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菠菜枯萎病是由致病性镰刀菌(F.o.f.sp.spinaciae)引起的,是菠菜生产中的重要病害之一。利用常规方法鉴定菠菜枯萎病病原菌需耗费大量时间,并且很难得到正确的结论。随机扩增多态性DNA序列标签(Randomly amplifiedpolymorphic DNA-sequence tagged sites,RAPD-STS)为病原菌鉴定提供了一种有效方法。本研究通过对供试菌株的RAPD分析,克隆出了1个菠菜枯萎病病原菌的特异片段(GenBank登录号:AY337463)。根据测序结果设计了1对菠菜枯萎病病原菌的特异引物,并利用常规PCR和实时定量PCR(real-time PCR)2种方法对病原菌进行了鉴定,并对2种方法的敏感性进行了比较。结果表明,2种PCR方法都可以鉴定菠菜枯萎病病原菌(F.o.f.sp.spinaciae),但二者对病原菌DNA敏感程度不同,常规PCR检测的最低DNA量100 pg,而实时定量PCR检测的最低DNA量是1 pg。同时,实时定量PCR还可以对病原菌DNA进行定量分析,并依此估算病原菌的数量。该方法可用于菠菜枯萎病病原菌的快速鉴定和病因诊断。
Spinach Fusarium wilt is caused by pathogenic Fusarium (F.o.f.sp. Spinaciae) and is one of the most important diseases in spinach production. It takes a lot of time to identify the pathogen of spinach wilt by conventional methods and it is difficult to get the correct conclusion. Randomly amplified polymorphic DNA-sequence tagged sites (RAPD-STS) provides an effective method for the identification of pathogenic bacteria. In this study, a specific fragment of the pathogen of Fusarium oxysporum f. Sp. (GenBank accession number: AY337463) was cloned by RAPD analysis of the tested strains. According to the sequencing results, a pair of specific primers were designed for the pathogen of Fusarium oxysporum f. Sp., And the pathogenic bacteria were identified by conventional PCR and real-time PCR. The sensitivity of the two methods were compared. The results showed that Fofsp.spinaciae could be identified by both PCR methods, but the sensitivity to pathogen DNA was different. The minimum amount of DNA detected by conventional PCR was 100 pg, while the lowest amount of DNA detected by real-time PCR Is 1 pg. At the same time, real-time quantitative PCR can also quantify the DNA of pathogenic bacteria and estimate the number of pathogens accordingly. The method can be used for the rapid identification and etiological diagnosis of pathogen of Fusarium oxysporum.