番茄灰霉病菌对杀菌剂的室内和田间抗性进化分子机制差异性解析

Because the decline of efficacy of fungicide to plant pathogen for the reason of resistance, serious losses were caused to the pesticide company and farmer. In order to delay and avoid the occurrence and development of resistance for new fungicides, it is necessary to carry out resistance risk assessment before the application, and to develop resistance risk management measures. Based on the previous research, the molecular mechanism of resistance among resistant mutants screened in laboratory and field resistant strains was differently sharp, which seriously affect the accuracy of resistance risk assessment. This project intends to use Botrytis cinerea as test pathogen, and three fungicides azoxystrobin, pyrisoxazole, boscalid as test fungicide, and their resistances have been already happened in the field and the molecular mechanism is clear. The resistant mutants for these fungicides were generated through physical and chemical mutagenesis, repeat selection with fungicide in vitro and in vivo. The fitness and molecular mechanism for these mutants and field resistant isolates were studied and compared to identify the difference of molecular evolution mechanism for laboratory and field resistance. Finally, find a method to screen resistant mutants in laboratory which with the same mechanism to field isolates. This study can improve the scientific aspects and accuracy of fungicide resistance risk assessment, and is benefit to understanding the fungicide resistance evolution mechanism in the field, so the production of this project has important practical significance and theoretical value.

由于植物病原菌抗药性导致杀菌剂防效下降，给农药研发者和使用者造成了严重损失。为延缓和避免新杀菌剂抗性的发生和发展，有必要在田间应用之前开展抗性风险评估，并制定抗性风险管理措施。前期研究发现，室内筛选获得的抗药性突变体与田间抗性菌株的抗性分子机制差异显著，严重影响抗性风险评估的准确性。本项目拟以番茄灰霉病菌作为供试菌，选择田间已出现抗性且抗性分子机制明确的嘧菌酯、啶菌恶唑及啶菌酰胺为供试药剂，通过物理和化学诱变，以及离体和活体下药剂驯化等方法获取抗药突变体，研究其适合度、克隆分析抗性相关基因，并与田间抗性菌株进行比较，解析番茄灰霉病菌对上述3种杀菌剂在室内和田间条件下的抗性进化分子机制差异，明确一种能获得与田间抗性菌株进化方式相近或相同的抗药突变体筛选方法。本研究对提高杀菌剂抗性风险评估的科学性和准确性，探明杀菌剂田间抗性进化机制，具有重要的生产实践意义和理论参考价值。

为筛选获得与田间抗性菌株的抗性机制相近和适合度相当的室内抗药性突变体的最适方法，本项目以灰霉病菌作为供试菌，以灰霉病防治中常用的三种药剂嘧菌酯、啶菌恶唑及啶菌酰胺为供试药剂，通过不同浓度的药剂训化、紫外诱变和诱变剂诱变方法，获取灰霉病菌对不同药剂的抗性突变体，研究其适合度和抗性分子机制。从田间采集灰霉菌株，检测其对三种药剂敏感性，选择不同抗性水平的突变体开展适合度和抗性机制研究。综合分析室内诱导抗性突变体和田间抗性菌株的适合度和分子抗性机制，结果表明对于嘧菌酯，采用药剂训化方法诱导抗药突变体，其适合度和抗性机制与田间抗性菌株相近；紫外诱变和诱变剂诱变获得菌株竞争力显著低于亲本菌株。对于啶酰菌胺药剂，采用低-中等抑制浓度进行药剂训化，可获得与田间抗性菌株的适合度和抗性机制相近的突变体。对于啶菌恶唑无论是室内还是田间都难以获得抗性菌株，说明该类药剂具有较低的抗性风险。同时发现药剂训化的代数是病原菌抗药性发展的主要因素。田间抗性菌株检测发现，对嘧菌酯和啶酰菌胺同时产生抗性的菌株普遍存在。通过交互抗药性研究，嘧菌酯、啶菌恶唑和啶酰菌胺之间无交互抗性，但是嘧菌酯与QoIs的其它药剂，啶酰菌胺与萎锈灵（SDHIs）之间有正交互抗药性。因此当田间病菌对嘧菌酯和啶酰菌胺同时产生抗性时，可以使用啶菌恶唑有效控制病害的发生与发展。灰霉病菌对嘧菌酯类杀菌剂产生抗性，主要与Cytb基因的143位氨基酸突变（G143A）有关。对于啶酰菌胺，其抗性与琥珀酸脱氢酶的多个基因点突变（SdhBh和SdhD）有关，也发现了未发生靶标基因点突变的啶酰菌胺抗性菌株。多抗菌株的抗性机制与运输体基因过量表达也有关系。本研究明确一种能获得与田间抗性菌株进化方式相近或相同的抗药突变体筛选方法。本研究对提高杀菌剂抗性风险评估的科学性和准确性，探明杀菌剂田间抗性进化机制，具有重要的生产实践意义和理论参考价值。