番茄枯萎病菌RT-PCR检测技术的建立与应用  

Establishment and Application of Real-Time PCR for Quantitative Detection of Fusarium oxysporum f.sp.lycopersici

作　　者：孟臻 张伟萍 王莹 李龙 姬小雪[1] 董贝 乔康[1] MENG Zhen;ZHANG Weiping;WANG Ying;LI Long;JI Xiaoxue;DONG Bei;QIAO Kang(College of Plant Protection,Shandong Agricultural University,Tai’an,Shandong 271018,China;Jinan Academy of Agricultural Sciences,Jinan 250316,China;Tai’an Institute for Food and Drug Control,Tai’an,Shandong 271000,China)

机构地区：[1]山东农业大学植物保护学院,山东泰安271018 [2]济南市农业科学研究院,济南250316 [3]泰安市食品药品检验检测研究院,山东泰安271000

出　　处：《园艺学报》2022年第11期2479-2488,共10页Acta Horticulturae Sinica

基　　金：山东省自然科学基金项目(ZR2021MC065);济南市农业科学院项目(yy201915)。

摘　　要：根据番茄枯萎病病原菌尖孢镰刀菌番茄专化型(Fusarium oxysporum f.sp.lycopersici,FOL)蛋白激酶基因序列,设计1对番茄枯萎病特异性引物209F/361R,构建番茄枯萎病菌的RT-PCR(real-time PCR)检测体系,并利用该体系定量检测盆栽番茄茎基部病原菌。RT-PCR结果表明该引物只对番茄枯萎病菌有唯一产物吸收峰,对其他供试菌株都未检到荧光信号。利用该引物建立的RT-PCR检测体系线性关系良好,灵敏度为5.76×10^(3)copies·μL^(-1),是普通PCR的10倍。人工接种番茄枯萎病菌定植后7 d即可在茎基部检测到病原菌;田间采集的24个自然发病样本中有16个能够检测到番茄枯萎病菌。基于RT-PCR技术构建的番茄枯萎病菌快速检测方法检测速度快、灵敏度高、特异性强、重现性好,能够为该病的早期诊断和流行监测提供理论依据。In this study,a pair of specific primers,209F/361R,were designed to establish a real-time PCR(RT-PCR)reaction system of Fusarium oxysporum f.sp.lycopersici(FOL)based on the protein kinase gene sequences of FOL.FOL was detected at the base of tomato stems using this assay.Results of RT-PCR assays showed that only one absorption peak was detected with the primers for F.oxysporum and no absorption peak was produced for other tested strains.The RT-PCR detection system established with the primers showed a good linear relationship.The detection sensitivity was 5.76×10^(3)copies·μL^(-1),which was 10 times more sensitive than the conventional PCR.Moreover,FOL was detected at the base of the tomato stems at the 7th day after artificial inoculation.Twenty-four naturally infected tomato samples were collected from field condition and the RT-PCR results showed that FOL was detected in 16 samples.The established RT-PCR detection system for F.oxysporum was fast,highly specific,sensitive,and reproducible,providing scientific tools for epidemic monitoring and early diagnosis of Fusarium wilt of tomato.

关 键 词：番茄枯萎病 尖孢镰刀菌番茄专化型 实时荧光定量PCR 早期检测技术 

分 类 号：S641.2[农业科学—蔬菜学]