中国农业科学 ›› 2015, Vol. 48 ›› Issue (1): 55-62.doi: 10.3864/j.issn.0578-1752.2015.01.06

• 植物保护 • 上一篇    下一篇

SYBR Green I 实时荧光定量PCR检测小麦纹枯病菌体系的建立和应用

孙炳剑,陈清清,袁虹霞,施艳,李洪连   

  1. 河南农业大学植物保护学院/河南省粮食作物协同创新中心/小麦玉米作物学国家重点实验室,郑州 450002
  • 收稿日期:2014-06-24 出版日期:2015-01-01 发布日期:2015-01-01
  • 通讯作者: 李洪连,Tel:0371-63558791;Fax:0371-63558170;E-mail:honglianli@sina.com
  • 作者简介:孙炳剑,E-mail:sbj8624@sina.com
  • 基金资助:
    国家“十二五”粮食丰产科技工程(2012BAD04B07)、中国科学院知识创新工程重大项目(KSCX2-EW-N-08)

Establishment of SYBR Green I Real-Time PCR for Quantitatively Detecting Rhizoctonia cerealis in Winter Wheat

SUN Bing-jian, CHEN Qing-qing, YUAN Hong-xia, SHI Yan, LI Hong-lian   

  1. College of Plant Protection, Henan Agricultural University/Collaborative Innovation Center of Henan Grain Crops/National Key Laboratory of Wheat and Maize Crop Science, Zhengzhou 450002
  • Received:2014-06-24 Online:2015-01-01 Published:2015-01-01

摘要: 【目的】纹枯病是小麦生产上重要的土传病害之一,早期准确定量检测病原是病害预测预报及实现有效防控的基础。传统组织分离鉴定方法费时、程序复杂,而且无法做到准确定量。为实现小麦纹枯病早期快速准确定量检测,研究旨在建立小麦纹枯病菌(Rhizoctonia cerealis)的SYBR Green I实时荧光定量PCR(real-time PCR)检测方法。【方法】根据小麦纹枯病菌的β-tubilin序列,设计1对特异性引物,建立并优化SYBR Green I real-time PCR反应体系,利用小麦纹枯病菌、立枯丝核菌(R. solani)、双核丝核菌AG-A和AG-F菌株、小麦全蚀病菌(Gaeumannomyces graminis var. tritici小麦根腐病菌(Bipolaris sorokiniana)、小麦茎基腐病菌假禾谷镰孢(Fusarium pseudograminearum)和禾谷镰孢(F. graminearum)等8种小麦根部或土壤病原物的菌丝DNA进行普通PCR和real-time PCR特异性检测,并对灵敏度、可重复性进行评价。利用该体系分别检测接种后5、10、60 d不同接种浓度的盆栽小麦病株。【结果】研究设计的引物特异性强,普通PCR检测结果仅小麦纹枯病菌DNA有扩增条带。Real-time检测结果表明该对引物对小麦纹枯病菌只有唯一的产物吸收峰,对其余供试菌株均未检测到荧光信号。普通PCR检测的灵敏度为6.5×103 copies/μL, real-time PCR的灵敏度可达到6.5×102 copies/μL。以携带目的基因片段的重组质粒为标准品,构建的real-time PCR标准曲线循环阈值与模板浓度呈良好的线性关系,熔解曲线的吸收峰单一,相关系数为0.997,扩增效率为0.91。对人工接种的盆栽小麦病株进行real-time PCR检测,结果表明与病情指数和接种量分别呈显著正相关。【结论】研究建立的基于real-time PCR技术的小麦纹枯病菌快速检测方法速度快、灵敏度高、特异性强、重复性好。可以用于小麦纹枯病菌检测、指导病害预测和防治。

关键词: 小麦纹枯病菌, SYBR Green I, 实时荧光定量PCR, 早期检测

Abstract: 【Objective】Wheat sharp eyespot (WSE) caused by Rhizoctonia cerealis is one of the most important soil-born diseases on wheat in China. Early accurate quantitative detection is a foundation of forecast and control. Traditional method of organization isolation and identification of pathogen is time consuming, complicated and can’t be accurately quantified. In order to implement the early and quick quantitative determination of wheat sharp eyespot, a SYBR Green I real-time PCR method of R. cerealis was established based on the pathogen sequence information.【Method】 Based on the β-tubilin of R. cerealis, a pair of specific primers was designed. The SYBR Green I real-time PCR reaction system was established and optimized. The sensitivity, specificity and repeatability of the system were also evaluated, and RBipolaris sorokiniana, Fusarium graminearum, F. pseudograminearum were used for control fungi. The indoor potted plants of wheat which infected by R. cerealis were detected with optimized reaction system after inoculated for 5, 10 and 60 days, respectively.【Result】The primers were of great specificity, the specific PCR fragment was amplified from the DNA of R. cerealis isolates, but not from the DNA of other fungal isolates by conventional PCR. The real-time PCR assays also did not amplify DNA from control fungi. The sensitivity of conventional PCR was 6.5×103 copies/μL plasmid, while the sensitivity of real-time PCR was 6.5×102 copies/μL. The standard curve established by recombinant plasmid showed a fine linear relationship between threshold cycle and template concentration. The melt curve was specific with the correlation coefficient of 0.997 and with high amplification efficiency (0.91). For the indoor potted experiments, the detection results of real-time PCR of infected wheat samples, were showed a significant positive correlation with disease index and inoculum, respectively. 【Conclusion】 The developed real-time PCR assay for R. cerealis is fast, highly specific, sensitive, and reproducible. This method can be used to detect R. cerealis in wheat, and guidance prediction and control of wheat sharp eyespot.. cerealis, R. solani, AG-A, AG-F, Gaeumannomyces graminis var. tritici,

Key words: Rhizoctonia cerealis, SYBR Green I, real-time PCR, early detection