Phytopathology. 2007 Nov;97(11):1458-66. doi: 10.1094/PHYTO-97-11-1458.
ABSTRACT The molecular mechanism of QoI fungicide resistance was studied using isolates of cucumber Corynespora leaf spot fungus (Corynespora cassiicola) and the eggplant leaf mold (Mycovellosiella nattrassii). In both pathogens, a mutation at position 143 from glycine to alanine (G143A) was detected in the cytochrome b gene that encodes for the fungicide-targeted protein. Moreover, the nucleotide sequence at amino acid position 143 was converted from GGT or GGA in sensitive (wild-type) to GCT or GCA in resistant (mutant-type) isolates. The methods of polymerase chain reaction restriction fragment length polymorphism commonly used for QoI resistance monitoring were employed successfully, leading to the amplified gene fragment from resistant isolates being cut with the restriction enzyme ItaI. However, heteroplasmy (the coexistence of wild-type and mutated alleles) was found when the resistant isolates of C. cassiicola, M. nattrassii, and Colletotrichum gloeosporioides (strawberry anthracnose fungus) were subcultured in the presence or absence of QoI fungicides. QoI resistance of cucumber powdery and downy mildew isolates persisted for a few years following the removal of the selection pressure imposed by the fungicide under both laboratory and commercial greenhouse conditions. The proportion of mutated sequences in cytochrome b gene decreased over time in the pathogen population. The protective efficacy of the full dose of azoxystrobin decreased when the populations of powdery and downy mildews contained resistant isolates at 10%. Using FMBIO, a fluorescence bio-imaging analyzer, the mutant allele from the QoI-resistant isolates could be detected at the level of 1%, whereas the detection sensitivity of ethidium-bromide-stained gels was approximately 10 times lower.
摘要 本研究以黄瓜炭疽病菌(Corynespora cassiicola)和茄子叶霉菌(Mycovellosiella nattrassii)分离株为研究对象,探讨了 QoI 类杀菌剂抗性的分子机制。在这两种病原菌中,均检测到编码杀菌剂靶标蛋白的细胞色素 b 基因第 143 位由甘氨酸突变为丙氨酸(G143A)。此外,在敏感(野生型)分离株中,氨基酸位置 143 的核苷酸序列为 GGT 或 GGA,而在抗性(突变型)分离株中则转换为 GCT 或 GCA。用于 QoI 抗性监测的聚合酶链反应限制性片段长度多态性方法成功应用,导致抗性分离株的扩增基因片段被限制酶 ItaI 切割。然而,当 C. cassiicola、M. nattrassii 和 Colletotrichum gloeosporioides(草莓炭疽菌)的抗性分离株在有或没有 QoI 杀菌剂存在的情况下进行继代培养时,发现了异质体(野生型和突变型等位基因共存)。在实验室和商业温室条件下,去除杀菌剂施加的选择压力后,黄瓜白粉病和霜霉病分离株的 QoI 抗性仍持续了几年。随着时间的推移,病原菌种群中细胞色素 b 基因的突变序列比例逐渐降低。当白粉病和霜霉病种群中含有 10%抗性分离株时,全剂量肟菌酯的保护效果降低。使用 FMBIO 荧光生物成像分析仪,可以检测到 QoI 抗性分离株的突变等位基因水平为 1%,而溴化乙锭染色凝胶的检测灵敏度约低 10 倍。