Department of Plant Pathology, Washington State University, Tree Fruit Research and Extension Center, Wenatchee, WA 98801, USA.
Phytopathology. 2011 Aug;101(8):986-95. doi: 10.1094/PHYTO-01-11-0016.
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.
从苹果园中分离到的葡萄孢菌对 boscalid 的抗性进行了筛选。根据抑制真菌生长 50%的浓度与对照相比，将 Boscalid 抗性（BosR）分离物分为四种表型。在测试的 220 个分离物中，有 42 个对 boscalid 具有抗性，其抗性表型从低到非常高。boscalid 和 carboxin 之间存在交叉抗性。对来自 13 个 BosR 和 9 个 boscalid 敏感（BosS）分离物的葡萄孢菌（BcSdhB）的铁硫亚单位琥珀酸脱氢酶基因的部分序列分析表明，导致密码子 272 处组氨酸到酪氨酸（H272Y）或精氨酸（H272R）的氨基酸取代的 BcSdhB 点突变与 boscalid 抗性相关。对 66 个 BosR 分离物（包括从腐烂苹果果实中获得的 24 个额外分离物）的等位基因特异性聚合酶链反应（PCR）分析表明，19 个携带点突变 H272Y，46 个携带点突变 H272R，但 1 个 BosR 分离物没有扩增产物。对该分离物的 BcSdhB 序列分析显示，在密码子 225 处发生了不同的点突变，导致脯氨酸（P）被苯丙氨酸（F）取代（P225F）。结果表明，在来自苹果的田间 BosR 分离物中，BcSdhB 中的 H272R/Y 是突变体的主要基因型。开发了一种多重等位基因特异性 PCR 检测方法，用于在单个 PCR 扩增中检测 H272R/Y 点突变。在携带 H272R 或 H272Y 突变的分离物中，boscalid 抗性的水平从低到非常高，表明在 BosR 分离物中，不同的 BosR 表型（抗性水平）与 BcSdhB 中的特定类型点突变（H272R 与 H272Y）无关。基于三个微卫星标记对 39 个 BosR 和 56 个 BosS 分离物的遗传关系进行分析表明，39 个 BosR 分离物和 30 个 BosS 分离物分为两组，第三组仅由 BosS 分离物组成，表明葡萄孢菌对 boscalid 的抗性的发展可能并非完全随机，抗性群体可能来自特定的遗传群体。
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