基于SYBR GreenⅠ实时荧光定量PCR技术检测烟草靶斑病发病菌量  

Using SYBR GreenⅠReal-Time Quantitative PCR System for Detection of Rhizoctonia solani

作　　者：李凤巍 裴悦宏 张晓枫 王智 陈海涛 徐宸 冉茂 孙现超[1] LI Fengwei;PEI Yuehong;ZHANG Xiaofeng;WANG Zhi;CHEN Haitao;XU Chen;RAN Mao;SUN Xianchao(College of Plant Protection,Southwest University,Chongqing 400715,China;Tobacco leaf Branch of Chongqing Company of China National Tobacco Corporation,Chongqing 400020,China;Shizhu Branch of Chongqing Company of China National Tobacco Corporation,Chongqing 409100,China)

机构地区：[1]西南大学植物保护学院,重庆400715 [2]中国烟草总公司重庆市公司烟叶分公司,重庆400020 [3]中国烟草总公司重庆市公司石柱分公司,重庆409100

出　　处：《西南大学学报（自然科学版）》2023年第11期96-105,共10页Journal of Southwest University(Natural Science Edition)

基　　金：国家自然科学基金项目(31870147);中国烟草总公司重庆市公司科技项目(B20212NY2312,B20221NY1307,B20211-NY1315);广西中烟工业有限责任公司项目(2021450000340029).

摘　　要：本研究根据烟草靶斑病菌(Rhizoctonia solani)与烟草赤星病菌(Alternaria alternata)、烟草棒孢霉叶斑病菌(Corynespora cassiicola)等田间常见烟草叶部病害病原菌的rDNA-ITS(ribosomal DNA Internal Transcribed Spacers,rDNA-ITS)序列差异位点,设计一对特异性检测引物RSW1F/RSW2R,建立烟草靶斑病菌实时荧光定量PCR(Real time fluorescence quantitative Polymerase Chain Reaction,RT-qPCR)快速准确检测体系,并分析烟草靶斑病最低发病菌量.结果表明,设计的引物特异性良好,灵敏度达到1×10^(-3) ng/μL.利用已获得的实时荧光定量PCR体系计算得出引起烟草靶斑病发病的最低菌量为12.03 pg/μL.此外,对重庆龚滩地区烟叶中烟草靶斑病菌含量进行计算,发现土壤以及周边杂草可能为该地区烟草靶斑病菌的侵染来源,并进一步确定该地区预防烟草靶斑病的最佳时期为5月15日至6月15日,为烟草靶斑病菌的田间预警预报及流行提供理论依据.In this study,a pair of specific primers RSW1F/RSW2R were designed according to the sequence differences of rDNA-ITS of common tobacco leaf disease pathogens in the field such as Rhizoctonia solani,Alternaria alternata and Corynespora cassiicola.Based on this,a real-time fluorescence quantitative PCR detection system were further developed for the rapid detection of R.solani and analysis of minimum pathogen content to cause tobacco target spot.The results demonstrated that the primers were designed with good specificity and sensitivity of 1×10^(-3) ng/μL.Using the developed real-time quantitative PCR system,the minimum content of R.solani DNA resulting tobacco target spot was 12.03 pg/μL.Furthermore,the content of R.solani in tobacco leaves in Gongtan area of Chongqing was calculated.The soil and surrounding weeds were found to be the possible sources of pathogens of tobacco target spot in this area.And the best prevention time of tobacco target spot was determined to be from May 15 to June 15,which provided a theoretical basis for the field early warning,forecast and epidemic occurrence of tobacco target spot.

关 键 词：烟草 烟草靶斑病 实时荧光定量PCR 

分 类 号：S432.44[农业科学—植物病理学]