辣椒疫霉对氟吡菌胺抗性分子机制研究

Plant pathogenic oomycetes could cause many devastating diseases. Currently, chemical control is still one of the most effective methods to control oomycete diseases. However, many systemic fungicides against oomycetes have generated serious resistance problems because of irrational and frequent applying in fields. Therefore, it is imperative to develop new classes of fungicides with new mode of action to control oomycete diseases and to manage resistance problems. Fluopicolide, as a novel benzamide class fungicide, has distinctive mode of action and exhibits a high bioactivity on oomycetes, it affects several stages of the oomycete life cycle such as the release and motility of zoospores, germination of cysts, mycelial growth as well as sporulation. Until now, in the study of interaction between the fungicide and oomycete, there is no any research report about the resistance molecular mechanism of the fungicide, the target’s gene and the exact action sites of target’s protein are unknown as well. The application of High-throughput sequencing provide new experimental considerations and effective method for this study. Based on the above, this project aims to: (i) obtain the fluopicolide-resistant mutants with different resistance factor in Phytophthora capsici by repeat selection on fluopicolide-amended plates, (ii) compare the genetic information distinctions between fluopicolide-resistant mutants and their parent isolate through Whole-genome resequencing, and identification of resistance related genes and validation by protoplast genetic transformation, scientifically clarify resistance molecular mechanism of P. capsici to fluopicolide. This project could provide a technical and academic support for rational use of fluopicolide, delaying emergence and development of resistance and further revealing the mechanism of action.

植物病原卵菌可导致多种毁灭性病害，目前化学防治仍是最为有效的防治方法之一。由于一些内吸性杀菌剂不合理频繁使用，许多卵菌已对其产生严重抗药性，亟需开发全新作用机制药剂用于防治和抗药性治理。氟吡菌胺是具有独特作用机理的新型苯甲酰胺类杀菌剂，对卵菌具有较好的抑制作用，可影响游动孢子释放、孢子萌发、菌丝生长、孢子囊产生等各生长阶段。在药剂与卵菌互作研究中，迄今该药剂的靶标蛋白基因、作用位点以及卵菌的抗药性分子机制尚不清楚，而高通量测序技术应用，为此研究提供了新的思路和手段。本项目拟针对上述内容开展研究，通过药剂驯化筛选获得辣椒疫霉对氟吡菌胺不同抗性水平突变体，应用全基因组重测序方法比较抗性突变体与亲本间遗传信息差异，确定抗性相关基因并通过原生质体转化进行验证，初步阐明其抗性分子机制，为田间合理用药、延缓抗药性产生和发展以及进一步揭示药剂作用机理提供技术和理论支持。

辣椒疫霉世界范围内广泛存在，可引起茄科、葫芦科等多种植物的疫病发生。化学防治仍然是目前生产上用于疫病防治的最主要措施之一。氟吡菌胺用于疫病等多种卵菌病害防治，目前该药剂抗性风险、靶标蛋白及抗性分子机制鲜有研究报道。本项目开展相关研究并取得如下结果：1、测定146株田间菌株对氟吡菌胺敏感性，供试菌株的平均EC50值为0.17 µg/mL，EC50值范围为0.07-0.34 µg/mL，敏感性频率分布呈单峰分布，表明自然条件下未检测到辣椒疫霉对氟吡菌胺的抗性亚群体。本研究获得的平均EC50值0.17 µg/mL可用于辣椒疫霉对氟吡菌胺的抗性检测和监测；2、通过菌饼药剂驯化方法，从6株辣椒疫霉敏感菌株中驯化获得了44株抗性突变体菌株，抗性突变频率为10-4，抗性突变体菌株继代培养10代后，仍保持相对稳定抗性倍数。生存适合度研究结果表明，抗性突变体与亲本敏感菌株相比，在菌丝生长、孢子囊产生、休止孢萌发和致病力等不同发育阶段均表现出较强的生存适合度。交互抗药性结果显示，氟吡菌胺与9种不同作用机制的卵菌杀菌剂之间不存在交互抗药性，而与氟醚菌酰胺存在正交互抗药性，综合以上结果，表明辣椒疫霉对氟吡菌胺具有中到高度的抗性风险；3、分析并克隆了辣椒疫霉敏感、抗性菌株的3个血影蛋白α-actinin 编码基因，测序和序列比对后未发现在Spec1、Spec2、Spec3 基因上存在与抗药性相关的点突变；运用全基因组重测序的方法对敏感菌株LT1534，高抗菌株LT-5、LT-10的遗传变异情况进行分析，共筛选到1216个基因作为进一步研究的候选基因。进一步分析氟吡菌胺处理敏感菌株后差异代谢物情况，发现16种氨基酸类、10种有机酸类、6种糖类和6种其他类代谢物发生显著变化。采用转录组学方法分析氟吡菌胺处理敏感菌株LT1534及其高抗菌株LT-10的基因差异表达情况，其中抗性菌株LT-10中发生差异表达的基因为589个，显著低于敏感菌株LT1534的1713个；4、综合代谢组、转录组分析结果，推测在氟吡菌胺处理辣椒疫霉后，氟吡菌胺主要作用于膜结构上，使得膜通透性相关蛋白、细胞膜及骨架相关蛋白发生了显著差异表达；同时影响了氨基酸、脂肪酸类物质的代谢，也影响了细胞信号转导功能。