Entomol., 1, 465-472. Goldberg, L.J. and Margalit, V.F. (1977). A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaenia unguiculata, Culex univitatus, Aedes aegypti, and Culex pipiens. Mosq. News, 37, 355-358. Heimpel, A.M. (1967). A critical review of Bacillus thuringiensis var. t aringiensis and other crystalliferous bacteria. Ann. Rev. Entomol., 12. 287-322.

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Hermstadt, C. ; Soares, G.G. ; Wilcox, E.R. and Edwards, D.L. (1986). A new strain of Bacillus thuringiensis with activity against coleopteran insects. Bio/Technology, 4, 305-308. Ibarra, J.E. and Federici, B.A. (1986). Parasporal bodies of Bacillus thuringiensis subsp. morrisoni (PG-14) and Bacillus thuringiensis subsp. israelensis are similar in protein composition and toxicity. FEMS Microbiol. Lett., 34, 79-84. IRRI Highlights. (1986). Bacillus thuringiensis : an indigenous microbial pesticide, 16-1'7. Krieg, A. ; Huger, A.M. ; Langenbruch, G.A. and Schnetter, W. (1983). Bacillus

thuringiensis var. tenebrionis : ein neuer, gegenuber Larven von Coeeopteren wirksamer Pathotyp. Z. angew. Ent., 96, 500-508. Lacey, L.A. and Singer, S. (1982). Larvicidal activity of new isolates of Bacillus sphaericus and Bacillus thuringiensis (H-1) against anopheline and culicine mosquitoes. Mosq. News, 42, 537-543. Lacey, L.A. and Oldacre, S.L. (1983). The effect of temperature, larval age, and species of mosquito on the activity of an isolate of Bacillus thuringiensis var. darmstadiensis toxic for mosquito larvae. Mosq. News, 43, 176-180. Lacey, L.A. ; Lacey, C.M. and Padua, L.E. (1988). Host range and selected factors influencing the mosquito larvicidal activity of the PG-14 isolate of Bacillus thuringiensis var. morrisoni. J. Am. Mosq. Cont. Assoc., 3, 39-43. Pacumbaba, E.P. and Padua, L.E. (1987). Screening of Bacillus thuringiensis isolates against coconut slug caterpillars (Lepidoptera: Limacodidae). Proc. 11th Internal. Cong. Plant Prot., Manila, Philippines. Padua, L.E. (1988). Development of microbial pesticide for mosquito control in the Philippines. Proc. Coordination Workshop in Bacterial Control of Agricultural Insect Pests and Vectors of Human Diseases. Ein Gedi, Israel. Padua, L.E. ; Ohba, M. and Aizawa, K. (1980). The isolates of Bacillus thuringiensis serotype 10 with a highly preferential toxicity to mosquito larvae. J. Invertebr. Pathol., 36, 180-186. Padua, L.E. ; Ohba, M. and Aizawa, K. (1984). Isolation of a Bacillus thuringiensis (Serotype 8a : 8b) highly and selectively toxic against mosquito larvae. J. Invertebr. Pathol., 44, 12-17. Padua, L.E. ; Ohba, M. and Aizawa, K. (1988). Comparative serology of crystals

produced by PG-14, Bacillus thuringiensis subsp. morrisoni (serotype H. 8a : 8b) and Bacillus thuringiensis subsp. israelensis (serotype H-14). J. Invertebr. Pathol., 52, 192-194. Padua, L.E. ; Gabriel, B.P. ; Aizawa, K. and Ohba, M. (1982). Bacillus thuringiensis isolated in the Philippines. Philipp. Entomol., 5, 199-208.

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Padua, L.E. ; Ebora, R.V. and Moran, D.G. (1987a). Screening of Bacillus thuringiensis against Asiatic corn borer, Ostrinia furnacalis (Guenee) and diamondback moth, Plutella xylostella (L.). Tech. Bull. No. 103. FETC, Taiwan, Rep. China, 1-7. Padua, L.E. ; Wen, C.X. ; Lizhi, L. and Margalit, V.F. (1987b). The susceptibility of the lepidopterous pests of rice to Bacillus thuringiensis. Proc. 11th Internal. Cong. Plant Prot., Manila, Philippines. Undeen, A.H. and Nagel, W.L. (1978). The effect of Bacillus thuringiensis ONR-60A strain (Goldberg) on Simulium larvae in the laboratory. Mosq. News, 38, 524-527. Identification and Purification of Differents Exotoxins from Nine Strains of Bacillus thuringiensis

B. A. AM , L. Ferid and B. Omrane Laboratoire de Biochimie, Faculte des Sciences de Tunis, Tunisia

ABSTRACT

The main toxin products of Bacillus thuringiensis (B. t.) include a protein endotoxin and a heat stable exotoxin which is released into the cultivation medium of the bacteria. By high performance liquid chromatography and inhibitory tests of the biosynthesis ofRNA, we have isolated five components from growth medium of nine different strains The of B. t. , two of these inhibit the synthesis of RNA in the yeast. component evaluated at 4 minutes by H. P. L. C. is the thermostable f3-exotoxin which is a potential inhibitor of transcription. (Ki=5, 1.10-6 for yeast RNA polymerase A). Another component was released only into the growth medium of T7 galleriae) and T9 (tolworthi) and has an inhibitory effect less important than ff-exotoxin (Ki=20, 4.10-6 for yeast RNA polymerase A). The other components are not effective on the RNA synthesis but show a toxic role on the yeast culture and immature flies.

INTRODUCTION

In the area of the biological products, Bacillus thuringiensis (B.t.) shows a large development in different sectors as agriculture, health, industry and fundamental research. B. t. can be used as a whole bacteria or a spore or a crystal protein by pulverisation, mixing with different media or integrating in plant by the genetic engineering. Different problems seem to be resolved to confront the practical contraints. - How we determine the toxicity in the range and site action? - How can we know the specificity and the tolerance of the toxins?

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- The purification and the problem of synergism between different components? -- Which toxin we can use and how it can be applied? - Which strains we can use easily and specifically? To contribute in the cocktail of these questions, we started to study the different components excreted by B. t., determine the corresponding structure, the biochemical effect and the biological action.

RESULTS The 13-exotoxin as purified by DeBarjac and DeDonder (1968) has been used as a model of study to focus our attention on the use of the bacteria in the general domain of pesticides.

It is known that the f3-exotoxin as an analogue of the ATP inhibits the synthesis of the RNA and particularly acts on the RNA polymerases. Using the three purified RNA polymerases of the yeast Saccharomyces cervisiae and the purified enzyme of Echerichi coli it has been found that :

1 - All the enzymes can be inhibited by the exotoxin. 2 - The rate of the inhibition depend on the enzyme. The more inhibited is the RNA polymerase A; 50% activity was obtained at 5pg/ml while the activity was 0% at 10 pg/ml. The RNA polymerase B is less inhibited. The polymerase C is slowly inhibited as the RNA polymerase of E. coli. 3 - The exotoxin action depends on the template used. 4 - In general the inhibition is competitive. 5 - A mix result was found with the RNA polymerase B.

When the exotoxin was labelled with the tritium, the autoradiochromatogram gives different labelled compounds. The proton NMR spectra shows a heterogenous resonancy. The re-chromatography of the exotoxin on H.P.L.C. system with C18 reversed phase column shows different nucleotides. The bacteria excretes different compounds. Nine serotypes of B. t. have been studied for the nucleotides components excreted, the phase of excretion and the relative action by each one. Five components have been detected, and found highly purified. The 13-exotoxin is excreted by the nine serotypes used, the others called 1,2,4 and 5 depends on the serotype. The fifth component excreted only by T, and T9 inhibits the RNA

- 190 - Endotoxins from Strains of B.t. polymerases with a low rate.

Ki = 5,6. 10-6M for RNA polymerase A of yeast.

Ki = 20,4.10-6M for RNA polymerase of E. coll.

The three other components have no effect on the RNA polymerase but they are toxic to biological system tested as the growth of the yeast (component No. 4) and the larvae developement of Ceratitis capitata (components No. 2,3,5) The production of each component has been studied with the different growth conditions and stadium. The high purification of the 8-exotoxin which gives a homogenous product permits the biochemical study of the nucleotide and the biological action site.

The 8-exotoxin acts on the RNA polymerases activity, it inhibits the initiation of the transcription in the tertiary complex enzyme-template-substrates. Table (1) and Figs. (1-3).

Table (1) Nucleotides production for 2 g.of bactrial cells.

Total Nucleotide Strains Nucleotides excretion production

No. 1 0,033 0,320 No. 2 0,003 0,030 TI 390 No. 3 0,013 0,126 No. 4 0;012 0,117

No. 1 0,044 1,100 No. 2 0,010 0,270 T7 1000 No. 3 0,039 0,987 No. 4 0,00 0,765 No. 5 0,230 5,750

No. 1 0,023 0,590 No. 2 0,037 0,940 T9 1000 No. 3 0,040 1,000 No. 4 0,008 0,210 No. 5 0,080 2,120

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Table (1) continued

Total Nucleotide Strains Nucleotides excretion production

No. 1 0,06 0,340 T10 230 No. 2 0,029 0,160 No. 3 0,076 0,438

No. 1 0,030 0,210 No. 2 0,016 0,104 T13 250 No. 3 0,017 0,111 No. 4 0,001 0,009

No. 1 0,071 0,445 No. 2 0,030 0,187 T14 250 No. 3 0,099 0,610

No. 4 0,027 . 0,173

No. 1 0,029 0,500 No. 2 0,023 0,410 T33 700 No. 3 0,082 1,440 No. 4 0,017 0,290

No. 1 0,042 1,010 No. 2 0,030 0,910 T44 950 No. 3 0,190 4,620 No. 4 0,123 2,930

No. 1 0,030 0,180 No. 2 0,020 0,110 T55 200 No. 3 0,030 0,190 No. 4 0,017 0,080 100

s0

wo

0 I i I I I I I 1 r-L I _1_ 1 0 10 20 30 40 s0 0 10 20 30 40 s0

Exotoxin (Ng/mL)

Figure 1. Effect of exotoxin on the RNA polymerases activity A B C (11 , ) of Yeast and RNA polymerase of E.coli ( after transcription "in vitro" in poly dAT and heated denatured DNA. 100

50

0 1 I 1 I 1 1 1 1 1 1 I i I I I I I I I 30 40 50 0 10 20 30 40 50 10 20 Exotoxin (Ng/mL)

Figure 2. Exotoxin effect on the synthesis of pApU dinucleotide ( o ---o ) and UpApU (,a-- ) trinucleotide. Endotoxins from Strains of B.t.

0

T7 a b c d 0.01. e

C a

E

IT 9 p V Q a

b A

0 2 4 6 8 10 Time min

Figure 3. High-performance liquid chromatogram of growing medium in which B.t. had been incubated at 30°C for 72 h. - Total extract of the T, serotype. - Different nucleotides elueted on H.P.L.C. - T.L.C. profil of the different fractions.

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CONCLUSION

8-exotoxin of B. t. was used as a model study of the effect of its different components. This model will serve as an example to judge the quality of the product. Application of Biotechnology in Pest Control using Bacillus thuringiensis Formulations

A. Merdan Faculty of Sciences, Ain Shams University, Cairo, Egypt.

ABSTRACT Prospects of application of genetic engineering techniques in pest control research using the microbial control agent Bacillus thuringiensis (B. t.) are discussed, with more emphasis in the target insect, the control agent B. t. and host plants of the insects. The risk factor of genetically engineered microbial pesticides has been dealt with, together with some recommendations and safety guidelines.

The use of biological agents to control pests has been known and practised for a long time. The Chinese used Pharaohs ants to control pests in their grain stores. Mynah birds were brought to Mauritius in 1762 to control red locusts, dogs and many other animals have been used to control insects and rodent pests. Domestic cats which are thought to be the first biological control agents, played such a significant role in rat control that their presecution in the Middle Ages may well have contributed to the spread of bubonic plague in Europe, However their effects on non-target organisms were unpredictable and in those days, no tests were required for registrations.

The use of microorganisms as pesticides is more recent and has had some notable successes. At present there are a number of bacteria, fungi and viruses which have been introduced as commercial pesticides, frequently after skillful production and formulation efforts. Bacteria are the most promising agents in this respect. Over 90 species of bacteria which infect insects have been described. Most belong to the families Pseudomonadaceae, Enterobaeteriaceae, Lactobacillaceae, Micrococcaseae and Bacillaceae. The majority of the commercial strains belong to the genus Bacillus and the most widely used products are made from Bacillus thuringiensis, (B. t.) of which there are over 22 serotypes ( determined

by flagellar antigen ) . Members of the group are toxic to lepidopteran, dipteran

- 197 - A. Merdan and coleopteran insects. B. t. was discovered in silkworms in Japan in the early part of the 20th contury. The first commercial product was produced in France in 1938 and was based on strain which also made a toxin dangerous to man, called the B-exotoxin. Various methods were developed to remove this toxin from the commercial product by processing the fermentation broth. Dulmage isolated B. t. HD-1, an exotoxin-free strain of B.t.kurstaki which is now the active ingredient of most commercial strains of B. t. used against Lepidoptera.

In spite of the available information of the efficacy of B. t. formulations under field conditions, yet there are restraints in the use of the existing bacterial larvicide formulations against insect pests. One of the major restraints is the difficulty to convince the industrial interest for developing improved formulations as a second tool since the market of B. t. is not yet large, representing a slightly over 0.1% of the world pesticide market, however ; it is over 90% of all microbial pesticide sales.

To overcome this difficulty beside other restraints such as spreading potential, power of search for host, propagation rate, virulence and stability of field, ways and cost of production as well as biosafety measures, the application of biotechnology to develop a new promising tool based on B. t. agent seems to be the solution.

The present goal of applying biotechnology is to explore scientific and technological concepts central to developing molecular biological strategies for insect control. Successful use of several microbial preparations applied against target pests has prompted active research on the enhancement of epizootics for long-term pest management strategies. Genetically engineered microbial organisms have been obtained, and new mass production technology has been developed. It became possible to synthesize biological molecules by implanting specific genes into various kinds of microorganisms. One of the most remarkable application of genetic engineering is the manufacture of B. t. toxins using bacteria as factories. The prospects of applying genetic engineering techniques in pest control research may start in three directions: insects, microbial agents and host plants.

L INSECTS

Studying the mode of action on the molecular point of view, understanding

- 198 - Biotechnology of B.t. the reasons of specificity of the toxins toward the insects tested, structural function relationship, metabolism of the toxin. Designing effective microbial pathogens requires an intimate understanding of B. t. mode of action and how it interacts with the target insect and environment. Examples of other avenues of insect-toxin gene manipulation in pest control programs are the decrease or elimination of the development of resistance towards bacterial insecticides and other toxic chemicals. It is well known that organisms resist the effects of toxic elements either by developing ways of preventing the toxins from reaching the target sites or by modifying the site so that its sensitivity to the toxin is decreased. The metabolism of the toxic substances is a common mechanism for preventing the toxin from reachig the site. Scientists have been recently able to clone the gene that control the synthesis of the insect resistance and this should help them to understand how gene works.

2. ENTOMOPATHOGENS (B.t. )

( a ) Direct Molecular biology affords the possibility not only of improving these natural pathogens to better suit their targets but also of converting a non-pathogenic microbe to a pathogen, thus creating new weapons for the arsenal against insect pests.

Placing B. t. toxin genes in E. coli would serve in mass production of the toxin in addition to studying the interaction of different toxins and site directed mutagenesis from the academic point of view. Transferring toxins among different Bacillus based hosts will serve us better in the field, thereby gaining the wider spectrum of B. t. in a bacterium capable of surviving, persisting, and perhaps even recycling in the environment. The hope would be to see synergisms occur and new larvicidal activities develop. Using the same goal but with a protoplast fusion may require a system for classifying (naming) the fusants.

We ought to keep in mind that we do not yet understand all of the nature tricks for balancing insect populations besides laboratory manipulations. The search for new strains, specially in tropical or developing countries, should be actively pursued.

Advances in bioassays of different crystals using tissue culture techniques were

- 199 - A. Merdan

developed. Insect tissue cells are a valid model system for studying the toxic response to activated protein from crystals of (B.t.) The genes for several B. t. toxins have been cloned, sequenced, expressed in bacteria and the minimum genetic sequence required to encode a fully active toxin protein has been defined. The stage is now set for ultimately engineering more potent B. t. toxins. So that smaller doses can be used in the field in order to offset the currently high usage cost of the product, wider host range, more safe for non-target organisms, easy mass produce and more persistant under field conditions. Also, molecular biologists having transferred B.t. toxin gene into non-pathogenic Pseudomonas, and thus converting it to a new microbial agent active against Lepidoptera.

( ) b Indirect ( Cloning in symbiotic and other agents ) :

Another potential useful approach for a more efficient application of the insecticidal toxins of B.t. is the introduction and the expression of the toxin gene in the yeast cells.

It is hoped that recombinant DNA technology would produce in a suitable bacterial agent toxic materials which can overcome most of the drawbacks of the already existing microbial control agents such as non-persistance under field conditions and irradiation inactivation of their pathogenicity.

The delta-endotoxin of Lepidoptera specific strains of B.t. are currently the most intensively studied microbial insecticides when tested on series of Lepidopteran species, crystal protein from different strains show distinct insecticidal spectra. Immunoassays with polyclonal and monoclonal antisera indicates that multiple, structurally distinct polypeptides of Mr 130-140 can be present in single strain. These proteins are proteolytically converted. DNA probes and plasmid curing have been used to show that the genes which control the synthesis of the crystal production are located on a small number of high molecular weight plasmid. The toxic protein has been cloned into E. coli and B. subtilis, and has been expressed even during the vegetative growth phase.

Proteins from different strains have been combined, thereby expanding the activity spectrum of the recombinant strain. Wholly new strains have been reported with activity against additional groups of insects such as Coleoptera infected by

- 200 - Biotechnology of B.t.

B. t. tenebrionis and var. San Diego.

The intial report of this strain included the cloning and expression of its toxin gene in E. coli and the comment that the entire assembles of molecular genetic technology can be brought to bear on the fragment of DNA that contains the nucleotide sequence coding for B.t.s.d. toxin protein with the aim of producing novel genes that have improved transcription translation properties, encode protein with enhanced toxicity or with altered host range specificity.

Several genes for toxic protein have been isolated, and the amino acid sequence for the active site of protein from the DNA sequence of the gene and elucidated the composition of the crystal has been determined. The mode of action at the molecular level is not yet proved but receptors such as phosphatidyl choline and N-acetyl galactosamine may be the target sites for the toxin.

It was speculated on the possibility of using computer graphics to model the interaction between the toxin and insect receptors which could lead to prediction of insecticidal protein structures. After the secondary and tertiary structure of the protein is known, the active site can be described; this could lead eventually to custom-built biological insecticides.

New biotechnological techniques may make a contribution through increases in potency by modification of the plasmid complement of the bacterium which control the synthesis of the protein. This would both reduce the cost of production and provide the starting place for new, more easily used formulations. Alternatively, production of asporogenic strains may reduce production costs by avoiding the waste metabolic energy on spore production and may make the product more acceptable in certain countries.

In the shorter term, a variety' of more specifically targeted products may be developed. In 1985, Sandoz successfully field-tested a new product, Javelin, which was based on NRD-12, a 3A 3B strain discovered by Norman Dubois of the United States Forest Service. Although the strain was discovered and characterized for its enhanced effect against gypsy moths, Javelin's improved effects are against Spodoptera littoralis a pest of vegetables and in some countries of cotton. New products for control of Coleoptera based on the two strains cited above find a variety of markets, especially for control of Colorado potato beetle .

- 201 - A. Merdan

3. CROP PLANTS Engineering plants for insect resistance could one day be a safe and inexpensive alternative to using toxin chemicals for insect control. The development of smaller Ti plasmid vectors containing multiple dominant selectable markers, cloning sites, efficient promoters and poly A signals has greatly stream line agrobacterium mediated plant transformation. Additional techniques including microinjection, electroporation and particle acceleration will most likely make possible the transformation of plants which have thus far resistant transformation by the agrobacterium approach. In addition to transformation, another formidable challenge will be learning how to regenerate mature plants from transformed cells or protoplast, in those cases that have to date been refractory.

At the present time sufficiently good model plant system exist that can be used to set genetic engineering strategy for insect control. The successful introduction of B. t. toxin genes into Tobacco, resulting in otherwise normal plants resistant to Tobacco horn worm was reported.

Risk Factor of Genetically Engineered Microbial Pesticides

In testing and evaluating for risk associated with these pesticides, it is possible to identify a set of general factors which may contribute to risk. Because each product and its pattern of use is unique, these risk factors must be considered on a case-by-case basis respecting the regulatory and testing precedents for microbial pesticides identified above.

1. Because of their inherent pesticidal nature, these organisms are intended to have biological activity. They may control pests by a variety of modes of action : lethality; pathogenicity; competitiveness or displacement; deterrence of feeding; inhibition of growth; or adverse reproductive effects. Furthermore, the organisms are designed for environmental application; they may be applied to food crops, forests, rangeland or for vector control, close to homes. Therefore, exposure to non-target organisms and human may be widespread.

2. Unlike chemical pesticides, microbes are living entities, which may survive and replicate. Once releases into the environment, absolute containment is not possible. Thus, prior to any release of genetically engineered microbes, their capacity to flourish and find new ecological niches or to compete successfully

- 202 - Biotechnology of B.t. against other microorganisms in the environment must be carefully evaluated. Any assessment of environmental impact must take into consideration expected levels of the organisms in the environment but, in addition, must include reliable predictions of the effect of unusual environmental conditions and their influence on localized increases or blooms in the microbial population.

3. Any testing scheme for characterizing genetically engineered microbes or for screening their effects on or exposure to non-target organisms provides data for characterizing risk. However, placing an engineered organism into a new habitat can lead to unforeseen consequences. Thus, the ecological scenario for any proposed application of engineered organisms must be carefully evaluated, using approaches from a variety of scientific disciplines.

4. Certain types of engineered vectors have a high probability of genetic transfer. For example, transposons with broad host - range spectra are highly moveable chromosomal genetic elements which are able to transfer readily from one organism to another. This can lead to two problems: inserted genes might be expressed in other species of microorganisms in the environment and, in addition, multiple copies of the gene might possibly be produced in an individual species of microorganism, thereby increasing the activity.

5. Certain genetically engineered microbial pesticides may have enhanced efficacy against the target pest or may incorporate two toxins in one organism. Even though the host range of the non-engineered organism and its effect on beneficial insects may be known, it is worthwhile to re-evaluate the effects of the engineered microorganism by screening pathogenicity and infectivity in non-target and beneficial insects.

6. The genetically engineered microbial pesticide, by virtue of a unique combination of traits, may be able to persist in the environment in new niches in sufficient numbers to cause adverse effects on non-target species.

7. Increased environmental persistance may be a problem under certain circumstances. For example, the B. t. toxin gene can be inserted into a long-lasting or readily reproducing recipient or host, such as blue-green algae. This can be desirable for enhancing the useful lifetime and availability of the B. t. toxin to mosquito larvae, but may also lead to unforeseen problems, especially if the

- 203 - A. Merdan engineered strain is not narrowly focused in its target pest specificity.

8. With widespread use, after an engineered microbial agent passes all safety screens, the target pest may develop resistance to microbial toxins to the extent that they persist for long periods or continuously in the environment at high levels.

RECOMMENDATIONS To develop methods to assess and reduce the risk of introducing genetically engineered larvicidal organisms into the environment, the following should be considered: (a) In addition to the existing guidelines for safety testing of microbial agents for insect control, standards and methods for a stepwise approach to testing the efficacy and safety of genetically engineered microorganisms from the laboratory to the field should be developed. Testing beyond the laboratory should move sequentially to (i) simulated microcosms, (ii) containment greenhouses or growth chambers, (iii) restricted small-scale field evaluations. (b) The engineered product should be precisely defined and selected with emphasis on desirable traits to allow for adequate assessment of ecological impact. (c) Quality control of engineered strains should be well developed. In microorganisms developed for environmental application, the potential for genetic drift should be eliminated or minimize to the extent technically feasible. Transferred genetic elements should be immobilized, and, as noted before mechanisms for- self-containment or self-destruction should be engineered into recombinant microbial pesticides. Utilization of Bacillus thuringiensis for Crop Protection in Egypt, Emphasizing Costraints

F. N. Zaki Pests and Plant Protection Dept., National Research Centre, Dokki, Cairo, Egypt.

ABSTRACT

The entomopathogenic bacteria Bacillus thuringiensis (B. t.) represents a good example for the new methods of biological control. The successful use of B. t. to control some insect pests specially the cotton leafworm, Spodoptera littoralis, the black cutworm Agrotis ypsilon, the lesser cotton leaf worm Spodoptera exigua and other lepidopterous insects was repeatedly demonstrated infield experiments till 1991. The fields were cultivated with soybean, okra, cotton, Egyptian clover and some other crops. The area in which B. t. was applied increased from small plots, less than one feddan, to about 100 feddans in 1991.

INTRODUCTION

The successful use of Bacillus thuringiensis (B. t.) to control lepidopterous insect pests such as the cotton leaf worm, Spodoptera linoralis, the black cutworm, Agrotis ypsilon, and the lesser cotton Spodoptera exigua was repeatedly demonstrated in field experiments in Egypt.

MATERIALS AND METHODS In all field experiments carried out by Salama and his collaborators ( unpublished work ) molasses was added to B. t. suspensions to serve as a spreader-sticker, at the rate of 4 L/f. Two hundred liter water per feddan was applied on using knap-sack sprayers and 500-600 liter water/feddan on using spray motors. Spraying was always carried out before sunset to avoid the adverse effect of the ultra-violet light during the day and to give the larvae the chance to feed on the sprayed leaves all the night. One kilogram of calcium carbonate or calcium

- 205 - F. N. Zaki

oxide was added to the spray of each feddan to enhance the effectiveness of B. t. ( Salama et al., 1985, 1986, 1989 ).

For the application of baits against Agrotis ypsilon the desired amount of Dipel with or without the additives was added to molasses ( 4 L/f ) and wheat bran ( 25 kg./f ) moistened with water. The components were thoroughly mixed to .insure homogenous distribution of the ingredients in the baits before spreading between the plant seedlings. The baits were prepared just before field applications. In large scale field applications, 1 kg. of calcium carbonate or calcium oxide was added ( Salama et al., 1989 ).

The potential effect of B.t. in suppressing the population of Pieris rapae on different varieties of cabbage in Egypt has been evaluated by Salama et al., ( 1991 ). The commercial product Dipel 2X ( B. t. var. kurstaki HD-1 ) when used as a dust, scored the best results in controlling Pieris rapae attacking the winter cabbage, Brassica oleracea var. brunswick. One or two spray applications of Dipel 2X at the rate of 200 g/feddan, or 3 spray applications at one week intervals, at the rate of 100 g/feddan provided satisfactory protection for the cabbage when infested with P. rapae. The yield of marketable heads from Dipel 2 X treated plots was equal to that harvested from plots treated with the chemical insecticides (Lannate or Gardona).

Salama et al. ( 1990 ) found that the effective threshold rate of application of Dipel 2X to control A. ypsilon on some vegetable crops was 250 g/f. Incorporation of some chemical additives such as CaSO4 or CaO, significantly potentiated the effectiveness of Dipel 2X on larval population. They found that the addition of CaCo3 and ZnSO4 to Dipel 2X greatly reduced the larval population and led to a significant increase in the yield of some vegetable crops. Dipel 2X baits at 250 g/f. were almost as effective as the chemical insecticide (Hostathion) when used at 1.5 L/f.

Field trials were conducted to evaluate the effect of two commercial preparations of B. t. ( Dipel and Thuricide-HP ) on the population of Phthorimia operculella on potato plants in Egypt, compared with two chemical insecticides ( Sevin and Gusathion ). Dipel and Thuricide treatments as well as Gusathion treatment were efficient in reducing potato tuber moth infestation and thus led to a high potato yield ( Haydar and EI-Sherif, 1987 ).

- 206 - Utilization of B.I. in Egypt

In 1981, El-Husseini, found that Bactospeine application in clover fields was efficient against S. littoralis without any efficacy on the existing predators ( Coccinella undecimpunctata and Labidura riparia ), while Nuvacron application suppressed the populations of the pest and benefical insects. Larvae of S. littoralis that lived for more than 10 days were developmentally retarded.

Field Experiments

In 1990 Dipel 2X, wettable powder ( 32.000 IU ), Dipel ES emulsifiable suspension ( 17.600 IU ) and a local product B. t. var. entomocidus were used in 70 feddans cultivated with cotton against A. ypsilon as baits and spraying against S. littoralis, Pectinophora gossypiella and Earias insulana. Percent of reduction in A. ypsilon reached 98%, which is equal to that obtained when the chemical insecticide (Hostathion) was used. The percentage of mortality in S. littoralis ranged from 57 to 71% when different preparations of B. t. were sprayed. The percentage of infested bolls with the two bollworms was 16%, 21% and 65% in areas treated with B. t. , chemical insecticides and those untreated areas, respectively. The obtained yield showed no significant difference between areas treated with B. t. or chemical insecticides. During the same season, B. t. was used against A. ypsilon and S. littoralis in an area of 60 feddans cultivated with soybeans. More than 90% reduction was obtained in the population of A. ypsilon treated with B. t., percentage of reduction in the population of S. littoralis ranged between 82.7 - 91.3% in areas treated with B. t., compared with 95.7% in areas treated with chemical insecticides. Results obtained showed that the yield increased when two sprays were carried out using B. t. against S. littoralis.

In 1991, three experiments were carried out by Salama and his collaborators on cotton and soybeans using B. t. An area of 28 feddans in Dakahlia governorate, cultivated with cotton were sprayed with Dipe12X against S. littoralis at the rate of 750 g/f. One feddan of cotton was sprayed with the local product (B. thuringiensis var. entomocidus ). Three applications were carried out at two weeks intervals.

In Fayoum governorate on area of 28 feddans of cotton were also sprayed wish Dipel 8L ( 17.600 IU ), at the rate of 750 cc/f against bollworms. In this area 3-5 applications were made and compared with the chemical insecticide

- 207 - F. N. Zaki

treatments. In Menofia governorate, an area of 15 feddans cultivated with soybean were sprayed one or two times with Dipel 2X against S. linoralis. Results obtained shoved that the percentages of mortality of S. littoralis in the first experiment were

100, 95 and 80% for the 1 st, 2 nd and 3 rd sprays on cotton, respectively. The yield of cotton in this experiment was higher than other areas where the egg hatch of S. littoralis was hand - picked. It has been also found that 4 or 5 applications led to a low infection with the bollworms, compared to those areas treated with chemical insecticides and the yield was much higher. Experiments on soybean showed that the percentage of mortality in areas treated with B.t. was less than the areas treated with Lannate ( carbamate insecticide ). Insignificant differences were recorded in the yield of areas treated twice with B. t. and those treated with Lannate.

PERSISTANCE IN THE FIELD The low field persistance of B.t. is a major problem regulating its effective use for pest control. In 1983, Salama et al. studied the persistance of different formulations of B. t. spores after spray application in cotton cultivations in Egypt. They found an obvious reduction after one day of weathering. The decay in the spores viability is progressively correlated with the time of exposure in the field. The spores half life of the tested B. t. formulations ranged between 75 and 256 hours and cannot be correlated with the temperatures attained on the surface of sunny cotton leaves. Ultraviolet radiation seemed to be the dominant factor affecting the viability of spores.

A measure of the viable spores of B. t. preparations at various intervals after spray application in cotton cultivations was carried out during two successive seasons 1980-1981. Salama and Zaki ( 1985 ) determined the mortality of neonate larvae of S. littoralis in correlation with the field persistance and subsequent decay of spores of B.t. preparations on the cotton plant leaves. Various preparations of B.t. differ in their degree of protection from sunlight and their efficacy against S. littoralis though there was inconsistency in some cases. The decay in the spore viability showed to be proportional to larval mortality, but this does not necessarily mean that insect intoxication is only correlated to the spores viability. The same authors reported that sunlight seems to be the main factor affecting the spores viability. They mentioned that the use of adjuvants or phagostimulants such as

- 208 - Utilization of B.t. in Egypt

of coax, extracts of cotton plant leaves or jew's mallow, increased the activity the subsequent the B. t. formulations vs. S. littoralis and they can compensate for spores decay through increased ingestion and hence higher mortalities but they did not act as protectants from inactivation by sunlight.

COMBINATIONS OF B.t. AND CHEMICAL INSECTICIDES

The combined use of biological and chemical insecticides depends on their compatability. In 1984, Salama et al studied the effect of selected chemical insecticides on the sporulation of B.t.var. entomocidus, and the effect of those combinations on the cotton leafworm, S. littoralis. Results showed that among the carbamates tested, carbaryl had a more deleterious effect on the sporulation process of B. t. than methomyl. Within the organophosphorous group, phoxin inhibited sporulation less than profenofos. The pyrethroid group, represented by fenvalerate, cypermethrin, and permethrin, showed less deleterious effects on sporulation of B. t. than other groups.

The same authors found that the pyrethroids and most organophosphorous compounds tested potentiated the activity of B.t. applied against S. littoralis. They found also that the carbamates, diflubenzuron, and a combination of methomyl and diflubenzuron showed an additive effect when applied jointly with B. t. varieties. The mild effect of pyrethroids on sporulation processes of B. t., compared to effects of other classes of chemical insecticides, suggested little or no interference with the ecology and perpetuation of this useful bacterium at the site of application. Synergistic interactions suggest that application of pyrethroids with B. t. may be a safe and effective means for controlling S. littoralis. Investigations have been made on the effect of combinations of sublethal dosages of the nuclear polyhedrosis virus and B.t. on the second instar larvae of B. t. var. S. littoralis ( Salama et al., 1987 ). Bioassays showed that the potency of entomocidus HD-635 was higher than that of B.t. var. galleriae HD-129. Combinations of B.t. HD-129 and SLNPV showed an antagonistic effect, while combinations of B. t. HD-635 and SLNPV showed an additive effect. The median lethal doses ( LD50 ) of B. t. varieties decreased with the increase of the viral dose in the mixture. The simultaneous application of SLNPV and B. t. especially with lower concentrations that showed an additive effect on S. littoralis larvae may be

- 209 - F. N. Zaki useful and effective for controlling this harmful pest.

REFERENCES El-Husseini, M.M. ( 1981 ). New approach to control the cotton leafworm, Spodoptera littoralis by Bacillus thrungiensis in clover fields. Bull. ent. Soc. Egypt, Econ. Ser., 12 , 1-6. Haydar, M.F. and El-Sherif, L.S. ( 1987 ). Microbial control of the potato tuber worm, Phthoriniae operculella in the field. Bull. ent. Soc. Egypt. Econ Ser., 16, 127-132. Salama, H.S. ; Matter, M. ; Zaki, F.N. and Salem, S. ( 1991 ). Field evaluation of Bacillus thuringiensis for control of Pieris rapae on two varieties of cabbage in Egypt. Discovery and Innovation, 3 (1), 71-76. Salama, H.S. and Zaki, F.N. ( 1991 ). Utilization of Bacillus thuringiensis to control the cotton leafworm in cotton and soybean and the bollworms in cotton in Dakahlia, Fayoum and Menofia governorates, Report from N.R.C., Cairo. Salama, H.S. ; Salem, S. ; Zaki, F.N. and Matter, M. ( 1990 ). Control of Agrotis ypsilon on some vegetable crops in Egypt using the microbial agent Bacillus thuringiensis. Anz. Schadlingskde, Pflanzenschutz, Umweltschutz, 63, 147-151. Salama, H.S. ; Moawad, S.M. and Zaki, F.N. ( 1987 ). Effects of nuclear polyhedrosis virus, Bacillus thuringiensis combinations on Spodoptera littoralis. J. Appl. Ent., 104, 23-27. Salama, H.S. and Zaki, F.N. ( 1985 ). Application of Bacillus thuringiensis and its potency for the control of Spodoptera littoralis. Z. ang. Ent., 99, 425-431. Salama, H.S. ; Foda, M.S. ; Zaki, F.N. and Moawad, S. ( 1984 ). Potency of combinations of Bacillus thuringiensis and chemical insecticides on Spodoptera littoralis. J. Econ. Entomol., 77, 885-890. Salama, H.S. ; Foda, M.S. ; Zaki, F.N. and Khalafallah, A. ( 1983 ). Persistance of Bacillus thuringiensis spores in cotton cultivations. Z. ang. Ent., 95, 321 - 326. Farmers Acceptability of the Microbial Control Application in Egypt

S. A. Salem National Research Centre, Dokki, Cairo, Egypt.

ABSTRACT

The application of pesticides depends on a number of factors particularly the technical knowledge and official support. Close collaboration between the scientists and farmers, has fostered the development of IPC approach and insect microbial control as one of its components.

INTRODUCTION Among the important activities in the area of agriculture, in Egypt, food production occupies a very important place and must be increased in rder to keep needs. pace with the progressive increase in human population and to fulfil their Attack by insect pests cause severe pre-harvest losses in the field and post-harvest losses during storage. Various control methods mainly the use of chemical pesticides are being adopted in agriculture in Egypt. The hazards of these pesticides to the environment, human-beings and animals are well known. Therefore, the shift towards using biological approaches or pest management systems is highly encouraged. In this for concern, the development of the microbial control agent, Bacillus thuringiensis possible use against some of our key lepidopterous insect pests received a great attention during the last 10 years from the scientists of the National Research Centre and other institutions in Egypt. Based on the investigations that have been carried out by Salama and his collaborators (1984, 1985, 1989), a series of large-scale field experiments was carried out using the microbial control agent Bacillus thuringiensis against the major lepidopterous insect pests attacking cotton, soybeans and other oilseed crops. The results obtained were compared with those of traditional chemical insecticides.

- 211 - S. A. Salem

RESULTS The farmers in various governorates are well aware of the major importance of the lepidopterous insect pests, Spodoptera littoralis, Pectinophora gossypiella on cotton and oilseed crops in Egypt. They also believed of the great hazards caused by chemical pesticides. The farmers knowledge of biological control approaches is very low, with the exception of their awareness of some birds predating upon the insect pests of their crops. This fact was similarly reported in many African countries (Saxena et al., 1989).

The application of B. t. formulations on various crops led to successful results. The experiments have been run for 3 successive years. The participation of the farmers in these pilot scale experiments from the beginning till harvest time is very essential to get convinced.

In the first season, the farmers were so cautious on using B. t. formulations since they were unable to get the immediate killing effect caused by the chemical insecticides. However, after repeating the experiments for 3 seasons, and based on the evaluation of final crop yield, the farmers accepted the idea of using these biological control agents and prefer it to chemical insecticides.

REFERENCES

Saxena, K.N. ; Pala OKeyo, A. ; Seshu Reddy, K. ; Emolo, E. and Ngode, L. (1989). Insect pest management and socio-economic circumstances of small scale farmers for food crop production in Western Kenya, A case study. Insect Sci. Applic., 10 (4), 443 - 462. Salama, H. S. (1984). Bacillus thuringiensis Berliner and its role as a biological control agent Z. ang. Ent., 98, 206 - 220. Salama, H. S. and Zaki, F. (1985). Application of Bacillus thuringiensis for the control of Spodoptera liuoralis. Z. ang. Ent., 99 (4), 425 - 431. Salama, H. S. ; Foda, M.S. and Sharaby, A. (1989). Potentiation of Bacillus thuringiensis endotoxin against he greasy cutworm Agrotis ypsilon Z. ang. Ent., 108, 372 - 380. Metabolic Characteristics of Bacillus thuringiensis during Submerged Fermentation

Me Tianjiant, Ma Tianlian2, Me Xinzhu3 and Yang Ziwen4

Hubei Academy of Agriculture Science' & 4. and East China Chemical Technology College2 & 3.

ABSTRACT

Metabolic characteristics were investigated during Bacillus thuringiensis ( B. t. ) submerged fermentation which was inoculated with spores directly. 0-4 hr. was tested for spores germination but almost no metabolism was involved. Metabolism became very active within acceleration phase of6-8 hrs. and log phase of 8-10 hrs., concentration of nucleic acid and CFU grown exponently, NH2 - N NH+4 raised gently. Residual sugar and ATP raised first and dropped later, phosphorus and total sugar dropped gently and dissolved oxygen dropped sharply, viscosity and pH dropped first and then raised later. It seems that catabolized glucose played an important role during vegetative cell growth 1-2 hrs after entering stationary phase, appearence of exoprotease and dropping of NH2 . N and NH+4 demonstrated that nitrogen metabolism was at main position.

INTRODUCTION

Bacillus thuringiensis (B.t.) insecticide is safe to human beings and animals, and it does not pollute environment or damage natural enemies. More research work has been done on B. t. production and utilization. In China, the submerged fermentation has been adopted for large scale production for a long time. Medium of low concentration was used early with spore count of 2.5 x 109 spores/ml. There is great potential with improving composition of media and fermentation process. Several parameters were determined among the different growth stages of B.t. and their relationships were analysed.

- 213 - Me Tianjian et al.

RESULTS AND DISCUSSION

1. Cell Population, CNA ( total nucleic acid content ) and DO ( dissolved oxygen )

The pattern of nucleic acid content was almost the same as that of B. t. growth ( see Fig. 1 ). It was reasonable to use CNA to reflect the cell population. Spores were used directly as inoculum. During zero to four hour, the endospores germinated and developed to vegetative cells. The CNA and viable count were among the lowest levels. The cells metabolized did not divide, and almost no respiration was observed. DO remained at 100% level. During 4 to 6 hrs. vegetative cell started to reproduce, and entered lag phase. The log phase was from 8 to 10 hrs. and the cell achieved the full rate of growth. The increase of CNA was corresponding to viable count.The maximum value of CNA and viable count was 1.114 mg/ml and 4.25 x 108 cells ( or CFU ) / ml, respectively. After 9 hr. of culturing, the DO dropped sharply and reached the lowest level of 5%, for two hours, it was much lower than 25.2%u of critical DO. Along with the population increase, the deficiency of DO inhibited the cells from continuous growing. The growth rate decreased and ceased, then stationary phase was observed. The growth rate decreased and ceased, then stationary phase was observed. The CNA and CFU remained constant at its maximum value. Within the vegetative cytoplasm forespore and crystal were formed and consumed less oxygen. The DO raised again and maintained around 70% up to the cell lysis stage. It showed that the metabolism was still going slightly.

2. Ts ( Total sugar ), Rs ( Residual sugar ) and pH Very little total sugar ( Ts ) was consumed during the germination of spores ( Fig. 2 ). Ts and Rs didn't change obviously during 0 to 4 hours. After that, the vegetative cells appeared and released a large amount of extracellular enzyme. Carbohydrate was decomposed into glucose. The Rs content increased quickly and reached its maximum value of 3.4 mg/ml. Glucose was further converted into pyruvic acid and acetic acid through EMP and PP pathways. The accumulation of organic acid led to a drop of pH and it reached the lowest value of 6.3 at 7 hour. Along with the further conversion of pyruvic acid and acetic acid through a revised TCA cycle to produce ATP, pH raised and reached the value of 7.4 at 10 hour, which was suitable for the sporulation. pH value was going up steadily

- 214 - B.t. Characters during Fermentation

SC._- L.---+ 100-

Figure 1. The patterns of CNA, CFU and DO curves.

Ts (mg/ml) 28 Ts E 26 pH 24 32 8.5 H 22 2.8 8.0 2.4 20 3 2.0 7.5 1.6 18 7.0 1.2 16 - 6.5

1 I i t 4 6 8 10 12 14 16 18 20 22 (hr.)

Figure 2. The patterns of Ts, Rs and pH curves.

GP = Germination Phase LaP = Lag Phase LoP = Log Phase SP = Stationery Phase SC = Sporulation Ce I I L = Lysis

- 215 - Xie Tianjian et al. along with the accumulation of amino acids and amonia. At harvest, pH value was over 8. Sugar was consumed both in cell growth and in sporulation, but the use rate was higher in exponential growth. The consumption of sugar supplied energy and intermediate metabolite for construction of cell and formation of spore and crystal. Metabolism of sugar played a very important role in reproduction of cells. At harvest, the Ts content was 17.1 mg/ml. The rate of utilization was 3907o. It indicated that there was still a room for further use of carbohydrates.

3. NH2 - N ( amino acid ), NH4 ( amonia ) and E ( extracellular protease ) Along with the reproduction of vegetative cells, the accumulation of extracellular protease increased steadily during lag and log phase (Fig. 3). Protein sources were partly decomposed into amino acids, Part of the L-amino acid was converted to a-ketonic acid and entered the revised TCA cycle. At the same time, amonia was released. The content of amino acid and amonia was increased and reached its maximum value at the end of log phase. During stationary phase, the reproduction ceased and spores and crystals were formed. Extracellular protease content reached its peak value two hours after the beginning of stationary phase. Protein sources were decomposed greatly. Large amounts of amino acids and amonia were utilized for synthesizing the protein part of spores and crystals. The continuous use led to a decrease in their accumulation. Nitrogen metabolism was very active in stationary phase. Before harvest, the formation of spore and crystal stopped and the cell lysis occurred. The released amino acids and ammonia led to an increase in their concentration.

4. DP ( dissolved phosphorus ) and ATP During the growth of B.t., glucose was converted through EMP and PP pathways, then entered TCA cycle. Phosphorus was a necessary element. Dissolved phosphorus content decreased steadily along with the reproduction of cells through lag phase to log phase ( Fig. 4 ). The residue of dissolved phosphorus at 12 hour was only 0.15 mg/ml, which was less than half of the original content. It was not clear whether the deficiency of phosphorus inhibited carbohydrate metabolism and caused the cease of reproduction while the Ts still maintained a high level. But the addition of KH2PO4 into medium did not show obvious effect on the enhancement of fermentation level.

- 216 - B.t. Characters during Fermentation

GP.4LaP4---LoP-I-- SP- -SC_4-- L

E (V/mL) 80

60

40

20 0

NH J I 1 A I I_I I i 1 1 i 0 2 4 6 8 10 12 14 16 18 20 22 (hr.)

Figure 3. The patterns of NH2-N, NH 4 and E curves.

,-, GP-4LaP4- L -I

80 x M, E E 60 rn rn c E 0.35 40

0 0.30 20 Q 0.25 0.20 0.15 0 4 6 8 10 12 14 16 18 20 22 (hr.)

Figure 4. The patterns of DP and ATP curves.

GP = Germination Phase LaP = Lag Phase LoP = Log Phase SP - Stationery Phase SC = Sporulation Ce I I L = Lysis

- 217 - Me Tianjisn et al.

ATP curve showed that catabolism was the major metabolic pattern before 8 hours, generation of ATP was more than the consumption. The content of ATP increased steadily and reached its peak value at 8 hours. During the log phase, vegetative cells reproduced exponentially and biosynthesis increased. The consumption rate was obviously faster than the generation rate of ATP. The accumulation decreased sharply. At the end of log phase, the growth ceased and the content of ATP increased soon. During the formation of spore and crystal, there was still a certain consumption of energy for the ATP curve drop.

5. Viscosity

Viscosities of the culture beer were measured and the results are shown in Figure (5). After 4 hours) the germinated cells secreted starch-converting enzymes and starch was converted into subunits. The viscosity decreased quickly. During the lag phase, the beer viscosity increased along with the cell population increase and remained relatively constant in later phases. It should be mentioned that vegetative cells needed a large amount of oxygen in log phase, but the increased viscosity enhanced the transfer resistance of oxygen and gave more difficulty to oxygen supply.

-GP.4LOP SC-4---L--1

1

v0

6 5 4 3 2

0 2 4 6 8 10 12 14 16 18 20 22 (hr.)

Figure 5. The patterns of viscosity curve.

GP = Germination Phase LaP = Lag Phase LoP = Loq Phasc j

SP = Stationery Phase SC = Sporulation Ce!! L = Lysis I I

- 218 - B.t. Characters during Fermentation

CONCLUSIONS

1. It was feasible to use nucleic acid content to reflect the cell population. With the advantages of rapidity, easiness and accuracy, the determination of nucleic acid content could be used for middle analysis of fermentation process. 2. During log phase, all kinds of metabolic processes were very actively going on. It was the key stage for the improvement of fermentation level. The adjustment of carbohydrate metabolism was conducted mainly in this phase. Oxygen supply should be strengthened. Further research should be conducted to reveal the role of DP.

3. The mass biosynthesis of protein crystal was the final goal of B. t. fermentation. The clear interrelationships among nitrogen nutrition, protease and crystal might provide a basis for high fermentation toxicity. So, research on nitrogen metabolism should be strengthened, especially from stationary phase to sporulation stage.

Study on Optimization of Dissolved Oxygen to Raise Spore Count of Bacillus thuringiensis Berl.

Xie Tianjiant, Ma Tianliang2, Xie Xinthu3 and Ding Qiumei4 Hubei Academy of Agriculture Science, Wuhan, P.R.C.,1&4 East China Chemical Technology College283,

ABSTRACT

Recently media of high concentration (about 9% ) was very popular for industrial Bacillus thuringiensis (B. t.) production in China. Hindrance occurred with enhancing media concentration, because synchronization was poorer and spore count was lower. Dissolved oxygen (DO ) was detected with DO probe during whole fermentation process in 12 liter glass fermentor. It shows clearly that DO could not meet the demand of growing during log phase, the critical dissolved oxygen was 25.2 % and DO of beer was only 5 %. Several factors were checked and agitating speed was found to affect DO most obviously. With changing agitating speed from 300 r. p. m. to 600 r. p. m. , spore count was raised 30% more.

INTRODUCTION In 1980's, high concentration medium was effectively used for production of Bacillus thuringiensis (B. t.) insecticide in China and the fermentation level was nearly 3 times more. It played a very important role in the improvement of production quality and the reduction of production costs. In high concentration medium, the analysis of fermentation parameters showed that DO ( dissolved oxygen ) declined to a very low level during log phase. DO deficiency inhibited the further improvement of B. t. fermentation level. In these studies, the great consumption of oxygen and the need to verify the influence of DO on the cellular growth was evidenced. The influence of the main operational variables on DO transfer coefficient was determined and assured proper oxygen supply.

- 221 - Me Tianjian et al.

RESULTS AND DISCUSSION 1. CDO (critical dissolved oxygen) and OUR (oxygen uptake rate) of Different Growth Phases of B.t.

CDO can give a very improtant information for oxygen supply in fermentation process. When the DO of beer is below it, cells cann't grow normally, and extra supply of oxygen is unnecessary and also causes the waste of energy. CDO in different B. t. growth stage was determined by motional way (Figs. 1 and 2). OUR was calculated according to critical value of oxygen concentration (Fig. 2). During early and late growth stage, B. t. consumed less oxygen. The critical values of oxygen concentration at 4 and 22 hours of fermentation were 13.2076 and 13.0%, respectively. During log phase, especially at the end of log phase, the CDO reached its peak value of 25.2%, and the oxygen uptake rate was 27.8 mol/ 1 min. It showed that log phase was the key stage of oxygen supply.

DO (%)

100 Air off

80

60

40

20

0 20 40 60 80 100 120 (min.)

Figure 1. The critical dissolved oxygen during different stages of fermentation.

2. The Patterns of DO Curves of Different Concentration Media The consumption of carbon sources is combined with the uptake of oxygen at the same time to generate ATP and intermediate metabolite for construction of cells. High concentration medium usually contained a high portion of carbon sources, which caused the increase of uptake rate of oxygen. The patterns of DO

- 222 - Spore Count of B.t. curves of two concentration media, which were 4.2% and 9.5%, respectively, are shown in Figure (2). During the germination stage, almost no oxygen, was consumed and DO remained at 100%. level. Then, along with the reproduction of B. t. cells, the oxygen uptake rate increased, and DO curves began to decline. During log phase, DO curves declined sharply, the lowest value of medium of 4.2% was 23%, which was near to the CDO. The lowest value of medium of 9.5% was 501o, below which the critical value of oxygen concentration occurred for 3.6 hours. Obviously, the deficiency of oxygen during log phase was very heavy by use of high concentration medium. In the stage of formation of spores and crystals, oxygen uptake rate decreased and DO curves of the two media rised quickly.

Figure 2. The patterns of CDO level and DO curves of media of different concentrations.

3. Improvement of DO Supply to Enhance Fermentation Level To improve the oxygen supply by using high concentration medium, four factors of agitation speed, aeration rate, termperature and pressure were studied by the orthogonal test. Viable count was indirectly used to reflect the DO improvement. The relationship between viable count and each of the four factors was given in Figure (3). It showed that vaible count could be greatly influenced

by agitation speed. So, agitation speed was the major factor affecting DO .

- 223 - Me Tianjian et al.

CFU (106/ml)

20

15

10

I i 1 i I 1 1 1 1 5 1 1 1- 0.6 1.0 1.6 200 300 400 0.2 0.4 0.6 30 32.5 35 ( rrm ) ( rpm ) ( kg/cm2 ) (0c) Aeration rate Agitation Pressure Temperature

Figure 3. The relationship between CFU and each of the four factors.

The patterns of DO curves of different agitation speeds are shown in Figure (4). Along with the raise of agitation speed, the lowest value of DO curves rised. The period below the CDO was shortened. At the agitation speed of 300 rpm, 400 rpm and 500 rpm throughout the fermentation, the lowest value of DO was 5016, 18% and 25%, respectively. When the agitation speed was over 500 rpm, the DO curve was above the critical value of oxygen concentration, which indicated that oxygen supply was sufficient. The raise of agitation speed increased the dissolved oxygen concentration.

The viable cell count of the different agitation speed was determined (Fig. 5). Along with the agitation speed raising, the viable cell count increased, and the use rate of total sugar increased, too. It revealed that the raise of agitation speed could increase DO concentration, which led to the enhancement of viable cell count. But the effect of different regions, agitation speed on fermentation level varied significantly. When agitation speed raised from 300 rpm to 400 rpm, the viable cell count increased by 7.47, while from 400 rpm to 500 rpm and 500 rpm to 600 rpm the counts were 31.4% and 4.2%, respectively. Agitation speed from 400 rpm to 500 rpm was the best adjustment region. Extra supply of oxygen showed little effect on the enhancement of viable cell count.

- 224 - Spore Count of B.t.

Figure 4. The patterns of DO curves of different agitation speed.

65

60

45

300 400 500 600 rpm

Figure 5. The relationship between CFU and sugar consumption rate of different agitation speeds.

- 225 - Me Tianjian et al.

CONCLUSIONS

1. The oxygen uptake rate of B.t. in different growth stages varied significantly. The highest oxygen uptake rate occurred at the end of log phase. Oxygen supply in log phase should be strengthened especially by the use of high concentration medium. Agitation speed could effectively influence the dissolved oxygen concentration.

2. During the log phase, the deficiency of oxygen supply inhibited the reproduction of cells. When the concentration of dissolved oxygen was above the critical value, B. t. got sufficient oxygen supply, which led to an increase of fermentation level. Extra supply of oxygen showed little effect on viable cell count. 3. Reasonable oxygen supply could be achieved by changing agitation speed. During log phase, the agitation speed can maintain at a high level to assure that the DO was over the critical value of oxygen concentration, while the other stage adjusted to a relatively low speed. It could be achieved both in high fermentation level and with less energy consumed. Production and Utilization of Bacillus thuringiensis for Crop Protection in Brazil

I.O. Moraes Food Eng. and Tech. Dept./UNESP CP 136, CEP 15055, S.J. Rio Preto, SP, BRAZIL

ABSTRACT

In Brazil the first use of Bacillus thringiensis (B. t.) started in 1960, but the state of the art in that time was not sufficient to promote its use. Till now the difficulties in the use, arise because the farmer did not understand the big difference between a chemical and a biological insecticide, its mode of action and efficiency. In 1970 in the State University of Campinas ( UNICAMP ) in the new graduated program in Food Engineering, a group of researchers began to study all aspects of B. t. fermentation. In this concern, four Master Science theses were developed, most of them related to endotoxin production, some of them related to the exotoxin, both by submerged or semi-solid fermentation. Two patents BR 7608688 and BR 8500663 were deposited and technology transfer is being tried. There are many applications of B. t. mainly in big plantations ( cotton, soybean, vegetables ) and in forest areas. Last years, an interest was deviated to the development of the exotoxin to be used against mosquitoes ( Aedes aegyptii ). Some activities were concerned with protecting the plants through genetic engineering, introducing the B. t. gene into the plants (cotton, tomato, brassicae, tobacco ). This paper deals with the use of B. t. in Brazil, the possibilities of mass production in large scale, using different substrates, sub products of agro-industries that are available in the country.

In Brazil the first use of Bacillus thuringiensis was in 1960 in the Institute of Agriculture of Campinas (IAC) that is a centennaire institution, but the state of the art was not sufficient to promote its use, and the farmer did not understood the big difference between chemical and biological insecticide action and effectiveness.

- 227 - I. O. Moses

They were confused because chemical insecticides killed the insect-pests rapidly, but biological insecticides did not, Problems related to toxicity caused by the indiscriminate use of chemical insecticides were not discussed till that time, but now, there is a big effort in Brazil to reduce and to control the pollution level, as well as to encourage the use of the insecticides, in integrated control programmes. With the increasing public concern about environmental safety, many groups are proposing studies to get a better environment. Next year there will be the ECO 92 in Brazil, the most important conference in environment, so the actions towards the better management of pollutants are being adopted, and the use and production of biological insecticides are fundamental in this context. Since 1990, selling insecticides need an agronomic prescription, passed by an agricultural or forestal engineer, and this is stated by the law 7802/90, the Brazilian law against agrotoxics. Some multinationals alone or in joint-ventures with Brazilian industries are studying the large scale introduction of biological insecticides. Among the problems encountered are the market size which has never been large to justify the introduction of a new plant dedicated exclusively to B. t.

Products that are sold in Brazil, are imported and marketed by Merck Sharp Dohme ( Dipel, from Abbott Lab. USA ) and Sandoz ( Thuricide ). In the 70's, Rhodia marketed Bactospeine, from Rhone Poulenc. These are insecticides against Lepidoptera, but in recent years the discovery of the israelensis strain which is active against mosquito larvae led some Brazilian institutional laboratories to try to develop this product against Aedes aegypti and Culex quinquefasciatus, mosquitoes of public health importance. Du Pont, Monsanto, Ciba Geigy and Shell are intending to produce elsewere to sell in Brazil, or to produce in Brazil too.

B.t. RESEARCH IN BRAZIL In 1970, in the State University of Campinas, in the graduate program in Food Engineering, a group of workers began to study this type of bacterial insecticide production by submerged fermentation.

From 1970 to 1976, two thesis, M.Sc. and Ph.D. were developed and a patent of process production was deposited in Brazil. These studies were related to the endotoxin production, by batch fermentation, and the culture medium was

- 228 - Production of B.t. in Brazil

when composed of sugar cane molasses ( 10 g/L ) and corn steep liquor ( 25 g/L ), were done using minifermenters of 1 liter capacity or 20 liters. Some experiments in 250 liters fermenters, using the same culture media composition. Aeration and agitation rates were studied to state parameters and variables of the process. These sources of carbon, nitrogen, amino acids and vitamins are of low cost and available in our country.

From 1976 to 1981, the study that was turned to the exotoxin work was developed. Thus, work was patented and the Governor Prize of 1985 was awarded for this. During the same period, two theses were developed, one on the influence of aeration and agitation rates on the growth and product formation and the second on the continuous fermentation process ( 1982 ). The influence of the cost of substrates in the composition of the culture media led the researchers to study different types of raw material, mainly residues or wastewaters of food, beverages and agroindustries. The semi-solid fermentation poved to be interesting and many papers and a Ph.D. thesis were published. Since 1989, the research work is in another State University - UNESP, with the support of FAPESP (Foundation to Support Research in the State of Sao Paulo), CNPq (National Research Council) and OAS (Organization of American States). With the installation of the National Center of Research in Agricultural Defense ( CNPDA/EMBRAPA ) many achievements on B. t. are developed. In addition to that, other groups of researchers in many public or private national organizations are trying to develop both endo or exotoxins of B. t. and B. t. israelensis.

B.t. APPLICATIONS

Some papers on B. t. and B. t. israelensis applications were published in Brazilian Journals and elsewhere. The imported products are used because there is no local production. Researchers can produce B. t. and B. t. israelensis at a laboratory scale, in shakers or small fermentors. Many problems have been met when they decided to produce B. t. at the commercial scale, particularly those related to quality control of the product, standardization, formulation, registration and cost/benefit ratio.

- 229 - 1. O. Moraes

REFERENCES

Benetoli, I. ; Donaires, F.S. ; Yamamoto, P.T. and Gravena, S. (1991). Biological control of soybean catterpillar Anticarsia gemmatalis, with Bacillus thuringiensis and influence against natural enemies. XIII Cong. Bras. Ento. Recife, Brazil. Abstracts. Capalbo, D.M. (1982). A contribution to the study of continuous fermentation of Bacillus thuringiensis. M.Sc. thesis, 81 pp. UNICAMP. Brazil (Advisor MORAES, I.0.). Capalbo, D.M. and Moraes, I.O. (1984). Study of continuous fermentation for obtaining bacterial insecticide to control agricultural pests. IV Japan Brazil Symposium on Science and Technology. V. 2, 248-255. Capalbo, D.M. (1989). Development of a semi-solid fermentation process to get Bacillus thuringiensis. Ph.D. thesis. 221 pp. UNICAMP. Brazil. (Advisor MORAES, 1.0.). Capalbo, D.M. and Moraes, I.O. (1991). Semi-solid fermentation for obtaining spores of Bacillus thuringiensis. In: International Conf. on B.t. Oxford. England. Abstracts. Capalbo, D.M. and Moraes, I.O. (1991). A study on the production of Bacillus

thruingiensis spores in different culture media for insect control. In : Insect Plant Protection Congress 11 Rio de Janeiro. Brazil. Abstracts. Cardoso, V.A. ; Rios, E.M. and Lopes, C.E. (1990). Production of Bacillus thuringiensis var. kurstaki in liquid culture medium. An. I I Simp. Controle Biologico. Brasilia. Brazil. Abstracts. Costa, H. ; Ventura, J.A. and Batista, M.G. (1990). Simplified methodology for production of Bacillus thuringiensis. An. 11 simp. Controle Biologico. Brasilia. Brazil. Abstracts.

Costa, H. ; Ventura, J.A. and Batista, M.G. (1991). Biological Control : An alternative to controlling mosquitoes in urban areas. XVI Cong. Bras. Eng. Sanit. Ambiental. Goiania. Brazil. 11 pp. Gravena, S. ; Bara, J.R. and Sanguido, J.R. (1980). Efficiency of insecticides and Bacillus thuringiensis Berliner to control the sugar cane borer Diatraea saccharalis and their effects under arthropods predators. Proc. Entom. Soc. Brazil. 9 (1), 97 - 103. Gravena, S. ; Sanguino, J.R. and Bara, J.R. (1980). Biological control of the sugar cane borer Diatraea saccharalis using egg predators and Bacillus thuringiensis, Proc. Entom. Soc. Brazil. 9 (1), 87 - 95. Gravena, S. et al. (1983 ). Cotton integrated pest management strategies in Jaboticabal - SP, with Bacillus thuringiensis and beneficid arthropods. Proc. Ent. Soc. Brazil, 12 (1), 17 - 29.

- 230 - Production of B.t. in Brazil

on cotton Gravena, S. et al. (1984). Heliothis spp. integrated management strategies arthropods in the region of Guaira ; with Bacillus thuringiensis and native predators. Ecossistema (9), 5 - 22. Martucci, E.T. and Moraes, I.O. (1982). Substrate sterilization for Bacillus thuringiensis. Process Biochemistry, 17 (6), 35 - 36. Moraes, I.O. (1973). Bacterial insecticide production using submerged fermentation. M.Sc. thesis. UNICAMP. Brazil. 70 pp. bacterial Moraes, I.O. ( 1976 ). Studies of submerged fermentation to get insecticide in minifermenters. Ph.D. thesis. UNICAMP. Brazil. 77 pp. para Moraes, I.O. ( 1976 ). BR PI 7 608 688 processo de fermentacao submersa producao de um and insecticida bacteriano. Patente. Moraes, 1.0. and Chaib, M.A. ( 1978 ). Bioassay technique modified for Plodia interpunctella. Process Biochemistry. 13 ( 10 ), 23 - 24. The influence of oxygen Moraes, I.O. ; Santana, M.H. and Hokka, C.O. (1981). concentration on microbial insecticide production. Advances in Biotechnology. (1), 75 - 79. Moraes, I.O. (1981). Production, separation and bioassay of the thermostable Brazil. exotoxin of Bacillus thuringiensis. Post Doctorate thcsis. UNICAMP. 100 pp. de toxina Moraes, I.O. ( 1985 ). BR PI 8 500 663 Processo de producao termoestavel de Bacillus thuringiensis. Patente. Moraes, I.O. and Capalbo, D.M. ( 1986 ). The use of agricultural by-products as culture media for bioinsecticide production. In: Food Engineering and Processes Applications. V 2, Chap. IV, 377 - 381. Elsevier. Bacterial insecticide Moraes, I.O. ; Capalbo, D.M. and Moraes, R.O. ( 1989 ). production: potential use of waste sludges from pulp and paper industries. and utilization of Biomass. In : Biomass for energy and Industry : Conversion London. Elsevier., (2), 1036 - 1040. and liquid waste Moraes, I.O. ; Capalbo, D.M. and Moraes, R.O. (1990). Solid utilization in fermentation processes to get bacterial insecticide. In: 3, 785 - 792. Engineering and Food : Advanced Processes. London. Elsevier. insecticide Moraes, 1.0. ; Capalbo, D.M. and Moraes, R.O. ( 1990 ). Bacterial 4 (1), 282. production by Bacillus thuringiensis . In : Food Biotechnology. of Bacillus Moscardi, F. ; Gomes, D.R. and Morales, L. (1991). Field evaluation thuringiensis isolates for control of the velvet bean caterpillar ( VBC ) in soybean and their relative virulence to VBC and to the soybean looper under laboratory conditions. XII Intern. Plant Protection Congress. Rio de Janeiro. Brazil. Abstracts.

- 231 - I. O. Moraes

Villani, H.C. ; Campos, A.R. and Gravena, S. ( 1980 ). Efficiency of Bacillus thuringiensis and phenitrothion plus phenvalerate to control Dione juno juno in passion flower. Proc. Entom. Soc. Brazil., 9 (2), 255 - 260. Optimization of Process Parameters for an Economic Production of Biocide-T, Active against Lepidopteran Agricultural Pests, by the Use of Continuous Culture Studies

R. Sachidanandhaml, N. Rajendran2, E. Sivamani, Kunthala Jayaraman4, K. Jenny5, R. Laforce6 and A. Fiechter7 Center for Biotechnology, Anna University, Madras, India1-4 and Institut fur Biotechnologie, ETH, Zurich, Switzerland.5"7

ABSTRACT

In order to be competitive in the production of biopesticides, the cost involved with various aspects of the process technologies have to be looked into in great detail. The principal factor that contributes to the cost ofproduction is the efficient conversion of carbon and nitrogen sources to biomass as well as the insecticidal factors produced by the cells of Bacillus thuringiensis (B.t.) strains. Addition of appropriate precursors at the time of onset of crystal formation combined with the selected growth kinetics have been shown to contribute to the efficiency ofproduction of Biocide T. (A commercial name adopted by us for B. t. formulations). By employing chemostat cultures, several growth supporting compounds for B. t. have been tested and an xD-diagram developed for glucose and yeast extract medium. The correlation of biomass to evolved C02 and 02 has clearly shown that glucose serves both as carbon and energy source. Addition of amino acids to glucose yeast extract medium has shown that some amino acids may act as growth factors and may act by substitution as carbon .sources. A major observation made was on the relative instability of the B. t. strains in chemostat cultures as regards their ability to form spores and crystals. However, this was not noticed in batch cultures. Some details of the economics of fermentation will be presented.

INTRODUCTION

In the past 15 years, a subtle shift in the use of pesticides has occurred in

- 233 - R. Sachidanandham et al.

agriculture. Environmental hazards, resistant pests and mushrooming cost of standard chemical crop control agents, have made biological insecticides more attractive to growers. When biological insecticides were first used in the mid-1960s, chemical pesticides were cheap. But, today the cost of chemical pesticide is three to four fold higher. Besides, the molecular mechanism of action of the biocontrol agents has been more clearly understood and these are applied for development of better biocontrol agents.

The bio-alternatives include specific narrow spectrum chemicals like pheromones and entomopathogens i.e. viruses, bacteria and fungi. These bio-rational insecticides are unique because they have a narrow spectrum of action and literally no toxicity to non-target vertebrates and invertebrates.

Spore forming bacilli belonging to Bacillus thuringiensis (B.t.) species are a major group which produce protoxins with molecular weight ranging between 110 to 135 KDa, during sporulation. These proteins are deposited either as parasporal inclusions in these bacilli or in some cases found on the surface of spores. Different strains of B.t. are known to have different host range, a fact that was not fully realized during the early days of B. t. production (Aronson, et al., 1986). This has resulted in a somewhat lukeworm reception to the biopesticide in the agricultural market since one single strain of B. t. manufactured was not effective against several lepidopteron pests. Due to the applications of molecular biology, the crystal toxins of B.t. became better characterized and currently effective combinations of B.t. strains for diverse pests are evolving. In addition, in integrated pest management strategies, safer chemical antagonists are admixed with B. t. to reduce the concentration of chemicals in the environment. There are more than 600 registered formulations of B. t.

In as much most of the commercial data base on B. t. is unavailable except on payment basis, the need to develop indigenous strains and technology becomes unavoidable. During the last decade, extensive research was being undertaken by us in the following aspects, towards development of a safe and economical, biological pesticide for application in field. The studies include ; (a) Isolation and characterization of potential B. t. strains suitable for insect pests of agricultural crops in India. (b) Genetic engineering approaches for strain development (with multiple host range). - 234 - Production Optimization of B.T. in India

(c) Media optimization for economic scale up of the production, downstream processing for formulation and application in field conditions.

Selection of the strain was made by screening a number of B. t. isolates against the most difficult pests, Heliothis armigera and Spodoptera litura. A potent isolate was the derivative of B.t. var. gallaeriae and is termed as CBT-1. The results of our studies on the optimization of the growth requirements and economic production of CBT-1 based biopesticide are outlined in this presentation.

MEDIA OPTIMIZATION

Selection of Carbon Source With shake flask cultures, a semisynthetic medium, made up of glucose, yeast extract and tryptose (Table 1) was found to be optimal for endotoxin production of CBT-1 as assessed by toxicity and Dot blot immuno assays. The onset of synthesis of crystal antigen in this strain accompanying growth and sporulation is shown in Fig. (1). The appearance of 130 Kda peptide representing the protoxin can be seen in the cultures beginning from the stationary phase of growth. In this medium, optimal biomass was achieved when glucose was used as the carbon source at 1% level. Glycerol was less effective and also the biocide content was lower in these cells (Fig. 2).

A second semisynthetic medium was designed with glucose as the major carbon source (Table 2).

In this medium also several carbon sources were tested for their ability to support growth. It can be seen from the data outlined in Fig. (3) that again glucose was maximally effective in biomass production expressed as economic yield coefficient, which is calculated as the ratio of biomass production and substrate utilization. R. Sachidanandham et al.

Table (1) Katxnelson's tryptose broth (KTB) K- Medium

Components Concentration (g L-1)

Ammonium sulfate 15.0 Magnesium sulfate 0.2 Sodium chloride 0.1 Calcium chloride 0.1 Ferrous sulfate 0.01 Zinc sulfate 0.01 Manganeous sulfate 0.007 Potassium dihydrogen - ortho phosphate 0.403 Disodium hydrogen phosphate 2.96

Dissolve in 800 ml distilled water and boil for 15 minutes. Adjust pH to 7.5 and make up to 1000 ml.

TB-Medium

Components Concentration (g L'1)

Tryptose 10 Sodium chloride 5 Yeast extract 3 Dextrose 2

Dissolve in 1 litre adjust pH to 7.5.

The final KTB composition is derived by mixing 3 volumes of 1 x K medium to 1 volume of Tryptose broth.

- 236 - Production Optimization of B.T. in India

M.wt (KDa)

130

68

6 Lanes 1 2 3 4 5

Lane 1. 4.00 h. Lane 4. 14.00 h. Lane 2. 6.00 h. Lane 5. 24.00 h. Lane 3. 11.00 h. Lane 6. Control toxin.

Figure 1. Western blot analysis of time course appearance of toxin peptides of CBT-1 during growth and sporulation. Samples were collected at different time intervals. R. Sachidanandham et at.

Samples

Note : Dry weight is expressed in grams per liter.

Figure 2. Effective carbon source utilization by CBT-1 in shake flask experiments.

M3

ao Glu INN Fru Gal Suc Lac CSoure"

Figure 3. Economic yield coefficient (Yeco) of CBT-1 grown in ifferent C-sources.

- 238 - Production Optimization of B. T. in India

Table (2)

Components Concentration (g L-1)

Glucose 3.4 Yeast extract 1.0 (NH4)2 S04 0.2 KH2 P04 1.7 MgC12.6H2O 0.04 CaC12.2H2O 0.02 FeSO4 7H2O 0.0001 NaCl 0.02 ZnSO4.7H2O 0.0001 Trace elements 100 ul pH (controlled at) 7.0

Trace elements (Kuhn et al., 1979)

Components Concentration (g L-1)

MgC12'4H2O 15.13 ZnSO4.7H2O 0.25 H3BO3 2.5 CuS04.51-120 0.125 Na2MOO4.2H2O 0.125 CoN03.61-120 0.23 H2SO4 2.5 Dist. H2O 1000 ml

CHEMOSTAT STUDIES

(a) Construction of a x -D Diagram for B.t. In a batch culture, microorganisms are always in a transient state, where defining growth behavior is difficult and inaccurate. Whereas in continous culture, at the steady state there is always the limitation of a nutrient or substrate, which

- 239 - R. Sachidanandham et at.

can be deciphered by the addition of various components. Initially towards optimization of the substrate concentration that allows maximal growth rate in a continous culture, a x -D diagram was constructed for CBT-1. in a 2 L chemostat with a working volume of 1.5 L. At lower dilution rate, there was complete utilization of glucose and the residual glucose increased with increase in dilution rates (Fig. 4). The substrate saturation constant (Ks) was 0.13 g/L.

Figure 4. x D - Diagram of CBT-1 grown in a semisynthetic medium.

(h) Yield coefficient

Correlation of biomass yield with substrate utilization and dilution rates was arrived at by measuring the (unutilized) glucose concentration in the washouts of the chemostat cultures (Fig. 5). The increase in yield of biomass with increase in dilution rates, indicated a maintenance requirement by the organism for the substrate and the maintenance coefficient was calculated (Abbott and Clamen, 1973) to be as 0.1/h.

(c) Exhaust Gas Analysis Data Specific oxygen uptake rate (qo2) and specific carbon dioxide production rate (gco2) were determined from exhaust gas analysis data and it was found that, qo2

- 240 - Production Optimization of B. T. in India and gco2 remained constant at lower dilution rates and increased beyond the dilution rate of 0.3 h-1 (Fig. 6). This indicated that there was spillage of excess energy derived from the substrate required at higher specific growth rates and this has to be corrected to production of secondary metabolite (crystal) synthesis, so, that an effective utilization of the substrate can be ensured.

m X

Figure 5. Steady state biomass concentration (x) and yield coefficient (Y x is) as a function of dilution rate in a continuous culture of CBT-1.

1.0

0.8

0.4

0

Figure 6. Specific oxygen uptake (q02) and specific carbon dioxide production (gCO2) rates as a function of dilution rate in a continuous culture of CBT-1. - 241 - R. Sachidanandham et al.

(d) Pulsing of Growth Factors

In as much we could not arrive at a completely defined medium with yeast extract addition, the nitrogen and supplementary carbon requirements were studied at optimal glucose concentration. By the pulsing of different amino acids at the steady state. It can be seen that different combinations of amino acids had growth supporting effects (Fig. 7).

rn N

1 0 1 1 10 0 10 20 30 40 Time (hr.) Figure 7. Response of continuous culture of CBT-1 for combined amino acids pulse.

(e) Sporulation Status of CBT-1 Cells in Chemostat Cultures In order to obtain the sporulation frequency of the CBT-1 population at every dilution rate, samples were collected and plated on nutrient agar plates and incubated at 300C, two types of colony morphologies were observed in these plates, the pale colonies which are spo- mutants can be distinguished from the white colonies on the agar surface. Spo- mutants started appearing even at a vcry low dilution rate of 0.06 h-1. At higher dilution rates the population was completely replaced by the mutants (Table 3). Similar observations were made by Yousten, (1978) through induced mutagenic studies. Currently these mutants are being assessed for their endotoxin producing capabilities by ELISA methods.

- 242 - Production Optimization of B. T. in India

Table (3) Frequency of asporogenic mutants with different dilution rates in a chemostat culture.

D,h-t Fm

0.06 very low 0.13 0.1286 + /-0.0507 0.22 0.96 + /-0.0988 0.33 0.9753 + /-0.0308 0.36 1.0 0.44 1.0

D = Dilution rate I h-1 Fm = Frequency of mutation

This result has great implications in the production strategy of B.t. Since sporulating cultures might spread to locations where sericulture is an economic and important agriculture (Bombyx mori, the silkworm is a lepidopteran insect species and is the most vulnerable for most B. t. toxins), the development of SpO-cry+ mutants are valuable especially if they are stable in fermentations. Extraction of the toxin by simple chemical means or formulating the non viable cultures should provide a safe biopesticide.

BATCH EXPERIMENTS The Main Objective The above results were compared by batch experiments, which were conducted with glucose yeast extract medium and it was found that there was always residual glucose left out in the medium. When the batch was supplemented with amino acids at a concentration of 100 mg each, there was complete degradation of glucose followed by increase in yield coefficient (Fig. 8). A significant increase in biomass could be achieved by this addition (8 g dry weight /L.) (Fig. 9). This represented a four fold increase when compared to our earlier batch experiment with glucose and yeast extract.

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(a) semidefined medium without aminoacid supplement.

4 1.0

0.8

3 0.6 rn N

2

0.2

1 1 I I I I I 1 0 0 10 20 30 Time (hr.)

(b) semidefined medium supplemented with aminoacids.

14 4

1.2

3 1.0

0.8 rn *--x X 0.6

0.4 1 X

0.2 S

I 0 10 20 30 Time (hr.)

Figure 8. Growth of CBT-1 in batch culture.

- 244 - Production Optimization of B.T. in India

1.0

J 0.8

40.2

I i i

3 8 13 18 23

S0, g 1-1

Figure 9. Correlation between substrate concentration, biomass production and economic yield coefficient.

Scale-up Process Scaling up of this product upto 3000 liter was performed in a CSTR at ETH Zurich, Switzerland under a joint collaboration program in biotechnolgy. Figure (10) shows the schematic representation of the process scale-up. The process details and other parameters maintained during the production are presented in Table (4) and Fig. (11). The biomass obtained in all the batches were screened on Spodoptera litura and were found to have same level of toxicity (Fig. 12).

Field Application Plant phenolics such as gallic acid, resorcinol were found to have enhanced the activity of B. t. endotoxin when applied along with CBT-1. (Ananthakrishnan

et al., 1990 ; Sivamani et al., 1991) and also it reduces the time taken for mortality ,(Fig. 13).

- 245 - 4

I 1. Test tube stock. 2. Petridish culture.

I 3. Static culture 10 ml. I 4. Preinoculum 100 ml. 5. Seed fermenter (Color). 3 Ra 6. Batch fermenter KL. 7. Centrifuge. 8. Supernatant storage. P 9. Effluent sterilizer. 10. Settler. 11. Package (carboys).

Figure 10. Proposed process scheme. Production Optimization of B.T. in India

Table (4) in 3 Process parameters maintained during the pilot plant production of CBT-1 KL Bioreactor.

1 % 1. Media Composition Batch I Soy meal NaCl 0.5 % Batch II Soy meal I % NaCl 0.5 % Glucose 0.5 %

Batch III Soy meal 1 % NaCl 0.5 % Glucose 1 % 2. pH 7.0 3. Air 0.5 vvm 4. Agitation 200 rpm 5. Temp. 30 C. 6. Inoculum strength 2 % 7. Antifoam 0.1 % Polypropylene glycol P 2000

BATCH I BATCH II BATCH III Different batches

Batch 1 1 % soy meal alone Batch 2 : 1076 soy meal + 0.5% glucose

Batch 3 : 1016 soy meal + 1% glucose

Figure 11. Yield of CBT-1 biomass with soy meal and glucose. - 247 - R. Sachidanandham et al.

24 H 48 H 72 H Tlms,h

1 2 8 4 =6 C

1 : 100 jig of total protein 2 : 50 pg " 3 : 20 jig " 4 : 10 pg " 5 : 1 pg "

No mortality was observed in control set of experiments.

Figure 12. Toxicity for Heliothis artnigera of CBT-1 Biomass obtained in 3 KL bioreactor. Production Optimization of B. T. in India

L C

6 0

M CDT-14GAL. \\** CBT-I+RESOR. l CBT-1 ALONE

Appropriate concentration of resorcinol and or gallic acid were added to controls and no mortality was observed.

Lc 50 values are expressed as pg /larva/2 gram of diet.

Figure 13. Effect of plant phenolics on the potency of CBT-1 against Heliothis armigera.

Combination of 2 strains of B. t. is useful in tackling more problematic insect larvae such as Spodoptera where the two different protoxins of these 2 strains of B. t. have an impact on mortality (Fig. 14).

A small scale field trial was carried out with the formulation of CBT-1 on the Heliothis amdgera in cotton. An yield increase of about 60% in terms of fruit weight was observed in the plants sprayed with B. t. (Fig. 15).

- 249 - R. Sachidanandham et al.

48 72 Time, h

=1 2 3 4 E_18 ED 6 7

1. CBT-1 and CBT-2 1 : 1

2. CBT-1 and CBT-2 1 : 2

3. CBT-1 and CB'T-2 2: 1 4. CBT-1 and CBT-2 1 : 3 5. CBT-1 and CBT-2 3 :1 6. CBT-1 alone i volume = 650 pg/ 10 larvae 7. CBT-2 alone 1 volume = 650 pg/ 10 larvae

Figure 14. Effect of CBT-1 and CBT-2 combinations against Spodoptera litura in Synthetic diet. Production Optimization of B.T. in India

65 130 325 650 Protein concentration

Note : - The total average of 10 fruits for each sample is expressed in grams. - The protein concentrations of CBT-1 used are expressed in ug per plant per 10 larvae.

Figure 15. Effect of CBT-1 formulation on cotton against Heliothis armigera in field conditions.

REFERENCES Bernard J. Abbott and Clamen, A. (1973). The relationship of substrate, growth rate, and maintenance coefficient to single cell protein production. Biotech. Bioeng., 5, 117 - 129. Ananthakrishan, T. N. ; Senrayan, R. ; Annadurai, R. S. and Murugesan, S. (1990). Antibiotic effects of resorcinol, gallic acid and phloroglucinol on Heliothis armigera Hubner (Insecta : Noctuidae). Proc. Indian Acad. Sci. (Anim. Sci.) 99 (1). 39 - 52. Aronson, I. ; Beckman, W. and Dunn, P. (1986). Bacillus thuringiensis and related insect pathogens. Microbiol. Rev. 50, 1 - 24.

- 251 - R. Sachidanandham et al.

Kuhn, H. ; Friederich, U. and Fiechter, A. (1979). Defined minimal medium for Bacillus sp. developed by pulse and shift technique. European J. Appl. Microbiol. Biotechnol., 6, 341 - 349. Sivamani, E. ; Rajendran, N. ;, Senrayan, R. ; Ananthakrishnan, T.N. and Jayaraman, Kunthala. (1991). Influence of some plant phenolics on the activity of endotoxin of Bacillus thuringiensis var. galleriae on Heliothis armigera Hubner (Lepidoptera : Noctuidae). Entomol. exper. appl. (in press). Yousten A. A. (1978). A method for isolation of asporogenic mutants of Bacillus thuringiensis. Canadian J. Microbiol., 24. Hofte, Herman and Whiteley, H. R. (1989) : Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev. 53, 242 - 255. Bioassay of Bacillus thuringiensis

A. Sharaby Plant Protection Dept., National Research Centre, Dokki, Cairo, Egypt.

ABSTRACT

The Bacillus thuringiensis isolates were bioassayed against some insect pests larvae using the natural host by dipping technique that simulates natural conditions. A preliminary screening procedure was used as follows. The host plant leaves were dipped in water suspension containing 500 jig B. t. /ml water, then placed in transparent plastic jar (114 liter) its cover provided with small pin holes for ventilation. The jars were placed in conditioned room and larval mortalities were calculated and corrected after seven days. Isolates giving 80% larval mortality or more were only selected. We used the commercial preparation Dipel 2 x as a standared base line for comparison. If a formulation is active against an insect species, it will be valuable to determine its LC5Q .

Bacillus thuringiensis (B.t.) isolates were bioassayed against the major cotton pests in Egypt belonging to order Lepidoptera like the cotton leafworm Spodoptera littoralis, lesser cotton leafworm Spodoptera exigua and the black cutworm Agrotis ypsilon using the dipping leaf technique of their preferable natural host plant (castor oil plant or cotton plant leaves) that simulates the natural conditions more closely than artificial diet incorporation methods. For testing the effectiveness of an isolate, a preliminary screening was done. Fresh plant leaves were dipped in water suspension containing 500 pg B. t. /ml of water to which two drops of tween 80 had been added as a wetting agent. The treated plant leaves were left to dry for 10 minutes, then placed in transparent plastic jars (1/4 liter capacity), its cover provided with small pin holes for ventilation. A piece of filter paper was introduced in the jars to absorb the moisture. The jars were placed in a conditioned room at 26°C exposed to the natural day light and kept for seven days. Ten replicates with ten second instar larvae in each were used. For the control test, the plant leaves were treated with water combined with tween 80 only.

- 253 - A. Sharaby

Percentage of larval mortalities were calculated after 7 days from treatment and corrected according to Abbott's formula. T - C Corrected percent mortality = 100 100 - C T = No of dead larvae in treated replicates. C = No of dead larvae in control replicates.

Isolates giving 80076 larval mortality or more were only considered and selected. For each sample, assay was replicated three times in three separate days to obtain the mean acurate mortality. If a formulation is active against an insect species, it will be valuable to determine the LC50 of the formulation. Subsequent assays were, therefore based on the results of the preliminary screening. In order to standardize the B. t. preparations throughout the experimental work, their bioassay should be based on a comparison with reference to standard preparation (Dipel 2 X was used as a standard base line for comparison). For this purpose, seven serial dilutions of the test sample were used, each dilution was agitated properly just before making the next dilution from it and then a comparison has to be made on the percentage of kill at each dilution tested through probit analysis within the 95% confidence limits.

The potency of a test sample could be expressed as a simple ratio to the standarded which has a potency of 32,000 IU/mg. The potency was calculated by the following formula (Dulmage et al., 1971). Potency of the sample (IU/mg) _

LC50 standard (Dipel 2 X) Potency of standard IU/mg 32,000 LC50 sample

The marked resistance of Spodoptera spp. to endotoxins of Bacillus thuringiensis including the well recognized standards HD-1-1971 and HD-1-S-1980 has necessitated the search for a new active strain that can be used as a standard reference in the bioassays of these lepidopterous insects. The high activity of B. t. var. entomocidus HD-635 vs. Spodoptera linoralis and S. exigua was found to fulfill

- 254 - Bioassay of B.t. these requirements. The spore-endotoxin complex of this strain, designated as HD-635-5-1987, is proposed as a reference standard for bioassays against Spodoptera spp. and is assigned arbitrarily the value of 10,000 IU/mg. Potencies of an active strain, i.e. B.t. var. aizawai HD-133 and of a low-active preparation, i.e. HD-1-S-1980 were carried out in reference to the newly proposed standard. Comparative studies revealed the possible reduction of bioassay period to 4 days as time saving and for a priori judgement on the activity of certain bioinsecticide preparations derived from B.t. (Salama et al., 1989).

REFERENCES Dulmage, H. T.; Boening, O.; Rehenborg, C.; Hansen, G. (1971). A proposed standardized bioassay for formulations of Bacillus thuringiensis based on the international unit. J. Invertebr. Pathol. 15, 15 - 20. Salama, H. S. ; Foda, M. S. and Sharaby, A. (1988). A proposed new biological standard for bioassay of bacterial insecticides, vs. Spodoptera spp. Tropical Pest Management, 35, 326.

Bacillus thuringiensis and Environmental Safety

M. Matter National Research Centre, Dokki, Cairo, Egypt

ABSTRACT Insecticidal hazards and their grave pollutional consequences (development of resistance and toxicity to human and beneficial insects) now, remain problematical. On account of this and of the pressing need to advocate safe alternate in pest management, researches on the safety utilization of Bacillus thuringiensis (B. t.) based on experiments till now in different countries, were reviewed. The soluble crystal of B. t. var kurstaki has no in vivo or in vitro toxicity, cytopathological changes or hemolytic activity on mammalian cells. Acute administration of exotoxin produced some pathological effects. However, risks of cytogenetic damage to human by exotoxin in amounts normally used to control pests was considered negligible. No deleterious effects were observed on beneficial insects. Sometimes, synergistic effects were achieved in the host-parasite-pathogen interaction. Therefore, B. t. should receive its fair share of emphasis and strongly recommended in control programs dealing with susceptible pests.

INTRODUCTION

The over pollution of our environment, toxic residues, resistance problems and carcinogenicity of current chemical insecticides urged many researchers and industry to search for new safe alternates.

Entomopathogens and their relative specificity have attracted the attention of many investigators since several decades. Bacillus thuringiensis was considered a potential pathogen to numerous lepidopterous pests. There has always been the fear that entomopathogens might be selected in such a way and convert to vertebrate pathogen. However, Brown et al. (1958) claimed that B.t. strains can not be converted to become pathogens for vertebrates. Before manufacturing and introducing such bio-agent in different control programs, intensive bio-safety

- 257 - M. Matter studies have been carried out and given the priority in many countries. This text is reviewing different bio-safety studies based on experiments in different countries.

SAFETY STUDIES

Studies were designed to assess the degree of safety of B. t. to beneficial invertebrates, vertebrates (birds, animals and human) and plants. Also the guidelines for assessing the safety of bacterial agents adopted by the WHO were followed.

ASSESSMENT OF TOXICITY TO HUMAN

B.t. strains produce several toxins. The toxigenic activity of each was evaluated using the classic mammalian test animals as rat, mouse, rabbit or guinea pig.

The test animals were challenged with doses that ranged from the average field dose to as much as 100 times that dosage level. Administration route was either per os, intraperitoneal injection, subcutaneous, inhalation, topical exposure or dermal application.

In case of acute toxicity evaluations, the dose was administrated once followed by observation period of varying lengths that depend on the type of the test. Long-term administration (2 weeks up to 6 months) of sublethal doses was used to assess chronic toxicity.

Recently, physiological and histopathological studies were carried out in vivo or in vitro using slices from different organs to examine the effect of such toxins on different physiological processes and cellular functions of the animal.

EFFECT ON VERTEBRATES

Fisher and Rosner (1959), Brown et al. (1958), and others did not give any evidence of any infectious nature of B. t. preparations for mammals and birds whether administration was performed per os or through intra-peritoneal injection. Its spores remained viable in the faeces of the test animal. High doses induce bacterimia which disappeared 2 - 4 days after treatment. Godavaribi et al. (1962) noticed that no pathological changes or localized infections were observed in the liver, kidney and spleen tissues of rats previously treated per os for 3 months with

- 258 - Environmental Safety of B.t.

high doses of B. t. spores. Lammana and Jones (1963) considered that the filtrate growing culture of B. t. was 100 times more toxic than the sporulated culture.

Similar observations were recorded with birds (phaesant, patridge and chicken) treated for a long period (28 - 70 days) with daily dosage rate of B. t. which ranged between 0.2 - 570 x 107 spores/bird (Fisher and Rosner, 1959). Ignoffo (1973) stated that no adverse effects were observed on using exotoxin-free preparations, while weight loss and a reduction in food consumed were obtained when birds were treated with the B-exotoxins of B. t.

Experiments were also conducted using human volunteers. Fisher and Rosner (1959) reported that each volunteer inhaled 100 mg of the powder daily for 5 gays. After 4 - 5 weeks the individuals were thoroughly reexamined and they were found healthy and all the extensive laboratory tests were negative. Acute and oral toxicity of thuricide (B.t.) suspensions (33%) were tested. Doses up to 24 g of thur cide were placed directly into the stomachs of rats. There were no deaths or symptoms of toxicity up to one week after treatment.

In the last decade, gross pathological studies were achieved. The results almost confirmed previous conclusions. In Japan, Nishiitsutsuji et al. (1980) found that the crystal endotoxin of B.t. has neither cytotoxic effects nor showing, even at the ultrastructural level, any morphological changes or growth inhibition on mammalian cells. This is contrary to the classical effects on insect cells (swelling and bursting of the columner epithelial cells lining the mid gut). Thomas and Eller (1983) noticed that the alkali soluble crystals of B.t. var kurstakii has no in vivo or in vitro toxicity cytopathological changes or hemolytic activity to rat, mouse, horse, sheep and human erythrocytes.

On the other hand, exotoxins present in the supernatant of the growing cultures of some B. t. strains caused some pathological manifestations in mammalian cells when administrated at high dosage levels and for a long time. Meretoja et al. (1977) found that toxic concentrations of exotoxins induced significant increase in the incidence of chromosomal aberrations while low concetrations did not affect blood metaphases of the treated animal. Experiments carried out by the same authors in vivo indicated that clastogenic effect appeared, only, when the drinking water of the animal was substituted by 50 or 100% of the agent during 3 months or

- 259 - M. Matter

when rats were given lethal doses of serotype I supernatant. However, the treatment with serotype 3 supernatant revealed slight clastogenic effect. In 1979, Kahkonen et al., found that some pathological effects (C-mitotic influences) were observed in cells of the test animals when treated with higher doses of B-exotoxin of B.t. while exposure to field applied doses did not evaluate clastogenic or mutagenic effects. It was concluded that risks of cytogenetic damage to human caused by exotoxin in amounts used to control insect pests are considered negligible. Also, in some in vitro tests, it was found that phospholipase C, produced in the growing culture of some B.t. strains, affected the binding of alkaline phosphodiesterase I to the plasma membrane of intestine and pancreas (Nakabayashi and Ikezaiva, 1984). However, Taguchi et al. (1985) proved that the alkaline phosphatase in the presence of the toxin was released at significantly higher amounts than in the control but without cell lysis.

Although most of the previously mentioned bio-safety studies revealed that exotoxins were safe enough if used at the recommended field doses, almost all factories now restricted their production on exotoxin-free commercial preparations of B. t. The supernatant containing undesirable toxins was decanted and completely eliminated. Some B. t. varieties are not exotoxin producers like var. kurstaki and these are more attractive to the industry.

However, recent advances in B. t. control and the great progress in gene transfer technology has the advantage of introducing, only the completely safe toxic ingredient of B. t. delta-endotoxin to the environment.

EFFECT ON BENEFICIAL INSECTS Effect on Bees Krieg and Herfs (1963) reported that the spore crystal preparations were harmless to bees at the recommended doses while the supernatant of B. t. var thuringiensis was highly toxic to the worker bees. Krieg (1964) stated that recommended concentrations of B. t. in agriculture practices are completely inoffensive to worker bees. With respect to exotoxin, Krieg (1967) stated that the normal dosage rate in field applications is toxic for bees.

- 260 - Environmental Safety of B.t.

Effect on Natural Enemies Evidence of safety to numerous predaceous mites and entomphagous insects have been shown by many authors, (Salama et al., 1982, 1985., El-Banhawy and Matter, 1986). In some cases, B. t. slightly affected some biological aspects (prolongation of duration, reduction of feeding capacity, and slight decrease in fecundity (Salama et al., 1982, El-Banhawy and Matter, 1986). Dirimanev et al. (1980) consider that when B. t. was used with Trichogramma spp., the damage caused by the codling moth was reduced to 4% at harvest. Chemical insecticides reduce the damage to 1% but the reduction in insecticidal treatments resulted in greater densities of predacious syrphids, chrysopids, coccinellids, anthrocoids and predaceous finites.

Marchal (1975 a,b) consider that the parasite may have an adverse effect on the pathogen and vice versa (reduction of the amount of food taken by the host may reduce the infective dose while mortality of larvae due to the pathogen may kill the parasite). Synergistic effects were produced when B. t. is applied at low doses that allow enough time for the parasite to emerge before host death. Synergestic effects between parasite and B. t. was also evaluated in field experiments by Sneh (1983). Salama andLaki (1985) found that no deleterious effects were observed in the development of the immature stages of the parasite, T. evanescens or the percent of its emergence when the pathogen was applied to the host eggs before or after parasitism. Last but not the least, let us look forward to share in making better environment free from insecticidal hazards and pollutional consequences. It is time for B.t. and related safe biological control agents to receive their fair share of emphasis and to be registered and recommended for use in different control programs against target insects.

REFERENCES

Brown, E. R. ; Mady, M. D. ; Treece, E. L. and Smith, C. W. (1958). Differential diagnosis of Bacillus cereus, Bacillus anthracis and Bacillus cereus var mycoides. J. Bacteriol, 75, 499 - 509. Dirimanov, M. ; Angelova, R. and Sabrikova, T. (1980). State of the harmful entomo and acarofauna and predacious species of insects in apple orchards with some plant protection technologies. Nauchni Trudova, Ent. Microbiol. Fitopathol. 25 : 15 - 30.

- 261 - M. Matter

EI-Banhawy, E. M. and Matter, M. M. (1986). Reaction of the predaceous mite Amblyseus gossipi to three varieties of Bacillus thuringiensis under laboratory conditions. Bull, Fac.,,of Agric. Univ. of Cairo 37, 1067 - 1077. Fisher, R. A. and Rosner, L. (1959). Toxicology of the microbial insecticide Thuricide. J. Agric. Ed., Chem., 7, 636 - 688. Grodavaribi (1962). Bacterial spores with malathion for controlling Ephestia cautella. Pest Technol. London 4, 155 - 158. Ignoffo, C. M. (1973). Effects of entomopathogens on vertebrates. Ann. N.Y. Academy of Science, 217, 141 - 164. Kahkonen, M. ; Gnipenberg, V. ; Curllerg, G. ; Meretoja, 1. and Sorga, M. (1979). Mutagenicity of Bacillus thuringiensis exotoxin 3 sister chromatoid

exchange in rats in vivo. Hereditas 91, 1 - 4. Krieg, A. (1964). Ober die Binenvertraglichkeit verschiedener Industrie - preparate des Bacillus thuringiensis. Anz. Schadl-Kde, 37, 39 - 40. Krieg, A. (1967). Neues Ober Bacillus thuringiensis and seine Anwendung. Mitte. Biol. Bundesanstalt fur land and forstwirtschaft Berline-Dahlern Heft 125, 106 pp. Lammana, C. and Jones, L. (1963). Lethality for mice of vegetative and spore forms of Bacillus cereus and Bacillus cereus - like insect pathogens injected intraperitoneally and subcutaneously. J. Bacteriol 85, 532 - 535. Marchal, S. D. (1975a). Role des larves entomophages dans 1'infection a Bacillus thuringiensis des chenilles de "Pieris brassicae" L. et "Anagasta kuekniella" Zell. Rev. Zool. Agr. Pathol. Vegetale 74, 68 - 84. Marchal, S. D. (1975b). Development larvaire des Hymenopteres parasites, Apanteles glomeratus L. et Phanerotoma flavitestacea F. chez des chenilles infectees par Bacillus thuringiensis Berliner. Ann. Parasitologie. Humaine et comparee 50, 223 - 232. Meretoja, T. ; Carlberg, C. ; Cnipenberg, U.'; Linnainmera, K. and Sora, M. (1977). Mutagenicity of Bacillus thuringiensis exotoxin. Part 1. Mammalian tests. Hereditas 85, 105 - 112. Nakabayashi, T. and Ikezaiva, H. (1984). Release of alkaline phosphodiesterase from rat kidney by the specific phospholipase C of Bacillus thuringiensis Cell Struct. Funct. 9, 247 - 264. Nishiitsutsuji - Uwo, J. ; Endo, Y. and Himeno, M. (1980). Effect of Bacillus thuringiensis delta-endotoxin on insect and mammalian cells. Appl. Entomol Zool. 15, 133 - 139. Salama, H. S. ; Zaki, F. N. and Sharaby, A. (1982). Effect of Bacillus thuringiensis Berl. on. parasites and predators of the cotton leafworm Spodoptera littoralis (Boisd.). Z. ang. Ent. 94, 498 - 509.

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Salama, H. S. and Zaki, F. N. (1985). Biological effects of Bacillus thuringiensis on the egg parasitoid Trichogramma evanescens Westw. Insect Sci. Applic., 6 (2), 145 - 148. of Spodoptera Sneh, B. ; Gross, S. and Gasith, A. (1983). Biological control Bracon littoralis ( Boisd.) by Bacillus thuringiensis subsp. entomocidus and hebetor Say (Hymenoptera, Brachonidae). Z., ang. Ent. 96, 408 - 412. release from rat Taguchi, R. ; Asani, Y. and Ikezawa, H. (1985). Ectoenzyme liver and kidney by phosphatidylinositol - specific phospholipase C. J. Biochem. (Tokyo) 97, 911 - 922. Thomas, W. E. and Ellar, D. J. (1983). Bacillus thuringiensis var. israelensis crystal delta-endotoxin effects on insect and mammalian cells in vitro and in vivo". J. Cell Sci. 60, 181 - 189.

Part III

Commercialization of Bacterial Insecticides

Commercialization of Bacillus thuringiensis and other Bacterial Insecticides.

R. A. Daoast Ecogen Inc., 2005 Cabot Blvd West., Langhorne, PA

ABSTRACT Bacteria have long been associated with insects, but only a few species are pathogenic or produce toxins that kill their hosts. Even fewer bacteria have ever been commercialized for use as insecticides. The first and most prominent bacterium to be commercialized as an insecticide was the crystalliferous sporeformer Bacillus thuringiensis (B. t.). B. t. -based products were first sold in Europe in the late 1930'x. Soon thereafter, the causative agent of the milky spore disease of Japanese beetle grubs, Bacillus popilliae, was commercialized in the United States. There are many important aspects in the commercialization of bacterial insecticides. Strain identification and selection is the first stage in the development of a bacterial insecticide. After a strain has been selected for commercial development, production, through fermentation and downstream processing, and formulation are essential stages in the development of a commercial product. Other essential aspects in the commercialization process include, registration by the appropriate regulatory authorities in the countries in which the end product will be sold and quality control to ensure consistency and product quality for successful commercialization. Field development is the last stage in the commercialization process, generally conducted concurrently with safety testing required for registration. This paper will discuss these aspects of the development process, with specific examples of the commercialization of novel genetically-modified B. t. -based products.

HISTORICAL PERSPECTIVE Bacteria are commonly found in close association with insects in nature, but only a few, e.g. Pseudomonas aeruginosa, Streptococcus fecalis and Serratia marcescens, are pathogenic to their hosts. Entomopathogenic bacteria, on rare occasions, also cause epizootics, but only when pest infestations are dense and highly stressed. This, in turn, can lead to the complete collapse of the infected

- 267 - R. A. Daoust pest population. Rarely if ever, however, are epizootics caused by toxins or other metabolites produced by bacteria, but rather pathogens invade and multiply within their hosts, generally producing a fatal septicemia. Among bacteria that are pathogenic to pestiferous insects, only a few members of the genus Bacillus have ever been developed as commercial insecticides.

Bacterial insecticides account for less than 2 percent, or ca. 100 million U.S. dollars, of the 6 billion U.S. dollar worldwide insecticide market (Prudential Securities, 1991). Of this amount, Bacillus thuringiensis, or B. t. as it is commonly referred to, accounts for the vast majority of sales, while a small proportion of the total is due to sales of the agent of milky spore disease of the Japanese beetle, Bacillus popilliae.

Bacillus thuringiensis (B.t.) was the first bacterium used in a commercial insecticide (Luthy, et al., 1982). As early as 1938, a French company sold a B.t. preparation, under the trade name Sporeine, that was based on a strain (Serovar. 1, B.t. var. thuringiensis) that produced a thermostable exotoxin (later referred to as the beta-exotoxin), in addition to the parasporal crystal that is responsible for primary insecticidel activity in B. t. based products. In the early 1940s, several more products under-several trade names were introduced into the United States using the same serotype as was used in the French product. At about the same time, another French product, Bactospeine, and a Soviet product, Entobacterin-3, were introduced into France and the Soviet Union, respectively.

The exact mode of action had not been clearly elucidated in these early products. There were also several problems associated with these early products, not the least of which was their high cost, due to the expense of production in fermentors, and their relative lack of consistent performance in the field. In addition, it was later found that the beta-exotoxin contained in these products, was highly toxic to certain beneficial insects and also possessed mammalian toxicity, being a potent inhibitor of DNA-dependent RNA polymerase. Studies in the late 1960s confirmed the hazards of using strains that produced the beta-exotoxin by proving that this toxin could inhibit RNA biosynthesis in the liver in mice (Sebesta, et al., 1981).

Following World War 11, there was a surge of new pesticide chemistry and the introduction of many new synthetic organic insecticides into the market place

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(Luckmann and Metcalf, 1982). Products such as the chlorinated hydrocarbons (e.g. DDT, BHC and later toxaphene, aldrin, dieldrin, heptachlor and endrin), organophosphates (e.g. methyl parathion and EPN), and a few years later, carbamates (e.g. carbaryl) were highly effective, easy to use and inexpensive. This -resulted in many early manufacturers abandoning their B. t. products and selling their rights to others.

It was not until the late-1960's that a new isolate of B.t. was discovered that possessed sufficient potential to renew interest in B.t.-based products. This strain, called the HD-1 strain in the serotype 3a, 3b variety kurstaki, lacked the beta-exotoxin, had a far broader spectrum of activity against lepidopteran larvae than its predecessor, the "old" thuringiensis variety, and possessed higher inate potency against key target insect species (see Dulmage, 1981a). In 1971, a batch of HD-1 was produced in the United States and selected for use as a primary reference sample in that country (later became known as the international standard). This first batch, referred to a HD-1-S-1971, was assigned a potency of 18,000 IU/mg, based on assays against the cabbage looper, Trichoplusia ni, in comparison to the European standard, called E-61, that was based upon the "old" variety thuringiensis and assigned a potency of 1,000 IU/mg in 1966.

By the late-1960's, concerns began to mount over the unrestricted use of chemical insecticides. The ubiquitous presence of chemical pesticide residues in all parts of our ecosystem and in our foodstuffs gained widespread publicity, both in the scientific community and among the general populace. People began to worry about pesticide safety and many public interest groups began to clamor for alternative approaches to pest control. In addition to pesticide safety issues, the increasing incidence of insect resistance to nearly all classes of synthetic chemical insecticides contributed heavily to the renewed interest in alternative pest control strategies. Among the most dramatic examples of pesticide resistance was in the treatment of cotton with synthetic chemicals in countries such as Peru, Mexico, Egypt and parts of the United States (Luckmann and Metcalf, 1982). In some locations, pesticide applications increased dramatically to more than 30 sprays per season due to the resurgence of resistant pest populations. In certain areas of North America, the bollworm and tobacco budworm replaced the boll weevil as the major pest of cotton

- 269 - R. A. Daoust due to overuse of ineffective chemicals. These two caterpillar species virtually des'-xoyed cotton production in parts of Central America due to the development of multiple resistance to most classes of insecticides.

Among the major concerns over the use of chemical insecticides are the following :

(1) High cost of discovery and development of chemical insecticides. The cost has risen dramatically as it becomes more and more difficult to find new classes of chemicals. When new highly efficacious chemicals are discovered, there is no assurances that such products can be registered or that insect resistance will not occur quickly even if they are registered. Current estimates exceed 30 million U.S. dollars for registration of a new chemical in the United States.

(2) Further cost considerations related to the registration of chemical insecticides. The rapidly increasing cost of conducting long-term chronic toxicity, environmental fate and soil dissipation studies that are needed to ensure safety to mammals and other non target organisms, including beneficial insects and important plant species, are prohibitive to the registration of new chemicals, especially if intended for small market opportunities.

(3) Pesticide residues on foodstuffs and re-entry restrictions for chemical insecticides. Tolerances established by federal regulatory agencies can limit the use of chemicals prior to harvest. In addition, re-entry restrictions prevent farm workers from re-entering a treated field for a specified time following spraying.

(4) Environmental contamination caused by chemical insecticides. Ground water contamination, pollution of lakes, streams and other bodies of water, presence of residues in animals, such as birds and mammals, have all been associated with chemical pesticides.

(5) Safety hazards associated with chemical insecticides. Problems of oncogenicity, carcinogenicity and toxicity to animals and humans have plagued many chemical insecticides, often leading to their removal from the market.

(6) Rapidly developing incidence of resistance to chemical insecticides. As stated above, the incidence of target pest resistance is increasing dramatically to chemical insecticides, often leading to secondary pest outbreaks and resurgence of target

- 270 - Commercialization of Bacterial Insecticides pest species. In 1986, Georghiou reported that approximately 500 insect and mite species were resistant to at least one chemical insecticide.

The problems associated with chemical insecticides led to a heightened interest in the use of alternative methods of control by the early 1970's. Few alternative approaches, however, were actually available at that time. B. t. and other biorational approaches gained more prominence, but still accounted for a minor part of the pesticide market. By the early- to mid-1980's, much new information became available about B. t. and other bacteria through advances in genetics and genetic engineering procedures. This gave rise to many newly established biotechnology companies devoted to the use of biotechnological approaches to insect pest control. By far and away, major emphasis was placed on the use of B.t. or its toxin encoding genes, as insecticidal agents. This was expressed in many ways, including the development of spray on products and the incorporation of genes into plants or bacteria.

Much of the resurgence in interest in B. t. comes from the fact that B. t. and other biorational insecticides have several advantages that include :

(1) Low cost of research and development and registration of B.t. and other bioinsecticides, relative to the cost of chemical insecticides.

(2) Lack of environmental pollution by B. t. and other bioinsecticides.

(3) Lack of hazardous effects of B.t. and other bioinsecticides on birds, mammals, non target plants and beneficial insects.

(4) Ability to apply B. t. and other bioinsecticides up until the time of harvest and lack of worker re-entry restrictions.

(5) Lack of the development of resistance to B. t. and other bioinsecticides.

B. t. and B. popilliae have been sprayed throughout many parts of the world for more than 20 years with few documented incidences of resistance. Recently, however, one researcher has claimed that diamondback moth resistance developed to commercial B. t. formulations in Hawaii under field conditions (Tabashnik, 1990). Concerns have also been expressed that transgenic plants containing B. t. toxin encoding genes could lead to widespread insect resistance to B. t. in the future (Gould, 1988).

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GENETICS AND MODE OF ACTION OF B.t. B. t. is a common soil microbe that has rarely been found to cause infection in insects in nature. It can be distinguished from B. cereus by the large proteinaceous crystal inclusions, referred to as the parasporal crystal or delta-endotoxin, that are produced along with an endospore during sporulation. More than 30 varieties are recognized by the scientific community, based upon the serological classification of the flagellar antigen in the vegetative cell. Other factors, such as biochemical differences between strains or their toxin crystals, or differences in insecticidal activity between strains, have not been used to distinguish between different varieties of B. t. , due to the natural diversity that exists between strains within a given variety as well as between different varieties. Dulmage (1981a) pointed out that delta-endotoxins of different B. t. isolates, even if the isolates are serologically identical, often have insecticidal activities that are different, both with regard to their host spectrum and their potency against an individual pest species. This is due to the genetic complexity of the insecticidal toxin encoding genes that are present in B. t. This subject has been the subject of numerous recent reviews, such as that by Currier and Gawron-Burke (1989).

B. t. and other bacterial insecticides must be ingested by a susceptible insect host to be effective. A formulated preparation of B.t. that typically contains a mixture of spores, toxic protein crystals, debris from the fermentation process and inert formulation ingredients must first be sprayed onto the host plant. Upon ingestion by a susceptible insect host, the delta-endotoxin solubilizes into high molecular weight proteins in the insect's alkaline midgut. These solubilized proteins produce a toxic response in the insect's gut by binding directly to midgut epithelial cells, causing an osmotic imbalance across the gut barrier that separates the midgut from the hemocoel. Spores may also invade the compromised gut barrier, ultimately germinating and producing a septicemia within the hemolymph of the insect. The insect ceases to feed almost immediately after ingestion of the B. t. treated plant, ultimately starving to death within a few days.

Although spores aid in establishing infection in certain insect species, the primary basis of insecticidal activity of B. t. is the proteinaceous delta-endotoxin. This crystalline protoxin is produced during the vegetative phase of growth in the fermentor. Several genes that are harbored on extrachromosomal plasmids

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(non-essential circular strands of DNA found in many species of bacteria) encode for one or more protein products that constitute the delta-endotoxin of a given possess insecticidal B. t. strain. In the HD-1 strain, two distinct protein crystals that distinct activity are present. The larger bipyramidal crystal is composed of several proteins (designated P1 or Cry I) ranging in size from 130 - 140 kD, that are encoded by three distinct genes on two different plasmids. The smaller cuboidal size and is crystal contains a protein (designated P2 or Cry II) of ca. 65 kD in I toxin encoded by one gene on the same plasmid that contains one of the Cry encoding genes. Of importance is the fact that these distinct types of proteins (Cry of insects, the I and Cry II) have insecticidal activities against different spectra activity Cry I being active against lepidopteran species only while the Cry IIA has against both lepidopteran and some dipteran species.

During the last few years, many new strains of B. t. have been discovered and much work has been done to genetically modify both strains from existing culture has collections and newly discovered strains. Ecogen Inc. (Langhorne, PA, USA) been a leader in this area with its recent commercialization of three novel B. t. -based products that contain genetically-altered strains of B. t. var. kurstaki. One product, with the brand name Foil ® OF bioinsecticide, has been shown to have bifunctional activity against lepidopteran (e.g., European corn borer) and coleopteran (e.g., Colorado potato beetle) insect pest on potatoes. The bifunctional from activity of this product is a result of the combination of specific plasmids different parental strains into one background, using the conjugal transfer method, that encode for delta-endotoxin crystals toxic to both categories of pests. Other products containing natural isolates of B. t. var. morrisoni (also referred to as tenebrionis and san diego are also available for control of the Colorado potato beetle, but these products do not control lepidopteran pests. Several other commercial products contain a naturally-occurring variety of B. t. (B. t. var. israelensis) that kills mosquitoes and blaekflies.

At Ecogen Inc., we have established an extensive screening program to isolate novel B. t. strains from grain dust, soil and other ecological niches from around the world. Novel strains are identified using plasmid profiles and various genetic probes as well as certain morphological traits and insecticidal activity in bioassays against target insect species. Newly discovered strains and strains in our existing library are then used to develop genetically modified strains through two genetic - 273 - R. A. Woust

techniques, referred to as plasmid curing and conjugation. Plasmid curing involves the loss of a plasmid from a strain that often results in enhanced insecticidal activity. Conjugation, on the other hand, is the process whereby plasmids containing toxin encoding genes can be transferred through a natural mating process from one strain to another.

More recently, scientists at Ecogen have conducted field trials with a genetically engineered B. t. strain that contained a recombinant plasmid harboring delta-endotoxin genes from different parental B. t. strains. Results from these trials indicated that field efficacy against Colorado potato beetle larvae was at least as high as that for Foil ® OF bioinsecticide (discussed above) using half the rate. Since the mid-1980's, many advances have been made in the expression of B. t. toxin-encoding genes in alternate host delivery systems and in plants. Several companies have engineered B. t. toxin-encoding genes into epiphytic and endophytic bacteria, while others have focused more on using recombinant DNA technology to insert B. t. genes into plants. Mycogen Corporation (San Diego, CA) has developed a process whereby a delta-endotoxin encoding gene was engineered into a plant epiphyte, Pseudomonas, fZuorescens (P, fl (Brosten and Simmonds, 1989). The engineered Pf cells were then grown in a fermenter and chemically fixed, thereby encapsulating the crystal toxin within the dead Pf cell. Using this process, Mycogen claims that its products have an extended residual under field conditions.

PRODUCTION OF B.t.-BASED PRODUCTS A few bacteria in the genus Bacillus have been produced commercially, including B. t. , B. popilliae and B. moritai (Dulmage, 19816). Others have been proposed as potential candidates for commercial development, e.g. B. sphaericus, but no attempts have been made to produce them commercially. Among those species that have been produced commercially, by far and away the most important has been B. t.

Many early manufacturers of B. t. produced this bacterium on semisolid substrates, usually moist bran due to its ability to hold nutrient solutions on individual particles and act as "miniature fermenters" (Dulmage, 1981b). In recent years, however, this method has been largely abandoned due to the difficulty of controlling physical parameters during fermentation. Today, the more widely used, - 274 - Commercialization of Bacterial Insecticides and may be exclusive, method of production of B.t. is deep-tank liquid fermentation. Liquid fermentation, also referred to as submerged fermentation, is a highly efficient and available method for the production of B.t. and other microbes, especially those used in the production of drugs by the pharmaceutical industry. It is, however, an expensive process that requires highly sophisticated equipment that allows the producer to carefully control and monitor physical parameters during fermentation, e.g., temperature, pH, aeration. Medium ingredients can also be adjusted to optimize the production of individual B.t. strains that may vary in their nutritional and physiological requirements. Changes in medium components can also lead to increased quantities of delta-endotoxin protein being produced during the fermentation process. This, in turn, will lead to a cheaper product to the end user. This is important for expansion of the B. t. market, since B. t. is already perceived as expensive relative to chemical insecticides used to control the same insect species.

Although physical parameters and growth substrate are among the most important factors during fermentation, other factors, such as the genetic composition of the strain itself, may also influence the crystal toxin and spore yield from fermentation beers. Strains of B. t. can differ considerably when grown under similar conditions. Therefore, the growth substrate and fermentation conditions must be carefully defined for each strain to ensure product consistency. It is generally believed that the fermentation process can only be optimized using commercial scale fermenters, as scale up from research fermenters to commercial fermenters can cause strains to react differently. In addition, low quality fermentation substrates can lead to downstream processing problems, such as problems in formulating the technical powder produced from the fermentation concentrate.

DOWNSTREAM PROCESSING AND FORMULATION OF B.t.-BASED PRODUCTS Following fermentation, the beer is first concentrated, and usually spray dried, prior to being formulated into a final product. Fermentation concentrates must be handled in an aseptic manner or stabilized with chemicals prior to being made

- 275 - R. A. Woust

into a technical powder (i.e., spray dried). Contamination during this stage in the process could lead to deterioration of the active ingredients (spores and crystals). Prior to formulation of the technical powder, both the protein toxin concentration and biological activity should be measured to ensure high quality of the final product.

Ingredients added to the formulation are generally considered trade secrets by commercial manufacturers. They include such ingredients as dispersants, surfactants, humectants and diluents that stabilize spores and crystals, and enhance the physical characteristics of the final product. In other words, formulation components may not enhance the potency of a specific technical powder, but they will facilitate handling of the product. There has been considerable effort by many B.t. manufacturers to add components, such as U.V. stabilizers and feeding stimulants, to B. t. formulations to enhance the potency of their products. It is, however, not clear whether such additives have resulted in improved products, vis-a-vis insect control.

QUALITY CONTROL OF B.t.-BASED PRODUCTS

At various stages during fermentation, downstream processing and formulation of a B. t. product, as well as after the final formulation is made and during storage, the manufacturer must assess the quantity and quality of delta-endotoxin in the product. Until recently, manufacturers worldwide relied upon a biological assay against the cabbage looper to establish international potency units. This method was used to monitor both product quality and consistency from batch to batch. In the United States, the Environmental Protection Agency (EPA) has recently required B. t. manufacturers to measure the delta-endotoxin content in their products (as a % by weight of the final formulation). At least one manufacturer, Ecogen Inc., has labelled their products for the % lepidopteran and/or coleopteran active toxin, depending upon the product, that is available in the formulated product. This requirement however, does not preclude the continued need to establish biological activity against at least one target pest species for which the product will be used to ensure that the product is biologically active. In addition, some countries, such as Canada, require microbiological analyses of products to prevent products contaminated with high levels of non-B. t. bacteria from being sold.

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As the B. t. industry becomes more and more competitive, as we have seen in the last few years with the introduction of many new B. t. -based products into markets that were traditionally considered chemical insecticide markets, quality control will play an increasingly important role. Only those products that are of high quality with consistent performance in the field will survive.

REGISTRATION OF B.t.-BASED PRODUCTS Registration requirements for B. t. vary considerably from country to country. In the United States, a new strain or formulation of B. t. may be approved in less than one year from the date of submission to the EPA. The cost of registration and field development is usually less than U.S. 500,000 for a B. t. product compared to more than U.S. $ 30 million for a chemical insecticide. In other countries, e.g., many European countries, Japan and Canada, registration requirements are much more stringent and the registration of a B. t. -based product could cost considerably more.

In the United States, the EPA does not require notification for small-scale field trials conducted on less than 10 acres (per crop per annum at all locations combined) as long as the strain being tested is indigenous, non pathogenic and naturally occurring. For non indigenous, pathogenic and genetically-altered strains, notification must be made to the EPA. This could result in the EPA requiring an Experimental Use Permit (EUP) for such testing, even if total acreage does not exceed 10. Large-scale trials on 10 or more acres cumulative require an EUP. This usually requires that data be supplied to the EPA on the physical chemistry, characterization of the active ingredient, manufacturing process and safety to mammals and nontarget organisms for the trials to be approved. Generally, such data also fulfils the requirements for full registration by the EPA (for registration requirements of the U.S. EPA see Code of Federal Regulations 40, Part 158. 740).

In order to obtain registration of a B. t. -based product, a battery of Tier I tests must be completed including : 3 acute toxicity/pathogenicity studies in mice or rats, and an acute dermal toxicity study and primary eye irritation/infection study in rabbits. Hypersensitivity incidents must also be reported. In addition, nontarget organism testing must be conducted that includes : avian oral pathogenicity/toxicity studies in 2 species, freshwater fish and aquatic invertebrate

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toxicity/pathogenicity studies, nontarget plant studies, nontarget insect studies in 4 species and a honey bee toxicity/pathogenicity study. Registration is generally granted without further testing unless problems are encountered during the Tier I testing.

FIELD DEVELOPMENT OF B.t.-BASED PRODUCTS Field evaluation is an essential part of the development process for a pesticide. Field trials are initiated well before safety testing begins and continue even after product registration is obtained and sales begin. The first trials to be conducted are usually based upon data obtained in laboratory bioassay and greenhouse "on-plant" trials. Because of the high cost associated with field trials, only the most promising experimental strains or formulations of B. t. from laboratory and greenhouse bioassays will make it to the field. Following one or more years in which competitive product efficacy is shown in highly replicated, small-plot field trials, large-scale and commercial development trials are initiated. In these trials, an attempt is made to apply the product in its final approved, or soon to be approved, formulation using conventional agricultural equipment representative of the areas in which these trials are conducted. Information obtained from both small-plot and large-scale trials is used to confirm product efficacy under field conditions and to establish the product's use directions. These directions appear on the final approved label and include : application rates and timing, approved crops and pest species, mixing instructions, use of tank mix adjuvants, such as spreaker/stickers, and other similar information.

To further illustrate the process by which a B. t. -based product is developed, a short review of one recently registered (in the U.S.) product, Cutlass e WP (wettable powder) bioinsecticide, is provided. Cutlass (9) was developed by Ecogen Inc., a Pennsylvania-based agricultural biotechnology company that specializes in the development and commercialization of B. t. and other biopesticides. Cutlass ® was field tested over a period of three years (1987 - 1989) prior to its registration by the EPA on September 21, 1989. Field trials have continued with the product, both on crops for which it is already labelled and on new crops in an effort to obtain an expanded label. Cutlass 0 is a broad spectrum vegetable bioinsecticide that is labelled for the control of most

- 278 - Commercialization of Bacterial Insecticides lepidopterous pests, e.g., diamondback moth, cabbage looper, beet armyworm, imported cabbageworm, cabbage webworm and many more, occurring on vegetable in the U.S. The recommended use rates for CutlasAP are between 1.0 and 2.5 pounds per acre.

Cutlass ® WP bioinsecticide contains a genetically-altered strain of B.t. that was generated by the nonrecombinant methods of plasmid curing and conjugal transfer, referred to earlier. First, a B. t. variety kurstaki was cured of toxin-encoding plasmids that contribute little to insecticidal activity against the target pest species. Second, plasmids containing toxin-encoding genes from two other strains were transferred via conjugation into the cured kurstaki strain resulting in increased potency against a broader spectrum of insect pest species than that of any of the parental strains.

Small-plot research and large-plot commercial scale trials were conducted with Cutlass OF (an oil-based flowable) and Cutlass OF and WP in 1988 and 1989, respectively. Small-plot trials included more than 50 locations in 9 important vegetable-producing states in.the United States and in Canada and Mexico. Trials were conducted by university research and extension entomologists as well as private contract researchers with standard application equipment, such as In most trials, C02 -pressurized backpack sprayers, and standard spray schedules. commercially registered and representative chemical (e.g., Lannate and-B. t. -baseds (Dipel 0 2X WP and Javelir0) insecticides were also included. A diversity of vegetables, such as broccoli, cabbage, cauliflower, celery, collards, lettuce, mustard greens, tomatoes, peppers and others, were treated for control of lepidopterous pest species. The experimental design for these trials was generally 2 - 4 rows X 25 to 40 feet in length, replicated 4 or more times in a randomized complete block.

Commercial development trials were conducted under an EUP in several different states with trials ranging from less than 10 acres to more than 25 acres for each treatment in a given geographical region. When possible, at least 4 to 5 different spray blocks (not actually considered to be true replicates) that each measured between 2 and 5 acres were included for each treatment. Typically, Cutlass ® was compared to only one chemical insecticide in each region which was selected on the basis of local use patterns, i.e., considered to be a standard in that specific region. Commercial application equipment, such as ground rigs,

- 279 - R. A. Woust helicopters and fixed wing aircraft was used to apply the products.

Commercial development trials provided data that supplemented data obtained from small-plot research trials. In addition, these trials had several other objectives. The first was to confirm the efficacy of Cutlass applied through conventional commercial application equipment. The second was to familiarize farmers and extension agents with Cutlass prior to its introduction into the market. The third was to compare Cutlass to other commercial standards in each of the geographical regions in which Cutlass was tested. Data obtained from these and small-plot research trials was used to prepare final product labels and to support the registration of Cutlass ® WP bioinsecticide in the United States and Mexico.

During 1990 and 1991, many more trials were conducted with Cutlass ® WP bioinsecticide in an effort to develop an expanded label and to ensure consistent product quality, based not only on quality control analyses (see section on Quality Control), but also on field performance. At the time of preparation of this manuscript a revised label is being prepared that will include many new crops and new pest species. The expansion of Cutlass and other B. t. -based products into new markets and against new pest species will provide an important alternative to farmers that did not have these choices before.

In conclusion, field development is an integral, and perhaps the most important step, in the commercialization of a new insecticide, especially a bioinsecticide which may be sensitive to field degradation. Efficacy data obtained in field trials provides reliable expectations of how the product will perform in the hands of the farmer. It also gives the manufacturer the opportunity to establish proper use directions and to understand the subtleties of the product and its marketing opportunities.

REFERENCES Brosten, D. and Simmonds, B. (1989). The evolution of the biotech revolution. Agrichemical Age, 33 (9), 6, 7, 26 - 27A. Currier, T. C. and Gawron-Burke, C. (1989). Commercial development of Bacillus thuringiensis bioinsecticide products. In Biotechnology of Plant-Microbe Interactions, J. P. Nakas and C. Hagedorn (eds.). Mc Graw-Hill Publ., New York, 111 - 143.

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Dulmage, H. (1981a). Insecticidal activity of isolates of Bacillus thuringiensis and their potential for pest control. In Microbial Control of Pests and Plant Diseases 1970 - 1980, H. D. Burges (ed.). Academic Press, London, 193 - 222. Dulmage, H. (1981b). Production of bacteria for biological control of insects. In Beltsville Symposium in Agricultural Research 5, Biological Control in Crop Protection, G. C. Papavizas (ed.). Allanheld, Osmun & Co., Totowa, New Jersey, 129 - 141. Georghion, G. P. (1986). Pesticide Resistance : Strategies and Tactics for Management, National Academy Press, Washington, D.C., 14 - 43. design of Gould, F. (1988). Ecological-genetic approaches for the a genetically-engineered crops. In Biotechnology, Biological Pesticides and Novel Plant-Pest Resistance for Insect Pest Management. D. W. Roberts and R. R. Granados (eds.)., Boyce Thompson Institute for Plant Research, Ithaca, New York, 146 - 151. Luckmann; W. H. and Metcalf, R. L. (1982). The pest management concept. In "Introduction to Insect Pest Management", 2nd Edition, R. L. Metcalf and W. H. Luckmann (eds.). John Wiley & Sons, New York, 1 - 31. Luthy, P. ; Cordier, J. L. and Fischer, H. M. (1982). Bacillus thuringiensis as a bacterial insecticide : Basic considerations and application. In "Microbial and Viral Pesticides", E. Kurstak (ed.). Marcel Dekker, New York, 33 - 74. Prudential Securities Research, August 8, (1991). Ecogen Inc., Company Report. Prudential Securities Incorporated, One Seaport Plaza, New York, N. Y., 10292. Sebesta, K. ; Farkas, J. and Horska, K. (1981). Thuringiensin, the Beta-exotoxin of Bacillus thuringiensis. In Microbial Control of Pests and Plant Diseases 1970 - 1980, H. D. Burges (ed.). Academic Press, London, 249 - 281. Tabashnik, B. E. et al. (1990). Field development of resistance to Bacillus thuringiensis in diamondback moth (Lepidoptera : Plutellidae). J. Econ. Entomol. 83(5), 1671 - 1676. United States Federal Insecticide, Fungicide, and Rodenticide Act as Amended by Public Law 100 - 532, October 25, 1988, U.S. Government Printing Office, Washington, D.C. 20402.

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Development of Bacillus thuringiensis Insecticides in Ciba-Geigy as Exemplified with CGA 237'218

K. Bernhard Ciba-Geigy AG, Plant Protection Division, CH-4002 Basel, Switzerland

ABSTRACT

Based on the safety record of Bacillus thuringiensis (B.t.) insecticides, and law registration requirements, this allow rapid product development at relatively low cost. The development of some B. t. insecticides particularly CGA 237218 within Ciba-Geigy has been described including various aspects such as optimization of mass production, technical requirements for fermentation, formulation, quality control and field screening against lepidoptereus insects e.g., diamondback moth Plutella xylostella, armyworms Spodoptera spp., Heliothis armigera and Lobesia botrana. The registration requirements have been also discussed.

INTRODUCTION Bacillus thuringiensis (B.t.) is a ubiquitous insect pathogen first isolated from diseased lepidopteran larvae in Japan and Germany at the turn of the century. It is not only found in insect cadavers but also in soil and stored products (Travers and Martin, 1989 and Meadows et al., 1991). It is closely related to the common soil bacterium Bacillus cereus. Strains belonging to the B. cereus/thuringiensis group have vegetative cells of more than 0.9 um diameter and 3 - 5 pm length. The sporangia are not only slightly swollen. They may be differentiated by flagella antigens or biochemical characteristics, e.g. utilization or formation of acids from sugars and glycerides. The patterns observed can however not be correlated with either species. (Baumann et al., 1984 and Ohba and Aizawai 1986). The major difference between the two species is production of parasporal inclusion bodies by B. t. during sporulation (Buchanan and Gibbons, 1974). Sporulating cells of B. t. are therefore easy distinguishable from other bacilli by phase contrast

- 283 - K. Bernhard microscopy. The inclusion bodies consist of proteins called delta-endotoxins and are also referred to as parasporal crystals. Pathogenicity of B. t. is mainly due to production of delta-endotoxins, which act as selective stomach poisons. Because delta-endotoxins are synthesized as particles, B. t. insecticides are only useful for control of chewing insect pests which feed at plant surfaces and filter feeding aquatic larvae of mosquitos and blackflies. Mining insects cannot be controlled under field conditions even if they are sensitive to delta-endotoxins in laboratory tests, With most strains, the delta-endotoxin genes are located on plasmids, and may be transferred to other strains of B. t. and B. cereus by cell to cell contact, which is usually referred to as conjugation (Gonzalez et al., 1982). The potential of B.t. for biological pest control was first recognized by Berliner (1915). Since then it received considerable attention in the research community. So far, it is the most successful biological principle used as bioinsecticide and is estimated to account for 80% to 90076 of all biological pest control agents sold worldwide. Nevertheless, B.t. insecticides have not yet made much impact on the total insecticide market, taking only a 1% to 2% share.

Although B.t. is by no means a novel insecticidal principle, several developments in recent years led us to reexamine its potential for commercial insect control : (1) the work of Dulmage and collaborators (1981) showed that B. t. strains active against lepidopteran larvae differ considerably in potency and insecticidal spectra. (2) In some crop/pest situations, B. t. products are as efficacious as chemical standards. (3) Discovery of strains active against diptera (Goldberg and Margalit, 1977) and coleoptera (Krieg et al., 1983) demonstrated that the spectra of potential uses is wider than previously thought. (4) Increasing public concern about residues in food and in the environment increased awareness for integrated pest management, for which B. t. provides highly compatible insecticides. (5) Based on the impeccable safety record of B.t. insecticides over the past 30 years, registration requirements are lower in many countries, which allows rapid product development at relatively low cost. This situation can help to reduce hurdles for these selective insecticides to enter relatively small markets too.

DEVELOPMENT OF B.t. INSECTICIDES

B. t. can be regarded as a biological but also as a chemical insecticide, since its biological activity is mostly due to the toxicity of the protein .cons

- 284 - Development of B.t. Insecticides delta-endotoxins. The major difference to synthetic insecticides, however, is the mode of production: fermentation rather than chemical synthesis. In order to gain experience in areas like production, registration and sales with this class of insecticides, development within Ciba-Geigy followed two tracks : (1) Development of one strain, CGA 237'218, to the sales stage in order to gain expertise. (2) Implementation of strain search and screening programs in order to identify naturally occuring strains of B.t. with activity against specific pest insects superior to the current standard strains.

1. Development of CGA 237'218

Jarrett and Burges (1986) ; Burges and Jarrett (1988) described a strain designated GC-91, obtained by conjugation between two wild type strains, which produce delta-endotoxins with different insecticidal spectra. It produces delta-endotoxins of both parent strains and is useful for control of a range of lepidopteran insect pests, broader than the range of either parent. Although strain GC-91 harbours more than 2 different delta-endotoxins genes, its parasporal crystals consist mainly of two types of delta-endotoxins. One of them belongs to It the cry 1 C-class of delta-endotoxins and is active against Spodoptera littoralis. originates from a derivative of strain HD-135. The other one belongs to the crylA-class and is active against Heliotnis virescens. Its gene is located on a 50 mDal-plasmid which was transferred from the HD-191 A2 parent to the HD-135 parent, to obtain strain GC-91. To conform with established Ciba-Geigy nomenclature, the development product based upon this strain was designated as CGA 237'218.

1.1. Optimization of mass production An important pre-requisite for development of B. t. insecticides is an efficient method for mass production. The ultimate criterium for efficiency is the amount of delta-endotoxin produced per batch, which depends on cell density, sporulation rate and amount of delta-endotoxin produced per sporulating cell. Judged from size of the parasporal inclusion bodies, production of delta-endotoxin is much less sensitive to changes in environmental conditions than cell density and sporulation rate. Accordingly, high cell density and high sporulation rate are the primary criteria for production optimization. These criteria are easily measured

- 285 - K. Bernhard

by counting vegetative and sporulating cells in a counting chamber under the phase contrast microscope, which allows rapid examination of many samples. The method is not much hampered by the presence of other particles in the culture, e.g. non degradable media components. Delta-endotoxin may be directly quantified by bioassays or biochemically by ion exchange chromatography. Bioassays take one to six days, biochemical assay methods several hours and microscopic investigation only a couple of minutes. We therefore make extensive use of phase contrast microscopy, when large numbers of samples need to be analyzed, or rapid analysis is required.

As previously outlined, an efficient production process for B. t. aims primarily at high cell densities and high sporulation rates. These two criteria are not necessarily linked with each other. Using media which contain e.g. a high ratio of sugar, high cell densities may be achieved with little or no sporulation. For optimal sporulation, careful balance of substrates and synchronized growth must be achieved. Suitable culture media typically contain protein sources, sugars, yeast extract and mineral salts.

Media optimization is always accompanied by a search for inexpensive media components. Suitable protein sources can be found among agricultural byproducts used for production of animal feedstuffs. Typical substrates are soya flour, cotton seed flour (Proflo is probably the best known brand name), bean powders and corn steep liquor. Molasses are frequently considered as sugar source, although not all strains seem to utilize sucrose. Since B. t. produces amylases, glucose may be substituted by starch. Yeast extracts are available in many qualities, differing in yeast species, production method and price. Most suitable are extracts from baker's yeast, whereas brewer's yeast is unsuitable since many preparations contain compounds which inhibit growth of B. t. entirely. Sporulation is stimulated by

inorganic ions, particularly Ca2+ and Mn2+. Phosphate salts are usually added in larger quantities than required to sustain growth because of their buffering capacity. Adjusting concentrations of Mgt+, Cue+, Fe3+, Co+ and Zn+ ions may also improve growth and sporulation, even if complex substrates are used..

Apart from supporting growth, several other factors may also be important for selecting substrates. For mass production, availability of raw materials at the required quantity and price is critical as well as consistent physical and nutritional

- 286 - Development of B.t. Insecticides properties. Concentration of undegradable compounds e.g. cellulose in. the substrates should be considered, since they may dilute the delta-endotoxin content in the final product. All substrates should be finely ground flours, containing no coarse particles. Large particles can cause considerable difficulties during production due to clogging of pipes and nozzles.

1.2. Technical requirements for fermentation

B. t. fermentation is generally perceived as being simple. There are, however, several pitfalls which must be avoided : (a) B.t. does not grow faster than possible contaminants e.g. B. cereus. Therefore care has to be taken to properly sterilize fermenter vessels and culture media. The equipment should be sterilizable under constant agitation of the culture media. Media made from agricultural byproducts invariably contain large quantities of insoluble particles, which sediment quickly without agitation. In sediments complete heat inactivation of Bacillus spores may not be attainable, even if sterilisation time is increased. It is therefore important that these media are agitated during sterilization to prevent sedimentation. Even if heating is done electrically, steam should be available for sterilization of sample valves during fermentation. After a fermentation process is completed, the fermenter is heavily contaminated by endospores. Careful cleaning and sterilization are required to avoid cross contamination, particularly if different microorganisms are to be produced in the same facility.

(b) B.t. grows aerobically, sufficient aeration is therefore essential. Most fermenters are agitated by paddle blade stirrers together with static baffles at the inner wall of the fermenter vessel. Fermenters agitated by impeller and draught tube as well as airlift fermenters may also be used. Air for aeration must be sterilized by filtration. We have used membrane filters and ceramic filters and both gave satisfactory results. B.t. fermentations are short and air filters are frequently sterilized. Membrane filters deteriorate after a number of sterilization cycles and have to be replaced. Ceramic filters last much longer and are therefore preferable even for small scale production for experimental uses.

- 287 - K. Bernhard.

(c) With B.t. fermentations foaming is a problem. It occurs during sterilization and after inoculation throughout the early log-phase, when the medium still contains large amounts of undigested protein. Tow,:=rds the end of sporulation, foaming may become a problem again, because liberated spores may attach themselves to air bubbles and stabilize the foam. We have successfully used silicon based and polypropylene based antifoam agents. They may form lumps which cause trouble during harvesting and spray drying. The use of antifoam agents should therefore be minimized. Accordingly, foam buildup is monitored by a probe darin fermentation and sterilized antifoam emulsion pumped automatically into the fermenter vessel as required.

(d) Sufficient instrumentation of the fermenter vessels is important to monitor the status of the process. pH and oxygen concentration in particular should be continuously monitored by electrodes. If agitation and aereation rates remain unchanged during the fermentation process, oxygen concentration in the medium gradually declines during logarithmic growth. Entry into the stationary phase is characterized by gradual increase in oxygen concentration and pH. Beginning release of spores and parasporal crystals from the sporangia is indicated by an increase of pH above pH 8.0. As long as oxygen consumption is low, agitation may be kept low, which helps to reduce foaming.

For production of field trial quantities as well as further optimization of production'methods, we use fermenters measuring 15 L to 200 L in total vohime. Commercial production typically occurs in fermentation vessels of 50 m' and more. Media volume in a fermenter vessel usually does not exceed 2/3 of the total volume. A typical fermentation is shown in Fig. (1). Culture medium in a flask is inoculated with a loopful of bacteria from an agar plate and incubated on the shaker. At this stage spores germinate and the vegetative cells adapt to the culture medium. After 14 to 16 hours, the cells are in the mid-log phase. Then the culture is used to inoculate a 20 L fermenter containing the same culture medium. Growth in the fermenter is monitored by countir.° ,.ails microscopically. After 4 - 6 hours, the culture is in the late log-phase and transferred into the 200 L fermenter. If the transfer had not been delayed too long, the cells will continue to grow without lag-phase. Growth in the production fermenter is monitored again microscopically - 288 - Development of B.t. Insecticides until the sporangia start to lyse.

rpm pO2(%) cells/ml pH

5x 1010 -1 r 500

x100 1 x1010

F 50

1 x 109 I- 10

5

1 x 108 1 I -r -I 0 5 10 15 20 25 time (hrs)

Open circles: agitation (rpm). Open squares: cell density/ml. Crosses: Oxygen concentration in the medium (9'o saturation). Stars: pH in the medium.

Figure 1. Growth of Bacillus thuringiensis in submerged culture.

- 289 - K. Bernhard

1.3. Formulation Sporulated cultures are further processed to achieve physical properties suitable for commercial pest control. The objective is to provide a concentrate, which may be diluted with water and sprayed on the plants. The concentrate should mix readily with water to give a homogenous suspension without lumps. The suspension should be stable for some time without aggregation of particles or sedimentation and homogenous enough not to cause any blockage of spray nozzles. Storage stability is also important. For chemical insecticides two years storage stability is a standard requirement. With regard to biological activity, dry preparations of B.t. are normally stable for several years at ambient temperature and short time exposure to temperatures above 50°C, as may occur in warehouses, is also tolerated. In a first step towards formulation, the cultures are concentrated by centrifugation to reduce bulk. Reduction of pH in the culture can improve recovery of product with many strains. The collected sludge still contains nutrients which support growth of microorganisms other than B. t. It should therefore not be left more than 24 hrs. at room temperature, but should be stabilized as soon as possible, which may be done in different ways. With CGA 237'218, we employ spray drying which yields a fine stable powder, that may be stored and shipped for further formulation. In the spray drying process, the sludge is pumped as a fine spray into a large vessel, through which hot air is blown by a fan. The water evaporates quickly and the droplets are converted into dust particles. Particle size depends mainly upon the initial droplet size. Spraying occurs either by compressed air in a nozzle or on a disk spinning at high speed. Energy requirements during the evaporation process cools the air in the spray chamber preventing thermal denaturation of delta-endotoxin. In our laboratory spray driers for example, air inlet temperature in the spray chamber is 180°C to 200°C and outlet temperature 80°C to 90°C. The dust is separated from the air by a cyclone and collected in a container. It is referred to as technical product. Since milling is expensive and may also cause thermal denaturation of d-endotoxin, high quality spraying is desirable. The material is then mixed with equal amounts of inerts to obtain a wettable powder with the physical properties required for field application. The concentration given for the active ingredient in the final product is based upon delta-endotoxin content rather than technical product. Since the technical material contains 1.2% delta-endotoxin, the wettable powder is specified as a WP 0.6 rather

- 290 - Development of B.t. Insecticides than a WP 50. For control of the European Corn borer, Ostrinia nubilialis, a granular formulation was developed, which is applied directly to the plants without further dilution. Production and formulation are summarized in Fig. (2).

1.4. Quality Control Quality control aims at preventing manufacture and release of bad batches, which would fail to control insect pests in the field. In many countries, descriptions of the production process and quality control procedures are amongst the requirements for registration of biological pesticides. Quality control occurs at two levels (Fig. 3) : (a) The activity of CGA 237'218 is due to production of several delta-endotoxins. It is therefore important that the cultures used to start a fermentation process are not only microbiologically pure, but also genetically unaltered. Currently the safest way to test a starter culture is by bioassay against several insect species, supplemented by analysis of plasmid profiles. Spontaneously occuring changes of the genome, particularly plasmid loss, occur during cell proliferation. In order to minimize the potential for plasmid loss, unnecessary propagation of seed cultures must be avoided. It should be considered in this context, that only small amounts of cells are needed to start a fermentation process. Therefore, a small batch of cells, which does not need much storage space may be sufficient to start several years' fermentation cycles. With that approach, extensive testing is acceptable, since it occurs only once in several years. It requires suitable procedures for storage of the stock cultures. For long term storage of B. t. stock cultures, we take advantage of the persistance of endospores under dry and dark conditions. Permanent cultures are prepared by adding aliquots of sporulated B. t. culture to either sterilized quartz sand or pieces of sterilized filter paper contained in brown screw cap glass vials. They are thoroughly dried under a laminar flow hood and stored at room temperature in the dark.

(b) The objectives of the second level of quality control is to monitor the production process and performance of the final product. Since data supplied by manufacturers of raw materials for fermentation are not necessarily suitable to predict their ability to support growth, tests in shaker

- 291 - Iriolopicel ide

mixing with additives Centrifuge packaging

Spray dried powder

N N mixing with additives packaging

Figure 2. Schematic outline of Bacillus thuringiensis production and formulation. Development of B.t. Insecticides

Production 111111 11 Culture Collection/ Stock Quality Control: Stock Cultures

Bioassays Genetic Analysis Q-exotoxin conc. Plasmid profiles microbial purit y Southern blot ting PCR harvesting spray drying Quality Control: Techn.Product Bioassays S-endotoxin conc. microbial purity Mouse tox. Techn. Product

Figure 3. Quality control for production of Bacillus thuringiensis strains.

- 293 - K. Bernhard

flasks and even laboratory fermenters may be needed with new batches. Controls, needed to monitor the production process, are useful tools in quality control. They allow recognition of defects, e.g. contaminations, occurring early in the manufacturing process, which saves cost in downstream processing. The spray dried product is then tested for delta-endotoxin concentration and by bioassays against larvae of two lepidopteran species. Product specifications should be defined, which take into account normal batch to batch variations as well as confidence limits of the proposed quality control tests. In future biochemical tests may limit the need for bioassays. Regulating agencies, particularly in the US and in Canada, are increasingly concerned about presence of human pathogens in B. t. products, and request additional hygiene tests, particularly with products intended for use on food crops.

1.5. Field trials

Field development of CGA 237'218 was initially done at the different field stations maintained by Ciba-Geigy all over the world. At later stages external collaborators were also involved. The product is mainly intended for control of different lepidopteran pests like diamondback moth, Plutella xylostella, armyworms, Spodoptera spp. the tomato fruitworm, Heliothis armigera in vegetables and the grape berry moth. Lobesia botrana in wine grapes. Fig. (4) summarizes results of more than 20 trials against P. xylostella on cabbage. It clearly shows dosage dependend activity. Since 100 g product contain just 0.6% active ingredient, it also demonstrates that delta-endotoxins are highly potent insecticides. This becomes even more apparent in Figs. (5 and 6) which show efficacy of CGA 237'218 against H. armigera on tomatoes in Spain and L. botrana on wine grapes in Switzerland. Development of B.t. Insecticides

% Control

75

25

2.4 1.2 0.6 0.3 50 1.8 9 a.i./hl

400 200 100 50 g productml f CGA 237"218 I Abamectin Polo

Figure 4. Efficacy of CGA 237'218 against Plutella xylost,lla on cabbage.

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% Control 1001

801

60

20

24 12 6 500 g a.i./ha

4 2 1 Kg product/ha CGA 237'218 Selecron

Figure 5. Efficacy of CGA 237'218 against Heliothis armigera on tomatoes. Development of B.t. Insecticides

Parathion % Control Cascade I Supracide 901 CGA 237'218 Insegar

7S-

601

30

15

1.2 0.6 0.6 40 10 2.5 40 g a.i./hl 200 100 100 g product/h1 +sugar

Figure 6. Efficacy of CGA 237'218 against Lobesia botrana on wine grapes. K. Bernhard

2. Registration

Registration requirements for B. t. insecticides are generally much lower than for synthetic chemicals. In the past, they were frequently exempted from registration, or their use allowed under special permits. Today more and more countries are issuing specific requirements for registration of biologicals. Due to lacking experience on their part, currently regulatory authorities are often reluctant to be too specific and prefer to discuss details on a case to case basis with the applicants. The general issues are, however, in most countries the same. To facilitate discussion, the issues scrutinized in the registration process are described as follows :

(a) Identification of the strain : This set of information is supposed to describe the biological activity, properties upon which classification as B.t. is based as well as properties to differentiate it from other B. t. strains.

(b) Description of the production process : This set of information describes details of the production process, e.g. media composition, locations of potential fermentation plants, composition of the formulated product, storage stability data, quality control procedures and methods for measuring performance.

(c) Toxicology : Only acute toxicology testing is required. Usually only one high dosage is tested. The rationale is that a number of B.t. strains have already been extensively tested. If a new isolate shows no adverse effects at a high dosage either, it is assumed, that it does not toxicologically differ from the known strains. Testing with technical material is done to demonstrate that it is harmless to man. Tests with formulated material are to demonstrate that no synergystic effects exist (with regard to mammalian toxicity) between the technical material and inerts used to formulate the product. The technical material may either be a sludge obtained after centrifugation, or spray dried powder.

(d) Ecology : This set of data is referring to effects of the product on the environment. Tests are usually performed against several species of nontarget insects, particularly honey bees, birds, freshwater fish, marine fish and marine invertebrates. Again, whenever possible testing at higher doses than encountered during regular use is requested.

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(e) Efficacy data : It is interesting to note, that in some countries particularly attention is given to practical field efficacy data, whereas in other countries only information on possible environmental impact is required.

STRAIN SEARCH AND SCREENING Strains search and screening is done in collaboration with a team at Horticultural Research International Littlehampton/UK and the Agricultural Genetics Company Cambridge/UK. New strains are isolated from various kinds of samples collected worldwide. Much attention has been paid to obtain the greatest possible diversity of strains. New isolates are entered into a strain collection. Screening currently relies entirely upon bioassays. In a first tier of tests, sporulated cultures are tested against several species of model insects, which are easy to rear. They belong to the orders lepidoptera, diptera and coleoptera. Strains with interesting activity are then grown at the 10 L scale and spray dried. The powders obtained are entered into a collection of powders. Their delta-endotoxin concentrations are determined and all further testing is based upon delta-endotoxin concentration rather than amounts of powders used. The powders are tested in multidose assays against a wide range of test insects. At this level we also cooperate with external collaborators, to get tests done against species of insect pests which are not available within Ciba-Geigy. Strains which show interesting levels of activity are then grown at the 200 L scale and tested in small plot trials at the Ciba-Geigy field stations.

OUTLOOK Insecticides against chewing insect pests constitute a large segment of the total insecticides market. With regard to B. t., it is very much divided into submarkets, because of the selectivity of the delta-endotoxins. Based on the diversity of B.t. strains, it is expected that new submarkets will be entered, where currently available products cannot compete with synthetic chemicals. Accordingly a whole range of products need to be developed. Thus market volume will expand and is expected to increase to 50% - 10% of the total insecticides market by the year 2000. Considering that studies required for registration of a B. t. preparation cost less than 10 percent of what is required for a synthetic chemical, development of a whole range of selective B. t. insecticides appears economically attractive.

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In the short and mid term, we expect new strains to come from strain search and screening and conjugation experiments. At the laboratory scale, the use of recombinant DNA technology has already made considerable contributions to our understanding of B. t. activity against insect pests. In the longer term, we expect strains derived by recombinant DNA technology to be ready for entry into the market within the present decade. Whether improvements expected with recombined strains will materialize and whether products based upon such strains will be accepted by the farmers and the general public remains to be seen.

REFERENCES

Baumann, L. ; Okamoto, K. ; Unterman, B.M. ; Lynch, M. J. and Baumann, P. (1984) . Phenotypic characterization of Bacillus thuringiensis and Bacillus cereus. Invertebr. Pathol., 44, 329 - 341. Berliner, E. (1915) . Uber die schlaffsucht der mehlmottenraupe Ephestia kahniella Zell. and ihren erreger Bacillus thuringiensis n. sp. Z. ang. Ent., 2, 29 - 56. Bernhard, K. (1991). Quantitative determination of delta-endotoxin contents in spray dried preparations of Bacillus thuringiensis strain GC-91. World J. Microbiol. Biotechnol. (in press). Buchanan, R.E. and Gibbons, N. E. (Eds). (1974). Bergey's Manual of

Determinative Bacteriology Eighth Ed., 529 - 550 Baltimore : Williams and Wilkins Company. Burges, D.H. and Jarrett, P. (1988). Preparation of strains of Bacillus thuringiensis having improved activity against certain lepidopterous pests and novel strain produced thereby. UK Patent GB 2165261. The Patent Office, London. 28 pp. Dulmage, H. T. and Collaborators (1981). Insecticidal activity of isolates of Bacillus thuringiensis and their potential for pest control. In : Microbial Control of Pests and Plant Diseases 1970 - 1980, ed. Burges, H. D. 193 - 222. London : Academic Press. Goldberg, L. J. and Margalit, J. (1977) . A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaenia unguicultata, Culex univittatus, Aedes aegypti and Culex pipiens. Mosquito News, 37, 246 - 251. Gonzalez, J. M. ; Brown, B. J. and Carlton, B. C. (1982). Transfer of Bacillus thuringiensis plasmids coding for delta-endotoxin among strains of Bacillus thuringiensis and Bacillus cereus. Proc. Nat. Acad. Sci. USA., 79, 691 - 695. Jarrett, P. and Burges, H. D. (1986). Bacillus thuringiensis : tailoring the strain to fit the pest complex on the crop. BCPC Mono. No. 34 Biotechnology and

Crop Improvement and Protection, 259 - 264. Cambridge : British Crop Protection Council.

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Krieg, A. ; Huger, A. M. ; Langenbruch, G. A. and Schnetter, W. (1983). Bacillus thuringiensis var. tenebrionis : ein neuer, gegenuber Larven von Coleopteren wirksamer Pathotyp. Z. ang. Ent., 96, 500 - 508. Martin, P. A. W. and Travers, R. S. (1989). Worldwide abundance and distribution of Bacillus thuringiensis isolates. Appl. Environ. Microbiol. 55, 2437 -2242. Meadows, M. P. ; Ellis, D. J. ; Pethybridge, N. J. ; Bernhard, K. ; Burges, H. D. and Jarrett, P. (1990). Activity of new isolates of Bacillus thuringiensis against five insect species . In : Proceedings of the Vth International Colloquium on Invertebrate Pathology and Microbial Control, pp. 497.

Adelaide : Society of Invertebrate Pathology 1990.

Canadian Policy and Regulations for the Adoption of Naturally Occurring and Genetically Modified Bacillus thuringiensis

J. E. Hollebone Pesticides Directorate, Agriculture Canada, Ottawa, Ontario K1A OC6

ABSTRACT

Microbial pest control agents, such as Bacillus thuringiensis (B. t.) must be registered before sale and use in Canada in compliance with requirements established under the authority of the Pest Control Products Act and Regulations. In addition, before release to the environment is permitted, research permits must be approved which set the conditions and limitations forfield testing. Data to support field testing and registration requirements are outlined in appropriate guidelines, following a tiered approach (increasing requirements with increasing potential risk to human and environmental safety). Data requirements include : proposed use ; identification, manufacturing methods, quality control ; human health, safety testing including acute toxicity/infectivity/hypersensitivity tests ;food and feed residue studies ; environmental fate and toxicity to non-targets ; and performance (efficacy, phytotoxicity) data. Currently, guidelines for naturally occurring microorganisms are being revised to also include genetically engineered organisms (GEO's). The approach is to compare the GEO to its conventional relatives and evaluate any perturbations/ differences. Specifically for GEO's, the techniques of construction of new genetic material, the stability and specificity of the construct, the potential for gene transfer and environmental or human health impact will be evaluated and monitored.

INTRODUCTION The first registration of a biological pest control agent was granted in 1962 in Canada for the use of Bacillus thuringiensis (B.t.) var. kurstaki for control of agricultural lepidopteran pests. In the U.S., the first registration was for Bacillus

- 303 - J. E. Hollebone pop4lae in 1947. While immediately finding a small niche as a narrow spectrum, biological alternatives with low toxicity to humans and low impact on the environment, the early products, did not make a significant impact in the reduction of agricultural chemical insecticides. This was due primarily to low efficacy, product instability in sunlight and limited environmental persistance. In forestry, however, by the mid 1980's B.t. strains had made a major impact, virtually replacing the major chemical pesticides for spruce budworm and gypsy moth control in Ontario, Quebec and the Atlantic provinces. In the last 5 years, the use of microbial pest control has rapidly increased. This is due in part to the increasingly receptive climate for biologicals. The public is increasingly concerned over the continued use of chemical pesticides with their associated effects on human health and the environment. Several national governments, such as Sweden, the Netherlands and Australia have instituted programs aimed at a reduction in chemical dependency and use of alternative to agrochemicals. The Netherlands, for example, has called for a 50% reduction of chemical pesticides by the year 2000. The U.S. is in the process of introducing a "safer pesticides " program which encourages the development and use of biological pesticides.

The last decade has also seen a marked increase in new strains and new techniques which enhance the longevity and performance capability of B.t. products. The isolation and subsequent commercialization of new strains specific for other orders of insects have greatly increased the range of available products, e.g., B. t. aizawai (see H- 14) specific for nematocerous Diptera such as black flies and mosquitoes, B. t. tenebrionis and San diego strains for beetle control, B. t. aizawai for a wide range of Lepidoptera. In addition, using the new techniques of molecular biology, genetically improved and engineered B. t. 's are being developed, and the genes coding for the B. t. toxin have been sequenced and engineered into other microbes and plants to express B. t. endotoxins.

While microbial pest control agents such as B.t. provide narrow spectrum, fairly target-specific methods of pest control which are generally environmentally benign, nevertheless, regulatory agencies have required careful testing and subsequent evaluation to ensure that such products can be used safely. THE REGISTRATION PROCESS IN CANADA In Canada, all pest control products must undergo an evaluation and approval

- 304 - Registration Requirements for B.t. in Canada under the authority of the Pest Control Products Act before sale and commercialization. The purpose of the evaluation is to ensure the "safety, merit and value" of candidate products. Special guidelines have been developed to establish regulatory requirements for microbial pest control agents, such as bacteria

including B. t. , algae, fungi and viruses.

Data submitted by applicants (researchers and companies) are designed to Pesticides allow an assessment of these factors by the regulatory agency, the Directorate, in order to determine the conditions under which these products can be used safely. Specialists from the Departments of Health and Welfare, Environment Canada and Forestry Canada, provide assessments and expert advise to the Directorate on the human and environmental safety of these products.

DATA REQUIREMENTS In Canada, as in the United States, data requirements are tailor-made to different types of pesticides, those for microbial pest control agents are less extensive than those for chemicals (and cost about 10076), yet are designed to answer the same questions of safety, merit and value. Companies are asked to provide of the data for assessment. They include : general identification and manufacture organism (identification, techniques to isolate and grow the organism, taxonomy, fermentation process, quality control techniques) ; toxicology data ; metabolism and food residue data (if used on a food crop) ; environmental fate (e.g., persistance, survival, fate, spread) ; environmental toxicology (effects on nontarget timing of organisms) ; and efficacy (performance in field and laboratory, application, etc.).

These data allow evaluators to evaluate possible effects on users and bystanders, to determine whether residues may occur on harvested food, and if so, the safety of such food for consumption by humans or animals. As well, possible effects on environment and performance are evaluated.

THE REVIEW PROCESS Depending on the nature of the product to be evaluated, reviews are either conducted in-house (minor amendments, non-food uses) or in consultation with advisors from other government departments for human safety and environmental

- 305 - J. E. Hollebone impacts (all new biologicals, and all new food uses).

In both the Canada and the U.S., the review process consists of several checkpoints or steps, at which questions are asked and must be answered before proceeding to the next stage. Data requirements are similarly appropriate to these stages, increasing as the size of the trial, and the potential for exposure to humans and the environment increases. The stages of review occur at importation, before environmental release as small scale field trials, or precommercial (large scale) trials, and before registration.

Tiering of data is used where appropriate. The concept of tiering is risk-based, e.g., there is a basic set of tests that all products must undergo. If no adverse effects are noted, further testing may cease ; if adverse effects are identified, then the product may have to undergo further tests at a higher tier or level of testing.

GUIDELINES The Pesticides Directorate issues guidelines to outline the steps and data required to register products. R-memos (registrant memos) are used for guidelines in the development stage, and are issued for public comment. T-memos (trade memos) are used to issue final guidelines after consultation is complete. The U.S. EPA follows similar procedures, Sub Part M deals with requirements for registration of microbial pest control agents.

In both Canada and the U.S., guidelines for microbial pest control agents are still under development, at least for specific aspects. They have evolved in a manner parallel to those for chemical guidelines, but are tailor-made to deal with microbes such as B. t.

STAGES OF REGULATORY OVERSIGHT Importation

All unregistered biological or microbial pest control products imported into Canada require an import permit. For most B. t. products which have been reviewed in Canada, this is a fairly simple procedure, filling out a permit application which requires identification of the B. t. , its origin and the laboratory of destination, and returning it to the Plant Protection Division which issues an import permit. However, for non indigenous organisms which have the potential to become plant - 306 - Registration Requirements for B.t. in Canada pests in Canada, a pest risk assessment is carried out before the permit is granted. For strains isolated in Canada, a permit is not required. The current legislation, under the Plant Quarantine Act, is considered adequate to prevent the introduction of plant pests, however, it does not cover all possible cases and extending the legislation to cover such areas as insect pathogens is being considered.

Field Trials

1. Small-scale trials : Research permits are required in Canada for all field releases except small-scale trials for indigenous organisms. This exemption applies to field trials of less than

10 hectares (terrestrial) or 1 acre (closed aquatic system applied by ground equipment).

Exempted trials may be conducted by research staff only, with no cooperators, e.g., farmers, on the research facility.

2. Large-scale field trials : In larger trials, or trials using a non-indigenous organism, a research permit is required before trials may proceed. The following data are required and assessed

before the permit is granted : product specifications, (including product identification, taxonomy and morphology, mode of action, native habitat, ecological range in Canada, and production methods) ; human safety testing (acutes, infectivity-toxicity study, I.V, for bacteria) including acute studies before the product is allowed into the hands of cooperators ; experimental uses and labels. Food residue data are often gathered in small-scale trials and are required to support sale of any treated crop which will be used for human consumption. In general, for B. t. in Canada, an exemption from residue testing has been granted.

Environmental data are not required to support small-scale testing of indigenous B. t. in Canada, for non-indigenous B. t.'s host specificity tests and non-target organism tests are required as well as laboratory fate data. For all trials, field test designs, contingency plans, termination procedures, etc., and an experimental label are required. For forest and water applications, maps may be required.

- 307 - J. E. Hollebone

Registration Requirements Data requirements for registration purposes build on those required at research permit stage. In general, at this final stage of risk assessment, the loose ends of the registration review are completed ; manufacturing methods and quality control procedures are received and reviewed, human safety and testing is completed including acute toxicity (eye and dermal irritation), pathogenicity (maximum challenge) and infectivity studies (IV) are required. Hypersensitivity testing is required on the formulated products. Environmental fate and toxicity are completed and performance at several different locations to reflect climatic, soil and geographic variations are required for all microbials. Studies to check genotoxic potential of microbials producing toxins are checked at different sites across Canada.

U.S. and Canadian guidelines are overtly similar, requiring the same tests. However, the level of detail per study tends to be greater in Canada. Industry has argued strenuously that these requirements add additional costs, without adding significantly new data to assessments. These are now under discussion with advisors, in order to better harmonize with other international bodies.

The greatest differences occur regarding : (1) appropriate end points for safety testing and in the more detailed histopathology and organ culture for the recovery of test organisms. For example, OECD and EPA guideline are 21 days for acute infectivity studies, for Canada, 28 day studies are required. (2) requirements for food residue uses. A 30-day infectivity feeding study is required in Canada (in the U.S., it is 21 days).

(3) use of an ecozone concept for determining if an organism is indigenous or nonindigenous and requiring in Canada, a full set of environmental testing in each identified ecozone. (4) whether residue data should be required for all field crop use. (5) level of identification of possible contaminants.

GENETICALLY-ENGINEERED B.t. Genetically engineered microbes, including B.t. will be regulated under the

- 308 - Registration Requirements for B.t. in Canada same criteria and guidelines as non-engineered B. t. 's. Such GE - B. t. are not considered a "unique category ", but rather new strains, sharing basic characteristics common to the unmodified plant. The approach will be to compare the new genetic construct to appropriate unmodified B. t. strains, and to evaluate for any differences which may have potential impacts on safety or performance. Specifically for GE-B.t.'s, the techniques of construction of the new genetic material, the potential for gene transfer, the stability of the GEM and the construct will be reviewed and considered. Draft guidelines should be available for comment and discussion by late spring 1992.

CONCLUSION The development of regulatory guidelines must be flexible, appropriate to the organism being evaluated. Hence, regulatory requirements, while having a common core of studies which could be common to evaluations performed by any nation must also be flexible enough to allow requirements to be adapted to safety assessments appropriate to the proposed country of use. Similarly, the approach to regulation should be science-based, risk assessments. Since regulatory approaches in most countries are basically similar, due to the major role played by FAO/WHO and OECD in harmonizing regulatory guidelines and review procedures, a useful trend in this time of economic restraint, would be a move to greater acceptance of common standards and evaluations to reduce costs and maximum scarce resources.

Commercialization and Utilization of Bacillus thuringiensis for Crop Protection in China

Xie Tianjian, Huang Bingao, Zhong Liansen and Wu Gixin Hubei Academy of Agricultural Sciences, Wuhan, P.R.C.

ABSTRACT

Bacillus thuringiensis (B. t) is the most produced and widely used microbial insecticide in China. In 1964 the first workshop was established in Wuhan, since then Wuhan has been the main base for producing B. t. insecticide. B. t. output has grown steadily. For example, in Microbial Pilot Plant, Hubei Academy of Agricultural Sciences, the output was 260,000 kg in 1986 ; 360,000 kg in 1987; 472,000 kg in 1988 ; 732,000 kg in 1989 and 880,000 kg in 1990. B. t. is mainly used for control of agriculture pests, such as diamondback moth, cabbage looper, corn borer, rice leaf roller, canker worms and others. B. t. also is used for control offorest and public health pests. F,specially pest resistance to pyrethroid insecticide develop so fast that B. t. is well received by more and more farmers. Two formulations, liquid and wettable powder, are provided according to difference demand. Recently more research work was put on fermentation and formulation technology to lower the cost and to enhance the product quality. There is no doubt that the output of B. t. will keep growing.

INTRODUCTION

B.t. Insecticide is the most produced and the most widely used microbial insecticide in China. In 1964, the first workshop on B. t. was established in Wuhan, and since then Wuhan has been the main base for producing B. t. insecticide. During the past several years, B. t. output has grown steadily. For example, in Microbial Pilot Plant, Hubei Academy of Agricultural Sciences, the output was 260,000kg in 1986, 360,000 kg in 1987. 472,000 kg in 1988, and 732,000 kg in 1989. B.t.

- 311 - Xie Tianjian et al.

insecticide has been used for control of agriculture, forestry and public health pests among twenty-eight provinces and big cities. B. t. insecticide has shown good results and has been exported to South East Asia. Along with the development of B. t. commercial production and application, the Chinese government, research workers, producers and users have known more advantages of B.t. insecticide. Only agricultural byproducts are used as the main medium ingredients. B.t. does not pollut environment and does not destory ecological balance. There is no doubt that the output of B. t. insecticide will keep growing in 1990's.

COMMERCIAL PRODUCTION

1. Strain

B. t. galleriae was the strain used for production in 1960'x. B. t. galleriae, B. t. wuhannensis, B.t. dendrolimus, HD-1 and 7216 were used in 1970's, and HD-1 was the main strain in 1980's.

2. Fermentation

Agricultural by-products are used as the main medium component such as defatted soybean cake, peanut cake, cotton seed cake and others. Some kinds of waste materials are also used successfully for production of B. t. mosquitocide. At early stage, the concentration of nutrient ingredients was between 34%, beer spore count was about 20X 101/ml., but when the concentration was 7-9% the spore count was 50-70 x l0A/ml, Most fermenters used for B. t. commercial production are 5,000-7,000 liters. With the market growing, 20,000 liters fermenter has been used.

3. Phage

Phage once threatened B. t. commercial production seriously. The failure rate caused by phage was even as high as 30% in a typical factory which had to be closed finally. Now almost no damage is caused by phage. For example the failure rate was 1.5% in 1986 and zero in 1987. The main measures taken include strengthening air filter system, heating spore suspension before inoculation, changing two stages of fermentation to one stage of fermentation and so on. 4. Formulations and recovery procedures Different formulations must be processed with different recovery techniques.

- 312 - Commercialization of B.t. in China

In 1980's, flowable, formulation was produced mainly in China. The flow chart applied in Microbial Pilot Plant, Hubei Academy of Agricultural Sciences was as follows : Soil culture -- slant seed -- fermentation -- screening centrifugation -- formulation -- quality checking -- packing

Comparing with powder formulations, flowable formulations consume less energy to be produced and its recovery rate is higher. So, the production cost is lower. But the stablility is not as good as the powder. Usually, the storage period is one year. Flowable formulation is very popular in China. About70-80% of total amount of B. t. insecticide belongs to flowable formulations which is produced by Microbial Pilot Plant, Hubei Academy of Agricultural Sciences.

Wettable powder of 16,000 IU/mg has been produced using the spray drying technique. Wettable powder is easy to transport and is very stable. Membrane, granular and briquet formulations have been investigated for the control of mosquitos. Membrane formulations will be specially used to treat larvae habitat of water in South China.

5. Product standards

B. t. insecticide used to be measured with spore count. Even now, in flowable formulation, the spore count is still used to express its quality. Great progress has been made for bioassay in which cotton bollworm and diamondback moth are chosen as test insects.

Product standards are as follows = Wettable Powder 16,000 IU/mg - Flowable formulation : spore count 20 x 108/ml - Stability period one year

APPLICATION

Low cost stimulates farmers to use B. t. insecticide for the control of pests. During the past several years, the area treated with B. t. insecticide was about 300,000 hectares each year.

- 313 - Xie Tianjian et al.

1. Grain crop pests

In north China, granular B. t. formulations have been applied to control the corn borer for more-than twenty years. Flowable formulations are also sprayed by airoplane recently. It is very effective to control rice leaf tier, rice leaf folder and sorghun spotted borer. Progress has been made wih the mixture of B. t. and chemical insecticide for the control of rice stem bores.

2. Cash crop pests

Good results were obtained for the control of tobacco budworm, tea caterpillar, soybean caterpillar and cotton geometrid. High dosage of B. t. can work well for the control of cotton bollworm. Recently, it has been replaced by mixture of B. t. and chemicals which result in lowering the control cost. This method has been expanding in Hubei Province.

3. Vegetable pests

This is the most successful area in China. Usually more than 90% of B. t. is used in spring, summer and fall for the control of cabbage caterpillar and diamondback moth. Nearly 50% of those pests are controlled now in big cities such as Shanghai, Beijing and Lanzhou. The control cost of applying B. t. insecticide is now less than chemicals. It is also very effective against pickle worm and others.

4. Forest pests

Nearly 9010 of mortality can be obtained for the control of poplar looper, poplar caterpillar, oriental moth, and orange dog. It is about 80% for the control of pine caterpillar. B. t. also works well for the control of bag moth.

5. Mosquitoes

B.t. israelensis has been used for control of mosquitoes for five years in Hubei Province. It was highly toxic to Anopheles sinensis and Culex fatigans.

PROSPECT

The price of B.t. insecticide is so low that the control cost by using B.t. is cheaper than that of using chemicals for several pests, such as cahbµge iooper, crankworm, corn borer and others. The amount of natural enemies on using B.t.

- 314 -- Commercialization of B.t. in China area is obviousely more than that on using chemical areas so that less insecticide is used on using B. t. area. Those economic reasons will encourage farmers to use more and more B. t. insecticide. During 1990's the B. t. output will grow up steadily.

Investigations with Dipel ES and Dipel ES-Chemical Insecticide Mixtures for Control of Lepidoptera Pests in Cotton in the United States and Australia

R. A. Fusco Abbott Laboratories, U.S.A.

ABSTRACT Field programs initially developed in the United States with Dipel ES, (Bacillus thuringiensis) and its combinations with new ovicides and synthetic pyrethroids were successfully tested in Australia during the 1989-1990 season. A total of 20 trials was completed which demonstrated that the use of Dipel ES alone or combined with chemical insecticides would be a suitable material to include in the pyrethroid resistance management program in that countryfor control of their most serious lepidopterous pests in cotton, Heliothis armigera and Heliothis punctigera.

INTRODUCTION

In the last decade, cotton Heliothis/Helicoverpa resistance to several chemical insecticide groups has spread around the world. Increased resistance in several countries such as the USA, Australia, China, and Colombia has forced growers to adopt stringent resistance management strategies to protect existing products and maintain yields. For example, in the cotton growing belt in the USA, no pyrethroids are used during stage 1, 2 or 4 ; they are used only during the first big Helicoverpa egg lay and hatch- (stage 3 in the US) and (stage 2 in Australia) and a maximum of 3 sprays only.

In addition to these strategies, there is the need to use high potency Bacillus thuringiensis products such as Dipel 2 x or Dipel ES. Since their mode of action is different from the chemical insecticides and they have narrow spectrum of activity, they can be alternated with conventional insecticides or can substitute for them.

- 317 - R. A. Fusco

Since its introduction into the US cotton market in 1987, Dipel ES has been the mostly widely used biological insecticide on cotton due to its field performance, "fit" into resistance management programs and environmental safety. Dipel ES contains 17,600 IU's of potency per mg, the most potent biological insecticide registered for use on cotton. It's a unique oil-based formulation which has been designed for aerial or ground ULV or high volume applications.

The work presented was conducted in Australia in 1989-91 by members of the Abbott R & D staff and Agrisearch Co., a contract research group in Australia.

MATERIALS AND METHODS

Target Pests in Australia

The target pests in Australia were Helicoverpa armigera and Helicoverpa punctigera.

Treatments/Rates

The main treatment combinations were Dipel ES plus endosulfan 240, Dipel ES plus Larvin 175 (thiodicarb), and Dipel ES plus the synthetic pyrethroid Decis ULV (deltamethrin). These were compared against Dipel ES and the chemicals alone.

Rates of Dipel ES and Larvin were I and 2 liters/hectare. Rates of endosulfan and Decis were 1.5, 2.0, and 3.0 liters/hectare.

Location of Trials

The trials were located in the major cotton regions of eastern Australia in Queensland and New South Wales over a wide range of environmental and climatic conditions, pest pressures and resistance levels. A total of 40 aerial ULV trials were conducted.

Application Techniques/Equipment

Treatments in all trials were applied with commercial aircraft equipped with AU-5000 Micronair Rotary atomizers. Total spray volumes ranged from 3.0 to

- 318 - Potency of Dipel, Chem. Insecticide Mixtures

4.0 L/ha depending upon the treatment applied. Each site received up to 4 applications of each treatment.

Applications were timed to coincide with peak egg laying where possible. Applications were sometimes made on mixed size Helicoverpa larvae as the sequential application technique used in these trials allowed larvae to develop in these less efficacious treatments.

When necessary, D-C-Tron, a crop oil was added to bring the volume up to 3.0 - 4.0 L/ha.

Trial Design

All trials were set out as large plot unreplicated field trials with 4 internal subplots per treatment block. Block sizes ranged from 4-6 aircraft swaths wide (72-120 meters) by 350 to 1,100 meters long.

Evaluations

Evaluations were made pre-spray, then 2-3 days post-spray by counting the number of Helicoverpa eggs and larvae and the number of damaged bolls and squares on 10 randomly selected cotton terminals per subplot.

Helicoverpa Pressure Based on Egg Counts

Before reviewing the data, it is essential to show the basis for which Helicoverpa population pressure is largely determined in Australia.

Scouting and control decisions are made in large part on the egg density. We did not see much in the way of beneficials and it was apparent that egg parasitism and predation were not high ; therefore a high percentage of eggs were expected to hatch. R. A. Fusco

RESULTS

The data given in these tables represent counts per 10 terminals.

Table 1. Dipel + Larvin/Light Infestation

Dipel ES alone at either 1 or 2 liters/ha was commercially acceptable under low to moderate insect pressure as seen from Table 1 in one of the trials at Dalby in which Dipel/Larvin combinations were tested. As pressure increased, the lower rate of ES alone was starting to break in terms of fruit damage.

On the average from the total program, it was found that as pest pressure increased, the lowest point at which the 1 liter rate of ES separated from the 2L rate was at approximately 300% egg deposition. For 2 L/ha, the lowest density at which the treatment failed to give good control was at approximately 400% egg pressure.

Table (1) Mean No. Damaged Squares and Bolls. Dipel + Larvin, Light Infestation. Dalby, QL.

Treatment L/ha 5DAT1 2DAT3

Dipel ES 1.0 1.62 b 2.25 b D-C-Tron 2.0

Dipel ES 2.0 0.50 a 1.75 ab D-C-Tron 1.0

Dipel ES 1.0 0.38 a 1.62 ab Larvin 175 1.0 D-C-Tron 1.0

Dipel ES 2.0 1.00 ab 1.38 a Larvin 175 1.0

Larvin 175 3.0 0.12 a 1.50 a

- 320 - Potency or Dipel, Chem. Insecticide Mixtures

Table 2. Dipel + Larvin/Moderate Pressure

At moderate pressure, Dipel combinations with Larvin did well and in general were equal to or better than the Larvin alone treatment at 3 L/ha. In Table 2, as pressure increased, the combinations did well and ES alone failed to provide adequate control.

Table (2) Mean No. Damaged Squares and Bolls. Dipel + Larvin, Moderate Infestation. Dalby, QL.

Treatment L/ha 4DAT2 2DAT3

Dipel ES 1.0 10.62 c 8.00 b D-C-Tron 2.0

Dipel ES 2.0 7.88 b 6.62 b D-C-Tron 1.0

Dipel ES 1.0 2.38 a 3.62 a Larvin 175 1.0 D-C-Tron 1.0

Dipel ES 2.0 3.62 a 2.88 a Larvin 175 1.0

Larvin 175 3.0 5.88 b 4.00 a

Table 3. Dipel + Larvin/Heavy Pressure

Table (3) shows a situation of heavy pest pressure for sure would not be a situation where Dipel alone should be used. The 2 liter rate of Dipel + Larvin at 1 liter was the best combination, but Larvin alone was also doing well, indicating low resistance in this population.

- 321 - R. A. Fusco

Table (3) Mean No. Damaged Squares and Bolls. Dipel + Larvin, Heavy Infestation. Dalby, QL.

Treatment L/ha 5DAT3 7DAT3

Dipel ES 1.0 27.12 d 35.62 d D-C-Trop 2.0

Dipel ES 2.0 19.12 c 20.75 c D-C-iron 1.0

Dipel ES 1.0 8.75 b 11.25 b Larvin 175 1.0 D-C-Tron 1.0

Dipel ES 2.0 6.38 a 9.88 b Larvin 175 1.0

Larvin 175 3.0 6.62 ab 3.50 a

Table 4. Dipel + Endosulfan/Emerald

This is a trial with the Dipel/Endosulfan mixtures and is typical of the results we saw in a light infestatin. Control with the mixtures using half the full rate of Endosulfan was similar to that from the 3 liter rate of Endosulfan alone.

Table 5. Dipel + Endosulfan/Bongeen

The advantage of applying the Dipel ES + Endosulfan mixtures over applying Endosulfan alone in situations of high Endosulfan resistant to Helicoverpa annigera is illustrated in this trial.

With the exception of the low rate of 1 + 1.5 + 0.5. all of the other mixtures had significantly fewer damaged bolls and squares than the Endosulfan ULV at the 3.0 L/ha rate.

- 322 - Potency of Bipel, Chem. Insecticide Mixtures

The trial also showed that when the pressure was heavy, the 2.0 + 1.5 L/h rate of Dipel + Endosulfan was not as robust as the 2.0 + 2.0 L/ha rate. The 2.0 + 2.0 L/ha rate consistently provided superior control to all other treatments.

Table (4) Mean No. Damaged Squares and Bolls. Dipel + Larvin, Light Infestation. Emerald, QL.

Treatment L/ha 6DAT2 4DAT3

Dipel ES 1.0 0.62 a 2.12 b D-C-Trop 2.0

Dipel ES 2.0 1.38 b 1.38 ab D-C-Trop 1.0

Dipel ES 1.0 0.88 ab 0.88 ab Endosulfan 240 1.5 D-C-Tron 0.5

Dipel ES 2.0 0.38 a 0.50 a Endosulfan 240 1.5

Endosulfan 240 3.0 0.38 a 0.38 a R. A. Fusco

Table (5) Mean No. of Damaged Squares and Bolls. per 10 Terminals. Dipel + Endosulfan. Bongeen, QLD

Treatment L/ha PRE-TMT 7DAT1 6DAT2 CUM*

Dipel ES 1.0 2.3 a 6.8 c 4.8 be 6.9 d Endosulfan 1.5 D-C-Tron 0.5

Dipel ES 1.0 2.5 a 6.1 be 3.5 b 4.1 c Endosulfan 2.0

Dipel ES 2.0 3.9 a 3.9 a 3.6 b 3.5 b Endosulfan 1.5

Dipel ES 2.0 2.9 a 4.1 ab 2.0 a 2.8 a Endosulfan 2.0

Endosulfan 3.0 2.6 a 6.3 c 6.6 c 4.9 d

* CUM = Cummulative counts per 10 terminals not including pre-spray data from 1st application.

Table 6. Dipel ES + Decis/Emerald

Mixtures of Dipel + Decis provided better control of Helicoverpa larvae than the pyrethroid alone when pest pressure was low but resistance levels were high. This resulted in lower damage levels.

Table 7. Dipel ES + Decis/Boggabri

This Table illustrates the benefit of increasing the rate of both pyrethroid and Dipel. Cumulative damage was reduced by 30% by the 2.0 + 2.0 L/ha rate relative to Decis alone at 3.0 L/ha treatment. The next lowest damage score was obtained by the 1.0 + 2.0 L/ha followed by the lower rates of the mixture. All Dipel ES + Decis mixtures produced significantly less damaged squares and bolls than Decis alone at 3.0 L/ha in this trial.

- 324 - Potency of Dipel, Chem. Insecticide Mixtures

Table (6) Mean No. of Damaged Squares and Bolls per 10 Terminals. Dipel + Decis. Emerald, QLD

Treatment L/ha PRE-TMT 5DAT1 6DAT2 CUM*

Dipel ES 11) 0.38 a 0.25 a 0.25 ab 0.31 a Decis 1.5 D-C-Trop 0.5

Dipel ES 1.0 0.12 a 0.25 a 0.25 ab 0.29 a Decis 2.0

Dipel ES 2.0 0.25 a 0.38 a 0.25 ab 0.27 a Decis 1.5

Dipel ES 2.0 0.12 a 0.25 a 0.12 a 0.23 a Decis 2.0

Decis 3.0 0. 00 a 1.75 b 0.62 b 0.96 b

* CUM = Cummulative counts per 10 terminals not including pre-spray data from lst application.

Table (7) Mean No. of Damaged Squares and Bolls. per 10 Terminals. Dipel + Decis. Boggabri, NSW.

Treatment L/ha PRE-TMT 3DAT1 7DAT2 CUM*

Dipel ES 1.0 2.5 ab 6.5 be 5.9 ab 7.3 c Decis 1.5 D-C-Tron 0.5

Dipel ES 1.0 2.1 ab 5.4 b 6.5 b 6.9 b Decis ULV 2.0

Dipel ES 2.0 3.4 b 7.0 c 4.5 a 7.3 c Decis ULV 1.5

- 325 - R. A. Fusco

Table (7) Continued.

Treatment L/ha PRE-TMT 3DAT1 7DAT2 CUM*

Dipel ES 2.0 3.4 b 3.5 a 2.0 a 5.7 a Decis ULV 2.0

Decis ULV 3.0 1.5 a 7.5 c 5.1 ab 8.1 d

* CUM = Cummulative counts / 10 terminals not including pre-spray data from 1st application.

DISCUSSION The results indicate that Dipel ES is very effective in controlling Helicoverpa spp. regardless of the level of chemical resistance in the larval population.

From studies conducted in the United States and Australia, Dipel ES is able to control larvae resistant to other insecticides due to its completely different mode of action as a stomach poison. It can therefore be used at different degrees in all stages of the resistance management program. The ability of growers and consultants to alternate products with different chemistries and modes of action is a key component of a successful IPM program.

In addition to providing wider choice of Helicoverpa control products, Dipel ES relieves pressure from overuse of widely used chemical materials (in Australia, its Endosulfan) and adds to the number of alternatives. Endosulfan is a valuable product for growing cotton in Australia and it is in the industry's interest to retain its use and availability.

It was shown that by using Dipel ES in combination with Endosulfan, it is possible to improve control of resistant Helicoverpa armigera and to reduce the volume amount of Endosulfan being sprayed. Thus, by using Dipel ES it is possible to obtaion commercially acceptable Helicoverpa control while extending the life of Endosulfan.

Another important use of Dipel ES demonstrated in Australia during the 1990/9: season was a combination of Dipel ES with low label rates of synthetic

- 326 - Potency of Dipel, Chem. Insecticide Mixtures pyrethroids. Dipel ES used in combination with SP's (at reduced rates) has shown better control of resistant Helicoverpa than full pyrethroid rates alone. By acting as a stomach poison, Dipel ES complements the chemical mode of action of the SP and reduces the levels of resistant Helicoverpa which are hard to control with the SP alone. B. t. acting as a "de-facto" synergist, Dipel ES reduces the number of resistant larvae in the population, thus making the remaining population more susceptible to other insecticides.

Dipel ES combinations with Larvin at recommended rates provides insecticidal and ovicidal activity on eggs and hatching larvae, respectively. Both Dipel ES and Larvin are very selective products with minimal impact on beneficial insects.

SUMMARY AND CONCLUSIONS

Helicoverpa Control with Dipel ES 2 + Larvin

Taking into consideration all the trials from the total program, we can provide the following summary for the mixtures with Larvin : - Under low to moderate pressure, Dipel ES at 1-2 L/ha + Larvin at ovicidal rates were equivalent and sometimes gave faster knockdown than Larvin at the full rate of 3 L/ha.

- At moderate to high pressure, Dipel ES + Larvin at 2 + 1 L/ha generally gave better control than rates at 1 + 1 but generally more damage occurred than with Larvin at 3 L/ha.

Helicoverpa Control with Dipel ES + Endosulfan Mixtures

As a general statement concerning all the trials with Endosulfan mixtures - Under low pest pressure and resistance, the lowest rate mixtures (1.0 + 1.5 + 0.5) provided good control. It was not as efficacious as other treatments when the pest pressures were increased and / or resistance levels were elevated. - Under moderate to high pressure and resistance, Dipel ES + Endosulfan (2.0 + 1.5, 2.0 + 2.0) gave superior control compared with applications of Endosulfan alone at 3.0.

- 327 - R. A. Fusco

Helicoverpa Control with Dipel ES + Decis Mixtures

- Under low pest pressure and resistance, all treatments provided similar control.

- Mixtures of Dipel ES + Decis provided better control than Decis alone where pressure was low but resistance levels were high.

Under moderate to high pressure and resistance, Dipel ES + Decis 2.0 + 2.0 provided superior control than 2.0 + 1.5 L/ha or Decis at 3.0 L/ha. Conclusions and Recommendations

CONCLUSIONS AND RECOMMENDATIONS

A. The workshop succeeded in uniting the interests of scientists, policy makers and extension specialists and which aim to the necessity of reducing the use of pesticides in developing countries, through the intensified implementation of microbial control agents and particularly Bacillus thuringiensis. Efforts are essential to stimulate and co-ordinate the activities in this domain.

B. Some developing i o_untries have developed policies for crop protection based on the application of Bacillus thuringiensis and technologies for its production.

C. The roundtable discussions (directed by Dr. Dulmage) that took place following the workshop presentations of different scientists have led to the

following :

Five task forces were created, with members drawn from public sector research institutions in developed and developing countries, from two of the principal western-based producers (Abbott Laboratories and Ecogen Ltd., both of the US) and from a government-financed production initiative in China. The following areas were agreed on :

1. Feasibility Studies of B.t. Production in Developing Countries

Metnbers : Drs. O. Morris (Canada), M.S. Foda (Egypt), L.E. Padua (Philippines), E. Urquijo (Mexico), Xie Tianjian (China), R.A. Fusco (Abbott). A number of research institutions, including those benefitting from IDRC support, are experiencing difficulty in having the promising results they obtain in laboratory and pilot-scale field tests translated into products available to small farmers. These institutions generally lack experience in the planning and contracting (much less the execution) of realistic studies on the economics of production, on the potential domestic and international market, and on the various options for drawing on national, regional and/or international capital and/or expertise to exploit the developed technology. Dr. Egial Rached outlined the need for such studies, Earlier private conversations had confirmed, there is a recognition by many workshop participants of the importance of competent and independent studies of this kind.

- 331 -- 2. Facilities of Registration for B.t. Products

Members : Drs. K. A. Jones (U. K.), R. A. Fusco (Abbott), K. Jayaraman (India), F. N. Zaki (Egypt).

In some jurisdictions, such as the US, registration requirements for B.t. products have been significantly relaxed in view of their relative innocuousness to health and the environment. This means that the registration process is both less costly ($ 0.5 million in the US vs $ 30 million for a new synthetic pesticide, according to one industry representative) and quicker for B. t. products, with important consequences for their competitiveness and the speed with which they can reach the market. Some participants saw the failure of other countries, both developed (notably Canada) and developing, to ease registration for B. t. as an important constraint to wider use.

3. Standardization of Quality Control

Members : Drs. M. de la Torre (Mexico), O. Belhadj (Tunisia), R.A. Fusco (Abbott), H. S. Salama (Egypt), S. Pantuwatana (Thailand).

With the proliferation of B.t. products, the former international standard is becoming a less useful guide to their potency. Based on toxicity to a particular species (the wax moth), the system of International Units made sense when the great majority of products employed a particular B.t. strain (1113-1). However, new products are based on other strains or sub-species that are effective against a range of Lepidoptera as well as some Coleoptera and Diptera but that may have limited toxicity against the wax moth. The representatives from Abbott and Ecogen claimed that the pesticide industry in the US had already agreed on standards and that there was no need to reinvent the wheel. Developing country participants replied that these standards were not necessarily relevant to their conditions and that industry associations, such as GIFAP (Groupement International des Associations Nationales des Fabricants des Pesticides) at the international level, were not the ideal fora in which to resolve the matter as Third World producers, now or in the future, may not be pesticide companies.

4. Sensitization of Public Opinion Regarding B.t.

Members : Drs. K. Jayaraman (Indian), R.A. Daoust (Ecogen), S. Pantuwatana (Thailand), S. Salem (Egypt).

Some participants felt that lack of familiarity with B. t. and its advantages

- 332 - at the user, extension and policy levels is impeding adoption. Farmers may abandon these products when they observe insects living for several days after treatment, not recognizing that they cease feeding almost immediately. Decision-makers in government may be misled by short-term economic evaluations focusing on B. t. 's efficacy at the level of the field and farm that do not factor in its benefits in conserving natural enemies, avoiding the creation of secondary pests, reducing dangers to non-target organisms, workers and bystanders and improving ground water and food quality.

5. Integration of B.t. in IPM Programs and Relations with Relevant Organizations

Members : Drs. Aboulela, A.R. Hamed (Egypt). Some participants pointed out that the benefits derived from B.t., and their sustainability, can be increased when it is promoted as part of a programme of integrated pest management that seeks the best mix of practices in a given context. The particularities of B. t. can be more efficiently communicated within a wider framework of this sort. However in working in this and other areas it is vital for the Working Group to avoid duplicating the activities of other groups concerned with B. t. , notably the International Organization for Biological Control.

There is certainly potential for competition in terms of B. t. production among developing countries and between them and multinational or -developed country firms. (participants from all sides were cautious about disclosing details of their production processes, though the essentials of the technology are well known). There are nontheless good prospects for cooperation both between developing countries and between individual national programs and the national and international private sector. IDRC may be well placed to play a role in catalyzing certain of these relationships with a view to ensuring both that technology whose development the Centre has helped support is fully exploited and that the benefits developing countries and the rural poor draw from it are maximized. Michael Loevinsohn (Consultant in Applied Ecology - Butane - Rwanda) presented the following report's based on the roundtable discussions

B. t. 's Prospects in Developing Countries : Bacillus thuringiensis has considerable potential as an element in integrated pest management systems appropriate to small farmers. In terms of immediate efficacy in reducing pest numbers, it is often comparable and sometimes superior

- 333 - to synthetic insecticides ; its potency can be enhanced by selection and creation of strains with greater toxicity to target insects, delivered in customized formulations. The discovery of B.t. subspecies effective against Coleoptera (beetles) and Diptera (flies and mosquitoes) increases the range of pests against which specific products can be devised.

B. t. 's advantages over conventional pesticides are clearer when evaluated over a longer term and a wider scale. Because of its low toxicity to predators and parasites, it does not give rise to "resurgence", an explosion of pest numbers after their initial decline following treatment. Nor, for the same reason, does it lead to the creation of secondary pests, that had previously been contained by natural enemies. B. t. is of negligible toxicity to human; and vertebrates in general and its toxins are not magnified through the food chain. Particularly in situations where farmers find themselves caught on a "pesticide treadmill", the introduction of B.t. may prove an important initial step in breaking the addiction, allowing natural controls to reassert themselves.

It must be remembered that B.t. is itself an insecticide and may do little to attack the ultimate causes of pest abundance. Resistance to its effects has been reported in areas where it has been extensively used and where the ecological and agronomic conditions allow pests to breed throughout the year, as in some of the vegetable producing areas of Southeast Asia. Very little appears to be known at present about this resistance and, critically, to what extent it is strain-specific. It seems clear how, ver, that to conserve its utility, B. t. must be employed judiciously and in combination with other measures, particularly cultural controls that reduce the potential for pest multiplication.

Despite its advantage, the use of B. t. remains modest, accounting for some $ 300 - 500 million annually world-wide. The specificity of individual strains means that a number of products must be developed in order to have a broad impact on the market. This may be commercially feasible given the lower costs of development noted earlier, but conventional pesticides still often have an edge 'in price. B. t. has been most successful where insect resistance to conventional pesticides has reduced their profitability, as in Southeast Asia with the diamondback moth, and where environmental concerns have overridden the price differential, as in Canadian forests with the spruce budworm. Government policy clearly plays an important role : measures such as environmental taxes that would cause the external costs of conventional products to be reflected in their market

- 334 - price would prove a significant boost to B.t. and other bio-pesticides.

Yet even without such shifts in policy, B.t. may be made more competitive in developing countries through local production. Reduced delays between manufacture and use would alleviate some of the problems of deterioration that have been noted with imported products. Shortened lines of supply may also make it possible to market liquid formulations which, though not as stable as powders, avoid the costly process of drying. The Chinese participants at the workshop reported that they are able to sell their liquid products cheaper than conventional pesticides. A preliminary economic analysis that Jerry Rowe carried out several years ago for the IDRC-supported project in Nicaragua concluded as well that a liquid formulation could be marketed at a lower price than imports, whether synthetic products or B. t. wettable powders. Production costs can be lowered further by making use of certain agricultural and industrial residues as nitrogen and carbon sources in the fermentation.

Local production of B.t. would have benefits in terms of import substitution - conventional pesticides are often a significant drain on foreign exchange - and in generating employment. The fermentation techniques and equipment used in manufacturing B.t. are shared at least in part with other industries (antibiotics, rhizobium and yeast production) that are already operating in many developing countries. However, as considerable evidence attests, there are numerous ways that production can go wrong and avoiding these pitfalls requires careful process monitoring and quality control. Efficient and internationally competitive production may require joint ventures with enterprises in the North and possibly the South that have already developed expertise in the field.

Another strategy for exploiting B.t. is to transfer the genes coding for its toxins either into crop plants themselves or into associated micro-organisms (e.g. Rhizobium, Azotobacter, Pseudomouas, blue-green algae). Many laboratories in developed countries and increasingly in developing countries are pursuing such approaches with a growing number of plants. A widely voiced concern is that in these new hosts the toxin genes will be expressed continuously, even when insect pests are present at very low density, speeding the evolution of resistance, as well as needlessly diverting photosynthates from the economic parts of the plant. Some attempts are being made to ensure the genes are expressed only in the tissues at risk of attack and only after being "switched on" by insect feeding. It seems doubtful at present, however, that the measures being tried would permit a

- 335 - deployment of the toxins sufficiently selective to be considered "judicious" in the terms of integrated pest management.

Another drawback is that transformed plants rely on the toxin genes alone, whereas in nature the bacterial spore often plays an important synergistic role. However, from the Centre's perspective, a more serious concern with the genetic engineering approach is that it is being applied primarily to high value commodities of which farmers regularly purchase seeds. Its impact on the crops grown by poorer farmers whose seed is conserved from season to season is likely to be limited. Inserting the toxin genes into appropriate germplasm and assessing its performance under station and field conditions is also likely to be time consuming and would permit less flexibility as to strain than is possible through fermentation.

Priorities for IDRC Activities

The amount of support IDRC should allocate in future to B. t. relative to other aspects of IPM and how the Centre's activities in the pest management area should be coordinated will be considered in a later report ; here, I indicate what I see as some of the priorities in relation to B. t. in particular, in whose development the Centre has been involved for 6 years through two multi-recipient projects.

1. In light of what was said in the last few paragraphs, it would seem justified to put greatest emphasis on supporting B. t. 's production via fermentation, the approach, the Centre has thus far been following, rather than through the genetic engineering of plants or their associated micro-organisms. However, developments in this latter area should be followed carefully, particularly in terms of how they affect the concerns that have been described above.

2. The key constraint to wider utilization of B. t. that has emerged from Centre-supported projects is the difficulty in moving promising research into production. B. t. has for many years been produced commercially in China and

Cuba, with public financing ; in India, a joint venture between Anna University and a local company is actively developing two Bacillus pesticides.

IDRC should consider supporting two types of study that might prove of great benefit to institutions intent on developing B. t. for particular applications but which may have little in-house experience with technology transfer at this scale. The first would focus on the shared problems of a few, specific countries : it would describe the various options available for commercial production, examining their advantages and disadvantages and suggesting the best ways of pursuing them. The

- 336 - reasons for past difficulties in having technology exploited might also be considered. Options that should be examined include : (i) selling the developed technology to a company already engaged in production, most likely abroad ;

(ii) attempting to interest a company in a joint venture involving local

production ;

(iii) collaborating with a production project underway elsewhere in the region.

The Chinese approach of publicly-financed production based in or spun off from the research institute might also be examined, but would seem an unlikely prospect in developing countries at present. Another possibility suggested at the workshop is the creation of a regional production facility in Southeast Asia that would draw on research in several countries, with financing from private, national and international sources.

Both the Abbott and Ecogen representatives indicated their companies' interest in pursuing joint ventures, while Dr. Xie Tianjian of China (whose institute is collaborating with the Goodgene Corporation, Taiwan, in international trade) suggested that his facility might be used to produce B. t. formulations developed in Thailand, the Philippines and Malaysia. In both cases, local marketing expertise might well have to be supplied by a third partner. An international study could help clarify these options and suggest to individual institutions the kind of information (e.g. market studies, process design) they should bring to negotiations in order to secure the best deal. The assistance of UNIDO and possibly other international organizations may prove useful in planning and executing a study of this sort.

The second type of study, possibly integrated with the first, would focus on project-level feasibility. It would involve gathering the information that investors

and collaborators require : a model of the production process, the economics of production and the size and characteristics of the market. While international assistance may be required for certain aspects of these studies, expertise available within the country and opportunities for South-South collaboration should be tapped wherever possible. In particular, the SPIC Bioprocess Laboratory at Anna University's Centre for Biochemistry, Madras and Mayra de la Torres' group at CINVESTAV in Mexico City might be able to assist in relation to process design and modelling.

- 337 - IDRC should await the conclusions of the Working Group's task force on feasibility studies before launching any activity in this area. It would of course be pointless to commission such studies without an indication that they are truly of interest to the countries involved, that there is a real commitment at the research institutions and beyond to seeing B. t. technology exploited commercially. However, some preliminary contacts e.g. with UNIDO might be made immediately.

3. The working group identified the failure of some governments to recognize the innocuousness of B. t. products and to allow them quicker and less costly registration as an important impediment to wider use. However, there are some legitimate grounds for caution as regards B.t. Firstly, a number of new products have been developed using genetic engineering and hence contain novel organisms whose release into the environment should not be permitted lightly. Secondly, some B. t. strains are known to produce an exotoxin that is a potent inhibitor of RNA transcription and whose effects, unlike those of B.t. 's endotoxin, are anything but specific. In both Canada and the US, registered B. t. products cannot contain ail any exotoxin.

Pesticide regulatory agencies in many developing countries are technically weak and have difficulty enforcing their decisions. This is a major reason for the wide ranging problems observed with synthetic pesticides. Interventions aimed at improving the efficiency with which these agencies process the registration of B. t. products should also bolster their capacity to evaluate risks in a balanced and informed manner and to act effectively on their judgements. Again, IDRC should await the recommendations of the Working Group task force, but it may be able to play a useful role by making available regulators from agencies in developed countries to assist in reviewing the assumptions and procedures used in making regulatory decisions.

The introduction of genetically-engineered plants containing B. t. endotoxins may pose a problem of another sort. As noted earlier, one consequence may be a more rapid spread of resistance in pest populations, which could undermine the effectiveness of B.t. produced through fermentation. In Egypt for example, a B.t. toxin-containing cotton plant might hasten the evolution of resistance in Spodoptera, which would compromise the efforts of the NRC to produce B.t. formulations against the same insect when it attacks oilseed crops and which could also be used to protect maize and vegetables from this polyphagous pest. The susceptibility of insect pests to B.t. can be considered a common property resource that must

- 338 - not be squandered. It is not clear how such hazards can best be regulated - at the moment the only opportunity may be when the Ministry of Agriculture decides whether to approve release of the new crop variety. IDRC should seek to ensure that the issue is considered by the Working Group and the projects the Centre supports.

4. While the project at the Egyptian NRC has focused on the control of pests of oilseed crops, the financial viability of the technology it has developed will be enhanced if the B. t. formulations are shown to be effective as well against these same pests attacking other crops. As indicated above, Spodoptera is a wide-ranging insect and there is likely to be a greater market for B.t. products against it on cotton where pesticide use is currently far greater than on oilseed crops. The Centre should encourage and if necessary facilitate wider field testing of the products on cotton and other crops.