Method for rapid detection of the PvCesA3 gene allele conferring resistance to mandipropamid, a carboxylic acid amide fungicide, in Plasmopara viticola populations

BACKGROUND: The occurrence of carboxylic acid amide (CAA)‐fungicide‐resistant Plasmopara viticola populations is becoming a serious problem in the control of grapevine downy mildew worldwide. RESULTS: The authors have developed a method, which utilises PCR‐RFLP, for the rapid detection of resistance to the CAA fungicide mandipropamid in P. viticola populations. With this method, a glycine‐to‐serine substitution at codon 1105 of the cellulose synthase gene PvCesA3 of CAA‐fungicide‐resistant P. viticola was easily detected, although no resistant P. viticola was detected from 398 isolates in Japan. CONCLUSION: It is proposed that the PCR‐RFLP method is a reliable tool for the rapid detection of CAA‐fungicide‐resistant P. viticola isolates. Only 4 h was required from the sampling of symptoms to the phenotyping of fungicide resistance. Copyright © 2011 Society of Chemical Industry     

Rapid method for detecting resistance to a QoI fungicide in Plasmopara viticola populations

BACKGROUND: The increasing occurrence of Qo inhibitor (QoI)‐fungicide‐resistant Plasmopara viticola (Berk. & MA Curtis) Berl. & DeToni populations is becoming a serious problem in the control of grapevine downy mildew worldwide. RESULTS: The authors have developed a rapid method for detecting resistance to a QoI fungicide, azoxystrobin, in P. viticola populations using the nested PCR‐RFLP method. With this method, a glycine‐to‐alanine substitution was discovered at codon 143 in the cytochrome b gene of P. viticola populations found in Japan. CONCLUSION: It is proposed that the nested PCR‐RFLP method is a high‐speed, sensitive and reliable tool for detecting azoxystrobin‐resistant P. viticola populations. Copyright © 2009 Society of Chemical Industry     

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     

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     

Identification of the mutation responsible for resistance to QoI fungicides and its detection in Ascochyta rabiei (teleomorph Didymella rabiei)

This study characterized a fragment of the cytochrome b gene from Ascochyta rabiei isolates collected in North Dakota, USA, that varied in sensitivity to quinone‐outside inhibitor (QoI) fungicides. The sequenced genomic DNA fragment contained a group I intron immediately after codon 131. The size of the cytochrome b gene was estimated to be over 4·6 kb. Multiple alignment analysis of cDNA and protein sequences revealed a mutation that changed the codon for amino acid 143 from GGT to GCT, introducing an amino acid substitution from glycine to alanine (G143A), which is frequently associated with QoI resistance. Based on this mutation, a diagnostic PCR assay was developed using an approach called mismatch amplification mutation assay. This method was successfully validated by testing a total of 70 A. rabiei isolates, of which 38 isolates were found to be QoI‐resistant. This fast and accurate PCR assay provides a very useful and simple screening method for QoI resistance in A. rabiei isolates.     

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     

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.     

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     

Resistance mechanism to carboxylic acid amide fungicides in the cucurbit downy mildew pathogen Pseudoperonospora cubensis

BACKGROUND: Pseudoperonospora cubensis, the causal oomycete agent of cucurbit downy mildew, is responsible for enormous crop losses in many species of Cucurbitaceae, particularly in cucumber and melon. Disease control is mainly achieved by combinations of host resistance and fungicide applications. However, since 2004, resistance to downy mildew in cucumber has been overcome by the pathogen, thus driving farmers to rely only on fungicide spray applications, including carboxylic acid amide (CAA) fungicides. Recently, CAA‐resistant isolates of P. cubensis were recovered, but the underlying mechanism of resistance was not revealed. The purpose of the present study was to identify the molecular mechanism controlling resistance to CAAs in P. cubensis. RESULTS: The four CesA (cellulose synthase) genes responsible for cellulose biosynthesis in P. cubensis were characterised. Resistant strains showed a mutation in the CesA3 gene, at position 1105, leading to an amino acid exchange from glycine to valine or tryptophan. Cross‐resistance tests with different CAAs indicated that these mutations lead to resistance against all tested CAAs. CONCLUSION: Point mutations in the CesA3 gene of P. cubensis lead to CAA resistance. Accurate monitoring of these mutations among P. cubensis populations may improve/facilitate adequate recommendation/deployment of fungicides in the field. Copyright © 2011 Society of Chemical Industry     

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     

Characterization of European Plasmopara viticola isolates with reduced sensitivity to cymoxanil

Cymoxanil has been used for over 30 years to control grape downy mildew (Plasmopara viticola) in European vineyards, prevalently in mixture with other fungicides active on this disease. In the 1990’s cases of P. viticola resistant to cymoxanil were detected using a leaf disc assay. In this study, we establish that the presence of only 1 % of resistant isolates in a P. viticola population will allow the detection of cymoxanil resistance in the leaf disc assay. A poor correlation (R?=?0.194) was observed between the leaf disc assay and a whole- plant test for 38 P. viticola field populations collected in 2004. Over 60 % of these populations were characterized as fully sensitive in a whole-plant assay compared to 10 % in the leaf disc assay. Five P. viticola field isolates resistant to cymoxanil reverted to full sensitivity after six to nine transfers to untreated vines, indicating that cymoxanil resistance in P. viticola is unstable. Two European P. viticola populations sensitive to cymoxanil became resistant when transferred 12–14 times on vines treated with cymoxanil. In contrast, two populations originating from the USA and three monozoospore isolates from France retained full sensitivity to the fungicide after 13 cycles on cymoxanil-treated plants. Whole-plant experiments were conducted in the laboratory to compare the efficiency of spray programs to delay the development of cymoxanil resistance. Whereas the continuous use of cymoxanil alone quickly selected for resistance, the mixture of cymoxanil and folpet applied either continuously or in strict or block alternation effectively prevented the development of resistance over 10 generations of the fungus. These results demonstrate that resistance to cymoxanil in P. viticola can be managed with appropriate spray programs.     

Application of cycleave PCR to the detection of a point mutation (F167Y) in the β2‐tubulin gene of Fusarium graminearum

BACKGROUND: Resistance of Fusarium graminearum to the benzimidazole fungicide carbendazim is caused by point mutations in the β₂‐tubulin gene (FGSG_06611.3). The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field isolates in China. It is important to find a suitable method for rapid detection and quantification of this point mutation in the F. graminearum populations. RESULTS: A pair of primers, Codon167F/Codon167R, were designed to amplify a fragment containing the mutation site, and two cycling probes labelled with different fluorescent reporters were used to detect whether the mutation was present. A cycleave real‐time PCR method was developed for rapid determination of the frequency of this point mutation in 282 F. graminearum perithecia collected from different fields in Jiangsu Province, China. The mutation frequency in ascospores from the perithecia to carbendazim by a spore germination assay was 6.0%, while the frequency of the point mutation at codon 167 by the cycleave real‐time PCR assay was 3.9%. CONCLUSION: The cycleave real‐time PCR method is suitable for accurate detection of the codon 167 point mutation. The frequency of this mutation in the β₂‐tubulin gene represents the resistance frequency in F. graminearum populations to carbendazim. Copyright © 2011 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.     

An intron in the cytochrome b gene of Monilinia fructicola mitigates the risk of resistance development to QoI fungicides

BACKGROUND: The cytochrome b (Cyt b) gene is a key genetic determinant for quinone outside inhibitor (QoI) fungicide resistance in plant pathogenic fungi. A mutation at amino acid position G143 can cause qualitative resistance unless it is part of the recognition site for a self‐splicing intron. The objective of this study was to clone and sequence the Cyt b gene from Monilinia fructicola (Wint.) Honey, the causal agent of brown rot of stone fruits, and to assess the risk for the development of a mutation at position 143. RESULTS: The Cyt b gene of M. fructicola was 11 927 bp in size and contained seven introns located at cDNA positions (5′–3′) 204, 395, 430, 491, 507, 780 and 812 with sizes of 1592, 1318, 1166, 1252, 1065, 2131 and 2227 bp respectively. Sequence analysis revealed that the above‐mentioned 1166 bp intron, a self‐splicing group I intron, was located just downstream of the G143 codon. The Cyt b gene region covering the G143 location and the adjacent 1166 bp intron was PCR amplified and sequenced from Chinese and US isolates, indicating that the intron may be omnipresent in M. fructicola. CONCLUSION: This is the first complete Cyt b gene sequence published for M. fructicola or any other Monilinia species, forming the basis for molecular analysis of QoI fungicide resistance. Sequence analysis revealed that the G143A mutation responsible for high levels of QoI fungicide resistance in many plant pathogenic fungi may not develop in M. fructicola unless genotypes emerge that lack the 1166 bp intron. Copyright © 2010 Society of Chemical Industry     

Selection for fungicide resistance throughout a growing season in populations of Plasmopara viticola

A method for evaluating the potential threat of selection for resistance to organically-based fungicides in populations of P. viticola is needed to screen a large panel of products alternative to copper in organic viticulture. Populations from an unexposed plot were compared throughout one season with a population sprayed with azoxystrobin (Quadris), reported as engendering selection pressure and resistance, and a population sprayed with an organically-based fungicide (Mycosan). The evolution of the three populations was followed with neutral specific SSR markers and with the specific marker for strobilurin resistance, as control of selection for resistant mutants. A reduction in genetic diversity of the P. viticola population was observed in the population sprayed with azoxystrobin, consistent with directional selection toward higher resistance, confirmed by an enhanced frequency of resistant mutants with respect to the unexposed population. In contrast, a higher diversity and a reduced frequency of resistant mutants were observed in the population sprayed with the organically-based fungicide. Assessing a reduction of genotypic diversity allows the detection of selection for resistance and constitutes a valid instrument for screening a large panel of products with non-specific, different and possibly indirect modes of action.     

Using allele-specific oligonucleotide probes to characterize benzimidazole resistance in Rhynchosporium secalis

Benzimidazole fungicides are important mixture components in strategies to combat fungicide resistance in Rhynchosporium secalis Davis. To monitor the performance of these strategies, a rapid, accurate assay has been developed to detect point mutations in the β-tubulin gene which confers resistance of benzimidazoles. The β-tubulin gene of a benzimidazole-resistant strain of R. secalis has been cloned and sequenced. Except for the difference in the position of one of its six introns, this gene showed a strong homology with other β-tubulin genes from filamentous fungi. Resistance was related to a point mutation in codon 198 which caused a glutamic acid to glycine change in resistant field strains, but glutamic acid to lysine in a laboratory mutant. A DNA fragment surrounding codon 198 was amplified directly from diseased lesions using a ‘nested’ set of PCR primers. Combining PCR amplificiation of a target DNA sequence with hybridization of Allele-Specific Oligonucleotide probes (ASO_s, 15-mers) allowed accurate detection of benzimidazole resistance. Only two probes, one sensitive and one resistant, were sufficient to monitor current field populations. Detection was achieved using either ³²P-labelled probe, or non-radioactively using a biotin-labelled probe coupled to streptavidin/alkaline phosphatase. This rapid method using ASOS can detect benzimidazole resistance within 48 h compared with 6–8 weeks by conventional assay procedures.     

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     

Characterization of the cytochrome b (cyt b) gene from Monilinia species causing brown rot of stone and pome fruit and its significance in the development of QoI resistance

BACKGROUND: Quinone outside inhibitor (QoI) resistance as a consequence of point mutations in the cytochrome b (cyt b) gene has been reported in numerous plant pathogenic fungi. To examine the potential for QoI resistance development in those Monilinia species causing brown rot of stone and pome fruits [Monilinia fructicola (G Winter) Honey, M. laxa (Aderhold & Ruhland) Honey and M. fructigena (Aderhold & Ruhland) Honey], an examination was made of the sequence and exon/intron structure of their cyt b genes for the presence of any point mutations and/or introns commonly associated with resistance to QoIs in fungal plant pathogens. RESULTS: None of the point mutations typically linked to QoI resistance was present in any of the Monilinia isolates examined. Furthermore, the cyt b genes from M. fructicola and M. laxa, but not M. fructigena, possessed a group‐I‐like intron directly after codon 143. Based on the results obtained, a simple PCR assay using a single primer pair was developed, allowing discrimination between the three Monilinia species without the need for culturing. CONCLUSIONS: Results suggest that resistance to QoI fungicides based on the G143A mutation is not likely to occur in M. fructicola or M. laxa. Conversely, M. fructigena may be at higher risk for developing QoI resistance owing to the absence of a G143‐associated intron. Copyright © 2010 Society of Chemical Industry