Effects of fungicide dose and mixtures on selection for triazole resistance in Mycosphaerella graminicola under field conditions

The effects of varying doses of fungicides, alone or in mixtures, on selection for triazole resistance were examined under field conditions. Two experiments were conducted using the triazole fungicide fluquinconazole with the strobilurin fungicide azoxystrobin as a mixture partner. Inoculated wheat plots with a known ratio of more sensitive to less sensitive isolates of the leaf blotch fungus Mycosphaerella graminicola were sprayed with fungicide and sampled once symptoms had appeared. Selection for fluquinconazole resistance increased in proportion to the dose, up to one-half of the full dose (the maximum tested) in both experiments. At the higher doses of fluquinconazole, the addition of azoxystrobin was associated with a decrease in selection (nonsignificant in the first experiment) for triazole resistance. Control by low doses of fluquinconazole was increased by mixture with azoxystrobin, but at higher doses mixture with azoxystrobin sometimes decreased control, so that reduced selection was obtained at the cost of some reduction in control. The effects on resistance are not necessarily general consequences of mixing fungicides, and suggest that the properties of any specific mixture may need to be demonstrated experimentally. Selection was inversely related to control in the unmixed treatments in both experiments, but the relationship was weaker in the mixtures with azoxystrobin.     

Adaptation to fungicides in Monilinia fructicola isolates with different fungicide resistance phenotypes

The ability to develop fungicide resistance was assessed in Monilinia fructicola isolates with different fungicide sensitivity phenotypes by adapting mycelium and conidia to increasing concentrations of selective fungicides and UV mutagenesis. Results showed that adaptation to Quinone outside inhibitor (QoI) fungicide azoxystrobin and sterol demethylation inhibitor (DMI) fungicide propiconazole was more effective in conidial-transfer experiments compared to mycelial-transfer experiments. DMI-resistant (DMI-R) isolates adapted to significantly higher doses of azoxystrobin in both, mycelial- and conidial-transfer experiments compared to benzimidazole-resistant (BZI-R) and sensitive (S) isolates. Adaptation to propiconazole in conidial-transfer experiments was accelerated in BZI-R isolates when a stable, nonlethal dose of 50 microg/ml thiophanate-methyl was added to the selection medium. One of two azoxystrobin-resistant mutants from DMI-R isolates did not show any fitness penalties; the other isolate expired before further tests could be carried out. The viable mutant caused larger lesions on detached peach fruit sprayed with azoxystrobin compared to the parental isolate. The azoxystrobin sensitivity of the viable mutant returned to baseline levels after the mutant was transferred to unamended medium. However, azoxystrobin resistance recovered quicker in the mutant compared to the corresponding parental isolate after renewed subculturing on medium amended with 0.2 and 1 microg/ml azoxystrobin; only the mutant but not the parental isolate was able to adapt to 5 microg/ml azoxystrobin. In UV mutagenesis experiments, the DMI-R isolates produced significantly more mutants compared to S isolates. All of the UV-induced mutants showed stable fungicide resistance with little fitness penalty. This study indicates the potential for QoI fungicide resistance development in M. fructicola in the absence of a mutagen and provides evidence for increased mutability and predisposition to accelerated adaptation to azoxystrobin in M. fructicola isolates resistant to DMI fungicides.     

Assessment of QoI resistance in Plasmopara viticola oospores

QoI fungicides, inhibitors of mitochondrial respiration at the Qo site of cytochrome b in the mitochondrial bc(1) enzyme complex, are commonly applied in vineyards against Plasmopara viticola (Berk. & MA Curtis) Berl. & De Toni. Numerous treatments per year with QoI fungicides can lead to the selection of resistant strains in the pathogen population owing to the very specific and efficient mode of action. In order to evaluate the resistance risk and its development, two different methods, biological and molecular, were applied to measure the sensitivity of oospores differentiated in vineyards, both treated and untreated with azoxystrobin, from 2000 to 2004. Assays using oospores have the advantage of analysing the sensitivity of bulked samples randomly collected in vineyards, describing accurately the status of resistance at the end of the grapevine growing season. Both methods correlated well in describing the resistance situation in vineyards. QoI resistance was not observed in one vineyard never treated with QoI fungicides. In the vineyard where azoxystrobin had been used in mixture with folpet, the selection of QoI-resistant strains was lower, compared with using solely QoI. In vineyards where QoI treatments have been stopped, a decrease in resistance was generally observed.     

Molecular Characterization and Diagnosis of QoI Resistance in Cucumber and Eggplant Fungal Pathogens

ABSTRACT The molecular mechanism of QoI fungicide resistance was studied using isolates of cucumber Corynespora leaf spot fungus (Corynespora cassiicola) and the eggplant leaf mold (Mycovellosiella nattrassii). In both pathogens, a mutation at position 143 from glycine to alanine (G143A) was detected in the cytochrome b gene that encodes for the fungicide-targeted protein. Moreover, the nucleotide sequence at amino acid position 143 was converted from GGT or GGA in sensitive (wild-type) to GCT or GCA in resistant (mutant-type) isolates. The methods of polymerase chain reaction restriction fragment length polymorphism commonly used for QoI resistance monitoring were employed successfully, leading to the amplified gene fragment from resistant isolates being cut with the restriction enzyme ItaI. However, heteroplasmy (the coexistence of wild-type and mutated alleles) was found when the resistant isolates of C. cassiicola, M. nattrassii, and Colletotrichum gloeosporioides (strawberry anthracnose fungus) were subcultured in the presence or absence of QoI fungicides. QoI resistance of cucumber powdery and downy mildew isolates persisted for a few years following the removal of the selection pressure imposed by the fungicide under both laboratory and commercial greenhouse conditions. The proportion of mutated sequences in cytochrome b gene decreased over time in the pathogen population. The protective efficacy of the full dose of azoxystrobin decreased when the populations of powdery and downy mildews contained resistant isolates at 10%. Using FMBIO, a fluorescence bio-imaging analyzer, the mutant allele from the QoI-resistant isolates could be detected at the level of 1%, whereas the detection sensitivity of ethidium-bromide-stained gels was approximately 10 times lower.     

Delaying selection for fungicide insensitivity by mixing fungicides at a low and high risk of resistance development: a modeling analysis

This study used mathematical modeling to predict whether mixtures of a high-resistance-risk and a low-risk fungicide delay selection for resistance against the high-risk fungicide. We used the winter wheat and Mycosphaerella graminicola host-pathogen system as an example, with a quinone outside inhibitor fungicide as the high-risk and chlorothalonil as the low-risk fungicide. The usefulness of the mixing strategy was measured as the "effective life": the number of seasons that the disease-induced reduction of the integral of canopy green area index during the yield forming period could be kept <5%. We determined effective lives for strategies in which the dose rate (i) was constant for both the low-risk and high-risk fungicides, (ii) was constant for the low-risk fungicide but could increase for the high-risk fungicide, and (iii) was adjusted for both fungicides but their ratio in the mixture was fixed. The effective life was highest when applying the full label-recommended dose of the low-risk fungicide and adjusting the dose of the high-risk fungicide each season to the level required to maintain effective control. This strategy resulted in a predicted effective life of ≤ 12 years compared with 3 to 4 years when using the high risk fungicide alone.     

Monitoring of QoI fungicide resistance in Plasmopara viticola populations in Japan

BACKGROUND: The increasing occurrence of QoI fungicide resistance in Plasmopara viticola (Berk. & MA Curtis) Berl. & DeToni populations is becoming a serious problem in the control of grapevine downy mildew. In Japan, the existence of QoI‐fungicide‐resistant P. viticola was reported in 2009. RESULTS: The QoI fungicide resistance in P. viticola samples collected from vineyards in Japan in 2008 and 2009 was monitored. Resistant P. viticola were detected in the regions where QoI fungicides have been introduced in accordance with the pest management programme, whereas in Hokkaido vineyards, where QoI fungicides have not yet been introduced, QoI‐fungicide‐resistant P. viticola were not found. CONCLUSION: Japan comprises thousands of islands and is physically isolated from other countries by the sea. Monitoring the emergence, incidence and distribution of QoI fungicide resistance in P. viticola populations in Japan is necessary to improve pest management strategies for downy mildew disease in Japanese vineyards. Copyright © 2010 Society of Chemical Industry     

Emergence and early evolution of fungicide resistance in North American populations of Zymoseptoria tritici

Although fungicide resistance in crop pathogens is a global threat to food production, surprisingly little is known about the evolutionary processes associated with the emergence and spread of fungicide resistance. Early stages in the evolution of fungicide resistance were evaluated using the wheat pathogen Zymoseptoria tritici, taking advantage of an isolate collection spanning 20 years in Oregon, USA, and including two sites with differing intensity of fungicide use. Sequences of the mitochondrial cytb protein conferring single‐mutation resistance to QoI fungicides and the nuclear CYP51 gene implicated in multiple‐mutation resistance to azole fungicides were analysed. Mutations associated with resistance to both fungicides were absent in the 1992 isolates, but frequent in the 2012 collection, with higher frequencies of resistance alleles found at the field site with more intensive fungicide use. Results suggest that the QoI resistance evolved independently in several lineages, and resulted in significant mitochondrial genome bottlenecks. In contrast, the CYP51 gene showed signatures of diversifying selection and intragenic recombination among three phylogenetic clades. The findings support a recent emergence of resistance to the two fungicide classes in Oregon, facilitated by selection for mutations in the associated resistance genes.     

Evolution of Qol resistance in <Emphasis Type="Italic">Plasmopara viticola</Emphasis> oospores

QoI resistance in P. viticola was first detected in France and Italy in 1999. Molecular and biological assays have been carried out since 2000 in order to provide reliable methods of detecting and quantifying resistance. Oospores were collected in vineyards located in northern and southern Italy. QoI resistance was evaluated by the germination rate of oospores on azoxystrobin amended medium and the frequency of mutant alleles in the DNA extracted from oospores. Both methods correlated to each other and were used side by side to test QoI resistance. Due to the spontaneous occurrence of the G143A mutation in wild type populations and the immigration from surrounding vineyards, resistance frequencies up to 10% were found in samples collected from vineyards never treated with QoIs. Particularly high values, about 90%, were associated with the application of five to six QoI treatments within the same season, while lower percentages, about 30%, were detected in vineyards treated with QoI used in mixture with fungicides belonging to a different resistance group. A progressive decrease of resistance frequency was observed when QoI applications were reduced in number or completely suspended for at least one season. Therefore, a full recovery of sensitivity may be achieved even in vineyards characterized by high levels of resistance, if particular care is taken during disease control by using QoIs only in mixtures and reducing the number of QoI treatments.     

Target and non-target site mechanisms of fungicide resistance and their implications for the management of crop pathogens

Fungicides are indispensable for high-quality crops, but the rapid emergence and evolution of fungicide resistance have become the most important issues in modern agriculture. Hence, the sustainability and profitability of agricultural production have been challenged due to the limited number of fungicide chemical classes. Resistance to site-specific fungicides has principally been linked to target and non-target site mechanisms. These mechanisms change the structure or expression level, affecting fungicide efficacy and resulting in different and varying resistance levels. This review provides background information about fungicide resistance mechanisms and their implications for developing anti-resistance strategies in plant pathogens. Here, our purpose was to review changes at the target and non-target sites of quinone outside inhibitor (QoI) fungicides, methyl-benzimidazole carbamate (MBC) fungicides, demethylation inhibitor (DMI) fungicides, and succinate dehydrogenase inhibitor (SDHI) fungicides and to evaluate if they may also be associated with a fitness cost on crop pathogen populations. The current knowledge suggests that understanding fungicide resistance mechanisms can facilitate resistance monitoring and assist in developing anti-resistance strategies and new fungicide molecules to help solve this issue. © 2023 Society of Chemical Industry.     

Occurrence and distribution of resistance to QoI fungicides in populations of Podosphaera fusca in south central Spain

Cucurbit powdery mildew caused by Podosphaera fusca limits crop production in Spain. Since its management is strongly dependent on chemicals, the rational design of control programmes requires a good understanding of the fungicide resistance phenomenon in field populations. Fifty single-spore isolates of P. fusca were tested for sensitivity to three quinone-outside inhibiting (QoI) fungicides: azoxystrobin, kresoxim-methyl and trifloxystrobin. Minimum inhibitory concentration (MIC) values for QoI-sensitive isolates were found to range from 0.25 to 10 μg ml⁻¹ for azoxystrobin to 5–25 μg ml⁻¹ for kresoxim-methyl, using a leaf disc-based bioassay. High levels of cross-resistance to QoI fungicides were found. Eleven isolates showed resistance to the three QoI fungicides tested with MIC and EC₅₀ values >500 μg ml⁻¹ resulting in RF values as high as >715 and >1000 for trifloxystrobin and azoxystrobin, respectively. A survey of P. fusca QoI resistance was carried out in different provinces located in the south central area of Spain during the cucurbit growing seasons in 2002, 2003 and 2004. Examination of a collection of 250 isolates for QoI resistance revealed that 32% were resistant to the three fungicides tested; the provinces of Ciudad Real, Córdoba and Murcia being the locations with the highest frequencies of resistance (44–74%). By contrast, no resistance was found in Badajoz, and relatively low frequencies were observed in Almería and Valencia (10–13%). Nearly 50% of resistant isolates were collected from melon plants. Based on these data, recommendations about the use of QoI fungicides for cucurbit powdery mildew management in the sampled areas are made.     

QoI resistance emerged independently at least 4 times in European populations of Mycosphaerella graminicola

BACKGROUND: QoI fungicides or quinone outside inhibitors (also called strobilurins) have been widely used to control agriculturally important fungal pathogens since their introduction in 1996. Strobilurins block the respiration pathway by inhibiting the cytochrome bc1 complex in mitochondria. Several plant pathogenic fungi have developed field resistance. The first QoI resistance in Mycosphaerella graminicola (Fuckel) Schroter was detected retrospectively in UK in 2001 at a low frequency in QoI-treated plots. During the following seasons, resistance reached high frequencies across northern Europe. The aim of this study was to identify the main evolutionary forces driving the rapid emergence and spread of QoI resistance in M. graminicola populations.RESULTS: The G143A mutation causing QoI resistance was first detected during 2002 in all tested populations and in eight distinct mtDNA sequence haplotypes. By 2004, 24 different mtDNA haplotypes contained the G143A mutation. Phylogenetic analysis showed that strobilurin resistance was acquired independently through at least four recurrent mutations at the same site of cytochrome b. Estimates of directional migration rates showed that the majority of gene flow in Europe had occurred in a west-to-east direction.CONCLUSION: This study demonstrated that recurring mutations independently introduced the QoI resistance allele into different genetic and geographic backgrounds. The resistant haplotypes then increased in frequency owing to the strong fungicide selection and spread eastward through wind dispersal of ascospores.     

植物病原菌对杀菌剂抗性风险评估

植物病原菌对杀菌剂的抗性风险由基本风险和治理风险组成。杀菌剂使用之前或之初可根据 人工诱变、药剂选择或驯化实验、田间野生敏感菌株敏感性变异、抗药菌株的生物及遗传特 征、杀菌剂作用方式等进行基本抗药风险预测；杀菌剂使用数年之后可根据人工诱变、药剂 选择或驯化、田间药效与抗药性发生、抗药菌株的生物及遗传特征、杀菌剂作用方式与使用 对策等已有资料进行抗药风险评估。目前已有4种方法用于抗药风险评估。由杀菌剂与病害 共同决定的基本抗药风险可分成低、中和高度。基本抗性风险高的药剂合理使用可延缓田间 抗药性发生，中度基本抗性风险药剂不合理使用也可引发田间抗药性发生和药效明显降低。     

Development of a multiplex allele‐specific primer PCR assay for simultaneous detection of QoI and CAA fungicide resistance alleles in Plasmopara viticola populations

BACKGROUND: DNA‐based diagnosis has become a common tool for the evaluation of fungicide resistance in obligate phytopathogenic fungus Plasmopara viticola. RESULTS: A multiplex allele‐specific primer PCR assay has been developed for the rapid detection of fungicide resistance in P. viticola populations. With this assay, a glycine‐to‐alanine substitution at codon 143 of the P. viticola cytochrome b gene, which conferred QoI fungicide resistance, and a glycine‐to‐serine substitution at codon 1105 of the P. viticola cellulose synthase gene PvCesA3, which conferred CAA fungicide resistance, were detected simultaneously. CONCLUSION: It is suggested that the present assay is a reliable tool for the rapid and simultaneous detection of QoI and CAA fungicide resistance alleles in P. viticola populations. The assay required only 2 h from the sampling of symptoms to the detection of resistance alleles to both fungicides. Copyright © 2012 Society of Chemical Industry     

Evaluation of the incidence of the G143A mutation and cytb intron presence in the cytochrome bc-1 gene conferring QoI resistance in Botrytis cinerea populations from several hosts

BACKGROUND: Previous studies have shown that resistance of Botrytis cinerea to QoI fungicides has been attributed to the G143A mutation in the cytochrome b (cytb) gene, while, in a part of the fungal population, an intron has been detected at codon 143 of the gene, preventing QoI resistance. During 2005–2009, 304 grey mould isolates were collected from strawberry, tomato, grape, kiwifruit, cucumber and apple in Greece and screened for resistance to pyraclostrobin and for the presence of the cytb intron, using a novel real‐time TaqMan PCR assay developed in the present study. RESULTS: QoI‐resistant phenotypes existed only within the population collected from strawberries. All resistant isolates possessed the G143A mutation. Differences were observed in the genotypic structure of cytb. Individuals possessing the intron were found at high incidence in apple fruit and greenhouse‐grown tomato and cucumber populations, whereas in the strawberry population the intron frequency was lower. Cultivation of QoI‐resistant and QoI‐sensitive isolates for ten culture cycles on artificial nutrient medium in the presence or absence of fungicide selection showed that QoI resistance was stable. CONCLUSIONS: The results of the study suggest that a high risk for selection of QoI‐resistant strains exists in crops heavily treated with QoIs, in spite of the widespread occurrence of the cytb intron in B. cinerea populations. The developed real‐time TaqMan PCR constitutes a powerful tool to streamline detection of the mutation by reducing pre‐ and post‐amplification manipulations, and can be used for rapid screening and quantification of QoI resistance. Copyright © 2011 Society of Chemical Industry     

葡萄霜霉病有效药剂筛选及药效评价

采用叶盘漂浮法测定了保护性杀菌剂二硫代氨基甲酸盐类、咪唑类、吡啶类,内吸性杀菌剂酰胺类、烟酰胺类、甲氧基丙烯酸酯类、三唑类、氨基甲酸酯类、恶唑类及多种复配剂等对葡萄霜霉病菌的毒力,并进行了田间药效试验。室内药剂测定结果表明,当前生产上的主要药剂和新投入到市场中的部分药剂对葡萄霜霉病菌表现较高毒力;6种复配药剂对葡萄霜霉病菌菌株的毒力高于单剂。田间试验结果表明试验药剂均可有效地控制葡萄霜霉病的发生和危害。     

Site‐directed mutagenesis of the cytochrome b gene and development of diagnostic methods for identifying QoI resistance of rice blast fungus

BACKGROUND: It is possible that a single nucleotide polymorphism (SNP) (G143A mutation) in the cytochrome b gene could confer resistance to quinone outside inhibiting (QoI) fungicides (strobilurins) in rice blast fungus because this mutation caused a high level of resistance to fungicides such as azoxystrobin in Pyricularia grisea Sacc. and other fungal plant pathogens. The aim of this study was to survey Magnaporthe oryzae B Couch sp. nov. isolates in Japan for resistance to QoIs, and to try to develop molecular detection methods for QoI resistance. RESULTS: A survey on the QoI resistance among M. oryzae isolates from rice was conducted in Japan. A total of 813 single‐spore isolates of M. oryzae were tested for their sensitivity to azoxystrobin using a mycelial growth test on PDA. QoI fungicide resistance was not found among these isolates. The introduction of G143A mutation into a plasmid containing the cytochrome b gene sequence of rice blast fungus was achieved by site‐directed mutagenesis. Molecular diagnostic methods were developed for identifying QoI resistance in rice blast fungus using the plasmid construct. CONCLUSION: As the management of rice blast disease is often dependent on chemicals, the rational design of control programmes requires a proper understanding of the fungicide resistance phenomenon in field populations of the pathogen. Mutation of the cytochrome b gene of rice blast fungus would be specifically detected from diseased leaves and seeds using the molecular methods developed in this study. Copyright © 2009 Society of Chemical Industry     

Cytochrome b gene structure and consequences for resistance to Qo inhibitor fungicides in plant pathogens

The cytochrome b (cyt b) gene structure was characterized for different agronomically important plant pathogens, such as Puccinia recondita f sp tritici (Erikss) CO Johnston, P graminis f sp tritici Erikss and Hennings, P striiformis f sp tritici Erikss, P coronata f sp avenae P Syd & Syd, P hordei GH Otth, P recondita f sp secalis Roberge, P sorghi Schwein, P horiana Henn, Uromyces appendiculatus (Pers) Unger, Phakopsora pachyrhizi Syd & P Syd, Hemileia vastatrix Berk & Broome, Alternaria solani Sorauer, A alternata (Fr) Keissl and Plasmopara viticola (Berk & Curt) Berlese & de Toni. The sequenced fragment included the two hot spot regions in which mutations conferring resistance to QoI fungicides may occur. The cyt b gene structure of these pathogens was compared with that of other species from public databases, including the strobilurin-producing fungus Mycena galopoda (Pers) P Kumm, Saccharomyces cerevisiae Meyer ex Hansen, Venturia inaequalis (Cooke) Winter and Mycosphaerella fijiensis Morelet. In all rust species, as well as in A solani, resistance to QoI fungicides caused by the mutation G143A has never been reported. A type I intron was observed directly after the codon for glycine at position 143 in these species. This intron was absent in pathogens such as A alternata, Blumeria graminis (DC) Speer, Pyricularia grisea Sacc, Mycosphaerella graminicola (Fuckel) J Schr?t, M fijiensis, V inaequalis and P viticola, in which resistance to QoI fungicides has occurred and the glycine is replaced by alanine at position 143 in the resistant genotype. The present authors predict that a nucleotide substitution in codon 143 would prevent splicing of the intron, leading to a deficient cytochrome b, which is lethal. As a consequence, the evolution of resistance to QoI fungicides based on G143A is not likely to evolve in pathogens carrying an intron directly after this codon.     

Diversity and fitness of <Emphasis Type="Italic">Plasmopara viticola</Emphasis> isolates resistant to QoI fungicides

The effectiveness of Quinone outside Inhibitor (QoI) fungicides against grape downy mildew in European vineyards has significantly decreased in the last decade. One nucleotide polymorphism, G143A in the cytochrome b gene of Plasmopara viticola, is involved in resistance to QoIs. Previous genetic examination on the mitochondrial genomes showed four major haplotypes (IR, IS, IIR, IIS) coexisting in European vineyards. A resistant allele (G143A) was present in IR and IIR haplotypes. The purpose of the present study was to estimate the diversity of the different mitochondrial haplotypes and their distribution in QoI-resistant populations before evaluating the potential cost of the resistant mutation G143A in P. viticola population. From 2000 to 2004, the frequencies of resistant isolates ranged from 0% to 23.25% with an average of 4.64 % among the populations examined. To evaluate the fitness of sensitive and resistant isolates, a comparison of different biological parameters including latent period, spore production and infection frequency was performed, enabling a fitness index (F_I) to be determined. Resistant isolates exhibited greater infection frequency than sensitive isolates, whereas no significant difference was found in sporulation ability and latent period between sensitive and resistant isolates. To further investigate competitiveness among isolates, an assay including two resistant isolates in different proportion with a sensitive isolate was conducted on eight asexual growing cycles in the absence of a QoI fungicide. The competitiveness of resistant isolates varied according to their fitness parameters, suggesting that there is no noticeable cost of QoI resistance in controlled conditions in Plasmopara viticola.     

Identification of the G143A mutation associated with QoI resistance in Cercospora beticola field isolates from Michigan,United States

BACKGROUND: Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola, is the most serious foliar disease of sugar beet (Beta vulgaris L.) worldwide. Disease control is mainly achieved by timely fungicide applications. In 2011, CLS control failures were reported in spite of application of quinone outside inhibitor (QoI) fungicide in several counties in Michigan, United States. The purpose of this study was to confirm the resistant phenotype and identify the molecular basis for QoI resistance of Michigan C. beticola isolates. RESULTS: Isolates collected in Michigan in 1998 and 1999 that had no previous exposure to the QoI fungicides trifloxystrobin or pyraclostrobin exhibited QoI EC₅₀ values of ?0.006 µg mL^?1. In contrast, all isolates obtained in 2011 exhibited EC₅₀ values of > 0.92 µg mL^?1 to both fungicides and harbored a mutation in cytochrome b (cytb) that led to an amino acid exchange from glycine to alanine at position 143 (G143A) compared with baseline QoI‐sensitive isolates. Microsatellite analysis of the isolates suggested that QoI resistance emerged independently in multiple genotypic backgrounds at multiple locations. A real‐time PCR assay utilizing dual‐labeled fluorogenic probes was developed to detect and differentiate QoI‐resistant isolates harboring the G143A mutation from sensitive isolates. CONCLUSION: The G143A mutation in cytb is associated with QoI resistance in C. beticola. Accurate monitoring of this mutation will be essential for fungicide resistance management in this pathosystem. Copyright © 2012 Society of Chemical Industry     

Resistance profiles of Botrytis cinerea populations to several fungicide classes on greenhouse tomato and strawberry in Lebanon

Tomato and strawberry are the most important protected crops in Lebanon and are seriously affected by grey mould disease, caused by Botrytis cinerea. In the present study, the fungicide sensitivity assays revealed medium to high frequencies of B. cinerea isolates resistant to benzimidazoles, dicarboximides, and anilinopyrimidines on tomato and strawberry. Fludioxonil- and boscalid-resistant mutants were uncommonly found at generally low frequency on both crops. Resistance to fenhexamid was detected in only one site on tomato but in most sites on strawberry with high frequencies, and the occurrence of resistance to QoI fungicides was ascertained on both crops. The majority of the tested isolates (>90%) exhibited multiple fungicide resistance, and isolates resistant to the seven antibotrydial fungicide classes were detected on strawberry in three locations. A high level of resistance was shown by B. cinerea mutants resistant to boscalid, fenhexamid, and QoI fungicides, while two levels of moderate and high resistance to anilinopyrimidines were identified. Genetic analysis revealed point mutations in the target genes commonly associated with resistance in B. cinerea isolates, with all mutants resistant to dicarboximides, fenhexamid, boscalid, and QoI fungicides carrying single-nucleotide polymorphims in BcOS1 (I365S/N, Q369P, and N373S), Erg27 (F412V/I), SdhB (H272R/Y), and cytb (G143A) genes, respectively. The general incorrect use of fungicides has caused the development and spread of fungicide resistance as a widespread phenomenon on protected tomato and strawberry in Lebanon. The implementation of appropriate antiresistance strategies is highly recommended.