Sterol Demethylation Inhibitor Research Articles

DISTRIBUTION OF MYCLOBUTANIL FUNGICIDE SENSITIVITIES AMONG POPULATIONS OF VENTURIA INAEQUALIS, THE CAUSAL AGENT OF APPLE SCAB, IN ONTARIO

ISHS XXVI International Horticultural Congress: Sustainability of Horticultural Systems in the 21st Century DISTRIBUTION OF MYCLOBUTANIL FUNGICIDE SENSITIVITIES AMONG POPULATIONS OF VENTURIA INAEQUALIS, THE CAUSAL AGENT OF APPLE SCAB, IN ONTARIO

Cross-Resistance Patterns Among Sterol Biosynthesis Inhibiting Fungicides (SBIs) inCercospora 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.

Detection of resistance to sterol demethylation-inhibiting (DMI) fungicides inCercospora beticola 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.

Lack of influence of host plant disease resistance on the evolution of resistance to sterol demethylation-inhibiting (DMI) fungicides inCercospora beticola

Field experiments were conducted during 1997 and 1998 to determine the effects of sugar beet cultivar susceptibility to Cercospora leaf-spot on the sensitivity ofCercospora beticola isolates to the triazole fungicide flutriafol. Four cultivars with different levels of disease resistance were treated in experimental plots with six spray applications of flutriafol. Disease assessments were carried out at 15-day intervals. Sensitivity to flutriafol was measured on isolates collected from the plots ∼15 days after the last flutriafol application. Measurements of disease severity and calculations of AUDPC (area under disease progress curve) values showed a distinct differentiation among cultivars, reflecting their level of disease resistance. Disease severity was significantly lower in cvs. ‘Bianca’ and ‘Areth’ than in ‘Univers’ and ‘Rizor’ both in the untreated and in the flutriafol-treated plots. Fungal isolates from flutriafol-treated plots were less sensitive to the fungicide than were isolates from untreated plots. However, no differences in isolate sensitivity were observed among the cultivars, as regards their level of disease resistance. Despite the fact that the use of resistant cultivars cannot eliminate selectively the resistant strains, it can eliminate both resistant and sensitive isolates. Reducing the number of treatments with DMIs, by applying them only when environmental conditions are favorable for disease development, is a prerequisite for successful resistance management; therefore, the use of disease-resistant varieties could aid toward management of DMIs resistance inC. beticola.

Modulators of membrane drug transporters potentiate the activity of the DMI fungicide oxpoconazole against Botrytis cinerea.

Modulators known to reduce multidrug resistance in tumour cells were tested for their potency to synergize the fungitoxic activity of the fungicide oxpoconazole, a sterol demethylation inhibitor (DMI), against Botrytis cinerea Pers. Chlorpromazine, a phenothiazine compound known as a calmodulin antagonist, appeared the most potent compound. Tacrolimus, a macrolide compound with immunosuppressive activity, was also active. The synergism of chlorpromazine negatively correlated with the sensitivity of the parent strain and mutants of B. cinerea. The synergism was highest in a mutant that overexpressed the ATP-binding cassette transporter BcatrD, known to transport DMI fungicides such as oxpoconazole. The synergism of chlorpromazine positively correlated with its potency to enhance the accumulation of oxpoconazole in BcatrD mutants. These results indicate that chlorpromazine is a modulator of BcatrD activity in B. cinerea and suggest that mixtures of DMI fungicides with modulators may represent a perspective for the development of new resistance management strategies.

Control of Pear Scab by Sterol Demethylation Inhibitors (DMIs)

Control of Pear Scab by Sterol Demethylation Inhibitors (DMIs)

Decrease of Venturia inaequalis (Cook) Aderh. sensitivity to dodine and sterol demethylation inhibitor fungicides

The effectiveness of fenarimol and difeconazole, DMIs fungicides, and dodine was tested in two experimental orchards and in greenhouse in two seasons. In 2001 monitoring of resistance to dodine was conducted also in some commercial orchards located in main apple production. In both experimental orchards fenarimol gave significantly poorer control of apple scab than difenoconazole when curative treatments were applied. The same results were obtained in greenhouse tests using for inoculation the suspension of conidia of V. inaequalis from these orchards. In many monitored orchards, in each region, a high level of dodine resistant isolates in population of V. inaequalis (even more than 40%) was noted. Only in six from 65 tested orchards there was less than 1% resistant isolates in population of the fungus.

Bcmfs1, a novel major facilitator superfamily transporter from Botrytis cinerea, provides tolerance towards the natural toxic compounds camptothecin and cercosporin and towards fungicides.

Bcmfs1, a novel major facilitator superfamily gene from Botrytis cinerea, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant Camptotheca acuminata and the plant pathogenic fungus Cercospora kikuchii, respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of Bcmfs1 and the ATP-binding cassette (ABC) transporter gene BcatrD was more sensitive to DMI fungicides than a single-replacement mutant of BcatrD, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with Bcmfs1 expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of B. cinerea against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth.

Expression of the ABC transporter BcatrD from Botrytis cinerea reduces sensitivity to sterol demethylation inhibitor fungicides

The ATP-binding cassette (ABC) transporter gene BcatrD from Botrytis cinerea was cloned and characterized. The open reading frame of BcatrD contains seven introns and encodes a putative protein of 1502 amino acids. The function of BcatrD was analyzed by phenotypic characterization of gene-replacement and overexpression mutants. Replacement mutants of BcatrD displayed a higher sensitivity to sterol demethylation inhibitor (DMI) fungicides as compared to the parental isolate. Gene replacement mutants also showed a relatively high accumulation of the DMI fungicide oxpoconazole. Overexpression mutants showed increased levels of basal and oxpoconazole-induced expression of BcatrD. Mutants with the highest expression level displayed the highest decrease in sensitivity to oxpoconazole and a relatively low accumulation of the compound. These results indicate a relation between oxpoconazole sensitivity, expression of BcatrD, and accumulation of oxpoconazole and demonstrate that the ABC transporter BcatrD is a determinant of the sensitivity of B. cinerea to DMI fungicides.

Evidence for the Predisposition of Fungicide-Resistant Isolates of Venturia inaequalis to a Preferential Selection for Resistance to Other Fungicides

In the United States, populations of the apple scab pathogen Venturia inaequalis have progressed through three consecutive rounds of fungicide resistance development, first to dodine, then to the benzimidazoles, and most recently to the sterol demethylation inhibitors (DMIs). Analysis of extensive monitoring data have to date provided no indication of detectable cross-resistance or partial cross-resistance of V. inaequalis populations to the three unrelated classes of fungicides prior to the selection of resistant subpopulations. However, in this study, resistance to both benomyl and DMIs developed to significantly higher frequencies within the previously established dodine-resistant population than in the population sensitive to dodine. Accelerated selection of phenotypes double resistant to dodine and the DMI fenarimol was apparent over the course of distinct seasons of apple scab management with either dodine or fenarimol. The data provide evidence for an accelerated speed of resistance development among phenotypes of V. inaequalis already resistant to an unrelated fungicide. This finding represents a departure from the previous model, which assumed entirely independent rounds of resistance developments. The data indicate that phenotypes of V. inaequalis might not only be selected for the trait of fungicide resistance but also for traits allowing a more flexible response to changes in the environment where they compete.

PCR-based detection of sterol demethylation inhibitor-resistant strains of Penicillium digitatum.

A simple method for detecting sterol demethylation inhibitor (DMI)-resistant strains of the citrus green mould pathogen, Penicillium digitatum, has been developed. The method involves detection of a tandem repeat of a transcriptional enhancer in the promoter region of PdCYP51, which encodes the target enzyme of DMIs, by PCR, using conidia as template. The presence of the tandem repeat leads to overexpression of this gene and confers DMI resistance to the fungus. We examined the relationship between the presence of the tandem repeat and DMI resistance in 39 strains of P digitatum. The results suggested that the DMI resistance mechanism based on the tandem repeat is common in this fungus. Using this method, the presence of DMI resistance of this fungus can be detected within 5-6 h.

Evaluation of Tactics for Managing Resistance of Venturia inaequalis to Sterol Demethylation Inhibitors.

The impact on the selection and control of subpopulations of V. inaequalis resistant to the sterol demethylation inhibitor (DMI) fenarimol or to dodine were evaluated with respect to several tactics of apple scab control. Experiments were conducted in an experimental orchard with elevated levels of DMI and dodine resistance over a period of three consecutive seasons. The DMI-resistant subpopulation was poorly (14%) controlled at a fenarimol rate of 15 mg/liter (sprayed to run-off), whereas control was significantly improved (54%) at twice that rate. Mancozeb mixed with the low rate of fenarimol also improved the control of DMI-resistant isolates, but the improvement was due to the indiscriminate control of both the DMI-sensitive and -resistant populations provided by mancozeb. The selection of fenarimol-resistant isolates resulting from poor control of the resistant subpopulation by the low rate of fenarimol was equivalent whether fenarimol was applied singly or in mixture with mancozeb. Consequently, the use of high DMI rates in mixture with a protective fungicide is expected to delay the build-up of resistant subpopulations by limiting their increase through two separate principles of control. For dodine in mixture with fenarimol, it was found that each mixing partner applied alone selected both fe-narimol- and dodine-resistant isolates. This selection pattern was partly explained by the possibility that one of the multiple genes underlying fenarimol and dodine resistance confers resistance to both fungicides, in addition to the selection of double-resistant isolates. Regardless, a mixture of fenarimol with dodine each employed at a low rate controlled both the fenarimol-and the dodine-resistant subpopulation at least as effectively as the individual components at twice their mixture rate, and an accelerated selection of double-resistant isolates was not detected. In commercial orchard trials, mixtures of DMIs with either a protective fungicide or with dodine provided equivalent control even when levels of DMI resistance, dodine resistance, or both were moderately elevated. With the exception of orchards with high levels of DMI or dodine resistance, dodine might be an alternative to protective fungicides as a mixing partner with DMIs.

Sensitivity of Monilinia oxycocci to Fenbuconazole and Propiconazole in vitro and Control of Cranberry Cottonball in the Field.

The efficacy of fungicides in controlling cottonball disease of cranberry was tested during 1996 to 1998 at three locations in Wisconsin. For some fungicides, the efficacy of four applications, two each during shoot elongation and bloom, was compared with two applications during bloom only. Spraying twice during bloom was as effective in controlling secondary infection as spraying twice during shoot elongation plus twice during bloom. Azoxystrobin, cyprodinil, and propiconazole were equally effective. None of the treatments affected yield, fruit retention, or berry weight compared with the controls. Sensitivity of M. oxycocci, the cottonball pathogen, to fenbuconazole and propiconazole was tested in vitro by comparing the distributions of ED50 values of populations collected from three sites that differed in previous exposure to fungicides. Median ED50 values for fenbuconazole were significantly greater at sites where sterol demethylation inhibitor fungicides had been used compared with a site where fungicides had never been used, but median ED50 values for propiconazole did not differ among sites. There was no correlation between the sensitivities to fenbuconazole and propiconazole. The data will form the basis of recommendations aimed at delaying the onset of fungicide resistance and will provide a baseline for monitoring resistance to fenbuconazole and propiconazole in populations of M. oxycocci in the future.

Recent evolution of multiple resistance ofBlumeria(Erysiphe)graminisf.sp.triticito selected DMI and morpholine fungicides in France

Fungicides inhibiting sterol biosynthesis are frequently used for powdery mildew control and can be subdivided into sterol demethylation inhibitors (DMIs) and morpholines with different modes of action. Whereas fungicide resistance to DMIs (Rdmi) and morpholines (Rmor) has been continuously monitored, there are no data available on the combination of Rdmi and Rmor, which led us to ask whether multiple resistance to triadimenol (Rtria), representing DMIs and to fenpropimorph (Rfen), representing the morpholines, evolved in France from 1993 to 1996. The method used allowed testing of both chemicals simultaneously, with the same inoculum. In 1993, the resistance factor of the mean (RFM) of the French wheat mildew population was 9·59 for Rtria and 5·11 for Rfen. Resistance increased, leading to RFMs of close to 14 for Rtria and 8 for Rfen at the end of the study. From the analysis of single colony isolates (SCI) that are genetically uniform, the presence of multiple resistance and its increase were evident and in line with the results of bulk isolates. Covariance of resistance to both chemicals was close to one. In contrast to the increase of Rfen, the use of morpholines decreased. These effects are supposed to result from multiple selection due to the use of mixtures of DMIs and morpholines that have been favoured in recent years. Fungicide sensitivity is, in general, not normally but lognormally distributed in a population. A new way to evaluate and describe lognormal data is presented. It is easy and convenient to use and provides solutions for current problems in the literature with lognormal distributions. Multiple resistance, its evolution and persistance are discussed in relation to fungicide use and to implications for anti-resistance strategies. © 1998 Society of Chemical Industry

Reduced sensitivity to fenarimol in Japanese field strains ofVenturia nashicola

Monoconidial strains of Venturia nashicola Tanaka et Yamamoto were isolated from Japanese or Chinese white pear trees which had never been treated with sterol demethylation inhibitors (DMIs) and their baseline sensitivities to fenarimol were determined by mycelial growth tests on fungicide-amended culture media. Strains were also obtained from Japanese pear orchards, which had been intensively treated with DMIs for several years and monitored for the shifts of fenarimol sensitivity in comparison with the baseline sensitivity. Results suggested slight shifts to lower fenarimol sensitivity in strains isolated from DMI-treated Japanese pear orchards. However, in inoculation tests on pear seedlings, fenarimol still provided adequate control of V. nashicola strains with reduced sensitivity to fenarimol in vitro, suggesting that the performance of this fungicide will still be maintained in the field. © 1998 Society of Chemical Industry

Acquisition of Resistance to Sterol Demethylation Inhibitors by Populations of Venturia inaequalis

ABSTRACT Acquisition of resistance to sterol demethylation inhibitors (DMIs) by populations of Venturia inaequalis was investigated using a microscopical method developed by C. Siebels and K. Mendgen. Microscopical analysis of conidiophore formation enabled the earlier detection of resistance and a clearer distinction between DMI-resistant and DMI-sensitive populations than other in vivo methods commonly used to analyze inhibitory effects of fungicides. In addition, because observations were made on the level of individuals, quantitative measures of the composition of conidial populations were obtained. The development of DMI sensitivity was followed over a period of 3 years in control apple orchards that had never been treated with fungicides and in orchards with DMI history. The 50% effective dose values determined by microscopical evaluation of conidio-phore development for untreated populations revealed the baseline sensitivities of 0.3, 0.96, 0.09, 1.22, and 1.92 mg/liter for flusilazole, fenarimol, difenoconazole, tebuconazole, and pyrifenox, respectively. As compared with the baseline sensitivity, all populations with DMI history showed significant resistance to flusilazole. A strong nonlinear correlation (R = 0.96) was found between the resistance factors and the sum of all DMI treatments of the 3 years before taking the sample. According to this correlation, resistance can be expected in all apple orchards of the fruit-growing area along Lake Constance, Germany, in which more than two DMI treatments per season have been applied. Due to cross-resistance, the recently introduced DMI fungicides difenoconazole, tebuconazole, and pyrifenox did not allow the control of V. inaequalis populations resistant to flusilazole.

Distributions of Sensitivities to Three Sterol Demethylation Inhibitor Fungicides Among Populations ofUncinula necatorSensitive and Resistant to Triadimefon

ABSTRACT Single-conidial isolates of Uncinula necator from (i) a population representing two vineyards with no previous exposure to sterol demethylation inhibitor (DMI) fungicides ("unexposed," n = 77) and (ii) a population representing two vineyards in which powdery mildew was poorly controlled by triadimefon after prolonged DMI use ("selected," n = 82) were assayed to determine distributions of sensitivities to the DMI fungicides triadimenol (the active form of triadimefon), myclobutanil, and fenarimol. Median 50% effective dose (ED(50)) values (micrograms per milliliter) in the selected versus unexposed populations were 0.06 versus 1.9 for triadimenol, 0.03 versus 0.23 for myclobutanil, and 0.03 versus 0.07 for fenarimol, respectively. Isolates were grouped into sensitivity classes according to their ED(50) values, and those from the selected population were categorized as resistant if the frequency of their sensitivity class had increased significantly relative to levels found in the unexposed population (ED(50) values exceeding 0.56, 0.18, and 0.18 mug/ml for triadimenol, myclobutanil, and fenarimol, respectively). Of the 76 isolates defined as resistant to triadimenol, 64% were classified as cross-resistant to myclobutanil, 18% were classified as cross-resistant to fenarimol, and 17% were classified as resistant to all three fungicides; 25% of the isolates classified as resistant to myclobutanil also were classified as resistant to fenarimol. Similar cross-resistance relationships were revealed when all isolates were examined by regressing log ED(50) values for each fungicide against those for the remaining two fungicides to determine the correlation coefficients (e.g., r = 0.85 for triadimenol versus myclobutanil and 0.56 for triadimenol versus fenarimol). The restricted levels of cross-resistance indicated by these data, particularly between fenarimol and the other two fungicides, is in sharp contrast to the high levels of cross-resistance among DMIs reported for some other pathogens and has significant implications with respect to programs for managing grapevine powdery mildew and DMI resistance.

Cloning and sequence analysis of the eburicol 14 α-demethylase gene of the obligate biotrophic grape powdery mildew fungus

In order to obtain molecular data concerning field resistance of Uncinula necator, the causal agent of grape powdery mildew, to sterol demethylation inhibitors, a major group of fungicides, the gene encoding the target of these compounds (eburicol 14 α-demethylase) was cloned and sequenced from this obligately biotrophic phytopathogenic fungus. This single-copy gene encodes a 524 amino acid protein which displays high similarity to other known sterol 14 α-demethylases (CYP51s). The coding sequence is interrupted by two short introns at positions identical to introns in Penicillium italicum CYP51, which is the only other known CYP51 gene in which introns have been identified. Intron excision was verified by cDNA sequencing.

Detection and Quantification of Resistance of Venturia inaequalis Populations to Sterol Demethylation Inhibitors

ABSTRACT Monoconidial isolates of Venturia inaequalis were collected in 1990 and 1991 from orchards in New York, Michigan, and Nova Scotia that had never or only sporadically been treated with fungicides acting as sterol demethylation inhibitors (DMIs). Sensitivities of isolates to two representative DMIs (fenarimol and myclobutanil) were determined by a sensitivity test based on the relative growth (RG) of mycelial colonies at one discriminatory dose. Mean isolate sensitivities were not significantly different (P > 0.2) for the majority of the populations tested, and all sensitivity data obtained from these sites were combined to provide a baseline distribution of isolate sensitivities for both fenarimol and myclobutanil. The baseline distributions were compared with isolate sensitivities determined for an experimental orchard in Nova Scotia with a documented case of DMI resistance and for a commercial orchard in Michigan with a long history of DMI use and first evidence of practical DMI resistance. For both DMIs tested and in both treated orchards, frequencies of isolates with RG values <80 had decreased or only slightly increased compared to the baseline population. In contrast, frequencies of isolates with RG values >80 had increased more than 20-fold over baseline levels. Thus, isolates with RG values >80 were rated DMI resistant. The validity of a qualitative isolate classification was tested in controlled infection studies. At doses of fenarimol and myclobutanil recommended for commercial control of apple scab, reproduction of a typical sensitive isolate on treated apple leaves was suppressed completely. Lesions caused by a resistant isolate continued to expand and produced abundant conidia. Statistical analysis of orchard sensitivities revealed that the analysis of isolate counts grouped into the categories DMI sensitive or resistant was most indicative in comparisons of orchard sensitivities aimed at detection of practical DMI resistance. A high degree of cross-resistance between fenarimol and myclobutanil indicated that sensitivity tests with one of the DMIs employed as the diagnostic tool in this study can serve as a test for other DMIs.

Resistance to Sterol Demethylation Inhibitors inUstilago maydis. III. Cross-Resistance Patterns and Sterol Analyses

Two spontaneous triadimefon-resistant mutants of Ustilago maydis, 151ar/1 and 151ar/3, were investigated with regard to their extent of cross-resistance and their sterol composition to elicit indications about the specificity of the present resistance mechanisms. Testing resistance to various sterol biosynthesis inhibitors and toxicants with different modes of action, it could be demonstrated that, in the mutant 151ar/1, cross-resistance was limited to the sterol demethylation inhibitors (DMIs), whereas, in strain 151ar/3, resistance included most sterol biosynthesis inhibitors studied (DMIs, morpholines, piperidines, allylamines) as well as the unrelated compounds vinclozolin and cycloheximide. Sterol analyses showed that both mutants contained ergosterol as the main sterol component. In comparison with the sensitive reference strain, the mutant 151ar/1 had a slightly elevated content of C-14 methyl sterols, whereas in strain 151ar/3 the amount of ergosterol was increased. Triadimefon caused an accumulation of C-14 methyl sterols and a decrease in ergosterol content in the sensitive strain and the mutant 151ar/1, whereas the other strain 151ar/3 remained unaffected. The results indicate that several resistance mechanisms are probably operating in the two mutants.