采用EF-1α序列分析法对苜蓿根腐病病原菌——锐顶镰刀菌的鉴定  

Identification of Fusaruim acuminatum isolated from Medicago sativa root using the EF-1α sequence analysis method

作　　者：易铭[1] 梁嘉俊[1] 史建 李洪建[1] 程积民[1] 焦锋[1] 

机构地区：[1]西北农林科技大学动物科技学院,陕西杨凌712100 [2]彬县西坡乡动物卫生监督所,陕西彬县713500

出　　处：《草业学报》2017年第2期61-68,共8页Acta Prataculturae Sinica

基　　金：国家公益性行业(农业)科研专项经费项目(201303157);国家现代农业牧草产业技术体系(CRAS-35)资助

摘　　要：苜蓿根腐病对苜蓿生长和产量造成严重影响,为了解引起根腐病病原真菌的种类,本文对从陕西省定边县牧草草场紫花苜蓿根部分离得到的根腐病病原菌进行形态学观察、EF-1α序列分析鉴定及接种试验。结果表明:通过对病原菌在PDA培养基中的分离、纯化得到的单菌落形态学观察及显微镜下菌丝、孢子的观察,该病原菌属于引起苜蓿根腐病的镰刀菌属(Fusarium);用CTAB法提取病原菌基因组DNA,并对EF-1α序列进行PCR扩增、回收纯化、克隆测序,将测序结果进行Blast比对,构建系统发育树,分析与Genbank已知近缘种属亲缘关系,结果显示与锐顶镰刀菌(F.acuminatum)亲缘关系最近,可信度达99%;最后通过根部接种试验,接种苜蓿根部发病症状与田间根腐病发病症状一致,鉴定结果显示本试验研究的引起紫花苜蓿根腐病的病原菌为镰刀菌属锐顶镰刀菌(F.acuminatum)。Alfalfa root rot causes serious reductions in alfalfa growth and yield.To identify the species of pathogenic fungus causing root rot disease,the root rot pathogen of Medicago sativa（alfalfa） collected from pasture in Dingbian County,Shaanxi Province,was analyzed by morphological observations,EF-1α sequence analysis,and inoculation tests.The mycelium and spores of a single colony of the pathogen growing on culture medium were observed under a microscope.Based on its morphology,the pathogen was identified as a Fusariumfungus.The genomic DNA was extracted from the pathogen using the cetyl-trimethylammonium bromide method,and the translation elongation factor gene was selected for PCR amplification,gel recycling,purification,cloning,sequencing,and phylogenetic analyses.A phylogenetic tree constructed using MEGA and sequences available in Genbank showed that the pathogen had the closest relationship with Fusarium acuminatum（99%）.In the root inoculation test,the disease symptoms of roots inoculated with the isolated pathogen were the same as those characteristic of root rot disease of M.sativain the field.Together,these results verified that the pathogen causing root rot disease of M.sativain Shaanxi Province is F.acuminatum.

关 键 词：紫花苜蓿 根腐病 EF-1α 锐顶镰刀菌 

分 类 号：S435.4[农业科学—农业昆虫与害虫防治]