葡萄霜霉菌对仿生杀菌剂缬霉威的敏感基线及其抗性突变体生物学性状的研究

Baseline sensitivity of Plasmopara viticola to iprovaiicarb and characteristics of its iprovalicarb-resistant mutants

本文对葡萄霜霉菌对缬霉威的抗性风险评估进行了研究。采集尚未使用过缬霉威的105个葡萄霜霉菌株,建立其对缬霉威的敏感基线。葡萄霜霉菌对缬霉威的有效抑制中浓度EC_(50)为0.04~0.60μg·mL^(-1),其敏感性频率符合正态分布,表明这105株葡萄霜霉菌对缬霉威均表现敏感。取其中10株敏感菌的孢子囊经紫外线(UV)诱导,获得4个缬霉威抗性突变体,突变频率为6.8×10^(-7)。将这10株葡萄霜霉菌孢子囊在叶片上进行缬霉威药剂驯化处理获得7个抗性突变体。11个抗性突变体的EC_(50)值为4.37~14.11μg·mL^(-1),抗性水平17.4~71.6。在未用药处理的健康葡萄叶片上继代培养10代后,这11个抗性突变体抗性水平保持在17.1~71.0。其中药剂驯化获得的7个抗性突变体和1个紫外诱导获得的抗性突变体的抗性水平均表现稳定遗传。但是,其余3个紫外诱导获得的抗性突变体抗性水平明显下降。与各亲本菌株进行比较,11个抗性突变体的潜育期、侵染率、病斑扩展速度和产孢能力都有显著不同。其中2个药剂驯化获得的抗性突变体和3个紫外诱导获得的抗性突变体表现潜育期变长,2个药剂驯化抗性突变体和1个紫外诱导抗性突变体表现侵染率下降。表明在一些情况下葡萄霜霉菌抗性突变体的潜伏期和侵染率会发生改变。药剂驯化获得的抗性突变体产孢能力存在显著差异,但与抗性突变与否无关。缬霉威与嘧菌醋、甲霜灵、烯酰吗啉和霜脲氰之间无交互抗药性。推测:葡萄霜霉菌对缬霉威为中到高等抗性风险,今后应定期对葡萄霜霉菌对缬霉威的抗性发生动态进行监测。

The risk of Plasmopara viticola developing resistance to iprovalicarb was assessed under laboratory conditions in the study. Baseline sensitivity to iprovalicarb was determined by using 105 P. viticola isolates collected from grape in the open fields in China, where iprovalicarb had not been previously used for control of grape downy mildew. The effective concentrations for 50% inhibition （EC50） of sporulation ranged from 0.04 to 0.60 μg·mL-1 and followed a normal distribution, indicating that all the 105 isolates were sensitive to iprovalicarb. Sporangia of ten sensitive isolates were exposed to ultraviolet （UV） radiation, and four iprovalicarb-resistant mutants were obtained at a mutation frequency of 6.8 × 10-7. In addition, seven mutants resistant to iprovalicarb were obtained from ten isolates by sporangia adaptation to iprovalicarb-treated grape leaves. The EC50 values for all the eleven iprovalicarb-resistant mutants ranged from 4.37 to 14.11 μg·mL-1 with average resistance factors of 17.4 to 71.6. The mutants were cultured on fungicide-free grape leaves for 10 generations, and all the eleven resistant mutants remained resistant to iprovalicarb with average resistance factors of 17.1 to 71.0. Seven resistant mutants from iprovalicarb selection and one resistant mutant from UV mutagenesis exhibited stable resistance. However, resistance of the other three resistant mutants from UV radiation to iprovalicarb was significantly reduced. Compared to their corresponding sensitive parents, the eleven resistant mutants exhibited diversity in latent period, infection frequency （IF）, lesion extension and sporulation ability. Two resistant mutants from iprovalicarb selection and three resistant mutants from UV radiation exhibited prolonged latent period. The two resistant mutants from iprovalicarb selection and one resistant mutant from UV radiation showed decreased IF compared to their corresponding parents, indicating that in some cases, resistance mutation might affect the latent period and IF of P. viticola. The resistant mutants from iprovalicarb selection displayed significant differences in sporangial production, however, the differences were not associated with the iprovalicarb-resistant mutation. Cross-resistance was not detected between iprovalicarb and azoxystrobin, metalaxyl, dimethomorph, or cymoxanil. Taken together, there could be a moderate to high risk for the field P. viticola populations developing resistance to iprovalicarb, and the P. viticola populations should be monitored regularly for their shift of sensitivity over years of iprovalicarb application.