利用RAPD-STS和实时定量PCR鉴别菠菜枯萎病病原菌(Fusarium oxysporum f.sp.spinaci-ae)(英文)

Identification of the Pathogenic Fusarium oxysporum f.sp.spinaciae Using RAPD-STS and Real-time PCR

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。

Fusarium wilt of spinach incited by the pathogenic fungus F. o. f. sp. spinaciae is a very important disease of spinach worldwide. Classical diagnosis of this disease normally requires many days and often gives erroneous results. Differential detection assays employing randomly amplified polymorphic DNA-sequence tagged sites （RAPD-STS） were developed for F. o. f. sp. spinaciae. Unique fragments （GenBank accession number AY337463 ） from RAPD profiles that differentiated the pathogens and other isolates were cloned and sequenced. Then six pairs of primers were designed and used for two types of polymerase chain reaction （PCR） techniques （a classical PCR and a real-time PCR） to compare the sensitivity. Results demonstrated that only one of the primer sets amplified a clear single fragment specifically for isolates of F. o. f. sp. spinaciae, and worked well in both of the PCR techniques. Classical PCR was able to detect at least 100 pg of pathogen DNA, while the real-time PCR method using SYBR Green Ⅰ on the Smart Cycler was capable of detecting less than 1 pg of pathogen DNA, and was able to rapidly diagnose the disease and quantitatively assay pathogen DNA. This method was optimized for using DNA extracted from the microconidia of the fungus, and may be useful for field disease diagnosis and prognosis.