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Genetic, Biochemical, and Physiological Mechanisms of Resistance to Pesticides SIMILAR MECHANISMS FOR RESISTANCE to pesticides have been observed in insects, fungi, bacteria, plants, and vertebrates. These include changes at target sites, increased rates of detoxification, decreased rates of uptake, and more effective storage (compartmentalization) mechanisms. The relative importance of these mechanisms varies among the classes of pests. Most resistance to pesticides is inherited in a typical Mendelian fashion, but in some cases, resistance can be attributed to, or influenced by, relatively unique genetic and biochemical characteristics, e.g., extranuclear genetic elements in bacteria and higher plants. A thorough understanding of the genetic, biochemical, and physiological mechanisms of pesticide resistance is essential to the development of solutions to the pesticide-resistance problem. GENETIC BACKGROUND Insects, vertebrates, most higher plants, and fungi of the class Oomycetes are diploid, and some fungi are dikaryotic. Therefore, the gene or genes responsible for resistance may exist in duplicate. Multiple allelic forms are known for many resistance genes. These alleles often produce an effect that is greater than additive. In some cases a resistance gene may exist in multiple copies, a condition called gene amplification. This is known to occur, for example, in the insects Myzus and Culex. Several genes at different loci also can be involved in resistance. Most fungi are haploid in their vegetative state, as are bacteria generally, although multiple genomes are found in actively growing cultures. In a 45

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46 MECHANISMS OF RESISTANCE TO PESTICIDES haploid state' the expression of each gene involved in resistance is not m.od- if~ed by another allele as in the case of the d~ploid organism. Many fungal cells, however, are multinucleate and heterokaryotic with respect to resistance genes, and these genes can produce a modification of resistance expression analogous to that found in diploid organisms. Furthermore, the resistance level of the organism is frequently the result of the interaction of alleles of several genes at different loci. This interaction is known as polygen~c resis- tance. An additional complication in bacteria is the existence of ex~achro- mosomal genes, which can act alone, or in concert with chrom.oson~ genes, to confer resistance. In plants, herbicide resistance can be inherited In the- plast~d genome. Genes that can mutate to confer: resistance to a pesticide may be Bit-her structural or regulatory (Plapp' this volume). Some structural genes are francs lated into products (enzymes, receptors, and other cell components, such as r~bosomes and tubul.in.) that are targets for pesticides. The mutation of struc- tural genes can result in a critical modification of the gene products' such as decreases in target site sensitivity or increased ability to metabolize ~s- ticides. Regulatory gene products may contra! rams of structural gene tran- script~on. They may also recognize and hind. pesticides and thereby control induction of appropriate detoxifying enzymes A clear and detailed understanding Of the molecular genetic apparatus of the resistant organism can provide essential intonation for devising tools and strategies for avoidance and management of practical pesticide resistance problems. Specific examples of the utilization of genetic inf.ormabon for these purposes have been discusses} elsewhere in this volume (Gressel, Hardy' Plapp). Some examples include: (~) the construction. of genetically defined organisms for investigation of the hi~hemical mechanism of pesticide Avon. and for studies on population dynamics of biotypes tot are heterozygous or polygenic for pesticide resistance; (2) the rational design of synthetic antag- onists to combine with regulatory proteins and block the induction of detox- ifying enzymes; (3) genetic ~ginemng of herbicide-re~stant plows' insecticide- resistant beneficial insects, and microbial antagonists, and (4) preparation of monoclonal antibodies for; rapid and specific detection of resistance in a pew population. Ideally, this research should' lead to die isolations cloning, and sequencing of alleles conf.emog resistance and eluc~d~ation of their structure relative to their susceptible alleles.. BIOCtIE1~ICAL MECHANISMS In insects and plants the principal biochemical mechanisms of res~s-tance are (Plapp, Gre$sel, this volume). (1) reduction in. the sensitivity of target sites;. (2) metabolic detoxicadon of the pC$tiCi~ by enzymes such as ester- ase$, monooxy-g.enases, and glutath~one--$~1fot~ans.fera$e$; and (3) decreased

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MECHANISMS OF RESISTANCE TO PESTICIDES 47 penetration and/or translocation of the pesticide to the target site in the insect. Alleles involving alteration of target sites include altered acetylcholinesterase resistance to organophosphates and carbamates, alterations in the gene for the receptor protein target of DDT and pyrethroids, and changes in the receptor protein target for cyclodiene insecticides. Metabolic resistance in the house fly seems to be under the control of a single gene whose product is a receptor protein. This protein binds insecticides, and the protein: insecticide complex induces synthesis of multiple detoxifying enzymes. Whether or not similar metabolic receptor proteins exist in other insects is not known. Decreased penetration has a minor or modifying effect on the level of re- sistance. A minor change in penetration, however, may have a profound effect upon the pharmacokinetics of a toxicant. In plant pathogenic fungi, resistance has been attributed mainly to single gene mutations that (1) reduce the affinity of fungicides for target sites (e.g., ribosomes, tubulin, enzymes); (2) change the absorption or excretion of the fungicides; (3) increase detoxication, for example, reducing the toxicity of Hg+ + and captan by an increase in the thiol pool of the cell (see Georgo- poulos, this volume, for details). Most cases of practical fungicide resistance can be attributed to the first mechanism, which often results in a striking increase in resistance level brought about by mutation of a single gene. For this reason, fungicides that act at a single target site are at great risk with respect to the possibility of resistance development (Dekker, this volume). Resistance to other fungicides, such as ergosterol biosynthesis inhibitors and polyene antibiotics, occurs through a polygenic process. Each gene mutation produces a relatively small, but additive, increase in resistance. When many mutations are required to achieve a significant level of resistance, there is an increased likelihood for a substantial loss of fitness in the pathogen. There have been no major outbreaks of resistance to these fungicides in the field, but this situation is changing rapidly and problems are beginning to occur with the ergosterol biosynthesis inhibitors (Butters et al., 1984; Gullino and DeWaard, 19841. Three bactericides are used to control plant diseases in the United States: copper complexes, streptomycin, and oxytetracycline. Resistance to strep- tomycin in Erwinia amylovora, the pathogen of fireblight disease of pear and apple trees, has been a widespread problem. Resistance appears to be controlled by alteration (or mutation) of a structural chromosomal gene that reduces the affinity of the bacterial ribosome for streptomycin, an inhibitor of protein synthesis (Georgopoulos, this volume). In contrast, the most com- mon mechanism of streptomycin resistance in human bacterial pathogens is mediated by an extrachromosomal (plasmid) gene that regulates the produc- tion of an enzyme (phosphorylase) that detoxifies streptomycin. The appli- cation of oxytetracycline to control streptomycin-resistant strains of Erwinia amylovora on pear trees is a relatively new practice, and reports of tetracycline

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48 MECHANISMS OF RESISTANCE TO PESTICIDES resistance have not yet appeared. Oxytetracycline has been injected into palm trees and stone fruit trees for several years to control mycoplasmalike or- ganisms, apparently without the development of resistance. In Xanthomonas campestris pv. vesicatoria (which causes bacterial leaf spot of tomatoes and peppers), resistance to copper is conferred by a plasmid gene that appears to regulate the absorption of copper ion by the bacterial cell. Plants utilize the same general resistance mechanisms as insects. The efficacious use of herbicides on crops is made possible because many crop plants are capable of rapid metabolic inactivation of the chemicals, thereby avoiding their toxic action. Target weeds are notably deficient in this capacity. It is apparent, though, that the capability to metabolize herbicides to innoc- uous compounds constitutes a potentially important basis of evolved resis- tance to herbicides in weeds. Documented cases of resistance have been due to other mechanisms, however, such as alteration of the herbicide's target site. For example, newly appearing s-triazine-resistant weeds have plastid- mediated resistance that involves a reduced affinity of the thylakoids for triazine herbicides (Gressel, this volume). The herbicide paraquat disrupts photosynthesis in target weeds by inter- cepting electrons from photosystem I, part of the metabolic cycle that fixes energy from sunlight into plant constituents via a complicated flow of elec- trons. Transfer of electrons from paraquat to oxygen gives rise to highly reactive oxygen radicals that damage plant membranes. Paraquat-resistant plants have higher levels of the enzyme superoxide dismutase, which quenches the reactive oxygen radicals. The mechanisms of weed resistance to the dinitroaniline herbicides and to diclofop-methyl have not yet been identified. A number of herbicides act on the photosynthetic mechanism in the chlo- roplasts. Although the frequency of resistant plants arising from plastic mu- tations would normally be very low, a plastome mutator gene has been recognized that increases the rate of plastome mutation in weeds. This factor could be largely responsible for the plastic-level resistance to herbicides that has emerged in some weeds (Gressel, this volume). Resistance to anticoagulants is the most widespread and thoroughly in- vestigated heritable resistance in vertebrates. Warfarin resistance in rats has been observed in several European countries, and in 1980 more than 10 percent of rats were resistant to warfarin in 45 out of 98 cities surveyed in the United States (Jackson and Ashton, this volume). Warfarin interferes with the synthesis of vitamin K-dependent blood-clot- ting factors in vertebrates. Resistance in rats (Rattus norvegicus) appears to involve a reduced affinity of a vitamin K-metabolizing enzyme or enzymes for warfarin. The affinity of the target site is controlled by one (of four) allelic forms of a gene in linkage group I. In the mouse, there are indications that increased resistance to warfarin is due to metabolic detoxication and that

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MECHANISMS OF RESISTANCE TO PESTICIDES 49 the detoxication system (mixed function oxidase) is controlled by a gene cluster on chromosome 7 (MacNicoll, this volume). Our knowledge of re- sistance mechanisms in rodents and other vertebrate pests is fragmentary. PROMISING RESEARCH DIRECTIONS AND THEIR IMPLEMENTATION Synthetic chemicals will probably continue for some time as the major weapon against most pests because of their general reliability and rapid action, and their ability to maintain the high quality of agricultural products that is demanded by urban consumers today. Although new chemicals offer a short- term solution, this approach to pest control alone will rarely provide a viable, long-term strategy. Moreover, a few years of commercial exploitation may not justify the investment required to develop a new pesticide today, except where there are reasonable prospects that a pesticide's mode of action may be beyond the capability of the pest for genetic adaptation. Despite the continual threat of resistance, we may still be able to exploit our expanding knowledge of the genetic and biochemical makeup of pests by designing pesticides that can circumvent existing resistance mechanisms, at least long enough to provide chemical manufacturers a reasonable rate of financial return on the investment needed to develop a new pesticide. Real- istically, though, it is difficult to be optimistic on this point in practical situations where a synthetic pesticide is applied repeatedly to the same crop or environment to control a well-adapted pest. History promises no encour- agement, at least for most pests, for the discovery of a "silver bullet." On the other hand, it is indeed encouraging that there are examples of pesticides, both selective and nonselective (e.g., the polyene fungistat pimaricin, the widely used herbicide 2,4-D, and the insecticides azinphosmethyl and car- bofuran), that have been used for years in certain situations without setting off rapid, extensive resistance. The phenoxy herbicides (e.g., 2,4-D) and the broad-spectrum fungicides (captan, dithiocarbamates, and fixed coppers) have been used successfully for decades without serious resistance problems. Still, the wisest course for future research appears to be the integration of a diversity of approaches to pest control chemical, biological, and cultural (or ecological)- because an integrated application of multiple methods will produce minimum selection pressure for development of resistance to pes- ticides. Evolution of resistance to minimally selective or multitarget synthetic chemicals might be delayed indefinitely if the selection pressure were kept within "reasonable" limits. The pressure might be reduced with crop rota- tions and careful management, but may be virtually impossible in agricultural areas typified by repeated monocultures. The development of resistance is encouraged by pesticides that act upon single biochemical targets. Unfortunately, the modes of action of many sys- temic plant fungicides, and most modern synthetic insecticides and herbi

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so MECHANISMS OF RESISTANCE TO PESTICIDES cides, are biochemically site-specif~c. Many of these fungicides arid Insecticides have produced a rapid, major buildup of resistance genes in pest populations after just a few seasons of use. Undoubtedly, the potential for resistance development to such compounds will continue to be a limiting factor in the widespread use of these compounds, although compounds differ in the degree of risk for rapid development of resistance. In addition, some compounds lend themselves to relatively effective resistance management strategy. Oth- ers do not. The genetic and biological reasons that some compounds rapidly select for resistance, whereas others do not, are presently obscure in nearly all cases. Further research in this area will greatly facilitate the development of efficacious strategies to manage resistance. RECOMMENDATIONS RECOMMENDATION 1. A major increase in research on the genetics, bio- chemistry, and physiology of resistance is recommended for all pest classes- insects, fungi, bacteria, weeds, and vertebrates. Research support should not be restricted to or allocated primarily on the basis of the economic importance of crops. Research should include studies of genetic mechanisms in wild and resistant populations, with emphasis on common gene pools, gene flow between related species, gene sequencing, and population dynamics. Biochemical and physiological studies should be encouraged on pes- ticidal mode of action, characterization of target site enzymology, pharmacoki- netics, and the transport, metabolism, and excretion of xenobiotics in pest spe- cies. The compilation and dissemination of data in these areas is essential to the identification of unique target sites less apt to develop resistance. Such data are essential in designing novel pesticides that exploit genetic weaknesses and bypass genetic capabilities to develop resistance. It is reasonable to anticipate that agents could be developed, for example, that are superior to existing cholinesterase inhibitors for insect pests, or to chemicals that inhibit macromolecular synthesis integral to the function of microorganisms. The research agenda is formidable. For most plant pathogens, virtually nothing is known that would be useful in the rational design of new fungicides and bactericides. To a lesser extent, this also appears to be the case for insects, weeds, and rodent pests. Significant efforts are in progress for the design of herbicides, however. RECOMMENDATION 2. Use molecular biology and recombinant DNA tech- nology to isolate, identify, and characterize the genes and gene products (enzymes and receptors) conferring resistance to pesticides and to compare these products with their alleles in susceptible pests. Use of microbial models, as appropriate, may facilitate progress in this area. Molecular biology has much to offer as a tool for elucidating the nature of

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MECHANISMS OF RESISTANCE TO PESTICIDES 51 pesticide target sites, particularly in promins. These techniques can define re- sistance due to changes in structural genes, amplification of a structural gene, and alteration in regulation. Using bactena ~ clone ~struetura1 genes (or DNA fragments) coding for pesticide-metaling enzymes can provable ~ mews for determining how these genes are regulated. These techniques can hop determine the mechanism of operation of genes that appear to carry out common -regulatory functions in insects, such as controlling He coordinated expre~-mn for structure genes that code for different enzymes involved in pesticide d~radabon. Other applications of molecular biology techniques could involve the insertion of genes for toxin production into insect-inhab~ting bacteria, -fungi, or vimses. Genes for resistance to insects or plant pathogens based on the production of allelochemicals might also be transferred from nonhost species ~ crop plant hosts. RECOMMENDATION 3. Conduct research on pesticide Bet bi~h~is~y to identify unique sites in pests that can serve as models [or the design of new pesticides. The development of fungicides that inhibit erg-osterot biosynthesis is a good example of the successes Mat can evolve from such a research program. It may also be possible to design pesticides that aback more than one target site, at least for most pests. '`Target site" research shout reveal opportunities for He systematic combination of commands Hat Assess negatively corTelat~l cross- resistance traits that exploit ;structur~ differences ~ the "target site" In resistant biotypes. Several clear--cut examples of compounds that are negatively correlated with respect to cross-resistance can be found in some carbamate pesticides (Geor- gopoulos, Plapp, this volume). To further the development of new rodentic~des, research Is required to es- tablish the selective affinities -of ant~lant-s and substrates for the target site. Such understanding would gready facil~ta~ the rational design of chemical agents to potentiate the action of anticoagulants author minimize detoxication. A major focus of target biochemistry should be the identification of novel systems for exploitation, rather than exclusively smdy~ng and characterizing the -targets -of existing compounds. In the future, greater understanding of target site biochemistry may make it possible to design pesticides that are themselves r-es~stant to pests' detoxication mechanisms, as is already Beirut -d-~ne for some of the semisynthetic penicillins that inhibit bactenal ,`3-lac~mase (see Hardy, the volume). Also, possibilities for the development of new s-ynergist~ relatiionsh~ps would be greatly expanded by detailed information on receplorlinhibitor interactions and the -metabolism of pesticides in resonant mu~n-ts~ Bl:COMP~ENDATION 4. Conduct research on the enzymology and Chars macokinetics of Pesticides in both to; and nontarget species.

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52 MECHANISMS OF RESISTANCE TO PESTICIDES Classical enzyme kinetics does not accurately describe the behavior of potential xenobiotics that are reactive at extremely low concentrations. A slight reduction in the rate of penetration of the xenobiotic into the pest may result in a drastic reduction in the reaction with the enzyme. In addition to inhibitors of detoxifying enzymes, other potentially fruitful areas for synergist research include compounds that interfere with the induction of detoxifying enzymes, agents that block active secretion (e.g., the fungicide fenarimol), and compounds that inhibit binding of anticoagulants by serum albumin in rats. RECOMMENDATION 5. Initiate research on new pesticides and on new ways to use existing pesticides that emphasizes compounds and procedures that result in minimum selection pressure on the pest population. Pesticides with one or more of the following properties would be useful in resistance management: (1) compounds that suppress target pest populations while allowing predators and parasites to multiply; (2) compounds (such as insect growth regulators) that are not lethal, but which effectively prohibit normal reproduction; (3) microbial pesticides, including bacteria, fungi, and viruses; (4) compounds related to the broad-spectrum fungicides (e.g., multisite electro- philes) that have been used for many years under high selection pressure with few problems with resistance; and (5) agents that control fungus diseases of plants by intensifying the natural defense reactions of the plant, such as the localized death of plant cells when infection by the pathogen is attempted (e.g., probendazole). Furthermore, broad-spectrum fungicides give satisfactory control in many disease situations; selective systemic compounds should be restricted to use in situations where systemic activity or postinfection activity is essential to disease control. REFERENCES Butters, J., J. Clark, and D. W. Hollomon. 1984. Resistance to inhibitors of sterol biosynthesis in barley powdery mildew. Meded. Fac. Landbouwwet. Rijksuniv. Gent. 49/2a:143-151. Gullino, M. L., and M. A. DeWaard. 1984. Laboratory resistance to dicarboxim~des and ergosterol biosynthesis inhibitors in Penicillium expansum. Neth. J. Plant Pathol. 90:177-179. WORKSHOP PARTICIPANTS Genetic, Biochemical, and Physiological Mechanisms of Resistance to Pesticides JOSEPH W. ECKERT (Leader), University of California, Riverside HUGH D. S~s~ER (Leader), University of Maryland S. G. GEoRGoPoucos, Athens College of Agricultural Sciences, Greece JONATHAN GRESSEE, The Weizmann Institute of Science

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MECHANISMS OF RESISTANCE TO PESTICIDES 53 BRUCE D. HAMMOCK, University of California, Davis JOHN M. HOUGHTON, Monsanto Agricultural Products Company DALE KAUKEINEN, ICI Americas, Inc. ALAN MAcN~cHo~, Ministry of Agriculture, Fisheries and Food, Great Britain R. L. METCALF, University of Illinois TOM O'BRIEN, Brigham and Women's Hospital, Boston FREDERICK W. PEAPP, JR., Texas A&M University NANCY RAGSDAEE, U.S. Department of Agriculture JAMES E. TAVARES, National Research Council