烟草四种重要土传病原菌多重PCR检测方法的建立与应用

Establishment and application of a multiplex PCR assay for detection of four tobacco soil-borne pathogens

为快速准确地鉴定和区分烟草生产中的4种土传病害病原菌-青枯雷尔氏菌(Ralstonia solanacearum)、软腐果胶杆菌(Pectobacterium carotovorum)、寄生疫霉(Phytophthora parasitica)和尖孢镰刀菌(Fusarium oxysporum),通过设计和筛选特异性引物,建立多重PCR反应体系,并对体系中的引物用量和退火温度进行优化,检测优化后体系的特异性和灵敏度,验证其在烟草组织、土壤样品及种子样本中病原菌检测的实用性。结果表明,反应体系中最优的引物用量组合为RsRipNTF3/RsRipNTR3各0.9μL、PccTF4/PccTR4各0.6μL、PpRhTF1/PpRhTR1各1.2μL和FoP450TF1/FoP450TR1各0.8μL,最佳退火温度为61.1℃。该体系可分别扩增出4种病原菌的特异性片段,大小分别为511、361、669和840 bp,对病原菌DNA的检测灵敏度可达50 pg·μL^(-1)。植物组织和土壤样本检测结果表明,所建立的多重PCR检测方法能够准确地检测出其中的病原菌;采用该检测方法对80份烟草商品种进行检测,发现其中4个样本携带有青枯雷尔氏菌或尖孢镰刀菌,而未检测到软腐果胶杆菌和寄生疫霉。本研究建立的多重PCR检测方法可以灵敏、准确、高通量检测4种烟草土传病原菌,为烟草土传病害的准确诊断和早期防控提供可靠的技术方法。

To rapidly and accurately identify and differentiate four soil-borne pathogens of tobacco,including Ralstonia solanacearum,Pectobacterium carotovorum,Phytophthora parasitica,and Fusarium oxysporum,a multiplex PCR assay was established by designing and screening of specific primers for each pathogen.The primer concentration and annealing temperature of the reaction system were optimized.The specificity and sensitivity of the optimized system were tested,and its practicability in detecting pathogens in tobacco tissue,soil samples and seed samples was verified.The results showed that the optimal primer concentrations were 0.9μL for RsRipNTF3/RsRipNTR3,0.6μL for PccTF4/PccTR4,1.2μL for PpRhTF1/PpRhTR1,and 0.8μL for FoP450TF1/FoP450TR1,and the optimal annealing temperature was 61.1℃.In the quadruple PCR detection system,the specific target fragments of R.solanacearum,Pectobacterium carotovorum,Phytophthora parasitica and F.oxysporum with sizes of 511,361,669 and 840 bp could be amplified,respectively,and the detection sensitivity of pathogen DNA can reach 50 pg·μL^(-1).The applicability test showed that the assay could accurately detect the pathogens in infected plant tissues and soil samples.Using the optimized reaction system,80 commercial tobacco varieties were detected for carrying pathogen,it was found that four samples were positive for R.solanacearum or F.oxysporum,while no Pectobacterium carotovorum or Phytophthora parasitica was detected.In conclusion,the multiplex PCR detection method established in this study can sensitively,accurately and high-throughput detect four tobacco soil-borne pathogens,providing a reliable technical method for accurate diagnosis and early prevention and control of tobacco soil-borne diseases.