Novel Biopesticides

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National Institute of Science Communication(CSlR) Dr. KS. Krishnan M;arg New Delhi 110012

Novel Biopesticides M.V. Deshpande ©National Institute of ScienceCommunication (CSIR) First Edition:July 1998 ISBN:81-7236-186-6

CSIR Golden Jubilee Series Publication No. 23 Series Editor

Dr Bal Phondke

Volume Editor

Dr Sukanya Datta

Cover Design Illustrations

Pradip Banerjee Sushila Vohra, Neeru Vijan, Malkhan Singh, Mohan Singh, Yogesh Kumar

Production

Pamila Khanna, Seema, Rohini Raina, AshokKalra

Printing

Jawahar Lal, Sudhir C. Mamgain, Gopal C. Porel, Tika Ram, Rattan Lal, Om Pal

Designed, Printed and Published by National Institute of Science Communication (CSIR) Dr K.S. Krishnan Marg, New Delhi 110012

Price: Rs 30/- '

The Council of Scientific & Industrial Research (CSIR), established in 1942, is committed to the advancement of scientific knowledge, and economic and industrial development of the -country. Over the years CSIR has created a base for scientific capability and excellence spanning a wide spectrum of areas enabling it to carry out research and development as well as provide national standards, testing and certification facilities. It has' also been training researchers, popularizing science and helping in the inculcation of scientific temper in the country. The CSIR today is a well knit and action oriented network of 41 laboratories spread throughout the country with activities ranging from molecular biology to mining, medicinal plants to mechanical engineering, mathematical modelling to metrology, chemieals to coal and so on. While discharging its mandate, CSIR has not lost sight of the necessity to remain at the cutting edge of science in order to be in a position to acquire and generate expertise in frontier areas of technology. CSIR's contributions to high-tech and emerging areas of science and technology are recognised among others for precocious flowering of tissue cultured bamboo, DNA finger-printing, development of non-noble metal zeolite catalysts, mining of polymetallic nodules from the Indian Ocean bed, building an all-composite light research aircraft, high temperature superconductivity, to mention only a few. Being acutely aware that the pace of scientific and technolozical development cannot be maintained without a steady influx of bright young scientists, CSIR has undertaken a vigorous programme of human resource development which includes, inter alia, collaborative efforts with the University Grants Commission aimed at nurturing the budding careers of fresh science and technology graduates. However, all these would not yield the desired results in the absence of an atmosphere appreciative of advances in science

and technology. If the people at large remain in awe of science and consider it as something which is far removed from their realms, scientific culture cannot take root. CSIR has been alive to this problem and has been active in taking science to the people, particularly through the print medium. It has an active programme aimed at popularization of science, its concepts, achievements and utili»" by bringing it to the doorsteps of the masses through both print and electronic media. This is expected to serve a dual purpose. First, it would create awareness and interest among the intelligent layman and, secondly, it would help youngsters at the point of choosing an academic career in getting a broad-based knowledge about science in general and its frontier areas in particular. Such familiarity would not only kindle in them deep and abiding interest in matters scientific but would also be instrumental in helping them to choose the scientific or technological education that is best suited to them according to their own interests and aptitudes. There would be no groping in the dark for them. However, this is one field where enough is never enough. This was the driving consideration when it was decided to bring out in this 50th anniversary year of CSIR a series of profusely illustrated and specially written popular monographs on a judicious mix of scientific and technological subjects varying from the outer space to the inner space. Some of the important subjects covered are astronomy, meteorology, oceanography, new materials, immunology and biotechnology. It is hoped that this series of monographs would be able to whet the varied appetites of a wide cross-section of the target readership and spur them on to gathering further knowledge on the subjects of their choice and liking. An exciting sojourn through the wonderland of science, we hope, awaits the reader. We can only wish him Bon voyage and say, happy hunting.

re Competition for food between humans and plant pathogens is as old as agriculture. Day by day, the problem is becoming more severe because of urbanization. The chemical warfare against pathogens has a much shorter history. Their rapid action, low cost and broad spectrum have brought chemical pesticides into limelight very fast. However, incessant use of chemicals in agriculture creates serious health problems in humans. A more dreadful situation that concerns us all is th~ building up of toxic residues which disturb soil ecosystems. In 1962, Silent Spring signalled the beginning of a "biological control" era. Hasty generalization and too much expectations of the farming community have retarded the speed of the success and commercialization ofbiopesticides. Hopefully, their selectivity and specificity, low cost of production and environmental friendliness will make biocontrol agents successful in the near future. Researchers are identifying newer targets for the control of plant pathogenic fungi and insects. One of the favoured targets, discussed in the book is chitin, a structural polymer of fungal cell wall and insect cuticle. Its absence in vascular plants and mammals make the target agents relatively nontoxic. A two pronged attack on chitin is suggested. Chitin synthesis inhibition and/ or degradation stops the proliferation of the pathogen and can save the crop. Above all, the controlling agents developed against this target can very well work in the integrated pest management programme. Finally, I would like to quote a very effective analogy given by Rachel Carson in Silent Spring. "We stand now where two roads diverge. The road we have long been travelling is deceptively easy, a smooth super highway on which we progress with great speed but at its end lies disaster. The other fork of the road - the one 'less . travelled by' - offers our last, our only chance to reach a destination that assures the preservation of the earth".

Initial efforts to write a popular article in NCL Bulletin were encouraged and supported by Dr. K. R. Srinivasan and Mr. H.B-Singh, the then editors of our house magazine. Mr. S.K.Nag and Dr. Sukanya Datta, NISCOM were instrumental in crystallising the thought of writing this book. I am thankful to all of them for their help in making my entry in this fascinating world of popularizing science. My contact with the farmers was Drs. A.S.Patil, VSI, Pune and M.S. friendly discussions with them on the ers have certainly given me the feel pathogen control.

established through Gaikwad, NCL, The problems of the farmfor the need of plant

The best way to convey the message is through cartoons and pictures. My brother Mr. Govind Deshpande , Mr. Salil Lachke, my student, and Mrs. Iyoti Pathak, NCL have made the book delightful. Thanks are due to my students, Mrs.Vandna Ghormade,·Ms.Aradhana Amin and Ms.Manisha Chitnis, who have tried to bridge the gap betw~en popular science and research by way of valuable suggestions. Dr. M.C.Srinivasan, Scientist Emeritus and Dr. Paul Ratnasamy, Director, NCL, Pune have given rightful direction and placeto my project in the laboratory. My gratitude is due to them. Last but not the least, my thanks to the funding agencies, DST and DBT, New Delhi for their support to carry out research on chitin metabolism and its role in plant-pathogen interaction.

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Trees, fields and towns Common Glossary Executing Bio-protection the enemy in fje1ds Bugs againstbond bugs

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Trees,

fields and towns

he most important innovation in the structuring of human society happened as early as in 10,000 BC when man started growing plants in the field. This new life-style saved a lot of the energy and time spent in hunting for food. Humanity thus progressed from living in trees, to openland and eventually, settlements There are two starting points of human civilization. Southwestern Asia (near east) and Mesoamerica (northern Mexico to northern South America) are the two regions where agricultural societies domesticated wild b~lrley,wheat and legumes for the first time. In time, farmers from the near east migrated to western Europe. But nowhere in eastern North America did agriculture start until 2000 BC, when it was introduced initially as a part-time occupation for womenfolk. Soon the demand for food by urban populations exceeded the production capacity of the farmers. Three solutions offered themselves for meeting the increasing demand for food: one, to bring more land under cultivation; two, to increase the output from existing land under cultivation and three, bette.r utilization of the food produced. The best approaches to achieve these goals were im-

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NOVEL BIOPESTICIDES

How would man meet increasing demand for food

proved cultural practices, greater use of fertilizers and protection of crops from pathogens. With the advent of agriculture, plant diseases became a problem. Farmers grow one kind of crop over large areas of land. This means that pests literally have a field day. It thus necessitates great care in restricting the pathogen's entry in the field, otherwise it can wipe off entire crops, causing widespread famine. Literally tens of thousands of insects,

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fungi, viruses, bacteria, nematodes and other living forms are potential hazards to agricultural crops, forests and animals. One big nuisance in the agricultural fields is weeds. A weed is a plant that grows where it is not wanted. Weeds clog waterways, destroy wild life and hamper farming. Plant pathogens are commonly known as pests. The attack of fungal pests in crops is mentioned in some of man's earliest writings. Fungal diseases like rust and smut in wheat were reported in the Bible as curses for disobedience of the commandments. Theophrastus, follower of Aristotle and the father of botany portrayed a number of crop diseases quite accurately. The culprit behind the great Irish Famine of 1840s, was a fungus which attacked potatoes and caused them to rot. The disease spread rapidly throughout the country. As a result, one million people died and two million people migrated to other countries. Today, the control of plant diseases constitutes a broad, highly technical and rapidly developing field of study. Many aspects of plant development are plastic and environmentally responsive. This is true for resistance to pests. Most of the crop varieties can be divided into two groups. They are either susceptible or else, resistant to pathogen attack. Upon recognition of a pathogen, resistant plants utilize a large arsenal of defence mechanisms to protect themselves. These include enzymes and chemicals to kill the pathogens. The modem tendency for large farming units that are devoted to growing only a single species of crop increases the need for effective disease control as the spread of airborne diseases is encouraged by such uniformity. To counteract this, the cultural practice is crop rotation. This involves selection of a crop which is not susceptible to the prevailing pathogen. Other measures are burning of infected debris and removal of weeds which sometimes act as second hosts. For pest-susceptible species, there is no choice other than employing chemicals. Comparatively recent pest-control

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NOVEL BIOPESTICIDES

Germinating spore of late blight fungus (top) Potato affected by late blight

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technology uses pesticides. Agricultural pesticides are organic or inorganic chemicals. Most of the inorganic products are based on elemental sulphur or various salts of copper. As early as in 1878, P.M.A.Millardet formulated a spray mixture called Bordeaux. It contained copper sulphate and powdered limestone. Initially, this blue coloured mixture was applied along the road-side edges of vineyards to discourage

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thievery of the grapes. Fortuitously, it CJ.lso proved to be useful in controlling downy mildew on grapes, a disease i~ which a fluffy fungal growth on the lower surfa~e of leaves causes yellowing. Downy mildew also drastically affects yield of the grapes. During the 1930s, organophosphorus compounds like parathion and schradan were first developed as pesticides by Gerhard Schrader in Germany. Interest in them was generated because of their swift paralysing action on insects. An added advantage was that the residues which remained in the soil were not toxic. This' was a boon from an ecological point of view. Broad spectrum organophosphrous insecti-

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cides, marketed under the trade name parathi~n are used for quick action. Schradan and its family members, (compounds of same chemical nature) are used for selected purposes. Schradans kill sucking insects like aphids, whitefly and leaf hoppers, but caterpillars and beetles, which are chewing insects can survive'schradans. These chemicals are very toxic to mammals. Anew group of insecticides, the carbamates, came into the picture in the late 1940s. Carbamates are highly poisonous to aphids and house flies but possess limited toxicity toward other insects. Such a narrow spectrum of toxicity restricts commercial interest in them. Of all the synthetic organic insecticides, DDT is the best known and the most widely discussed pesticide. In 1873, O.Zeidler synthesized 2,2-bis (p-chlorophenyD-l,l,l-trichloroethane. Today, the chemical enjoys global recognition as DDT. The name is derived from its chemical composition. Because of its.molecular configuration it was called dichloro diphenyl trichloroethane, or DDT for short. But almost 70 years were to go by before Dr. Paul Mu.ller first discovered the insecticidal property of DOT. Until the 1960s, DDT occupied a significant position in the field of public health, especially for malaria control. In agriculture, DDT was used on more than three hundred different agricultural commodities as a general insecticide. Unfortunately, it affected the nervous system of animals. As a result DDT, the favourite weapon of exterminators, had to be replaced by other synthetic organic insecticides. However, the introduction of new insecticides was not without consequence. The indiscriminate use of chemicals created serious problems by causing health hazards. The International Development Research Centre (IDRC), Canada reported that, about 10,000people die every year and another 400,000 suffer from a variety of effects of pesticide poisoning. Most of these casualities are in the developing countries. Blindness, asthma, cancer, skin disorders, enlargement of

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NOVEL BIOPESTICIDES

liver, neural malfunction and to some extent even psychological problems are the commonly encountered results of exposure to pesticide residues. In India, about 25% of the cultivated crops are protected by pestk;des. However, in spite of the growing use of pesticides, the annual crop loss due to insect pests has not been contained. On the contrary, the losses are increasing. During

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Pests account for huge crop loss

the 1950s, pesticide consumption in India was a meagre 2000 tonnes per year. In 1990, the figure stood at 80,000 tonnes per year. Unfortunately, (though this was still inadequate to protectthe farmer's interests) it led to a backlash of another kind. The World Health Organization (WHO) recently recorded that chlorinated pesticides like DOT and benzene hexachloride (BHC) were increasingly being used in India. Therefore, food commodities, such as wheat, rice, and pulses, showed high residual contents. During the last decade, the daily Indian intake of DOT was 0.27 mg per person and the level of accumulated DOT in the body was 12.8 - 31 parts per million (ppm). A high level of pesticides in the body is not a healthy sign. However, a more dreadful situation that concerns us all is the build-up of toxic residues which disturbs the activities of soil microorganisms. Under normal conditions various microbes . make atmospheric nitrogen available to plants. This process of nitrogen fixation is hampered by the chemicals applied for

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NOVEL BIOPESTICIDES

crop protection. DDT and BHC especially have longer lasting detrimental effects. Even a seemingly moderate use of insecticides over a period of years may build up alarming quantities in the soil. The old saying that" a pound of DDT to the acre is harmless", means nothing if spraying is repeated over the years. Since DDT has enjoyed a long innings, over the years, the DDT content in agricultural fields has reached the level of 7 - 25 kgs of DDT per acre. Naturally, consumers would like to know just how much of this would ultimately be absorbed by the plants. The answer is not easy to give as the rate of uptake varies from plant to plant. Carrots are known to absorb more insecticide than any other crop. The consumers would then, no doubt, wish to know just how much of the insecticide in the plants would find its way into their bodies. It is a pertinent albeit vexing question to which scientists have for long paid close attention.

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In 1962, Silent Spring a book by Rachel Carson signalled the beginning of an era of intensive scrutiny of both the benefits and the risks of chemical pesticides. Her graphic portrayal of the hazards of these materials sensitized the public to this potential problem. At the same time, the "antipollution" movement was developing against toxic-waste dumps of pesticides. And a little over two decades later the Bhopal tragedy of 1984 ultimately served to underscore the hazard potential of chemical pesticides to even the most casual observer. The realization of potential dangers significantly affected the pesticide industry. The registration process for agrochemicals became more strict. Yet the fact remains that, the pesticide industry has not fully accepted a mandate to develop alternative products. There are several reasons for this slow response. The first is the very strong inclination to save their investment on inventories, process development and marketing. A minimum of 7-10 years of inputs are necessary to bring a new environment- friendly product into the market. So. not only is it an expensive proposition to junk existing infrastructure, research and development results and existing chemicals, it is also a time consuming proposHkm to launch a product that does not conform to the tried and tested formulae. But the writing is on the wall. It is time to rethink because more than 400 different insect pests are known to have developed pesticide resistance. Sooner or later, the conventional chemical pesticides will have to be replaced. For example, in the late 1960s, the insect pest on cotton crop in Mexico became tolerant to organophosphate insecticides. Consequently, the cotton industry in that region was ruined. Cotton acreage declined from 700,000 acres to about 1000 acres in a matter of 2- '3 years and the allied textile businesses went bankrupt. Eventually, in the late 19708pyrethroid, a new insecticide, gained phenomenal support from cotton growers. Alas! in a

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NOVEL BIOPESTICIDES

Pest damage to cotton (inset)

short span of 5-7 years the cotton bollworm, Heliothis arnzigcrrI developed resistan{:e to pyrethroids. Various figures reported from Australia, Thailand, and USA revealed that even ;noderate amounts of resistance led to increased costs of global insect control by 40% or nearly 240 million dollars. In 1987, farmers from various parts of India, particularly Andhra

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Pradesh faced a similar problem not only in the control of cotton bollworm, but also in the staple crops of pigeon-pea and chick-pea. Out of a total 529 000 ha land, the cotton crop from 286 000 ha land in the coastal districts of Andhra was ruined by H. armigera. Similar incidences have been reported from other parts of the world. In Taiwan, bedbugs used to nonchalantly carry a deposit of DDT powder on their bodies. DDT-resistant flies, possess enzymes for detoxifying the DDT that enters their bodies. Given the rather depressing scenario of pesticide tolerance, the common man is justified in asking, are there means to displace chemical pesticides from the current agricultural practices? The answer, thankfully, is a reassuring 'Yes'. Some of the most fascinating of the new methods are those that seek to turn the strength of a pest species against itself. That- is, those that use the drive of an insect's life forces to destroy it. The most spectacular approach is the male sterilization technique using radiation or chemicals. The chemicals tested fall into two groups. The first one consists of chemicals which mimic some of the chemicals found in the insect's body as a result of its physiological cycles. However, the mimics cannot carry out the functions of the natural molecule and thus misguide the insect. The second group consists of chemicals that act on insect chromosomes. Sound is also being tested as an agent of direct destruction. Ultrasonic sound can kill all mosquito larvae in a laboratory tank. In nature, it will kill other aquatic organisms also . . However, such experiments are only the first step towards a totally new concept of using electronics for insect control. RachelCarson introduced the concept of biological control methods by providing a very effective analogy. In Silent Spring, she.says,"We stand now where two roads diverge. The road we have long been travelling is deceptively easy, a smooth superhighway on which we progress with great

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speed but at its end lies disaster. The other fork of the roadthe one "less travelled by"- offers our last, our only chance to reach a destination that assures the preservation of the earth". We can only exclaim, 'how true'. It is gratifying that in the nineties at least, scientists are exploring multipronged approaches to pest control. Higher plants are considered as the reservoir of effective drugs that can provide valuable sources of useful biopesticides. They are largely non-phytotoxic which means they will not harm plants, systemic and easily biodegradable. Extracts of onion, garlic, eucalyptus and tobacco, are reported to control many plant- pathogenic fungi and insects. There are many more plants awaiting a chance to help man in his war against pests. Pyrethrum is one of the oldest insecticides known. It was used in Iran (Persia) in 400 B.C. Finely ground,

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dried flowers of chrysanthemums were marketed as "insecticides". Today it is known that their efficacy was derived from the active ingredient pyrethrin. Pyrethrins have a rapid paralytic action on insects, which is usually reversible if fatal doses have not been administered. Although a number of plants produce metabolites which are active against insects, the ultimate natural arsenal seems to have been bestowed on the neem tree. It thrives in arid regions and is widely grown in India, Burma and Africa, as a fast-growing source of fuel wood. For centuries, neem leaves and seeds have helped people control insects in the tropics. In India, woollens, quilts and blankets are routinely stored with dried neem leaves to discourage insects during the summer months. Grain was also traditionally kept pest free by using neem leaves. Now we know the science behind grandmother's wisdom. Amongst a host of constituents, a number of triterpenoids, flavonoids, amino acids, and sulfur containing compounds, have been isolated from various parts of the tree. Azadirachtin, which has a disruptive effect on the feeding and growth of insects is a key constituent of the neem seeds and leaves. A non-toxic insect repellent, thioremone, is produced in the seed kernels. It is suggested that seed-extracts are highly effective against more than 100 species of crop pests, including the gypsy moth, Mexican bean beetle, confused flour beetle, citrus mealy bug, navel or orangebug, striped cucumber beetle, Japanese beetles, aphids and tobacco budworms. Initial experiments by American scientists had shown that Japanese beetles would rather starve than eat some of their favourite plants that had been treated with neem. Neem-formulations are mostly non-toxic and biodegradable. Margosan-O is the first neem seed-preparation approved by the US Environmental Protection Agency for its use in crop protection. The added incentive for using neem seed cake for pest control is its nutritive value f6r crops. It has

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very high contents of nitrogen, phosphorus, potassium, calcium and magnesium and therefore can double as a fertilizer. In nature, no species exists alone. Not even pests. Plant pathogenic fungi, insects and nematodes have various relationships with co-existing microbes. These interactions are either beneficial to both the parties, or else harmful to one of them. Insects, for example, are parasitized by viruses, bacteria, fungi and microscopic worms. Herein lies the key to another avenue that may be explored in our quest for a safer, more 'natural' insecticide. Mankind's use of natural enemies for pest control dates back to the ancient Chinese, whu used ants to control caterpillars ·and large boring beetles in their citrus trees. But it was not till the 18th and 19th centuries, that

Natural enemy. The Spiny soldier bug feeds on Mexican bean beetle larvae

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the necessary biological foundations were laid for the scientific development of biological pest control. One of the first scientist to envisage use of microbes for pest control was the 19th century zoologist Elie Metchnikoff. Some microbes produce antibiotics for their own defence. These antibiotics can help man to combat viral, bacterial and fungal diseases of crops. For example, streptomycin can be used to centro bean blight caused by the bacterium, Erwinia. Soaking of paddy seeds in aureofungin helps reduce incidence of seed-borne fungal infection. This can be used as a control measure. Actinomycin D. and blasticidin, are also being roped in to reduce viral infections in plants. It is apparent that nature has provided the means to effectively control pests.

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It is equally apparent that time is running out for the chemical- insecticides. It is not possible to continue to pay the price in terms of its undesirable side effects. But then, has the fledgling natural-insecticide industry really dented the synthetic pesticide industry? Has it made an impact? The answer is - marginally. Of the total worldwide pesticide market of more than $ 20 billion, bio-based materials account for only $ 50-60 million. This works out to less than one percent. To understand the reas~:mfor such a low acceptance of microbial products, one needs to compare the merits and demerits of both types of agents. There are several reasons for low acceptance. The foremost is that the growth requirements of microbes are not cheap enough to effectively compete in the pesticide marketplace. And if the producers of bio-insectides cannot be grown cheaply, how can non-expensive products flood the market? Biodegradability for the detoxification of chemicals is a must. But for biopesticides, this leads to an inherent inconsistency in their performance in nature. Nevertheless, it is vwrthwhile to use biological agents for pathogen control.

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Merits and demerits of pesticides

Chemical

Biological

Safety

Little or none

Often high

Environmentfriendliness

Not at all

High

Activity spectrum

Can be broad

Selective and specific

IField efficaC1{

High

Environmental ersistence

High-toxic residues

Little

Moderate to hi2:h

None to low

INon-targeted action

May be high

None

Probability of pest resistance

High

Less

I Pesticides

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IGenetically manipulative User cost

Possible Acceptable Low

Icosts Development time and

Compatibility with other control

High Possible

he broad field of pest control includes not only chemical pesticides but also methods involving development of pest- resistant varieties and the use of agricultural practices which are unfavourable to a particular pest, and application of pest repellants. The strategy highlights the current and potential capitalization on the age-old rivalry of organisms among themselves to serve our purpose, i.e. to exert control over the harmful species.

Bugs Against Bugs

Most of the fights for survival started over millions of years ago in natural ecosystems. They are therefore, rather specific and less likely to produce undesirable side-effects. In 1800, Erasmus Darwin, grandfather of Charles . Darwin, in his book, Philosophy of Agriculture and Gardening hypothesized about the use of natural predators to minimize pests. However, credit for the first experimental proof for such a strategy goes to an Italian scientist, Agostino Bassi. He demonstrated the parasitism of the fungus Beauveria bassiana on the silk worm, Bombyx mori. Later other scientists proposed that microbes could be sprayed over pest infested fields. Louis Pasteur, was the first to use

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BUGS AGAINST BUGS

Fungus Beauveria bassiana

a fungus on grape vines in the vineyards to control the tiny root- inhabiting insect Phylloxera . In the mid-1800s, interest in biological pest control received a tremendous boost from the successful control of the cottony-cushion scale insect on citrus trees in California. Charles Valentine Riley of the US Agriculture Department imported the Australian ladybird or Vedalia beetle (Rodolia cardinalis) for the control of the cottony-cushion scale insect. This novel approach to save the multimillion-dollar Californian citrus industry was the beginning of modem biological pest control. It was for all practical purposes a declaration of war at a microscopic level. For the farmers it spelt good news.

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Vedalia beetle

In the living world the largest number of known species, about 800,000 are of insects, which is just 13 per cent of the estimated number. How many species exist on earth?

BUGS AGAINST BUGS

The two faces of Class Insecta. Weevil used to decimate the pest snakeweed (top) Grain weevil coming out of a grain of wheat.

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The second largest group is of fungi of which only 5 per cent species are known. In this roster, the 'known bacterial species count reaches around 5000. Among the pathogenic organisms a rough rule is that generally, bacteria are dangerous to human beings and fungi and insects to plants. So, in agriculture, the control of insects and fungi is of vital importance and much attention is being given to their control. Their natural enemies are being studied extensively in the hope that someday these natural enemies may strengthen the hands of the farmers in their fight against the pests. Viruses, bacteria and fungi can function as biocontrol agents against insect pests. Many of these microorganisms have a narrow host range. This means that they are 'choosy' or selective about the insect species they attact. Therefore, they do not randomly destroy beneficial or non-target insects.

This armyworm has been killed by virus.

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Cell nucleus of killed insect shows presence of virus.

is seen that even after 2-3 days of infection, when all other tissues are infected, the midgut region is surprisingly free of viruses. This temporary recovery of the midgut is because the midgut epithelial cells are routinely sloughed off and replaced. Thus, although a potentially life- threatening viral infection has established itself in the insect's body, it goes on eating and damaging crops. Usually, 4- 6 days elapse between the time of ingestion of virus and the death of the host. Farmers therefore, are understandably unhappy about this delayed action biopesticide. There is another inherent drawback too. These viral preparations are not stable under the

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The viral and bacterial control agents infect insects via their digestive tract while fungi make their entry into the host through the cuticle which is the superficial non-cellular layer covering plants and certain lower animals. There are more than 1600 different viruses which infect some 1100 species of insects and mites. However, it was not till the beginning of the 20th century, that it was recognised that viruses can infect insects. Today we know that there is a special group of virus called baculovirus to which about 100 insects are susceptible. This group accounts for more than 60 per cent of all insect pathogenic viruses. These are not harmful to mammals and plants. Baculoviruses are rod-shaped particles which contain DNA. Most viruses are enclosed in a protein coat to make up a virus inclusion body. An intact chitinous cuticle restricts the entry of the virus. But sometimes the cuticle may be damaged when the insect is injured. Viruses find their wcty into the body cavity of such insects quite easily. Or else, an infection cycle of the virus could begin when an insect ingests virus inclusion bodies. Alkaline conditions of insect's midgut dissolve the protein covering and the viral particles are released. These particles fuse with the midgut epithelial cells to start an infection. The virus multiply rapidly and eventually pack the body of the host. ELCAR the first commercial viral pesticide, was marketed by the pharmaceutical giant Sandoz in the 1970s. The preparations were made as wettable powder spray. However, the mass production of the virus from either infected larvae or adult and its purification is time consuming as well as labourintensive. As a result, viral pesticides are more expensive than chemical agents. Furthermore many baculoviruses are host specific. Therefore, they cannot be used to control several different pests. The action of baculoviruses on insect larvae is too slow to satisfy most farmers. This is because of a physiological process which extends the life of the virus-infected pest with unfortunate consequences for the farmer. It

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ultraviolet rays of the sun. Spraying the virus on the under surface of the leaves helps to alleviate the. problem of its degradation under ultraviolet light. Efforts are being made to encapsulate baculoviruses with UV protectants, like tinopal, aluminium powder or egg albumin to ensure a longer field-life. There are also a number of technical means being considered to compensate for the shortcomings of baculoviruses as insecticides. The foremost attempt is to improve the speed of kill by genetic engineering. Employing virus to deliver harmful proteinaceous toxin into the larvae of pests could greatly increase the efficiency of kill. The possible candidates for genetic engineering are caterpillar-specific toxin, and neurohormones. This strategy would entail/stitching' of a selected gene governing the production of the proteinaceous toxin into the genetic make-up of the virus. This genetically engineered virus would now produce the toxin along with the other proteins that it naturally makes. So once inside the insect host, the virus would not only overwhelm its system but also destroy it with the toxin. Of course, precautionary measures are taken to make the engineered virus "safe" . That is to say steps are taken to ensure that the engineered virus does not run wild in the ecosystem or attack unintended targets. Many bacteria, particularly the Bacillus species cause disease in insects. As early as in 1939, Bacillus popilliae spores were used to control Japanese beetle. The bacterial cells after being ingested by the larvae invade the insect's body cavity. The organism does not produce any toxin but by sheer number suffocates the larvae. However, the constraints of growing B. popilliae restricts its use on a large scale. The most popular bacterial pesticide is BT, a mixture of Bacillus thuringiensis spores and its toxin. As a pesticide, B. thuringiensis accounts for over 90 per cent of the total share

of today's bioins'ecticide market. The credit for discovering B. thuringiensis goes to a Japanese scientist, S. Ishiwata. He isolated the bacterium from silk worm larvae suffering from

NOVEL BIOPESTICIDES

28

Gene of choice

Genetic Engineering

BUGS AGAINST BUGS

29

Spraying BT insecticide

a disease called flacherie. A similar organism was isolated later in Thuringen, (Germany) by E. Berliner from a diseased moth. He named it B. thuringiensis after the province of Thuringen. Up to 1976, only the strains pathogenic to butterflies and moths were known. These strains were not effective against blackflies, mosquitoes and beetles. Subsequently scientists from Israel isolated a strain pathogenic to mosquitoes. Thus the strain, B. thuringiensis variety. israelensis was of immediate practical importance. Now the insecticidal preparations of this strain are being used all over the world for pest control. But then, how does the BT insecticide work? What is the secret of its efficiency? The sporulating cells of B. thuringiensis contain insecticidal crystal protein (Iep) which accounts for the commercial value of BT as a biopesticide. Actively growing cells lack the crystalline inclusions and thus are not toxic to insects. However, some strains exude small molecules of toxin which are appropriately enough, called exotoxins and

30

NOVEL BIOPESTICIDES

Scientists have 12,145 cultures of different varieties of B. thuringiensis in this American laboratory

which affect the insect host. It has been used in Russia to control cattle fly larvae. However, its use is discouraged because of its toxicity to mammals. The BT preparations remain stable over several years of storage and even in the presence of UV rays of the sun.

BUGS AGAINST BUGS

31

Reviving BT spores just takes a drop of water

However, the major drawback of BT is that the ICP acts only after ingestion by the insect. As the insect feeds on the foliage, the crystals too are eaten up. These are solubilized in the insect's midgut to produce an active endotoxin. Some B. thuringiensis strains produce a single endotoxin, while others have a family of endotoxins of different specificities. Several

32

NOVEL BIOPESTICIDES

aspects of the structure-function relationships in the crystal proteins have been tackled using molecular techniques. The ability of the individual toxins to kill the larvae of pests has been extensively investigated using molecular techniques. Sometimes the toxin is secreted as an inactive 'protoxin' that needs to be activated before it can kilL The activated toxin binds to receptor sites on gut epithelial cells and creates imbalance in the ionic make-up of the celL This is seen by the swelling and bursting of the cells due to osmotic shock. Subsequent symptoms are paralysis of the insect's

BUGS AGAINST BUGS

33

mouth parts and gut. So obviously the feeding process is disrupted. An added advantage of the BT insecticide is that it is harmless if touched. It also needs an alkaline gut to show results. This means that insects with their alkaline guts are at risk, because the human stomach is acidic. Thus safety measures are inherent to this insecticide. Today, B. thuringiensis is perhaps the best possible alternative to chemical pesticides. The most favourable characteristic of BT is the demonstrated safety of prepara~ions in over 30 years of field application around the world. However,

34

NOVEL BIOPESTICIDES

repeated exposure of an insect population to B. thuringiensis induces the emergenc of resistant pests. Treating the target pest with a mixture of toxins usually reduces the likelihood that the insect species will develop resistance to the toxins. Scientists around the globe are trying to develop better, more effective strains of the Bacillus species. Several factors influence the strain improvement strategies. The number of toxin genes, the qualitative and quantitative differences among them and the properties of the resulting toxin, affect the quality of the strains developed. The range of commercial products include Thuricide, Dipel, Bug Time, and Bathurin. Apart from virus and bacteria, fungi also affect insects. Many insects have fungi in their guts, use fungi as food or are attacked by fungal pathogens. Man's association with insectpathogenic fungi is also almost equally old and varied. :\

!

11\

!li!:!1 III I:I,:!; ': !;!. :111

I

iIi I i,I.

: "

: : j~

1;1:!

:(I~I'I'Iil'(/~~';j;: . .""

'I ; I

'"

1\

I

\.

~

I \V~·

,-'

.~

Jr}

('J .\(~~tF,'~

\t!~\,'~;~(

Louis Pasteur first recognised the potential of fungi for insect control

BUGS AGAINST BUGS

35

The potential of fungi for insect control was first recognised by Louis Pasteur. Two Russian scientists E. Metchnikoff and J. Krassilstchik used Metarrhizium against two species of beetles, the wheat chafer and sugarbeet carculio respectively. Shortly afterwards, in USA, F. Snow made an attempt to control chinch bug using fungi. In both the cases, success was sporadic and the blame was placed on the changing weather. Thus even before the advent of chemical pesticides in the 1940's, the use of fungi for pest control had been tried albeit with only marginal su,ccess. Over 400 different species of fungi have been shown to parasitize living insects. The host range varies with the fungus species concerned. For example, M. anisopliae can parasitize as many as 200 different insects. An interesting use of M. anisopliae has been discovered by scientists. Termites are notorious pests. From a scientific and practical perspective, a fungus based pesticide targetted at termites can be developed using M. anisopliae. It would be interesting to know how far this fungal anti termite compound could work in the fields. Most fungi that attack insects first make contact with the host in the form of spores. A few fungi have slimy conidia while some species use sticky mucus for attachment. A few insect pathogens, such as M. anisopliae and Beauveria bassiana produce very dry spores. They have special components for adhesion to insect cuticles. Once it attaches to the host, the fungus penetrates the insect body wall with the help of hyphae produced from the spores. The invasion of the hyphae in the cuticle is through wounds, joints between segments or through sense organs. The physical process of penetration is accelerated by the fungal enzymes that weaken the cuticle. Once the hypha enters the body's circulatory system, a number of options are available. The cause of the insect's death is extensive fungal growth in the haemolymph and poisoning .by fungal toxin. All insect pathogenic fungi cannot be cultivated on artificial media. Therefore, fungi such as Entomophthora grylli is

NOVEL BIOPESTICIDES

36,

Ant parasitized by fungi

grown either by infecting healthy insects in the laboratory or collected from the diseased insects in the field. Fungal formulations are usually made up of growing hyphae and spores. These active ingredients are mixed with bentonite, kaolin clay or a carrier base. The forJIlulations are supplied as a wettable powder or as a dust for spraying. The fungi which are being used as fungal-insecticides in the field are: B. bassiana (trade name, Boverin), Hirsutella thompsonii (Mycar), M. anisopliae (Metaquino, Combio, Biomax, Metapol), Verticillium lecanii (Vertalec, Mycotal, Thriptal) to name a few.

BUGS AGAINST BUGS

Grasshoppers are voracious feeders (top) Grasshopper killed in the field by B. bassiana

37

38

NOVEL BIOPESTICIDES

Now, apart from insect pests, fungi too often play havoc with crop plants, so scientists have developed a policy in which they use ':l fungus to battle another. It is almost like setting a thief to catch a thief and it is one of the most promising ways to control plant pathogenic fungi. Around 50 different fungal genera cause plant infections. A number

Pears ruined by fungus (top) Pears treated with Blue mold fungus remain unaffected

BUGS AGAINST BUGS

39

of saprophytic fungi produce antibiotics, enzymes and toxins to fight the pathogens. However, although these products are known to be useful in eradicating pathogens, very few are registered as commercial agents. Therefore, efforts largely empirical, were made to identify selective biocontrol agents. Trichoderma harzianum is a potential biocontrol agent for a

number

of soil borne plant pathogens.

Application

of

Trichoderma to fields loaded with root infecting pathogens such as Rhizoctonia and Sclerotium successfully reduced dis-

Spores ofTrichodenna begin to send threads around a lettuce leaf

Thesl! coil around Rhizoctonia, burrow into it and kill it.

Fungal friends

40

NOVEL BlOPESTICIDES

ease incidences. Some of the Trichoderma species are parasitic on fungi and grow inside the hyphae of pathogens. In addition to the invasion, these fungi produce antibiotics and cell wall degrading enzymes. That means, it mounts a two pronged attack on the pathogen. Another fungus, Pythium oligandrum is effective against Verticillium damping-off diseas in sugar beet. Such farmer friendly fungus can be used as seed dressings. Before sowing, seeds are mixed with fungal spores erswell as mycelia along with a suitable adhesive so that they adhere well the seeds. Various fungi and bacteria can effectively control post-harvest diseases of fruits, vegetables and grains. For seed dressing fungi such as Trichoderma, Myrothecium, Pichia and bacteria such as Bacillus, and Pseudomonas, are used. Seedling blight of maize caused by Fusarium can be controlled by seed dressing, with Bacillus .spores. Pichia guilliermondii is highly active against citrus fruit pathogens as well as against spoilage fungi of soyabean. However, the protection does not last long. Therefore, more research is needed to increase the duration of protection. Then and only then will it see the light of commercialization. Nematodes playa significant role in the soil ecosystem. Some are parasitic on crop plants. Plant parasitic nematodes spend part of their life cycle in soil or on the root surface. The biological control of nematodes is of two types, - natural and induced. Nematophagus fungi or literally, nematode eating fungi prevent susceptible nematode species from multiplying. These fungi are naturally associated with nematodes on perennial crops. More than 150 fungal species have been isolated from cyst and root-knot nematodes. Fungi in the soil may colonize the nematode female through its natural openings or by penetrating its cuticle. Fungi, such as Verticillum chlamydosporium, Paecilomyces lilacinus and Dactylella oviparasitica attack young females. These are all opportunistic parasities, while Catenaria auxiliaris and Nematophthora species are obligate parasites on nematodes. Some charac-

BUGS AGAINST BUGS

41

Larva of root nematode (x500) burrowing into plant cell

teristics of obligate and opportunistic parasitic fungi greatly affect their biological control potential. Obligate parasites are host specific, and difficult to culture in vitro. Facultative parasites like Dactyella have wide host range and can be· cultured easily in the laboratory. Application of the nematicide, carbofuran along with a nematode- trapping fungus, Arthrobotrys irregularis gives good results in controlling Meloidogyne species on cucumber. A commercial preparation called Bioact R,using P lilacinus is being produced in Phillipines for nematode control. Once a biocontrol fungus has shown potential for disease control, production of an effective biomass becomes a major concern. Low cost and high productivity of top quality inocu-

42

NOVEL

BIOPESTICIDES

lum are the goals of production. The next step after production is formulation. The desirable characteristics of formulations are ease of preparation and application, stability and adequate shelf life. This can be achieved by mixing the organisms with agricultural materials like wheat bran.

Verticillium

lecanii pellets

Microorganisms, often unobserved by man, do a good job of controlling various crop pathogens. Therefore, integrated pest control programme which use all forms of pest control, may be developed to ensure the success of a strategy which is environmentally friendly, and ecologically stable.

Effective use of biological agents requires considerable knowledge of the biology of the pest, the agent to be used and their interactions within the ecosystem. Strategies used can be grouped as importation, conservation and augmentation. In most cases, the pest is introduced in the field accidentally. Obviously, us natural enemies are absent in that area. Importation, as the name indicates, implies a situation when natural enemies are introduced to infested fields. Of course, strict quarantine rules are followed. This method has gained lot of success in controlling more than 200 species of insect pathogens in various countries of the world. Irrespective of whether the natural enemy is imported or native to the area, its conservation in the chosen habitat is necessary. This is usually achieved by plantation of alternate,

BUGS AGAINST BUGS

43

Thanks giving monument to a beetle that helped Californian ranchers

less troublesome host plants, and cultural practices that create favourable environment for the bio-control agent. The third approach, augmentation, involves mass production and periodic distribution of these control organisms. These three approaches are not mutually exclusive. In fact, their combination is always desirable.

he physiology of growth in plants, bacteria, fungi and insects is profoundly influenced by the properties of their protective covers. Plants are well protected by the cell walls, so are the bacteria and fungi. In insects, cuticle is the protective cover.

•

Common Bond

Bacterial cell walls are made of proteins and sugars, which make a tough barrier, rather like a three dimensional wire mesh. Such a wire mesh can, of course, be weakened by antibiotics such as penicillin. Cell walls of terrestrial plants contain large amounts of microscopic cellulose fibrils embedded in a matrix made up of lignin and hemicellulose. Lignins are phenolic compounds but their chemical structure varies from species to species. Hemicelluloses are heterogeneous polysaccharides made up of a mixture of sugars. Physical properties of plant cell walls depend on the interactions among these components. By and large plant cell walls are mainly cellulose in composition. The purity of cellulose ranges from 20 per cent in some grasses to 90 per cent in cotton fibre. The protective covers, that is, cell wall in fungi and cuticle in

COMMON BOND

45

Golgi body nudeus

Endoplasmic reticulum Plasma membrane Mitochon dria

Plant cell (top) Animal cell

46

NOVEL BIOPESTICIDES

insects, share common structural components. The cell wall is not an inert protective shell but rather, ~ dynamic organelle required for the viability of the cell. Lts complex structure serves many functions, such as osmotic protection to the protoplast, transport of macromolecules, growth, conjugation and spore formation. This means, any major disruption in its organization or metabolism will be deleterious to the cell. Like bacteria and plants, fungal cell walls are primarily composed of polysaccharides. These are divided into two groups on the basis of their physical form, such as skeletal and matrix components. Fungal cell wall components Skeletal components Chitinpolymer of N-acetylglucosamine with B-l,41inkages R-Glucanspolymer of glucose with B-l,3 and B1,6 linkages

Other components (skeletal/matrix) Chitosan polymer of glucosamine with B-l,4 linkages uronides,Melanins The skeletal components provide strength to the cell wall. These are the highlycrystalline polymers, chitin and glucans. Chitin is a very interesting and widely distributed com-

COMMON BOND

H N-Acety

. 41

NH-CO-CH3 1- D-glucosamine

Chitin is a polymer of N-A-G (inset)

48

NOVEL BIOPESTICIDES

pound. Chemically it is a nitrogen containing polysaccharide with long fibrous molecules. It forms material of considerable mechanical strength and resistance to chemicals. The chitin content of fungal cell walls varies widely from 3-5 per cent to about 60 per cent In Sclerotium rolfsii, a root infecting peanut pathogen, 60 per cent of cell walls is made of chitin. The matrix components are polysaccharides called mannans linked with proteins which form a sort of cementing material in the cell and which also contribute to the flexibility of the cell walls. Other constitutents, such as lipids and pigments such as melanins, though quantitatively minor, contribute a lot to the protective mechanisms. Of course, the first step towards cell wall formation would be the synthesis of chitin. This is immediately followed by certain modifications to form the cell wall. Chitin either forms bonds with matrix components or get chemically transformed to chitosan. All these operations are enzymatically controlled. During this period, chitin remains in loose, amorphous state. This is particularly true in regions at the growing tips. In cross-walls, chitin is crystalline in nature, tough and resistant to chemicals and enzyme attack. While fungal cell wall can boast of protective chitin, insect cuticle is not lacking in it either. Insect cuticle may be visualized in a simplified manner as containing alternate layers of protein and chitin. It has also been regarded as a plasiticized, protein sheet, variously subdivided to give a layered structure. The outer surface has waxes and the inner parts chitin. The hardening of the cuticle is due to its impregnation with calcium salts or further polymerization with certain chemical compounds or both. The thick cuticle consists of three layers. From the outside going in, the layers are called epicuticle, exocuticle, and endocuticle, respectively. The epicuticle is a thin, dark pigmented multilayered membrane made up of cuticulin (lipoprotein), wax and cementing material of uncertain composition. The exocuticle is amber or black coloured. Its

COMMON BOND

49

Epicuticle

Lipoprotein+ Wax+ Cementing material

Exocuticle

Chitin+ Protein

Protein+ Chitin

Endocuticle

Melanin

Glucan

Glucan-MannanProtein

-- --

~-

- -- -

- -- -:.. ----- --

-:.. -

--

--

-:.

_.

•.•..

•. ...

Outer mixed matrix components

~~·i!~~~~~:~~~;~~. ~..... .....

Matrix components

Skeletal components

Chitin

Insect cuticle (top) Fungal chitin

50

NOVEL BIOPESTICIDES

main constitutents are chitin and protein. The endocuticle is double the thickness of the exocuticle, but colourless and is made of chitin and protein. Chitin usually accounts for only 25- 50 per cent of the dry weight of the cuticle. Below the cuticle are present the cells involved ill the synthesis of cuticle. These are bound to it by a membrane. The rigidity of the cuticle is due to the exocuticle. Since the cuticle is incapable of growth, it must be shed from time to time as the insect grows, and a new and larger cuticle laid down in its place. This is what is commonly called 'shedding the skin'. Scientists prefer to call it moulting or ecdysis. The moulting process uses glycogen reserves for chitin synthesis. When a new cuticle is being formed, there is a certain order that the body follows. The epicuticle is laid down first, then the exo-and endo-cuticles are laid down.

52

NOVEL BIOPESTICIDES

tough. So enzymatic breakdown is not so easy. Gamma-chitin has properties which are more or less the same as beta-chitin. The distribution of chitin types in an organism has functional significance through chitin from fungal and animal sources are indistinguishable by X-ray and elemental analysis. Characteristics of chitin and chitosan

ICharacteristic parameter

IMonomeric su~ar

INature

l'Molecular wei~ht

Chitin

Chitosan

N- Acetyl~lucosamine

Glucosamme

Crystalline

Less c!ystalline

1-5 x 105

1-5 X 105

600 -1800

600 -1800

10%

60 -80 %

Other than insects and fungi, chitin is present in shells of marine invertebrates, such as crabs, oysters, lobsters and shrimps. Billions of tons of chitin are produced annually by marine animals. Since 1970, dumping of chitin wastes into waters has been declared illegal. In an attempt to find alternative methods to dispose of chitin waste, it was seredipitously discovered that flooding agricultural land with this waste helped combat root-infecting fungi. A marked reduction of root-rot in beans as well as wilt in radishes, both caused by Fusarium species was achieved by this means. Scientists who delved deeper into the phenomenon found

COMMON BOND

51

Finally the structure is layered with wax and cement coating. Glycogen reserves are reaccumulated after the moulting is complete. Synthesis and breakdown of chitin are active processes in insects and intimately linked to survival.If an insect is starved, its cuticle becomes much thinner because it uses cuticular chitin as an energy source. In Greek, chitin means tunic or covering and as such, the name is very apt. In 1823, A. Odier proposed this name for a substance isolated from the wings of May beetles. Later it was discovered that though it is a main cuticular component of insects, it may also be found in gut linings, tracheae, muscle attachments, and internal skeletons. Chitin is regarded as a modified cellulose, of which the monomer is glucose. If the OH group on the second carbon atom in glucose is changed to an amino group then the corresponding polymer is called chitosan. Chitosan is strictly speaking, not a substance but is rather a family of partially modified chitin products. Chitosan is non-crystalline in nature. It occurs in the walls of a limited but medically important group of fungi. These fungi, named Zycomycetes are opportunistic invaders of man and can be major pathogens in burn wounds. Chitin occurs in the form of microfibrils, also called crystallites by electron microscopists. Microfibrils in an insect cuticle exhibit diameters around 2.8 nm (1000 nm = 1 micron). In most fungal cell walls, chitin micro fibrils are randomly oriented with the exception of cross- walls in the fungal-filaments where they are organised in a circular fashion. The neighbouring chains of chitin are usually linked by hydrogen bonds. There are three types of chain arrangements viz. alpha, beta, gamma, which define the structural integrity of chitin. The more easily penetrable chitin is beta-chitin. Chemicals and enzymes can easily penetrate beta-chitin. Alpha- chitin, is widely distributed in the animal and fungal world. It has higher degree of 'packing' of chains and is more

COMMON

BOND

53

that land filling by chitin has as indirect effect on plant pathogenic fungi. The addition of chitin suppresses total fungal population and stimulates lytic and antibiotic producing microbes. Chitosan is involved in regulation of fungal growth. Chitosan which is a normal component of the cell walls of Mucor-like fungi can activate specific genes in plants and at the same time can inhibit RNAsynthesis is some fungi. When a pathogenic fungus attacks a plant, the affected plant produces chitinases which break down fungal cell walls and thus protects the host from the pathogen. Although in laboratory, chitosans inhibit fungal growth more effectively than chitin, for practical purposes chitin is found to be more suitable. Chitinous material is also useful in controlling plant parasitic nematodes. Tomato root-knot nematodes can be effectively reduced by addition of chitin. The different level of susceptibility of fungi and nematodes to chitin suggested modifications in the land filling process. As the chitin/ chitosan has no direct killing effect, the antifungal or nematocidal activity is'not dose dependent. Instead, periodic topping up with chitin is recommended for longer lasting effect.

he term antibiotics is not new to us. Each one of us has had reason to gulp antibiotic capsules at some time or the other. But what might surprise some of us is that antibiotics are included in man's arsenal against pests. Farmers have been using antibiotics in agriculture since the discovery of penicillin by Alexander Fleming.

Executing The Enemy

Of course, penicillin is not the only antibiotic that can help us. A mixture of streptomycin and tetracycline has been tried for the control of bacterial diseases. A mixture of cycloheximide and griseofulvin has been tried to rein in fungal pathogens. It is an emerging trend that a variety of newer antibiotics are replacing chemical fungicides. Mercuric chloride has been successfully replaced by kasugamycin and blasticidin S in Japan in the treatment of blast disease of rice. In Japan again, sheath blight of rice has been effectively controlled by replacing arsenic fungicides with polyoxins and validamycin. In the field, these antibiotics are used either as wettable powders or as dust. The insecticidal antibiotic, tetranactin, has also been used to control carmine mites of fruits and tea. Most of these anti-

EXECUTING THE ENEMY

55

biotics impair membrane functioning thus impeding cellular functions by disrupting movement of molecules into and out of cells. Simultaneously they may also inhibit synthesis of important cellular components such as sterols, protein or nucleic acid. Antibiotics in agriculture Name

Diseases

Medical Antibiotics as Agrochemicals Antibacteriall{ntibiotics

Streptomycin ChloramphenicolNovobiocin -

Fruit and vegetable diseases Rice leaf blight Tomato canker

Antifungal antibiotics

Griseofulvin Cycloheximide (Actidione)-

Fusarium wilt of melon

Onion downy mildew

Antibiotics Developed as Agrochemlcals Antibacterial antibiotics

Cellocidin -

Rice leaf blight

Antifungal antibiotics

Blasticidin S, Kasugomycin and Nikkomycins Polyoxins -

Rice blast Rice sheath blight, diseases of fruits and vegetables

Ezomycin Validamycin -

Stern rot of kidney bean Rice sheath blight

Insecticidal antibiotics

Tetranactin -

Carmine mite of fruits and tea

56

NOVEL BIOPESTICIDES

Alexander Fleming discovered penicillin

Now the question arises as to how antibiotics act as biocides. The answer lies in the architecture of the pests body covenng. Among the various components of fungal cell wall and insect cuticle/it is chitin and glucan metabolism that have been targetted by most antibiotics. Antibiotics mount a two pronged attack on the respective protective covers of fungi and insects. Attacks are either an inhibition of the formation of chitin and/ or glucan or an acceleration of their degradation with the aid of enzymes. Chitin synthesis is a fairly a simple process. The key enzyme which is involved in the final step of chitin synthesis is chitin synthase. Actually both carbon and nitrogen metabolism pathways are brought together by the organism for chitin synthesis. A number of enzymes contribute significantly to the synthesis of chitin. But only the enzymes glucosamine phosphate synthase and chitin synthase are considered prime targets for biocontrol agents. To decide the

EXECUTING THE ENEMY

57

---

--

Carbon Metabolism

Nitrogen Metabolism

':

-

Fructose 6 -Phosphate

Glutamine

Glucosamine 6-phosphate

t

UDP-GlcNAc

t t

)

II

Chitin)

11I/ Links with other polymers

Chitin-protein complexes Chitin-glucan, J

~I~ Hydrolysis

V Deacetylation

EJ EJ Glc NAc

Chitin synthesis

Chitosan

58

NOVEL BIOPESTICIDES

strategy of antibiotic treatment, it is important to know how these enzymes control chitin synthesis and whether if one blocks their activity, will the synthesis be impaired? Freshly formed chitin synthase is not fully active. Researchers have put forth a number of suggestions as to how it acquires its 'active' form. This has to occur prior to the synthesis of chitin. There is no concrete evidence for what is happening in the cell but certain observations have yielded definite clues about chitin synthesis. With the aid of the electron microscope, some distinct changes during fungal growth can be seen. These changes can be correlated with chitin synthesis. As a first step, vesicles called chitosomes accumulate in the growing regions. These vesicles carry inactive chitin synthase enzyme to the site where chitin will be synthesized. Chitin formation is a polymerization reaction. The monomers, or acetylglucosamine (GlcNAc) residues donot link directly with each other. They are "activated" by binding to Uridine diphosphate (UDP) to form UDPGlcNAc. After synthesis, lot of modifications take place during cell wall or cuticle formation. Chitin synthesizing systems characteristically include chitin degrading enzymes. Chitin is hydrolysed to its monomer by the synergistic and consecutive action of three types of enzymes: endo- and exo-chitinases and chitobiase. This understanding of the process of chitin syn~ thesis was vital. Once the biosynthesis of chitin was understood, scientists had a potential target at which to hit. The biosynthesis of chitin is a promising target for pesticide action. It was a startling and quite unexpected observation that many currently used pesticides inhibit chitin synthesis directly or indirectly. It was found that a number of neurotoxins and biochemicals which affect respiration also affect the process of chitin biosynthesis either directly or indirectly

•

EXECUTING THE ENEMY

59

Inhibition of chitin biosynthesis N on-specific inhibitors - Kitazin-p, Parathion, etc., block chitin synthase action

Competitive inhibitors - Polyoxins and Nikkomycins, compete with the substrate, UDP- GlcNAc Various compounds including chlorinated hydrocarbons, triazines, and nitrophenols, and organophosphates, are now known to inhibit chitin synthase activity. The organophosphates, kitazin-P and parathion and sulfenimide, and cap tan selecively prevent chitin formation in insects. Bioactive metabolites of microbes have distinct advantages of selectivity, specificity and ease of production. Actinomycetes are the most favoured sources of a variety of antibiotics. Two classes of antifungal antibiotics, namely polyoxins and nikkomycins have been isolated from streptomycetous cultures. The polyoxins are characterised as a family of metabolites from Streptomyces cacaoi variety. asoensis. and the nikkomycins from Streptomyces tendtie. Both of them are structurally very similar to UDP-GlcNAc, an active monomer of chitin. Since they are structurally similar, the antibiotic can mimic the action of UDP- GlcNAc and bind to chitin synthase. This would block the active site of the enzyme and thus make the enzyme unavailable to the monomer for polymerization. Thus the synthesis of chitin would be blocked. Polyoxins are a group of closely related compounds designated as A to M. Polyoxins usually inhibit the growth of filamentous fungi but most of them are inactive against bacteria. Polyoxin C is the smallest in the group and it lacks

60

NOVEL BIOPESTICIDES

Molecular mimicry

EXECUTING THE ENEMY

61

antifungal activity. However, all other polyoxins are effective against a variety of fungal pathogens albeit to different extents. Polyoxin D is most effective against Pellicularia sasakii, a rice sheath blight pathogen, while polyoxin Band L inhibit pear black-spot and apple cork-spot fungi respectively. However, polyoxins have an inherent drawback. They are nontoxic to certain fungi. This is because, these antibiotics can not enter through the cell membrane. This problem has been tackled by number of researchers. One of the approaches being followed is the preparation of synthetic analogs of polyoxins which can enter the cell easily. But most synthetic derivatives face the problem of stability inside the cell as they are susceptible to enzymatic degradation. Nikkomycins are structurally closely related to polyoxins and are very specific inhibitors of the enzyme chitin synthase. Apart from polyoxins, there are other antibiotics too which we can use to fight fungal and insect pests. Nikkomycins were first reported from Germany in 1980s. Among the whole lot of nikkomycins, discovered, the two named X and Z activity, respectively have a high antifungal, insecticidal as well as acaricidal activity. This means that nikkomycins can be used with profit, against fungi, insects and spiders! The only drawback that nikkomycins have is that it is rather difficult to isolate the specific nikkomycin that we might need. Therefore, attempts are being made to isolate new organisms which will produce one type of nikkomycin exclusively. Another approach is to prepare synthetic or semisynthetic derivatives of nikkomycins. Interestingly, a Japanese group has reported two new polyoxins, namely neopolyoxin A, Band C of which polyoxins A and Care structually similar to nikkomycin X and Z,respectively. Both classes of antibiotics synergistically inhibit chitin synthesis. Both polyoxins and nikkomycins show activity against all groups of chitin synthesizing organisms. In case of fungi, the observed effect on species exposed to polyoxins or nikkomy-

62

NOVEL BIOPESTICIDES

Nonnal

Impaired cell wall synthesis

Defonned mycelia

No zygospore

No transition

Yeast mycelium transition

Fate of hyphal tip

Hyphal tip bursting

Screening inhibitors

EXECUTING THE ENEMY

63

cins is swelling and bursting of growing hyphae while in case of insects, egg or cyst formation is inhibited. Both polyoxins and nikkomycins donot show any significant toxicity to mammals and higher plants. In other words, these antibiotics are specifically targeted towards chitin synthase only. Thus it would seem that these two groups of antibiotics are relatively safe for higher plants and animals. The discovery of polyoxins and nikkomycins does not mean that the search for better and safer pesticides is over. Far from it. In fact the chase has only hotted up so to say. Two compounds namely, pseurotin Aand 8-0-demethylpseurotin Ahave been isolated from a fungus Aspergillus fumigatus. These have shown inhibition of chitin synthase activity. However, these compounds donot show any direct antifungal activity. These antibiotics cannot enter into the fungal cell without a facilitating compound easing their entry. Therefore, a combination with Amphotericin B is used to overcome this problem and to render chitin synthase accessible to pseurotins. The inhibition of chitin synthase enzyme has led to other interesting speculations. Might not antibiotic activity.be targeted at other enzymes with the same net effect that chitin synthesis is stopped? Research along these lines has been quite rewarding to say the least. Other than Streptomyces strains, antifungal activity is found in bacteria such as Bacillus too. Although these antibiotics inhibit chitin synthesis" their target of action is not the enzyme, chitin synthase. For example, Tetaine, a compound isolated from Bacillus pumilus, inhibits an enzyme which brings together carbon and nitrogen pathways for chitin synthesis. The classical name of the enzyme is glucosamine6 phosphate synthase. Tetaine, a dipeptide, as such is not an enzyme inhibitor. However, when it enters into the fungal cell, it is broken down by cellular enzymes, to alanine and anticapsin. Anticapsin is the compound responsible for en-

64

NOVEL BIOPESTICIDES

zyme inhibition. A range of synthetic analogues of anticapsin, have been reported as glucosamine-6-phosphate synthase inhibitors. However, as with other antibiotics, most of these are not effective directly. This is because, their targets are inside the cell. So to be able to kill the organism they have to overcome two big hurdles. The first is of course, to enter the cells. Once this hurdle is crossed there rises the possibility that the enzymes in the cell might break down or degrade the antibiotics, However, the fact remains that these enzymes inhibitors merit more research if we are to win the fight against pests. So the search goes on. Other antibiotics which are in the queue are antibiotic A-19009, from Streptomyces collin us s. Lindenbeim and antibiotic Sch 37157 from Micromonospora.

The work so far carried out on glucosamine-6-phosphate synthase target is exploratory. At this point successful com-. mercialization of such inhibitors as antifungal or insecticidal agents in agriculture appears to be difficult. Furthermore, this enzyme also plays an important role in the biosynthesis of essential proteins in human cells. Therefore, careful consideration is necessary in developing glucosamine-6- phosphate synthase inhibitors. However, if we could find an enzyme unrepresented in humans and non-target animals yet present in the pest, we could perhaps safely find inhibitor(s) for it and stop the pest in its tracks'. Chitin is one such complex molecule which is not found in man. Arthropods such as insects and crabs have chitin in their bodies and it is pertinent to recall that many insects are senous pests, Chitin synthesising enzymes or chitinases play an important role during fungal and insect development. They are equally necessary for protozoa during formation of cysts and for the hatching of nematode eggs. In view of this, these enzymes could be a prime potl'l1tial target for antifungal and antiparasitic drugs. Scientists ha\'e not ne'glected this line of attack.

EXECUTING THE ENEMY

6S

Actinomycetes, are a rich source of chitin synthase inhibitors as well as chitinase inhibitors. While screening actinomycete strains for new growth regulators, a Japanese research group isolated a compound subsequently named allosamidin. Laboratory research indicated that it worked well enough to inihibit the chitinase enzyme of silk worm. It also prevented Leucania separata from changing from larvae to pupae thus successfully interfering with its life cycle. The allosamidins, produced by Streptomyces species, is structurally very closely related to acetylglucosamine, a basic unit of chitin. In other words, allosamidine is a molecular mimic and therefore, is a powerful competitive inhibitor of chitinase.

66

NOVEL BIOPF5TICIDES

Actinomycetes have proved to be a rich source of anti chitin compounds. Methylallosamidin, an inhibitor of the enzyme chitinase has also been isolated from actinomycetes. Both, allosamidin and methylallosamidin are structurally the same and thus their modes of chitinase inhibition is similar. Chitinases are widely distributed in nature and have different roles to play in different systems. Surprisingly, allosamidins are specific inhibitors of insect chitinases. They seem to have only negligible effect (if at all) on bacterial and plant chitinases. Scientists feel that this could be because of differences in the sites on the enzymes from different species where the inhibitors bind prior to action. So if the enzyme from a species does not have the requisite binding site, the allosamidin would not be able to bind to it. Obviously, if it cannot bind, it cannot act either. Structural differertces in binding sites of insect, fungal and plant chitinases could well .explain why allosamidins work well on one group and not on the others. Therefore, it is important to carry out toxicity test of allosamidin on plants and soil microflora, in general, before its application as an insecticide. Though the chitin metabolism inhibitors are being studied in a big way, it is a great challenge to successfully use them as a replacement for chemical pesticides. The pivotal point on which the success of these pesticides hinges is that insect cuticle and fungal cell walls share common structural components. What actually kills pathogenic fungi and insects are a variety of enzymes and toxins that their natural enemies possess and use against them. These have been identified and turned into weapons in the hand of science. The cuticle degrading enzymes (CDE) and mycolytic enzymes (ME) facilitate the penetration and killing process of pathogenic insects and fungi and eventually save the crops. Instead of using live natural enemies, researchers have tried spraying CDE and ME complexes for biocontrol of agricultural pests. Under certain conditions, these mixtures have worked won-

T EXECUTING THE ENEMY

67

ders and therefore, the efforts to develop such biocontrol agents are considered worthwhile. In soil, microbes are present which hydrolyse chitin polymer to acetylglucosamine. But this is not a single step reaction. The enzymes which can degrade chitin, are endo- and exo-chitinases and dimer hydrolysing chitobiase. The plant and bacterial chitinases differ markedly in antifungal activity. For self defence, plants produce chitinases which breakdown the pathogen's cell wall. The sugar produced induces one more line of defence by stimulating the synthesis of phenolics and promoting the synthesis of lignin in plant cells. This process can be facilitated by spraying chitinase on infected plants. To control root infection caused by fungi like Sclerotium rolfsii and Rhizoctonia solani of peanut, beans and cotton The daily use of dilute Serratia marcescens chitinase was tried. The feasibilty of this method is yet to be analysed in the field. On similar lines the anti fungal enzymes of Myrothecium verruca ria have been applied to control S. rolfsii infection of peanut. However, for large scale application, one has to the fluctuations in soil conditions and the effect enzymes on beneficial microflora. Nevertheless, proach does have commercial potential and cannot out.

consider of these this apbe ruled

To be honest, however, this approach is a little neglected because success has been sporadic. Most fungi have tendency to go into "hibernation" under unfavourable conditions. That means, hydrolytic enzymes are more fungistatic than fungicidal. In other words, they donot kill the fungi, but merely limit its growth. The resistant structures produced by root pathogens, remain in the soil almost forever. The longevity of anti fungal preparations in the soil is uncertain. Still, this approach has lot of scope in integrated pest management programme and we may hope that further research will help produce better preparations.

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NOVEL BIOPESTICIDES

-----------

EXECUTING THE ENEMY

.--.-_._---

.----

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69

Fungi are reportedly very effective against the brown plant hopper and other pests of rice. The fungus, Pandora delphacis is more effective in treating rice pests. Two mycoinsecticide formulations have been tried in Tamil Nadu, a rice growing state of India. Between the two formulations, namely dust (10 per cent) and wettable powder (70 per cent), the latter performed better in killing rice pest. The cost of 1 kilogram of the product is very little. Since it is priced around Rupees ten or so, farmers will find it affordable. The time taken by one crop of sugarcane from sowing to reaching the factory is around 16-18 months. Around 50 tons of sugarcane is produced per acre for which the cultivation cost was Rs 12,000 in 1996. Under ideal conditions of disease- free seeds, climate and rain, the farmer gets a return of Rs 40,000/ acre. If the crop is infested with pest such as Pyrilla the farmer has to bear 30 per cent loss in the y.ield while for fungal infections, such as smut caused by Ustilago or grassy shoot by Mycoplasma his loss is 15- 20 per cent. To be on the safer side the seeds are washed with fungicidals before sowing. Usually this takes care of the fungal infections. However, in the later stages, chemical treatment is not desirable. For the protection of crops chemicals such as endosulfan are used. The cost of chemical treatment is around Rs 2000/ acre. In view of the hazardous effects of chemicals,· several attempts have been made to check the pest population through application of its natural enemy Epiricania melanoleuca. The only drawback with this parasite is that it does not multiply well under dry weather conditions. The green muscardian fungus, Metarrhizium anisopliae, which affects insects has also been tried but without much success. Nevertheless, it is a potential agent as a biopesticide in strain improvement programmes. As the chemicals are not much effective for the control of fungal pathogens, suitable b.iocontrol agents are necessary for seed dressing. The probable candidates can be Trichoderma, a mycoparasitic fungus and Myrothecium, a high chitinase producer.

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NOVEL BIOPESTICIDES

In the third world countries, including India, most of the farmers use chemicals because of their ease of application, affordable cost and gauranteed results. Unfortunately, the aspects like long term toxicity and environmental hazards, are overlooked. Most of the biocontrol research is confined to the laboratory because of lack of enthusiasm among users. Slowly the situation is changing. Of course, one cannot really, at this point, expect replacement of all the conventional methods of pest management. Chitin metabolism inhibitors and, chitin degrading enzymes, have a bright future. Sooner or later they are sure to emerge as important components of integrated pest management programme.

Bio-protection in fields

pplications of biotechndlogy in agriculture range from genetic improvement in cash-crops to production of environmentallyfriendly biopesticides. Novel biopesticides are being created and tested in the laboratories around the world. Apart from trying out viruses and bacteria or even toxins and enzy:(nes as biocontrol agents, scientists are also trying to genetically transform the plants so that tbey become inherently resistcllnt to pests. On paper, the idea is simple enough. All it takes is to identify the genes endowing resistance to a particular pest in a resistant species of plant and to transfer them into the genome of the vulnerable variety. Of course, this is not as easy as it sounds. Plants which have had a foreign geneI 'stitched' into their genome are called transgenic plants. One can have 'tailored' plants to suit one's requirements. Flavour, colour, ripening and other genetically determined characteristics may be altered at will. But how do scientists ferry genes in and 'out of plants?

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NOVEL BIOPESTICIDES

Microinjection

Microinjection is a method of introducing foreign DNA into plants. But as this often damages the protoplasm in the cells, this method is not very popular. Sometimes DNA is encapsulated in closed vesicles, called 'liposomes' which are then introduced into the celL The liposomes fuse with the plasma membrane and empty their contents into the protoplasm. Electroporation has also been used to introduce foreign DNA into the celL The process involves passing of short pulses of electric current which makes the cell membrane temporarily permeable to molecules such as DNA. Sometimes DNA is 'shot' into plant cells using what is called a 'gene gun'. Using virus as a molecular ferry to transfer genes is also an option that scientists have. The tumour inducing plasmid of Agrobacterium tumefaciens which is a circular piece

BIO-PROTECTION IN FIELDS

of DNA has also been used by scientists to suit their purpose. However, in all fairness, it must be admitted that the integration of foreign genes into plan t chromosomes during genetic manipulations occurs at very low frequencies. May be only one in a thousand or even million cells will carry the gene stably. The Flavr SavrR tomato, developed in USA to improve the flavour quality of tomato is one such transgenic plant.

73

Firing device

Stop

P/~

Gene gun

Several strategies have emerged for improving crop resistance. These include the manipulation of resistance specially if this characteristic is governed by a single gene. Resistance that is governed by more than one gene or regulatory mechanisms are of course, a bit more difficult to manipulate. For many years it was known that BT could be useful for biological control of insects because it produced toxins against insects. Therefore, it represented a potential transgenic solution for insect control. The "pesticidal" plants are the closest transgenic application to commercialization. In China, a lot Qf effort is being expended to develop virus-resistant rice. Hopefully, by the turn of the century, insect -resistant maize, wheat and rice will be available in the market. However, success of transgenic plants in the fields may pose the two potential problems of biosafety and destabilization of natural ecosystems. /

NOVEL BIOPESTICIDES

74

In view of the hazardous effects of chemicals, and an apparent danger from transgenic plants running wild, biopesticides are attracting big business. The total sale of agrochemicals is estimated at about $ 25,280 x 106of which nearly 1.5 per cent (approx. $ 380 x 106)is that ofbiopesticides.

85%

Ill'

Agrochemicals

•

Biopesticides

BIO-PROTECTION IN FIELDS

75

Currently the insect control market is dominated by BTtoxin. BT is considered to be environmentally benign, though some insects, for example, Cotton Bollworm are becoming resistant to its action. But there are vulnerable insects and the market remains vast. Before introducing new chemical pesticide in agro-industry, serious thought has to be given to the projected market. This covers the costs of discovery, production, registration and of course, the product should be reasonably profitable. Normal time required from identification of a new chemical to its marketing as a pesticide is around eight years. Registration alone can cost more than $ 20 million. On the other hand, for biopesticides the lead time can be less than five years and the registration cost is also very little. The advantages ofbiopesticides therefore speak for themselves. Essential characteristics of effective biocontrol agents SpeedPf6'enaBl~ pi:t1logentode~~iop r&istant?~truc~es.1 Longevity, enough to protect plant during its vulnerable period, whatever that may be. Environmental tolerance, to sustain activity under differept soil and dit:natic ..conditions. Mode of action, varies from pathogen to pathogen, physical contact, chemical nature of killing component. While using natural enemies, it is important to have fast growing biocontrol organism in the fields which can eventually make the conditions unfavourable for the pathogens prolifera tion.

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NOVEL BIOPESTICIDES

Requirements for commercialization

High performance and consistency, effect has to be comarable to that of chemical pesticides.

Application, should not change the present day agricultural practices. Longer lasting stability of the biopesticide under field conditions is essential to protect crops during its vulnerable period. Spraying or irrigation time depends on the site and mode of biopesticide attack on the pathogen. Preventive approach is no good for heavily infested fields. One of the significant factors in the success of newly developed biopesticides is the education and knowledge of the farming community about the pest and its developmental cycle. For biochemicals such as antibiotics and hydrolytic enzymes, the storage stability is the main concern. To get licence for genetically engineered biopesticide, strict regulations are followed. Quality control of agro-industry needs due consideration. To avoid marketing of substandard biocontrol agents, coor-

BIO-PROTECTION IN FIELDS

77

dinated effforts are necessary by the industry and food growers. To improve the quality of the biocontrol agents many different aspects require careful attention. Quality control of the biopesticides includes stringent regulation of production standard, production facility and field performance. Although lot of experimental evidence is available on biocontrol agents, very few of these, have actually seen the light of commercialization. Bacteria, viruses and fungi have been used as biocontrol agents in agriculture. However, the greatest impact has been made only by BT. At present more than 13 microbial pesticide agents, have been approved and registered with the Environ'mental Protection Agency. These registered organisms are marketed in around 75 different products for use in agriculture, forestry and insect control. Since 1975, co-ordinated efforts for the biocontrol of pest and weed have been made by the Indian Council of Agricultural Research and other Indian laboratories and Universities with some success. The future therefore, does seem replete with the possibility of safe biocides at last.

Actinomycetes: A group of bacteria which produce filamentous growth. Antibiotics: Chemicals that are made by certain microbes and which limit or kill the growth of other microbes. Bacteria: Microscopic, single-celled forms of life. Biocontrol: Control of pathogen by biological means. Biomass: Growth of the organism. Cell wall: A dynamic protective cover of plant, bacteria or fungal cell required for the viability of the cell. It serves many functions such as protection, transport of molecules, and growth. Conidia: Specialized, non motile, asexual propagules. Cyst: Resting form of an organism in a protective covering. Dipeptide: A combination of two amino acids. Electron microscope: A microscope which uses electrons to produce magnified images of objects. It can magnify up to one million times. Epithelial: Surface layer of cells. Enzyme: Protein which acts as biological catalyst. Fungi: A group of plants which typically form a mycelium (mass of threads). Fungi lack chlorophyll and cannot make their own food. Facultative parasite: Parasites that can survive outside the host. Genetic engineering: Technique for altering the genetic make-up of an organism to artificially endow it with desirable traits. Haemolymph: 'Blood" of insects. A clear fluid, sometimes colourless, often tinged with green or yellow pigment. It is

GlOSSARY

79

the medium through which all the chemical exchanges between the organs are effected. Hyphae: Fungal filaments, a mass of hyphae is called mycelium. In vitro: Literally in "glass", refers to the re-creation of biological processes in an artificial environment. Lytic: Refers to infection of bacteria by bacteriophage virus in which the bacterial cell is destroyed (lysed) and virus progeny released. Metabolism: Biochemical processes occurring within an orgamsm. Metabolites: Substances which takes part in metabolism. Monomer: Unit(s) that take part in polymerization to make up the compound or polymer Mycolytic enzymes: A mixture of enzymes which degrade fungal cell wall polymers such as, chitin, glucan, protein, etc. Mycoparasitic: A parasite which attacks a fungus. Obligate parasites: Parasites that cannot survive outside the host. Pest: Commonly used term for plant pathogenic fungi and insects. Plasmid: A small circular structure mae entirely of DNA, found in bacteria. It can replicate independently of chromonomes. Plasmids are widely used in genetic engineering. PPM: Parts per million, Le. one milligram in one litre. Polysaccharide: Any of a large number of long-chain polymeric compound formed by linking together a large number of simple sugars called monosaccharides. Starch and cellulose are common examples. Polymerization:'Process by which a compound is made up by joining together a large number of monomers.

\,\

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NOVEL BIOPESTICIDES

Saprophytic: An organism that obtains its nourishment from dead or decaying matter. Many fungi and bacteria are saprophytes; they play an important role in the recycling of nutrients. Species A group of organisms that interbreed with each other to produce fertile offspring. Viruses: Very small infectious particles. When outside a cell viruses are lifeless, inert particles made of nucleic acid, fats and proteins. When they invade a cell they take over the cell, making more viruses, usually destroying the cell in the process.

r

I

WAR

between man and pests predates documented history. With time the fight has intensified and man's arsenal has expanded to include deadly chemical weapons with unintended backlash on the user and the environment. Therefore, the development of specific, yet environmentfriendly, pesticides became imperative. The study of insects, fungi and bacteria gave scientists the clue to the Achilles heel of the pests and helped devise safe pesticides. This lucidly written, profusely illustrated book details the evolution of the pesticide industry and highlights the development of novel biopesticides.

About .the Author After obtaining his Ph.D, Dr Mukund V. Deshpande (b. 1952) went on to earn !:lis D.Sc from the University of Pune in 1994 for his research in the area of fungal differentiation. At present, he is Senior Scientist at National Chemical-Laboratory, Pune. For the last 22 years he has been working on carbohydrate-bas~d enzymes, biocontrol of plant pathogenic fungi and insects, fungal morphogenesis and biosensors .

.9

t

'"81 72 "3 6 1 86 a ISBN: 81-7236-186-6