QoI Resistance Research Articles

Genetic analysis and molecular characterisation of laboratory and field mutants of Botryotinia fuckeliana (Botrytis cinerea) resistant to QoI fungicides

QoI fungicides, inhibitors of mitochondrial respiration, are considered to be at high risk of resistance development. In several phytopathogenic fungi, resistance is caused by mutations (most frequently G143A) in the mitochondrial cytochrome b (cytb) gene. The genetic and molecular basis of QoI resistance were investigated in laboratory and field mutants of Botryotinia fuckeliana (de Bary) Whetz. exhibiting in vitro reduced sensitivity to trifloxystrobin. B. fuckeliana mutants highly resistant to trifloxystrobin were obtained in the laboratory by spontaneous mutations in wild-type strains, or from naturally infected plants on a medium amended with 1-3 mg L(-1) trifloxystrobin and 2 mM salicylhydroxamic acid, an inhibitor of alternative oxidase. No point mutations were detected, either in the complete nucleotide sequences of the cytb gene or in those of the aox and Rieske protein genes of laboratory mutants, whereas all field mutants carried the G143A mutation in the mitochondrial cytb gene. QoI resistance was always maternally inherited in ascospore progeny of sexual crosses of field mutants with sensitive reference strains. The G143A mutation in cytb gene is confirmed to be responsible for field resistance to QoIs in B. fuckeliana. Maternal inheritance of resistance to QoIs in progeny of sexual crosses confirmed that it is caused by extranuclear genetic determinants. In laboratory mutants the heteroplasmic state of mutated mitochondria could likely hamper the G143A detection, otherwise other gene(s) underlying different mechanisms of resistance could be involved.

Sensitivity of Mycosphaerella graminicola isolates from Tunisia to epoxiconazole and pyraclostrobin

Fifty two isolates of Mycosphaerella graminicola from Tunisia were analysed for their sensitivity to pyraclostrobin and epoxiconazole in microtitre tests and for mutations in the target genes of the modes of action, cytochrome b and CYP51, respectively. All isolates were highly sensitive to pyraclostrobin, thus no isolate carried the G143A mutation in the cytochrome b gene, the mutation which is the proved mechanism in M. graminicola for QoI resistance. The Tunisian M. graminicola population was more sensitive to epoxiconazole when compared with European populations based on previously published data. However, some mutations in the CYP51, which are known to be an adaptation response to DMIs have been found in a part of the population (e.g. mutations A379G, I381V), but there is still a portion with wild type isolates, which has already disappeared in Northern Europe. The current data indicate a favourable situation for QoI and DMI sensitivity, but it should be considered in any disease management strategy to include different modes of action to avoid QoI resistance and further adaptation to DMIs.

Q-PCR analysis of the resistance of Hungarian Botrytis cinerea isolates toward azoxystrobin

The genes being in the mitochondrial DNA primarily encode the enzymes of cellular respiration. Fungicides belonging to the family of quinol oxidase inhibitors (QoIs) play on important role in the protection against several plant diseases caused by fungi. These fungicides bind to the cytochrome bc1 complex so they block electron transport between cytochrome b and cytochrome c1. This way these fungicides inhibit the ATP synthesis consequently they inhibit the mitochondrial respiration. The QoI resistance has two mechanisms. One of them is the point mutation of the cytochrome b gene (CYTB), e.g. the substitution of a single glycine by alanine at position 143 results in high-resistance. The other is the cyanide-resistant alternative respiration sustained by the alternative oxidase.In a cell there are several mitochondria. The phenomenon when the genomes of all mitochondria in the cell are identical is called homoplazmy. If in the cell there is wild and mutant mitochondrial DNA this is called heteroplasmy. Whether the mutation in the mitochondria causes fenotypical diversity or does not depend on the dose, i.e. it depends on the percentage of the changed mitochondrials. During our work we investigated Botrytis cinerea single spore isolates which have been collected in 2008-2009 on different host plants. Our goal was to decide whether heteroplasmy influences the level of resistance. We managed to detect the change of the level of heteroplasmy, so the change the level of the resistance due to the treatment with fungicide.

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

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. 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. 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.

Guignardia bidwellii, the causal agent of black rot on grapevine has a low risk for QoI resistance

Sequence analysis of the cytochrome b gene of Guignardia bidwellii showed that the occurrence of the most important resistance mechanism for QoI fungicides, the G143A mutation, is relatively unlikely. This is due to the presence of an intron directly after codon 143. A mutation in this codon (from glycine to e.g. alanine) would presumably lead to incorrect mRNA maturation and a non-functional, lethal protein. Intron sequences have not been detected in immediate vicinity to the codons 129 and 137; therefore, the occurrence of those two mutations cannot be excluded. Since the effects of these mutations on field performance on QoI fungicides are rather low, the overall resistance risk of G. bidwellii to this fungicide class is estimated to be low.

Molecular diagnostic for detecting the cytochrome b G143S – QoI resistance mutation in Cercospora beticola

The mutation G143S has been associated with high-level strobilurin resistance in laboratory mutant strains of Cercospora beticola, one of the most destructive pathogens in sugar beet plants. By using allele specific primers (PASA-PCR) and agarose gel visualization, a molecular diagnostic was developed for the detection of the G143S resistance mutation. This assay is simple and applicable in low tech laboratory settings, with high reliability when a relatively large proportion of mutated mitochondrial alleles are present in the resistant strains. To achieve detection of resistant alleles at low frequencies, a more sensitive Real Time PCR based assay capable of discriminating resistant (S143) genotypes in frequencies as low as 1:10,000 resistant:sensitive alleles was developed. Both diagnostics were successfully validated in laboratory strains. Subsequently, a large number of C. beticola isolates from QoI-treated sugar beet experimental fields in Greece were screened for resistance to Qo fungicides using these diagnostics and classic bioassays. No proportion of the 143S resistant allele was detected in all field isolates tested, which was in agreement with the phenotypes revealed by the biotests confirming that the efficacy of QoIs against C. beticola has been sustained in Greece 7 years after their introduction.

Cytochrome b diversity of Hungarian Botrytis cinerea strains

In the mitochondrion of eukaryotes, cytochrome b is a component of respiratory chain complex III. Cytochrome b is encoded by thecytochrome b (CYTB) gene located in the mitochondrial genome. The fungicidal activity of QoIs relies on their ability to inhibit mitochondrial respiration by binding at the so-called Qo site (the outer quinol-oxidation site) of the complex III. Since their introduction, QoIs (like azoxystrobin) have become essential components of plant disease control programs because of their wide-ranging efficacy against many agriculturally important fungal diseases like grey mould on various crops. QoI resistance primarily arises from a target-site-based mechanism involving mutations in the mitochondrial CYTB. As the management of grey mould is often dependent on chemicals, the rational design of control programs requires the information about the diversity of genes connected with resistance in field populations of the pathogen.Monospore B. cinerea field isolates has been collected during 2008-2009 from different hosts in Hungary. PCR fragment length analysisindicated the high frequency presence of type large intron in the isolates while in a few strains G143A substitution could also be detected.These results indicated the heterogeneity of CYTB in the Hungarian B. cinerea populations, which possibly involve the heteroplasmy of thismitochondrial gene, moreover indicates the existence op azoxystrobin resistant populations in Hungary.This work was supported by NKFP-A2-2006/0017 grant. Erzsébet Fekete is a grantee of the János Bolyai Scholarship (BO/00519/09/8).

Evolution of Qol resistance in Plasmopara viticola 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.

Occurrence and Molecular Identification of Azoxystrobin-Resistant Colletotrichum cereale Isolates from Golf Course Putting Greens in the Southern United States.

Turfgrass anthracnose, caused by Colletotrichum cereale (≡C. graminicola), has become a common disease of creeping bentgrass and annual bluegrass putting greens throughout the southern United States. Strobilurin (QoI) fungicides such as azoxystrobin are single-site mode-of-action fungicides applied to control C. cereale. In vitro bioassays with azoxystrobin at 0.031 and 8 μg/ml incorporated into agar were performed to evaluate the sensitivity of 175 isolates collected from symptomatic turfgrasses in Alabama, Mississippi, North Carolina, Tennessee, and Virginia. Three sensitivity levels were identified among C. cereale isolates. Resistant, intermediately resistant, and sensitive isolates were characterized by percent relative growth based on the controls with means of 81, 23, and 4%, respectively, on media containing azoxystrobin at 8 μg/ml. The molecular mechanism of resistance was determined by comparing amino acid sequences of the cytochrome b protein. Compared with sensitive isolates, C. cereale isolates exhibiting QoI resistance had a G143A substitution, whereas isolates expressing intermediate resistance had a F129L substitution. C. cereale isolates displaying azoxystrobin resistance in vitro were not controlled by QoI fungicides in a field evaluation. The dominance of QoI-resistant C. cereale isolates identified in this study indicates a shift to resistant populations on highly managed golf course putting greens.

Characterisation of QoI‐resistant field isolates of Botrytis cinerea from citrus and strawberry

In 2004, field isolates of Botrytis cinerea Pers. ex Fr., resistant to strobilurin fungicides (QoIs), were first found in commercial citrus orchards in Wakayama Prefecture, Japan. Subsequently, QoI-resistant isolates of this fungus were also detected in plastic strawberry greenhouses in Saga, Ibaraki and Chiba prefectures, Japan. Biological and molecular characterisation of resistant isolates was conducted in this study. QoI-resistant isolates of B. cinerea grew well on PDA plates containing kresoxim-methyl or azoxystrobin at 1 mg L(-1), supplemented with 1 mM of n-propyl gallate, an inhibitor of alternative oxidase, whereas the growth of sensitive isolates was strongly suppressed. Results from this in vitro test were in good agreement with those of fungus inoculation tests in vivo. In resistant isolates, the mutation at amino acid position 143 of the cytochrome b gene, known to be the cause of high QoI resistance in various fungal pathogens, was found, but only occasionally. The heteroplasmy of cytochrome b gene was confirmed, and the wild-type sequence often present in the majority of resistant isolates, indicating that the proportion of mutated cytochrome b gene was very low. The conventional RFLP and sequence analyses of PCR-amplified cytochrome b gene are insufficient for molecular identification of QoI resistance in B. cinerea.

First occurrence of resistance to strobilurin fungicides in Microdochium nivale and Microdochium majus from French naturally infected wheat grains

Microdochium nivale (Fr.) Samuels & Hallet and Microdochium majus (Wollenweber) belong to the Fusarium ear blight (FEB) fungal complex affecting cereals. In 2007 and 2008, major Microdochium sp. infestations were observed in France, and the efficacy of strobilurins was found to be altered in some field trials. The aim of this study was to determine the sensitivity to strobilurins of French isolates of Microdochium and to characterise the possible mechanisms of resistance. Half of the strains collected in 2007 were resistant to strobilurins, and most also displayed strong resistance to benzimidazoles. Strobilurin resistance was found mostly in M. majus isolates. Positive cross-resistance was observed between all strobilurins tested, but not with the phenylpyrrole derivative fludioxonil and the various classes of sterol biosynthesis inhibitors (SBIs). In most strains, resistance was correlated with the G143A substitution in cytochrome b, the molecular target of strobilurins. Two other mechanisms were also detected at lower frequencies. This is the first report of strobilurin resistance in Microdochium. Several resistance mechanisms have evolved independently in populations and may have different impacts on field efficacy. This makes the accurate detection and quantification of QoI resistance difficult. The management of field resistance and efficacy must be adapted to take these findings into account.

Characterization of QoI resistance in Botrytis cinerea and identification of two types of mitochondrial cytochrome b gene

Botrytis cinerea field isolates collected in Japan were screened for resistance to Qo inhibitor fungicides (QoIs). Of the 198 isolates screened, six grew well on a medium containing azoxystrobin, a QoI, when salicylhydroxamic acid, an alternative oxidase inhibitor, was present. The resistance mutation in the cytochrome b gene (cytb) was characterized. All QoI‐resistant isolates had the same mutation (GGT to GCT) in cytb that led to the substitution of glycine by alanine at position 143 of cytochrome b, which is known to confer QoI resistance in plant pathogens. To detect this mutation, a hybridization probe assay based on real‐time PCR amplification and melting curve analysis was developed. Using DNA samples prepared from aubergines coinfected with QoI‐resistant and QoI‐sensitive B. cinerea isolates, two similar peak profiles with their corresponding melting temperatures were obtained. This result suggests that QoI‐resistant and QoI‐sensitive isolates may compete equally in terms of pathogenicity, and the assay may be used to assess the population ratio of mutant and wild‐type isolates. However, the hybridization probe did not anneal to PCR products derived from the DNA samples of some QoI‐sensitive isolates. Structural analysis of cytb revealed that B. cinerea field isolates could be classified into two groups: one with three introns and the other with an additional intron (Bcbi‐143/144 intron) inserted between the 143rd and 144th codons. All 88 isolates possessing the Bcbi‐143/144 intron were azoxystrobin‐sensitive, suggesting that the QoI‐resistant mutation at codon 143 in cytb prevents self‐splicing of the Bcbi‐143/144 intron, as proposed in some other plant pathogens.

Sequence conservation in the mitochondrial cytochrome b gene and lack of G143A QoI resistance allele in a global sample ofRhynchosporium secalis

Barley scald caused by Rhynchosporium secalis is often controlled by fungicides, most recently by those in the strobilurin-based (QoI) class. Since the launch of QoIs in 1996 a range of important plant pathogens, including Blumeria graminis, Mycosphaerella fijiensis and Plasmopara viticola, developed resistance. Present monitoring data indicate that R. secalis populations remain sensitive. The primary molecular mechanism of QoI resistance in several fungi is a point mutation at codon 143 in the mitochondrial-encoded cytochrome b gene (cytb), which causes the substitution of glycine by alanine (G143A). We characterised the cytb gene of R. secalis, assessed the intraspecific and interspecific sequence diversity, developed a PCR-RFLP diagnostic tool to detect the most common allele associated with QoI resistance, screened a global collection of 841 R. secalis isolates for this allele and tested a representative sample of isolates for QoI resistance in vitro. The results indicated a high degree of conservation for the cytb gene at both intra- and interspecific levels, and complete QoI sensitivity in all R. secalis populations tested.

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

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

Monitoring of Venturia inaequalis harbouring the QoI resistance G143A mutation in French orchards as revealed by PCR assays

Genetic resistance to QoI fungicides may account for recent failures to control Venturia inaequalis (Cooke) Winter in French orchards. Two PCR-based assays were developed to detect the G143A point mutation in the fungal mitochondrial cytochrome b gene. The mutation is known to confer a high level of resistance to QoI fungicides. Occurrence of the G143A mutation in French field isolates collected from 2004 to 2007 was monitored. The QoI-resistant cytochrome b allele was specifically detected either following the cleavage of the amplified marker by a restriction endonuclease (CAPS assay) or its amplification using an allele-specific PCR primer. Using either method, the G143A mutation was found in 42% of the 291 field samples originating from French orchards in which apple scab proved difficult to be controlled. Monitoring of the G143A mutation in orchards located in 15 French administrative regions indicated that the mutation was detected at least once in nine of the regions, and its presence ranged from 33% to 64% of the orchards analysed in 2004 and in 2007 respectively. The PCR-based methods developed in this study efficiently reveal the presence of the G143A mutation in French V. inaequalis field populations.

Frequency of different CYP51-haplotypes of Mycosphaerella graminicola and their impact on epoxiconazole-sensitivity and -field efficacy

After development of QoI resistance in Mycosphaerella graminicola in intensive European wheat growing areas, demethylation inhibitors (DMIs) have become the most important fungicides for control of this disease. The sensitivity of M. graminicola towards DMIs has been monitored in recent years and a shift in the population to slightly reduced sensitivities in vitro was determined, which has now reached a plateau. As one reason for this shift, mutations in the target protein 14α-demethylase (CYP51) have been discussed, mainly the amino acid exchanges V136A, A379G, I381V, and mutations or deletions at the amino acid positions 459–462 of CYP51. Comparison of the CYP51-haplotypes of 615 isolates from different European regions for their in vitro sensitivity towards the triazole epoxiconazole nevertheless showed that the influence of the CYP51-haplotypes on sensitivity is limited. There was no correlation between in vitro sensitivity or CYP51-haplotype pattern and field performance of epoxiconazole detectable at different trials sites. Equally high levels of efficacy were achieved at sites where the mutations I381V or A379G, i.e. mutations conferring the highest reduction of efficacy in vitro, were dominant as well as where they were less frequent. Consequently, epoxiconazole treatments did not differentiate between isolates with different mutations. The European survey provides an overview of the distribution of the frequency of different CYP51-haplotypes in Europe indicating a heterogeneous population in Europe and the different regions, and even in a single field.

Mechanisms of resistance to QoI fungicides in phytopathogenic fungi.

The major threat to crops posed by fungal diseases results in the use by growers of enormous amounts of chemicals. Of these, quinol oxydation inhibitors (QoIs) are probably the most successful class of agricultural fungicides. QoIs inhibit mitochondrial respiration in fungi by binding to the Qo site of the cytochrome bc1 complex, blocking electron transfer and halting ATP synthesis. Unfortunately, the rapid development of resistance to these fungicides and consequent control failure has become increasingly problematic. The main mechanism conferring resistance to QoIs is target site modification, involving mutations in the cytochrome b gene CYTB, such as the substitution of glycine by alanine at position 143 (G143A) that occurs in several phytopathogenic fungi. The impact of other mechanisms, including alternative respiration and efflux transporters, on resistance seems to be limited. Interestingly, in some species QoI resistance is not supported by mutations in CYTB, while in others the structure of the gene is such that it is unlikely to undergo G143A mutations. Better understanding of the biological basis of QoI resistance in a single pathogen species will facilitate the development of resistance diagnostic tools as well as proper anti-resistance strategies aimed at maintaining the high efficacy of these fungicides.

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

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. 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. 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.

QoI Resistance of Plasmopara viticola and Erysiphe necator in the Mid-Atlantic United States

Of 20 Plasmopara viticola isolates collected in four locations in Virginia and northwest North Carolina in 2005, 16 were resistant to QoI fungicides. The resistance factor was over 100, and label rates of formulated azoxystrobin and pyraclostrobin provided little or no control of these isolates. Additional sampling in 2006 revealed at least 15 additional vineyards with QoI-resistant P. viticola in Virginia, Maryland, and Pennsylvania. Of 22 isolates of Erysiphe (Uncinula) necator collected in 2005 from five Virginia locations, 20 isolates from 4 locations showed resistance to QoI fungicides. The G143A mutation for resistance was detected in several isolates of both pathogens. This is the first detection of this type of resistance in P. viticola in North America, and the second North American report of QoI resistance in E. necator. Accepted for publication 26 November 2007. Published 11 February 2008.

Occurrence and Distribution of QoI-Resistant Isolates of Colletotrichum cereale from Annual Bluegrass in California.

Turfgrass anthracnose, caused by Colletotrichum cereale (ex. Colletotrichum graminicola), is an important disease of turf used on golf course putting greens. Recent management of the disease has become increasingly difficult, partly due to the possible development of practical resistance to the QoI fungicides. In all, 558 single-conidia isolates of C. cereale were collected from 10 California golf courses, 8 of which had been exposed to QoI fungicides and 2 where no fungicides had been used. Isolates were tested using a mycelial expansion assay on azoxystrobinamended media. For the two nonexposed populations, in vitro 50% effective dose (ED50) values ranged from 0.0060 to 0.089 μg/ml. All isolates from the exposed populations could not be fully inhibited by doses of azoxystrobin as high as 8.0 μg/ml. A subset of these isolates were tested in vitro with the QoI fungicides pyraclostrobin and trifloxystrobin and found to be similar in response, indicating that these isolates were fully cross-resistant to all three fungicides. In greenhouse pot experiments, three isolates nonresponsive to QoI fungicides in vitro were not controlled by label rates of the fungicides. Spore germination assays also were examined; for 10 isolates identified as sensitive by mycelial expansion assays, ED50 values for axoystrobin ranged from 0.0040 to 0.0047 μg/ml; for 25 isolates identified as QoI-resistant, 93 to 100% of the conidia germinated at azoxystrobin concentrations as high as 8.0 μg/ml relative to the nonamended check treatments. Mitochondrial cytochrome b genes from a subset of 15 isolates (12 resistant and 3 sensitive) were partially cloned and sequenced; all resistant isolates had an alanine substitution that corresponded to position 143 of the gene product. These results indicate that QoI resistance is present in California populations of C. cereale and is contributing to the difficulty in controlling this disease.