甘蔗褐锈病菌单管巢式PCR检测体系的建立  

Establishment of Single Tube Nested PCR Detection System for Puccinia melanocephala

作　　者：吴伟怀[1] 刘宝慧 鹿鹏鹏 梁艳琼[1] 贺春萍[1] 李锐[1] 易克贤[1] WU Weihuai;LIU Baohui;LU Pengpeng;LIANG Yanqiong;HE Chunping;LI Rui;YI Kexian(Key Laboratory of Integrated Pest Management on Tropical Crops,Ministry of Agriculture and Rural Affairs,P.R.China/Environment and Plant Protection Institute,Chinese Academy of Tropical Agricultural Sciences,Haikou 571101;College of Plant Protection,Nanjing Agricultural University,Nanjing 210095;College of Plant Protection,Hainan University,Haikou 570228)

机构地区：[1]中国热带农业科学院环境与植物保护研究所/农业农村部热带作物有害生物综合治理重点实验室,海口571101 [2]南京农业大学植物保护学院,南京210095 [3]海南大学植物保护学院,海口570228

出　　处：《中国糖料》2022年第3期1-7,共7页Sugar Crops of China

基　　金：国家重点研发计划项目(2018YFD0201100)资助。

摘　　要：由黑顶柄锈菌(Puccinia melanocephala Sydow)引起的甘蔗褐锈病是甘蔗生产中较为严重的叶部病害之一。建立该病原菌的高效检测与特异性鉴定技术体系,对于该病害的监测、预警以及及时采取必要的防治措施均具有重要的意义。为此,本文基于甘蔗褐锈病菌ITS序列保守区域位点设计了该病原菌的单管巢式PCR引物对,对外内引物退火温度、外内引物浓度比两个方面进行筛选与优化,并对所建立的单管巢式PCR检测体系的特异性和灵敏度进行了分析。结果表明,所设计的甘蔗褐锈病菌单管巢式PCR外引物PmF1/R1、内引物PmF2/R2的退火温度分别为63℃与52℃,以及外引物PmF1/R1与内引物PmF2/R2最佳终浓度比例为0.5 pmol/L∶0.5μmol/L时,单管巢氏PCR检测体系较佳。所建立的甘蔗褐锈病菌单管巢式PCR检测体系仅从含有甘蔗褐锈病菌DNA模板中检测出预期大小的特异性条带,而从含甘蔗黄锈病菌、咖啡驼孢锈菌、鸡蛋花鞘锈菌、葡萄层锈病菌、甘蔗黑穗病菌、橡胶炭疽病菌、咖啡褐斑病菌、稻瘟病菌、剑麻茎腐病菌的DNA中并未扩增出任何条带。通过模板梯度稀释法证实,所建立的单管巢氏PCR检测体系的最低检测限为10 fg/μL,是普通PCR灵敏度的1000倍。田间19份疑似病样单管巢式PCR检测结果,有18份样品DNA中含有甘蔗褐锈病菌。本检测方法可以用于甘蔗褐锈病病原菌的快速诊断及其病害监测,为该病害预测、预报提供技术支持。Brown rust of sugarcane caused by Puccinia melanocephalaH.&P.Syd is one of the most serious leaf diseases in sugarcane production.The establishment of a high-efficiency detection and specific identification system of the pathogen is of great significance for the disease monitoring,early warning and timely taking necessary control measures.In this study,single-tube nested primer pairs were designed based on the conservative region of ITS sequence of Puccinia melanocephala,which were screened and optimized by annealing temperature and concentration ratio of outer and inner primers.The specificity and sensitivity of the established single-tube nested PCR system were analyzed.The results showed that the optimum annealing temperatures of the outer primers PmF1/R1 and internal primers PmF2/R2 of single-tube nested PCR for Puccinia melanocephala were 64℃ and 52℃,respectively,and the optimum final concentration ratio of the outer PmF1/R1 and internal primers PmF2/R2was 0.5 fmol/L:0.5μmol/L.The single-tube nested PCR detection system only detected the expected size of specific bands from the DNA template containing the pathogen Puccinia melanocephala,but any bands were not amplified from the DNA of Puccinia kuehnii,Hemileia vastatrix,Colletotrichum gloeosporioides,Coffee coffeicola,Magnaporthe oryzae,Coleosporium plumierae,Phakopsora ampelopsidis,Sporisorium scitamineum,Aspergillus niger.The detection limit of the established single tube nested PCR system was 10 fg/μL,which was 1000 times higher than the traditional PCR sensitivity.Nineteen suspected samples were tested by single-tube nest PCR,and18 samples had DNA containing sugarcane brown rust bacteria.The method can be used for rapid diagnosis and disease monitoring of sugarcane brown rust,and provide technical support for disease prediction and forecast.

关 键 词：甘蔗 褐锈病 黑顶柄锈菌 单管巢式PCR 检测 

分 类 号：S566.1[农业科学—作物学] S534.661