五种烟草根茎病害病原菌多重PCR检测方法的建立与应用

Establishment and Application of Multiple PCR Detection Methods for Five Pathogens of Tobacco Root and Stem Diseases

为了快速准确鉴别烟草根腐病菌(Fusarium oxysporum)、黑胫病菌(Phytophthora nicotianae)、立枯病菌(Rhizoctonia solani)、根黑腐病菌(Thielaviopsis basicola)和鸢尾丝囊霉菌(Aphanomyces iridis)等5种烟草根茎病害病原菌,利用RAPD分子标记等方法筛选和设计特异性扩增引物,优化多重PCR扩增体系中各引物添加量、退火温度、循环数等条件,建立多重PCR检测体系,并对其检测的可行性进行验证。本研究筛选并设计出F.oxysporum、P.nicotianae、R.solani、T.basicola和A.iridis的特异性引物对LD141 F/R、YM1002 F/R、LK111 F/R、GHFT F/R、AiT7 F/R,PCR反应体系中最佳引物组合浓度分别为1.0、1.0、0.2、0.5、0.5μmol/L,最适退火温度为54℃,最佳循环数为28,可同时扩增出大小分别为370、240、536、783、138 bp的特异性片段,能同时检出5种病原菌DNA的最低限值为0.5 ng/μL,可实现对烟草育苗基质和烟株中5种病原菌的快速检测,对烟草苗期根茎病害的早期预防具有重要意义。

In order to identify five tobacco root and stem disease pathogens,including Fusarium oxysporum,Phytophthora nicotianae,Rhizoctonia solani,Thieleaviopsis basicola,and Aphromyces iridis rapidly and accurately,specific amplification primers were screened and designed.The primer concentration,annealing temperature and number of cycles of the reaction system were optimized to establish a multiple PCR detection system,and its practicability in detecting pathogens was verified.The specific primer pairs of LD141 F/R,YM1002 F/R,LK111 F/R,GHFT F/R,AiT7 F/R for F.oxysporum,P.nicotianae,R.solani,T.basicola and A.iridis were screened by RAPD molecular markers and designed.The optimal primer concentration in the PCR reaction system was 1.0,1.0,0.2,0.5,0.5μmol/L,respectively,and the optimal annealing temperature was 54℃,the number of optimal cycles was 28.The specific fragments with sizes of 370,240,536,783,138 bp could be amplified simultaneously and the detection sensitivity of five pathogens DNA reached 0.5 ng/μL.The detection system could quickly detect F.oxysporum,P.nicotianae,R.solani,T.basicola and A.iridis in seedling substrate samples and infected tobacco plants,which might be useful for early prevention of tobacco root and stem diseases.