基于代谢组学的灰葡萄孢菌多药抗性调控机制研究

Gray mold is one of the top ten farmland diseases. The pathogen of Botrytis cinerea has a a wide range of host plants and a high hereditary variability. It is easy to produce resistance to fungicides, and even multi-drug resistance, which has directly brought serious harm to the agricultural production and posed a great deal of pressure on the environment safety. The resistance management of gray mold became a worldwide problem that plagued plant protection. Based on the regulation mechanism of efflux pump leading to multidrug resistance of B. cinerea, we plan to screen inhibitors of the efflux pump to restore the sensitivity of resistant Botrytis cinerea by blocking efflux. Furthermore, a metabolic flux based on metabolomics combined with 13C tracer will be applied to clarify metabolic pathway and target enzymes that mediate inhibition on efflux pump. It is expected that the results of this study will be able to effectively solve the problem of multidrug resistance modulated by the efflux pump in B. cinerea. The method of metabolomics depth analysis will provide a comprehensive and rapid way to explain the mechanism of efflux pump inhibitors. The management strategy of multidrug resistance created and the research results obtained in this project are expected to fill the gaps of domestic and foreign research field of multidrug resistance and enrich the application of metabolomics in fungicides research field. It will also provide theoretical basis and application reference for multidrug resistance management of gray mold and reduce pesticides with increasing efficacy.

灰霉病是农田十大病害之一，其病原菌Botrytis cinerea寄主范围广、遗传变异性强，对防治药剂极易产生多药抗性，直接给农业生产带来严重危害，对环境安全亦造成极大压力。多药抗性治理一直是灰霉病防治所面临的世界性难题。项目拟针对导致灰霉病菌多药抗性的外排泵调控机制，筛选ABC和MFS活性功能抑制剂，力求通过阻断抗性菌株对药剂的外排以恢复其敏感性；进一步通过代谢组学结合13C示踪的代谢通量分析，深度解析抑制剂对外排泵作用的代谢途径及靶标酶系。预期研究结果将有效解决外排泵介导的灰霉病多药抗性问题，代谢组学深度解析方法将为外排泵抑制剂作用机理的阐释提供全面快速的途径。创建的灰霉病多药抗性治理策略及其防病机理研究结果，有望丰富杀菌剂代谢组学和多药抗性研究体系，为灰霉病抗性治理中老药新用及农药减施增效技术提升提供理论依据和应用借鉴。

灰霉病是农田十大病害之一，其病原菌Botrytis cinerea寄主范围广、遗传变异性强，对防治药剂极易产生多药抗性，直接给农业生产带来严重危害，对环境安全亦造成极大压力。多药抗性治理一直是灰霉病防治所面临的世界性难题。项目针对导致灰霉病菌多药抗性的外排泵调控机制，建立了外排泵抑制剂的发现及活性筛选验证体系，为灰霉病防治药剂协同增效剂的发现和创制提供技术支持。并筛选获得了外排蛋白ABC活性功能抑制剂，发现了3种天然产物：法尼醇、小檗碱和利血平可对苯并咪唑、三唑类、甲氧基丙烯酸酯类、SDHI类等杀菌剂产生增效性，恢复了多药抗性灰葡萄孢对上述杀菌剂的敏感性。对多菌灵活性提升52.6倍，嘧菌酯提升11.7倍，啶酰菌胺提升15倍。研究发现，这些增效剂通过阻断抗性菌株对药剂的外排恢复了药剂的敏感性，进一步通过代谢组学、靶标蛋白分子对接和遗传转化验证等技术，探明了外排抑制剂小檗碱等通过与杀菌剂竞争外排蛋白atrB、atrD和atrK促进了杀菌剂的靶内积累，实现对灰霉病菌多药抗性有效治理的机制。发表科研论文12篇，获得了5项专利授权，并完成了专利成果的转让1项，培养硕士研究生5人。创建的灰霉病多药抗性治理策略有效解决了外排泵介导的灰霉病多药抗性问题，突破了植物病害抗性治理中只能“防”不能“治”的瓶颈，丰富了多药抗性研究体系，为从源头上治理多药抗性做出创新性贡献，为灰霉病抗性治理中老药新用及农药减施增效技术提升提供理论依据和应用借鉴，并为生产中已发生多药抗性的杀菌剂减量高效利用提供了重要依据，研究成果在果蔬灰霉病绿色防控中获得了推广应用。