Department of Plant Pathology, Washington State University, Tree Fruit Research and Extension Center, Wenatchee, WA 98801, USA.
Phytopathology. 2012 Mar;102(3):315-22. doi: 10.1094/PHYTO-08-11-0234.
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.
从苹果园中分离得到的灰葡萄孢菌被筛选出对醌外抑制剂（QoI）吡唑醚菌酯的抗性。在所测试的 220 个分离物中，有 43 个（19.5％）对吡唑醚菌酯具有抗性。对 5 个吡唑醚菌酯抗性（PR）和 5 个吡唑醚菌酯敏感（PS）分离物的细胞色素 b 基因（cyt b）部分序列分析表明，PR 分离物携带导致密码子 143 处甘氨酸被丙氨酸取代的点突变（G143A）。基于该点突变设计了两对等位基因特异性引物，用这些引物进行的等位基因特异性聚合酶链反应分析表明，所有 73 个 PR 分离物（包括 30 个从腐烂苹果果实中分离的）均携带 G143A 突变，但 PS 分离物没有。设计了六对引物来分析 cyt b 中各种内含子的存在。在从华盛顿州各个苹果生产区采集的 247 个灰葡萄孢菌分离物中存在六种类型（I 至 VI）的 cyt b。在 247 个分离物中，有 23 个具有包含所有四个内含子（Bcbi-67/68、Bcbi-131/132、Bcbi-143/144 和 Bcbi-164）的 I 型 cyt b，176 个具有包含三个内含子（Bcbi-67/68、Bcbi-131/132 和 Bcbi-164）的 II 型 cyt b，6 个具有包含两个内含子（Bcbi-67/68 和 Bcbi-131/132）的 III 型 cyt b，1 个具有包含两个内含子（Bcbi-131/132 和 Bcbi-164）的 IV 型 cyt b，1 个具有仅包含 Bcbi-131/132 内含子的 V 型 cyt b，40 个具有不包含内含子的 VI 型 cyt b。这是首次报道灰葡萄孢菌中存在 III 至 VI 型 cyt b。所有 73 个 PR 分离物均不携带 cyt b 中的 Bcbi-143/144 内含子。在所测试的 247 个分离物中，>90％的分离物不携带 cyt b 中的 Bcbi-143/144 内含子，这表明来自苹果的灰葡萄孢菌种群对 QoI 产生抗性的固有风险很高，因为 cyt b 中存在该内含子可防止发生 G143A 介导的抗性。基于三个微卫星引物的遗传背景分析表明，PR 分离物源自不同的谱系，并且 cyt b 类型（I、II 和 III）与分离物的遗传背景之间没有相关性；然而，携带 VI 型 cyt b 的分离物可能源自同一谱系。
