Pyricularia grisea Isolates Causing Gray Leaf Spot on Perennial Ryegrass (Lolium perenne) in the United States: Relationship to P. grisea Isolates from Other Host Plants

ABSTRACT Gray leaf spot of perennial ryegrass (prg) (Lolium perenne), caused by the fungus Pyricularia grisea (teleomorph = Magnaporthe grisea), has rapidly become the most destructive of all turf grass diseases in the United States. Fungal isolates from infected prg were analyzed with several molecular markers to investigate their relationship to P. grisea strains found on other hosts. All of the molecular markers used in this study revealed that isolates from prg are very distantly related to those found on crabgrass. Fingerprinting with MGR586 (Pot3) revealed zero to three copies of this transposon in the prg pathogens, distinguishing them from isolates pathogenic to rice, which typically have more than 50 copies of this element. RETRO5, a newly identified retroelement in P. grisea, was present at a copy number of >50 in isolates from rice and Setaria spp. but only six to eight copies were found in the isolates from prg. The MAGGY retrotransposon was unevenly distributed in the prg pathogens, with some isolates lacking this element, some possessing six to eight copies, and others having 10 to 30 copies. These results indicated that the P. grisea isolates causing gray leaf spot are distinct from those found on crabgrass, rice, or Setaria spp. This conclusion was supported by an unweighted pair-group method with arithmetic average cluster analysis of single-copy restriction fragment length polymorphism haplo-types. Fingerprints obtained with probes from the Pot2 and MGR583 transposons revealed that the prg pathogens are very closely related to isolates from tall fescue, and that they share similarity with isolates from wheat. However, the wheat pathogens had fewer copies of these elements than those found on prg. Therefore, I conclude that P. grisea isolates commonly found on other host plant species did not cause gray leaf spot epidemics on prg. Instead, the disease appears to be caused by a P. grisea population that is specific to prg and tall fescue.     

A temperature and leaf wetness duration-based model for prediction of gray leaf spot of perennial ryegrass turf

ABSTRACT Gray leaf spot is a serious disease of perennial ryegrass (Lolium perenne), causing severe epidemics in golf course fairways. The effects of temperature and leaf wetness duration on the development of gray leaf spot of perennial ryegrass turf were evaluated in controlled environment chambers. Six-week-old Legacy II ryegrass plants were inoculated with an aqueous conidial suspension of Pyricularia grisea (approximately 8 x 10(4) conidia per ml of water) and subjected to four different temperatures (20, 24, 28, and 32 degrees C) and 12 leaf wetness durations (3 to 36 h at 3-h intervals). Three days after inoculation, gray leaf spot developed on plants at all temperatures and leaf wetness durations. Disease incidence (percent leaf blades symptomatic) and severity (index 0 to 10; 0 = leaf blades asymptomatic, 10 = >90% leaf area necrotic) were assessed 7 days after inoculation. There were significant effects ( alpha = 0.0001) of temperature and leaf wetness duration on disease incidence and severity, and there were significant interactions ( alpha = 0.0001) between them. Among the four temperatures tested, 28 degrees C was most favorable to gray leaf spot development. Disease incidence and severity increased with increased leaf wetness duration at all temperatures. A shorter leaf wetness duration was required for disease development under warmer temperatures. Analysis of variance with orthogonal polynomial contrasts and regression analyses were used to determine the functional relationships among temperature and leaf wetness duration and gray leaf spot incidence and severity. Significant effects were included in a regression model that described the relationship. The polynomial model included linear, quadratic, and cubic terms for temperature and leaf wetness duration effects. The adjusted coefficients of determination for the fitted model for disease incidence and severity were 0.84 and 0.87, respectively. The predictive model may be used as part of an integrated gray leaf spot forecasting system for perennial ryegrass turf.     

Expression Analysis of Rice Defense-Related Genes in Turfgrass in Response to Magnaporthe grisea

ABSTRACT Magnaporthe grisea (anamorph = Pyricularia grisea) causes blast on rice (Oryza sativa) and gray leaf spot on turfgrass. Gray leaf spot is a serious disease on St. Augustinegrass (Stenotaphrum secundatum), perennial ryegrass (Lolium perenne), and tall fescue (Festuca arundinacea). Virulence assays performed in this study revealed that M. grisea collected from rice could also cause disease on St. Augustinegrass and tall fescue. One rice isolate, Che86061, caused similar disease reactions on susceptible cultivars of rice and St. Augustinegrass and an incompatible interaction on resistant cultivars of both species. To explore whether similar defense-related genes are expressed in rice and St. Augustinegrass, a rice cDNA library was screened using pooled cDNAs derived from M. grisea-infected St. Augustinegrass. Thirty rice EST (expressed sequence tag) clones showing differential expression in St. Augustinegrass following M. grisea inoculation were identified and classified into six putative functional groups. Northern blot analyses of seven EST clones that collectively represented each putative functional group confirmed that the expression of five out of seven EST clones was similar in both rice and St. Augustinegrass. This study represents one of the first attempts to use a broad-scale genomic approach and resources of a model monocot system to study defense gene expression in St. Augustinegrass following M. grisea infection.     

Cytochrome b gene sequence and structure of Pyrenophora teres and P. tritici-repentis and implications for QoI resistance

Resistance to QoI fungicides in Pyrenophora teres (Dreschsler) and P. tritici-repentis (Died.) Dreschsler was detected in 2003 in France and in Sweden and Denmark respectively. Molecular analysis revealed the presence of the F129L mutation in resistant isolates of both pathogens. In 2004, the frequency of the F129L mutation in populations of both pathogens further increased. The G143A mutation was also detected in a few isolates of P. tritici-repentis from Denmark and Germany. In 2005, the F129L mutation in P. teres increased in frequency and geographical distribution in France and the UK but remained below 2% in Germany, Switzerland, Belgium and Ireland. In P. tritici-repentis, both mutations were found in a significant proportion of the isolates from Sweden, Denmark and Germany. The G143A mutation conferred a significantly higher level of resistance (higher EC50 values) to Qo inhibitors (QoIs) than did the F129L mutation. In greenhouse trials, resistant isolates with G143A were not well controlled on plants sprayed with recommended field rates, whereas satisfactory control of isolates with F129L was achieved. For the F129L mutation, three different single nucleotide polymorphisms (SNPs), TTA, TTG and CTC, can code for L (leucine) in P. teres, whereas only the CTC codon was detected in P. tritici-repentis isolates. In two out of 250 isolates of P. tritici-repentis from 2005, a mutation at position 137 (G137R) was detected at very low frequency. This mutation conferred similar resistance levels to F129L. The structure of the cytochrome b gene of P. tritici-repentis is significantly different from that of P. teres: an intron directly after amino acid position 143 was detected in P. teres which is not present in P. tritici-repentis. This gene structure suggests that resistance based on the G143A mutation may not occur in P. teres because it is lethal. No G143A isolates were found in any P. teres populations. Although different mutations may evolve in P. tritici-repentis, the G143A mutation will have the strongest impact on field performance of QoI fungicides.     

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     

Contrasting genetic structure between Magnaporthe grisea populations associated with the golf course turfgrasses Lolium perenne (perennial ryegrass) and Pennisetum clandestinum (kikuyugrass)

Gray leaf spot (GLS) disease of perennial ryegrass (Lolium perenne) and kikuyugrass (Pennisetum clandestinum) in golf courses in California was first noted in 2001 and 2003, respectively, and within 5 years had become well established. The causal agent of the disease is the fungus Magnaporthe grisea, which is known to consist primarily of clonal lineages that are highly host specific. Therefore, our objective was to investigate host specificity and population dynamics among isolates associated primarily from perennial ryegrass and kikuyugrass since the disease emerged at similar times in California. We also obtained isolates from additional hosts (tall fescue, St. Augustinegrass, weeping lovegrass, and rice) and from the eastern United States for comparative purposes. A total of 38 polymorphic amplified fragment length polymorphism makers were scored from 450 isolates which clustered by host with high bootstrap support (71 to 100%). Genetic structure between kikuyugrass and perennial ryegrass isolates differed significantly. Isolates from kikuyugrass were genotypically diverse (n = 34), possessed both mating types, and some tests for random mating could not be rejected, whereas isolates from perennial ryegrass were less genotypically diverse (n = 10) and only consisted of a single mating type. Low genotypic diversity was also found among the other host specific isolates which also only consisted of a single mating type. This is the first study to document evidence for the potential of sexual reproduction to occur in M. grisea isolates not associated with rice (Oryza sativa). Moreover, given the significant host specificity and contrasting genetic structures between turfgrass-associated isolates, the recent emergence of GLS on various grass hosts in California suggests that potential cultural practices or environmental changes have become conducive for the disease and that the primary inoculum may have already been present in the state, despite the fact that two genotypes associated with perennial ryegrass and St. Augustinegrass in California were the same as isolates collected from the eastern United States.     

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.     

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.     

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.     

Molecular characterization and biological response to respiration inhibitors of Pyricularia isolates from ctenanthe and rice plants

The molecular profile and the biological response of isolates of Pyricularia oryzae Cavara obtained from ctenanthe to two strobilurins (azoxystrobin, kresoxim-methyl) and the phenylpyridinamine fungicide fluazinam were characterized, and compared with isolates from rice plants. Five different isozymes (alpha-esterase, lactate, malate, isocitrate and sorbitol dehydrogenases) and five random decamer primers for RAPD-PCR were used to generate molecular markers. Using unweighted pair-group with arithmetic average analysis, ctenanthe isolates were found to form a separate group distinct from that of the rice isolates for both sets of markers. Amplified polymorphic sequences of mitochondrial cytochrome b that were digested with Fnu4HI or StyI revealed no differences among Pyricularia isolates at amino acid positions 143 or 129 which confer resistance to strobilurins in several fungi. In absence of the alternative respiration inhibitor salicylhydroxamic acid (SHAM) the three fungicides showed inferior and variable efficacy, with a trend toward the rice isolate being less sensitive. The addition of SHAM enhanced the effectiveness of all fungicides against isolates regardless of their origin. Appressorium formation was the most vulnerable target of action of the respiration inhibitors and azoxystrobin the most effective. This is the first report of a comparison between the molecular profiles and sensitivities to respiration inhibitors for Pyricularia oryzae isolates from a non-gramineous host and from rice.     

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.     

Assessing the Belgian potato <Emphasis Type="Italic">Alternaria</Emphasis> population for sensitivity to fungicides with diverse modes of action

Early blight and brown spot, caused by respectively Alternaria solani and Alternaria alternata, can lead to severe yield losses in potato-growing areas. To date, fungicide application is the most effective measure to control the disease. However, in recent years, a reduced sensitivity towards several active ingredients has been reported. To shed light on this issue, Alternaria isolates were collected from different potato fields in Belgium during two growing seasons. Subsequently, the sensitivity of these isolates was assessed using four widely used fungicides with different modes of action. Demethylation inhibitors, quinone outside inhibitors, a dithiocarbamate and a carboxylic acid amide were included in this study. Although all fungicides reduced spore germination and vegetative growth of Alternaria species to some extent, the interspecies sensitivity was very variable. In general, A. solani was more suppressed by the fungicides compared to A. alternata. The effectiveness of the dithiocarbamate mancozeb was high, whereas the quinone outside inhibitor azoxystrobin showed a limited activity, especially towards A. alternata. Therefore, a subset of the A. alternata and A. solani isolates was tested for the presence of, respectively, the G143A substitution and the F129L substitution in the cytochrome b. The frequency of A. alternata isolates bearing the resistant G143A allele (approximately 65%) was comparable in both sampling years, although sensitivity of isolates decreased during the growing season. This finding points to a shift of the population towards resistant isolates. Both the European genotype I and American genotype II were present in the A. solani population, with genotype I being the most prevalent. None of the genotype I isolates carried the F129L substitution, whereas in 83% of the genotype II isolates this substitution was present. Our results demonstrate for the first time that the Belgian Alternaria population on potato comprises a considerable broad spectrum of isolates with different sensitivity to fungicides.     

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.     

Role of Ascospores in Further Spread of QoI-Resistant Cytochrome b Alleles (G143A) in Field Populations of Mycosphaerella graminicola

ABSTRACT Strobilurin fungicides or quinone outside inhibitors (QoIs) have been used successfully to control Septoria leaf blotch in the United Kingdom since 1997. However, QoI-resistant isolates of Mycosphaerella graminicola were reported for the first time at Rothamsted during the summer of 2002. Sequence analysis of the cytochrome b gene revealed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Extensive monitoring using real-time polymerase chain reaction (PCR) testing revealed that fungicide treatments based on QoIs rapidly selected for isolates carrying resistant A143 (R) alleles within field populations. This selection is driven mainly by polycyclic dispersal of abundantly produced asexual conidia over short distances. In order to investigate the role of sexually produced airborne ascospores in the further spread of R alleles, a method integrating spore trapping with real-time PCR assays was developed. This method enabled us to both quantify the number of M. graminicola ascospores in air samples as well as estimate the frequency of R alleles in ascospore populations. As expected, most ascospores were produced at the end of the growing season during senescence of the wheat crop. However, a rapid increase in R-allele frequency, from 35 to 80%, was measured immediately in airborne ascospore populations sampled in a wheat plot after the first QoI application at growth stage 32. After the second QoI application, most R-allele frequencies measured for M. graminicola populations present in leaves and aerosols sampled from the treated plot exceeded 90%. Spatial sampling and testing of M. graminicola flag leaf populations derived from ascospores in the surrounding crop showed that ascospores carrying R alleles can spread readily within the crop at distances of up to 85 m. After harvest, fewer ascospores were detected in air samples and the R-allele frequencies measured were influenced by ascospores originating from nearby wheat fields.     

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 of the F129L mutation in Alternaria solani populations in Germany in response to QoI application,and its effect on sensitivity

Early blight caused by Alternaria solani is a highly destructive disease of potatoes. Control of early blight mainly relies on the use of preventive fungicide treatments. Because of their high efficacy, azoxystrobin and other quinone outside inhibitors (QoIs) are commonly used to manage early blight. However, loss of sensitivity to QoIs has previously been reported for A. solani in the United States. Two hundred and three A. solani field isolates collected from 81 locations in Germany between 2005 and 2011 were screened for the presence of the F129L mutation in the cytochrome b gene; of these, 74 contained the F129L mutation. Sequence analysis revealed the occurrence of two structurally different cytb genes, which differed in the presence (genotype I) or absence (genotype II) of an intron, with genotype I being the most prevalent (63% of isolates). The F129L mutation was detected only in genotype II isolates, where it occurred in 97%. Sensitivity to azoxystrobin and pyraclostrobin was determined in conidial germination assays. All isolates possessing the F129L mutation had reduced sensitivity to azoxystrobin and, to a lesser extent, to pyraclostrobin. Early blight disease severity on plants treated with azoxystrobin was significantly higher for A. solani isolates with reduced fungicide sensitivity in the conidial germination assay compared with sensitive isolates. Data suggest an accumulation of F129L isolates in the German A. solani population over the years 2009–2011. It is assumed that the application of QoIs has selected for the occurrence of F129L mutations, which may contribute to loss of fungicide efficacy.     

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.     

Fungicide resistance in Cercospora species causing cercospora leaf blight and purple seed stain of soybean in Argentina

Cercospora species cause cercospora leaf blight (CLB) and purple seed stain (PSS) on soybean. Because there are few resistant soybean varieties available, CLB/PSS management relies heavily upon fungicide applications. Sensitivity of 62 Argentinian Cercospora isolates to demethylation inhibitor (DMI), methyl benzimidazole carbamate (MBC), quinone outside inhibitor (QoI), succinate dehydrogenase inhibitor (SDHI) fungicides, and mancozeb was determined in this study. All isolates were sensitive to difenoconazole, epoxiconazole, prothioconazole, tebuconazole, and cyproconazole (EC₅₀ values ranged from 0.006 to 2.4 µg/ml). In contrast, 51% of the tested isolates were sensitive (EC₅₀ values ranged from 0.003 to 0.2 µg/ml), and 49% were highly resistant (EC₅₀ > 100 µg/ml) to carbendazim. Interestingly, all isolates were completely resistant to azoxystrobin, trifloxystrobin, and pyraclostrobin, and insensitive to boscalid, fluxapyroxad, and pydiflumetofen (EC₅₀ > 100 µg/ml). The G143A mutation was detected in 82% (53) of the QoI-resistant isolates and the E198A mutation in 97% (31) of the carbendazim-resistant isolates. No apparent resistance mutations were detected in the succinate dehydrogenase genes (subunits sdhB, sdhC, and sdhD). Mancozeb completely inhibited mycelial growth of the isolates evaluated at a concentration of 100 µg/ml. All Argentinian Cercospora isolates were sensitive to the DMI fungicides tested, but we report for the first time resistance to QoI and MBC fungicides. Mechanism(s) other than fungicide target-site modification may be responsible for resistance of Cercospora to QoI and MBC fungicides. Moreover, based on our results and on the recent introduction of SDHI fungicides on soybean in Argentina, Cercospora species causing CLB/PSS are insensitive (naturally resistant) to SDHI fungicides. Insensitivity must be confirmed under field conditions.     

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.     

Following the dynamics of strobilurin resistance in Blumeria graminis f.sp. tritici using quantitative allele-specific real-time PCR measurements with the fluorescent dye SYBR Green I

Strobilurin-resistant isolates of Blumeria ( Erysiphe ) graminis f.sp. tritici , the cause of wheat powdery mildew, were more than 10-fold less sensitive to azoxystrobin than sensitive isolates. In all resistant isolates, a mutation resulting in the replacement of a glycine by an alanine residue at codon 143 (G143A) in the mitochondrial cytochrome b gene was found. Allele-specific primers were designed to detect this point mutation in infected wheat leaves. Using quantitative fluorescent allele-specific real-time polymerase chain reaction (PCR) measurements, strobilurin-resistant A143 alleles could be detected amongst strobilurin-sensitive G143 alleles at a frequency of at least 1 in 10 000, depending on the amount of target and nontarget DNA. Most isolates tested were dominant homoplasmic for either the A143 or G143 allele, although mixed populations of alleles could be detected in some isolates. In some of these isolates, strobilurin resistance was not always stable when they were maintained for many generations in the absence of selection. The allele-specific real-time PCR assay was also used to follow the dynamics of A143 alleles in field populations of B . graminis f.sp. tritici before and after application of fungicides. As expected, the A143 allele frequency only increased under selection pressure from a strobilurin fungicide. After three sprays of azoxystrobin, a pronounced selection for the strobilurin-resistant allele, with an increase in average frequency from 2·2 to 58%, was measured. The use of quantitative real-time PCR diagnostics for early detection of fungicide resistance genes at low frequency, coupled with risk evaluation, will be invaluable for further resistance risk assessment and validation of antiresistance strategies.