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Mechanisms of fungicide resistance in phytopathogenic fungi
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Mechanisms of fungicide resistance in phytopathogenic fungi James J Steffens*, Eva J Pellt and Ming Tien The disciplines traditionally used to investigate the mode of action of fungicides have been biochemistry and physiology. Over the past decade, classical and molecular genetics have been brought to bear on this problem with increasing success. Recently, genetic studies of fungicide resistance have led to advances in our understanding of the site of action of chemicals active against plant pathogens and, in some cases, to an appreciation of additional mechanisms of resistance to fungicide action.
Addresses *DuPont Agricultural Products, Experimental Station, PO Box 80402, Wilmington, Delaware 19880-0402, USA; e-mail: steffejj@al .esvax.umc.dupont.com tDepartment of Rant Pathology, PennsylvaniaState University, University Park, Pennsylvania16802, USA $Department of Biochemistryand Molecular Biology, Pennsylvania State University, University Park, Pennsylvania16802, USA Current Opinion in Biotechnology 1996, 7:348-355 © Current Biology Ltd ISSN 0958-1669 Abbreviations OHO dihydro-orotatedehydrogenase DMI demethylationinhibitor
Introduction T h e appearance of fungicide resistance among agronomically important fungal pathogens is sometimes a key factor in limiting the efficacy and lifetime of important disease control strategies [1,2]. Even so, resistance may also be an important aid to our understanding, at a molecular level, of the fungicidal mechanism of action of a particular class of chemicals [3,4"']. Once understood, target site based resistance may become useful as a selection mechanism in fungal DNA transformation protocols. Finally, fungicide resistance, when based upon multi-drug resistance genes, is becoming a target in its own right for fungicide screening and design.
T h e intent of this review is to summarize recent advances in our knowledge of fungicide resistance, with an emphasis on examples where molecular genetics has contributed substantially to this understanding. We concentrate on examples of various fungicides where target site based resistance is understood, and finally the case of triazole resistance, where multiple mechanisms of resistance have been described. F u n g i d d e r e s i s t a n c e d u e t o m u t a t i o n s in t h e target gene It is generally assumed that most fungicides exert their effect by interacting with a specific protein target molecule. In the past, identification of this target has depended
on the collation of biochemical and physiological evidence. Because fungicides can often produce effects that are only indirectly linked to the immediate site of action, the determination of direct cause-and-effect relationships can prove very difficult. Increasingly, researchers are turning to the genetics of fungicide resistance to understand the mechanism of action of a particular chemical or of a class of fungicidal chemicals. It is first necessary to identify a resistant mutant, in which the resistance is due to mutation in a single gene, producing alterations most likely at the site of fungicide action, rather than changes in uptake, efflux, or metabolism of the fungicide. A gene that confers resistance upon a wild-type strain can then, in principle, be isolated using the techniques of fungal DNA transformation. High-efficiency transformation protocols are available in a number of fungi, including several agronomically important plant pathogens (e.g. Alternaria,
Cercospora, Cladosporium, Cochliobolus, ColletotHchum, Gaeumannomyces, Magnaporthe, and Ustilago). T h e availability of DNA sequence databases and the capability to search them rapidly make gene identification increasingly straightforward, at least to the level of protein family by means of motif homology. T h e final step in identification is to demonstrate that transformation of a wild-type strain with a single mutant gene is sufficient to confer resistance. Several shortcuts are often available for identification of a target gene. If a particular molecular target is strongly suspected, then homologous or heterologous probes for the suspected gene will aid in its isolation from the target organism. T h e use of an origin of replication from Aspergillus nidulans [5-8] or Ustilago maydis [9] makes it possible to recover the transforming DNA as a self-replicating plasmid. T h e Aspergillus origin of replication may be employed on a helper plasmid in a co-transformation protocol. Apparently, a recombination event occurs between the two plasmids following transformation, leading to a self-replicating recoverable plasmid. Although the origin of replication from A. nidulans appears to function successfully only in closely allied species [10], it is possible to co-transform Aspergillus using heterologous DNA and the origin of replication on separate plasmids [11]. Thus, it should be possible to utilize Aspergillus for the selection of a fungicide resistance gene from another species of ascomycete, as long as Aspergillus is sensitive to the fungicide and the phenotype of resistance is dominant. T h e Ustilago origin of replication has been used to clone a gene responsible for dicarboxamide resistance (vide infra). Benzimidazoles Studies to elucidate the mode of action of the benzimidazole class of fungicides were the first to utilize classical
Mechanisms of fungicide resistance in phytopathogenic fungi Steffens, Pell and Tien 349
Figure 1 Classical and molecular genetics have contributed greatly to our understanding of the mechanism of action of the fungicides whose chemical structures are shown to the right. (a) Benomyl, a benzimidazole fungicide, interferes with the cell cycle by preventing the polymerization of et,l~-tubulin dimers into microtubules. (b) Carboxin is an inhibitor of succinate dehydrogenase. (c) Vinclozolin, an example of a dicarboximide fungicide, appears to interact with a serine/threonine protein kinase. (d) ICIA5504, a stobilurin analog, is a specific inhibitor of mitochondrial respiration by binding to cytochrome b. (e) LY214352, a phenoxyquinoline, interferes with pyrimidine biosynthesis by inhibiting DHO. (f) Pyrimethanil, an example of a anilinopyrimidine fungicide, acts by inhibiting the infection process. (g) The natural product soraphen A inhibits acetyl-CoA carboxylase. (h) Triadimenol is an example of a broad class of fungicides that interfere with sterol biosynthesis by inhibiting the P-450 sterol 14ct-demethylase.
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genetics and later the methods of molecular genetics, using benzimidazole-resistant mutants. At the outset, there was considerable evidence that benzimidazoles, such as benomyl (Fig. la), interfere with fungal cell division and bind to proteins with molecular weights similar to that of tubulin [12]. T h e analysis of benzimidazole-resistant mutants of Aspepgillus demonstrated that resistance could be correlated with changes in benzimidazole binding to tubulin. Gene isolation and sequence analysis then firmly established that resistance to benzimidazoles is due to specific mutations in the gene coding for I~-tubulin. T h e understanding that has emerged from these and subsequent studies is that fungicidal benzimidazoles bind specifically to 13-tubulin and inhibit the non-covalent polymerization of ct,13-tubulin dimers into stable microtubules
[121. T h e 13-tubulin genes from a variety of organisms have been isolated and sequenced [13,14]. Comparison of these sequences helps to explain not only why compounds such as benomyl are specific inhibitors of fungal tubulin polymerization, but also why species of yeasts and oomycetes are resistant; in general, resistant organisms contain amino acid substitutions in I~-tubulin at the critical positions that are found in benzimidazole-resistant ascomycete isolates. Subsequently, genes encoding benzimidazole-resistant [3-tubulin have been used as
dominant selectable markers for homologous [15-17] and heterologous fungal transformation [18]. Although many different 13-tubulin mutations have been created through chemical and UV-mutagenesis [12], very few of these mutations appear to be viable and/or pathogenic under field conditions [19,20"]. This phenomenon has allowed the development of methods based on PCR technology for the detection of specific field isolates that are resistant to benzimidazoles [21-24]. These methods are fairly rapid and are designed to aid in making decisions regarding field applications of fungicides.
Carboxin Carboxin (Fig. lb) is another comparatively old fungicide, with commercial levels of activity, particularly against basidiomycete pathogens. Recently, a gene from a carboxin-resistant strain of U. maydis has been cloned, sequenced, and shown to be homologous to known genes encoding the iron-sulfur subunit of succinate dehydrogenase [25]. Transformation of wild-type strains with this gene was sufficient to confer carboxin resistance. Subsequent comparison of sequences from wild-type and resistant strains demonstrated that mutation of two contiguous base pairs, within the codon for a single amino acid of a highly conserved region, was responsible for the resistant phenotype [26,27]. Carboxin has, at least in vitro,
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activity against Aspergillus and other ascomycetes. T h e r e have been no reports of the use of the carboxin-resistant genes as selectable markers for transformation, either in Ustilago or in other species.
Dicarboximides T h e dicarboximide fungicides are a class with several commercially successful examples that are active against Botrytis cinerea and numerous pathogens affecting vegetable crops. Vinclozolin (Fig. lc) is one such dicarboximide. Studies over several years have been carried out to define the mechanism of action [28], without convincing success. A comparatively low level of resistance to dicarboximides has often been reported among field isolates of pathogenic fungi, whereas isolation of resistance in the laboratory routinely results in more resistant strains [28]. High-level resistance shows some pleiotropic effects: pronounced sensitivity to high osmotic stress and cross-resistance to other fungicides, including the newer cyanopyrrole class [29,30]. It is highly probable that resistant strains isolated in the laboratory do not possess sufficient fitness to be pathogenic in the field. Neither of these two resistant biotypes has provided evidence concerning the mechanism of action of the dicarboximides. To elucidate the mode of action of the dicarboximides in U. maydis, the mechanism of resistance to vinclozolin has been investigated [31]. A large number of resistant mutants were isolated, which could be grouped into three complementation groups by subsequent genetic analysis. One of the mutants, U. maydis VR43, carrying resistance gene adr-1, was further characterized [32]. A cosmid DNA library was constructed from this mutant in an autonomously replicating vector and pooled DNA was used for transformation of wild-type U. maydis. A 32 kb cosmid conferring resistance to vinclozolin was isolated after four rounds of sib selection. Restriction analysis of the cosmid led to isolation of an 8.7kb fragment. Sequence analysis of this fragment revealed a 1218bp open reading frame coding for a serine/threonine protein kinase. Residues essential for kinase catalytic function are conserved within this gene. T h e role of the protein kinase gene adr-1 in conferring resistance was further demonstrated by deleting a 384bp NarI fragment from the coding region. Transformation of wild-type U. maydis with this modified construct did not result in fungicide resistance, confirming the role of the protein kinase gene. There are several explanations for how a modified protein kinase gene might confer vinclozolin resistance in U. maydis. It has been suggested that vinclozolin and other dicarboximide fungicides have structural similarity to the substrates of protein kinases [32]. This group of fungicides contains amide bonds and methyl groups positioned equivalently to the alcohol side chain of serine. Serine is the protein amino acid residue that is phosphorylated by the serine/threonine protein kinases; vinclozolin may be toxic by competing at the active site of the targeted protein
kinase with its natural substrate. An alternate explanation is that the protein kinase in the mutant U. maydis strain VR43 confers resistance indirectly. T h a t is, the protein kinase itself may not be the target of the fungicide; rather, its altered activity could circumvent inhibition by the fungicide at another site in the cell.
Strobilurin analogs T h e strobilurin analogs represent the first broad-spectrum class of fungicides since the development of the demethylation inhibitor (DMI) fungicides. Already, three development candidates have been announced in this chemical class [33,34"], one of which is A5504 (Fig. ld). Their structure is derived from a series of natural products--particularly strobilurin, oudemansin and myxothiazole - - found in certain basidiomycetes and myxobacteria. Aside from somewhat lower activity against the eukaryotic organisms from which some of these natural products are isolated, the strobilurin analogs have remarkable efficacy against a broad range of ascomycetes, basidiomycetes, and oomycetes. It was recognized early in the study of the original natural products that these compounds owe their fungicidal activity to inhibition of mitochondrial respiration at the level of complex III [35,36]. Subsequently, a series of elegant experiments was carried out involving yeast mutants resistant to the natural products, in which it was demonstrated that resistance is due to mutations in the mitochondrially encoded gene for apocytochrome b [37,38"]. More recent data have confirmed that synthetic compounds, designed for optimized fungicidal activity, selectivity and stability, also interact specifically with cytochrome b [39]. To date, all reported resistance studies on strobilurin analogs have been carried out in yeast, and there have not yet been any reports of resistant strains of phytopathogenic fungi, either from the field or in the laboratory. Because this is the first known case where the target protein of a fungicide is mitochondrially e n c o d e d - - a l t h o u g h the target of carboxin is a mitochondrial protein, the iron-sulfur subunit is encoded in the n u c l e u s - - i t will be interesting to see whether, and to what extent, target site based resistance develops to this class of fungicides.
Miscellaneous fungicides There exist several additional e'xamples of fungicidally active materials that deserve some mention. T h e phenoxyquinolines, such as LY214352 (Fig. le), are a group of compounds with appreciable in vitro activity, although whole-plant disease control is best against Botrytis and VentuHa. Although, to date, no development candidate has been announced from this class, it is notable because of the early and successful use of classical and molecular genetics to determine the site of action. In these studies, mutants of A. nidulans resistant to LY214352 were developed [40], and a cosmid library
Mechanisms of fungicide resistance in phytopathogenicfungi Steffens, Pell and Tien 351
was prepared from one of them [41,42]. A cosmid conferring resistance to a wild-type strain was found and sub-cloned to yield an open reading frame with homology to prokaryotic dihydro-orotate dehydrogenase (DHO), an enzyme involved in pyrimidine biosynthesis. Enzyme assays confirmed that the DHO enzymes from the resistant strains had diminished sensitivity to the inhibitors. Concomitant feeding studies showed that inhibition by LY214352 could be reversed by the addition of uridine or uracil. Significantly, fungicidal effects could also be reversed by co-enzyme Q6, a co-substrate in the DHO-catalyzed reaction, but not by dihydro-orotate itself. These results suggest that the phenoxyquinolines may bind to the co-enzyme domain of the active site. Genes encoding resistant forms of DHO have been proposed as selectable markers for fungal transformation [P1]. Acetyl-CoA carboxylase has long been a target for herbicide design. Several chemical classes are active against this target, with high selectivity for the enzyme from gramineous species. Recently, an antifungal natural product named soraphen A (Fig. lg) was isolated from a species of myxobacteria [43]. Biochemical studies have shown that the effects of soraphen A are reversed by fatty acids [44]. Using extracts of Ustilago zeae in in vitro experiments, it has been demonstrated that the natural product blocks fatty acid biosynthesis when acetyl-CoA is used as precursor, but that synthesis is unaffected when malonyl-CoA is employed. Subsequently, acetyl-CoA carboxylase has been purified from U. zeae and shown to be highly sensitive to soraphen A. Additional experiments in yeast have confirmed that mutants resistant to soraphen A are tightly linked to the accl locus, which codes for acetyl-CoA carboxylase [45]. Although highly active against both fungi and mites in field experiments, and inactive against the acetyl-CoA carboxylase from higher plants, soraphen A was dropped from development because of toxicological concerns [44]. Nevertheless, this work confirms acetyl-CoA carboxylase as a potential fungicide target. T h e ACCI gene from U. maydis has recently been cloned [46]. Blasticidin is a complex natural product, obtained by fermentation, that is used against rice blast disease caused by Magnaporthe gtisea. There have been no reports of resistance to blasticidin in isolates of Magnaporthe in the field. Even so, a gene that encodes an enzyme catalyzing the deamination of blasticidin has been cloned from Aspepgillus terreus isolated from rice paddy soil, and this has been used as a selectable marker for transformation of M. gtisea and Schizosaccharomyces pombe [47,48]. Because blasticidin is a selective fungicide (with no activity against Aspergillus, for example), the utility of this vector in filamentous fungi other than Magnaporthe remains to be demonstrated. Mention should be made of the anilinopyrimidines. No fungi resistant to these compounds have been reported;
this is a mixed blessing as fungi exhibiting target site based resistance Would be extremely informative and useful. Three examples of anilinopyrimidine fungicides, such as pyrimethanil (Fig. If), are now at or nearing commercialization, with activity against cereal diseases as well as Botrytis and Venturia. A series of studies have shown that these compounds have little effect on conidial germination and germ-tube growth; instead, they appear to inhibit the infection process (summarized in [49]). Subsequent investigations have demonstrated that the secretion of enzymes involved in the infection process, such as polygalacturonase, pectinase, cellulase, and proteinase, is significantly reduced by fungicide treatment and, furthermore, that the intracellular level of enzymes normally secreted dramatically increases [50,51]. Control experiments demonstrate that enzyme glycosylation is not affected by fungicide treatment. Seemingly in line with the inhibitory action of anilinopyrimidine fungicides on protein secretion is the finding that fungi are more susceptible to these compounds when they are grown on gelatin as a source of nitrogen than when they are grown on potato dextrose agar, which contains many free amino acids [51]. There are data, however, suggesting that specific sulfur-containing amino acids [52], or valine and leucine in another study [53], may specifically counteract growth inhibition by these fungicides. No experiments with fungicide-resistant mutants have been reported as yet. Such mutants might be chosen by selecting for strains that are able to grow on protein substrates in the presence of normally inhibitory concentrations of fungicide. An analysis of such mutants may resolve the existing uncertainties in our understanding of these compounds. Furthermore, if the hypothesis concerning inhibition of protein secretion is correct, the anilinopyrimidines may provide a means of unraveling the processes of fungal protein secretion, much as benzimidazoles have increased our appreciation of the importance of microtubule formation in the cell cycle. Demethylation Mode of action
inhibitor (DMI) fungicides
T h e DMI group of fungicides comprises a large number of commercially successful compounds, such as triadimenol (Fig. lh), which have activity at comparatively low use rates against a wide variety of cereal, vineyard, and orchard pathogens [54]). Other analogs are used to treat human and animal mycoses. As a class, these compounds act by inhibiting the cytochrome P450 dependent oxidative demethylation of eburicol in filamentous fungi (or lanosterol in yeasts) in the ergosterol biosynthetic pathway; thus, members of this class are designated DMIs. T h e bulk of the evidence in support of this site of action was obtained from investigations of the effects of DMI fungicides on the levels of sterol intermediates isolated from treated fungi, from spectral measurement of fungicide binding to cytochrome P450 at physiologically relevant concentrations [55,56], and from studies of the
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effects of D M I fungicides on ergosterol biosynthesis in cell-free systems [57,58]. From these studies, we can be quite confident that these compounds act by binding a heterocyclic r i n g - - w h i c h all DMI fungicides contain as a structural u n i t - - t o the sixth ligand position of the P450 heine iron, simultaneously preventing substrate binding and oxygen activation [59].
Resistance resulting from mutation in the genes encoding the target site and other steps in sterol biosynthesis Several reports have reported the successful cloning and sequencing of lanosterol 14et-demethylase genes from yeast [60-64]. T h e corresponding eburicol 14et-demethylase has been characterized from a filamentous fungus only recently, however [65]. In this work, multiple copies of the gene, isolated from Penicillium italicum, were introduced by transformation into Aspergillus niger. T h e resulting transformants showed reduced sensitivity to D M I fungicides, indicating that over-expression of the demethylase gene is at least a potential mechanism of resistance. Subsequent analysis of one DMI-resistant laboratory mutant of P italicum has shown that a point mutation in the demethylase gene is responsible for the resistance phenotype [66].
Resistance to DMI fungicides has been documented in a variety of plant-pathogenic fungi [67], and cases of monogenic [68] and polygenic [67,69] resistance are known. No examples of target site based resistance have been conclusively proven in strains isolated from the field. Among species of yeast pathogenic in immunocompromised patients, cases of resistance due to gene over-expression and target site based resistance have been recorded [70]. A variety of mechanisms of resistance have been encountered in laboratory strains selected upon fungicide challenge with or without mutagenesis. In both yeasts [69,70] and U. maydis [72,73], mutant isolates are obtained in which an alteration in the gene encoding sterol A5,6-desaturase must have occurred. This result can be explained on the basis of the presumed toxicity resulting from the accumulation of sterol 3,6-diols, the product appearing when 14ct-demethylation is blocked either by a D M I fungicide or by mutation in the 14ct-demethylase gene. As a result, the A5,6-saturase enzyme acts upon an alternative substrate. Thus, a change in a gene responsible for a different step in sterol biosynthesis appears to compensate for the effect of DMI inhibition.
Resistance resulting from an altered uptake or efflux of fungicide There is increasing evidence for the involvement of active efflux mechanisms in DMI fungicide resistance. Early results indicated that, in some DMI-resistant laboratory isolates, resistance could be correlated with levels of fungicide accumulation within fungal cells [74].
These results have been extended in other fungi, along with the observation that inhibitors of mitochondrial respiration affect the levels of fungicide accumulation in both sensitive and resistant strains [75]. This suggests that energy-dependent efflux mechanisms are already operative in sensitive strains, and perhaps enhanced in resistant ones. Plasmid membrane proton pumps, often called P-glycoproteins, have been implicated in resistance in human cell lines to a wide variety of anticancer drugs, and increasingly to human antifungals [70,76°]. Where this mechanism is operative, pleiotropic resistance to other unrelated inhibitors is often observed. In order to extend the efficacy of traditional chemotherapies, P-glycoproteins are now receiving attention in their own right as targets for inhibition, with the rationale that co-inhibition of the efflux pump may restore or improve the activity of a drug. A fungicide strategy based on the inhibition of efflux mechanisms has application to plant disease control as well. If fungicide level is, at least in some instances, affected by efflux mechanisms, even in wild-type strains, then combination treatment with an inhibitor of P-glycoprotein action will increase intracellular concentration of the fungicide. Moreover, efflux mechanisms may naturally play a role in pathogenesis mechanisms, both as a means to reduce the intracellular levels of natural plant defence compounds, and to export fungal pathogenesis factors and toxins. If this is correct, then inhibitors of membrane proton pumps themselves may be fungistatic. Research is currently under way to clone genes coding for P-glycoproteins in Aspergillus and Botrytis and to understand their function [77].
Conclusions T h e techniques of molecular genetics have significantly accelerated the rate at which sites of fungicide action can be positively identified. Any such study must begin with a careful classical genetic characterization of a fungicideresistant mutant in an organism that is amenable to transformation. Once the target gene has been identified, its fungicide-resistant form may be used as a selectable marker in transformation experiments, and the resistant organism itself may be used in fungicide screens, to select fungicide candidates active on the resistant phenotype. Finally, we have seen how non-target site resistance can offer insights into further aspects of a biosynthetic pathway or, in the case of multi-drug resistance, into potentially novel target sites for fungicide action. Thus, in coming years, we can expect many interesting applications of molecular genetics to the study of fungicide resistance.
Acknowledgement The authors wish to express their appreciation to IVlAde Waard and to Gi) Gustafson for providing copies of manuscripts before publication.
Mechanisms of fungicide resistance in phytopathogenic fungi Steffens, Pell and Tien
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This chapter and [33], which are written from a chemical and biological viewpoint, are the best introductions to strobilurin analogs, a rapidly emerging class of fungicides. They document, from the perspectives of two different research organizations, how crop protection chemicals with commercial levels of activity were developed from photolytically unstable natural product leads, which themselves were largely inactive in whole-plant fungicide screens.
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Becker WF, Von Jagow G, Anke T, Steglish W: Oudemansin, strobilurin A, stroblluin B and myxothlazoh new inhibitors of the b¢ 1 segment of the respiratory chain with an E-I~methoxyacrylate sstem as common structural element. FEBS Lett 1981, 132:329-333.
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Brandt U, Sch~gger H, Von Jagow G: Characterisation of binding of the methoxyscrylate inhibitors to mitochondrial cytochrome c reductase. Eur J Biochem 1988, 173:499-506.
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Geier BM, Haase U, Von Jagow G: Inhibitor binding to the • Qp-site of bcl complex: comparative studies of yeast mutant and natural inhibitor resistant fungi. Biochem Soc Trans 1994, 22:203-209. This and [37] summarize considerable data on resistance to strobilurin analogs in yeast and other fungi. The data are all the more interesting in that the resistance factors are often quite low, and the folding model for cytochrome b is theoretical, based on the hydrophobicity of membrane-spanning helices. Nevertheless, the resultant model for binding of the inhibitors is satisfyingly consistent. 39.
Mansfield RW, Wiggins TE: Photoaffinity labelling of the ~-methoxyacrylate binding site in bovine heart mitochondrial cytochrome bcr complex. Biochim Biophys Acta 1990, 1015:109-115.
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Gustafson G: Nucleic acid metabolism as a target for antifungals: the mechanism of action of LY214352. In Antifungal Agents. Discovery and Mode of Action. Edited by Dixon GK, Copping LG, Hollomon DW. Oxford: BIOS Scientific Publishers; 1995:111-117.
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Gustafson G, Davis G, Waldron C, Smith A, Henry M: Identification of a new antifungal target site through a dual biochemical and molecular genetics approach. Curt Genet 1996, in press. Gerth K, Bedorf N, Irschik H, HTfle G, Reichenbach H: The soraphens: a family of novel antifungal compounds from Sorangium cellulosum (myxobacteria). I Soraphen AI~: fermentation, isolation, biological properties. J Antibiot 1994, 47:23-31. PridzunL, Sasse F, Reichenbach H: Inhibition of fungal acetyI-CoA cerboxylase: a novel target discovered with the myxobacterial compound soraphen. In Antifungal Agents. Discovery and Mode of Action. Edited by Dixon GK, Copping LG, Hollomon DW. Oxford: BIOS Scientific Publishers; 1995:99-109. Vahlensieck HI:, Pridzun L, Reichenbach H, Hinnen A: Identification of the yeast ACCl gene product (acetylCoA cerboxylase) as the target of the polyketide fungicide soraphen A. Curt Genet 1994, 25:95-100.
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KirnuraM, Kamakura T, Tao QZ, Kaneko I, Yamaguchi h Cloning of the blasticidin S deaminase 9ene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae. Mol Gen Genet 1994, 242:121-129.
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Kimura M, Izawa K, Yoneyama K, Arie T, Kamakura T, Yamaguchi I: A novel transformation system for Pyricularia oryzae: adhesion of regenerating fungal protoplasts to collagen-coated dishes. Biosci Biotechnol Biochem 1995, 56:1177-1180.
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Milling RJ, Daniels A, Pillmocr JB: The mode of action of the anilinopyrimidlnes: a new fungicide target. In Antifungal Agents. Edited by Dixon GK, Copping LG, Hollomon DW. Oxford: BIOS Scientific Publishers; 1995:201-209.
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mepanipyrim, a novel fungicide. Pestic Biochem Physiol 1994, 48:222-228.
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This review, which is written with reference to human fungal pathogens, presents useful comparative data on deduced structure and amino acid sequences of P-glycoproteins from a variety sources. It will also probably provide a valuable starting point for research to isolate and sequence the corresponding genes from filamentous fungi. 77
De Waard MA, Van Nistelrooy JGM, Langeveld CR, Van Kan JAL, Del Sorbo G: Multldrug resistance in filamentous fungi. In Modern Fungicides and Antifungal Compounds. Edited by Lyr H, Russell P, Sisler HD. Andover, UK: Intercept: 1996:293-300.
Patents • of special interest oe of outstanding interest
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Mechanisms of fungicide resistance in phytopathogenic fungi James J Steffens*, Eva J Pellt and Ming Tien The disciplines traditionally used to investigate the mode of action of fungicides have been biochemistry and physiology. Over the past decade, classical and molecular genetics have been brought to bear on this problem with increasing success. Recently, genetic studies of fungicide resistance have led to advances in our understanding of the site of action of chemicals active against plant pathogens and, in some cases, to an appreciation of additional mechanisms of resistance to fungicide action.
Addresses *DuPont Agricultural Products, Experimental Station, PO Box 80402, Wilmington, Delaware 19880-0402, USA; e-mail: steffejj@al .esvax.umc.dupont.com tDepartment of Rant Pathology, PennsylvaniaState University, University Park, Pennsylvania16802, USA $Department of Biochemistryand Molecular Biology, Pennsylvania State University, University Park, Pennsylvania16802, USA Current Opinion in Biotechnology 1996, 7:348-355 © Current Biology Ltd ISSN 0958-1669 Abbreviations OHO dihydro-orotatedehydrogenase DMI demethylationinhibitor
Introduction T h e appearance of fungicide resistance among agronomically important fungal pathogens is sometimes a key factor in limiting the efficacy and lifetime of important disease control strategies [1,2]. Even so, resistance may also be an important aid to our understanding, at a molecular level, of the fungicidal mechanism of action of a particular class of chemicals [3,4"']. Once understood, target site based resistance may become useful as a selection mechanism in fungal DNA transformation protocols. Finally, fungicide resistance, when based upon multi-drug resistance genes, is becoming a target in its own right for fungicide screening and design.
T h e intent of this review is to summarize recent advances in our knowledge of fungicide resistance, with an emphasis on examples where molecular genetics has contributed substantially to this understanding. We concentrate on examples of various fungicides where target site based resistance is understood, and finally the case of triazole resistance, where multiple mechanisms of resistance have been described. F u n g i d d e r e s i s t a n c e d u e t o m u t a t i o n s in t h e target gene It is generally assumed that most fungicides exert their effect by interacting with a specific protein target molecule. In the past, identification of this target has depended
on the collation of biochemical and physiological evidence. Because fungicides can often produce effects that are only indirectly linked to the immediate site of action, the determination of direct cause-and-effect relationships can prove very difficult. Increasingly, researchers are turning to the genetics of fungicide resistance to understand the mechanism of action of a particular chemical or of a class of fungicidal chemicals. It is first necessary to identify a resistant mutant, in which the resistance is due to mutation in a single gene, producing alterations most likely at the site of fungicide action, rather than changes in uptake, efflux, or metabolism of the fungicide. A gene that confers resistance upon a wild-type strain can then, in principle, be isolated using the techniques of fungal DNA transformation. High-efficiency transformation protocols are available in a number of fungi, including several agronomically important plant pathogens (e.g. Alternaria,
Cercospora, Cladosporium, Cochliobolus, ColletotHchum, Gaeumannomyces, Magnaporthe, and Ustilago). T h e availability of DNA sequence databases and the capability to search them rapidly make gene identification increasingly straightforward, at least to the level of protein family by means of motif homology. T h e final step in identification is to demonstrate that transformation of a wild-type strain with a single mutant gene is sufficient to confer resistance. Several shortcuts are often available for identification of a target gene. If a particular molecular target is strongly suspected, then homologous or heterologous probes for the suspected gene will aid in its isolation from the target organism. T h e use of an origin of replication from Aspergillus nidulans [5-8] or Ustilago maydis [9] makes it possible to recover the transforming DNA as a self-replicating plasmid. T h e Aspergillus origin of replication may be employed on a helper plasmid in a co-transformation protocol. Apparently, a recombination event occurs between the two plasmids following transformation, leading to a self-replicating recoverable plasmid. Although the origin of replication from A. nidulans appears to function successfully only in closely allied species [10], it is possible to co-transform Aspergillus using heterologous DNA and the origin of replication on separate plasmids [11]. Thus, it should be possible to utilize Aspergillus for the selection of a fungicide resistance gene from another species of ascomycete, as long as Aspergillus is sensitive to the fungicide and the phenotype of resistance is dominant. T h e Ustilago origin of replication has been used to clone a gene responsible for dicarboxamide resistance (vide infra). Benzimidazoles Studies to elucidate the mode of action of the benzimidazole class of fungicides were the first to utilize classical
Mechanisms of fungicide resistance in phytopathogenic fungi Steffens, Pell and Tien 349
Figure 1 Classical and molecular genetics have contributed greatly to our understanding of the mechanism of action of the fungicides whose chemical structures are shown to the right. (a) Benomyl, a benzimidazole fungicide, interferes with the cell cycle by preventing the polymerization of et,l~-tubulin dimers into microtubules. (b) Carboxin is an inhibitor of succinate dehydrogenase. (c) Vinclozolin, an example of a dicarboximide fungicide, appears to interact with a serine/threonine protein kinase. (d) ICIA5504, a stobilurin analog, is a specific inhibitor of mitochondrial respiration by binding to cytochrome b. (e) LY214352, a phenoxyquinoline, interferes with pyrimidine biosynthesis by inhibiting DHO. (f) Pyrimethanil, an example of a anilinopyrimidine fungicide, acts by inhibiting the infection process. (g) The natural product soraphen A inhibits acetyl-CoA carboxylase. (h) Triadimenol is an example of a broad class of fungicides that interfere with sterol biosynthesis by inhibiting the P-450 sterol 14ct-demethylase.
CI
C,oc._cH (a) Benomyl
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~
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...,,~CH3 CH 3
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genetics and later the methods of molecular genetics, using benzimidazole-resistant mutants. At the outset, there was considerable evidence that benzimidazoles, such as benomyl (Fig. la), interfere with fungal cell division and bind to proteins with molecular weights similar to that of tubulin [12]. T h e analysis of benzimidazole-resistant mutants of Aspepgillus demonstrated that resistance could be correlated with changes in benzimidazole binding to tubulin. Gene isolation and sequence analysis then firmly established that resistance to benzimidazoles is due to specific mutations in the gene coding for I~-tubulin. T h e understanding that has emerged from these and subsequent studies is that fungicidal benzimidazoles bind specifically to 13-tubulin and inhibit the non-covalent polymerization of ct,13-tubulin dimers into stable microtubules
[121. T h e 13-tubulin genes from a variety of organisms have been isolated and sequenced [13,14]. Comparison of these sequences helps to explain not only why compounds such as benomyl are specific inhibitors of fungal tubulin polymerization, but also why species of yeasts and oomycetes are resistant; in general, resistant organisms contain amino acid substitutions in I~-tubulin at the critical positions that are found in benzimidazole-resistant ascomycete isolates. Subsequently, genes encoding benzimidazole-resistant [3-tubulin have been used as
dominant selectable markers for homologous [15-17] and heterologous fungal transformation [18]. Although many different 13-tubulin mutations have been created through chemical and UV-mutagenesis [12], very few of these mutations appear to be viable and/or pathogenic under field conditions [19,20"]. This phenomenon has allowed the development of methods based on PCR technology for the detection of specific field isolates that are resistant to benzimidazoles [21-24]. These methods are fairly rapid and are designed to aid in making decisions regarding field applications of fungicides.
Carboxin Carboxin (Fig. lb) is another comparatively old fungicide, with commercial levels of activity, particularly against basidiomycete pathogens. Recently, a gene from a carboxin-resistant strain of U. maydis has been cloned, sequenced, and shown to be homologous to known genes encoding the iron-sulfur subunit of succinate dehydrogenase [25]. Transformation of wild-type strains with this gene was sufficient to confer carboxin resistance. Subsequent comparison of sequences from wild-type and resistant strains demonstrated that mutation of two contiguous base pairs, within the codon for a single amino acid of a highly conserved region, was responsible for the resistant phenotype [26,27]. Carboxin has, at least in vitro,
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activity against Aspergillus and other ascomycetes. T h e r e have been no reports of the use of the carboxin-resistant genes as selectable markers for transformation, either in Ustilago or in other species.
Dicarboximides T h e dicarboximide fungicides are a class with several commercially successful examples that are active against Botrytis cinerea and numerous pathogens affecting vegetable crops. Vinclozolin (Fig. lc) is one such dicarboximide. Studies over several years have been carried out to define the mechanism of action [28], without convincing success. A comparatively low level of resistance to dicarboximides has often been reported among field isolates of pathogenic fungi, whereas isolation of resistance in the laboratory routinely results in more resistant strains [28]. High-level resistance shows some pleiotropic effects: pronounced sensitivity to high osmotic stress and cross-resistance to other fungicides, including the newer cyanopyrrole class [29,30]. It is highly probable that resistant strains isolated in the laboratory do not possess sufficient fitness to be pathogenic in the field. Neither of these two resistant biotypes has provided evidence concerning the mechanism of action of the dicarboximides. To elucidate the mode of action of the dicarboximides in U. maydis, the mechanism of resistance to vinclozolin has been investigated [31]. A large number of resistant mutants were isolated, which could be grouped into three complementation groups by subsequent genetic analysis. One of the mutants, U. maydis VR43, carrying resistance gene adr-1, was further characterized [32]. A cosmid DNA library was constructed from this mutant in an autonomously replicating vector and pooled DNA was used for transformation of wild-type U. maydis. A 32 kb cosmid conferring resistance to vinclozolin was isolated after four rounds of sib selection. Restriction analysis of the cosmid led to isolation of an 8.7kb fragment. Sequence analysis of this fragment revealed a 1218bp open reading frame coding for a serine/threonine protein kinase. Residues essential for kinase catalytic function are conserved within this gene. T h e role of the protein kinase gene adr-1 in conferring resistance was further demonstrated by deleting a 384bp NarI fragment from the coding region. Transformation of wild-type U. maydis with this modified construct did not result in fungicide resistance, confirming the role of the protein kinase gene. There are several explanations for how a modified protein kinase gene might confer vinclozolin resistance in U. maydis. It has been suggested that vinclozolin and other dicarboximide fungicides have structural similarity to the substrates of protein kinases [32]. This group of fungicides contains amide bonds and methyl groups positioned equivalently to the alcohol side chain of serine. Serine is the protein amino acid residue that is phosphorylated by the serine/threonine protein kinases; vinclozolin may be toxic by competing at the active site of the targeted protein
kinase with its natural substrate. An alternate explanation is that the protein kinase in the mutant U. maydis strain VR43 confers resistance indirectly. T h a t is, the protein kinase itself may not be the target of the fungicide; rather, its altered activity could circumvent inhibition by the fungicide at another site in the cell.
Strobilurin analogs T h e strobilurin analogs represent the first broad-spectrum class of fungicides since the development of the demethylation inhibitor (DMI) fungicides. Already, three development candidates have been announced in this chemical class [33,34"], one of which is A5504 (Fig. ld). Their structure is derived from a series of natural products--particularly strobilurin, oudemansin and myxothiazole - - found in certain basidiomycetes and myxobacteria. Aside from somewhat lower activity against the eukaryotic organisms from which some of these natural products are isolated, the strobilurin analogs have remarkable efficacy against a broad range of ascomycetes, basidiomycetes, and oomycetes. It was recognized early in the study of the original natural products that these compounds owe their fungicidal activity to inhibition of mitochondrial respiration at the level of complex III [35,36]. Subsequently, a series of elegant experiments was carried out involving yeast mutants resistant to the natural products, in which it was demonstrated that resistance is due to mutations in the mitochondrially encoded gene for apocytochrome b [37,38"]. More recent data have confirmed that synthetic compounds, designed for optimized fungicidal activity, selectivity and stability, also interact specifically with cytochrome b [39]. To date, all reported resistance studies on strobilurin analogs have been carried out in yeast, and there have not yet been any reports of resistant strains of phytopathogenic fungi, either from the field or in the laboratory. Because this is the first known case where the target protein of a fungicide is mitochondrially e n c o d e d - - a l t h o u g h the target of carboxin is a mitochondrial protein, the iron-sulfur subunit is encoded in the n u c l e u s - - i t will be interesting to see whether, and to what extent, target site based resistance develops to this class of fungicides.
Miscellaneous fungicides There exist several additional e'xamples of fungicidally active materials that deserve some mention. T h e phenoxyquinolines, such as LY214352 (Fig. le), are a group of compounds with appreciable in vitro activity, although whole-plant disease control is best against Botrytis and VentuHa. Although, to date, no development candidate has been announced from this class, it is notable because of the early and successful use of classical and molecular genetics to determine the site of action. In these studies, mutants of A. nidulans resistant to LY214352 were developed [40], and a cosmid library
Mechanisms of fungicide resistance in phytopathogenicfungi Steffens, Pell and Tien 351
was prepared from one of them [41,42]. A cosmid conferring resistance to a wild-type strain was found and sub-cloned to yield an open reading frame with homology to prokaryotic dihydro-orotate dehydrogenase (DHO), an enzyme involved in pyrimidine biosynthesis. Enzyme assays confirmed that the DHO enzymes from the resistant strains had diminished sensitivity to the inhibitors. Concomitant feeding studies showed that inhibition by LY214352 could be reversed by the addition of uridine or uracil. Significantly, fungicidal effects could also be reversed by co-enzyme Q6, a co-substrate in the DHO-catalyzed reaction, but not by dihydro-orotate itself. These results suggest that the phenoxyquinolines may bind to the co-enzyme domain of the active site. Genes encoding resistant forms of DHO have been proposed as selectable markers for fungal transformation [P1]. Acetyl-CoA carboxylase has long been a target for herbicide design. Several chemical classes are active against this target, with high selectivity for the enzyme from gramineous species. Recently, an antifungal natural product named soraphen A (Fig. lg) was isolated from a species of myxobacteria [43]. Biochemical studies have shown that the effects of soraphen A are reversed by fatty acids [44]. Using extracts of Ustilago zeae in in vitro experiments, it has been demonstrated that the natural product blocks fatty acid biosynthesis when acetyl-CoA is used as precursor, but that synthesis is unaffected when malonyl-CoA is employed. Subsequently, acetyl-CoA carboxylase has been purified from U. zeae and shown to be highly sensitive to soraphen A. Additional experiments in yeast have confirmed that mutants resistant to soraphen A are tightly linked to the accl locus, which codes for acetyl-CoA carboxylase [45]. Although highly active against both fungi and mites in field experiments, and inactive against the acetyl-CoA carboxylase from higher plants, soraphen A was dropped from development because of toxicological concerns [44]. Nevertheless, this work confirms acetyl-CoA carboxylase as a potential fungicide target. T h e ACCI gene from U. maydis has recently been cloned [46]. Blasticidin is a complex natural product, obtained by fermentation, that is used against rice blast disease caused by Magnaporthe gtisea. There have been no reports of resistance to blasticidin in isolates of Magnaporthe in the field. Even so, a gene that encodes an enzyme catalyzing the deamination of blasticidin has been cloned from Aspepgillus terreus isolated from rice paddy soil, and this has been used as a selectable marker for transformation of M. gtisea and Schizosaccharomyces pombe [47,48]. Because blasticidin is a selective fungicide (with no activity against Aspergillus, for example), the utility of this vector in filamentous fungi other than Magnaporthe remains to be demonstrated. Mention should be made of the anilinopyrimidines. No fungi resistant to these compounds have been reported;
this is a mixed blessing as fungi exhibiting target site based resistance Would be extremely informative and useful. Three examples of anilinopyrimidine fungicides, such as pyrimethanil (Fig. If), are now at or nearing commercialization, with activity against cereal diseases as well as Botrytis and Venturia. A series of studies have shown that these compounds have little effect on conidial germination and germ-tube growth; instead, they appear to inhibit the infection process (summarized in [49]). Subsequent investigations have demonstrated that the secretion of enzymes involved in the infection process, such as polygalacturonase, pectinase, cellulase, and proteinase, is significantly reduced by fungicide treatment and, furthermore, that the intracellular level of enzymes normally secreted dramatically increases [50,51]. Control experiments demonstrate that enzyme glycosylation is not affected by fungicide treatment. Seemingly in line with the inhibitory action of anilinopyrimidine fungicides on protein secretion is the finding that fungi are more susceptible to these compounds when they are grown on gelatin as a source of nitrogen than when they are grown on potato dextrose agar, which contains many free amino acids [51]. There are data, however, suggesting that specific sulfur-containing amino acids [52], or valine and leucine in another study [53], may specifically counteract growth inhibition by these fungicides. No experiments with fungicide-resistant mutants have been reported as yet. Such mutants might be chosen by selecting for strains that are able to grow on protein substrates in the presence of normally inhibitory concentrations of fungicide. An analysis of such mutants may resolve the existing uncertainties in our understanding of these compounds. Furthermore, if the hypothesis concerning inhibition of protein secretion is correct, the anilinopyrimidines may provide a means of unraveling the processes of fungal protein secretion, much as benzimidazoles have increased our appreciation of the importance of microtubule formation in the cell cycle. Demethylation Mode of action
inhibitor (DMI) fungicides
T h e DMI group of fungicides comprises a large number of commercially successful compounds, such as triadimenol (Fig. lh), which have activity at comparatively low use rates against a wide variety of cereal, vineyard, and orchard pathogens [54]). Other analogs are used to treat human and animal mycoses. As a class, these compounds act by inhibiting the cytochrome P450 dependent oxidative demethylation of eburicol in filamentous fungi (or lanosterol in yeasts) in the ergosterol biosynthetic pathway; thus, members of this class are designated DMIs. T h e bulk of the evidence in support of this site of action was obtained from investigations of the effects of DMI fungicides on the levels of sterol intermediates isolated from treated fungi, from spectral measurement of fungicide binding to cytochrome P450 at physiologically relevant concentrations [55,56], and from studies of the
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Environmental biotechnology
effects of D M I fungicides on ergosterol biosynthesis in cell-free systems [57,58]. From these studies, we can be quite confident that these compounds act by binding a heterocyclic r i n g - - w h i c h all DMI fungicides contain as a structural u n i t - - t o the sixth ligand position of the P450 heine iron, simultaneously preventing substrate binding and oxygen activation [59].
Resistance resulting from mutation in the genes encoding the target site and other steps in sterol biosynthesis Several reports have reported the successful cloning and sequencing of lanosterol 14et-demethylase genes from yeast [60-64]. T h e corresponding eburicol 14et-demethylase has been characterized from a filamentous fungus only recently, however [65]. In this work, multiple copies of the gene, isolated from Penicillium italicum, were introduced by transformation into Aspergillus niger. T h e resulting transformants showed reduced sensitivity to D M I fungicides, indicating that over-expression of the demethylase gene is at least a potential mechanism of resistance. Subsequent analysis of one DMI-resistant laboratory mutant of P italicum has shown that a point mutation in the demethylase gene is responsible for the resistance phenotype [66].
Resistance to DMI fungicides has been documented in a variety of plant-pathogenic fungi [67], and cases of monogenic [68] and polygenic [67,69] resistance are known. No examples of target site based resistance have been conclusively proven in strains isolated from the field. Among species of yeast pathogenic in immunocompromised patients, cases of resistance due to gene over-expression and target site based resistance have been recorded [70]. A variety of mechanisms of resistance have been encountered in laboratory strains selected upon fungicide challenge with or without mutagenesis. In both yeasts [69,70] and U. maydis [72,73], mutant isolates are obtained in which an alteration in the gene encoding sterol A5,6-desaturase must have occurred. This result can be explained on the basis of the presumed toxicity resulting from the accumulation of sterol 3,6-diols, the product appearing when 14ct-demethylation is blocked either by a D M I fungicide or by mutation in the 14ct-demethylase gene. As a result, the A5,6-saturase enzyme acts upon an alternative substrate. Thus, a change in a gene responsible for a different step in sterol biosynthesis appears to compensate for the effect of DMI inhibition.
Resistance resulting from an altered uptake or efflux of fungicide There is increasing evidence for the involvement of active efflux mechanisms in DMI fungicide resistance. Early results indicated that, in some DMI-resistant laboratory isolates, resistance could be correlated with levels of fungicide accumulation within fungal cells [74].
These results have been extended in other fungi, along with the observation that inhibitors of mitochondrial respiration affect the levels of fungicide accumulation in both sensitive and resistant strains [75]. This suggests that energy-dependent efflux mechanisms are already operative in sensitive strains, and perhaps enhanced in resistant ones. Plasmid membrane proton pumps, often called P-glycoproteins, have been implicated in resistance in human cell lines to a wide variety of anticancer drugs, and increasingly to human antifungals [70,76°]. Where this mechanism is operative, pleiotropic resistance to other unrelated inhibitors is often observed. In order to extend the efficacy of traditional chemotherapies, P-glycoproteins are now receiving attention in their own right as targets for inhibition, with the rationale that co-inhibition of the efflux pump may restore or improve the activity of a drug. A fungicide strategy based on the inhibition of efflux mechanisms has application to plant disease control as well. If fungicide level is, at least in some instances, affected by efflux mechanisms, even in wild-type strains, then combination treatment with an inhibitor of P-glycoprotein action will increase intracellular concentration of the fungicide. Moreover, efflux mechanisms may naturally play a role in pathogenesis mechanisms, both as a means to reduce the intracellular levels of natural plant defence compounds, and to export fungal pathogenesis factors and toxins. If this is correct, then inhibitors of membrane proton pumps themselves may be fungistatic. Research is currently under way to clone genes coding for P-glycoproteins in Aspergillus and Botrytis and to understand their function [77].
Conclusions T h e techniques of molecular genetics have significantly accelerated the rate at which sites of fungicide action can be positively identified. Any such study must begin with a careful classical genetic characterization of a fungicideresistant mutant in an organism that is amenable to transformation. Once the target gene has been identified, its fungicide-resistant form may be used as a selectable marker in transformation experiments, and the resistant organism itself may be used in fungicide screens, to select fungicide candidates active on the resistant phenotype. Finally, we have seen how non-target site resistance can offer insights into further aspects of a biosynthetic pathway or, in the case of multi-drug resistance, into potentially novel target sites for fungicide action. Thus, in coming years, we can expect many interesting applications of molecular genetics to the study of fungicide resistance.
Acknowledgement The authors wish to express their appreciation to IVlAde Waard and to Gi) Gustafson for providing copies of manuscripts before publication.
Mechanisms of fungicide resistance in phytopathogenic fungi Steffens, Pell and Tien
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