币斑病菌对咯菌腈和异菌脲抗性分子机制及其调控路径研究

Fludioxonil and Iprodione are widely used to control many agricultural diseases, the mechanism of resistance and action of the two fungicdes are not very clear, although they have been put on the market for several decades. We obtained three resistant types (resistance to fludioxonil only, resistance to iprodione only and resistance to both fungicides) in field isolates of Sclerotinia homoeocarpa from turfgrass based on previous study, field isolates resistance to fludioxonil only and the two fungicides were never reported in S. homoeocarpa. In this project, methods of biological phenotypes analysis, protoplast transformation and comparative transcriptomics were combined to understand phenotypes, gene loci and gene expresion changes related to fungicide resistance, clarify the molecular mechanisms of resistance in the three resistant types, and furthermore, transcriptional profiles of the transformants carrying the resistant genes of the above three resistant types were analysed to explore key regulatory factors responsible for resistance development, in order to clarify their different regulatory pathways associated with resistance. The expected results will not only lay scienctific basis for quick dection of field resistance in S. homoeocarpa populations and illustrating the action mechanisms of fludioxonil and iprodione, but also provide new ideas for designing a series of drugs with novel molecular targets.

咯菌腈和异菌脲在农业生产中被广泛地用于多种病害的防治，虽然已投入市场多年，但其抗性机制和作用机制仍不明确。本研究前期获得分别仅对咯菌腈和异菌脲抗性以及同时对两种杀菌剂高抗的三种类型草坪草币斑病菌田间抗性菌株，其中对咯菌腈单抗和对两种杀菌剂双抗的类型尚未在币斑病菌上报道。本项目拟在此基础上采用生物学表型分析、原生质体转化以及转录组学等研究手段和技术，明确与抗性相关的表型、基因位点及基因表达水平变化，系统阐明三种抗性类型币斑病菌对两种杀菌剂的抗性分子机制，进一步分析上述三种类型抗性转化子转录表达谱，探寻抗性形成的关键调控因子，明确其调控路径是否存在差异。以上研究结果将为币斑病菌田间抗性的快速诊断及进一步揭示咯菌腈和异菌脲的作用机制奠定科学基础，为系列以分子靶标为导向的药物设计与创制提供新思路。

币斑病是造成草坪上经济损失最为严重的一种病害，化学防治仍然是币斑病防治的主要方式，当前我国草坪草币斑病菌对杀菌剂的抗药性现状尚不明确，基于此，本项目开展了系列工作，（1）明确了中国草坪草币斑病菌对杀菌剂抗药性发展态势，结果显示中国不同地区币斑病菌对甲基硫菌灵已产生一定程度的抗药性，对异菌脲和丙环唑则均呈现出敏感性下降的趋势；且田间存在多药抗药性问题，需要密切关注抗药性发展动态；（2）建立了一种币斑病菌高质量DNA提取方法，该方法可有效应用于常规PCR扩增、基因组测序等领域。通过使用MM培养基降低币斑病菌菌丝组织中的多糖含量，从而提高病原菌基因组DNA的提取质量，无需昂贵的仪器设备或DNA提取试剂盒，DNA提取质量显著提高、稳定性好且经济高效；（3）建立了一种新的鉴别浓度体系，可用于准确区分对甲基硫菌灵敏感、中抗和高抗的币斑病菌。国际上首次揭示了中抗菌株是在β-tubulin基因的200位发生了TTC-TAC的突变。根据这些突变，本研究还设计了AS-PCR引物，用于特异性扩增这些点突变引起的抗药性。研究结果将有助于加快币斑病菌田间抗药性的检测效率，为延缓抗药性发展提供技术支持；（4）建立了币斑病菌对咯菌腈的敏感基线，可用于币斑病菌对咯菌腈的抗药性监测。采用药剂驯化的方法筛选获得币斑病菌对异菌脲和咯菌腈的抗性突变体。突变体生物学性状研究表明，币斑病菌对咯菌腈具有低等抗性风险。克隆并比对了突变体与其亲本菌株的Shos1的基因序列，结果显示该基因上存在13bp的碱基缺失可能与抗药性相关，进一步通过原生质体转化验证了其与抗药性的关系。币斑病菌对两种药剂还存在其它未知的抗性分子机制，需要进一步研究以探明。