我国葡萄白粉病菌对吡唑醚菌酯的敏感性分析

Sensitivity analysis of Erysiphe necator to pyraclostrobin in China

【目的】建立我国葡萄白粉病菌对QoI类杀菌剂吡唑醚菌酯的敏感基线,明确葡萄白粉病菌对吡唑醚菌酯的抗性水平。【方法】采用孢子萌发法测定了吡唑醚菌酯对来自我国北京、湖南、宁夏、云南、江苏5个葡萄种植地区的145株葡萄白粉病菌的EC_(50)值,并利用荧光定量PCR法检测cytb基因上G143A的突变情况。【结果】吡唑醚菌酯对43株当年未施用过化学杀菌剂的葡萄白粉病菌菌株EC_(50)值呈连续性单峰且符合正态分布,其均值(1.387±0.107)μg·mL^(-1)作为葡萄白粉病菌对吡唑醚菌酯的敏感基线。采用孢子萌发法分析的结果表明,供试菌株对吡唑醚菌酯的抗性频率为45.52%,采用荧光定量PCR(G143A)法分析的结果显示供试菌株对QoI类杀菌剂的抗性频率为57.69%。在RF>2时,采用荧光定量PCR检测结果与孢子萌发法测定结果趋势一致,Pearson相关性分析结果显示二者存在显著相关性。【结论】我国葡萄白粉病菌对吡唑醚菌酯的抗药性发生较为普遍,应注意与不同作用机制的药剂轮换使用或混用,以延缓抗药性发展。

【Objective】Grape powdery mildew(Erysiphe necator)is a worldwide fungal disease occurring 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.However,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 mildew 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 purified from 5 areas in Beijing,Hunan,Ningxia,Yunnan and Jiangsu.Among them,the 51 isolates collected 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 EC_(50) value was determined by the spore germination method,The conidia were spread onto a serial concentration gradient(0.1,0.5,1,5,10,25,50,100μg·mL^(-1))of pyraclostrobin-containing plates with 100μg·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 universal reverse primers.The DNA of grape powdery mildew was diluted to 5-10 ng·μL^(-1) for detection.The EC_(50) value of each isolate and the percentage of G143A in total mitochondrial DNA were calculated using Microsoft Excle2016 software.For the determination of EC_(50) values of the 51 isolates,the normality test was carried out using the Shapiro-Wilk method of SPSS software to establish the sensitive baseline of E.necator to pyraclostrobin,and classify the level of E.necator resistance in China.【Results】The EC_(50) range of the 51 isolates against pyraclostrobin was 0.034-13.219μg·mL^(-1).Among them,the EC_(50) values of the 43 isolates showed a continuous single peak and conformed to a normal distribution,the average value of 1.387±0.107μg·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 pyraclostrobin 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,coordinating with agronomic measures and biological control to formulate comprehensive control strategies.