| 两种葡萄溃疡病菌双重PCR检测方法的建立与应用 |
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| 引用本文：张玮,李兴红,郭飞飞,刘梅,黄金宝,燕继晔.两种葡萄溃疡病菌双重PCR检测方法的建立与应用.植物保护学报,2017,44(4):636-642 |
| DOI：10.13802/j.cnki.zwbhxb.2017.2015235 |
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| 中文摘要: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。 |
| 中文关键词:葡萄 葡萄座腔菌 小新壳梭孢 双重PCR检测 |
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| Establishment and application of duplex PCR assay for grape canker pathogens Botryosphaeria dothidea and Neofusicoccum parvum |
| Author Name | Affiliation | E-mail | | Zhang Wei | Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China
College of Pant Protection, China Agricultural University, Beijing 100193, China | | | Li Xinghong | Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China | | | Guo Feifei | College of Pant Protection, China Agricultural University, Beijing 100193, China | | | Liu Mei | Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China | | | Huang Jinbao | Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China | | | Yan Jiye | Institute of Plant and Environment Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing 100097, China | jiyeyan@vip.163.com |
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| 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-1α and β-tubulin sequences of six main species of grapevine Botryosphaeria dieback fungi reported in China. A duplex PCR assay was established and applied on the grape diseased samples collected from vineyard. A 324 bp band was amplified specifically from the genomic DNA of B. dothidea isolates, while the size of specific band was 212 bp in N. parvum isolates. The detection sensitivity for both B.d-F/B.d-R and N.p-F/N.p-R was 10 pg. In the final duplex PCR system, the optimal usage of Taq DNA polymerase was 0.05 U/μL, the optimal concentration for both B.d-F/B. d-R and N.p-F/N.p-R primers were 0.2 μmol/L, and the optimal annealing temperature and time were 58℃ and 30 s. The detection results obtained by the duplex PCR method were consistent with the traditional detection method in vivo on naturally infected grape disease samples collected from vineyard. The duplex PCR method could detect the B. dothidea and N. parvum simultaneously and could be helpfully used for the rapid and sensitive detection of these two fungi in nursery propagating materials. |
| keywords:grape Botryosphaeria dothidea Neofusicoccum parvum duplex PCR assay |
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