Resistance to inhibitors of sterol biosynthesis in field isolates or laboratory strains of the eyespot pathogen Pseudocercosporella herpotrichoides

Strains of Pseudocercosporella herpotrichoides collected in France on winter wheat give either fast-growing mycelial colonies with regular margins or slow-growing mycelial colonies with irregular margins. Most of the fastgrowing isolates were sensitive to triadimenol (EC₅₀ below 2mg litre^?1), but some of them were resistant to this inhibitor of sterol C-14 demethylation. In contrast, all the slow-growing strains were highly resistant to triadimenol (EC₅₀ greater than 100 mg litre^?1). This resistance was also expressed in inhibition of germ-tube elongation. Positive cross-resistance was observed between most of the inhibitors of sterol C-14 demethylation, with the exception of some imidazole derivatives (clotrimazole, prochloraz). All the fast-growing strains were tolerant to fenpropimorph and fenpropidin whereas the slow-growing ones were susceptible; the reverse was true with piperalin and tridemorph. All the field isolates were inhibited to the same extent by the inhibitors of squalene-epoxidase, nafifine and terbinafine. Two types of mutant resistant to triadimenol have been induced under laboratory conditions from sensitive fast-growing strains. The most common mutants were resistant to all the inhibitors of sterol C–14 demethylation and also in some conditions to fenpropimorph, tridemorph and the inhibitors of squalene-epoxidase. The other mutants were characterised by a reduced spectrum of cross-resistance between triadimenol and the other inhibitors of sterol biosynthesis. The field isolates and laboratory mutants resistant to triadimenol and propiconazole were also resistant to each of the four enantiomers of these two fungicides.     

Resistance of Erysiphe graminis on barley and wheat to sterol C-14-demethylation inhibitors1

Isolates of Erysiphe graminis f. sp. hordei and tritici with decreased sensitivity to triadimefon showed cross-resistance to other inhibitors of sterol C-14-demethylation, such as triadimenol, propiconazol, diclobutrazol, prochloraz and nuarimol. The isolates exhibited a moderate degree of resistance to these compounds. No cross-resistance was detected to tridemorph, fenpropimorph and pyrazophos. The resistant hordei isolates were more sensitive to ethirimol than the sensitive isolate. The competitive abilities of resistant hordei and tritici isolates were inferior to that of the sensitive isolates. In the presence of the fungicides no differences in germination, appressorium formation and penetration between the sensitive and resistant isolates were observed; 48 h after inoculation the sensitive isolate showed several morphological alterations and further fungal development was arrested. At four to five times higher doses of triadimefon, similar morphological alterations were detected in the resistant isolate. Low concentrations of triazole fungicides which slightly affected mycelium growth of both the sensitive and the resistant isolate of f.sp. hordei severely inhibited development of conidiophores of the sensitive isolate whereas that of the resistant isolate was hardly affected.     

Resistance in mutagen-induced mutants of Ustilago maydis to fungicides which inhibit ergosterol biosynthesis

Resistance to a number of inhibitors of sterol C-14 demethylation, (clotrimazole, imazalil, miconazole, fenarimol, nuarimol and triadimefon), as well as resistance to inhibitors of sterol C-14(15) double bond reduction, (tridemorph and fenpropi-morph), was readily induced in Ustilago maydis. Resistant mutants were obtained after mutagenic treatment by ultraviolet irradiation, or by treatment with 1-methyl-3-nitro-1-nitrosoguanidine, of sporidia of the wild-type strain, followed by selection in the presence of the toxicant. The level of resistance of these mutants varied appreciably. Although not always reciprocal, cross-resistance to fungicides which inhibit ergosterol biosynthesis (EBIs) appeared to be present in most cases. Several of the U. maydis mutants which were resistant to inhibitors of sterol C-14 demethylation lacked cross-resistance to tridemorph and fenpropimorph, or displayed increased sensitivity to fenpropimorph (negatively correlated cross-resistance). Cross-resistance between EBIs and the antimicrobial agents climbazole and lombazole was also established. It is suggested that fungal mutants that possess a resistance mechanism based on a deficiency in sterol C-14 demethylation or sterol C-14(15) double bond reduction, have a greatly reduced chance of survival.     

New Cases of Negative Cross-resistance between Fungicides,Including Sterol Biosynthesis Inhibitors

A survey of fungicide resistance in Mycosphaerella graminicola and Tapesia acuformis, two major pathogens of winter wheat in France, respectively responsible for speckled leaf blotch and eyespot, led to the characterization of two types of resistant strains to sterol 14α-demethylation inhibitors (DMIs). Most of the strains of M. graminicola collected in France in 1997–1998 were resistant to all DMIs, and only in a few strains was the resistance to several triazoles associated with increased susceptibility to pyrimidine derivatives (i.e., fenarimol, nuarimol) and triflumizole. On the other hand, in T. acuformis the most prevalent strains were those which exhibited negative-cross resistance between DMIs. In both fungi such a phenomenon could be related to changes in cytochrome P450 sterol 14α-demethylase, the target site of these fungicides. For Botryotinia fuckeliana, the causal agent of grey mould, the extensive monitoring conducted in French vineyards before the marketing of fenhexamid revealed the presence of highly resistant strains to this promising botryticide (only in tests involving mycelial growth measurements). Negative cross-resistance to edifenphos and several sterol biosynthesis inhibitors, such as prochloraz and fenpropimorph, was observed in fenhexamid resistant strains. Synergism of the antifungal action of fenhexamid by cytochrome P450 inhibitors, such as the DMI fungicides, was only recorded in fenhexamid resistant strains. These data and those previously obtained with edifenphos resistant strains of Magnaporthe grisea (rice blast pathogen) suggest that in fenhexamid resistant strains of B. fuckeliana the same cytochrome P450 monooxygenase could be involved in detoxification of fenhexamid and activation of edifenphos. Received 6 September 1999/ Accepted in revised form 13 September 1999     

Sterol content and other characteristics of pimaricin-resistant mutants of Aspergillus nidulans

Pimaricin-resistant mutants of Aspergillus nidulans were selected on a medium containing the polyene-antibiotic. Some resistant mutants contained markedly reduced amounts of ergosterol, but others contained almost normal levels of this sterol. Most resistant mutants which lacked ergosterol had a biochemical lesion in sterol C-22 desaturation. Analysis of sterols in one of these isolates showed the presence of 5,7-ergostadienol, 5,7,24(28)-ergostatrienol, and 5,8-ergostadienol. The sterol C-14 demethylation inhibitor, fenarimol, was more toxic to this mutant than to the wild type. On the other hand, mutants inactive in sterol C-22 desaturation were resistant to oligomycin but showed wild type sensitivity to carboxin. Attempts to select sterol C-14-demethylation-deficient mutants of Aspergillus nidulans, Monilinia fructicola, and Pyricularia oryzae on polyene-containing media were unsuccessful. Apparently C-14-methyl sterols do not support growth of these filamentous fungi.     

Resistance to fungicides which inhibit ergosterol biosynthesis in laboratory strains of Botrytis cinerea and Ustilago maydis

The strains of Botrytis cinerea or Ustilago maydis selected on fenarimol, triarimol, or triadimefon were also resistant to the other inhibitors of sterol C-14 demethylation; the sterol composition of the strains was normal. Among the isolates of U. maydis resistant to dodemorph, fenpropidin, fenpropimorph and tridemorph, some were resistant to the 15-azasteroid A 25822B and did not contain ergosterol. The other strains remained sensitive to A 25822B and had a normal sterol composition. All the resistant isolates and the wild-type were inhibited to the same extent by nystatin and pimaricin.     

Genetics of responses to morpholine-type fungicides and of avirulences inErysiphe graminis f. sp.hordei

The genetics of the responses of the barley powdery mildew pathogen,Erysiphe graminis f.sp.hordei, to three morpholine-type fungicides were studied. Resistances to a phenylpropylamine fungicide, fenpropidin, and to a morpholine, fenpropimorph, co-segregated in crosses of a sensitive isolate, DH14, with each of two resistant ones, CC151 and CC152. In the cross CC151×DH14, the results were consistent with resistance to both fungicides being controlled by a single gene, at a locus namedFenl. In the other cross, CC152×DH14, the genetics of resistance were more complicated; the data were consistent with the segregation of two complementary, unlinked genes which each conferred resistance to both fungicides. Fenpropidin-resistant progeny of CC151×DH14 were significantly more resistant to fenpropimorph than were fenpropidin-resistant progeny of CCI 52×DH14, although the resistant progeny of the two crosses did not differ significantly in their level of fenpropidin resistance. Fenpropidin-resistant progeny of CC151×DH14 were significantly more resistant to another morpholine, tridemorph, than were fenpropidin-sensitive progeny, but this was not the case for CC152×DH14. Resistance to triadimenol, a C14 demethylation-inhibitor (DMI) fungicide, segregated in both crosses. Triadimenol resistance appeared to be controlled by one gene in each cross and was not associated with morpholine resistance. CC151×DH14 also segregated for eight avirulence genes. Two of these matched theMla6 resistance, while one gene matched a previously unknown resistance in a Pallas near-isogenic line, P17, which also carries a known resistance gene,Mlk. Fenl was not significantly linked to the triadimenol resistance gene,Tdl(a), or to any of the eight avirulence genes.Avr _a6 1, Avr _a12 ,Avr _La ,Avr _p17 andTdl(a) were linked, as wereAvr _a 10 andAvr _k .Abbreviations ED₅₀ median effective dose - Fpd fenpropidin - Fpm fenpropimorph - PCA principal components analysis - Tdm tridemorph     

Relationship between chemical structure and biological activity of triazole fungicides against Botrytis cinerea

The inhibitory activity of commercial and experimental triazole fungicides on the target enzyme, sterol 14α-demethylase (P450_14DM), was studied in a cell-free sterol synthesis assay of Botrytis cinerea Pers. ex Fr. In order to assess structure-activity relationships, the inhibitory activities of the compounds on radial growth of the fungus were tested as well. The EC₅₀ values (concentrations of fungicide inhibiting radial growth of B. cinerea on PDA by 50%) of all triazoles tested ranged between 10^?8 and 10^?5 m. IC₅₀ values (concentrations of fungicide inhibiting incorporation of [2-¹⁴C]mevalonate into C4-desmethyl sterols by 50%) generally ranged between 10^?9 and 10^?7 M and correlated with inhibition of radial mycelial growth. However, differences in IC₅₀ values did not reflect quantitatively the observed differences in EC₅₀ values, since the ratio between EC₅₀ and IC₅₀ increased with decreasing fungitoxicity. For a limited number of compounds the correlation between intrinsic inhibitory activity and fungitoxicity was low. Both in-vitro tests were used to investigate structure-activity relationships for stereoisomers of cyproconazole, SSF-109 and tebucona-zole. Fungitoxicity and the potency to inhibit cell-free C4-desmethyl sterol synthesis correlated for all stereoisomers tested. Mixtures of isomers of tebucona-zole or cyproconazole were slightly less active than the most potent isomer. The high activity of several commercial triazoles in both experiments implies that poor field performance of triazole fungicides against B. cinerea is due neither to insensitivity of the P450_14DM nor to low in-vitro sensitivity of the fungus.     

Evolution of fungicide resistance in the cereal eyespot fungi Tapesia yallundae and Tapesia acuformis in France

Field isolates of the cereal eyespot pathogen can be divided into two groups which are now considered as two species: Tapesia yallundae and Tapesia acuformis. In both species the first case of acquired resistance was observed with benzimidazole fungicides in the early 1980s. At the same time, a number of sterol C-14 demethylation inhibitors (DMIs), such as the imidazole prochloraz and several triazoles, including flusilazole, were introduced. Surprisingly T. acuformis appeared intrinsically resistant to the triazole derivatives in comparison to T. yallundae, but both species were sensitive to prochloraz. The intensive use of these DMIs led to the development of acquired resistance towards triazoles in T. yallundae and towards prochloraz in T. acuformis. Today all the strains in both species appear equally sensitive to the anilinopyrimidine cyprodinil. ©1997 SCI     

Analysis of the CYP51 gene and encoded protein in propiconazole‐resistant isolates of Mycosphaerella fijiensis

BACKGROUND: Mycosphaerella fijiensis Morelet causes black sigatoka, the most important disease in bananas and plantains. Disease control is mainly through the application of systemic fungicides, including sterol demethylation inhibitors (DMIs). Their intensive use has favoured the appearance of resistant strains. However, no studies have been published on the possible resistance mechanisms. RESULTS: In this work, the CYP51 gene was isolated and sequenced in 11 M. fijiensis strains that had shown different degrees of in vitro sensitivity to propiconazole, one of the most widely used DMI fungicides. Six mutations that could be related to the loss in sensitivity to this fungicide were found: Y136F, A313G, Y461D, Y463D, Y463H and Y463N. The mutations were analysed using a homology model of the protein that was constructed from the crystallographic structure of Mycobacterium tuberculosis (Zoff.) Lehmann & Neumann. Additionally, gene expression was determined in 13 M. fijiensis strains through quantitative analysis of products obtained by RT‐PCR. CONCLUSION: Several changes in the sequence of the gene encoding sterol 14α‐demethylase were found that have been described in other fungi as being correlated with resistance to azole fungicides. No correlation was found between gene expression and propiconazole resistance. Copyright © 2009 Society of Chemical Industry     

Cross-Resistance Patterns Among Sterol Biosynthesis Inhibiting Fungicides (SBIs) in Cercospora beticola

Thirty single-spore isolates of Cercospora beticola, collected from several fields in northern Greece, representing a broad spectrum sensitivity to the sterol demethylation-inhibiting (DMIs) fungicide flutriafol, were tested for sensitivity to eleven other sterol biosynthesis-inhibiting (SBI) fungicides and to the guanidine fungicide dodine. Sensitivity was measured as EC₅₀ values for each fungicide and log-transformed EC₅₀ values to each fungicide were pairwise correlated and the correlation coefficient estimated. These pairwise comparisons showed high correlation coefficients between the DMIs suggesting a cross-resistance relationship between these fungicides. However, the degree of cross-resistance between DMIs varied greatly. Conversely, low correlation coefficients were obtained for the pair-wise comparisons with the morpholine fungicide fenpropimorph suggesting a lack of cross-resistance between morpholines and DMIs in C. beticola. Similarly, there was no correlation between the sensitivity (EC₅₀ values) to dodine and all the other fungicides tested, indicating that there was no negative cross-resistance relationship between dodine and SBIs in C. beticola. Based on these results, combinations or alternations of fungicides which show no cross-resistance relationship should be used to control the disease in areas where reduced sensitivity to DMIs has been already observed.     

A mutant of Ustilago maydis deficient in sterol C-14 demethylation: Characteristics and sensitivity to inhibitors of ergosterol biosynthesis

An ergosterol-deficient mutant of Ustilago maydis was compared to the wild type in regard to morphology, growth rate, lipid content, and sensitivity to ergosterol biosynthetic inhibitors. Morphology of mutant sporidia is abnormal and resembles that of fenarimol-treated wild-type sporidia. Doubling time of mutant sporidia is 6.3 hr compared to 2.5 hr for the wild type. The mutant produces 24-methylenedihydrolanosterol, obtusifoliol, and 14α-methylfecosterol; ergosterol is absent. The sterols of the mutant are the same as those which accumulate in wild-type sporidia treated with the sterol C-14 demethylation inhibitors fenarimol, etaconazole, and miconazole. The level of free fatty acids is higher in the mutant than in wild-type cells. Growth of mutant sporidia is not inhibited by fenarimol, etaconazole, and miconazole, or by the sterol Δ¹⁴-reductase inhibitor azasterol A25822B at low concentrations which inhibit growth of wild-type sporidia. The residual growth rate of wild-type sporidia treated with low concentrations of the sterol C-14 demethylation inhibitors is about the same as that of untreated mutant sporidia. Therefore, the mutant would not be recognized as resistant in a wild-type population. The mutant is deficient in sterol C-14 demethylation and is similar in all properties studied to wild-type sporidia treated with sterol C-14 demethylation inhibitors. These findings support the contention that inhibition of sterol C-14 demethylation in U. maydis is the primary mode of toxicity of fenarimol, etaconazole, and miconazole. A secondary mode of toxicity is evident for miconazole and etaconazole at higher concentrations but is doubtful for fenarimol.     

Multiple resistance of Botrytis cinerea from kiwifruit to SDHIs,QoIs and fungicides of other chemical groups

BACKGROUND: Botrytis cinerea Pers.: Fr. is a high‐risk pathogen for fungicide resistance development that has caused resistance problems on many crops throughout the world. This study investigated the fungicide sensitivity profile of isolates from kiwifruits originating from three Greek locations with different fungicide use histories. Sensitivity was measured by in vitro fungitoxicity tests on artificial nutrient media. RESULTS: Seventy‐six single‐spore isolates were tested for sensitivity to the SDHI fungicide boscalid, the QoI pyraclostrobin, the anilinopyrimidine cyprodinil, the hydroxyanilide fenhexamid, the phenylpyrrole fludioxonil, the dicarboxamide iprodione and the benzimidazole carbendazim. All isolates from Thessaloniki showed resistance to both boscalid and pyraclostrobin, while in the other two locations the fungal population was sensitive to these two fungicides. Sensitive isolates showed EC₅₀ values to boscalid and pyraclostrobin ranging from 0.9 to 5.2 and from 0.04 to 0.14 mg L^?1 respectively, while the resistant isolates showed EC₅₀ values higher than 50 mg L^?1 for boscalid and from 16 to > 50 mg L^?1 for pyraclostrobin. All QoI‐resistant isolates carried the G143A mutation in cytb. Sensitivity determinations to the remaining fungicides revealed in total eight resistance phenotypes. No isolates were resistant to the fungicides fenhexamid and fludioxonil. CONCLUSION: This is the first report of B. cinerea field isolates with resistance to both boscalid and pyraclostrobin, and it strongly suggests that there may be a major problem in controlling this important pathogen on kiwifruit. Copyright © 2010 Society of Chemical Industry     

Impact of fludioxonil resistance on fitness and cross-resistance profiles of Altrenaria solani laboratory mutants

Sensitivity and inherent resistance risk of Alternaria solani to fludioxonil, cross-resistance profiles and the potential implications of resistance mutations on fitness parameters were investigated. Fludioxonil was highly effective against a wild type A. solani field strain both in vitro (EC₅₀?=?0.05 μg/mL) and in preventive applications on artificially inoculated tomato fruit. Mutants with low [Resistance factor (Rf): 15 based on EC₅₀], medium (Rf: 150–300) and high (Rf: > 1000) levels of phenylpyrrole resistance were isolated from the wild type strain at high frequencies following mutagenesis with UV irradiation and selection on fludioxonil containing medium. Resistant isolates retained their resistance levels even after 9 subcultures on fungicide-free growth medium while they could express their resistant phenotypes in planta. Investigation of cross-resistance relationships showed that fludioxonil resistance mutations also reduce the sensitivity of mutant strains to the aromatic hydrocarbon fungicide quintozene as well as the dicarboximides iprodione and vinclozolin. No cross-resistance was observed between fludioxonil and fungicides with different modes of action such as the sterol biosynthesis inhibitors (DMIs) imazalil and flusilazole and the carboxamide boscalid. All fludioxonil resistant isolates were more sensitive to the anilinopyrimidine pyrimethanil, while only two isolates were less sensitive to the QoI pyraclostrobin compared to the wild-type strain. Study of fitness determining parameters showed that resistance mutation(s) had no adverse effects on mycelial growth, conidial germination and sensitivity to osmotic stress while they had a pleiotropic effect on virulence and conidia production in resistant mutants. Results of the present study indicate that fludioxonil is a highly effective fungicide against A. solani, while the risk of resistance development to this fungicide is considered to be medium making fludioxonil an ideal alternative to high risk fungicides such as boscalid and pyraclostrobin whose performance against early blight has already been compromised by resistance development.

     

Fungitoxicity and growth regulation involving aspects of lipid biosynthesis

Triarimol and triforine inhibit ergosterol biosynthesis in fungi and cause accumulation of free fatty acids, 24-methylenedihydrolanosterol, obtusifoliol and 14α-methyl-δ^8,24(28)-ergostadienol. Triparanol also inhibits ergosterol synthesis and causes accumulation of free fatty acids, but not of the latter 3 sterols. Triparanol appears to inhibit prior to lanosterol in the sterol biosynthetic pathway of Ustilago maydis and at unidentified sites subsequent to lanosterol which lead to the accumulation of a sterol which migrates with desmethylsterols on TLC plates. Quantitative abnormalities in sterols and free fatty acids in U. maydis are not produced by the fungicides carbendazim, chloroneb, carboxin and cycloheximide. A deficiency in nitrogen leads to a marked increase in triglycerides, but a normal distribution pattern for other lipids.Inhibition of oxidative demethylation of the sterol 14α-methyl group is probably the prime mechanism of inhibition of ergosterol biosynthesis by triarimol. Rates of formation of obtusifoliol and 14α-methyl-δ^8,24(28)-ergostadienol in triarimol-treated U. maydis cells suggest that C-4 demethylation occurs along an abnormal pathway which operates effectively only at high substrate concentrations. The growth retardant action of triarimol and ancymidol in higher plants most likely results from inhibition of a reaction in the gibberellin biosynthetic pathway analogous to sterol C-14 demethylation.Free fatty acid accumulation in U. maydis cells treated with inhibitors of sterol synthesis are derived mainly from polar lipid degradation and from de novo synthesis as a consequence of the disproportionality between fatty acid synthesis and utilization. The free fatty acids may play a significant role in the lethality of these inhibitors in this organism.     

Fungicide resistance status in French populations of the wheat eyespot fungi Oculimacula acuformis and Oculimacula yallundae

Eyespot, caused by Oculimacula acuformis and Oculimacula yallundae, is the major foot disease of winter wheat in several European countries, including France. It can be controlled by chemical treatment between tillering and the second node stage. The fungicides used include antimicrotubule toxicants (benzimidazoles), inhibitors of sterol 14α‐demethylation (DMIs) or of succinate dehydrogenase (SDHIs), the anilinopyrimidines cyprodinil and the benzophenone metrafenone. Since the early 1980s, a long‐term survey has been set up in France to monitor changes in the sensitivity of eyespot populations to fungicides. Resistance to benzimidazoles has become generalised since the early 1990s, in spite of the withdrawal of this class of fungicides. In the DMI group, resistance to triazoles is generalised, whereas no resistance to the triazolinethione prothioconazole has yet developed. Resistance to the imidazole prochloraz evolved successively in O. acuformis and O. yallundae and is now well established. Specific resistance to cyprodinil has also been detected, but its frequency has generally remained low. Finally, since the early 2000s, a few strains of O. yallundae displaying multidrug resistance (MDR) have been detected. These strains display low levels of resistance to prothioconazole and SDHIs, such as boscalid. Knowledge of the spatiotemporal distribution in France of O. acuformis and O. yallundae field strains resistant to fungicides allows resistance management strategies for eyespot fungi in winter wheat to be proposed.© 2012 Society of Chemical Industry     

Effect of triadimefon and triadimenol on growth of various plant species as well as on gibberellin content and sterol metabolism in shoots of barley seedlings

The fungicides triadimefon and triadimenol markedly reduced growth of coleoptiles, primary leaves, and roots of barley seedlings when grown for 7 days in petri dishes in the dark. The addition of gibberellins (A₁, A₃, A₄, A₇, A₉) alleviated growth retardation of primary leaves and coleoptiles induced by the fungicides. While fungicide-induced growth retardation of the shoots was partly relieved by kinetin, IAA did not show an alleviating activity. Triadimefon and triadimenol also substantially retarded the elongation of shoots of tomato and cotton plants and simultaneous application of GA₃ nullified the retardation. The fungicides only slightly interfered with both α-amylase production of intact germinating barley seed and the GA₃-induced α-amylase synthesis in barley endosperm. On the other hand, extracts of triadimefon- and triadimenol-treated shoot tissue of 10- to 12-day-old barley plants contained substantially lower gibberellin-like activity than control shoots. Both compounds also interfered in sterol metabolism of shoots of barley seedlings when compared to control plants, treatment resulted in lower amounts and altered proportions of C-4,4-desmethyl sterols. While both fungicides inhibited synthesis of C-4,4-desmethyl sterol fraction, sterols possessing C-4 and C-14-methyl groups were accumulated. It is assumed that triadimefon and triadimenol interfere in gibberellin and sterol biosynthesis in barley seedlings by inhibiting oxidative demethylation reactions.     

Detection of resistance to sterol demethylation-inhibiting (DMI) fungicides in<Emphasis Type="Italic">Cercospora beticola</Emphasis> and efficacy of control of resistant and sensitive strains with flutriafol

Single-lesion isolates ofCercospora beticola (n=150) were collected in 1998 from sugar beet fields in the area of Serres, N. Greece. In this area, sterol demethylation-inhibiting (DMI) fungicides have been used for almost 20 years to control sugar beet leaf spot. The sensitivity of these isolates to the DMI fungicides flutriafol and difenoconazole (EC₅₀ values) was determined on the basis of inhibition of mycelial growth at several fungicide concentrations. The relative growth (RG) of isolates was correlated at all tested concentrations with the respective EC₅₀ values, indicating that RG provides a reliable estimate for the sensitivity of the isolates. The highest correlation coefficients were obtained for concentrations of 1 μg ml⁻¹ flutriafol and of 0.05 μg ml⁻¹ difenoconazole, respectively. Consequently, they are proposed for monitoring of DMI sensitivity inC. beticola populations, as single discriminatory concentrations in a simplified test method. Based on the RG values at the discriminatory concentration of 1 μg ml⁻¹ flutriafol,C. beticola isolates were classified as either resistant or sensitive. The efficacy of flutriafol, applied at the commercially recommended dose, in controlling Cercospora leaf spot was examined in field experiments conducted during 1999 and 2000. Disease incidence in plots artificially inoculated with resistant isolates and treated with flutriafol was significantly higher than in similar plots inoculated with sensitive strains. These results suggest that poor disease control after application of flutriafol may be based on the presence of resistant strains within the pathogen population in northern Greece. This emphasizes the risk of the development of practical resistance if there is increased frequency of such strains within the population. http://www.phytoparasitica.org posting July 13, 2003.     

Mutations in the CYP51 gene correlated with changes in sensitivity to sterol 14 alpha-demethylation inhibitors in field isolates of Mycosphaerella graminicola

In France, as in many other European countries, Mycosphaerella graminicola (Fuckel) Schr?ter in Cohn (anamorph Septoria tritici), the causal agent of wheat leaf blotch, is controlled by foliar applications of fungicides. With the recent generalization of resistance to strobilurins (QoIs), reliable control is mainly dependent upon inhibitors of sterol 14 alpha-demethylation (DMIs). To date, strains with reduced sensitivity to DMIs are widespread, but disease control using members of this class of sterol biosynthesis inhibitors has not been compromised. In this study, sensitivity assays based on in vitro effects of fungicides towards germ-tube elongation allowed the characterization of seven DMI-resistant phenotypes. In four of them, cross-resistance was not observed between all tested DMIs; this characteristic concerned prochloraz, triflumizole, fluquinconazole and tebuconazole. Moreover, the highest resistant factors to most DMIs were found only in recent isolates; according to their response towards prochloraz, they were classified into two categories. Molecular studies showed that DMI resistance was associated with mutations in the CYP51 gene encoding the sterol 14 alpha-demethylase. Alterations at codons 459, 460 and 461 were related to low resistance levels, whereas, at position 381, a valine instead of an isoleucine, in combination with the previous changes, determined the highest resistance levels to all DMIs except prochloraz. Mutations in codons 316 and 317 were also found in some isolates exhibiting low resistance factors towards most DMIs.     

Mechanisms of resistance to fungicides in field strains of Botrytis cinerea

Field strains of Botrytis cinerea Pers ex Fr, the causal agent of grey mould diseases, were collected from French vineyards between 1993 and 2000. Several phenotypes have been characterized according to the inhibitory effects of fungicides towards germ-tube elongation and mycelial growth. Two types of benzimidazole-resistant strains (Ben R1 and Ben R2) could be detected; negative cross-resistance to phenylcarbamates (e.g. diethofencarb) was only found in Ben R1. Benzimidazole resistance was related to point mutations at codon 198 (Ben R1) or 200 (Ben R2) of the beta-tubulin gene. Most dicarboximide-resistant strains were also weakly resistant to aromatic hydrocarbon fungicides (e.g. dicloran) but remained sensitive to phenylpyrroles (e.g. fludioxonil). These resistant field strains (Imi R1) contained a single base pair mutation at position 365 in a two-component histidine kinase gene, probably involved in the fungal osmoregulation. Three anilinopyrimidine-resistant phenotypes have been identified. In the most resistant one (Ani R1), resistance was restricted to anilinopyrimidines, but no differences were observed in the amino-acid sequences of cystathionine beta-lyase (the potential target site of these fungicides) from Ani R1 or wild-type strains. In the two other phenotypes (Ani R2 and Ani R3), resistance extended to various other groups of fungicide, including dicarboximides, phenylpyrroles and sterol biosynthesis inhibitors. This multi-drug resistance was probably determined by over-production of ATP-binding cassette transporters. The hydroxyanilide fenhexamid is a novel botryticide whose primary target site is the 3-keto reductase involved in sterol C-4 demethylations. Apart from the multi-drug-resistant strain Ani R3, three other fenhexamid-resistant phenotypes have been recognized. For two of them (Hyd R1 and Hyd R2) fenhexamid-resistance seemed to result from P450-mediated detoxification. Reduced sensitivity of the target site could be the putative resistance mechanism operating in the third resistant phenotype (Hyd R3). Increased sensitivity to inhibitors of sterol 14 alpha-demethylase recorded in Hyd R1 strains was related to two amino-acid changes at positions 15 and 105 of this enzyme.