INRA, UMR Santé Végétale 1065, ISVV, Villenave d'Ornon, France.
Pest Manag Sci. 2011 Jan;67(1):60-9. doi: 10.1002/ps.2032. Epub 2010 Oct 14.
Management of grapevine powdery mildew Erysiphe necator Schw. requires fungicide treatments such as sterol demethylation inhibitors (DMIs) or mitochondrial inhibitors (QoIs). Recently, reduction in the efficacy of DMIs or QoIs was reported in Europe and the United States. The aim of the present study was to develop real-time qPCR tools to detect and quantify several CYP51 gene variants of E. necator: (i) A versus B groups (G37A) and (ii) sensitive versus resistant to sterol demethylase inhibitor fungicides (Y136F).
The efficacy of the qPCR tools developed was better than the CAPS method, with a limit of 2 pg for E necator DNA, 0.06 ng for genetic group A and 1.4 ng for the DMI-resistant allele. The detection limits of qPCR protocols (LOD) ranged from 0.72 to 0.85%, and the quantification limits (LOQ) ranged from 2.4 to 2.85% for the two alleles G47A and Y136F respectively. The application of qPCR to field isolates from French vineyards showed the presence of DMI-resistant and/or QoI-resistant alleles in French pathogen populations, linked to genetic group B.
The real-time PCR assay developed in this study provides a potentially useful tool for efficient quantification of different alleles of interest for fungicide monitoring and for population structure of E. necator.
葡萄白粉病病原菌（Erysiphe necator Schw.）的防治需要使用杀菌剂处理，如甾醇脱甲基抑制剂（DMIs）或线粒体抑制剂（QoIs）。最近，在欧洲和美国报道了 DMIs 或 QoIs 功效降低的情况。本研究的目的是开发实时 qPCR 工具来检测和量化几种 E. necator 的 CYP51 基因变异体：（i）A 与 B 组（G37A）和（ii）对甾醇脱甲基酶抑制剂杀菌剂敏感与抗性（Y136F）。
开发的 qPCR 工具的功效优于 CAPS 方法，对于 E necator DNA 的检测限为 2 pg，A 组遗传群的检测限为 0.06 ng，对 DMI 抗性等位基因的检测限为 1.4 ng。qPCR 协议的检测限（LOD）范围为 0.72%至 0.85%，两个等位基因 G47A 和 Y136F 的定量限（LOQ）范围分别为 2.4%至 2.85%。qPCR 技术在法国葡萄园的田间分离物中的应用表明，法国病原菌群体中存在对 DMI 和/或 QoI 具有抗性的等位基因，与遗传群 B 有关。
本研究开发的实时 PCR 检测方法为杀菌剂监测和 E. necator 种群结构的不同感兴趣等位基因的有效定量提供了一种潜在有用的工具。
