芸薹根肿菌与榨菜根系微生物组生态互作研究

The clubroot disease, caused by the obligate biotrophic protist Plasmodiophora brassicae, is the most damaging disease of tumorous stem mustard. The happening of this disease is related to the rhizosphere microbes and endophytic microbes of tumorous stem mustard. The root microbiome including rhizosphere microbes and endophytic microbes have an important role in plant growth, nutrient absorption and resistance to pathogen. In this program, the transcriptome of root microbiome is analyzed in depth and breadth by metatranscriptome combined with third generation DNA sequencing. The ribosomal RNA sequences are screened from metatranscriptome sequences to reveal the changing characteristic of diversity of root microbes. The sequences encoded protein are also screened to annotate microbial function and to locate to KEGG pathway and to reconstructure the network of metabolic pathway. The result will uncover the effect of P. brassicae infection on the diversity and function of root microbiome. Meanwhile, the microbes with biocontrol potential are also searched from the root microbiome for developing biocontrol fungicide for control clubroot. In a conclusion, the program will clarify the mechnism of clubroot disease at microbial ecology level and built a theoretical basis for controling the disease.

芸薹根肿菌侵染榨菜造成生产上重要病害-根肿病。榨菜根肿病发生与榨菜根际土壤微生物和根内生菌之间存在密切关系。植物根系微生物组包括根际土壤微生物和根内生菌，在植物生长、营养吸收和抗病过程中起着重要作用。本项目采用第三代DNA测序结合宏转录组技术对根肿菌侵染榨菜后的根系微生物组群落进行宏转录组测序，从转录组数据中筛选核糖体RNA序列分析微生物群落结构的变化特征，筛选蛋白质编码序列进行微生物群落功能注释和KEGG定位，重建微生物群落的代谢途径网络，从而揭示根肿菌侵染对榨菜根系微生物组群落结构和生态功能的影响。同时从榨菜根系微生物组中分离筛选拮抗根肿菌的生防菌，开发生防菌剂应用到根肿病防治。本项目能够在微生物生态角度阐明根肿菌暴发成灾的机制，为生态防治该病奠定理论基础。

芸薹根肿菌导致的根肿病是榨菜（茎瘤芥）的主要病害，造成榨菜严重减产。榨菜根肿病发生与榨菜根际土壤微生物和根内生菌之间存在密切关系。本项目采用第三代DNA测序Miseq技术对健康根系和根肿菌侵染的发病根系内生细菌和真菌进行研究，并筛选根肿病生防菌，应用到生产。结果表明，健康根系内生真菌群落的Alpha多样性显著高于发病根系，健康根系以Phoma、Mortierella、Penicillium和Pseudallescheria为主，发病根系以根肿菌为主。从Beta多样性来看，层次聚类分析、PCA、PCOA和ANOSIM等结果表明，健康和发病根系内生真菌群落也存在差异。从网络结构来看，健康根系内生真菌群落网络节点多，结构复杂，比发病根系内生真菌群落稳定。发病根系中可培养镰刀菌数量最多，为优势菌群。特别是导致植物病害的病原镰刀菌数量多，表明镰刀菌与根肿菌存在复合侵染现象。健康和发病根系内生细菌群落也存在差异，健康根系内生细菌以假单胞为主，其次为根瘤菌、未分类链霉菌和伦茨氏菌。发病根系内生细菌群落以假单胞、嗜甲基菌、根瘤菌、杜擀氏菌为主。从Beta多样性来看，健康和发病根系内生细菌群落差异显著，表明根肿菌侵染改变了茎瘤芥内生细菌群落。健康根系中甲醇含量为0.174 ng/g，显著低于发病根系的0.632 ng/g，表明根肿菌侵染诱导根系内甲醇含量增加。相关性分析发现嗜甲基菌与甲醇含量明显正相关（相关系数0.986），表明发病根系甲醇含量可能诱导嗜甲基菌属细菌繁殖，从而改变内生细菌群落结构。此外，本项目筛选3种对根肿病具有良好抑制效果的细菌，开发为根肿病生防菌剂。目前，本项目发表sci文章1篇，审稿1篇，获得国家发明专利3项。本项目揭示了根肿菌侵染改变茎瘤芥内生微生物群落的结构，同时获得的成果能够用于茎瘤芥根肿病的防治，具有重要的理论和实践意义。