番茄根际劳尔氏属菌(Ralstonia spp.)种间竞争机制研究

Rhizosphere competition of plant growth promoting rhizobacterium (PGPR) with soilborne phytopathogens, such as Ralstonia solanacearum (Rs), the causative agent of bacterial wilt of tomato (BWT) is an important prerequisite for efficient disease suppression. Our previous study showed that pre-inoculation of tomato plants with Ralstonia pickettii strain QL-A6 (Rp) successfully reduced disease incidence of BWT and rhizosphere populations of Rs. Further study showed that Rp colonized as aggressively as Rs in tomato rhizosphere, indicating intense interspecific competitions, which includes exploitation competition (spatial and nutritional competitions) and interference competition (antagonism) may occur between Rp and Rs. To illustrate the interspecific competitions, first Rp and Rs strains will be tagged with green and red fluorescent proteins, respectively, which facilitate us to directly investigate their distributions in/on tomato (c.v. Micro-tom) root systems using a confocal laser scanning microscopy. The effects of inoculation rates (Rp:Rs) designed using de Wit replacement series model on disease incidence of BWT will be studied in a gnotobiotic microcosm and their populations in tomato rhizosphere will be assessed by combining of using a modified semi-selective medium (M-SMSA) and a fluorospectro photometer. Second, 52 chemical carbons previously reported to be existing in tomato tissue will be selected and assumed as components of tomato root exudates. The abilities of sole carbon utilization of Rp and Rs charactered through tetrazolium reduction reaction will be analyzed by using microplates- ELIASA system.The ability of competition between Rp and Rs for the carbon sources that can be utilized by both strains as well as tomato root exudates will be quantified by using the fluorospectro photometer. Third, the effects of cell-free liquid fermentation of Rp cultivated using various carbons and tomato root exudates as sole energy source on the growth of Rs will be evaluated by using the microplates- ELIASA system. Through the second and third steps we may clarify the key carbon sources that are responsible to nutritional or antagonistic competition between Rp and Rs. Last, we check whether these key carbon sources exist in tomato root exudates by using the High Performance Liquid Chromatography. The results of this study will clarify the interspecific competition between Rp and Rs in tomato rhizosphere and supply a technique support for improving the rhizocompetence and biocontrol efficacy of Rp.

研究表明Ralstonia pickettii (Rp)根际定殖竞争能力强，能显著降低番茄根际土传青枯病致病菌Ralstonia solanacearum (Rs)的数量和青枯病的发病率，但Rp与Rs的根际竞争机制尚不清楚。通过对Rp和Rs分别进行绿色和红色荧光蛋白基因标记，在限菌系统中考察Rp和Rs不同接种比例处理的发病率，利用荧光分光光度计和激光共聚焦显微镜等手段研究两者在番茄根际的定殖数量和分布规律，探明两者的竞争位点；用化学碳源模拟番茄根系分泌物组分，利用微孔板-酶标仪系统研究两者利用和竞争根际分泌物的能力，以及Rp利用番茄根系分泌物产拮抗物质能力，探明两者营养或拮抗竞争的关键碳源；收集番茄不同时期根系分泌物，用高效液相色谱检测根系分泌物中存在的关键碳源种类，验证营养和拮抗竞争关系。从空间、营养和拮抗三个方面揭示Rp和Rs在番茄根际的竞争机制，为调控Rp根际竞争能力提供理论支持。

根际微生物是保护植物根系免受病原菌入侵的第一道防线，阐明根际有益微生物与病原菌竞争、抑制病原菌入侵对于制定有效的土传病害防控体系非常重要。本研究主要围绕根际有益菌与青枯菌的竞争机制这一主题，从营养竞争、拮抗竞争和空间竞争三个方面开展工作。营养竞争方面主要引入了三个生态学参数：资源利用连接度（Connectance）、资源利用嵌套性（Nestedness）和资源利用重叠度（Niche Overlap），并分析了资源浓度和环境温度对有益菌（群）与青枯菌竞争的影响；拮抗竞争方面主要采用HPLC-MS、定量PCR等定量分析检测有益菌的主要拮抗物质；空间竞争方面主要采用荧光蛋白标记方法，在限菌条件下分析有益菌和青枯菌在根系的空间分布。结果发现：.1）资源利用连接度高、内部竞争小，与青枯菌营养竞争能力强的群落（非致病性青枯菌同属菌群）能更好地抑制病原菌的入侵和病害的发生。这一发现有助于指导构建高效防控土传青枯病的有益菌群，并且可以延伸到其他病害防控。.2）资源浓度影响着微生物的生长速率，进而影响菌株及群落（非致病性青枯菌同属菌群）与青枯菌的竞争关系。这一发现使得我们可以根据环境中的资源水平，有针对性选择功能微生物进行生物防控。.3）环境温度能调节有益菌Ralstonia pickettii QL-A6和青枯菌的竞争关系和结果，温度较高时有利于青枯菌，温度较低时有利于有益菌。这一结果对田间应用生防产品起到指导作用，温度对有益菌的功能发挥起到重大影响，需要因地制宜，在恰当的时间应用生防产品。.4）有益菌Bacillus amyloliquefaciens T-5主要产生Bacillaene和Macrolactin两种抑制青枯菌的拮抗物质，这两种物质的产生受根系分泌物的种类和多样性影响。这一研究对开发高效生物源拮抗型生防产品具有重要意义，尤其是碳源对生防菌拮抗物质产生的影响研究可以用于指导后续规模化发酵生产。.5）通过荧光蛋白标记发现有益菌B. amyloliquefaciens T-5能在根表能形成生物膜,与青枯菌很好地进行空间竞争。这一研究直接证明了生防菌在根际与青枯菌竞争分布特征，为产品功能机制的描述提供重要证据。