Sensitivity analysis of Erysiphe necator to pyraclostrobin in China

Abstract:【Objective】Grape powdery mildew (Erysiphe necator) is a worldwide fungal disease occur-ring in all grape-growing regions around the world. At present, the effective way to prevent powdery mildew is spray of fungicides. Strobilurin (QoI) fungicides are commonly applied on this disease. How-ever, the frequent use of fungicides can lead to increase of fungicide resistance, inducing a decline of control efficacy. At present, pyraclostrobin is often used in vineyards for controlling grape powdery mil-dew in China, whereas the sensitivity of grape powdery mildew to pyraclostrobin keeps unclear. The purpose of this study was to determine the sensitivity baseline and resistance level of E. necator to the QoI fungicide pyraclostrobin.【Methods】145 isolates of E. necator were collected, isolated and puri- fied from 5 areas in Beijing, Hunan, Ningxia, Yunnan and Jiangsu. Among them, the 51 isolates collect-ed from the wine yards where chemical fungicides had never been applied were used to establish the baseline sensitivity of E. necator to pyraclostrobin. The isolates EC50 value was determined by the spore germination method, The conidia were spread onto a serial concentration gradient (0.1, 0.5, 1, 5, 10, 25,50, 100 μg·mL^-1) of pyraclostrobin-containing plates with 100 μg·mL^-1 of SHAM to reduce the effects of bypass respiration. The Q- PCR (G143A) method was used to detect the mutation information of G143A on cytb. The G143A mutation was detected by point mutation detection using the Amplification Retention Mutation System (ARMS) including wild-type allele primers, mutant allele primers and uni-versal reverse primers. The DNA of grape powdery mildew was diluted to 5-10 ng · μL^-1 for detection.The EC50 value of each isolate and the percentage of G143A in total mitochondrial DNA were calculat-ed using Microsoft Excle2016 software. For the determination of EC50 values of the 51 isolates, the nor-mality test was carried out using the Shapiro-Wilk method of SPSS software to establish the sensitivebaseline of E. necator to pyraclostrobin, and classify the level of E. necator resistance in China.【Re-sults】The EC50 range of the 51 isolates against pyraclostrobin was 0.034-13.219 μg·m^L-1. Among them,the EC50 values of the 43 isolates showed a continuous single peak and conformed to a normal distribu-tion, the average value of 1.387±0.107 μg · mL^{- 1} was used as the sensitivity baseline of E. necator to pyraclostrobin. The spore germination method showed the resistance frequency of the isolates to pyra-clostrobin was 45.52%, and the qPCR (G143A) method showed the frequency was 57.69%. When RF＞2, the results of qPCR (G143A) was consistent with the results of the spore germination method, and the Pearson correlation was significantly correlated.【Conclusion】At present, the resistance frequency of E. necator to pyraclostrobin is relatively high in China, and the total resistance frequency detected by the two detection methods is about 50%, indicating a high level of resistance risk. In order to prevent and control grape powdery mildew effectively and delay the occurrence of fungicide resistance, the monitoring of the resistance level of grape powdery mildew to pyraclostrobin should be strengthened in the future, and the usage of fungicides should be optimized by mixing and rotating of fungicides, coordi-nating with agronomic measures and biological control to formulate comprehensive control strategies.