两种葡萄溃疡病菌双重PCR检测方法建立与应用.PDFVIP

植物保护学报Journal of Plant Protection ，2017 ，44 （4 ）：636-642 DOI ：10.13802/ki.zwbhxb.2017.2015235 两种葡萄溃疡病菌双重PCR 检测方法的建立与应用 1,2 1 2 1 1 1* 张 玮 李兴红 郭飞飞 刘 梅 黄金宝 燕继晔 （1. 北京市农林科学院植物保护环境保护研究所，北京 100097；2. 中国农业大学植物保护学院，北京 100193） 摘要：为快速准确地鉴定出2 种不同的葡萄溃疡病菌——葡萄座腔菌Botryosphaeria dothidea 和小 新壳梭孢Neofusicoccum parvum ，根据GenBank 中已报道的引起我国葡萄溃疡病的6 个主要种的延 伸因子- 1序列及β-微管蛋白序列分别设计了葡萄座腔菌和小新壳梭孢的特异性引物B.d-F/B.d-R 及N.p-F/N.p-R ，建立了双重PCR 检测方法，并对田间病样进行检测。结果表明，引物B.d-F/B.d-R 和N.p-F/N.p-R 可以分别在葡萄座腔菌和小新壳梭孢中扩增到324 bp 和212 bp 的特异性条带，检测 灵敏度均为10 pg 。双重PCR 检测体系中Taq DNA 聚合酶的最佳用量为0.05 U/μL ，引物B.d-F/B.d-R 和N.p-F/N.p-R 的最佳终浓度均为0.2 μmol/L ，最适退火温度和退火时间分别为58℃和30 s。对田 间葡萄病样的检测结果与室内病样常规病原菌分离结果一致。表明该双重PCR 检测方法能够同 时检测葡萄座腔菌和小新壳梭孢，有助于快速、灵敏地检测葡萄苗木的带菌情况。 关键词：葡萄；葡萄座腔菌；小新壳梭孢；双重PCR 检测 Establishment and application of duplex PCR assay for grape canker pathogens Botryosphaeria dothidea and Neofusicoccum parvum Zhang Wei1,2 Li Xinghong1 Guo Feifei2 Liu Mei1 Huang Jinbao1 Yan Jiye1* （1. Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China; 2. College of Pant Protection, China Agricultural University, Beijing 100193, China） Abstract: To establish a rapid and accurate molecular detection method for Botryosphaeria dothidea and Neofusicoccum parvum, which were two main pathogens of grapevine Botryosphaeria dieback in China, the specific primers B.d-F/B.d-R for B. dothidea and N.p-F/N.p-R for N. parvum were designed based on differences among the EF- 1αand β-tubulin sequences of six main species of grapevine Botryo- sphaeria dieback fungi reported in China. A duplex PCR assay was established and applied on the grape diseased samples collected from vineyar