土壤中生防菌粉红粘帚霉67-1的荧光定量PCR检测方法  

Development of Real-time PCR Quantitative Assay for Rapid Detection of Gliocladium roseum 67-1,an Effective Biocontrol Agent,in Soil

作　　者：陶萌[1] 李世东[1] 张拥华[1] 

机构地区：[1]中国农业科学院植物保护研究所/植物病虫害生物学国家重点实验室,北京100081

出　　处：《中国生物防治》2009年第1期35-40,共6页Chinese Journal of Biological Control

基　　金：国家"863"项目(2006AA10A211;2006AA06Z357);公益性行业(农业)科研专项(nyhyzx07-049)

摘　　要：为了建立重要植病生防真菌粉红粘帚霉67-1菌株的荧光定量PCR检测方法,收集了目标菌株、粘帚霉属其它多个种及近缘木霉属的多个种等共18个菌株,并进行了ITS区测序。以200bp左右差异较大区段设计出探针和引物。该引物及探针能有效扩增目标菌株,而其它17株非目标菌株没有扩增,表明所设计的引物和探针具有高度特异性。以目标菌株的阳性克隆质粒作为标准物质,建立了标准曲线,相关系数为0.9989,且扩增效率较高(95.0%)。经过土壤样品试验,得出标准曲线相关系数为0.9979,表明所建立的粘帚霉67-1菌株荧光定量PCR检测方法合理有效、快速实用,适合生态学研究的要求。Gliocladium roseum 67-1 selected by the authors has been demonstrated to be a highly effective biocontrol agent of the diseases caused by Sclerotinia sclerotiorum and Rhizocotonia solani. A rapid and accurate quantification of the agent is urgently needed for understanding its interaction with disease pathogens in soil and improving its effectiveness for disease management. Primers and probes for quantification in soil by real-time PCR were designed from ITS regions of the fungi belonging to several taxon. Specificity tests of the primers and probes using genomic DNA indicated that they could specifically amplify ITS rDNA of the biocontrol agent. Standard curve developed by using the plasmid containing the ITS rDNA of the agent gave rise to 0. 9989 as linear correlation coefficient between DNA concentrations and Ct values, and 95 % as amplification efficiency. Quantification of the agent added in natural soil by amplifying soil DNA obtained with Mobio UhraClean soil DNA kit showed that the value of copies was highly correlated with the value of density of the fungus （coefficient 0.9979）.

关 键 词：粉红粘帚霉 土壤 快速检测 荧光定量PCR 

分 类 号：S476.8[农业科学—农业昆虫与害虫防治]