Resistance to Sterol Biosynthesis-Inhibiting Fungicides - ACS


Feb 23, 1990 - Inhibition of sterol biosynthesis has proved a fertile area for discovery of broad spectrum fungicides (SBI's), many of which are activ...
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Chapter 13

Resistance to Sterol Biosynthesis-Inhibiting Fungicides Current Status and Biochemical Basis

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D. W. Hollomon, J. A. Butters, and J. A. Hargreaves Department of Agricultural Sciences, University of Bristol, AFRC Institute of Arable Crops Research, Long Ashton Research Station, Long Ashton, Bristol BS18 9AF, United Kingdom

Inhibition of sterol biosynthesis has proved a fertile area for discovery of broad spectrum fungicides (SBI's), many of which are active against foliar diseases of small grain cereals, and especially against powdery mildews. Two groups of SBI's are important agriculturally. The morpholine group inhibit Δ8-Δ7 isomerase or Δ14(15) reductase, and have not encountered resistance problems despite twenty years intensive use. The second group of diverse chemicals all inhibit sterol 14 αdemethylase (DMI's), and although resistant mutants have been generated to DMI fungicides in the laboratory in many different fungi, resistance has only occurred in practice in five cases, mainly involving powdery mildews. Genetic analysis of both laboratory and field resistant mutants suggests that DMI resistance is controlled by many independent genes, and the biochemical data reflect this, pointing to several posssible mechanisms of resistance. Failure to accumulate sufficient fungicide, tolerance of abnormal sterols, or a lack of sterol 14 α demethylase, are just a few of the resistance mechanisms considered. Uncertainty surrounds the possibility that changes in sterol 14 αdemethylase may alter fungicide or substrate binding, but definitive data on this may be obtained through cloning and sequencing of the demethylase. Sufficient homology exists between a sterol 14 αdemethylase probe from Saccharomyces cerevisiae and Erysiphe graminis DNA, and we have identified a 3.0 KB fragment of Eyrsiphe graminis f.sp. hordei DNA, which appears to contain the demethylase sequence. 0097-6156/90/0421-0199$06.00/0 © 1990 American Chemical Society

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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MANAGING RESISTANCE TO AGROCHEMICALS

Sterols are important membrane lipids in Eukaryotes. Various functions have been identified for sterols within the membrane (1), but in general a planar molecule with a 3-hydroxyl group is required to modulate movement of phospholipids, to ensure correct membrane fluidity, and to control both permeability and the activity of at least some membrane bound enzymes. In addition, sterols may have a hormonal (so-called "sparking") function, which can only be satisfied by A sterols (2). The biosynthesis of sterols from lanosterol involves a matrix of reactions (3), and although the chemical changes are limited (demethylation, desaturation, isomerization, reduction), different enzymes generally catalyse each step in this matrix. Consequently, there is ample scope for specifity in this region of the pathway where most sterol biosynthesis inhibiting (SBI) fungicides act; for example, inhibition of oxidative demethylation of the C-14 methyl group of lanosterol in fungi does not generally interfere with the corresponding step in plant sterol biosynthesis, where obtusifoliol is the substrate for this demethylation. The last twenty years has witnessed the successful development of a number of fungicides that inhibit different steps in the sterol biosynthesis pathway (Table I). Table I.

Sites of Action of SBI Fungicides

Fungicides Allylamines Triazoles, imidazoles, pyrimidines, pyridines, piperazines, Triazoles, pyrimidines Ethylazoles, pyrimidines Tridemorph, fenpropimorph Fenpropimorph, fenpropidin

Target site Squalene epoxidase Lanosterol C-14 demethylase C-22 desaturase Ag reduction A.7A--isomerase 14(15; , A reductase A

fc

Reference 8 10 4 5 7, 36 7, 36

Twenty-six different SBI fungicides are currently used in agriculture and horticulture, and these involve 150 different products. By far the major target are diseases of small grain cereals (30m ha treated annually; Williams, R . J . , Ciba-Geigy Basle, personal communication, 1988) followed by vine diseases (2.2m ha treated) and many other crops. More than 75% SBI fungicides are used in Europe, although as a result of new registrations, use is expanding in other areas such as North America. Inhibition of sterol C-14 a demethylase (14DM) is the target site for a diverse, but widely used, group of DeMethylation Inhibiting (DMI) fungicides which generally are

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

13. HOLLOMON ETAL.

Sterol Biosynthesis-Inhibiting Fungicides 201

systemic, and have a broad spectrum of activity. Some of these fungicides may inhibit other steps in the pathway, such as C-22 desaturase (4) or A reductase (5,6), although more detailed work is needed in some instances to confirm these observations. DMI fungicides account for at least 75% of a l l SBI's used. A second group of SBI fungicides, the morpholines and 3-phenylpropylamines, are especially active against cereal powdery mildews, but seem tOgblock more than °J}fi§^ P pathway. They may inhibit A - A isomerase, A * ' reductase, or both depending on the target fungus (7). A third SBI group, the allylamines, inhibit squalene epoxidase (8) but although these reactive compounds have been successfully used as medical antimycotics, none are yet used in agriculture. Downloaded by TUFTS UNIV on November 27, 2015 | http://pubs.acs.org Publication Date: February 23, 1990 | doi: 10.1021/bk-1990-0421.ch013

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Despite a good understanding of the site of action of SBI fungicides, no clear reason has emerged as to why fungal growth is stopped. Methylated sterols are apparently not toxic since mutants lacking 14DM activity accumulate these sterols, but can grow normally under certain conditions (9). Mucor rouxii accumulates methylated sterols after propiconazole or etaconazole treatment, but is apparently unaffected by these fungicides (10). In many cases SBI fungicides reduce the total amount of sterols, and this may restrict membrane synthesis. Changes in membrane sterols will affect fluidity and alter the integrity of membranes which may become leaky. Activity of membrane bound enzymes, such as chitin synthase, may be altered (11), and it is perhaps these effects that limit growth. Current Status of Practical Resistance to SBI Fungicides Some variation in sensitivity to SBI fungicides seems inevitable in a l l target pathogens. The extent of this variation, and whether monitoring, mutation or recombination can expose i t , depends on the pathogen, and the sensitivity of the assay system available. It is perhaps not too surprising that resistance to a l l SBI fungicides has been identified in at least some fungi, either in laboratory or field studies. The practical significance of any variation will depend on many factors, including its association with relative fitness, which are discussed elsewhere in this volume. Despite early indications to the contrary (13), there are now five plant pathogens against which DMI fungicides have failed to sustain adequate control (Table II), because of resistance. In three other diseases, available information suggests that significant shifts towards less sensitive populations has occurred following repeated use of DMI fungicides (14). As yet these shifts can not be correlated with decreased performance, but similar situations were encountered when the first signs of DMI resistance emerged in cereal powdery mildews. Field resistance to DMI's was first detected in Erysiphe graminis f.sp. hordei in the UK in 1981 (15), some three years, or 90 mildew generations, after the first use of

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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MANAGING RESISTANCE

TO AGROCHEMICALS

triadimefon. Since then, gradual evolution towards higher levels of resistance, and their greater frequency within populations, has eroded the effectiveness of a l l current DMI fungicides in regions where their use remains high. Elsewhere mildew populations have remained more sensitive, and effective disease control is s t i l l possible. A similar pattern of change has occurred in wheat powdery mildew in the Netherlands (16). Table II. Resistance to DMI fungicides

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Pathogen

Resistance in practice Erysiphe graminis f. sp. hordei Sphaerotheca fuliginea Pyrenophora teres Erysiphe graminis f. sp. t r i t i c i Penicillium digitatum

Year resistance first reported

Crop

Reference

1982

barley

15

1982 1985 1986

cucurbits barley wheat

21 52 16

1987

citrus

53

Significant shifts in sensitivity not yet leading to loss of control Rhynchosporiura secalls 1986 barley 14 Uncinula necator 1986 apple 14 Venturia inaequalis 1987 grape 14 Field resistance

Cross resistance generally extends to a l l DMI fungicides, although "resistance factors" may not be identical for a l l DMI's in a l l fungal strains. No cross resistance was observed to morpholine fungicides, and to the non-SBI fungicide, ethirimol, evidence of negatively correlated cross resistance was reported in several studies (17, 18, 19). These observations on cross resistance patterns form the basis of strategies to combat further spread of DMI resistance, which use mixtures of a DMI fungicide with either a morpholine or a hydroxypyrimidine fungicide. Resistance to DMI fungicides has also emerged gradually in cucurbit powdery mildew (Sphaerotheca fuliginea), and has been identified as the cause of poor performance, especially on indoor crops (20, 21). Any lack of fitness associated with resistance is less significant in glasshouses and polythene tunnels, since the enclosed environment largely excludes fitter, more sensitive strains. Similarly, in citrus packing sheds Penicillium digitatum resistant to imazalil has caused control difficulties (53, Eckert, J . N . , University of California, Riverside, personal communication), despite the lower relative

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

13. HOLLOMON ETAL.

Sterol Biosynthesis-Inhibiting Fungicides 203

fitness of these strains (van Gestel, Janssen Pharmaceuticals, personal communication, 1988). Although one report identified field isolates of E. graminis f.sp. hordei resistant to tridemorph (22) no practical case of resistance to morpholine fungicides has emerged in any disease. This is remarkable given the specificity in their mode of action, and their continuous and increasing use against cereal mildews since tridemorph was introduced in 1970.

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Genetics of Resistance to DMI Fungicides Once stable variation in sensitivity to SBI fungicides was identified, either in field strains or laboratory induced mutants, genetic analysis was possible. Only a few studies, however, have attempted to unravel the genetic basis for control of DMI resistance, partly because of the difficulties of genetic analysis with obligate powdery mildews. No attempt seems to have been made to analyse genetically morpholine resistant mutants of Ustilago maydis that have been generated in laboratory studies. From a study of 21 u/v induced mutants of Aspergillus nidulans selected for resistance to imazalil, resistance was shown to be multigenic (23). Eight different genes were identified, one having 11 different alleles, giving an eight to ten-fold increase in resistance. Two additional genes were identified in other work (cited in 23), so that 10 unlinked genes were recognised as contributing to DMI resistance. The effects of two of these genes, ima A and ima B, together with a modifier gene of ima A, were additive. Pleiotropic effects were associated with some of these genes, which also conferred increased sensitivity to cycloheximide, indicating that they were possibly permeability mutants. Similar low levels of resistance to DMI fungicides in Venturia inaequalis were found to be controlled by a single gene (24). It is not yet known how many genes are involved in the higher levels of DMI resistance now present in field isolates of the apple scab pathogen. In obligate powdery mildews, fungicide assays are more variable, and multigenic resistance is likely to be blurred into continuous variation, and equated with polygenes. The distinction between multigenic and polygenic i s , in any case, a poor one (25), but DMI resistance in barley powdery mildew is not simply controlled by one or two genes. Crosses between two DMI sensitive barley mildew strains yielded continuous variation in the progeny (26), with some progeny distinctly less sensitive than either parent, indicating that they apparently did not have the same genetic architecture. Other crosses between DMI resistant and sensitive strains confirmed the polygenic nature of resistance. All available genetic evidence points, therefore, to more than one mechanism of resistance operating against DMI

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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fungicides. Whilst i t is possible to construct A. nidulans strains with readily identified single gene resistance, in order to study the biochemical mechanism of resistance, similar constuction of strains with a single identifiable resistance gene is not yet possible in powdery mildews.

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Mechanisms of Resistance to DMI Fungicides Decreased Fungicide Uptake. Genetic analysis of DMI resistant mutants of A. nidulans paved the way for studies in the biochemical mechanisms involved (27), and these have been extended to include resistant Penicillium italicum mutants (28). Less fungicide accumulated within resistant cells of both fungi than in sensitive cells, and consequently insufficient fungicide reached the site of action to exert a fungistatic effect. Dissection of the accumulation process into passive influx, and an energy dependent efflux, revealed that efflux was constitutative in resistant mutants, but needed to be induced in wild-type strains. Efflux was induced by many, seemingly unrelated toxicants including some SBI fungicides. The process was inhibited by ATP-ase inhibitors, which synergised the action of DMI's against resistant strains. It was suggested that efflux of DMI's is mediated by an ATP-dependent proton gradient across the fungal membrane, and this is more efficient in resistant strains. In other experiments, accumulation of imazalil sulphate was similar in both resistant and sensitive strains (29), although this may be because imazalil is likely to be protonated at physiological pH (29,30). Reduced uptake was also implicated as the mechanism of resistance to ketoconazole in clinical isolates of Candida albicans (31), although whether "active efflux" was involved was not established. Fungicide Transfer to Barley Powdery Mildew. Similar experiments with powdery mildews are not possible,^ but we have attempted to measure the transfer of C triadimenol from barley to mildew strains differing in their sensitivity to DMI fungicides. A total of 1.75 uCi of [C !?] triadimenol (sp. act. 6.5 uCi/limol) and 70 uCi of L-[methylH ] methionine (sp. act. 75 curies/mM) were taken up by 40 heavily infected 6.0 cm long leaves for 4 h. at 20°C in daylight. Radio-labelled triadimenol was kindly prepared by Dr. K. Chamberlain (Rothamsted Experimental Station) and i t was a 50 : 50 mixture of the two diastereomers. Mycelium and conidia were removed from the apical 4.0 cm of each leaf with nail varnish, which was then dissolved in 60 : 40 (v/v) ether : ethanol. Insoluble material was washed twice with ethanol : acetic acid and the supernatants combined with the ether : ethanol fraction. Insoluble material was dried, digested overnight in N-Chlorosuccinimide at 40°C, and bleached with benzoyl peroxide in toluene (51), and radio­ activity counted by liquid scintillation spectrometry. Treatment of this insoluble residue with proteinase rendered a l l radio­ activity soluble in 10% trichloroacetic acid, indicating that methionine was incorporated into protein.

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

13. HOLLOMON ETAL.

Sterol Biosynthesis-Inhibiting Fungicides

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The ether : ethanol fraction was reduced in volume to 0.5 ml, and ether (4.0 ml) : water (3.0 ml, pH 4.0) added to precipitate the nail varnish. The ether supernatant was removed and washed twice with watej. This water fraction contained no signifj-gant amounts of [C ] radioactivity. After determining the [C ] radioactivity in the ether fraction, the remainder was dried down, chromatographed on Silica Gel 60 F254 zone concentrating plates (Merck 13794) using hexane, ethylacetate, acetic acid ( 9 : 4 : 1 ) and autoradiographed to detect possible triadimenol metabolites. To allow for inevitable differences in infection levels between experiment^, transfer was expressed relative to the incorporation of H methionine into trichloroacetic acid insoluble material (= protein), which provided a measure of mildew growth. This technique only accounted for radioactivity in conidia and surface mycelium, not haustoria, but significant differences in the transfer of triadimenol were apparent between strains (Table III). These differences were not, however, correlated with triadimenol sensitivity, so that if reduced accumulation is involved in DMI resistance in barley powdery mildew, i t must not operate in a l l resistant strains. In the same experiments we looked at the possibility that detoxification of triadimenol might account for resistance, but were unable to find any evidence of triadimenol metabolites in the six strains examined. 14 Table III. Transfer of C triadimenol from barley to Erysiphe graminis f.sp. hordei strains differing in sensitivity to DMI fungicides

Strain DH49 DH14 23D5 JB115 JB152 JB214

Triadimenol Sensitivity (ED50 ug/ml) 0.002 ± 0.004 ± 0.061 ± 0.391 ± 1.034 ± 1.380 ±

0.0002 0.0006 0.008 0.042 0.108 0.140

C Triadimenol accumulation* 0.66 1.52 1.37 0.18 1.03 0.44

± ± ± ± ± ±

0.01 0.54 0.60 0.10 0.26 0.28

expressed as total incorporation of C~ triadimenol into mildew relative to incorporation of H methionine into mildew protein. Values are derived from at least two separate determinants for each strain. Sterol Changes and Resistance. DMI-resistant mutants U. maydis lacking 14DM have been isolated (32). These which were i n i t i a l l y selected for polyene resistance, detectable demethyl sterols and grow more slowly than

of mutants, have no wild-type

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

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strains. Attempts to select similar mutants in filamentous fungi have failed (26, 27, 33), and so it seems unlikely that a similar mechanism would operate in many plant diseases. Indeed, other DMI resistant mutants of U. maydis have unaltered sterol composition (34, 35), as do triadimenol resistant strains of powdery mildews (36), Rhynchosporium secalis (37) and Cladosporium cucumerinum (38) (Figure 1). In a recent study of nine ketoconazole-resistant rautantg of S. cerevisiae, a l l were found to have a lesion in the A -A desaturase suggesting that this somehow compensated for the 14-methyl sterols that otherwise accumulated in the presence of ketoconazole (39). Changes in sterol 14 a demethylase. Changes in 14DM may also account for resistance to DMI fungicides. A DMI resistant clinical isolate of C. albicans also appeared to have an altered 14DM from both spectral and kinetic analysis of crude cell homogenates (40). Binding of both the pyridine DMI fungicide buthiobate, and the substrate lanosterol, to isolated 14DM was abolished in one DMI resistant mutant of S. cerevisiae, although the imidazole ketoconazole s t i l l bound normally to this altered enzyme (41). Another mutant was obtained in which ketoconazole did not bind to the 14 a demethylase. The primary structures of 14DM from a ketoconazole resistant mutant (SGI), and its wildtype parent, were deduced from the DNA sequences of their structural genes. A single amino acid substitution of aspartic acid to glycine so altered the protein, that a neighbouring histidine residue gained access to the heme iron as the sixth ligand, blocking entry of molecular oxygen, and presumably ketoconazole (42). Changes influencing the sterol substrate binding site are less likely to have such a dramatic impact, since considerable chemical diversity contained in the non-polar part of DMI fungicides, suggests that the configuration of the sterol binding region is not too c r i t i c a l . Changes of this type may account for the low levels of resistance often observed to DMI fungicides In many plant pathogens. In U. avenae, the CO difference spectra of the target cytochrome P450 was almost the same in sensitive and triadimenol resistant strains, indicating that fungicide binding was not impaired in the resistant strain (43). Although this may indicate that resistance is not caused by a mutation in the target demethylase, until binding studies can be done with purified enzymes, this conclusion is perhaps premature. A summary of possible mechanisms of resistance to SBI fungicides is given in Table IV. Detoxification. A ten minute incubation of bojfjj triadimenol resistant and sensitive U. avenae strains in C acetate, labelled 4,4 dimethyl sterols equally in both strains, when triadimenol was present. After further incubation in the absence of radio-isotope, methyl sterols were no longer labelled in the resistant strain, suggesting that rapid turnover

In Managing Resistance to Agrochemicals; Green, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.

Sterol Biosynthesis-Inhibiting Fungicides 207

13. HOLLOMON ETAL.

Erysiphe

graminis

Cholestanol

100

f.sp. horde!

24 methylene cholesterol

80

PEAK AMPLITUDE

-RESISTANT

STRAIN

-SENSITIVE

STRAIN

60

40A 24(28) ergostadlenol 7

20

J

1

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100

1

150

RELATIVE RETENTION

100 ui Q D H IJ

Q.