三唑类手性杀菌剂对映体选择性调控镰刀菌产真菌毒素及机理研究

Mycotoxins, which seriously endanger to the safety of agricultural products, are kinds of toxic secondary metabolites produced by some filamentous fungi. Chiral triazole fungicides were widely used to control the crop fungal diseases. The enantiomers often show different bioactivity to the target fungi. However, it is no reports about enantiomers can selectively regulate the production of mycotoxins by toxigenic fungi. Our study showed that tebuconazole and myclobutanil enantiomers had different bioactivity to Fusarium graminearum and F. moniliforme and could selectively effect on mycotoxins production. In this study, two important toxigenic pathogens Fusarium graminearum and F. moniliforme are selected to study as target strains. Firstly, stereoselective bioactivity of enantiomers to two toxigenic Fusarium spp. and selective affect on production of mycotoxins are determined. The in vivo changes of the strains, the expression and interaction of genes and proteins related to mycotoxin synthesis, and the docking of enantiomers and proteins were studied to clarify the mechanism of selective regulation by means of metabolomic, genomics and proteomics methods. The most important significance of the study is the first systematic studies on chiral triazole fungicides enantiomers differentially regulate the mycotoxins synthesis by toxigenic fungi. The results will provide more informations about the mechanism of mycotoxin production and provide new ideas for control the mycotoxins contamination in agricultural products and theoretical basis for screening safe and efficient fungicides, and provide more datas for the risk assessment of chiral triazole fungicides and mycotoxins in agro-products. It is very important theoretical and practical significance to ensure the safety of agricultural products.

真菌毒素是由某些丝状真菌产生的有毒代谢产物，严重危及农产品安全。三唑类手性杀菌剂广泛用于真菌病害防治，其对映体具有选择性活性，但是否选择性调控真菌产毒素尚不清楚。项目前期研究表明，戊唑醇、腈菌唑对映体会选择性影响镰刀菌产真菌毒素。基于此，本项目选取重要产毒性病原禾谷镰刀菌和串珠镰刀菌为目标菌株，在测定三唑类杀菌剂对映体生物活性基础上，结合环境因素开展对映体选择调控镰刀菌产毒素研究，采用组学等技术，研究菌株氧化应激、毒素合成相关基因和蛋白的表达与互作、特定蛋白与对映体结合等，结合代谢产物分析，阐明选择性调控机制。本项目创新之处在于首次系统研究三唑类手性杀菌剂对映体选择性调控真菌产毒素及其机制。项目结果将有助于深入解析真菌毒素合成机制，为真菌毒素污染控制提供新思路，为筛选安全高效杀菌剂提供理论指导，完善三唑类手性杀菌剂和真菌毒素风险评估数据，对于确保农产品安全具有重要的理论和现实意义。

手性农药对映体往往在活性、毒性、环境行为等方面存在差异。目前，我国市场上的手性农药比例约占40%，且大部分手性农药仍以外消旋体形式生产和销售。三唑类杀菌剂是目前世界上广泛应用的杀菌剂，而绝大部分三唑类杀菌剂属于手性农药。三唑类手性杀菌剂常用于防治由镰刀菌引起的作物病害，而某些镰刀菌如串珠镰刀菌、禾谷镰刀菌等在侵染农作物时还会产生真菌毒素。但三唑类手性杀菌剂对映体是否差异调控镰刀菌产真菌毒素尚不清楚。本项目首先研究三种典型手性三唑类杀菌剂叶菌唑、粉唑醇和丙硫菌唑对产毒性病原串珠镰刀菌和禾谷镰刀菌的生物活性。活性结果发现，三种三唑类杀菌剂对映体在不同培养条件下表现不同的生物活性，且对映体之间具有活性差异，具体为（1S,5R）-叶菌唑>外消旋体>（1R,5R）-叶菌唑≈（1S,5S）-叶菌唑>（1R,5S）-叶菌唑，R-(-)-粉唑醇高于S-(+)-粉唑醇，R-(-)-丙硫菌唑高于S-(+)-丙硫菌唑。基于生物活性结果，研究了不同浓度对映体在不同培养条件下对串珠镰刀菌和禾谷镰刀菌产真菌毒素的差异影响。结果表明，三种三唑类杀菌剂对映体可抑制或诱导镰刀菌产生真菌毒素，且表现出对映体之间的差异，但与培养条件如药剂浓度、温度、水活度等有关。相关基因表达分析发现，对映体可能通过差异影响基因表达，从而差异影响真菌毒素的产生。最后，从转录组和代谢组二个方面解析了丙硫菌唑对映体差异影响禾谷镰刀菌合成DON的机理；筛选到部分与致病性、生长和产毒素能力相关的差异基因，并进行了初步验证。项目结果为筛选高效安全杀菌剂提供了基础数据与理论参考，丰富了真菌毒素合成机制，为真菌毒素污染控制提供新思路，完善了三唑类手性杀菌剂和真菌毒素风险评估数据。