Department of Biological Chemistry and Crop Protection, Rothamsted Research, Harpenden, Hertfordshire, UK.
Pest Manag Sci. 2013 Sep;69(9):1040-8. doi: 10.1002/ps.3585. Epub 2013 Jul 9.
Methyl benzimidazole carbamate (MBC) fungicides are used to control the oilseed rape pathogen Pyrenopeziza brassicae. Resistance to MBCs has been reported in P. brassicae, but the molecular mechanism(s) associated with reductions in sensitivity have not been verified in this species. Elucidation of the genetic changes responsible for resistance, hypothesised to be target-site mutations in β-tubulin, will enable resistance diagnostics and thereby inform resistance management strategies.
P. brassicae isolates were classified as sensitive, moderately resistant or resistant to MBCs. Crossing P. brassicae isolates of different MBC sensitivities indicated that resistance was conferred by a single gene. The MBC-target encoding gene β-tubulin was cloned and sequenced. Reduced MBC sensitivity of field isolates correlated with β-tubulin amino acid substitutions L240F and E198A. The highest level of MBC resistance was measured for isolates carrying E198A. Negative cross-resistance between MBCs and the fungicides diethofencarb and zoxamide was only measured in E198A isolates. PCR-RFLP was used to screen isolates for the presence of L240F and E198A. The substitutions E198G and F200Y were also detected in DNA samples from P. brassicae populations after cloning and sequencing of PCR products. The frequencies of L240F and E198A in different P. brassicae populations were quantified by pyrosequencing. There were no differences in the frequencies of these alleles between P. brassicae populations sampled from different locations or after fungicide treatment regimes.
The molecular mechanisms affecting sensitivity to MBCs in P. brassicae have been identified. Pyrosequencing assays are a powerful tool for quantifying fungicide-resistant alleles in pathogen populations.
甲基苯并咪唑氨基甲酸酯（MBC）杀菌剂用于防治油菜病原菌核盘菌。已报道核盘菌对 MBC 产生了抗性，但在该物种中尚未验证与敏感性降低相关的分子机制。阐明导致抗性的遗传变化，假设为β-微管蛋白的靶标突变，将使抗性诊断成为可能，并为抗性管理策略提供信息。
将核盘菌分离株分为对 MBC 敏感、中度抗性和抗性。不同 MBC 敏感性的核盘菌分离株的杂交表明，抗性由单个基因决定。克隆并测序了 MBC 靶标编码基因β-微管蛋白。田间分离株对 MBC 的敏感性降低与β-微管蛋白氨基酸取代 L240F 和 E198A 相关。携带 E198A 的分离株表现出最高水平的 MBC 抗性。仅在 E198A 分离株中测量到 MBC 和杀菌剂 diethofencarb 和 zoxamide 之间的负交叉抗性。PCR-RFLP 用于筛选分离株中是否存在 L240F 和 E198A。在对 PCR 产物进行克隆和测序后，还在核盘菌种群的 DNA 样本中检测到 E198G 和 F200Y 取代。通过焦磷酸测序定量不同核盘菌种群中 L240F 和 E198A 的频率。在不同地点或在杀菌剂处理方案后采样的核盘菌种群中，这些等位基因的频率没有差异。
确定了影响核盘菌对 MBC 敏感性的分子机制。焦磷酸测序测定是定量病原体种群中抗杀菌剂等位基因的有力工具。
