药用植物连作障碍土壤病原菌定向分子调控及其作用机制研究

The rhizosphere soils under consecutively monocultured Chinese medicinal plants of Rehmannia glutinosa in Henan province and Pseudostellariae heterophylla in Fujian province are planed to be used as the research object to explore the approach to effectively overcome the continuous cropping problems resulting from the disordered structure of microbial flora and the explosive growth of pathogenic bacterial quorum sensing in common mediated by root exudates from the two medicinal plants in the consecutive monoculture system. Accordingly, in this study, the quorum quenching (QQ) is used as a new strategy to control in-common pathogenic bacteria in the rhizosphere soil of monocultured medicinal plants. Based on the targets of the in-common pathogenic bacteria, the Quorum Sensing Inhibitor Selected in Peptide Aptamers Library (QSI-SPAL) is used to screen the QSI with high efficiency to inhibit the QS of pathogenic microorganisms. Then the molecular mechanism of quorum quenching (QQ) to pathogenic microorganisms in rhizhospere soil of medicine plants is studied by the technologies of quantitative transcriptomics and quantitative real time proteomics. And qRT-PCR is used to analyze the ecological effects of QSI selectively inhibiting the pathogenic microorganisms to further understand the rhizosphere ecological processes in which how QSI oriently regulates the structure of microbial communities after the QSI is added into rhizosphere soil of medicine plants. The result of this investigation is expected to deeply explore the molecular ecological process and its underlying mechanisms of QQ in the rhizosphere soil of medicine plants, and the resultant approach could be provided to tackle the continuous cropping problems of medicinal plants.

本项目以河南怀地黄和福建柘荣太子参根际土壤为研究对象，着力解决连作药用植物根系分泌物介导的土壤微生物结构失衡,病原微生物群体效应的“爆发式增长”而导致的连作障碍问题,采取群体感应淬灭策略,以连作土壤中存在的大量共性病原细菌为靶标，利用QS报告菌株结合肽适体文库技术，筛选高效能病原菌群体效应抑制子（QSI），通过转录组学与定量蛋白组学，探明群体效应抑制子对药用植物根际病原微生物的分子作用机制；并借用荧光定量PCR分析QSI添加到连作根际土壤后对靶标病原菌选择性抑制的生态效果，阐明QSI定向分子调控药用植物微生物群落的根际生态学过程，为深入揭示植物根际病原菌群体效应淬灭的分子生态学机制，破解中药材生产中连作障碍难题提供理论依据与技术支持。

针对太子参根际土壤中病原微生物群体效应的“爆发式增长”而导致的连作障碍问题, 本研究采取群体感应淬灭策略，建立高效筛选病原菌群体效应抑制子策略，探明其对药用植物根际病原微生物的分子作用机制。QSI策略体系构建结果表明，通过全基因组测序技术解析太子参致病菌-粘质沙雷氏菌SP95的QS系统，并基于该QS系统构建具有响应AHL的报告元件和报告基因元件的QS报告菌株，基于该报告菌株，构建噬菌体展示文库与微生物来源的群体效应抑制子（QSI）双筛选策略。噬菌体展示文库策略可以在短时内获得多个QSI候选多肽，并且适用于任何已知的QS基因调控背景的病原QS菌。微生物来源的QSI策略中微生物来源广，QSI易提取，大规模运用潜力强。侯选QSI抑制太子参连作根际土壤特异病原菌的作用机制研究的结果表明，QSI在一定程度上抑制粘质沙雷氏菌SP95产生AHL，并对其生物膜形成和涌动性均具有显著地抑制，进而影响粘质沙雷氏菌SP95的QS系统调控的相关基因表达和生物学特性。QSI的根际土壤添加与生态效应评价结果表明，QS能够阻断病原微生物的群体效应，压制病原微生物群体效应的“爆发式增长”，平衡根际土壤微生物群落结构，促进根际土壤的物质循环与能量流动。此研究不仅定向又能从根本上修复连作根际土壤环境，并提高药用植物的整体抗性，从而实现安全、生态、有效防控药用植物连作障碍的目的。