浙贝母两种主要病害病原菌巢式多重PCR检测方法的建立

Nested Multiplex PCR to Detect Two Major Fungal Pathogens of Fritillaria thunbergii

浙贝母Fritillaria thunbergii炭疽病与干腐病作为浙贝母上常见的病害,严重影响浙贝母的质量和产量。因此,亟需建立一种快速准确的检测手段,这对病害早期诊断、早发现、早防治具有重要意义。本文以宁波市海曙区章水镇的浙贝母为材料,在前期明确引起上述两种真菌病害的病原菌分别为细线炭疽菌Colletotrichum lineola和尖孢镰刀菌Fusarium oxysporum的基础上,根据ITS序列,分别设计了两对特异性引物FO1-F/FO1-R和BMTJ4-F/BMTJ4-R,建立了针对浙贝母炭疽病与干腐病的巢式多重PCR检测方法。试验结果表明,建立的巢式多重PCR检测体系特异性好,同时检测2种病原菌的灵敏度高达100pg DNA/μL,对10株田间患病的浙贝母植株进行检测,4个样品检测出细线炭疽菌,2个样品检测出尖孢镰刀菌,另有4个样品同时检测出2种病原菌,检出率高达100%,表明该方法可以用于浙贝母炭疽病与干腐病的鉴定,且快速、准确、灵敏,研究结果为浙贝母炭疽病与干腐病的田间检测提供技术支持。

Anthracnose and dry rot diseases are common diseases in Fritillaria thunbergii,which seriously affect the quality and output of F.thunbergii.It is urgent to establish a rapid and accurate detection method of the two diseases,which is of great significance for early diagnosis,effective prevention and control of the disease.In this study,diseased F.thunbergii were collected from Zhangshui Town,Haishu District,Ningbo City.On the basis of previous results,two pairs of specific primers FO1-F/FO1-R and BMTJ4-F/BMTJ4-R were designed according to the ITS sequence of Colletotrichum lineola and Fusarium oxysporum on NCBI,respectively.The nested multiplex PCR method for detection of anthracnose and dry rot of F.thunbergii was established.The results showed that the nested multiplex PCR system had a high specificity and could detect C.lineola and F.oxysporum in infected plant tissues rapidly with a sensitivity of DNA 100 pg/μL.To verify the feasibility and accuracy of the detection system,10 diseased samples were randomly collected from the field.The results showed that C.lineola was detected in four samples,F.oxysporum was detected in two samples,and two pathogens were simultaneously detected in four samples,the detection rate was as high as 100%,indicating that this method can be used to identify anthracnose and dry rot in F.thunbergii quickly,accurately and sensitively.Our research results provided technical support for field detection of anthracnose and dry rot diseases of F.thunbergii.