灰葡萄孢菌对二甲酰亚胺类杀菌剂抗性的AS-PCR快速检测技术  

Rapid Detection of the Resistance of Botrytis cinerea to Dicarboximide Fungicides by AS-PCR

作　　者：刘梦晴 屠紫娟 余洋[1,2] 杨宇衡[1,2] 方安菲 田斌年 王静[1,2] 毕朝位 LIU Mengqing;TU Zijuan;YU Yang;YANG Yuheng;FANG Anfei;TIAN Binnian;WANG Jing;BI Chaowei(Yibin Academy of Southwest University,Yibin Sichuan 644000,China;College of Plant Protection,Southwest University,Chongqing 400715,China)

机构地区：[1]西南大学宜宾研究院,四川宜宾644000 [2]西南大学植物保护学院,重庆400715

出　　处：《西南大学学报(自然科学版)》2025年第4期101-112,共12页Journal of Southwest University(Natural Science Edition)

基　　金：国家重点研发计划项目(2022YFD1400901);四川省宜宾市双城协议保障科研经费项目(XNDX2022020011);国家自然科学基金项目(31871990)。

摘　　要：灰霉病是一种由灰葡萄孢菌引起、果蔬上常见的真菌病害,其发生对农业生产造成巨大损失。由于二甲酰亚胺类杀菌剂(DCFs)的广泛使用,灰葡萄孢菌对其已经产生了抗药性,为快速检测灰葡萄孢菌对DCFs抗性,基于灰葡萄孢菌BOS1基因的点突变I365S和I365N,建立了一种灰葡萄孢菌对DCFs抗性的等位基因特异性PCR(AS-PCR)快速分子检测方法。首先,以BOS1基因为靶序列设计特异性引物,然后对PCR体系的退火温度、内参引物与特异性引物浓度配比进行优化,并对引物灵敏度和特异性进行评价,最后利用快速抽提法提取田间病原菌DNA进行抗药性检测。结果表明:正向特异性引物I365S-7F、I365S-8F和I365N-8F分别与引物I365-R结合组成的引物对可以检测出I365S和I365N抗性菌株,最适退火温度分别为57℃、58℃和58℃;内参引物与特异性引物浓度比例为1∶2,其灵敏度分别为7 ng/μL、70 ng/μL和70 ng/μL,并具有特异性;利用NaOH裂解法快速提取田间菌株DNA进行抗药性检测,其结果与测序结果一致。以上结果说明该AS-PCR技术具有特异性强、操作简单等优势,可为田间灰葡萄孢菌对DCFs类抗性菌株的快速检测提供一种较为可行的方法。Gray mold is a common fungal disease in fruits and vegetables caused by Botrytis cinerea,which causes great losses to agricultural production.Due to the widespread use of dicarboximide fungicides(DCFs),B.cinerea has developed resistance to this fungicide.In order to rapidly detect resistance of B.cinerea to DCFs,we developed an allele-specific PCR(AS-PCR)rapid molecular detection method for the resistance of B.cinerea to DCFs based on the point mutations I365S and I365N of BOS1 gene.First,we designed specific primers with BOS1 gene as the target sequence,then optimized the base mismatch,annealing temperature,the concentration ratio of internal reference primers and specific primers of PCR system,and evaluated the specificity and sensitivity of primers.Finally,we used rapid extraction method to extract DNA from field samples for detection and application.The results showed that the primer pairs composed of the forward specific primers I365S-7F,I365S-8F and I365N-8F respectively with reverse primer I365-R could distinguish the resistant and sensitive strains of I365S and I365N mutation,and the optimal annealing temperatures was 57℃,58℃and 58℃,respectively.The concentration ratio of internal reference primers and specific primers was 1∶2,the sensitivities were 7 ng/μL,70 ng/μL and 70 ng/μL,respectively,and the amplifications were specific.The results of PCR detection with DNA extracted from field strains by NaOH lysis method were consistent with the sequencing results.In conclusion,AS-PCR technology has the advantages of strong specificity and simple operation,which provides a feasible and rapid detection method for monitoring the resistance of gray mold to DCFs kind of fungicides.

关 键 词：灰葡萄孢菌 二甲酰亚胺类杀菌剂 等位基因特异性PCR 抗药性 

分 类 号：S436.631[农业科学—农业昆虫与害虫防治]