尖孢镰刀菌分子检测技术的建立与应用

尖孢镰刀菌从形态学和分子生物学上与属内多种菌难于区分,为准确鉴定豆科牧草上的尖孢镰刀菌,本研究基于尖孢镰刀菌与其他镰刀菌的核糖体DNA基因间间隔区IGS(intergenic spacer)序列间的差异,设计了一对特异性引物P₁/P₂,检测体系可以特异地从尖孢镰刀菌中扩增出一条1081 bp的条带;引物P₁/P₂对尖孢镰刀菌基因组DNA的检测阈值为100 pg,对土壤中尖孢镰刀菌分生孢子的检测灵敏度为100个孢子/g土;同时,引物P₁/P₂也可以对发病牧草根部组织中尖孢镰刀菌的存在进行特异性检测。检测体系可不经过室内病原菌的分离纯化即可有效的从土壤和病株中提取的DNA进行PCR检测鉴定,是一种快速有效的豆科牧草根腐病病原菌分子检测技术。

Establishment and application of rapid molecular detection for Fusarium oxysporum

It is difficult to distinguish Fusarium oxysporum from other Fusarium species using traditional mor-phological observations or molecular analysis based on rDNA-ITS sequencing.In order to accurately identify F .oxysporumin legumes,a pair of primers named P1/P2 was designed based on differences in ribosomal DNA intergenic spacer (rDNA-IGS)sequences of the Fusarium genus,which can be used to amplify DNA from F . oxysporum by conventional PCR.More than 23 species of root rot pathogens were used to verify the specificity of the primers.P1/P2 could amplify a unique 1081 bp sequence from different biotypes of F .oxysporum while it could not amplify from other root rot pathogens.The sensitivity of P1/P2 was 100 pg for genomic DNA and 100 conidia/g soil for the root rot pathogens.Furthermore,this pair of primers could directly amplify se-quences from the genomic DNA of F .oxysporum diseased plant samples without pathogen isolation,indicating that this is a rapid and effective legume root rot pathogen detection technology.