Evaluation of Erysiphe graminis f sp tritici field isolates for resistance to strobilurin fungicides with different SNP detection systems

A single nucleotide polymorphism (snp) in the cytochrome b gene confers resistance to strobilurin fungicides in Erysiphe graminis DC fsp tritici Marchal. On the basis of this point mutation three different types of molecular markers have been developed. Cleaved amplified polymorphic sequences and allele-specific PCR were used to score resistant and sensitive isolates from specifically selected regional populations across Europe. The results of molecular tests were in total agreement with the resistance phenotypes revealed by in vivo tests. Serial dilutions of mixed samples (resistant/sensitive) delimited the detection for strobilurin-resistant alleles to a range of 10-50% for both marker classes. Due to these detection limits no mixture of mitochondria within individual isolates was found. Denaturing high performance chromatography was used to increase the detection sensitivity for the mutant allele. Although the detection limit was lowered to 5-10%, there was no evidence for the existence of mixed mitochondrial genotypes.     

2009年我国部分麦区小麦白粉菌群体对三唑酮和嘧菌酯的敏感性测定

自1976年以来,三唑酮一直就是我国防治小麦白粉病的主要药剂.1991年首次在山东省发现抗性菌株,之后其他地区也相继有抗药性菌株的报道.本实验室从1995年就开始监测小麦白粉菌对三唑酮的抗药性,研究结果表明,平均抗性水平和抗性菌株的频率均总体呈上升趋势[1-3].     

Dynamics of QoI Sensitivity in Mycosphaerella fijiensis in Costa Rica During 2000 to 2003

ABSTRACT From 1997 onward, the strobilurin fungicide azoxystrobin was widely used in the main banana-production zone in Costa Rica against Mycosphaerella fijiensis var. difformis causing black Sigatoka of banana. By 2000, isolates of M. fijiensis with resistance to the quinolene oxidase inhibitor fungicides were common on some farms in the area. The cause was a single point mutation from glycine to alanine in the fungal target protein, cytochrome b gene. An amplification refractory mutation system Scorpion quantitative polymerase chain reaction assay was developed and used to determine the frequency of G143A allele in samples of M. fijiensis. Two hierarchical surveys of spatial variability, in 2001 and 2002, found no significant variation in frequency on spatial scales <10 m. This allowed the frequency of G143A alleles on a farm to be estimated efficiently by averaging single samples taken at two fixed locations. The frequency of G143A allele in bulk samples from 11 farms throughout Costa Rica was determined at 2-month intervals. There was no direct relationship between the number of spray applications and the frequency of G143A on individual farms. Instead, the frequency converged toward regional averages, presumably due to the large-scale mixing of ascospores dispersed by wind. Using trap plants in an area remote from the main producing area, immigration of resistant ascospores was detected as far as 6 km away both with and against the prevailing wind.     

Mechanisms influencing the evolution of resistance to Qo inhibitor fungicides

Fungicides inhibiting the mitochondrial respiration of plant pathogens by binding to the cytochrome bc1 enzyme complex (complex III) at the Qo site (Qo inhibitors, QoIs) were first introduced to the market in 1996. After a short time period, isolates resistant to QoIs were detected in field populations of a range of important plant pathogens including Blumeria graminis Speer f sp tritici, Sphaerotheca fuliginea (Schlecht ex Fr) Poll, Plasmopara viticola (Berk & MA Curtis ex de Bary) Berl & de Toni, Pseudoperonospora cubensis (Berk & MA Curtis) Rost, Mycosphaerella fijiensis Morelet and Venturia inaequalis (Cooke) Wint. In most cases, resistance was conferred by a point mutation in the mitochondrial cytochrome b (cyt b) gene leading to an amino-acid change from glycine to alanine at position 143 (G143A), although additional mutations and mechanisms have been claimed in a number of organisms. Transformation of sensitive protoplasts of M fijiensis with a DNA fragment of a resistant M fijiensis isolate containing the mutation yielded fully resistant transformants, demonstrating that the G143A substitution may be the most powerful transversion in the cyt b gene conferring resistance. The G143A substitution is claimed not to affect the activity of the enzyme, suggesting that resistant individuals may not suffer from a significant fitness penalty, as was demonstrated in B graminis f sp tritici. It is not known whether this observation applies also for other pathogen species expressing the G143A substitution. Since fungal cells contain a large number of mitochondria, early mitotic events in the evolution of resistance to QoIs have to be considered, such as mutation frequency (claimed to be higher in mitochondrial than nuclear DNA), intracellular proliferation of mitochondria in the heteroplasmatic cell stage, and cell to cell donation of mutated mitochondria. Since the cyt b gene is located in the mitochondrial genome, inheritance of resistance in filamentous fungi is expected to be non-Mendelian and, therefore, in most species uniparental. In the isogamous fungus B graminis f sp tritici, crosses of sensitive and resistant parents yielded cleistothecia containing either sensitive or resistant ascospores and the segregation pattern for resistance in the F1 progeny population was 1:1. In the anisogamous fungus V inaequalis, donation of resistance was maternal and the segregation ratio 1:0. In random mating populations, the sex ratio (mating type distribution) is generally assumed to be 1:1. Therefore, the overall proportion of sensitive and resistant individuals in unselected populations is expected to be 1:1. Evolution of resistance to QoIs will depend mainly on early mitotic events; the selection process for resistant mutants in populations exposed to QoI treatments may follow mechanisms similar to those described for resistance controlled by single nuclear genes in other fungicide classes. It will remain important to understand how the mitochondrial nature of QoI resistance and factors such as mutation, recombination, selection and migration might influence the evolution of QoI resistance in different plant pathogens.     

Using real-time PCR to survey frequency of azoxystrobin-resistant allele G143A in Alternaria populations from almond and pistachio orchards in California

Alternaria spp. cause leaf spot of almond and Alternaria late blight of pistachio in California, and azoxystrobin is a strobilurin fungicide that has been registered for the control of these diseases. To date, only a single point mutation of G143A in cytochrome b resulting to azoxystrobin resistance in Alternaria spp. was found in California. Based on this single point mutation, a real-time PCR assay was developed to quantify the frequency of the resistant allele G143A (FA) in pathogen samples taken from orchards. Forty-one almond and pistachio orchards were arbitrarily selected in eight counties of California. Fifty leaf lesions caused by Alternaria spp. per orchard were cut to extract the fungal DNA for a real-time PCR assay to determine the FA. About 88% of 41 surveyed orchards had Alternaria spp. with FA > 0.90, while six pistachio orchards showed a FA < 0.90. Therefore, azoxystrobin-resistant Alternaria populations are predominant in almond and pistachio orchards in California, and sprays of azoxystrobin to control Alternaria diseases are not recommended in these orchards. This study shows a potential use of a real-time PCR assay to efficiently quantify the frequency of azoxystrobin-resistant Alternaria spp. from large number of samples.     

2007年我国部分麦区小麦白粉菌对三唑酮的抗药性监测

采用拌种离体叶段法测定了我国9个省（市）的112个小麦白粉菌株对三唑酮的抗性。结果表明,供试菌株的平均EC50为57.25mg/L,平均抗性水平为27.39倍,其中86.61%供试菌株已经产生抗性,抗药性比敏感菌株高出10~40倍的菌株占47.32%,四川、山东、甘肃和贵州等地菌株的抗性高于其他地区。此结果可为三唑类杀菌剂在我国的推广应用和制定合理的抗药性治理措施提供依据。     

2011年我国主要麦区小麦白粉病菌群体对三唑酮和苯锈啶的敏感性

 小麦白粉病(Wheat Powdery Mildew)是我国小麦生产上常发性病害之一^[1]。小麦白粉病的防治主要采用抗病品种和化学药剂,辅之以栽培措施的综合防治技术。由于目前生产上抗病品种相对缺乏,药剂防治成为我国小麦白粉病防治的主要措施之一^[2]。自20世纪80年代以来三唑类杀菌剂一直是我国防治小麦白粉病的主要药剂,由于长期、大范围、单一的使用导致小麦白粉病菌对三唑类杀菌剂的抗药性大大提高。监测结果表明,2009年我国小麦白粉病菌群体对三唑酮的平均抗性水平已经达到56.58倍,抗性频率达到99.09%,其中高抗菌株占49.09%^[3]。目前小麦白粉病菌对三唑酮的抗性形势十分严峻,寻找三唑类杀菌剂的替代药剂成为控制该病害的迫切需求。     

Validation of a real-time PCR for the quantitative estimation of a G143A mutation in the cytochrome bc1 gene of Pyrenophora teres

A single nucleotide polymorphism (SNP) in the cytochrome b gene confers resistance to strobilurin fungicides for several fungal pathogens. Therefore, on the basis of a change at amino acid position 143 from glycine to alanine, a real-time PCR assay was established for the quantitative detection of the analogous SNP in the cytochrome b sequence of Pyrenophora teres Drechsler, which causes barley net blotch. Allelic discrimination was achieved by using allele specific primers with artificially mismatched nucleic acid bases and minor groove binding probes. Validation parameters for the lower limits of the working range, namely limits of detection (LOD) and limits of quantification (LOQ), were statistically determined by the variance of calibration data, as well as by the variance of the 100% non-strobilurin-resistant allele DNA sample (blank values). It was found that the detection was limited by the variance of blank values (five in 801 458 copies; 0.0006%), whereas the quantification was limited by the variance of calibration data (37 in 801 458 copies; 0.0046%). The real-time PCR assay was finally used to monitor strobilurin-resistant cytochrome b alleles in barley net blotch field samples, which were already classified in in vivo biotests to be fully sensitive to strobilurins. All signals for strobilurin-resistant cytochrome b alleles were below the LOD, and therefore the results are in total agreement with the phenotypes revealed by biotests.     

Genetic Diversity in Australian Populations of Puccinia graminis f. sp. avenae

ABSTRACT Sequence-tagged microsatellite profiling was used to develop 110 microsatellites for Puccinia graminis f. sp. tritici (causal agent of wheat stem rust). Low microsatellite polymorphism was exhibited among 10 pathogenically diverse P. graminis f. sp. tritici isolates collected from Australian cereal growing regions over a period of at least 70 years, with two polymorphic loci detected, each revealing two alleles. Limited cross-species amplification was observed for the wheat rust pathogens, P. triticina (leaf rust) and P. striiformis f. sp. tritici (stripe rust). However, very high transferability was revealed with P. graminis f. sp. avenae (causal agent of oat stem rust) isolates. A genetic diversity study of 47 P. graminis f. sp. avenae isolates collected from an Australia-wide survey in 1999, and a historical group of 16 isolates collected from Australian cereal growing regions from 1971 to 1996, revealed six polymorphic microsatellite loci with a total of 15 alleles. Genetic analysis revealed the presence of several clonal lineages and subpopulations in the pathogen population, and wide dispersal of identical races and genotypes throughout Australian cereal-growing regions. These findings demonstrated the dynamic population structure of this pathogen in Australia and concur with the patterns of diversity observed in pathogenicity studies.     

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.     

小麦白粉病菌对甲氧基丙烯酸酯类杀菌剂敏感性及其交互抗性

 20世纪70年代后期以来,小麦白粉病在我国小麦产区上升为主要病害,严重制约小麦生产。沿海北部及淮北地区,小麦白粉病常年发生4级左右。目前麦类白粉病的防治药剂主要有三唑类、嘧啶类、苯并咪唑类、甲氧基丙烯酸酯类及硫代氨基甲酸盐类等。但小麦白粉病菌变异快,繁殖迅速,以及药剂长期单一使用,造成小麦白粉病菌易产生抗药性。1995年我国小麦白粉菌对三唑酮的平均抗性水平在16.8倍以上。因而,急需寻找和开发替代三唑酮防治小麦白粉病的新型药剂。甲氧基丙烯酸酯类杀菌剂继三唑类杀菌剂后出现, 是一类内吸保护型杀菌剂,主要作用于真菌的线粒体呼吸链中细胞色素bc1复合物,阻止电子传递,抑制真菌生长。此类药剂于1996年投入德国市场,尚未在我国小麦白粉病防治上大量使用。     

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.     

Pyraclostrobin reduces germ tube growth of QoI‐resistant Mycosphaerella graminicola pycnidiospores and the severity of septoria tritici blotch on winter wheat

The effect of the quinone outside inhibitors (QoI) azoxystrobin and pyraclostrobin on yields of winter wheat where QoI resistant Mycosphaerella graminicola isolates were dominant was investigated in field trials in 2006 and 2007. Pyraclostrobin significantly increased yields by 1·57 t ha^?1 in 2006 and 0·89 t ha^?1 in 2007 when compared to the untreated controls, while azoxystrobin only provided a significant increase of 1·28 t ha^?1 in 2006. These yield increases were associated with reduction in septoria tritici blotch (STB) development as determined by weekly disease assessments over a 7 week interval. The effect of pyraclostrobin on STB was studied in controlled environment experiments using wheat seedlings inoculated with individual M. graminicola isolates. Pyraclostrobin significantly reduced STB symptoms by up to 62%, whether applied 48 h pre‐ or post‐ inoculation with resistant M. graminicola isolates containing the cytochrome b mutation G143A. Extremely limited disease (<1%) was observed on similarly treated seedlings inoculated with an intermediately resistant isolate containing the cytochrome b mutation F129L, while no disease was observed on seedlings inoculated with a wild‐type isolate. Germination studies of pycnidiospores of M. graminicola on water agar amended with azoxystrobin or pyraclostrobin showed that neither fungicide inhibited germination of spores of resistant isolates containing the mutation G143A. However, pyraclostrobin significantly reduced germ tube length by up to 46% when compared with the untreated controls. Although the QoIs can no longer be relied upon to provide effective M. graminicola control, this study provides an insight into why QoIs still provide limited STB disease control and yield increases even in situations of high QoI resistance.     

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     

50个小麦生产及后备品种(系)的抗白粉病基因推导

为明确我国小麦品种(系)中抗白粉病基因的组成,利用25个不同毒性的小麦白粉菌菌株对50个小麦生产及后备品种(系)进行抗白粉病基因推导,结果表明,参试的50个小麦品种(系)中有8个小麦品种(系)对供试的25个菌株全部感病,5个品种含有抗病基因Pm8,2个品种含有Pm4a,9个品种含有Pm2+6,4个品种含有Pm2,22个品种(系)可能含有供试基因之外的其他抗性基因或新基因。此研究结果可为小麦抗病育种以及品种利用提供依据。     

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.     

Structure and evolution of a population of Erysiphe graminis f.sp. hordei

Samples of single colony isolates of Erysiphe graminis f.sp. hordei were collected in Cambridge on three dates in 1985, and tested for the presence of 12 virulence alleles and resistance to two fungicides, triadimenol and ethirimol. The frequency of the virulence V-(La) fell during 1985, while the frequency of V-h. virulence on cv. Triumph and higher levels of resistance to each fungicide and combined resistance to both fungicides rose. Two phenotypes, both of which possessed virulence on cv. Triumph and three unnecessary virulence alleles and had similar sensitivities to ethirimol, but differed in their level of resistance to triadimenol, accounted for 35.9% of all isolates. The high frequency of these phenotypes accounted for most of the observed gametic phase disequilibria between pathogenicity characters. Most individuals with these two phenotypes may be members of the same clone. It was estimated that 25% of the spore population which initiated the autumn epidemic of E.g. f.sp. hordei originated from ascospores formed by sexual reproduction in the summer. It is proposed that genetic drift followed by hitch-hiking selection, due to intense selection for a clone virulent on a newly-introduced cultivar, is a major factor influencing the frequency of unnecessary virulence alleles.     

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.     

Field Resistance to Strobilurin (Q(o)I) Fungicides in Pyricularia grisea Caused by Mutations in the Mitochondrial Cytochrome b Gene

ABSTRACT Gray leaf spot caused by Pyricularia grisea is a highly destructive disease of perennial ryegrass turf. Control of gray leaf spot is dependent on the use of preventative fungicide treatments. Strobilurin-based (Q(o)I) fungicides, which inhibit the cytochrome bc(1) respiratory complex, have proven to be very effective against gray leaf spot. However, in August 2000, disease was diagnosed in Q(o)I-treated perennial ryegrass turf on golf courses in Lexington, KY, Champaign, IL, and Bloomington, IL. To determine if resistance was due to a mutation in the fungicide target, the cytochrome b gene (CYTB) was amplified from baseline and resistant isolates. Nucleotide sequence analysis revealed an intronless coding region of 1,179 bp. Isolates that were resistant to Q(o)I fungicides possessed one of two different mutant alleles, each of which carried a single point mutation. The first mutant allele had a guanine-to-cytosine transition at nucleotide position +428, resulting in a replacement of glycine 143 by alanine (G143A). Mutant allele two exhibited a cytosine-to-adenine transversion at position +387, causing a phenylalanine-to-leucine change (F129L). Cleavable amplified polymorphic sequence analysis revealed that neither mutation was present in a collection of baseline isolates collected before Q(o)I fungicide use and indicated that suspected Q(o)I- resistant isolates found in 2001 in Indiana and Maryland possessed the F129L mutation. The Pyricularia grisea isolates possessing the G143A substitution were significantly more resistant to azoxystrobin and trifloxystrobin, in vitro, than those having F129L. DNA fingerprinting of resistant isolates revealed that the mutations occurred in just five genetic backgrounds, suggesting that field resistance to the Q(o)I fungicides in Pyricularia grisea is due to a small number of ancestral mutations.     

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.