根系分泌物介导的连作太子参根际互作与作用机制研究

Chinese medicinal plants including Radix Pseudostellariae L., have been shown to suffer from serious consecutive monoculture problems that cause significant decline in both yield and quality. But little is known about its underlying molecular mechanism. In this work, Radix Pseudostellariae 'Zherong 2' was used and its tissue culture seedlings were used to study the following aspects in green house: (1) The contents of the key root exudates of Radix Pseudostellariae were analyzed by using HPLC after abiotic and biotic elicitation and their selective effects on the growth of the obligate harmful and beneficial bacteria were determined. (2) Drop assay and swarm plate assay were used to screen rhizospheric bacteria with chemotactic response. The root pathogenicity of these strains was tested and their effects on the release of Radix Pseudostellariae key root exudates were detected. (3) Agar disk diffusion method was used to study the changes in Pseudomonas sp. antagonistic activity against Fusarium oxysporum mediated by root exudates. As mentioned above, this study was aimed to uncover the underlying molecular ecological mechanism of Radix Pseudostellariae consecutive monoculture problems and provide a theoretical basis for solving replant disease existing in Chinese medicine materials production.

连作障碍问题长期困扰中药农业生产，其化感自毒作用的分子机理至今未解。本研究以"柘荣太子参2号"为材料，构建其无菌快繁体系，拟在室内模拟连作环境，主要研究：（1）利用HPLC技术分析连作胁迫下太子参根系分泌物组分含量变化，锁定关键分泌物并分析其对已获得专化型有害菌、有益菌生长选择性促进或抑制的生态效应。（2）运用滴定分析法（Drop assay）、群游扩散平板法（Swarm plate assay）筛选连作太子参根际趋化性微生物并分析其对太子参组培苗、盆栽脱毒苗的致病性，同时验证分析它们对太子参根系关键分泌物释放量的影响。（3）借助平板对峙试验分析太子参根系分泌物介导下有益菌-病原菌互作关系变化，探讨拮抗菌抑菌能力减弱而病原菌生长受促进的根际生态学过程。综上，以揭示太子参连作障碍形成的分子生态学机理，为解决中药材生产中连作障碍难题提供理论依据。

太子参为石竹科多年生草本植物，药用价值高，市场需求大，但其栽培中存在严重的连作障碍问题，连作导致产量品质严重下降，病虫害猖獗，探索太子参连作障碍形成的机制及其消减措施意义重大。本项目根据研究计划，经三年研究，完成了申请书提出的研究内容，具体结果如下：首先采用HPLC技术分析胁迫下太子参根系分泌物组分含量变化，发现太子参病原菌即专化型尖孢镰刀菌细胞壁诱导因子和几丁质糖处理均能明显调控根系分泌物中酚酸类、有机酸类物质的合成与释放，尤其是对多数酚酸类化合物的合成释放起促进作用，同时发现关键分泌物如酚酸类、有机酸类物质对课题组前期分离的专化型有害菌和有益菌生长有选择性促抑的不同生态效应。进一步，本项目运用滴定分析法、群游扩散平板法筛选连作太子参根际趋化性微生物，发现头茬、重茬太子参根际土壤经富集培养后趋化菌菌群差异较大，头茬太子参根际主要筛选到短小芽孢杆菌、巨大芽孢杆菌、新洋葱伯克氏菌等常见报道的有益菌，而重茬太子参根际主要筛选到Enterobacter sacchari、Enterobacter hormaechei、Enterobacter sp.等肠杆菌属或肠杆菌目的细菌，而且大部分肠杆菌对太子参组培苗、盆栽苗有较强的致病性。研究还发现特异趋化病原菌如Enterobacter sacchari处理下太子参根系分泌物中多数抗菌物质包括酚酸类、有机酸类物质的合成释放增强，然而Enterobacter sacchari对模拟连作环境的混合酚酸、混合有机酸均表现出较强的趋化特性。除此之外，发现Enterobacter sacchari能够利用香兰素这一酚酸底物代谢产生3,4-二羟基苯甲酸，该代谢产物却能显著抑制有益菌短小芽孢杆菌的生长。最后，借助平板对峙试验分析太子参根系分泌物介导下有益菌-病原菌互作关系变化，发现关键分泌物酚酸类物质介导下拮抗菌如Pseudomonas protegens、新洋葱伯克氏菌等对专化型病原菌尖孢镰刀菌的抑菌能力减弱，这促进了重茬土壤中病原菌的生长繁殖。综上，本项目深入揭示了太子参根系分泌物介导下植物-微生物、微生物-微生物根际互作及其参与连作障碍形成的根际生物学过程与机制，本研究成果可为最终实现有效控制药用植物连作障碍问题提供理论依据。