植烟土壤中枯萎病菌和黑胫病菌的快速检测方法

Method for Rapid Detection of Fusarium oxysporum and Phytophthora parasitica var.nicotianae in Tabacco Planting Soil

【目的】枯萎病和烟草黑胫病是植烟土壤中最为常见的主要土传病害,为害严重,建立植烟土壤中两种病原菌尖孢镰刀菌(Fusarium oxysporum)和烟草疫霉菌(Phytophthora parasitica var.nicotianae)快速检测方法,为烟草种植过程中病害流行与治理提供技术支持,保障烟草行业的稳定生产。【方法】以采集广西百色地区土传病害土壤与健康土壤为测试材料,以标准病原菌为参考模板建立标准曲线,利用尖孢镰刀菌和烟草疫霉菌的保守区域设计的引物,根据目标条带及荧光定量PCR(qPCR)的溶解曲线与循环阈值(Ct值)筛选出特异性引物;同时,改进土壤中微生物DNA试剂盒提取方法,建立PCR和qPCR检测体系。【结果】分别筛选出一对尖孢镰刀菌和烟草疫霉菌的特异性的引物Pn3和JBR,模板浓度在1×10^(2)~1×10^(-4)ng之间,具有良好的线性关系,两种病原菌的DNA数量在发病土壤高于健康土壤。风干土壤并研磨再加两次洗脱有助于提取土壤中病原菌的DNA,建立了以普通PCR预扩增反应得到的产物作为模板的qPCR检测方法。【结论】PCR和qPCR联用技术能快速地分辨和检测尖孢镰刀菌和烟草疫霉菌,对枯萎病和烟草黑胫病实时监测和前期预警具有重要作用。

【Objective】Fusarium wilt and tobacco black shank,caused by Fusarium oxysporum and Phytophthora parasitica var.Nicotianae,are common soil-borne diseases in tobacco planting soil,leading to severe damages.Therefore,a rapid detection method for the two pathogens was established to provide technical support for disease epidemic and treatment in the process of tobacco planting and ensure the stable production of tobacco industry.【Method】Taking the soil with soil-borne disease and healthy soil in Baise area of Guangxi were used as test materials,and the standard curve was established with the standard pathogen as reference template.The primers were designed by the conservative regions of the two pathogens,and the specific primers were screened according to the target bands and the dissolution curve and Ct value of fluorescence quantitative PCR.At the same time,the extraction method of DNA kit of microbial in soil was improved,and the detection systems of PCR and qPCR were established.【Result】A pair of specific primers PN3 and JBR for F.oxysporum and P.parasitica var.nicotianae were screened respectively,and the template concentration ranged from 1×10^(2) to 1×10^(-4) ng,with good linear relationship.The number of two pathogens in diseased soil was higher than that in healthy soil.The method of combination of soil drying,grinding and eluting for twice was helpful to extract the DNA of pathogens in the soil.A qPCR detection method with the products obtained by ordinary PCR pre-amplification reaction as a template was established.【Conclusion】The combination of PCR and qPCR can quickly distinguish and detect F.oxysporum and P.parasitica var.nicotianae,which plays an important role in real-time monitoring and early warning of fusarium wilt and tobacco black shank.