马铃薯块茎和土壤样品中粉痂病菌快速检测方法的建立  

Establishment of a rapid detection method for Spongospora subterranea f.sp.subterranea on potato tubers and in soil samples

作　　者：李磊 赵昱榕 黄艺烁 吴凤芝[2] 李庆全[3] 石延霞[1] 谢学文[1] 柴阿丽[1] 李宝聚[1] LI Lei;ZHAO Yu-rong;HUANG Yi-shuo;WU Feng-zhi;LI Qing-quan;SHI Yan-xia;XIE Xue-wen;CHAI A-li;LI Bao-ju(Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China;Department of Horticulture,Northeast Agricultural University,Harbin 150030,China;Potato Research Institute,Heilongjiang Academy of Agricultural Sciences,Harbin 150086,China)

机构地区：[1]中国农业科学院蔬菜花卉研究所,北京100081 [2]东北农业大学园艺学院,哈尔滨150030 [3]黑龙江省农业科学院马铃薯研究所,哈尔滨150086

出　　处：《植物病理学报》2021年第6期987-995,共9页Acta Phytopathologica Sinica

基　　金：国家重点研发计划(2018YFD0200807);中国农业科学院科技创新工程项目(CAAS-ASTIP-IVFCAAS);农业部园艺作物生物学与种质创制重点实验室项目。

摘　　要：马铃薯粉痂菌(Spongospora subterranea f.sp.subterranea)是引起马铃薯粉痂病的病原。本研究根据粉痂菌内部转录间隔区和线粒体DNA的保守区域,分别设计了2对适用于普通PCR的引物A5/A9、C3/C8和1对适用于荧光定量PCR的引物QF/QR,用于检测块茎和土壤样品中的粉痂菌。特异性检测结果表明:引物对A5/A9和C3/C8,以马铃薯粉痂菌DNA为模板,能分别扩增出264和367 bp大小的单一条带,而对其他非靶标DNA无扩增;引物对QF/QR对马铃薯粉痂菌有单一的熔解峰,说明三对引物特异性良好。灵敏性检测结果表明:荧光定量PCR灵敏度为13.8 fg·μL^(-1),是普通PCR灵敏度的1000倍。进一步建立循环域值(Ct)与质粒DNA含量的曲线关系,获得标准曲线y=-3.8939 x+35.228,R;=0.9966,呈良好线性关系。通过对不同地区采集的18份带菌种薯和18份带菌土壤进行普通PCR和荧光定量PCR检测,引物A5/A9、C3/C8和QF/QR对带菌种薯检测率均为100%,对带菌土壤的检测率分别为44.44%、66.67%和100%。本研究建立的马铃薯粉痂病菌快速检测方法,能及时、准确地检测带菌种薯和土壤,为马铃薯粉痂病的早期诊断和防治提供依据。Spongospora subterranea f.sp.subterranea(S.subterranea)is the pathogen of powdery scab disease on potatoes.For the detection of S.subterranea in potato tubers and soil,two pairs of specific primers A5/A9,C3/C8 and one pair of specific primers QF/QR were designed from the conserved region of internal transcribed spacer(ITS)regions and mitochondrial DNA to perform the conventional PCR and quantitative PCR,respectively.The results of specificity assay showed that the primers A5/A9 and C3/C8 amplified 264 bp and 367 bp products from S.subterranea DNA but did not amplify DNA from healthy potato or a range of soil-borne microbes including related species.The primers QF/QR had only one absorption peak for S.subterranea.These results indicated that the three pairs of primers have good specificity.The results of sensitivity assay indicated that diluted S.subterranea DNA was detected at a minimum concentration to 13.8 fg·μL^(-1)using real-time PCR,which was 1000 times higher than that of the conventional PCR.In addition,the curve relationship between different content of plasmid DNA and corresponding(Ct)value is y=-3.8939 x+35.228,R;=0.9966,showing a good linear relationship.A total of 18 samples of infected symptomatic potato tubers and 18 soil samples collected from different areas were detected by conventional PCR and real-time PCR.The detection rates of primers A5/A9,C3/C8,and QF/QR to diseased tissues were 100%,and the detection rates to soil samples were 44.44%,66.67%,and 100%,respectively.In this study,the PCR-based methods were developed for the detection and quantification of the potato pathogen S.subterranea in infected tuber and soil,which will help to provide a scientific basis for early diagnosis and prevention of potato powdery scab.

关 键 词：马铃薯 粉痂菌 快速检测 应用 

分 类 号：S435.32[农业科学—农业昆虫与害虫防治]