双基因联合PCR检测水稻叶鞘网斑病菌

[目的]建立水稻叶鞘网斑病的快速PCR检测体系,为该病害的早期诊断提供技术支持。[方法]以水稻叶鞘网斑病病原菌帚梗柱孢霉(Cylindrocladium scoparium Morgan)DNA为模板,分别以factor 1-α(tef1)和β-tubulin gene两个保守性极强的特定区域序列作为检测靶标,针对C.scoparium设计了EF-S-4/EF-A-4和BT-S-9/BT-A-9两对特异性引物,建立了水稻叶鞘网斑病的双基因联合PCR检测技术。[结果]该体系的最佳退火温度为58 ℃,DNA灵敏度检测限达到550 fg？μL-1,病原菌可扩增出大小分别为272 bp和157 bp的2条条带,而其他对照组均未有扩增条带。[结论]田间时效检测结果显示:该体系能准确检测出不同发病地区的水稻叶鞘网斑病病原菌,为农业生产早期诊断水稻叶鞘网斑病害提供技术支持。
[Objectives]To establish a rapid PCR technique for the detection of rice leaf sheath spot disease. [Methods]The method of double gene-jointed rapid PCR technique was established to detect rice leaf sheath spot disease caused by the pathogen Cylindrocladium scoparium Morgan, which was based on both factor 1-α(tef1)gene and β-tubulin gene and was established with the total DNA as templates. Two pairs of specificity primers(EF-S-4/EF-A-4, BT-S-9/BT-A-9)for C.scoparium on their factor 1-α(tef1)and β-tubulin gene were designed, respectively. [Results]The optimized conditions were an annealing temperature at 58 ℃, the minimum amount of DNA could be detected was as low as 550 fg？μL-1, and two bright strips of 272 bp and 157 bp were amplified only on the pathogen C.scoparium, while there was no amplification product found in positive controls, the rest Cylindrocladium genus isolates, and other tested ones. [Conclusions]The wild field effectiveness showed that the rapid PCR technique could accurately detect the diseased tissue of Oryza sativa, which fits entirely in the field test, and which provide technical support to the agricultural industry of Oryza sativa in the early diagnosis on rice leaf sheath spot