基于EF-1α序列位点特异性PCR快速鉴定小麦茎基腐病优势病原菌假禾谷镰孢菌  

Rapid Identification of Main Pathogen Fusarium pseudograminearum of Wheat Crown Rot Using Site-specific PCR Based on EF-1αSequence

作　　者：刘国霞[1] 谭晴晴[1] 齐军山[2] 王福玉[3] 陈雪燕[1] 范阳阳[1] 胡悦 步迅[1] 张全芳[1] LIU Guo-Xia;TAN Qing-Qing;QI Jun-Shan;WANG Fu-Yu;CHEN Xue-Yan;FAN Yang-Yang;HU Yue;BU Xun;ZHANG Quan-Fang(Biotechnology Research Center,Shandong Academy of Agricultural Sciences,Jinan 250100,China;Institute of Plant Protection,Shandong Academy of Agricultural Sciences,Jinan 250100,China;Jining Academy of Agricultural Sciences,Jining 272031,China)

机构地区：[1]山东省农业科学院生物技术研究中心,济南250100 [2]山东省农业科学院植物保护研究所,济南250100 [3]济宁市农业科学研究院,济宁272031

出　　处：《农业生物技术学报》2021年第5期985-994,共10页Journal of Agricultural Biotechnology

基　　金：山东省重点研发计划(2019GSF107085);山东省现代农业产业技术体系小麦创新团队(SDAIT-01-10);山东省农业科学院农业科技创新工程产业团队(CXGC2016A11);国家自然科学基金(31901543)。

摘　　要：小麦茎基腐病(wheat crown rot,WCR)是影响小麦(Triticum aestivum)产量的重要病害,假禾谷镰孢菌(Fusarium pseudograminearum)是其优势病原菌,对病原菌快速准确的鉴定对于制定合理的防治措施及选育抗病品种具有重要意义。本研究通过分析常见镰孢菌延伸因子-1α(elongation factor-1α,EF-1α)基因序列,针对假禾谷镰孢菌的SNP位点设计了特异引物F.pseudo-F3/R1,利用降落PCR(touchdown PCR,TD PCR)和该特异PCR引物,可在假禾谷镰孢菌中扩增到334 bp单一条带,而在禾谷镰孢菌(F.graminearum)、三线镰孢菌(F.tricinctum)、层出镰孢菌(F.proliferatum)、变红镰孢菌(F.incarnatum)和尖孢镰孢菌(F.oxysporum)中均无扩增。利用2019~2020两年在山东济宁和济阳采集的小麦茎秆中分离的病菌对该位点特异性诊断PCR引物和扩增方法进行了验证。研究发现,2019年山东济宁小麦疑似茎基腐病样品中分离到28株真菌,使用F.pseudo-F3/R1仅在假禾谷镰孢菌中扩增到334 bp单一条带,其他小麦真菌如禾谷镰孢菌、木贼镰孢菌(F.equiseti),链格孢菌(Alternaria alternate)、根腐离蠕孢菌(Bipolaris sorokiniana)和禾谷丝核菌(Rhizoctonia cerealis)中均无扩增。对2020年济宁田间表现为茎基腐病的小麦样品直接提取茎秆DNA,利用上述特异引物可扩增出假禾谷镰孢菌的特异条带,而在表现根腐、叶锈、白粉和枯白穗症状的小麦茎秆中则无扩增。经分离病菌测序鉴定,在茎基腐病小麦样品中有假禾谷镰孢菌和禾谷镰孢菌,而其他症状小麦中分离到禾谷镰孢菌和禾谷丝核菌。同时,在2020年济阳人工接种和自然感染的茎基腐病小麦茎秆的DNA中也可扩增到假禾谷镰孢菌的特异条带。上述研究表明,该方法特异性强,可直接用于小麦茎秆的假禾谷镰孢菌准确检测,为快速鉴别假禾谷镰孢菌提供了一种新方法。Wheat stem rot(WCR)is an important disease affecting wheat yield,Fusarium pseudograminearum is the main pathogen.The rapid and accurate identification of pathogens is significant for making reasonable control measures and resistant varieties breeding.In this study,elongation factor 1α(EF-1α)gene sequences of common Fusarium spp.were compared and specific primers F.pseudo-F3/R1 for F.pseudograminearum were designed,touchdown PCR were used to specifically amplify a 334 bp band from F.pseudograminearum,and no band amplified in F.graminearum,F.tricinctum,F.proliferatum,F.incarnatum and F.oxysporum.Wheat samples collected from Jining and Jiyang in Shandong province in 2019 and 2020 were detected using the site-specific PCR method which was just developed to test its validity.It was found that 28 fungi strains were isolated from infected samples from Jining in 2019,in which F.pseudograminearum was amplified with aimed band of 334 bp using primers F.pseudo-F3/R1,while no band from other fungi including F.graminearum,F.equiseti,Alternaria alternate,Bipolaris sorokiniana and Rhizoctonia cerealis.Samples were also collected from Jining in 2020 and DNA isolated directly from wheat stem with symptom of crown rot,then amplified using primers F.pseudo-F3/R1 and got a 334 bp aimed band of F.pseudograminearum,while other samples with symptom of root rot,leaf rust,powdery mildew or white-headed got no amplification band.The fungi isolated from stem of samples used above from Jining in 2020 were identified by sequencing analysis,and the result showed that F.pseudograminearum and F.graminearum existed in crown rot wheat sample,while F.graminearum and R.cerealis existed in other samples.The wheat stems of artificially and naturally infected by F.pseudograminearum from Jiyang in 2020 were amplified using primers F.pseudo-F3/R1 and got a 334 bp aimed band of F.pseudograminearum.The above results indicated that the specific PCR method could be used to detect F.pseudograminearum in stem directly,which provides a new method to rapidly

关 键 词：小麦茎基腐病 假禾谷镰孢菌 病原菌 快速鉴定 

分 类 号：S512.1[农业科学—作物学] S435.1