Molecular characterization and diagnosis of QoI resistance in cucumber and eggplant fungal pathogens
Molecular characterization and diagnosis of QoI resistance in cucumber and eggplant fungal pathogens
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DOI：
10.1094/phyto-97-11-1458
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发表时间：
2007-11-01
期刊：
影响因子：
3.2
通讯作者：
Hasama, W.
中科院分区：
文献类型：
--
作者：
Ishii, H.;Yano, K.;Hasama, W.
利用黄瓜棒孢叶斑病（Corynespora acumicola）和茄子叶霉病（Mycovellosiella nattrassii）菌株研究了QOI杀菌剂抗性的分子机制。在这两种病原体中，在编码杀真菌剂靶向蛋白的细胞色素B基因中检测到143位从甘氨酸突变为丙氨酸（G143 A）。此外，氨基酸位置143处的核苷酸序列从敏感（野生型）中的GGT或GGA转化为抗性（突变型）分离株中的GCT或GCA。聚合酶链反应-限制性片段长度多态性方法通常用于QoI耐药监测，成功地采用，导致从耐药分离株扩增的基因片段被切割的限制性内切酶Ital。然而，异质性（野生型和突变等位基因的共存）时，耐药菌株的C。M. tagicola，M. nattrassii和Colletotrichungloeosporioides（草莓炭疽病真菌）在存在或不存在QoI杀真菌剂的情况下传代培养。黄瓜白粉病和霜霉病菌株的QOI抗性持续了几年后，在实验室和商业温室条件下的选择压力的杀菌剂施加的去除。细胞色素B基因中突变序列的比例在病原体群体中随时间的推移而降低。当白粉病和霜霉病的种群中含有10%的抗性菌株时，全剂量嘧菌酯的保护效力降低。使用FMBIO，荧光生物成像分析仪，突变等位基因从QoI耐药菌株可以检测到在1%的水平，而溴化乙锭染色凝胶的检测灵敏度低10倍。
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 (wildtype) 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 Ital. However, heteroplasmy (the coexistence of wild-type and mutated alleles) was found when the resistant isolates of C. cassiicola, M. nattrassii, and Colletotrichunt 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 bioimaging 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 10 times lower.