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5 Plant Diseases and Insect Pests The damage to plants caused by competition from weeds and by other pests including viruses, bacteria, fungi, and insects greatly impairs their productivity and in some instances can totally destroy a crop. Today, dependable crop yields are obtained by using disease- resistant varieties, biological control practices, and by applying pesticides to control plant diseases, insects, weeds, and other pests. In 1983, $1.3 billion was spent on pesticides--excluding herbicides--to protect and limit the damage to crops from plant diseases, nematodes, and insects. me potential crop losses in the absence of pesticide use greatly exceeds that value. For about 100 years, breeding for disease resistance has been an important component of agricultural produc- tivity worldwide. But the successes achieved by plant breeding are largely empirical and can be ephemeral. mat is, because of a lack of basic information about the function of genes for resistance, studies are often ran- dom rather than specifically targeted explorations. In addition, any results can be short-lived because of the changing nature of pathogens and other pests as new genetic information is introduced into complex agro-ecol ogical systems. An excellent example of the effect of genetic change is the sterile pollen trait bred into most major corn varieties to aid in the production of hybrid seed. Plants containing Texas (T) cytoplasm transfer this male sterile trait via the cytoplasm; it is associated with a particular type of mitochondrion. Unknown to breeders, these mitochondria also carried vulnerability to a toxin produced by the pathogenic fungus Helminthosporium 81 -

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82 maydis. The result was the corn leaf blight epidemic in North America in the summer of 1970. The methods used in the discovery of pesticide chem- icals also have largely been empirical. With little or no prior information on mode of action, chemicals are tested to select those that kill the target insect, fun- gus, or weed but do not harm the crop plant or the environment. Empirical approaches have produced enormous successes in controlling some pests, particularly weeds, fungal diseases, and insects, but the struggle is continuous, since genetic changes in these pests can often restore their virulence over a resistant plant variety or render the pest resistant to a pesticide. What is missing from this apparently endless cycle of susceptibility and resistance is a clear understanding of both the organisms and the plants they attack. As knowledge of pests--their genetics, biochemistry, and physiology, their hosts and the interactions between them--increases, better-directed and more effective pest control measures will be devised. This chapter identifies several research approaches to a better understanding of the fundamental biological mechanisms that might be exploited to control plant path- ogens and insects. Molecular biology offers new tech- niques for isolating and studying the action of genes. me existence of susceptible and resistant host plants and virulent and avirulent pathogens can be exploited to identify and isolate the genes that control the inter- actions between host and pathogen. Studies of the fine structure of these genes can lead to clues about the biochemical interactions that occur between the two organisms and to the regulation of these genes in the pathogen and in the tissues of the plant. It should be possible in the future to improve the methods and opportunities for the transfer of desirable traits for resistance into crop plants and, conversely, to create pathogens that will be virulent against selected weeds or arthropod pests. An increased understanding of insect neurobiology and the chemistry and action of modulating substances, such as the endocrine hormones that regulate metamorphosis, diapause, and reproduction, will open new avenues for controlling insect pests by disrupting their physiology and behavior at critical stages in the life cycle.

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83 Molecular Bases of Plant-Pathogen Interactions The existence of susceptible and resistant cultivars implies specificity in plant diseases. One explanation for this high specificity is a "recognition" mechanism between pathogen and host. Understanding the molecular bases that determine this specificity in recognition or in the pathogen's ability to alter the host's metabolism should yield new, definitive, and more efficient ways to prevent attacks on crop plants or to mitigate disease symptoms. Based on our current, limited understanding of the types of interactions that occur between host plants and pathogens, the mechanisms involved are varied and complex. Theoretically, a minimum of two criteria are involved. The first is recognition. There may be preformed molecules in both host and parasite that can interact. Second, there must be metabolic changes in the host or pathogen or both that are triggered by the initial interaction step. Genetic mutations in either host or pathogen can change the specificity of molecular interactions or their ability to trigger metabolic change. The following presents discussions on research directed toward possible mechanisms involved in recog- nition between host and pathogen and the metabolic changes that cause disease symptoms. Molecular Determinants of Resistance and Susceptibility It is widely held that some forms of resistance to fungal and bacterial pathogens are the result of a host plant's ability to synthesize chemicals that inhibit the growth and development of the pathogen. During infection by a pathogen, metabolic pathways in the plant are acti- vated, leading to the detectable biosynthesis of the inhibitors. A major class of inhibitors, called phyto- alexins, are primarily low-molecular-weight, secondary plant metabolites that possess wide-ranging activity against fungi and, to a lesser extent, bacteria. In the last two decades, more than 100 phytoalexins have been identified. The induction of the biosynthesis of phytoalexins, however, does not follow the specificity that most pathogens have for a specific cultivar. For example, phytoalexin synthesis can be induced by abiotic agents, such as wounding or other stress conditions, in both resistant and susceptible plants. Phytoalexins can

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84 be toxic to both virulent and avirulent pathogens. It appears that phytoalexin synthesis might be a general, nonspecific type of active resistance. An alternate approach, the study of susceptibility, has revealed mechanisms that show a high degree of specificity. _ ~ ~ ~ Many Pathogens Possess specific agents for virulence, sucn as toxins or enzymes, that determine the course of events in susceptible plants. In the last five years, six host-specific or host-selective toxins have been chemically characterized. These toxins affect only susceptible cultivars and are produced only by specific pathogens that can attack these same susceptible culti- vars. One well-studied example is the toxin produced by the fungus Helminthosporium maydis, mentioned earlier. The H. mavdis toxin disrupts enerav Generation in susceD tible mitochondria that characterize the T cytoplasm of corn. Normal mitochondria are resistant and are unaffected by the toxin because they apparently lack a receptor site for it. Genetic specificity also exists for resistance and susceptibility to plant viruses, but there is no infor mation on how such genes act. With respect to plant viruses the term resistance is used rather loosely. Quite often only the appearance of disease symptoms is considered. Thus, a plant that supports virus repli- cation but shows no symptoms is considered to be - resistant because it superf~cially appears to be so. More correctly, that plant should be called tolerant. Recent observations suggest that one type of resistance may involve the ability of viruses to spread from cell to cell in their hosts. The continuum of responses ranges from rapid and complete invasion of the whole plant by the virus to slow invasion to circum- stances where the virus is unable to spread from an infected cell, even though it might replicate well there. Accumulating evidence indicates that viruses induce the synthesis of proteins that are necessary for the movement of viruses from cell to cell. The host, however, depending on its genotype, can in some way interfere with this protein. Although the process is poorly understood, it may be, in part, the basis of resistance of plants to viruses. In a sense, viruses might be thought of as packages of genes; they are composed primarily of RNA or DNA, and they can replicate only in a favorable host cell environment. Studies of the interactions between viral

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85 RNA or DNA and genes in the host cell can lead not only to an understanding of how viruses function but also to the development of viruses as gene-carrying vectors for genetic engineering. An improved understanding of the basic concepts con- trolling resistance and susceptibility will result from research based on interrelated approaches to the analysis of the genetics of these traits, the gene products, the structure of the genes, and the methods that will permit their transfer between organisms. Genetics Continued breeding studies and genetic analysis of resistance traits in host plants and viru- lence traits in pathogens provide the experimental systems needed to isolate and determine the properties of recognition molecules involved in susceptibility or active resistance, such as phytoalexin biosynthesis. Single-gene changes that confer resistance against a pathogen exist and are used in crop breeding to develop improved cultivars. In other cases multiple genes appear to be involved in resistance, and complicate crop breeding. m e growing collection of data on the genetics of host plants and particularly of pathogens needs to be strengthened. Such data are essential for identification of the genes that control the specificity of receptor molecules, which determine resistance or susceptibility to bacteria, fungi, or viruses. Genetic analysis of some important fungal pathogens, however, will be difficult because sexual reproduction does not occur, and the modes of genetic reassortment and inheritance are unknown. Many genetic approaches are now being initiated. For example, single-pathogen genes responsible for disease reactions in two bacterial leaf-spot diseases, soybean blight and bacterial spot of tomato, are being isolated and cloned. mese techniques have potential for wide application. Gene Products ~ =~ -_ __ __ ~ .-_ tein. There is little direct evidence for the role of any {rho end product of most genes is a oro- specific proteins in controlling interactions between a host and a pathogen. Many potential candidates, however, can be hypothesized. By analogy with animal systems, surface molecules, such as membrane glycoproteins, may interact with low-molecular-weight messenger molecules, such as small carbohydrates released from cell walls. Cell wall extracts from both hosts and pathogens have

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86 been shown to elicit some resistance responses. Both the hydrolytic enzymes that release carbohydrate fragments from cell walls and the enzymes involved in the biosynthesis of toxins or phytoalexins are gene products that may be selected for study. Additional basic information is needed about the cellular interactions between host and pathogen during the onset of resistance reactions. For example, the precise mechanisms employed by phytoalexins to exert their effects on pathogens are unknown and need to be actively studied. Metabolic pathways for the biosyn- thesis of phytoalexins must be clarified, and other com- pounds associated with resistance need to be identified. The regulation and coordinated synthesis of the enzymes involved in these pathways must be detailed. In addition, the phenomenon of acquired resistance in plants needs further study. Resistance can be localized or can occur throughout the plant. Systemic resistance, however, may be of more practical value. m is phenomenon can appear after a host plant is inoculated with an avir- ulent strain of the bacterial, fungal, or viral pathogen. This exposure somehow induces resistance properties so that when the plant is subsequently challenged by one or more pathogenic strains, it will resist infection or exhibit only mild disease symptoms. Acquired resistance is most actively being studied using Pseudomonas solanacearum, some strains of which cause wilt and stem rot in tobacco, ginger, potato, tomato, and banana. resistance. Other avirulent strains only induce resistance. The experimental approach is to find mutants of the avirulent strains that fail to induce the acquired A comparison of the gene libraries of the active with the inactive mutants could lead to the iden- tification of the genes and gene products responsible for triggering the acquired resistance. Gene Structure Once the genes and gene products are identified, it is feasible to alter their activity by changing the structure of the gene itself. The tools of molecular genetics can be used to study both the struc- ture and activity of pathogen genes for virulence and avirulence and host genes for resistance and suscep- tibility. Some progress has been made recently with bacterial pathogens, particularly in characterizing some virulence factors such as pectolytic enzymes. Much of the basic information on the molecular biology of fungal pathogens, however, is yet to be acquired.

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87 The functions of proteins coded for by viral genomes must also be established to aid in the understanding of their possible roles in replication and pathogenesis. It may now be possible to isolate genes for specific types of resistance, such as that characterized by so- called hypersensitive lesions. For example, certain plant species and cultivars respond to infection by a pathogen by rapidly undergoing cell necrosis at the site of infection. The hypersensitive lesion can effectively stop the spread of a virus or confine the bacterial or fungal pathogen. In the latter two cases, the pathogen then dies. m is response is controlled in most cases by a single, dominant gene in the host plant. One approach to study of the mechanism controlling development of the hypersen- sitive lesion would be to first isolate messenger RNA from infected plants--those induced to give a hypersen- sitive response and those with a suppressed hypersen- sitive response. The mRNA from the suppressed plants could be used to prepare complementary DNA. This complementary DNA should recognize and hybridize with all the mRNAs from induced plants, except for those involved in the hypersensitive response. In principle, the remaining free mRNAs could then be used to probe a gene library of the hypersensitive plant for the gene that they can hybridize with. This gene should be the one responsible for inducing the hypersensitive lesion. Gene Transfer The ultimate goal of research discussed in this section on genetics, gene products, and gene structure is the routine transfer and expression of genes for resistance in agriculturally useful plants. As noted in the earlier chapter on genetic engineering, some bac- terial and viral pathogens may be developed as suitable carriers for the transfer of genes into host plants. Current and prospective vectors take advantage of natu- rally occurring, intimate associations between micro- organisms and plants, both pathogenic and beneficial. An appreciable effort is needed to identify and obtain suit- able vectors in addition to the one successful vector, the Agrobacterium Ti plasmid that can be used in some dicotyledonous plants. m e techniques necessary to manipulate vectors are available and will likely be refined and improved within the next few years. It is, unfortunately, the lack of knowledge of the basic biology of plants and of the function, transfer, and expression of genes that restricts progress in this area.

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88 Molecular Basis of Cellular Drainage in Susceptible Bosts Although it may appear that research on cellular dam- age and disease symptoms is a subset of the research discussed previously on resistance and susceptibility, its intent is distinct, but of equal major importance. Research emphasis in this area will yield insights into the biochemical mechanisms that result in cellular dam- age, or disease, following successful pathogenic inva- sion. As yet there is no clear explanation of how major symptoms, such as the yellowing and loss of chlorophyll in chlorosis or the tumors, galls, and morphological changes caused by cellular growth distortion, are induced once a virulent pathogen becomes established in a tissue. It may be possible to ameliorate symptoms or prevent crop damage directly by treatment, if the biochemical details are known. m e little-understood phenomenon of natural tolerance to disease is evidence that such treatment should be possible. Indeed, the study of natural toler- ance may be a valuable guide for developing disease protection traits for crop improvement. Easily observable disease symptoms, such as chlorosis, necrosis, and cellular growth distortions, can have a number of diverse causes. Therefore, it is not possible to make progress on such generalized disease symptoms without some indication of the kinds of pathogens involved. Some research approaches hold promise for establishing general scientific principles of host- pathogen interactions. Mode Of Antinn Of Taxi no Research in the last decade on purification and structural characterization has led to an acceptance of the concept that toxins are the potent chemical agents of virulence in many important diseases caused by bacteria and fungi. Only a small number of toxins have been chemically identified. Even fewer have a postulated target or receptor site in the host cell, as was described earlier for Helminthosporium maydis. But even in these few cases, it is not known how interference with the target site leads to cell damage. Much additional research is needed on toxins--on their genetics, such as chromosomal versus plasmid inheritance; on their chemical structure; on the pathways of biosyn- thesis in pathogens; and on their biochemical effects and role in pathogenesis.

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89 Nucleic Acid Interactions It is clear that the mere replication of a virus or viroid within a plant does not determine whether that plant will be diseased. mere are many examples of strains that produce a great deal of virus, but with very little damage to the host. On the other hand, some of the most serious plant diseases are caused by viruses that replicate very sparingly. Viruses, with their small genomes, have too little genetic information to code for the variety of proteins necessary to account for the almost infinite number of symptom types. Thus, it seems likely that interactions between the nucleic acid of the pathogen and that of the host initiate the disease process. Viroids, which are RNA molecules that do not code for a protein product, can cause symptoms similar to those caused by viruses. This lends support to the supposition that viruses as well as viroids interact directly with the genome of the host plant. Complete nucleic acid sequences are now available for several viroids; for satellite RNAs, which modify the symptoms of their carrier viruses; and for a few plant viruses. Complete complementary DNA clones have been made for some of these RNA agents and have been shown to be infectious. Because DNA is technically easier to modify than RNA, such DNA clones provide the opportunity to make site-specific modifications in the sequence of the nucleic acid by inserting or deleting short stretches of DNA. me effect of such changes on the agent's ability to infect and on the symptoms produced can then be determined. Using current methods the nucleotide sequences respon- sible for the disease syndrome should be identified. Furthermore, these complementary DNA clones could also be used in hybridization studies to locate regions in the host genome where the host and the virus, satellite RNA, or viroid sequences interact. As knowledge of the fine structure of the host's genes increases, future studies should enable researchers to determine the specific genes and processes that are perturbed by the presence of the pathogen. If the DNA clones themselves are not infectious, the cloned viral or viroid DNA can be transcribed back to RNA using any of several in vitro systems. Thus, site- specific modifications made in the DNA clone can be tran- scribed into the RNA to test the effect of such changes on infectivity and disease symptoms. In this manner, critical regions of the genome could be identified,

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90 which would aid in the understanding of their functions and possibly the functions of viral-coded proteins. Bacterial Interactions Bacteria that cause diseases in plants cause symptoms, at least in part, by the produc- tion of various metabolites. Relatively few of these substances have been identified. The metabolites in- clude, but are not limited to, toxins, polysaccharides, pec tic enzymes, and plant hormones. All the bacterial toxins identified to date appear to be general toxins affecting a wide spectrum of plants. Many of these plants are not considered to be host species for the bacterial pathogen producing the toxin. Other bacterial metabolites appear to have specific effects on host plant species. charides, which are associated with wilting of plants, can be released in amounts great enough to clog up transport between plant cells, and may act by disrupting plant cell membrane functions. . , . _ . . . ~ , _ _ _, Bacterial polysac- Soft rots, for example J are the result of bacterial enzymes that degrade the cementing pectin layer between plant cells. m e pro- duction of plant hormones by bacteria disrupts the endogenous hormone balance in the host plant and can be part of the mechanism leading to crown gall tumors and other abnormal growths. The molecular and genetic bases of the synthesis of these pathogen metabolites and the basis of the symptoms they cause in the host plant are largely unknown. There is increasing knowledge, however, about the genetics of some of the bacterial virulence factors that contribute to the severity of a disease. For example, in crown gall, which is caused by Agrobacterium, both bacterial chromosomal and plasmid genes are known to be required for pathogenicity. The molecular Genetics of crown nail is the most thoroughly studied of any plant disease. In the genetic analyses of virulence in bacteria, two different approaches are currently being used. One is the introduction of transposons into virulent strains of bacteria to create avirulent mutants. The transposon is used as a probe to locate and isolate the turned-off virulence gene. DNA clones of virulence genes can be used for an analysis of gene products. The second approach is molecular and genetic analyses of known or suspected determinants of pathogenicity, such as cell surface components, hormones, toxins, and extracellular enzymes. Both approaches hold promise for the elucida-

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91 tion of the biochemical steps in pathogenesis. There is an essential need to have basic knowledge about the structure, function, and regulation of virulence factors in the pathogen to provide a basis for directed plant breeding and to design effective inducers of plant resistance. Research Status It is important to recognize that considerable exper- tise and training in molecular biology are necessary for many of the research approaches discussed in this section of the report. Progress is facilitated by individuals working together in groups. Interactions with researchers in other laboratories are important sources of intellectual stimulation as well as sources of tech- nical expertise. The tools of genetics and molecular biology offer some new methods for understanding the highly specific inter- actions between host and pathogen. Studies of the molecular aspects of plant pathology must receive high priority and emphasis within the ARS research programs on plant diseases. Currently the ARS research centers are undertaking relatively little basic work in molecular plant path- ology. The ARS does have a few strong research programs in virology and in viroids, but very little work at the molecular level is being conducted with bacteria or fungi. A single laboratory, at Beltsville, is studying plant mycoplasmas. To strengthen programs in the molecular basis of plant diseases, research investigations should emphasize: The molecular bases of the factors that determine whether a host-pathogen pair will result in a resistant or a susceptible interaction. me basic concepts of the interaction between the host and the invading pathogen that result in disease. This should lead to novel methods of preventing damage from disease, including natural plant tolerance. o The transfer of resistance traits to normally susceptible plants through the development and subsequent exploitation of vector systems that allow for gene transfer between plant species. It is significant to note that very few laboratories in the world have undertaken studies to understand

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94 The use and genetic manipulation of insect pathogenic bacteria and viruses constitute a promising but compara- tively underdeveloped approach to insect control. me potential exists for genetically improving these organ- isms to increase their pathogenicity, either by enhancing existing pathogenic traits or by introducing desirable pathogenic characteristics. Basic knowledge about potentially useful pathogens must be acquired. This includes identification of the pathogen and characterization of the insect host. The specificity between pathogen and host and the techniques for production and storage of candidate pathogens must also be studied. With this information the physiology, biochemistry, and genetics of the host-pathogen inter- action can then be investigated. More specific areas of study include the molecular basis of processes such as recognition, virulence, and toxicity and the mechanisms regulating gene function during these interactions. Progress in this line of research is apparent from the work of many laboratories worldwide. Candidate micro- organisms identified by this research include baculo- viruses and Bacillus thuringiensis. With recent devel- opments in insect cell culture, some of the fundamental processes detailed here, in principle, can be directly probed in vitro with any of these microorganisms. Control of Nematodes Control of plant parasitic nematodes has been largely accomplished through the use of chemical nematocides, many of which have now been shown to be harmful to the environment and have been withdrawn from use. Biological control measures using resistant plant varieties and trap crops have been effective in some cases. A trap crop can stimulate the hatching of nematode eggs but does not support nematode growth, thus reducing nematode populations to harmless levels. More information is needed on the basic biology of nematodes to provide directed approaches to their control, using less toxic, target-specific substances. This might include the use of the hatching stimulants that are apparently produced by plants and trigger nema- tode eggs to hatch. The growing nematodes then perish in the absence of a suitable host plant. Studies of nema- tode pheromones and hormones could lead to methods for controlling reproduction or development.

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as Plant Bealth Microorganisms In recent years some information has been gathered on soil microorganisms, specifically, certain bacteria, that can improve plant vigor and contribute to increased yields. me mechanisms by which such bacteria exert these effects are essen- tially unknown, nor are their relationships to pathogens or other microorganisms in the environment well under- stood. Indeed, candidate organisms suited for particular crops remain to be identified and characterized. Such bacteria contribute a desirable and perhaps essential microflora for optimal plant growth. While a range of microflora is known to be essential for human health, virtually nothing on a comparable basis is known for plants. Several mechanisms have been suggested that describe the effects of soil microorganisms on plant health. Beneficial microbes may produce antibiotics that inhibit the growth of pathogens, or they may be involved in the acquired resistance phenomenon. Recent evidence suggests that some plant growth-promoting bacteria produce sidero- phores, iron-chelating molecules, that restrict the availability of this essential element to pathogens and other members of the microflora. Biological Degradation of Organic Pesticides Timely and appropriate disposal of pesticide residues in water and soils is an important and attainable goal in routine agricultural production practices. The biological degra- dation of pesticides is theoretically feasible. For example, pseudomonads have been identified as being able to degrade the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) to innocuous compounds. Lack of knowledge of the chemistry, the fate of breakdown products, and the ecology of the organisms involved, however, is still a constraint to their use. Both waste disposal of agricultural by-products and biomass reduction on an industrial scale are under inten- sive investigation. The processes are not commercially feasible as yet, however, because of low yields and organism management problems. These problems can be overcome using genetically engineered organisms, espe- cially bacteria, that are currently more amenable to manipulation than other microorganisms. Research Status The ARS laboratories are among those contributing to progress in biological control of plant pathogens and

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96 insect pests. With increased emphasis, the ARS could be at the forefront of this research. The potential return for the ARS extends beyond the control of plant pathogens and insect pests; it would involve the development of general methodologies for gene transfer, cloning, and gene expression using microbial and insect systems. Basic research on the microflora of the rhizosphere is also an area that ARS can strengthen. There is enormous potential for the identification, development, and application of microorganisms that can degrade pesticide residues and other toxic wastes. The ARS should expand its efforts--some of which are exemplary --in these areas. It is high-risk, long-range research and requires the multidisciplinary base that is already in place in some locations. Specifically, the ARS should focus research toward: Exploring and identifying microbial agents that can control plant diseases and insect pests. Further, the agency should seek conventional genetic or recombinant-DNA technologies to make these agents more effective; Generating more knowledge of the basic biology of plant pathogenic nematodes to develop novel, nonpesticide means of control by perturbing reproduction and devel- opment; and Developing unique microorganisms that will pro- mote plant health and others that can be used to detoxify or destroy organic pesticide pollutants. Molecular Basis of Pesticide Action Pesticides are major tools in the production of food and fiber and in the maintenance of high standards of veterinary, human, and plant health. Better pesticides are needed, relative to cost effectiveness, potency, selectivity, persistence, environmental impact, and safety for domestic animals, humans, and plants. Most of the early pesticides were discovered in industrial pro- grams involving the synthesis and screening of thousands of synthetic chemicals for safe and effective molecules. The emphasis in current discovery efforts favors research on the natural chemicals produced by plants and micro- organisms, such as occurred for the pyrethroids. Equally important are investigations into the molecular basis of pesticide action.

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97 Advances in bioregulation research provide new vistas in seeking enzyme or receptor targets for pesticide action. Increasing fundamental knowledge of the function and regulation of communication systems within living organisms focuses attention on new targets and greatly facilitates the molecular design of optimal compounds for pest control. Greater diversity is needed in the targets for future pesticides, such as insecticides, herbicides, nematocides, and fungicides to avoid or minimize the impact of pesticide resistance and toxicity against non- target species. Susceptible and tolerant species often differ only in the sensitivity of their pesticide recep- tor site or their facility for detoxifying the pesticide. A clear definition of the mechanisms involved will provide the background for the next generation of improved pesticides. New pesticides, in turn, provide unique probes to explore cellular entities such as enzymes, receptors, and membranes. me molecular basis for metabolic activation and detox- ification must be defined. Using this background know- ledge genetic engineering can provide opportunities for modifying receptor sites and detoxification mechanisms for improved animal and crop safety. Research Status Research on the molecular basis of pesticide action is carried out in many laboratories within industry, univer- sities, and the ARS. Industrial labs tend to focus on the modes of action of their proprietary compounds. Universities more often use pesticides as probes for physiological and pharmacological investigations. me ARS has placed considerable emphasis on the mechanism of pesticide action. The laboratory defining a new target often reaps the benefit of finding alternative agents working at the same site or in the same way. Research on pesticide mode of action requires the creative teamwork of biochemists, chemists, and geneti- cists with adequate instrumentation and the appropriate environment to stimulate communication. This multi- disciplinary approach and the requisite personnel are now in place in several ARS laboratories. The ARS should increase its emphasis on the molecular basis of pesticide action, using the available expertise in microbial, plant, and insect physiology, biochemistry, and natural

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98 products chemistry. Success in this program will serve as the basis for improving animal health and for reducing crop losses during production and storage. More specifically, the ARS should emphasize: Definition of the molecular basis for metabolic activation and detoxification of pesticides; Study of new targets for selective pesticide action; Identification of new natural chemicals important in regulating pest populations; Investigation of the basic molecular biology of vectors for gene transfer and elucidation of gene regulation in insects; and Continued research on both insect genetics and on natural products chemistry. Insect Neurobiology and the Regulation of Development and Reproduction The functional responsiveness of an insect is depen- dent on rapid chemical communications among its own cells and between the individual and other insects. Intercel- lular communication is mediated primarily by the nervous system, through substances such as neurotransmitters, neurohormones, and neuromodulators as well as by the endocrine system, through hormones. me endocrine system is closely coupled to the functioning of the nervous system. Communication between individuals is achieved through volatile chemicals called pheromones. Their production and action is mediated by the nervous system. Insect Neurobiology The function of the nervous system makes it a logical focus for investigations of alternative means of insect control that could potentially have considerable selec- tivity. Before investigations can be initiated, however, basic information about the function of the insect nervous system must be obtained, specifically, infor- mation about nervous processes involving chemical communication. This approach is the only potentially successful avenue to the solution of applied research problems. For this reason a research emphasis in

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99 fundamental insect neurobiology should be developed by the ARE. Insect neurobiology is now experiencing a period of exponential growth. Despite the fact that the insect nervous system has been used for many years as a model for studying certain neurophysiological processes, basic research using modern techniques has only recently begun on the neurochemistry, neuroendocrinology, neurogenetics, and neuropharmacology of the insect nervous system. For example, the number of identified insect peptides with neurohormonal activity is fewer than 20. Only 4 of these insect neurohormones have been purified and sequenced. These include the neurotransmitter/neuro- modulator proctolin, the two adipokinetic hormones, and cardiac accelerator peptides. Proctolin is important in the stimulation of muscle contraction and is co-released with other neurotransmitters. me adipokinetic hormones mobilize lipid for its metabolism by muscle in insect flight, and the cardiac accelerator peptides control the heartbeat of the insect. It now appears that the struc- tures of the prothoracicotropic hormones, the primary effecters of insect metamorphosis and the first hormone of neural origin described for any animal (1917), are finally being resolved. In addition, a new brain peptide that regulates the production of pheromones has been described and promises to introduce a renaissance in pheromone research. Study of these and of yet-undiscovered hormones will aid in an understanding of the physiology of the insect, its growth and development. Such studies will also define the mechanisms by which the central nervous system integrates and regulates these processes. This under- standing may allow scientists eventually to selectively manipulate the neuroendocrine system, and thus control insects by altering their ability to fly, curtailing metamorphosis, or disrupting sexual recognition. The study of neurohormones may not provide an immediate answer to insect control. The resulting knowledge, how- ever, will provide scientists with the sound foundation necessary to propose and pursue new directed and applied research on the neural regulation of insect growth and development. The top scientific priority for neurobiological research on insects is the elucidation of the mechanisms by which chemical communication directs and coordinates the growth, development, homeostasis, and reproduction of

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100 insects. me basic information still lacking includes the identification of neural regulators and an eluci- dation of their chemistry, synthesis, secretion, and metabolism. Other opportunities for manipulation of insect pests include the neurohormones bursicon, diuretic hormone, and egg development neurotropic hormone. Bursicon causes the insect skeleton to harden. Inhibition of the secretion of this hormone would cause death. Manipulation of the diuretic hormone, which regulates water and salt balance, might also result in death, through ionic imbalance and dehydration. Secretion of egg development neurotropic hormone from the brain of the female mosquito is stim- ulated following a blood meal. The hormone indirectly causes the ovary to mature the eggs. Manipulation of this reproductive hormone would prevent the development of generations of mosquitoes. These hormones are examples of the potential in this field. To realize this potential the hormones must be studied extensively at the chemical, molecular, and physiological level. At this point a major reesarch program encompassing the physiology, biochemistry, and molecular biology of these regulators can be initiated. Research should in- clude the study of mechanisms of communication within the nervous system, between organs and organ systems, and between individuals of the same species. Studies of interorganismal communication should emphasize the neuroendocrine and neural bases of this process and relate this communication to behavioral patterns in nature. Knowledge gained from such a fundamental research program in insect neurobiology could be used in con- junction with genetic engineering methodologies to investigate the basic molecular biology of vectors for gene transfer and to elucidate gene regulation in insects. These new technologies could also aid in mapping the insect genome, particularly the genes for regulatory peptides. Peptides offer researchers an extremely important direct line of study; they probably are all products of single genes. An understanding of these gene products or polyprotein precursors and their posttranslational pro- cessing to a bioactive peptide is essential for the potential control of insects. (Posttranslational pro- cessing, which follows the translation of RNA, is proving to be a fundamental mechanism that determines the protein

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101 nature of the neurosecretion from a given cell.) A dis- ruption of the synthesis or processing of neurohormones would be lethal. The long-term goal of this research is modification of the normal function of the insect nervous system to affect viability. A research program on insect pathogens as vectors for gene transfer would clearly be important in achieving this objective. Endocr ine Regulation of Metamorphosis, Diapause, and Reproduction The postembryonic development of the insect involves a series of dramatic physiological and biochemical trans- formations that culminates in its emergence from a pupa as an adult form with its own unique function. It is generally accepted that these transformations and their associated metabolic processes all are directly or indirectly under endocrine control, including production of hormones by neural tissue. The full extent of the role of the endocrine system is not completely known, mainly because of a lack of knowledge of the hormones involved, the molecular basis of the developmental and reproductive processes these hormones control, and their mechanisms of action. m e progress made in this field in recent years has largely been at a descriptive level. Thus, basic research is needed to identify and chemically characterize insect hormones and to define at the molec- ular level both their physiological function and their mechanism of action. Although some insect hormones, such as the sesquiter- penoid juvenile hormones and the ecdysteroids, have been intensively investigated, the extent of their involvement in regulating insect development and reproduction is only now being realized. They are known to exist as struc- tural and functional families of molecules, each acting at a specific time during the life cycle of the insect. The multiple functions of these hormones provide multiple avenues for pursuing control of the insect. Substantial stantially more research is needed, both in the above- cited areas as well as on the mechanisms of their inter- action at the level of the target gland and interendo- crine feedback control. Research studies must be designed to show how these hormones regulate one another's synthesis and secretion to drive development and growth.

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102 A virtually unknown family of insect regulators that control metamorphosis, diapause, and reproduction is the peptides. Only a few have thus far been identified, and as has proved to be the case with vertebrates, there are numerous peptide hormones involved in the control of embryogenesis, postembryonic development, reproduction, and homeostasis. These peptides need to be charac- terized, their physiological functions defined, and mechanisms of action elucidated. The regulation of the synthesis, secretion, and metabolism of these insect hormones, whether peptide, steroid, or other chemical structure, is another rela- tively unexplored research area of considerable signif- icance and potential application to the control of insects. The secretion of these hormones has consis- tently been shown to be precisely regulated, frequently in response to discrete environmental cues such as photo- period, temperature, and stress. The mechanisms by which these cues are transduced by the nervous system to elicit an endocrine response are important areas for basic research in insect neurobiology. Knowledge of the regulation of insect development and reproduction is applicable to the manipulation of these systems for improved pest control. Some natural and synthetic chemicals, including insecticidal compounds, alter growth and development by inhibiting the biosyn- thesis or action of juvenile hormones or ecdysteroids and by governing the initiation and termination of diapause. Certain antibiotics and the highly insecticidal benzoyl- phenyl ureas interrupt chitin synthesis necessary for the formation of the insect cuticle or skeleton. Studies on insect genetics indicate the possibility of breeding sterile hybrids for use in pest control. Bacteria and other microorganisms producing insecticidal materials and the plant itself may also be modified by selection and genetic engineering to increase the impact of natural toxicants or feeding deterrents in host-insect pest interactions. Further development of insect cell cultures and vectors for gene transfer in insects may permit the introduction of deleterious effects into pest populations. The benefit from research in insect neurobiology is not the potential control of insect pests alone. Although the insect is a relatively simple system structurally, it is functionally complex, much like that of vertebrates. An understanding of the insect endocrine system will lead to a further understanding of similar processes in all eukaryotic organisms.

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103 Research Status The ARS is recognized worldwide for developing the sterile insect release method of control and for investi- gations on insect genetics and ecdysteroids. The agency also is internationally recognized for natural products research, particularly pheromone chemistry, and the application to insect development and reproduction. This type of interdisciplinary research requires a coordinated team of entomologists, physiologists, biochemists, and chemists. There are a number of ARS laboratories currently conducting excellent research on the physiological and chemical aspects of endocrine control of insect devel- opment and reproduction. By bolstering these existing programs with the appropriate additions of scientists skilled in protein chemistry, basic biochemistry, and the study of nuclear and membrane proteins as receptors, the ARS should be able to make substantial contributions to this research area. Although the ARS is becoming increasingly more involved in fundamental insect neurobiological research, this program is not developing in a focused manner. While most of the research skills necessary for a major program in insect neurobiology--chemistry, neuro- physiology, behavior, biophysics, and physiology--are already in place within the ARS, additional expertise in neurochemistry, peptide chemistry, and biochemistry (mechanistic aspects or chemical regulation), and immu- nology must be added. Generally, adequate instrumen- tation for this research exists within the ARS. Analytical facilities are needed, however, for peptide and neurotransmitter structural identification. Of the few laboratories worldwide engaged in insect neurobiological research, a number are emerging as centers of excellence. me comparative paucity of such centers, however, means that relatively few neuro- biological systems are currently being explored. Thus, the scientific opportunities in this field are enormous. Unfortunately, the lack of basic information has created a situation wherein the most important areas of research are high risk and will require considerable effort and resources. Such high-risk research is well suited for government-supported organizations like the ARS. To date, a multidisciplinary program in insect neuro- biology does not exist. me ARS has an opportunity to establish the first program of this kind. The success of such a program greatly depends upon the centralization of

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104 research at a single site, preferably near a university or another research institute that has a strong program in neurobiology. ARS research should specifically focus on the following: Chemistry of the brain factors that control pheromone production and release, and their mechanisms of action; Neural regulation of the synthesis, processing, and secretion of cerebral pheromonotropic peptides; The endocrine basis of insect reproduction, in particular, identification of the cerebral neuropeptides involved and their target glands, and identification of the mechanisms regulating these glands; Mechanisms that regulate the synthesis of ecdysteroids and juvenile hormones, and the biosynthetic pathways of these two hormome families; and Interhormonal endocrine feedback; regulation of insect growth, development, and reproduction; and the roles and molecular mechanisms of the principal devel- opmental hormones in regulating one another's synthesis.