一种基于LAMP技术快速检测小麦赤霉病菌对多菌灵抗药性方法的建立及应用  

Establishment and application of a loop-mediated isothermal amplification method for detection of carbendazim-resistant mutants in Fusarium graminearum

作　　者：段亚冰[1] 效雪梅 杨莹[1] 曹君红[1] 施一渊 张晓柯[1] 王勇[1] 王建新[1] 周明国[1] 

机构地区：[1]南京农业大学植物保护学院/绿色农药创制与应用技术国家地方联合工程研究中心,江苏南京210095

出　　处：《南京农业大学学报》2016年第1期97-105,共9页Journal of Nanjing Agricultural University

基　　金：国家公益性行业(农业)科研专项(201303023);国家大学生创新创业训练计划项目(201510307023)

摘　　要：[目的]传统的植物病原菌抗药性检测方法存在检测周期长、操作繁琐、效率低等诸多缺点,建立一种小麦赤霉病菌(Fusarium graminearum)对多菌灵抗药性快速检测的方法尤为重要。[方法]基于环介导等温扩增技术(LAMP),以小麦赤霉病菌对多菌灵抗药性基因型F167Y的β2微管蛋白基因为靶标,经过人工错配,设计并筛选LAMP特异引物,对检测体系各组分浓度配比进行优化,并通过人工接种试验验证该检测技术的可靠性。[结果]建立了一种小麦赤霉病菌对多菌灵抗性的快速、高通量的分子检测技术,该检测技术能特异性鉴定小麦赤霉病菌对多菌灵的抗药性菌株;证实了该检测技术在生产中可靠、稳定、可操作性强,具有广阔的应用前景。[结论]该检测方法是LAMP技术在小麦赤霉病菌抗药性监测中的首次应用,可用于抗药性群体的动态监测与抗性风险评估,为小麦赤霉病的抗药性治理提供用药指导。[Objectives]Traditional detection methods of fungicide resistance of plant pathogens are always time-consuming,tedious and inefficient. Therefore,it is important to establish a rapid method for detecting carbendazim-resistant mutants in Fusarium graminearum. [Methods]Based on the β2-tubulin gene of carbendazim-resistant genotype F167 Y in F. graminearum,the primers were designed and optimized to specially distinguish the carbendazim-resistant mutants according to loop-mediated isothermal amplication（ LAMP）. [Results]Through optimization of the concentration of components,a rapid,high-through detection method for carbendazim-resistant mutants of F. graminearum based on loop-mediated isothermal amplification was established. When hydroxynaphthol blue dye（ HNB） was added prior to amplification,samples with DNA of the F167 Y mutation developed a characteristic sky blue color after the reaction but those without DNA or with different DNA did not. Results of HNB staining method were reconfirmed by gel electrophoresis. In addition,artificial inoculation test was performed. The results further confirmed that the developed LAMP had good reliability,stability and maneuverability and had a broad application prospect in the production. [Conclusions]It is the first time that LAMP was used for monitoring the carbendazim resistance in F. graminearum. It is also used for dynamic monitoring of carbendazim resistance and risk assessment and will provide some reference data for controlling Fusarium head blight.

关 键 词：小麦赤霉病菌 多菌灵 环介导等温扩增技术 抗药性 

分 类 号：S482.2[农业科学—农药学]