Characterisation of benzimidazole resistance of Cercospora beticola in Serbia using PCR-based detection of resistance-associated mutations of the β-tubulin gene

A survey to detect and characterise benzimidazole resistance within populations of Cercospora beticola in Serbia was performed. From 52 field isolates collected from sugar beet and beet root, only eight were found to be benzimidazole-sensitive based on the inhibition of mycelial growth by discriminatory concentrations of carbendazim and thiophanate-methyl. Sensitivity tests revealed the presence of three resistant phenotypes among the tested isolates: high-resistance (HR), low-resistance (LR) and moderate-resistance (MR). The benzimidazole resistant isolates were characterised based on the DNA sequence of the β-tubulin gene and temperature sensitivity. The HR isolates showed no temperature sensitivity regardless of carbendazim concentration, whereas the LR and MR isolates were sensitive at lower temperatures. Analysis of the β-tubulin gene sequence revealed two amino acid replacements in the benzimidazole-resistant isolates of C. beticola. One was a glutamic acid to alanine change at position 198 (codon GAG to GCG) that was identified in HR isolates; this mutation has previously been reported to be associated with the development of benzimidazole resistance in C. beticola. The second replacement was a novel point mutation of phenylalanine (TTC) to tyrosine (TAC) at position 167, identified in low and moderate benzimidazole-resistant isolates, sharing a single LR/MR β-tubulin genotype. A diagnostic PCR-RFLP assay utilising a BsaI restriction site present in the benzimidazole sensitive and LR/MR genotypes but absent in the HR genotype was developed for the routine detection of high resistance. A mutation-specific PCR assay was developed for the diagnosis of LR/MR genotype based on a mutation from T to A at codon 167, which is unique to this genotype.     

Studies on thiabendazole resistance of Penicillium expansum of pears: pathogenic fitness and genetic characterization

Isolates of Penicillium expansum recovered from stored pears were scored for resistance to the fungicide thiabendazole (TBZ) and for pathogenic fitness. Out of 50 isolates, nine were sensitive (S) and 41 resistant (R or RR). Seven of these resistant isolates (RR) germinated with a higher percentage on TBZ-amended medium than on unamended medium. Six S isolates and six RR isolates were chosen at random for further analysis. S and RR isolates had similar in vitro growth fitness, although RR isolates were characterized by higher infection severity on fruits. Laboratory-induced resistant isolates were generated by UV-irradiating S strains, and a similar correlation between the induced TBZ resistance and pathogenic fitness was observed. The β-tubulin gene of RR and S isolates was amplified and sequenced; mutations correlating with TBZ resistance were identified at residues Phe 167 and Glu 198. Analogous mutations were detected in the laboratory-induced resistant isolates.     

Monitoring and characterization of resistance development of strawberry Phomopsis leaf blight to fungicides

From 2005 to 2009, a total of 479 single-conidial isolates of Phomopsis obscurans were collected from strawberry. The isolates were characterized for their resistance to benzimidazole fungicides, diethofencarb, and sterol demethylation inhibitors (DMIs). Low-level DMI resistant isolates (DMI-LR) and two types of benzimidazole-resistant (Ben R) isolates, Ben R1 (benzimidazole-resistant and diethofencarb -sensitive) and Ben R2 (benzimidazole-resistant and diethofencarb -resistant), were detected. Both Ben R and DMI-LR isolates exhibited comparable growth, sporulation, and pathogenicity with the sensitive isolates. No significant difference in growth at low temperature was observed between Ben R and benzimidazole-sensitive (Ben S) isolates. Ben R1 was caused by a point mutation from GAG to GTG at codon 198 in the β-tubulin gene in Ben S isolates, predicted to cause a change from glutamic acid to valine. Ben R2 was induced by a point mutation from TTC to TAC at codon 200 in the β-tubulin gene in Ben S isolates.     

Molecular characterization of benzimidazole-resistant B. cinerea field isolates with reduced or enhanced sensitivity to zoxamide and diethofencarb

Sensitivity profiles of Botrytis cinerea field isolates to zoxamide and the molecular basis of the resistance mechanism involved in cross-resistance relationships between benzamides, benzimidazoles and N-phenylcarbamates were investigated. B. cinerea isolates collected from southern, central and northern Greece were characterized based on their sensitivity to zoxamide, the benzimidazole carbendazim and the N-phenylcarbamate diethofencarb. Isolates exhibiting baseline sensitivity to carbendazim and zoxamide but no sensitivity to diethofencarb were considered wild type (S phenotype) and accounted for 44% of the total strains sampled. Thirty-three percent of the isolates had increased sensitivity (HS phenotype) to zoxamide and diethofencarb and were highly resistant to carbendazim compared to S isolates. Eight percent of the sample was highly resistant (HR phenotype) to all anti-tubulin agents studied. The rest of the isolates were moderately resistant to zoxamide (MR phenotype) and equally sensitive to benzimidazoles and N-phenylcarbamates compared to isolates of the S phenotype. Fungitoxicity tests with botrycides belonging to other chemical classes revealed no cross-resistance relationships between zoxamide and the phenylpyrrole fludioxonil, the dicarboximide iprodione, the hydroxyanilide fenhexamid, the anilinopyrimidine cyprodinil, the carboxamide boscalid and the strobilurin-type fungicide pyraclostrobin. Study of fitness characteristics did not show any significant difference between zoxamide resistant and sensitive isolates with respect to the parameters tested. PCR-RFLP analysis of a part of the β-tubulin gene sequence detected mutations in position 198 for both HS and HR zoxamide-sensitivity phenotypes. DNA sequence analysis of the B. cinerea β-tubulin gene revealed two previously described benzimidazole-resistance-conferring mutations. The first one was the glutamic acid (GAG) to alanine (GCG) change at position 198 (E198A), which was identified in all HS isolates. The second mutation (E198K) was a GAG-to-AAG substitution resulting in the replacement of glutamic acid with lysine present in all B. cinerea isolates highly resistant to all three anti-tubulin classes of fungicides. A number of mutations in other positions of the β-tubulin gene were detected in the moderately zoxamide-resistance phenotype.     

Using allele-specific oligonucleotide probes to characterize benzimidazole resistance in Rhynchosporium secalis

Benzimidazole fungicides are important mixture components in strategies to combat fungicide resistance in Rhynchosporium secalis Davis. To monitor the performance of these strategies, a rapid, accurate assay has been developed to detect point mutations in the β-tubulin gene which confers resistance of benzimidazoles. The β-tubulin gene of a benzimidazole-resistant strain of R. secalis has been cloned and sequenced. Except for the difference in the position of one of its six introns, this gene showed a strong homology with other β-tubulin genes from filamentous fungi. Resistance was related to a point mutation in codon 198 which caused a glutamic acid to glycine change in resistant field strains, but glutamic acid to lysine in a laboratory mutant. A DNA fragment surrounding codon 198 was amplified directly from diseased lesions using a ‘nested’ set of PCR primers. Combining PCR amplificiation of a target DNA sequence with hybridization of Allele-Specific Oligonucleotide probes (ASO_s, 15-mers) allowed accurate detection of benzimidazole resistance. Only two probes, one sensitive and one resistant, were sufficient to monitor current field populations. Detection was achieved using either ³²P-labelled probe, or non-radioactively using a biotin-labelled probe coupled to streptavidin/alkaline phosphatase. This rapid method using ASOS can detect benzimidazole resistance within 48 h compared with 6–8 weeks by conventional assay procedures.     

Molecular characterization of boscalid resistance in field isolates of Botrytis cinerea from apple

Botrytis cinerea isolates obtained from apple orchards were screened for resistance to boscalid. Boscalid-resistant (BosR) isolates were classified into four phenotypes based on the levels of the concentration that inhibited fungal growth by 50% relative to control. Of the 220 isolates tested, 42 were resistant to boscalid, with resistant phenotypes ranging from low to very high resistance. There was cross resistance between boscalid and carboxin. Analysis of partial sequences of the iron-sulfur subunit of succinate dehydrogenase gene in B. cinerea (BcSdhB) from 13 BosR and 9 boscalid-sensitive (BosS) isolates showed that point mutations in BcSdhB leading to amino acid substitutions at the codon position 272 from histidine to either tyrosine (H272Y) or arginine (H272R) were correlated with boscalid resistance. Allele-specific polymerase chain reaction (PCR) analysis of 66 BosR isolates (including 24 additional isolates obtained from decayed apple fruit) showed that 19 carried the point mutation H272Y and 46 had the point mutation H272R, but 1 BosR isolate gave no amplification product. Analysis of the BcSdhB sequence of this isolate revealed a different point mutation at codon 225, resulting in a substitution of proline (P) by phenylalanine (F) (P225F). The results indicated that H272R/Y in BcSdhB were the dominant genotypes of mutants in field BosR isolates from apple. A multiplex allele-specific PCR assay was developed to detect point mutations H272R/Y in a single PCR amplification. Levels of boscalid resistance ranged from low to very high within isolates carrying either the H272R or H272Y mutation, indicating that, among BosR isolates, different BosR phenotypes (levels of resistance) were not associated with particular types of point mutations (H272R versus H272Y) in BcSdhB. Analysis of genetic relationships between 39 BosR and 56 BosS isolates based on three microsatellite markers showed that 39 BosR isolates and 30 BosS isolates were clustered into two groups, and the third group consisted of only BosS isolates, suggesting that the development of resistance to boscalid in B. cinerea likely is not totally random, and resistant populations may come from specific genetic groups.     

Application of cycleave PCR to the detection of a point mutation (F167Y) in the β2‐tubulin gene of Fusarium graminearum

BACKGROUND: Resistance of Fusarium graminearum to the benzimidazole fungicide carbendazim is caused by point mutations in the β₂‐tubulin gene (FGSG_06611.3). The point mutation at codon 167 (TTT → TAT, F167Y) occurs in more than 90% of field isolates in China. It is important to find a suitable method for rapid detection and quantification of this point mutation in the F. graminearum populations. RESULTS: A pair of primers, Codon167F/Codon167R, were designed to amplify a fragment containing the mutation site, and two cycling probes labelled with different fluorescent reporters were used to detect whether the mutation was present. A cycleave real‐time PCR method was developed for rapid determination of the frequency of this point mutation in 282 F. graminearum perithecia collected from different fields in Jiangsu Province, China. The mutation frequency in ascospores from the perithecia to carbendazim by a spore germination assay was 6.0%, while the frequency of the point mutation at codon 167 by the cycleave real‐time PCR assay was 3.9%. CONCLUSION: The cycleave real‐time PCR method is suitable for accurate detection of the codon 167 point mutation. The frequency of this mutation in the β₂‐tubulin gene represents the resistance frequency in F. graminearum populations to carbendazim. Copyright © 2011 Society of Chemical Industry     

胶孢炭疽菌及两种疫霉菌对苯酰菌胺的敏感性与

选择对多菌灵、乙霉威和苯酰菌胺具有不同敏感性的胶孢炭疽菌Colletotrichum gloeosporioides、辣椒疫霉菌Phytophthora capsici及恶疫霉菌P. cactorum,分析其β-微管蛋白氨基酸突变与敏感性的关系。结果表明,胶孢炭疽菌对苯酰菌胺、多菌灵和乙霉威的敏感性与β-微管蛋白198位或200位氨基酸突变有关：对多菌灵敏感,对苯酰菌胺和乙霉威不敏感的胶孢炭疽菌β-微管蛋白氨基酸198位为谷氨酸（E）,200位为苯丙氨酸（F）;对多菌灵已产生抗性而对苯酰菌胺和乙霉威不敏感的菌株,其氨基酸200位由苯丙氨酸（F）突变为了酪氨酸（Y）;对多菌灵高抗,对苯酰菌胺和乙霉威敏感的菌株其氨基酸198位由谷氨酸（E）突变为了丙氨酸（A）。辣椒疫霉菌和恶疫霉菌对苯酰菌胺敏感,对多菌灵和乙霉威均不敏感。检测疫霉菌菌株β-微管蛋白未发现氨基酸突变,但发现其β-微管蛋白氨基酸在196~200位与胶孢炭疽菌差异较大,这可能是导致苯酰菌胺仅对疫霉菌有抑制效果的原因。     

胶孢炭疽菌及两种疫霉菌对苯酰菌胺的敏感性与β-微管蛋白氨基酸突变的相关性分析

选择对多菌灵、乙霉威和苯酰菌胺具有不同敏感性的胶孢炭疽菌 Colletotrichum gloeosporioides、辣椒疫霉菌 Phytophthora capsici 及恶疫霉菌 P.cactorum,采用菌丝生长速率抑制法及氨基酸序列比对法分析了其 β-微管蛋白氨基酸突变与敏感性的关系。结果表明,胶孢炭疽菌对苯酰菌胺、多菌灵和乙霉威的敏感性与 β-微管蛋白198位或200位氨基酸突变有关:对多菌灵敏感、对苯酰菌胺和乙霉威不敏感的胶孢炭疽菌 β-微管蛋白氨基酸198位为谷氨酸(E),200位为苯丙氨酸(F);对多菌灵已产生抗性而对苯酰菌胺和乙霉威不敏感的菌株,其 β-微管蛋白氨基酸200位由苯丙氨酸(F)突变为了酪氨酸(Y);对多菌灵高抗、对苯酰菌胺和乙霉威敏感的菌株其 β-微管蛋白氨基酸198位由谷氨酸(E)突变为了丙氨酸(A)。辣椒疫霉菌和恶疫霉菌对苯酰菌胺敏感,对多菌灵和乙霉威均不敏感。检测疫霉菌菌株 β-微管蛋白未发现氨基酸突变,但发现其 β-微管蛋白氨基酸在196~200位与胶孢炭疽菌差异较大,这可能是导致苯酰菌胺仅对疫霉菌有抑制效果的原因。     

Identification of a novel point mutation in the <Emphasis Type="Italic">β</Emphasis>-tubulin gene of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> and detection of benzimidazole resistance by a diagnostic PCR-RFLP assay

The molecular basis of resistance to benzimidazole fungicides with laboratory and field mutant isolates of Botrytis cinerea was investigated. After chemical mutagenesis with N-methyl-N-nitrosogouanidine (NMNG) two different benzimidazole-resistant phenotypes were isolated on media containing carbendazim or a mixture of carbendazim and diethofencarb. The mutant isolates from the fungicide-mixture-containing medium were moderately resistant to carbendazim with wild-type tolerance to diethofencarb while mutant isolates from carbendazim-containing medium were highly resistant to carbendazim but sensitive to diethofencarb. The studied field isolates were highly resistant to benzimidazoles and sensitive to diethofencarb. Study of fitness characteristics of benzimidazole highly-resistant isolates showed that the resistance mutation(s) had no apparent effect on fitness-determining parameters. Contrary to this, the moderately benzimidazole-resistant strains, with no increased diethofencarb sensitivity, had a significant reduction in certain ecological fitness-determining characteristics. Analysis of the sequence of the β-tubulin gene revealed two amino acid replacements in the highly benzimidazole-resistant mutants compared to that of the wild-type parent strain. One was the glutamic acid (GAG) to alanine (GCG) change at position 198 (E198A), identified in both laboratory and field highly benzimidazole-resistant isolates, a mutation previously implicated in benzimidazole resistance. The second was a novel benzimidazole resistance mutation of glutamic acid (GAG) to glycine (GGG) substitution at the same position 198 (E198G), identified in a highly benzimidazole-resistant laboratory mutant strain. Molecular analysis of the moderately benzimidazole-resistant strains revealed no mutations at the β-tubulin gene. A novel diagnostic PCR-RFLP assay utilising a BsaI restriction site present in the benzimidazole-sensitive (E198) but absent in both resistant genotypes (E198G and E198A) was developed for the detection of both amino acid replacements at the β-tubulin gene.     

浙江省西(甜)瓜蔓枯病菌对甲基硫菌灵和啶酰菌胺的抗性检测及抗性机制

从浙江省5地采集了112株西 (甜) 瓜蔓枯病菌Stagonosporppsis citrulli,采用区分剂量法检测其对苯并咪唑类杀菌剂甲基硫菌灵 (以下简称Ben) 和琥珀酸脱氢酶抑制剂类 (SDHIs) 杀菌剂啶酰菌胺 (简称Bos) 的抗性。结果显示:112株西 (甜) 瓜蔓枯病菌对Ben和Bos的抗药性频率分别为100%和28.6%,其中对甲基硫菌灵产生高水平抗性 (BenHR) 的菌株达100%,对啶酰菌胺产生低水平抗性 (BosLR) 和高水平抗性 (BosHR) 的菌株分别为18.8%和9.8%。抗药性分子机制研究表明:BenHR菌株中β-tubulin的第198位氨基酸由Glu (E) 突变成了Ala (A);BosHR菌株中Sdh B的第277位氨基酸由His (H) 突变成了Tyr (Y),但BosLR的抗性机制还需进一步研究。研究结果表明,浙江省西 (甜) 瓜蔓枯病菌对苯并咪唑类杀菌剂甲基硫菌灵的抗性已十分严重,尽管大多数菌株对啶酰菌胺仍表现敏感,但在一些地区已存在高水平抗性菌株,应在加强抗药性监测的同时,注意SDHIs类杀菌剂的科学使用。     

Genotyping of benzimidazole-resistant and dicarboximide-resistant mutations in Botrytis cinerea using real-time polymerase chain reaction assays

Botrytis cinerea, an economically important gray mold pathogen, frequently exhibits multiple fungicide resistance. A fluorescence resonance energy transfer-based real-time polymerase chain reaction assay has been developed to detect benzimidazole- and dicarboximide-resistant mutations. Three benzimidazole-resistant mutations-(198)Glu to Ala (E198A), F200Y, and E198K-in beta-tubulin BenA were detected using a single set of fluorescence-labeled sensor and anchor probes by melting curve analysis. Similarly, three dicarboximide-resistant mutations-I365S, V368F plus Q369H, and Q369P-in the histidine kinase BcOS1 were successfully distinguished. Unassigned melting profiles in BenA genotyping assay resulted in the identification of a new benzimidazole-resistant BenA E198V mutation. This mutation conferred resistance to carbendazim as do E198A and E198K mutations. The isolates with BenA E198V mutation showed a negative cross-resistance to diethofencarb, but to a lesser extent than the E198A mutants. A survey of 210 B. cinerea field isolates revealed that most of benzimidazole-resistant isolates possessed the E198V or E198A mutation in the BenA gene, and the I365S mutation in the BcOS1 gene was also frequently observed in Japanese isolates. However, benzimidazole-resistant isolates with BenA F200Y or E198K mutations, which confer the diethofencarb-insensitive phenotype, were rare. Our BenA and BcOS1 genotyping is a rapid and reliable method that is suitable for monitoring the fungicide-resistant field population.     

Simultaneous detection of benzimidazole-resistant strains of Fusarium head blight using the loop-mediated isothermal amplification-fluorescent loop primer method

The loop-mediated isothermal amplification (LAMP)-fluorescent loop primer (FLP) method detects genetic polymorphisms by using a LAMP amplicon and measuring the peak temperatures of fluorescence resonance energy transfer between an FLP and a quencher probe, which is specifically hybridized to a sequence including a single nucleotide polymorphism (SNP). In the present study, the LAMP-FLP method was used to detect mutant genotypes F167Y, E198Q, and F200Y in the β2-tubulin gene region of causal pathogens of Fusarium head blight of wheat that result in methyl benzimidazole carbamate (MBC) resistance, proving its usefulness for monitoring strains with SNPs in target regions of MBC resistance.     

Characterization of fludioxonil-resistant and pyrimethanil-resistant phenotypes of Penicillium expansum from apple

Penicillium expansum is the primary cause of blue mold, a major postharvest disease of apple. Fludioxonil and pyrimethanil are two newly registered postharvest fungicides for pome fruit in the United States. To evaluate the potential risk of resistance development in P. expansum to the new postharvest fungicides, one isolate of each of thiabendazole-resistant (TBZ-R) and -sensitive (TBZ-S) P. expansum was exposed to UV radiation to generate fungicide-resistant mutants. Four fludioxonil highly-resistant mutants (EC(50) > 1,000 microg/ml) and four pyrimethanil-resistant mutants (EC(50) > 10 microg/ml) were tested for sensitivities to thiabendazole, fludioxonil, and pyrimethanil, and fitness parameters including mycelial growth, sporulation on potato dextrose agar (PDA), sensitivity to osmotic stress, and pathogenicity and sporulation on apple fruit. The stability of resistance of the mutants was tested on PDA and apple fruit. Efficacy of the three fungicides to control blue mold incited by the mutants was evaluated on apple fruit. Six fungicide-resistant phenotypes were identified among the parental wild-type isolates and their mutants based upon their resistance levels. All four fludioxonil highly-resistant mutants were sensitive to pyrimethanil and retained the same phenotypes of resistance to TBZ as the parental isolates. All four pyrimethanil-resistant mutants had a low level of resistance to fludioxonil with a resistance factor >15. The two pyrimethanil-resistant mutants derived from a TBZ-S isolate became resistant to TBZ at 5 microg/ml. After 20 successive generations on PDA and four generations on apple fruit, the mutants retained the same phenotypes as the original generations. All mutants were pathogenic on apple fruit at both 0 and 20 degrees C, but fludioxonil highly-resistant mutants were less virulent and produced fewer conidia on apple fruit than pyrimethanil-resistant mutants and their parental wild-type isolates. Compared with the parental isolates, all four fludioxonil highly-resistant mutants had an increased sensitivity to osmotic stress on PDA amended with NaCl, while the pyrimethanil-resistant mutants did not. Pyrimethanil was effective against blue mold caused by fludioxonil-resistant mutants at both 0 and 20 degrees C. Pyrimethanil and fludioxonil reduced blue mold incited by pyrimethanil-resistant mutants during 12-week storage at 0 degrees C but were not effective at 20 degrees C. TBZ was not effective against pyrimethanil-resistant mutants derived from TBZ-S wild-type isolates at room temperature but provided some control at 0 degrees C. The results indicate that: (i) a fitness cost was associated with fludioxonil highly resistant mutants of P. expansum in both saprophytic and pathogenic phases of the pathogen but not pyrimethanil-resistant mutants; (ii) pyrimethanil possessed a higher risk than fludioxonil in the development of resistance in P. expansum; and (iii) triple resistance to the three apple-postharvest fungicides could emerge and become a practical problem if resistance to pyrimethanil develops in P. expansum populations.     

Detection and characterization of benzimidazole resistance of <Emphasis Type="Italic">Botrytis cinerea</Emphasis> in greenhouse vegetables

From 2003 to 2006, a total of 426 single-conidial isolates of B. cinerea collected from greenhouse vegetables in China were characterized for resistance to benzimidazole fungicides and diethofencarb according to inhibition of mycelial growth. Rapid development of double-resistance to benzimidazoles and diethofencarb was observed. Three types of benzimidazole-resistant isolates, Ben R1, Ben R2 and Ben R3 were detected. A new phenotype, Ben R3, which showed low level of resistance to benzimidazole fungicides and resistance to diethofencarb, was detected with frequencies of 6.8%, 10.0%, 13.2% and 12.4% from 2003 to 2006, respectively. Further studies indicated that Ben R3 was caused by a point mutation from GAG in sensitive(S) isolates to GTG at codon 198 in the β-tubulin gene, predicted to cause a change from glutamic acid to valine. Ben R3 isolates had comparable growth, sporulation and pathogenicity ability as isolates of other phenotypes but were more sensitive at lower temperatures.     

樱桃采后灰霉病菌对甲基硫菌灵、乙霉威和腐霉利的抗性

本文采用单孢分离法对四川汉源和山东烟台等地采集的樱桃果实进行了采后灰霉病的病原菌分离和鉴定;采用区分剂量法分别测定了菌株对苯并咪唑类杀菌剂甲基硫菌灵、乙霉威和二甲酰亚胺类杀菌剂腐霉利的敏感性,并进一步分析了抗药性菌株的分子机制。结果表明,分离得到的54株樱桃采后灰霉病菌均为灰葡萄孢Botrytis cinerea,对甲基硫菌灵的总抗性频率高达79.6%,其中甲基硫菌灵抗性-乙霉威敏感(BEN R1)菌株频率为25.9%;甲基硫菌灵-乙霉威双重抗性菌株(BEN R2)频率为53.7%;检测到腐霉利抗性菌株(DCF R) 9株,频率为16.7%。甲基硫菌灵抗性菌株在β-tubulin基因上的突变共有2种类型：BEN R1抗性菌株中,第198位密码子发生点突变(GAG→GCG),编码氨基酸由Glu (E)突变成缬氨酸Ala (A);在BEN R2抗性菌株中,第198位密码子发生点突变(GAG→GTG),编码氨基酸由Glu (E)突变成缬氨酸Val (V)。DCF R菌株在BcOS1的第365位密码子由ATC突变成AAC或AGC,导致编码的氨基酸由异亮氨酸Ile (I)突变成天冬酰胺Asn (...     

Biological and molecular characterization of field isolates of <Emphasis Type="Italic">Alternaria alternata</Emphasis> with single or double resistance to respiratory complex II and III inhibitors

Field isolates of Alternaria alternata collected from tomato processors were characterized for sensitivity to respiration inhibitors using in vitro mycelial growth assays. Pyraclostrobin (QoI), boscalid, fluopyram and isopyrazam (SDHIs) mean EC₅₀ values were 0.32, 1.43, 2.21, and 3.53 μg/ml respectively. Of the 42 isolates, 36 were sensitive to all respiration inhibiting fungicides tested whereas three isolates were less sensitive to boscalid, one to pyraclostrobin and two were simultaneously resistant to both inhibitors and isopyrazam. Correlation analysis between fungicide sensitivities revealed a positive cross-resistance between pyraclostrobin and tebuconazole, and between cyprodinil and mancozeb. There was no cross-resistance between QoIs, SHDIs or any other mode of action. Sequencing of the QoI and SDHI targets revealed the G143A cytochrome b resistance mutation in all pyraclostrobin-resistant isolates while analysis of the succinate dehydrogenase coding gene revealed point mutations in two of three of the gene subunits analyzed in boscalid-resistant isolates. Specifically, two isolates carried the H277Y and three the H133Q resistance mutations located in the sdhB and sdhD subunits of the respiration complex II, respectively. Isolates bearing the H277Y mutation also carried the G143A cytochrome b resistance mutation. Boscalid and pyraclostrobin-resistant isolates exhibited greater pathogenicity and sporulation compared to sensitive isolates, respectively. Isolates with cross-resistance exhibited greater pathogenicity and sporulation but slower mycelial growth compared to sensitive isolates. This is the first report of field isolates of A. alternata with single or double resistance to QoIs and SDHIs in Greece and should be considered in planning and implementing effective anti-resistance strategies.     

浙江衢州地区柑橘绿霉病菌对抑霉唑和多菌灵的抗性水平及其分子机制

研究了采自浙江衢州地区,包括柯城区、衢江区和开化县12个贮藏库的70个柑橘绿霉病菌Penicillium digitatum菌株对抑霉唑和多菌灵的抗性频率、抗性水平及其抗性分子机制。结果表明:柯城区和衢江区的抑霉唑抗性菌株(最低抑制浓度MIC≥0.5 μg/mL)的比例分别为77.1%和62.5%,两地抗性菌株的平均EC50值分别为2.07±1.04 μg/mL和2.35±0.73 μg/mL,分别是当地敏感菌株EC50值的41.4和47.0倍;而采自开化县的菌株均对抑霉唑敏感(MIC≤0.1 μg/mL),平均EC50值为0.04±0.02 μg/mL。柯城区和衢江区的多菌灵抗性菌株(MIC≥10 μg/mL) 的比例分别为54.3%和54.2%,而开化县的抗性菌株比例仅为9.1%。即来自柯城和衢江两个柑橘主产区的绿霉病菌群体对抑霉唑和多菌灵的抗性频率均远高于非柑橘主产区的开化县群体,说明抗药性群体的形成与药剂使用历史有关。进一步研究发现,衢州地区柑橘绿霉病菌对抑霉唑的抗性均属于IMZ-R3型,即与抑霉唑靶标基因 CYP51B 启动子区的插入突变有关,而对多菌灵的抗性则与 β-微管蛋白基因的992位核苷酸点突变(T→A)导致对应的200位点的氨基酸突变(F→Y)有关。     

Molecular characterization of pyraclostrobin resistance and structural diversity of the cytochrome b gene in Botrytis cinerea from apple

Botrytis cinerea isolates obtained from apple orchards were screened for resistance to the quinone outside inhibitor (QoI) pyraclostrobin. Of the 220 isolates tested, 43 (19.5%) were resistant to pyraclostrobin. Analysis of partial sequences of the cytochrome b gene (cyt b) in five pyraclostrobin-resistant (PR) and five pyraclostrobin-sensitive (PS) isolates showed that PR isolates harbored the point mutation leading to the substitution of glycine by alanine at codon position 143 in cyt b (G143A). Two pairs of allele-specific primers were designed based on this point mutation, and allele-specific polymerase chain reaction analysis with these primers showed that all 73 PR isolates (including 30 collected from decayed apple fruit) harbored the G143A mutation but PS isolates did not. Six pairs of primers were designed to analyze the presence of various introns in cyt b. There were six types (I to VI) of cyt b present in 247 isolates of B. cinerea collected from various apple-production areas in Washington State. Of the 247 isolates, 23 had type I cyt b containing all four introns (Bcbi-67/68, Bcbi-131/132, Bcbi-143/144, and Bcbi-164), 176 had type II cyt b containing three introns (Bcbi-67/68, Bcbi-131/132, and Bcbi-164), six had type III cyt b containing two introns (Bcbi-67/68 and Bcbi-131/132), one had type IV cyt b containing two introns (Bcbi-131/132 and Bcbi-164), one had type V cyt b containing only the Bcbi-131/132 intron, and 40 had type VI cyt b containing no introns. This is the first report of types III to VI cyt b present in B. cinerea. All 73 PR isolates did not carry the Bcbi-143/144 intron in cyt b. Of the 247 isolates tested, >90% did not carry the Bcbi-143/144 intron in cyt b, suggesting that B. cinerea populations from apple pose a high inherent risk for the development of resistance to QoIs because the presence of this intron in cyt b prevents the occurrence of G143A-mediated resistance. Analysis of genetic background based on three microsatellite primers showed that PR isolates originated from different lineages, and there was no correlation between cyt b types (I, II, and III) and the genetic background of the isolates; however, isolates carrying type VI cyt b might originate from the same lineage.     

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.