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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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. Copyright © 2012 Society of Chemical Industry     

Resistance of Corynespora cassiicola from soybean to QoI and MBC fungicides in Brazil

Fungicide sprays on soybean in Brazil have contributed to the selection of less sensitive isolates of Corynespora cassiicola. We collected 59 isolates of C. cassiicola from three Brazilian states and two isolates from Paraguay. We investigated their EC₅₀ to quinone outside inhibitors (QoI) and methyl benzimidazole carbamate (MBC), any cross-resistance to compounds within QoI and MBC groups, and characterized the polymorphisms in their cytb and β-tubulin genes. Local associations of polymorphisms identified in each gene were statistically correlated with assays results. In total, 79% and 74% of the isolates were classified as resistant to QoI and MBC fungicides, respectively. There was positive cross-resistance to active ingredients within QoI and MBC groups. For QoI, all isolates presented heteroplasmy in G143A of cytb gene; the mutations F129L and G137R were not found. For MBC, 63% of isolates possessed E198A and 21% possessed F200Y mutations, associated with reduced control by MBC fungicides. Heteroplasmy was identified in two and one isolates from Brazil with E198A and F200Y mutations, respectively. The resistance factor for isolates with E198A (10.9) was statistically similar to the isolate with F200Y (8.8) mutation. Genic association analysis of the in vitro assays using discriminatory doses proved them to be accurate. Reduced sensitivity of C. cassiicola to QoI and MBC was also identified in isolates from Paraguay and resistance to QoI and MBC was widely present in C. cassiicola isolates from the main soybean-producing states in Brazil. Thus, integrated management measures should be adopted to manage soybean target spot in these countries.     

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     

新疆鹰嘴豆壳二孢疫病病菌Ascochyta rabiei交配型、致病力及其对3种杀菌剂的敏感性测定

新疆是我国鹰嘴豆主要生产地,然而随着鹰嘴豆种植面积的扩大,集约化种植模式的发展以及广泛从国内外引种,鹰嘴豆壳二孢疫病(Ascochyta blight)开始发生。由于该病害在新疆发生历史较短,相关的研究还甚少。本研究对新疆鹰嘴豆壳二孢疫病病菌Ascochyta rabiei的交配型、致病力及对3种杀菌剂敏感性进行测定,结果表明,A.rabiei的交配型MAT1-1和MAT1-2在新疆木垒地区的分离频率明显偏离1∶1,以MAT1-2为优势种群,但其致病力与MAT1-1无显著差异,而MAT1-1对30%醚菌酯EC的敏感性显著低于MAT1-2。     

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.     

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

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

Monitoring of Brazilian wheat blast field populations reveals resistance to QoI,DMI, and SDHI fungicides

Wheat blast is one of the most important and devastating fungal diseases of wheat in South America, South-east Asia, and now in southern Africa. The disease can reduce grain yield by up to 70% and is best controlled using integrated disease management strategies. The difficulty in disease management is compounded by the lack of durable host resistance and the ineffectiveness of fungicide sprays. New succinate dehydrogenase inhibitor (SDHI) fungicides were recently introduced for the management of wheat diseases. Brazilian field populations of the wheat blast pathogen Pyricularia oryzae Triticum lineage (PoTl) sampled from different geographical regions in 2012 and 2018 were shown to be resistant to both QoI (strobilurin) and DMI (azole) fungicides. The main objective of the current study was to determine the SDHI baseline sensitivity in these populations. Moderate levels of SDHI resistance were detected in five out of the six field populations sampled in 2012 and in most of the strains isolated in 2018. No association was found between target site mutations in the sdhB, sdhC, and sdhD genes and the levels of SDHI resistance, indicating that a pre-existing resistance mechanism not associated with target site mutations is probably present in Brazilian wheat blast populations.     

Occurrence and distribution of resistance to QoI fungicides in populations of Podosphaera fusca in south central Spain

Cucurbit powdery mildew caused by Podosphaera fusca limits crop production in Spain. Since its management is strongly dependent on chemicals, the rational design of control programmes requires a good understanding of the fungicide resistance phenomenon in field populations. Fifty single-spore isolates of P. fusca were tested for sensitivity to three quinone-outside inhibiting (QoI) fungicides: azoxystrobin, kresoxim-methyl and trifloxystrobin. Minimum inhibitory concentration (MIC) values for QoI-sensitive isolates were found to range from 0.25 to 10 μg ml⁻¹ for azoxystrobin to 5–25 μg ml⁻¹ for kresoxim-methyl, using a leaf disc-based bioassay. High levels of cross-resistance to QoI fungicides were found. Eleven isolates showed resistance to the three QoI fungicides tested with MIC and EC₅₀ values >500 μg ml⁻¹ resulting in RF values as high as >715 and >1000 for trifloxystrobin and azoxystrobin, respectively. A survey of P. fusca QoI resistance was carried out in different provinces located in the south central area of Spain during the cucurbit growing seasons in 2002, 2003 and 2004. Examination of a collection of 250 isolates for QoI resistance revealed that 32% were resistant to the three fungicides tested; the provinces of Ciudad Real, Córdoba and Murcia being the locations with the highest frequencies of resistance (44–74%). By contrast, no resistance was found in Badajoz, and relatively low frequencies were observed in Almería and Valencia (10–13%). Nearly 50% of resistant isolates were collected from melon plants. Based on these data, recommendations about the use of QoI fungicides for cucurbit powdery mildew management in the sampled areas are made.     

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

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

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: The PCR-based methods developed in this study efficiently reveal the presence of the G143A mutation in French V. inaequalis field populations.     

灰葡萄孢抗二甲酰亚胺类杀菌剂研究进展

综述了灰葡萄孢对二甲酰亚胺类杀菌剂抗性的产生及现状,抗性菌株的生物学特性、生理生化特性以及抗药性机制,并就灰葡萄孢对二甲酰亚胺类杀菌剂抗性研究进行了展望。     

安徽省番茄灰霉菌抗药性测定和治理

测定了安徽省番茄灰霉病菌对多菌灵、腐霉利、乙霉威的抗性。结果表明,田间有B_SD_SN_S、B_SD_SN_LR、B_SD_SN_HR、B_HRD_SN_S、B_MRD_LRN_S、B_HRD_LRN_S、B_HRD_LRN_LR7种抗性类型,对多菌灵、腐霉利、乙霉威具有抗性的菌株分别占试验总菌株数的28.3%、20%和73.3%。     

Characterization of the cytochrome b gene fragment of Puccinia species responsible for the binding site of QoI fungicides

The fragment of the cytochrome b (cyt b) gene responsible for the binding site of QoI fungicides was sequenced for different Puccinia species by using DNA and RNA as template for PCR and RT-PCR, respectively. Degenerated primers for the cyt b gene amplified in P. recondita f.sp. tritici a 450 bp fragment, which was cloned and sequenced. At cDNA level, several Thermal Asymmetric InterLaced (TAIL)-PCR cycles were needed to produce a 996 bp long fragment, which corresponded to almost the whole cyt b gene (about 1160-1180 bp, without introns). This fragment was sequenced and specific primers were designed. Amplification with cyt b specific primers using genomic DNA as template revealed the presence of an intron of about 1500 bp length after the codon for glycine at amino acid position 143. By using the same primer pair, the cyt b gene fragment was amplified and sequenced both at cDNA and genomic DNA level also for other rust species, including P. graminis f.sp. tritici (length: 506 bp), P. striiformis f.sp. tritici (755 bp), P. coronata f.sp. avenae (644 bp), P. hordei (660 bp), P. recondita f.sp. secalis (687 bp), P. sorghi (709 bp), and P. horiana (478 bp). At the same position as for P. recondita f.sp. tritici, an intron of about 1500-1600 bp length was detected also in all other Puccinia species. High homologies were observed among all Puccinia species for both the exonic and intronic fragments of the cyt b gene. Specific primers for the cyt b gene of all eight Puccinia species were developed, which easily amplified the fragment of the gene including all possible mutations known to confer resistance to QoIs in several plant pathogens. However, in all tested isolates of the Puccinia species included in this study, the sequence of cyt b gene fragment did not contain any point mutations.     

四川猕猴桃灰霉病菌对4种杀菌剂的抗药性检测

为明确猕猴桃灰霉病菌对常见杀菌剂的抗药性状况,本试验于2016年-2018年调查统计了四川猕猴桃生产中防控灰霉病所用的药剂种类和施药频次,并从四川8个猕猴桃主产区采集病叶、病花和病果,经单孢分离获得122个灰葡萄孢Botrytis cinerea菌株,采用最低抑制浓度法(MIC)测定其对4种化学药剂嘧霉胺、咯菌腈、腐霉...     

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     

Sensitivity to azoxystrobin in Didymella bryoniae isolates collected before and after field use of strobilurin fungicides

BACKGROUND: Isolates of Didymella bryoniae (Auersw.) Rehm, causal agent of gummy stem blight on cucurbits, developed insensitivity to azoxystrobin in the eastern United States 2 years after first commercial use in 1998. Baseline sensitivity of this fungus to azoxystrobin has never been reported. The objectives were to compare baseline sensitivities of D. bryoniae from South Carolina and other locations to sensitivities of isolates exposed to azoxystrobin for one or more seasons, and to compare sensitivity in vitro and in vivo. RESULTS: Sixty‐one isolates of D. bryoniae collected before 1998 were sensitive. Median EC₅₀ was 0.055 mg L^?1 azoxystrobin (range 0.005 to 0.81). Forty isolates collected after exposure during 1998 also were sensitive. Fifty‐three of 64 isolates collected in South and North Carolina between 2000 and 2006 were insensitive to 10 mg L^?1 azoxystrobin. Sensitive and insensitive isolates were distinguished by disease severity on Cucumis melo L. seedlings treated with azoxystrobin (20 or 200 mg L^?1). CONCLUSIONS: An azoxystrobin baseline sensitivity distribution was established in vitro for isolates of D. bryoniae never exposed to strobilurins. Baseline values were comparable with those of other ascomycetes. Insensitive isolates were found in fields with a history of strobilurin applications. An in vivo method distinguished sensitive and insensitive isolates. Copyright © 2009 Society of Chemical Industry     

Characterization of chickpea differentials for pathogenicity assay of ascochyta blight and identification of chickpea accessions resistant to Didymella rabiei

Forty-eight chickpea germplasm lines, including 22 differentials used in previous studies, were characterized for disease phenotypes following inoculation with six isolates of Didymella (anamorph Ascochyta ) rabiei , representing a wide spectrum of pathogenic variation. Representative isolates were also directly compared with six previously identified races on eight chickpea genotypes. Many of the chickpea differentials reacted similarly to inoculation with each isolate of D. rabiei , and several previously identified races caused similar levels of disease on the differentials. This indicates that the number of differentials can be reduced significantly without sacrificing accuracy in describing pathogenic variation of D. rabiei on chickpea. Pathogenic variation among samples of US isolates allowed classification of the isolates into two pathotypes. The distribution of disease phenotypes of the 48 germplasm lines was bimodal after inoculation with pathotype I isolates, whereas the distribution of disease phenotypes was continuous after inoculation with pathotype II isolates. Such distinct distribution patterns suggest that chickpea plants employ different resistance mechanisms to each pathotype and that the two pathotypes may have different genetic mechanisms controlling pathogenicity. The advantages of using the two-pathotype system in assaying pathogenicity of the pathogen and in studying resistance mechanisms of the host are discussed. Three chickpea accessions, PI 559361, PI 559363 and W6 22589, showed a high level of resistance to both pathotypes, and can be employed as resistance sources in chickpea breeding programmes for resistance to ascochyta blight.     

山东蓬莱葡萄灰霉菌对7种杀菌剂的抗药性检测

为了明确葡萄灰霉菌对啶酰菌胺、多菌灵、咯菌腈、异菌脲、腐霉利、嘧霉胺的抗药性和对抑霉唑的敏感性,本试验采用菌丝生长速率法和孢子萌发法检测了采自山东蓬莱地区的69株葡萄灰霉菌对上述前6种杀菌剂的抗药性、对抑霉唑的敏感性及抑霉唑与其他6种杀菌剂的交互抗性关系。结果表明,抑霉唑对这69株葡萄灰霉菌的EC₅₀分布在0.403～28.76μg/mL之间,平均值为(9.34±10.34)μg/mL;葡萄灰霉菌菌株中抗啶酰菌胺(BosR)、多菌灵(CarR)、咯菌腈(FluR)、异菌脲(IprR)、嘧霉胺(PyrR)、腐霉利(ProR)的比例分别为100%、100%、9.47%、97.18%、100%、89.20%,测试菌株的抗药性均为多抗类型,没有单抗菌株,其中对3种杀菌剂(啶酰菌胺、多菌灵、嘧霉胺)、对4种杀菌剂(啶酰菌胺、多菌灵、异菌脲、嘧霉胺)、对5种杀菌剂(啶酰菌胺、多菌灵、异菌脲、嘧霉胺、腐霉利或啶酰菌胺、多菌灵、咯菌腈、异菌脲、嘧霉胺)和对6种杀菌剂(啶酰菌胺、多菌灵、咯菌腈、异菌脲、嘧霉胺、腐霉利)的抗性频率分别为2.33%、9.30%、79.07%、2.33%、6.97%,表明啶酰菌胺、多菌灵、嘧霉胺对测试葡萄菌株完全丧失防效,建议在该葡萄产区停止使用这些药剂,测试菌株对腐霉利、异菌脲的抗性频率高,建议采取限制使用、禁止单独使用等措施,测试菌株对咯菌腈的抗性频率较低,可以继续使用但需按照科学使用规则进行。抑霉唑与其他6种杀菌剂间不存在交互抗性关系,说明其可以和其他药剂同时使用但建议减少使用。     

Development of a multiplex allele‐specific primer PCR assay for simultaneous detection of QoI and CAA fungicide resistance alleles in Plasmopara viticola populations

BACKGROUND: DNA‐based diagnosis has become a common tool for the evaluation of fungicide resistance in obligate phytopathogenic fungus Plasmopara viticola. RESULTS: A multiplex allele‐specific primer PCR assay has been developed for the rapid detection of fungicide resistance in P. viticola populations. With this assay, a glycine‐to‐alanine substitution at codon 143 of the P. viticola cytochrome b gene, which conferred QoI fungicide resistance, and a glycine‐to‐serine substitution at codon 1105 of the P. viticola cellulose synthase gene PvCesA3, which conferred CAA fungicide resistance, were detected simultaneously. CONCLUSION: It is suggested that the present assay is a reliable tool for the rapid and simultaneous detection of QoI and CAA fungicide resistance alleles in P. viticola populations. The assay required only 2 h from the sampling of symptoms to the detection of resistance alleles to both fungicides. Copyright © 2012 Society of Chemical Industry