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.     

Orchard factors associated with resistance and cross resistance to sterol demethylation inhibitor fungicides in populations of Venturia inaequalis from Pennsylvania

Orchard management practices, such as destroying of overwintered inoculum and limiting the number of fungicide applications, are often recommended as tactics for slowing the development of resistance to sterol demethylation-inhibitor (DMI) fungicides in populations of Venturia inaequalis. However, there is little quantitative evidence relating the use of such practices to levels of resistance in orchards. The aim of this study was to evaluate the sensitivity of V. inaequalis isolates from Pennsylvania to DMI fungicides, and to identify orchard management factors related to the incidence of resistant isolates. In total, 644 single-spore V. inaequalis cultures obtained from 20 apple orchards in 2008 or 2009 were tested for sensitivity to myclobutanil, fenbuconazole, or difenoconazole. Growers provided management history of the sampled plots. Widespread shifts toward resistance to the three fungicides were noted, with mean effective concentration for 50% inhibition (EC(50)) values of 2.136, 0.786, and 0.187 μg/ml for myclobutanil, fenbuconazole, and difenoconazole, respectively. Cross resistance to the three fungicides was documented in high correlation (Spearman's r > 0.6) between mean EC(50) values for 14 orchards. Based on a 0.5-μg/ml threshold, 66 and 26% of isolates were resistant to myclobutanil and fenbuconazole, respectively, and 22% were cross resistant to the two fungicides. A significant between-year shift toward increased resistance was noted in two of three orchards surveyed in both years. Failure to use dormant copper sprays, older trees, larger orchards, orchards with ≤10 cultivars, and application of >4 DMI sprays were positively correlated (0.0001 < P < 0.05) with the incidence of resistant isolates. Isolates from orchards with >4 DMI sprays were four times as likely to be resistant to fenbuconazole (odds ratio = 4.57; P = 0.015). Isolates from orchards without dormant copper sprays were twice as likely to be cross-shifted toward resistance to all three fungicides (odds ratio = 1.76; P = 0.048). Results identify management practices that can reduce the risk of V. inaequalis developing resistance to DMI fungicides.     

The 14alpha-Demethylasse(CYP51A1) Gene is Overexpressed in Venturia inaequalis Strains Resistant to Myclobutanil

ABSTRACT We identified the cytochrome P450 sterol 14alpha-demethylase (CYP51A1) gene from Venturia inaequalis and optional insertions located upstream from CYP51A1 and evaluated their potential role in conferring resistance to the sterol demethylation-inhibitor (DMI) fungicide my-clobutanil. The CYP51A1 gene was completely sequenced from one my-clobutanil sensitive (S) and two myclobutanil-resistant (R) strains. No nucleotide variation was found when the three sequences were aligned. Allele-specific polymerase chain reaction (PCR) analysis indicated that a previously described single base pair mutation that correlated with resistance to DMI fungicides in strains of other filamentous fungi was absent in 19 S and 32 R strains of V. inaequalis from Michigan and elsewhere. The sequencing results and PCR analyses suggest that resistance in these strains was not due to a mutation in the sterol demethylase target site for DMI fungicides. Expression of CYP51A1 was determined for strains from an orchard that had never been sprayed with DMI fungicides (baseline orchard), and the data provided a reference for evaluating the expression of strains collected from a research orchard and from three commercial Michigan apple orchards with a long history of DMI use and a high frequency of R strains. Overexpression of CYP51A1 was significantly higher in 9 of 11 R strains from the research orchard than in S strains from the baseline orchard. The high expression was correlated with the presence of a 553-bp insertion located upstream of CYP51A1. Overexpression of the CYP51A1 gene was also detected in eight of eight, five of nine, and nine of nine R strains from three commercial orchards, but the insertion was not detected in the majority of these strains. The results suggest that overexpression of the target-site CYP51A1 gene is an important mechanism of resistance in some field resistant strains of V. inaequalis, but other mechanisms of resistance also appear to exist.     

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.     

Baseline sensitivity and cross-resistance to demethylation-inhibiting fungicides in Ontario isolates of Sclerotinia homoeocarpa

Four hundred and thirty-five isolates of Sclerotinia homoeocarpa from eight populations in southern Ontario were tested for sensitivity to the demethylation-inhibiting (DMI) fungicides, propiconazole, myclobutanil, fenarimol and tebuconazole. The isolates were collected in summer 1994 just prior to legal DMI fungicide use on turfgrass in Ontario. There were wide variations in sensitivities, and seven of the eight populations were very sensitive to the fungicides. Based on mean EC₅₀ and the distribution of DMI sensitivity, one population near the U.S. border was suspected of having been previously exposed to DMI fungicide. Pairwise comparisons of EC₅₀ values for the different fungicides showed low to moderate correlations between fungicides. EC₅₀ values of myclobutanil and propiconazole had the best correlation, followed by the pair of tebuconazole and fenarimol. Other pairwise comparisons were not statistically significant except for a barely significant relationship between EC₅₀ values of myclobutanil and tebuconazole. For field populations of plant pathogens, cross-resistance to different DMI fungicides may not be as strong as conventionally thought. The data collected here will allow comparison to subsequent years to look for detectable shifts in S. homoeocarpa sensitivity to DMI fungicides as they become more frequently used in Ontario.     

甘肃省枸杞炭疽病菌对4种甾醇脱甲基抑制剂的敏感性

为明确甘肃省枸杞炭疽病菌对甾醇脱甲基抑制剂类药剂 (DMIs) 的敏感性,采用菌丝生长速率法测定了采自甘肃省靖远县3个地区及景泰县3个地区共102株枸杞炭疽病菌对苯醚甲环唑、戊唑醇、丙环唑及氟硅唑的敏感性,分别就不同年份、不同地区间胶孢炭疽复合种和尖孢炭疽复合种对4 种 DMIs 杀菌剂的敏感性差异进行了分析。结果表明:供试46 株枸杞胶孢炭疽复合种整体上对苯醚甲环唑、戊唑醇、丙环唑和氟硅唑仍表现为敏感,EC₅₀值分别在0.28~1.20、0.11~2.98、0.32~2.84和0.35~3.85 μg/mL之间;而56株尖孢炭疽复合种对4种药剂的敏感性则出现了不同程度分化,部分菌株疑似已出现敏感性下降现象,其中,对苯醚甲环唑、戊唑醇、丙环唑和氟硅唑敏感性最低的菌株EC₅₀值分别为1.63、3.80、6.21和4.74 μg/mL。不同年份间采集的枸杞胶孢炭疽复合种和尖孢炭疽复合种对4种杀菌剂的敏感性均存在显著差异,2017年采集的菌株敏感性相对更低,4种杀菌剂对胶孢炭疽复合种的平均EC₅₀值分别为 (0.84 ± 0.03)、(1.23 ± 0.13)、(1.19 ± 0.09) 和 (1.69 ± 0.17) μg/mL,对尖孢炭疽复合种的平均EC₅₀值分别为 (1.06 ± 0.03)、(2.25 ± 0.15)、(2.43 ± 0.20) 和 (2.85 ± 0.19) μg/mL。不同地区枸杞炭疽病菌对4种杀菌剂的敏感性表现不同,其中靖远县五合镇的胶孢炭疽复合种对4种杀菌剂敏感性最低,平均EC₅₀值分别为 (0.79 ± 0.12)、(1.28 ± 0.87)、(1.39 ± 1.05) 和 (1.74 ± 1.04) μg/mL,景泰县草窝滩镇的胶孢炭疽复合种对苯醚甲环唑和丙环唑敏感性最高,平均EC₅₀值为 (0.28 ± 0.10) 和 (0.46 ± 0.10) μg/mL,对戊唑醇和氟硅唑敏感性最高的胶孢炭疽复合种来自景泰县寺滩乡,平均EC₅₀值为 (0.42 ± 0.16) 和 (0.65 ± 0.09) μg/mL;不同地区间采集的尖孢炭疽复合种对4种杀菌剂的敏感性则不存在显著差异。研究结果可为甘肃省枸杞炭疽病防治中杀菌剂的合理使用及延缓抗药性发展提供依据。     

Sensitivity of Lasiodiplodia theobromae from Brazilian papaya orchards to MBC and DMI fungicides

Stem rot caused by Lasiodiplodia theobromae is an important postharvest disease of papaya in Brazil, responsible for reducing the quality and quantity of fruits. Fungicide use is one of the main disease management measures. However, there are no estimates available of pathogen sensitivity to commonly employed fungicides. Therefore, the EC₅₀ from 120 isolates of L. theobromae from northeastern Brazil, representative of six populations of the pathogen, was estimated in vitro for fungicides of the methyl benzimidazole carbamates—MBC (benomyl and thiabendazole) and demethylation-inhibiting—DMI (imazalil, prochloraz, tebuconazole) groups. Mycelial growth on fungicide-free media and virulence on papaya fruits of the MBC-sensitive and non-sensitive isolates were compared. For MBCs, 8.4% of isolates were non-sensitive to fungicides. For the remaining 91.6%, the mean EC₅₀ ranged from 0.002 to 0.13 μg ml⁻¹ and 0.36 to 1.27 μg ml⁻¹ for benomyl and thiabendazole, respectively. For DMIs, the mean EC₅₀ range for imazalil was 0.001 to 2.27 μg ml⁻¹, 0.04 to 1.75 μg ml⁻¹ for prochloraz, and 0.14 to 4.05 μg ml⁻¹ for tebuconazole. The EC₅₀ values of non-sensitive isolates were significantly (P≤0.05) higher those for the sensitive isolates for each of the DMI fungicides. Differences (P≤0.05) were found in the levels of sensitivity to DMI fungicides among the isolate populations associated with orchards. The populations from two orchards were less sensitive to DMIs. No solid evidence was found for fitness costs relating to MBC non-sensitive isolates because mycelial growth in fungicide-free media and virulence on papaya fruits were similar to those of sensitive isolates.     

Baseline sensitivity of Monilia yunnanensis to the DMI fungicides tebuconazole and triadimefon

Monilia yunnanensis was recently identified as a new species causing brown rot of peach in China. Sterol 14α-demethylase inhibitors (DMIs) continue to be important in the management of brown rot of Monilinia spp. worldwide. Tebuconazole and triadimefon are two kinds of DMI fungicides that may be used for brown rot control in China. To establish the baseline sensitivity of M. yunnanensis to these two DMI fungicides, 203 M. yunnanensis single spore isolates were collected. Measurements of sensitivity to the two fungicides were based on inhibition of mycelial growth. For both fungicides, the sensitivity distribution was a unimodal curve, with an EC₅₀ range (the effective concentration to inhibit mycelial growth by 50 %) of 0.0001–0.0644 μg/ml for tebuconazole and 0.2311–1.7477 μg/ml for triadimefon. The M. yunnanensis isolates were obtained from orchards where DMI fungicides have not been used for peach brown rot control, thus the fungicide sensitivity distribution established in this study can be considered as the baseline for monitoring the resistance development in M. yunnanensis once the DMI fungicides are used to control peach brown rot.     

山核桃干腐病菌对7种杀菌剂的敏感性比较及其 对苯醚甲环唑敏感基线的建立

测定了甲基硫菌灵、百菌清、腐霉利、戊唑醇、啶酰菌胺、苯醚甲环唑和醚菌酯对山核桃干腐病菌Botryosphaeria dothidea的离体抑菌活性。结果表明,上述7种杀菌剂抑制该病原菌菌丝生长的活性依次为苯醚甲环唑＞戊唑醇＞腐霉利＞甲基硫菌灵＞百菌清＞醚菌酯＞啶酰菌胺。进一步研究表明,苯醚甲环唑对采自安徽和浙江两省从未使用过甾醇脱甲基抑制剂类(DMIs)杀菌剂的150株山核桃干腐病菌菌丝生长的平均EC50值为0.43( ±0.11) μ g/mL,可作为其敏感基线用于以后的抗药性监测。     

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.     

Effects of cherry leaf spot on photosynthesis in tart cherry 'Montmorency' foliage

Results described here span a total of three field seasons and quantitatively depict the effects of an economically important fungal pathogen (Blumeriella jaapii) on tart cherry (Prunus cerasus 'Montmorency') leaf physiology. For the first time, leaf photosynthesis, stomatal conductance (g(s)), maximum ribulose-1,5-bisphosphate carboxylation rate (V(cmax)), and maximum electron transport (J(max)) were measured as functions of visible cherry leaf spot disease (CLS) severity. Defined as the proportion of chlorotic and necrotic tissue per leaf, CLS severity was estimated from leaves of mature 'Montmorency' trees in 2007, 2008, and 2009. Briefly, as visible disease severity increased, all of the leaf-level physiological parameters decreased significantly (P < 0.01) and disproportionately. Thus, the effects of visible symptoms on leaf photosynthetic metabolic function encroached upon asymptomatic tissue as well. Impairment of photosynthetic metabolism in 'Montmorency' tart cherry leaves due to CLS appears to be mediated through disproportionately large perturbations in g(s), V(cmax), and J(max). These findings offer a new perspective on the amount of damage that this serious disease can inflict.     

Sensitivity of Mycosphaerella graminicola (anamorph: Septoria tritici) to DMI fungicides across Europe and impact on field performance

Since the occurrence and spread of resistance to quinone outside inhibitors (QoI) in Mycosphaerella graminicola in the early 2000s in Europe, demethylation inhibitors (DMIs) form the backbone for control of Septoria leaf blotch. European monitoring studies, carried out by various research institutes and DMI manufacturers, have shown a shift of the European M. graminicola population towards increased ED₅₀ values for DMI fungicides. Populations of M. graminicola consist of very heterogeneous isolates within a region, and even within a field, in terms of DMI sensitivity. Sensitivity to DMIs is influenced by the haplotype of CYP51, the target of DMIs. New CYP51‐haplotypes have emerged and the frequency of less sensitive haplotypes in Europe has increased in recent years. Studies with efflux transporter inhibitors showed that not only CYP51, but also enhanced efflux, may play a role in the DMI sensitivity response. Sensitivity studies with 5 DMIs registered for Septoria leaf blotch control indicated that sensitivity of isolates to the 5 DMIs is heterogeneous and the overall correlation of sensitivity to the different DMIs is poor. A key requirement for sustainable control and resistance management of Septoria leaf blotch is therefore the continued availability of different DMIs.     

Characterization of CbCyp51 from field isolates of Cercospora beticola

The hemibiotrophic fungus Cercospora beticola causes leaf spot of sugar beet. Leaf spot control measures include the application of sterol demethylation inhibitor (DMI) fungicides. However, reduced sensitivity to DMIs has been reported recently in the Red River Valley sugar beet-growing region of North Dakota and Minnesota. Here, we report the cloning and molecular characterization of CbCyp51, which encodes the DMI target enzyme sterol P450 14α-demethylase in C. beticola. CbCyp51 is a 1,632-bp intron-free gene with obvious homology to other fungal Cyp51 genes and is present as a single copy in the C. beticola genome. Five nucleotide haplotypes were identified which encoded three amino acid sequences. Protein variant 1 composed 79% of the sequenced isolates, followed by protein variant 2 that composed 18% of the sequences and a single isolate representative of protein variant 3. Because resistance to DMIs can be related to polymorphism in promoter or coding sequences, sequence diversity was assessed by sequencing >2,440 nucleotides encompassing CbCyp51 coding and flanking regions from isolates with varying EC(50) values (effective concentration to reduce growth by 50%) to DMI fungicides. However, no mutations or haplotypes were associated with DMI resistance or sensitivity. No evidence for alternative splicing or differential methylation of CbCyp51 was found that might explain reduced sensitivity to DMIs. However, CbCyp51 was overexpressed in isolates with high EC(50) values compared with isolates with low EC(50) values. After exposure to tetraconazole, isolates with high EC(50) values responded with further induction of CbCyp51, with a positive correlation of CbCyp51 expression and tetraconazole concentration up to 2.5 μg ml(-1).     

Sensitivity of populations ofBotrytis cinerea to triazoles,benomyl and vinclozolin

Sensitivity of field isolates (121) ofBotrytis cinerea from France (1992), Germany (1979–1992), Israel (1990) and the Netherlands (1970–1989) to the triazoles tebuconazole and triadimenol, the benzimidazole benomyl and the dicarboximide vinclozolin were tested in radial growth experiments. Resistance to benomyl (in 21 to 100% of isolates tested) and vinclozolin (in 25 to 71% of isolates tested) was common in most countries. EC₅₀s (concentrations of fungicides inhibiting radial mycelial growth ofB. cinerea on B5-agar by 50%) for tebuconazole and triadimenol ranged between 0.01–1.64 and 0.4–32.6g ml^–1, respectively, and were log-normally distributed. The variation factor (ratio between EC₅₀s of the least and most sensitive isolate tested) amounts 164 and 82 for tebuconazole and triadimenol, respectively. These values are comparable to those for azole fungicides applied in control of other pathogens. Hence, variation in sensitivity to triazoles can probably not explain limited field performance of triazoles towardsB. cinerea. Isolates from south west Germany (1992) were significantly less sensitive to tebuconazole than isolates collected earlier in Germany, Israel and the Netherlands. Such less sensitive populations may contribute to the limited field performance of DMI fungicides towardsB. cinerea. The sensitivity of isolates from south west Germany to tebuconazole was similar to that of DMI-resistant mutants generated in the laboratory. These mutants displayed stable resistance with Q-values (ratio between EC₅₀ of resistant mutant and wild type isolate) between 5 and 20. Sensitivity of field isolates and laboratory mutants to tebuconazole and triadimenol was correlated.     

Monitoring and characterization of resistance development of strawberry Phomopsis leaf blight to fungicides

From 2005 to 2009, a total of 479 single-conidial isolates of Phomopsis obscurans were collected from strawberry. The isolates were characterized for their resistance to benzimidazole fungicides, diethofencarb, and sterol demethylation inhibitors (DMIs). Low-level DMI resistant isolates (DMI-LR) and two types of benzimidazole-resistant (Ben R) isolates, Ben R1 (benzimidazole-resistant and diethofencarb -sensitive) and Ben R2 (benzimidazole-resistant and diethofencarb -resistant), were detected. Both Ben R and DMI-LR isolates exhibited comparable growth, sporulation, and pathogenicity with the sensitive isolates. No significant difference in growth at low temperature was observed between Ben R and benzimidazole-sensitive (Ben S) isolates. Ben R1 was caused by a point mutation from GAG to GTG at codon 198 in the β-tubulin gene in Ben S isolates, predicted to cause a change from glutamic acid to valine. Ben R2 was induced by a point mutation from TTC to TAC at codon 200 in the β-tubulin gene in Ben S isolates.     

Discontinuance of tebuconazole in the field restores sensitivity of Monilinia fructicola in stone fruit orchards

Demethylation inhibitor (DMI) fungicides are used to control brown rot in stone fruit worldwide. However, their specific mode of action can select resistant isolates of Monilinia fructicola. Monilinia fructicola resistant to DMI fungicides are associated with a fitness cost in the absence of selective pressure, indicating that the sensitive population can be re-established when discontinuing the fungicide in the field. This work aimed to build up the sensitive population of M. fructicola after discontinuing the use of tebuconazole for successive crop seasons. The sensitivity of M. fructicola to tebuconazole was assessed in four commercial peach orchards in Paraná and São Paulo States from 2012/13 to 2015/16. Different fungicide programmes were used and DMI fungicides were discontinued from 2013/14. The sensitivity of M. fructicola to tebuconazole was assessed by a mycelial growth assay in vitro and by determining the frequency of the G461S mutation in the MfCYP51 gene. The isolates from Paraná had high sensitivity to the fungicide across all seasons and the frequency of the G461S mutation remained below 5%. The isolates from São Paulo were highly resistant in the 2012/13 season; however, there was a gradual decline until 2015/16. In addition, the G461S mutation frequency in Sao Paulo State was about 80% in the 2012/13 season, but reduced until it was completely undetectable in 2015/16. These results provide evidence that resistance can be managed in orchards with high selective pressure to tebuconazole after discontinuing the use of the fungicide for at least 3 years.     

Heterologous expression of the P450 sterol 14α-demethylase gene from Monilinia fructicola reduces sensitivity to some but not all DMI fungicides

The cytochrome P450 sterol 14α-demethylase gene (MfCYP51) from Monilinia fructicola (G. Wint.) Honey was cloned and sequenced. The gene was 1680 bp in length (including introns) and was predicted to have two introns of 54 and 57 bp. The nucleotide sequence was 82.1, 53.4, 47.1, 45.1, and 33.6% and the amino acid sequence was 89.7, 76.1, 76.1, 71.8, and 66.9% identical to the CYP51 genes from Botrytis cinerea, Tapesia yallundae, T. acuformis, Erysiphe graminis, and Uncinula necator, respectively. Expression of MfCYP51 in PDR5::TN5 deficient Saccharomyces cerevisiae resulted in reduced sensitivity of the yeast transformants to myclobutanil but not to propiconazole, fenbuconazole or tebuconazole. A wildtype population of 33 M. fructicola isolates was significantly less sensitive to myclobutanil than to propiconazole, fenbuconazole, and tebuconazole. The sensitivity of the isolates to myclobutanil and the three other DMI fungicides included in this study was correlated positively, suggesting a similar or identical mode of action. The low sensitivity in M. fructicola wildtype isolates to myclobutanil could result from a less effective binding potential of the fungicide to the 14α-demethylase.     

Distributions of Sensitivities to Three Sterol Demethylation Inhibitor Fungicides Among Populations of Uncinula necator Sensitive 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.     

Protective and curative efficacy of prothioconazole against isolates of Mycosphaerella graminicola differing in their in vitro sensitivity to DMI fungicides

BACKGROUND: Septoria leaf blotch is the most important disease of wheat in Europe. To control this disease, fungicides of the 14α‐demethylase inhibitor group (DMIs) have been widely used for more than 20 years. However, resistance towards DMIs has increased rather quickly in recent years. The objective of this study was to evaluate, on plants and under controlled conditions, the protective and curative efficacy of the DMI fungicide prothioconazole against three current isolates of M. graminicola, chosen to belong to different DMI‐resistant phenotypes. Fungicide efficacy was assessed by visual symptoms and by quantitative real‐time polymerase chain reaction (PCR). RESULTS: With a protective fungicide application, prothioconazole was always effective against each isolate. This was in accordance with the EC₅₀ results. However, curative efficacy differed between the isolates. It remained at a good level, between 60 and 70% against one isolate, whereas it was strongly affected by late applications from 7 days post‐inoculation with the two other isolates. CONCLUSION: A protective application of prothioconazole in wheat crops could be the best strategy to keep a high efficacy against Septoria leaf blotch. Copyright © 2011 Society of Chemical Industry     

Assessment of fungicide resistance and pathogen diversity in Erysiphe necator using quantitative real-time PCR assays

BACKGROUND: Management of grapevine powdery mildew Erysiphe necator Schw. requires fungicide treatments such as sterol demethylation inhibitors (DMIs) or mitochondrial inhibitors (QoIs). Recently, reduction in the efficacy of DMIs or QoIs was reported in Europe and the United States. The aim of the present study was to develop real‐time qPCR tools to detect and quantify several CYP51 gene variants of E. necator: (i) A versus B groups (G37A) and (ii) sensitive versus resistant to sterol demethylase inhibitor fungicides (Y136F). RESULTS: The efficacy of the qPCR tools developed was better than the CAPS method, with a limit of 2 pg for E necator DNA, 0.06 ng for genetic group A and 1.4 ng for the DMI‐resistant allele. The detection limits of qPCR protocols (LOD) ranged from 0.72 to 0.85%, and the quantification limits (LOQ) ranged from 2.4 to 2.85% for the two alleles G47A and Y136F respectively. The application of qPCR to field isolates from French vineyards showed the presence of DMI‐resistant and/or QoI‐resistant alleles in French pathogen populations, linked to genetic group B. CONCLUSION: The real‐time PCR assay developed in this study provides a potentially useful tool for efficient quantification of different alleles of interest for fungicide monitoring and for population structure of E. necator. Copyright © 2010 Society of Chemical Industry