植物根际促生菌Bacillus pumilus WP8在“土壤-植物”系统中穿梭定殖机制研究

Bacillus pumilus WP8, a plant growth-promoting rhizobacterium (PGPR), is capable of responding to changes in environment and adjusting the spaces of colonization between rhizosphere and endorhizophere, and triggering different promoting effects. We thus proposed a hypothesis that WP8 can enhance the performance of promoting effect via shuttling colonization in soil-plant system with the inducement of C/N. Thus, three issues need to be studied in this proposal. (i) How does WP8 respond to the changes in environment; (ii) How does WP8 perform the process of shuttling colonization, and (iii) what is the relationship between the spaces of colonization with the promoting effects. Based on the analysis of bacterial artificial chromosome (BAC) sequences, we plan to focus on two possible related genes, tlpC and pelB, as well as an inducer C/N. Gene knockout and complementary strategy plus GFP-labelled technology will be adopted to generate different mutants and complementary strains, with which we use to compare the spaces colonization by WP8 and analyse the relationship between the spaces of colonization with the promoting effects. Besides, the transcriptomic profilings of WP8 under the inducement of root extract and intercellular tissue fluid will be compared to uncover the molecular mechanism of the difference of promoting effects when under colonization of different spaces. This study will help to discover novel growth promoting related genes.

我们前期工作发现Bacillus pumilus WP8定殖空间因环境条件不同而迥异，并产生不同的促生效果，本项目提出“WP8响应碳氮比（C/N）在土壤-植物系统中穿梭定殖以增强促生效果”的假设，拟解答三个与此相关的问题：一是WP8如何响应环境条件变化；二是WP8如何实现穿梭定殖；三是定殖空间与促生效果的关系。根据前期工作结果，我们聚焦趋化性和果胶裂解酶相关基因（tlpC、pelB）以及环境C/N，采取敲除和回补策略、结合GFP标记技术，设置不同N素水平，考察WP8在敲除前后定殖空间的变化规律，解析土壤C/N、定殖空间与WP8促生效果的相关性，明确目标基因在WP8穿梭定殖事件中的作用及其对土壤C/N的响应。此外基于高通量转录组测序技术，探究WP8对根系分泌物和根内胞间液的转录组响应差异，解析WP8在不同定殖空间促生效果差异的分子机制。本研究为挖掘PGPR促生相关基因奠定基础。

通过本项目的实施，我们重点开展了以下研究，获得了一些原创性结果：（1）解析根际促生菌Bacillus pumilus WP8在不同C/N土壤中对植物的促生效应。明确了随着C/N升高，WP8对于番茄植株地上、地下组织的干物质量促生效应增大，C/N（15-30）土壤环境中，WP8显著提高果实产量，而在土壤C/N（45-60）环境中，该促生效应下降；（2）土壤C/N变化对WP8在“土壤-植株”的定殖空间产生影响，环境C/N升高， WP8定殖空间由植物组织根外转至根内，在植物根内和茎部定殖数量不断增加；（3）土壤C/N（15-45）环境下，WP8对植物促生效应与其在植株不同部位的定殖量相关，土壤C/N（60）环境下，没有发现相同规律；（4）在环境C/N（15-45）土壤环境中，根系分泌物显著提高了WP8生物膜形成与稳定性；（5）环境C/N升高，根系分泌物中柠檬酸浓度上升，WP8在植物根部定殖下调了根系分泌物中柠檬酸含量，诱发植物根系分泌草酸；（6）WP8在定殖过程中响应不同效应物时具有浓度依赖性，草酸诱导WP8趋化、生物膜与定殖不受其浓度的影响，高浓度（100 μM）柠檬酸及混合酸（100 μM）主要通过抑制WP8生物膜途径抑制其定殖；（7）转录组测序结果表明参与WP8趋化、芽孢形成、鞭毛马达蛋白的3个基因 cheA、spoA、motA与其定殖密切相关；（8）WP8对番茄青枯病的拮抗作用表现为抑制Ralstonia solanacearum Rs1115运动性，通过产耐高温次级代谢产物抑制Rs1115典型毒力基因phcA、phcB、egl、pehC、pilT的表达而实现。.本项目通过对根际促生菌WP8在不同定殖空间促生效果差异的研究，初步揭示了WP8 在“土壤-植物”系统中穿梭定殖机制，为 WP8农用制剂的施用策略提供理论。通过项目实施，发表SCI论文3篇，申请发明专利2项。.