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.     

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     

Effect of dose rate and mixtures of fungicides on selection for QoI resistance in populations of Plasmopara viticola

Resistance to QoI fungicides (strobilurins, famoxadone and fenamidone) in populations of Plasmopara viticola (Berk & Curt) Berlese & de Toni developed soon after their introduction in France and Italy. Current resistance management strategies include limitation of the number of applications, use of mixtures and alternation of fungicides with different modes of action. The selection pressure resulting from QoI fungicides applied alone or in mixtures with non-QoI fungicides was investigated in whole plant experiments under controlled conditions. QoI-resistant populations of P. viticola gradually reverted to full sensitivity following consecutive transfers to untreated plants, suggesting that resistant phenotypes were less competitive than sensitive ones. When cycled on QoI-treated plants, reduction in sensitivity was greater for the QoI fungicide which had greater intrinsic activity on P. viticola. Sensitivity decreased at each subsequent cycle, resulting in almost full resistance after four generations. Mixture experiments indicated that selection pressure was affected most by the dose of the QoI fungicide and the nature of the partner fungicide. Folpet delayed selection pressure most effectively when it was associated with famoxadone or azoxystrobin. Mancozeb was least effective at reducing the rate of selection compared with the QoI alone, and fosetyl-aluminium was intermediate. Higher rates of selection were recorded when the dose of the QoI fungicide, solo or in a mixture, was increased from 1 to 4 microg ml(-1). Increasing the dose of the non-QoI partner fungicide in the mixture from 10 to 30 microg ml(-1) resulted in reduced selection pressure. These results suggest that the choice of the fungicide partner and its dosage in the mixture can significantly affect the success of QoI resistance management strategies under practical conditions.     

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     

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.     

Molecular and biochemical characterization of Colletotrichum gloeosporioides isolates resistant to azoxystrobin from grape in China

Anthracnose, caused by Colletotrichum gloeosporioides, is one of the most important diseases in grape-growing regions worldwide. In Jiangsu Province of China, quinone-outside inhibitor fungicides (QoIs) have been extensively sprayed as disease control for more than 10 years. A spore germination assay of 64 isolates obtained from 32 commercial vineyards was used to assess isolate sensitivity to azoxystrobin and 62 were found to be resistant to azoxystrobin. The biological fitness of QoI-resistant (QoI^R) isolates was significantly lower than the sensitive isolates (QoI^S) in terms of mycelial growth and conidiation. Nucleotide sequence alignment of CgCytb genes from the QoI^R and QoI^S isolates revealed that two point mutations (F129L and G143A) are involved in the QoI resistance. Isolates with the G143A mutation expressed high resistance to azoxystrobin, whereas isolates carrying the F129L mutation exhibited moderate resistance. Positive cross-resistance was observed between azoxystrobin and kersoxim-methyl, pyraclostrobin, or benzothiostrobin, but not with fluazinam. This study provides important information for management of QoI^R populations of C. gloeosporioides in the field.     

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.     

Field resistance to QoI fungicides in Podosphaera fusca is not supported by typical mutations in the mitochondrial cytochrome b gene

BACKGROUND: A single nucleotide polymorphism in the mitochondrial cytochrome b gene confers resistance to strobilurin (QoI) fungicides in phytopathogenic fungi. Recent studies have revealed worrying levels of resistance to strobilurins in Podosphaera fusca (Fr.) U Braun & N Shishkoff comb. nov. [ = Sphaerothecafusca (Fr.) S Blumer], the main causal agent of cucurbit powdery mildew in Spain. In the present study the underlying resistance mechanism to QoI fungicides in the Spanish populations of P. fusca was investigated. RESULTS: Analysis of the Q(o) domains of cytochrome b in a collection of isolates revealed that none of the typical mutations conferring resistance to QoI, including the G143A and F129L substitutions, was present in the QoI-resistant isolates. Moreover, although different amino acid polymorphisms were observed in the two regions spanning the Q(o) site, none of them consistently distinguished QoI-resistant from QoI-sensitive strains. Exposure to salicylhydroxamic acid (SHAM), a specific inhibitor of alternative oxidase, in the presence of trifloxystrobin did not have any effect on QoI resistance, ruling out alternative respiration as the mechanism of resistance. Sensitivity tests to a battery of respiration inhibitors revealed high levels of cross-resistance to all Qo-inhibitors tested but not to Qi-inhibitors, these features resembling those of a target-site-based resistance. CONCLUSIONS: The results indicate that the mechanism responsible for QoI resistance in P. fusca is not linked to typical mutations in cytochrome b gene and that the absence of the G143A substitution cannot be explained by an intron following codon 143. These are important observations, especially in relation to the possible molecular diagnosis of resistance.     

Characterization of moderate resistance to QoI fungicides in Pestalotiopsis longiseta and polymorphism in exon–intron structure of cytochrome b gene

Qo inhibitor (QoI) fungicides are used to control gray blight caused by Pestalotiopsis longiseta in Japanese tea cultivation. However, field isolates of P. longiseta highly resistant to QoI fungicides were found in 2008, resulting in failure of QoI fungicidal control. This resistance was attributed to a mutation in the cytochrome b gene (cytb) in which alanine was substituted for glycine at position 143 (G143A). In 2009–2010, we detected field isolates that had an intermediate reaction between sensitive and resistant isolates in a preliminary assay. These isolates showed intermediate sensitivity to azoxystrobin and kresoxim-methyl on PDA plates. The intermediate reaction to azoxystrobin was also confirmed on detached tea leaves. Consequently, they were considered moderately resistant to QoI fungicides. Nucleotide sequencing of cytb showed that moderate resistance correlated with a single point mutation; leucine was substituted for phenylalanine at amino acid position 129 (F129L). Sequence analysis also revealed two types of cytb, with or without an intron between codons 131 and 132, in P. longiseta. F129L and G143A mutations were detected in both types of cytb according to their QoI resistance. This result suggests that G143A and F129L mutations have each occurred at least twice in the P. longiseta population.     

An hypothesis to account for the behaviour of dicarboximide-resistant strains of Botrytis cinerea in vineyards

Ten trials were established in the 1986 vintage year on vineyards in three vine-growing districts of New Zealand, to examine changes in the frequency of resistance to dicarboximide fungicides of the Botrytis cinerea population under different fungicide programmes. The vineyards chosen encompassed sites with pre-season resistance frequencies ranging from 0 to 97%. Under a non-dicarboximide programme, using dichlofluanid and chlorothalonil, resistance frequency had decreased at harvest irrespective of the initial frequency. In programmes incorporating dicarboximides (chlozolinate, iprodione, procymidone, vinclozolin), the resistance frequency tended to increase if pre-season resistance frequency was low, and decrease if it was high. The hypothesis is presented that for a given dicarboximide programme, resistance frequency tended to one value, the balance value, where selection for resistant strains due to fungicide application balanced selection against the resistant strains due to their low fitness. This value was estimated for the 1986 vintage year to be 38% for a programme where application of dicarboximides was restricted to the period from the veraison (sugar rise), and 64% where dicarboximides were applied from the pre-bunch closure phase. Actual values observed at harvest 1987, after these same programmes had been applied for two consecutive years, were 39% (S.E. 8-5%) and 68% (S.E. 8-8%), respectively.     

Detection and characterization of QoI resistance in Pyrenophora tritici-repentis populations causing tan spot of wheat in Argentina

Tan spot, caused by the fungus Pyrenophora tritici-repentis (Ptr), is a disease that has become more prevalent and intense in wheat crops in Argentina in recent years. Failure to control the disease with strobilurin fungicides, which were once effective, has been observed in different zones where wheat is grown. However, whether or not true resistance is present in the pathogen population in the region is not scientifically confirmed. This study evaluated the sensitivity of numerous Ptr isolates to representative QoI fungicides used in Argentina through in vitro and in planta assays, as well as through molecular analysis. Eighty-two monosporic isolates obtained in different locations in the north and south of Buenos Aires province in 2014, 2016, and 2018 were tested to determine sensitivity to selected QoI fungicides in conidial germination and mycelial inhibition assays, as well as in molecular analysis. Conidial germination was not inhibited at 1 µg/ml of azoxystrobin, trifloxystrobin, and pyraclostrobin. On the other hand, mycelial growth was inhibited by 59%, 56%, and 86% at 100 µg/ml of azoxystrobin, trifloxystrobin, and pyraclostrobin, respectively. The molecular analysis detected the G143A mutation in the cytb gene of all the 82 Ptr isolates, but the F129L and G137R substitutions were not present. This study documents the G143A mutation conferring QoI resistance in Ptr in South America. The findings of this study are key for future decisions regarding use of fungicide and rotation in the region.     

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.     

Effect of the new pyrazole carboxamide fungicide penthiopyrad on late leaf spot and stem rot of peanut

BACKGROUND: Management of early leaf spot (Cercospora arachidicola Hori.), late leaf spot [Cercosporidium personatum (Berk. & MA Curtis) Deighton] and stem rot (Sclerotium rolfsii Sacc.) of peanut (Arachis hypogaea L.) in the southeastern USA is heavily dependent upon sterol biosynthesis inhibitor (SBI) and quinone outside inhibitor (QoI) fungicides. Effective new fungicides with different modes of action could improve overall disease control and extend the utility of the current fungicides. Penthiopryad is a pyrazole carboxamide fungicide being evaluated for use on peanut. Field experiments were conducted from 2004 to 2007 to determine the effect of a range of rates (0–0.36 kg AI ha^?1) of penthiopyrad on leaf spot and stem rot and the relative efficacy of penthiopyrad and current fungicide standards chlorothalonil, tebuconazole and azoxystrobin. RESULTS: Leaf spot control in plots treated with penthiopyrad at 0.20 kg AI ha^?1 or higher was similar to or better than that for the chlorothalonil standard. The incidence of stem rot for all penthiopyrad treatments was usually less than that for the tebuconazole or azoxystrobin standard treatments. Pod yields for all penthiopyrad treatments were similar to or higher than those for the respective standards. CONCLUSION: Penthiopyrad has excellent potential for management of late leaf spot and stem rot of peanut, and may complement current SBI and QoI fungicides. Copyright © 2008 Society of Chemical Industry     

A non-Mendelian inheritance of resistance to strobilurin fungicides in Ustilago maydis

Mutants of Ustilago maydis (DC) Corda with high resistance to azoxystrobin (RF 164 to 4714, based on EC50 values), an inhibitor of mitochondrial electron transport at the cytochrome bc1 complex, were isolated in a mutation frequency of 2.3 x 10(-7) after nitrosoguanidine mutagenesis and selection on media containing 1 microgram ml-1 azoxystrobin in addition to 0.5 mM salicylhydroxamate (SHAM), a specific inhibitor of cyanide-resistant (alternative) respiration. Oxygen uptake in whole cells was strongly inhibited in the wild-type strains by azoxystrobin (1.5 micrograms ml-1) in addition to SHAM (1 mM), but not in the mutant isolates. Genetic analysis with nine such mutant isolates resulted in progeny phenotypes which did not follow Mendelian segregation, but satisfied the criteria of non-Mendelian (cytoplasmic) heredity. In crosses between three mutant isolates with the compatible wild-type strains, the sensitivity was inherited by progeny maternally from the wild-type parent strain (criterion of uniparental inheritance). In crosses between wild-type strains and remaining mutant isolates, a continuous distribution of sensitivity in the progeny was found (criterion of vegetative segregation). The third criterion of cytoplasmic resistance (criterion of intracellular selection) was fulfilled by experiments on the stability of resistance phenotypes. With two exceptions, a reduction of resistance was observed in the mutant strains when they were grown on inhibitor-free medium. Recovery of the high resistance level was observed after they were returned to the selection medium. Cross-resistance studies with other fungicides, which also inhibit electron transport through complex III of respiratory chain, showed that mutations for resistance to azoxystrobin were also responsible for reduced sensitivity to kresoxim-methyl (RF 18 to 1199) and to antimycin-A (RF 20 to 305), which act at the Qo and Qi sites of the cytochrome bc1 complex, respectively. Studies of the fitness of azoxystrobin-resistant isolates showed that these mutations appeared to be pleiotropic, having significant adverse effects on growth in liquid culture and pathogenicity on young corn plants.     

Derivation and testing of a model to predict selection for fungicide resistance

A mathematical model was derived to predict selection for fungicide resistance in foliar pathogens of cereal crops. The model was tested against independent data from four field experiments quantifying selection for the G143A mutation conferring resistance to a quinone outside inhibitor (QoI) fungicide in powdery mildew (Blumeria graminis f.sp. hordei) on spring barley (Hordeum vulgare). Fungicide treatments with azoxystrobin differed in the total applied dose and spray number. For each treatment, we calculated the observed selection ratio as the ratio of the frequency of the resistant strain after the last and before the first spray. The model accurately predicted the variation in observed selection ratios with total applied fungicide dose and number of sprays for three of the four experiments. Underprediction of selection ratios in one experiment was attributed to the particularly late epidemic onset in that experiment. When the equation representing epidemic development was modified to account for the late epidemic, predicted and observed selection ratios at that site were in close agreement. On a scatter plot of observed selection ratios on predicted selection ratios, for all four experiments, the 1:1 line explained 89–92% of the variance in the mean of observed selection ratios. To our knowledge, this is the first fungicide resistance model for plant pathogens to be rigorously tested against field data. The model can be used with some degree of confidence, to identify anti‐resistance treatment strategies which are likely to be effective and would justify the resources required for experimental testing.     

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.     

Population dynamics of dicarboximide-resistant strains of Botrytis cinerea on grapevine in New Zealand

Changes in the proportion of dicarboximide-resistant strains of Botrytis cinerea were monitored in two vineyard trials conducted during the period from October 1985 to November 1987. Dicarboximide resistance frequency in samples taken prior to flowering from wood (dead cane-ends, dead tendrils, old bunch remains) was high (66%) in the first trial, and low (12%) in the second trial. In treatments where resistance frequency was high on wood in spring, it decreased during the phase of disease establishment in new bunches irrespective of whether or not dicarboximide fungicides were applied. In the absence of dicarboximide applications, resistance frequency showed little change during the phase of disease development in bunches. The resistance frequency increased, however, if dicarboximide fungicides were applied, the extent of increase depending on the number and timing of applications and on disease severity at the time of application. Where resistance frequency was high on the wood at harvest, it decreased over the following winter but increased again in spring even though no dicarboximides had been applied. The best disease control in both trials was obtained with a programme in which dicarboximide fungicides were only applied during the period from veraison until harvest.     

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     

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.     

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.