青枯病菌对氟啶胺的抗性机制研究

Bacterial wilt is a destructive soil-borne vascular disease, and it is lack of effective control bactericides. Fluazinam is a typical uncoupler，which has broad spectrum and commonly used, but the action site of fluazinam is unknown. The resistance mechanism of fluazinam has become a bottleneck problem because plant pathogenic fungi and oomycetes are difficult to obtain resistant strains. Preliminary experiments show that the control efficacy of fluazinam to bacterial wilt in field reached 76%~88%. Bacteria have the characteristics of more mutagenesis and evolution. One the hand that lead to bactericide use blindly and face more challenges, on the one hand it also provides good materials to study the resistance mechanism of fluazinam. In order to solve the above problems, 7 mutants with resistance factors of 7.02~62.50 times have been screened to ensure the feasibility of the project. This study will compare and screen resistant mutation sites by genome resequencing. The regulatory pathway of the target gene on resistance will be explored by transcriptional expression profile comparison between the mutants and the wild strain using transcriptome sequencing. The molecular detection method will also be established. This study will provide a new bactericide for bacterial wilt control, delaying the development of fluazinam-resistance, and also provide a reference to study the mode of action and resistance mechanism of uncoupler.

青枯病是毁灭性土传维管束病害，生产中匮乏高效的防治药剂。氟啶胺防治谱广泛，是最具代表性、生产应用最多的解偶联抑制剂，但其作用位点尚未明确，植物病原真菌、卵菌很难获得对氟啶胺的抗性菌株，成为研究其抗性机制的瓶颈。前期试验表明氟啶胺对青枯病的田间防效达76~88%，细菌更易突变、进化的特性，一方面导致药剂使用盲目，面临抗药性等诸多挑战，一方面也为氟啶胺的抗性机制研究提供了很好的试验材料。试验前期获得了7株抗性倍数在7.02~62.50倍的突变体，保障了后续研究的切实可行。本研究拟通过基因组重测序技术，比对、筛选关键候选抗性基因的突变位点，并通过RNA-seq分析抗感菌株在药剂处理前后的转录表达谱，明确基因表达量与抗性的关系。同源转化验证，解析青枯病菌对氟啶胺的抗性分子机制，建立分子检测方法。本研究将为青枯病防治提供新型药剂，延缓氟啶胺抗药性产生，也为解偶联剂的作用机制及抗性机制研究提供参考。

青枯病是毁灭性土传维管束病害，生产中匮乏高效的防治药剂。氟啶胺防治谱广泛，是最具代表性、生产应用最多的解偶联抑制剂，但尚未应用与细菌病害且其抗性机制尚未明确。项目将杀真菌药剂氟啶胺、双苯菌胺应用于细菌病害，明确了氟啶胺和双苯菌胺对10种病原细菌的抑菌效果。通过烟草青枯病菌对氟啶胺、双苯菌胺的突变体获得情况、抗感菌株的生物学性状、交互抗药性等，综合评价认为烟草青枯病菌对氟啶胺的抗性风险为中等水平，对双苯菌胺的抗性风险为低等水平，为今后药剂的科学使用及延缓抗性性产生提供了科学依据。通过转录组和蛋白组联合分析，明确了关键代谢通路与主要差异基因，ABC转运蛋白介导的药剂外排作用是导致烟草青枯病菌对氟啶胺产生抗性的主要原因，转化验证表明ATP结合蛋白RSc0164基因参与了抗性表达，并建立了基于RSc0164基因的抗性菌株qRT-PCR快速检测方法。为明确氟啶胺抗性机制及反抗性药剂开发奠定了基础。根据交互抗药性结果，将氟啶胺和双苯菌胺分别与不同作用机制药剂进行复配研究，明确了不同复配比例药剂的联合毒力作用，为烟草黑胫病防治提供更多备选药剂。