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Pesticide Resistance: Strategies and Tactics for Management. 1986. National Academy Press, Washington, D.C. Biotechnology in Pesticide Resistance Development RALPH W. F. HARDY The role and potential of biotechnology in pesticide resistance development is projected to be quite large but has been minimally used. Relevant biotechnology techniques are numerous, including cell and tissue culture and genetic and biochemical techniques. The classic case of the role of biotechnology in resistance is an- tibiotic resistance. Biotechnology identified the basis of resistance and is guiding synthesis of novel antibiotics to circumvent resistance; antibiotic resistance provided a critical process for genetic engi- neering. In the area of pesticide resistance, the only well-developed application of biotechnology is for three different classes of herbi- cides. The sulfonylurea herbicides are presented as an example of the role and potential of biotechnology in any pesticide resistance case. Biotechnology has not been applied to fungicide, insecticide, or rodenticide resistances. The opportunity for biotechnology is large, but will require a multiplicity of skills beyond those used by scientists who are working at the organismallphysiological and biochemical levels of pesticide resistance. This opportunity should be pursued aggressively, since it can provide new directions to alleviate or minimize pesticide re- sistance where the benefits from additional organismal, physiolog- ical, and biochemical studies may be limited. INTRODUCTION The new biotechnology is providing biology with a powerful array of techniques that are advancing molecular understanding of biological pro- cesses and phenomena at an unprecedented rate. Outstanding examples are 130

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BIOTECHNOLOGY IN PESTICIDE RESISTANCE DEVELOPMENT 131 antibody formation and oncogenes and cancer. From this understanding and these techniques, useful new products, processes, and services are being and will be generated. The generation will be direct in terms of biological prod- ucts, processes, and services and indirect in terms of chemical products, processes, and services. Agrichemical and pharmaceutical discoveries will become increasingly driven by biotechnology or biotechnology-chemistry rather than by the current dominant process of empirical chemical synthesis coupled with biological screening. Tagamet@, an antiulcer drug that has produced the highest sales for any single pharmaceutical, is an early example of a biotechnology-chemistry-based innovation. Since the health care field has been quicker in using the new biotechnology than the field of agriculture, such a product has yet to be produced for agriculture. For example, the application of the new biotechnology has only recently begun and is limited in the area of pesticide resistance (Brown, 1971; Dekker and Georgopoulos, 1982; Georghiou and Saito, 1983; Hardy and Giaquinta, 1984~. BIOTECHNOLOGY TECHNIQUES Biotechnology comprises cell and tissue culture techniques and genetic and biochemical-chemical techniques. Cell and tissue culture techniques range from microbial culture through higher organism cell and tissue culture to somatic cell fusion and regeneration. Somatic cell fusion has become es- pecially useful for antibody production, where an antibody-producing cell with a limited life is fused with a transformed cell with an infinite life to produce a hybrid cell (hybridoma) that produces over an almost infinite period of time a single type of antibody called a monoclonal antibody (MAB). These MABs could become very useful in both qualitative and quantitative diagnosis of pesticide resistance, as they are becoming useful as in vitro health care diagnostics. Several start-up companies have been established for health care MAB diagnostics. Cell culture techniques will also be useful in developing and/or selecting resistance in model systems. Resistance development may use microorgan- isms or cells or tissues of higher organisms. In the latter, regeneration of plants from culture often increases phenotypic variability, such as possible herbicide resistance, over that shown in the parental cells. This phenomenon is called somaclonal or gametoclonal variation, depending on the cell source. Genetic techniques, especially molecular genetic techniques, have ex- panded greatly during the last decade and are propelling our understanding at the molecular level. Several of these techniques are the basis of a major biotechnology called genetic engineering, in which defined genes are intro- duced into foreign host cells. In theory any gene can be moved from a microbe to a plant, a plant to an animal or human, a human to a microbe, eliminating the barriers of sexual plant and animal breeding.

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132 MECHANISMS OF RESISTANCE TO PESTICIDES Production of gene fragments is the initial technique used to generate understanding and to perform genetic engineering. Restriction enzymes, of which there are about 100, cleave DNA at specific sites dictated by the DNA base sequence. The restriction enzymes cut the DNA of organisms such as fungi, insects, plants, and animals into useful fragments called gene libraries. These fragments are useful, since they are of a small size in which specific genes can be identified. A gene library is the starting point. There are few if any gene libraries available for agricultural pests, although the techniques needed are available. Separating the fragments produced by the restriction enzymes enables characterization of the genotype for polymorphisms. This technique, called restriction enzyme mapping, could be used to diagnose and characterize resistance at the genetic level so as to establish the similarities or differences of observed resistances. Study of a gene of interest, such as a resistance gene, requires its iden- tification, isolation, biosynthesis or chemical synthesis, and cloning, usually in a genetically well-characterized microorganism such as E. coli, to produce adequate quantities for characterization or further use. The gene can be isolated from the gene library, biosynthesized as a complementary DNA (cDNA) from its messenger RNA (mRNA), or chemically synthesized di- rectly if the DNA sequence is known. If the sequence is not known, powerful DNA sequencing techniques exist for rapid sequencing. DNA sequencing will identify the similarity or difference of resistant versus susceptible genes. Genetic engineering of organisms requires these steps so as to obtain a source of the desired gene and to generate genetic constructions with appro- priate replication sites and control elements so that they can be introduced into the desired host, retained, and replicated to produce the gene product at an appropriate rate. Techniques have been developed to introduce func- tional foreign genes into microorganisms, embryos of mammals, and cells of at least dicotyledonous plants. Human insulin produced by microorgan- isms, antibiotic-resistant model plants, and super rodents with additional copies of the growth hormone gene are examples. We are beginning to understand the molecular basis of how gene expression is regulated. Recent studies on Drosophila are a major example in a model sytem. As this knowledge becomes known, it should be very useful in ex- ploring resistance on the basis of regulatory-based changes. Overall, genetic techniques will be useful to understand, manage, circum vent, and exploit pesticide resistance. These genetic techniques, however will need to be coupled with chemical and biochemical techniques. The biochemical and chemical techniques of biotechnology include syn- thetic and analytical methods. Synthetic oligonucleotides for use as DNA probes to identify genes can be made readily with automated commercial instruments. These DNA probes will succeed MABs as even more useful diagnostic agents for pesticide resistance. Micro quantities of proteins can

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BIOTECHNOLOGY IN PESTICIDE RESISTANCE DEVELOPMENT 133 be sequenced with commercial instruments, and synthetic peptides up to about 20 + amino acid residues can be synthesized routinely. Biophysical techniques utilizing X ray, nuclear magnetic resonance (NMR), and other methods will provide three-dimensional structures of biological macromo- lecules such as proteins; thus, we will be able to correlate structure with pesticide activity or resistance. Gene sequences and resultant protein sequences will be changed by design, using site-specific mutagenesis to change the DNA sequence. For example, p-lactamase, the antibiotic-resistant gene in bacteria, was altered to place a cysteine at the active site in place of the naturally occurring serine. The designed gene produced a novel active ,8-thiollactamase (Sigal et al., 19821. By combining this wealth of information (generated from chemical, bio- chemical, and genetic techniques) with computer graphics, we will be able to design novel pesticides and genes. BIOTECHNOLOGY AND XENOBIOTICS Biotechnology has been intimately involved in antibiotic resistance re- search and development. The techniques of biotechnology identified the basis of resistance, which provided a critical resource for genetic engineering. For example, penicillin and cephalosporins are widely used antibiotics. The ,8- lactam ring of these molecules is essential for their antibiotic activity. Bac- teria, however, have developed resistance to these molecules. The resistance is located on small extrachromosomal circular pieces of DNA called plasmids, and the resistance is specifically due to a gene that makes an enzyme called ,B-lactamase, which cleaves the ,8-lactam ring of these antibiotics and in- activates them. Antibiotic resistance has provided essential selectable markers for follow- ing genetic constructions introduced into cells. Cells containing the new functional genetic material are selected for their antibiotic resistance. The markers have enabled genetic engineering of microorganisms to develop rapidly. Understanding these antibiotics and antibiotic resistances facilitated the knowledge of microbial cell-wall synthesis. The problem of antibiotic resistance has led to several ways to circumvent it. An empirical approach such as the use of clavulinic acid (a naturally occurring suicide inhibitor of ,8-lactamase) in combination with an antibiotic, amoxacillin, is one way to circumvent the resistance problem. Another ap- proach is to develop commercial semisynthetic ,B-lactam antibiotics, which have incorporated within them the ability to also inhibit ,B-lactamase. Of possible greater significance, based on the understanding generated by bio- technology, are current efforts to design drugs to which resistant bacteria are susceptible. In pesticide resistance management, biotechnology can play a key role,

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134 MECHANISMS OF RESISTANCE TO PESTICIDES but much more research is necessary before we can fully exploit these ben- efits. For example, herbicide research and development offers opportunities and limitations. We little understand the mechanisms of action of herbicides; therefore, informed decisions on research and development, safety, and use are limited. The empirical synthesis-screening approach through which al- most all herbicides are discovered is becoming increasingly inefficient; re- searchers must synthesize some tens of thousands of new chemical structures to find a commercial product. Crops usually have inadequate tolerance to herbicides; thus, herbicides are selected for tolerance to specific crops. The lack of broad crop tolerance limits broad crop use of most herbicides, as do soil residues. More herbicide- resistant crops are desirable for broad use of low-cost herbicides and crop rotation. Finally, a few weeds have developed resistance to herbicides such as atrazine, and it may be desirable to manage or circumvent this resistance. The earliest products of crop biotechnology will probably be crops with specific herbicide resistance, followed by designed herbicides. The sulfo- nylurea herbicides illustrate the major role that biotechnology can play in generating understanding of pesticides and, in this case, resistance. The sulfonylurea herbicides demonstrate the integrated role of a number of tech- niques and disciplines. Using empirical synthesis and screening, the du Pont Company developed a novel class of herbicides, some examples of which are Allying, Classics, Gleans, Londax~, and Oust@. These herbicides are very potent, with un- usually low application rates. Plant physiological investigations on the active sulfonylurea compounds in Gleans and Ousts showed that these sulfonylureas rapidly inhibited cell division. Tobacco cell cultures grown on media containing the sulfonylureas yielded cell lines and regenerated plants with a chromosomally localized single resistant gene and a greater than 100-fold increase in resistance to sulfonylureas (Chaleff and Ray, 19841. Further mechanistic studies utilized less complex, more defined microorganismal systems. The sulfonylureas also inhibited the growth of several, but not all, bacteria. The biocidal target of these herbicides was an enzyme, acetolactate synthase (ALS II and III), that is involved in the synthesis of the branched-chain essential amino acids valine, isoleucine, and leucine (LaRossa and Schloss, 19844. Physiological, bio- chemical, and genetic analyses confirmed the target site. Along these same lines a molecular biological characterization showed that a major form of resistance in yeast arises from an altered structural gene for ALS, in which a praline amino acid residue in the sensitive ALS is replaced by a serine in the resistant ALS. Other forms of resistance were also found. The structural ALS resistance gene may be useful as a selectable marker for genetic engineering in higher organisms, as antibiotic resistance has been in bacteria.

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BIOTECHNOLOGY IN PESTICIDE RESISTANCE DEVELOPMENT 135 This rapidly generated base of information in model microorganismal sys- tems led to the identification of ALS as the site of herbicidal activity in plants (Ray, 19841. A less-sensitive ALS was shown to be the basis of herbicidal resistance in resistant tobacco. Other plant studies showed that herbicide selectivity in crop plants arose from metabolism of the sulfonylureas to a nonherbicidal form in the tolerant crops, not to a less-sensitive ALS. Her- bicidal activity can be evaluated directly on the ALS target, thus providing more rigorous structure/activity information than whole plant screens, where activity is the result of ALS activity and penetration, translocation, and detoxification of the sulfonylureas. Biophysical studies on sulfonylureas and ALS at the kinetic and structural levels can provide information on the specific mechanism of inhibition. Opportunities for designed herbicides, designed resistance genes, and the genetic engineering of herbicide-resistant crops come from this multidisci- plinary and multitechnique generation of understanding. Without microor- ganismal techniques and development of model resistance, the time required to generate this level of understanding on the sulfonylureas would have taken several additional years. Although sulfonylureas were used in the above study, similar examples exist for the s-triazine (Arntzen and Duesing, 1983) and glyphosate herbicides (Coma) et al., 19831. The time required to reach an understanding of the s-triazines and glyphosate was much longer than for the sulfonylureas, because the newer biotechnology techniques were not available or not initially used for most of the former studies. BIOTECHNOLOGY IN PESTICIDE RESISTANCE Schematics of the role and potential of biotechnology in pesticide research and development, understanding, management, circumvention, and exploi- tation and in pesticide resistance and development are presented in Figures 1 and 2. The following sections will consider biotechnology in all phases of pesticide resistance. Resistance Development Xenobiotics select or generate resistance broadly in organisms (Georghiou and Mellon, 19831. Fungi, acarina, and insects have shown resistance to fungicides. Bacteria, fungi, nematodes, acarina, insects, crustacea, fish, frogs, rodents, and higher plants have shown resistance to insecticides. Bacteria, yeast, and higher plants have shown resistance to herbicides. This broad occurrence of resistance suggests that by using model systems, we can un- derstand the molecular process of resistance. The model system should be biochemically and genetically well-characterized and as simple as possible, such as a microorganism, although some problems will require more complex

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136 MECHANISMS OF RESISTANCE TO PESTICIDES Empirical Synthesis Pest Screening Biocide discovery Model (Microbial) Systems Mechanism of biocide action Biocide target genes and gene products Surrogate screens Pest-Host Systems Biocide target in pest Molecular biological characterization of target and biocide target interaction In vitro biocide structure target activity relationships Designed biocides Host tolerance/resistance FIGURE 1 Biotechnology pesticide research and development. systems. Development of model resistance in defined organisms will accel- erate the understanding, management, circumvention, and exploitation of resistance. Resistance Understanding Most if not all resistances result from one of three genetic changes. With qualitative change a structural gene is altered so that its protein product is less affected by the pesticide, such as the sulfonylurea resistance gene with its altered ALS enzyme. The other genetic changes are quantitative: gene Empirical Synthesis Resistant Pest Screening Biocide discovery Model (Microbial) Systems Resistance development Resistance types Resistance genetic and gene products Surrogate screens MABs, DNA probes, restriction maps as diagnostics Pest-Host Systems Pest resistance types PesVhost resistance genes and gene products MABs, DNA probes, restriction maps as diagnostics In vitro biocide structure resistance relationships Designed biocides Resistant hosts by somaclonal variation Genetically engineered resistant hosts Natural biocides Synergists Agriregulators FIGURE 2 Biotechnology- pesticide resistance research and development.

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BIOTECHNOLOGY IN PESTICIDE RESISTANCE DEVELOPMENT 137 regulation and gene amplification, in which increased amounts of gene prod- uct make the organism less sensitive to the pesticide. Structural gene changes usually produce stable resistance, while gene amplification changes may be less stable. To understand resistance we need to address the number of types; identify the general types such as target site, metabolism, penetration, reproduction, excretion, storage, and feeding; and define the genetic change responsible. Biotechnology has provided this level of understanding for at least three herbicides-glyphosates, sulfonylureas, and s-triazines where altered structural genes, gene amplification of target-site genes, and altered regu- lation of target-site genes have been demonstrated. In some the product of the altered structural gene is as active as the unaltered (sulfonylureas and glyphosates) or highly fit; in others (s-triazines) the product is less active or less fit. Biotechnology should provide similar definition for other pesticide resistances where an adequate physiological, biochemical, and genetic base exists in an appropriate experimental organism. Effective programs will be highly interdisciplinary using a breadth of biotechnology techniques. Bio- technology can expand our understanding in most if not all areas of pesticide resistance. Unfortunately, biotechnology has been little used in this field. Obvious opportunities are cytochrome P4so in cases of some insecticide re- sistances and ,B-tubulin in the case of benomyl fungicide resistances. Resistance Management In the short term, biotechnology can provide the reagents and techniques for qualitative and quantitative diagnosis of pesticide-resistant organisms. MABs may be useful for measuring structurally altered gene products and an altered quantity of gene products. Restriction maps and DNA probes should be useful, but they will require an expanded base of information. These techniques should enable researchers to define the similarities or dif- ferences of observed pesticide resistances in the same or different labora- tories. They would be used first as research diagnostics, but could become field diagnostics to guide pesticide use practices. Also in the short term, biotechnology would help researchers to establish rigorous pesticide structure/resistance relationships that may differ from pes- ticide structure/activity relationships, especially for altered target sites. Pes- ticide use practice could be guided by this base of understanding. In the midterm, increased understanding of multiplicity, type, and genetic change will result in informed, early decisions on agronomic use practices that will minimize the impact of resistance. For example, gene amplification- based resistances are probably less stable than altered structural-gene resis- tances, suggesting alternation of pesticide use as a desirable practice in the first case. Further, an expanded use of biotechnology will provide significant new opportunities for the more effective management of pesticide resistance.

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138 MECHANISMS OF RESISTANCE TO PESTICIDES Resistance Circumvention Circumvention of resistance may be sought through new pesticides, natural pesticides, synergists, and agriregulants. New pesticides could be discovered by empirical synthesis or design. Empirical synthesis could be coupled with a screen using resistant organisms under continuous pesticide selection pres- sure to discover chemical structures that inhibit critical, nonalterable enzyme or protein sites. This approach realistically assumes the existence of critical sites that cannot be changed and still maintain an adequately fit activity for the pest. Designed synthesis would use target-site knowledge and computer graphics to guide synthesis of novel pesticides to be tested. Target sites could be selected that have low opportunity for change and retention of adequate fitness for the pest. A highly conserved gene such as the quinone-binding protein that is inhibited by the s-triazines is an example of a target site with limited opportunity for change and retention of adequate fitness. Additional critical catalytic sites unique to pests need to be identified. Natural pesticides may circumvent synthetic pesticide resistance. For ex- ample, biocontrol organisms could be genetically engineered to produce natural pesticides. Beneficial organisms such as plants could be genetically engineered for endogenous production of natural pesticides (Schneiderman, 19841. In both, methods for timed bioproduction of the pesticide would be needed, since continuous production would facilitate the development of pesticide resistance. Agriregulators, as described later in this subsection, may be developed for temporal control of biopesticide biosynthesis. Synergists may also circumvent pesticide resistance. These molecules are inactive as pesticides, but they synergize the activity of pesticides. As such they may decrease metabolic detoxification by inhibiting the detoxification system. Genetic, biochemical, and chemical biotechniques may improve our understanding, so that scientists can design synergists or produce quantities of the cloned detoxification system for use as in vitro screens for potential synergists. Genetic engineering may produce naturally occurring synergists, and biotechniques may synthesize modified synergists. Biotechnology tech- niques have been applied to several cytochrome P4so systems but not to any involved in pesticide detoxification. Synthetic compounds that regulate gene expression will be major oppor- tunities for agrichemicals and pharmaceuticals. One or more model examples already exist. The genes for biological nitrogen fixation are not expressed when N2-fixing organisms are grown in an environment containing adequate fixed nitrogen or ammonia. A synthetic molecule, methionine sulfoxamine (MS), causes the expression of the biological N2-fixing genes in the presence of adequate ammonia. Synthetic compounds such as MS will become im- portant useful future agriregulator agrichemicals. They will be discovered by empirical synthesis screening and by designed synthesis as our knowledge . . . O: gene expression increases.

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BIOTECHNOLOGY IN PESTICIDE RESISTANCE DEVELOPMENT 139 The opportunity for agriregulators is expected to be large. Plants may already contain the genetic information for natural pesticides, or genetic engineering will introduce the genetic information into crop plants. Agrire- gulators will be used to turn on the expression at the time of need. The idea that many of the detoxification systems for insecticides are regulated by a common genetic system suggests a major opportunity for an agriregulator that inhibits the genetic system regulating detoxification genes. Resistance Exploitation Xenobiotic resistance genes are and will be useful selectable markers to enable researchers to track and select organisms containing genetic construc- tions. Antibiotic resistance is the most common example, but pesticide- resistant genes will be increasingly used. Herbicide resistance may be used to follow genetic introductions into higher plants. Introducing herbicide-resistant genes into crop plants to increase tolerance and enable crop rotation and the use of herbicides on a broader group of crops is being pursued aggressively and may be the first major practical example of genetic engineering in crop agriculture. Similar approaches may be used to introduce rodenticide and insecticide resistance genes into pets and food animals and insecticide resistance genes into beneficial insects such as bees. With a dynamically expanding base of understanding of basic biological processes, researchers should be able to identify many exploitable targets, not only in agriculture (such as pest control and yield and quality improve- ment) but also in health care, food, energy, pollution control, and chemicals. Application to Pesticides Other than Herbicides Examples of the comprehensive application of biotechnology to fungicide, insecticide, or rodenticide resistance do not exist. An outline for such a study follows, using the rodenticide, warfarin, as the example. Model studies would use microbes to develop warfarin resistance, with emphasis on identifying resistance in a microbe for which the biochemical and genetic information is greatest. The type of resistances and the resis- tance genes and gene products would be identified as previously described for the sulfonylurea resistance microbes. Such resistances for warfarin may involve the biosynthetic pathway for vitamin K. The resistant microbes may provide useful screens to evaluate members of this class of rodenticides for ability to circumvent resistance. Diagnostic approaches such as MABs, DNA probes, and restriction maps may be developed to identify each type of resistance. Information and diagnostics from these model studies should facilitate studies of resistance in the more complex rodent pests. The rodent resistance

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140 MECHANISMS OF RESISTANCE TO PESTICIDES genes and gene products would be identified. Diagnostics would be developed to identify each type of resistance. The rodenticide structure/resistance re- lationships could be measured in vitro, eliminating effects of the nontarget components in the rodent. Rodenticides may be designed to circumvent or minimize the resistance using in vitro tests. Resistant animals such as pets could be developed by genetic engineering so as to decrease effects of ro- denticides on nontarget animals. Natural rodenticides may be produced by biotechnology. Synergists may be developed on the basis of the understanding generated by biotechnology. Similar approaches could be used for fungicides such as benomyl, where an altered p-tubulin is the site of resistance, or for insecticides, where in many cases detoxification by cytochrome P4so systems generates resistance. CONCLUSION Biotechnologies have been used very little in pesticide resistance research and development. Biotechnology has tremendous potential in almost all phases of pesticide resistance investigations and applications, as shown in the sul- fonylurea herbicide example. Biotechnology research and development with this and other herbicides has been useful in resistance development, under- standing, and exploitation. If desirable, biotechnology would also be useful in pesticide resistance management and circumvention. The most successful biotechnology efforts in pesticide resistance, as in almost all other areas, will integrate a multiplicity of biotechnologies by a group of multidiscipli- narians. REFERENCES Arntzen, C. J., and J. H. Duesing. 1983. Chloroplast-encoded herbicide resistance. Pp. 273-294 in Advances in Gene Technology: Molecular Genetics of Plants and Animals, K. Downey, R. W. Voellmy, F. Ahmand, and J. Schultz, eds. New York: Academic Press. Brown, A. W. A. 1971. Pesticide resistance to pesticides. Pp. 457-552 in Pesticides in the Envi- ronment, Vol. 1, Part II, R. H. White-Stevens, ed. New York: Marcel Dekker. Chaleff, R. S., and T. B. Ray. 1984. Herbicide resistant mutants from tobacco cell cultures. Science 223:1148. Comai, L., L. D. Sen, and D. M. Stalken. 1983. An altered aroA gene product confers resistance to the herbicide glyphosate. Science 221:370. Dekker, J., and S. G. Georgopoulos. 1982. Fungicide Resistance in Crop Protection. Wageningen, Netherlands: Centre for Agricultural Publishing and Documentation. Georghiou, G. P., and R. B. Mellon. 1983. Pesticide resistance in time and space. Pp. 1-46 in Pest Resistance to Pesticides, G. P. Georghiou and T. Saito, eds. New York: Plenum. Georghiou, G. P., and T. Saito, eds. 1983. Pesticide Resistance to Pesticides. New York: Plenum. Hardy, R. W. F., and R. T. Giaquinta. 1984. Molecular biology of herbicides. BioEssays 1:152. LaRossa, R. A., and J. V. Schloss. 1984. The sulfonylurea herbicide sulfometuron methyl is an extremely potent and selective inhibitor of acetolactate synthase in Salmonella typhimurium. J. Biol. Chem. 259:8753.

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BIOTECHNOLOGY IN PESTICIDE RESISTANCE DEVELOPMENT Ray, T. B. 1984. Site of action of chlorsulfuron inhibition of valine and isoleucine biosynthesis in plants. Plant Physiol. 75:827. Schneiderman, H. A. 1984. What entomology has in store for biotechnology. Bull. Entomol. Soc. Am. 1984:55-62. Sigal, I., B. G. Harwood, and R. Arentzen. 1982. Thiol p-lactamase: Replacementofthe active- site serine of RTEM p-lactamase by a cysteine residue. Proc. Natl. Acad. Sci. 79:7157. 141

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