施用荧光假单胞菌对羊肚菌白霉病高发土壤中微生物区系的影响及病害压制研究

The white mold caused by pathogenic Paecilomyces penicillatus is an awful disease for morel cultivation.Since there is still no effective chemical fungicide against this pathogen at present, manipulating microflora at ecosystem level to reach a disease suppressive effect is a strategy worth trying. Based on initial positive results obtained from our previous work, this project hypothesizes: For morel-cultivating soils suffering from highly prevalent white mold, enhancing the abundance of Pseudomonas fluorescens can descrease the occurrence rate of white mold in morel fruiting bodies. To validate this hypothesis, Morchella importuna, as a representative of the cultivable morel varieties, is cultivated in a soil with severe white mold occurrence for years. Living bacterium of P. fluorescens is supplemented into the soil in order to create microflora with enhanced abundance of P. fluorescens. Using high-throughput metagenomics as the major approach, soil microflora as well as fruiting body-associated microbiota are analyzed at multiple time-points. This project might be able to remedy the present situation that the highly-profitable morel cultivation is highly vulnerable to white mold disease, providing potential guidance for ecological manipulation to alleviate an “ascomycete pathogen of an ascomycete mushroom” as well as for development of applied microorganisms and ecological friendly approaches for biocontrol protecting artificially cultivated mushrooms.

长毛拟青霉引起的白霉病是羊肚菌栽培的严重病害。在暂缺有效防治药物的现状下，从生态系统层面利用微生物区系进行病害压制是值得尝试的策略。基于前期工作初步得到的正面结果，本项目提出假设：对于白霉病高发的羊肚菌栽培土壤，提高荧光假单胞菌丰度可降低羊肚菌子实体白霉病发生率。为验证假设，以梯棱羊肚菌作为羊肚菌栽培品种的代表，栽培于已连续多年发生严重白霉病的土壤中，通过施用荧光假单胞菌活菌以期构建具有更高丰度荧光假单胞菌的微生物区系。主要借助微生物宏基因组高通量测序手段解析多个时间点的土壤微生物区系和子实体伴生微生物区系，寻找与子实体白霉病发生率有关联的生态因素，探究荧光假单胞菌在梯棱羊肚菌所处微生态系统中压制长毛拟青霉的机制。本研究有望改变高经济效益但易因病害受损的羊肚菌栽培现状，为减轻一种“子囊菌蘑菇的子囊菌病害”提供田间生态控制的启示，为研发面向食用菌的生防菌株和绿色防控手段提供参考。

白霉病是严重威胁羊肚菌栽培产业的土传病害。本项目向白霉病高发的羊肚菌栽培土壤施用假单胞菌AF04菌株，以期调控土壤微生物区系结构与功能，实现对土壤中长毛拟青霉的生态压制，减轻白霉病严重程度。分别选择已栽培一茬和连续三茬栽培羊肚菌的白霉病高发土壤作为测试环境，处理组施用假单胞菌AF04活菌体，对照组施灭活菌。羊肚菌产量与病害统计结果表明：一茬土壤与三茬土壤施用假单胞菌AF04均可显著提高羊肚菌子实体单位面积产量，降低羊肚菌子实体白霉病发病率，具有病害压制效果。. 扩增子测序结果揭示：一茬土壤与三茬土壤的真菌群落中，拟青霉属相对丰度均远高于其他真菌类群。施用AF04活菌体后，栽培羊肚菌的一茬土壤和和三茬土壤均出现了拟青霉相对丰度的显著下降，而施用灭活菌体的对照组无此效果。施用AF04活菌体的土壤中，假单胞菌群体持续存在，且相对丰度较高，细菌群落中假单胞菌属相对丰度与真菌群落中拟青霉属相对丰度呈现显著负相关性。拟青霉属相对丰度与羊肚菌产量呈现显著负相关性，与羊肚菌子实体白霉病发病率呈现显著正相关性。这些耦合规律体现了通过调控土壤微生物区系对白霉病实现生态压制的有效性。. 微生物区系鸟枪法宏基因组测序结果与扩增子测序结果对于微生物区系组成变化的表现一致。功能分析结果揭示：施用AF04的土壤微生物区系与对照组相比，在羊肚菌栽培中期和出菇前后出现氨基糖与核苷酸糖代谢、链霉素合成等代谢途径丰度的显著上调。. 研究结果有望改变高经济效益但易因病害受损的羊肚菌栽培现状，为减轻一种“子囊菌蘑菇的子囊菌病害”提供田间生态控制的启示，为研发面向食用菌的生防菌株和绿色防控手段提供科学依据。发表第一/通讯作者SCI论文5篇（均为中科院一区、二区）、中文核心期刊论文7篇、中文科普文4篇，作为第一发明人获发明专利授权7项，培养5名学生。在微生物学和生态学领域国家级学会学术会议上做口头报告5次。