高效PGPR菌群构建及其稳定抵御青枯菌入侵番茄根际的机制研究

Biodiversity is widely recognized as a crucial driver of ecosystem functioning. Previous studies have shown that diverse plant growth-promoting rhizobacteria (PGPR) communities might be more efficient than species-poor assemblages in biocontrol of soilborne phytopathogen, such as Ralstonia solanacearum (Rs), the cause of bacterial wilt of tomato (BWT) disease. However, the mechanisms of stable resistances to pathogen invasion by PGPR communities have not been clearly understood. In present study eight Rs-antagonistic PGPRs isolated from the rhizosphere soils of tomato plants in field with a high disease incidence of BWT and an Rs strain QL-Rs1115 tagged with red fluorescent protein (Rs-rfp) were employed to understand their interactions, especially the their competition for root exudates in tomato rhizosphere. Forty Eight PGPR communities with diverse community gradients (1, 2, 4 and 8) used in the following experiments were designed from the 8 PGPRs by using stick broken model. Tomato (c.v. Micro-tom) will grow in a gnotobiotic microcosm and the root exudate will be collected at different growth stages. The components of tomato root exudate will further be identified by using HPLC-MS and Amino Acid Analyzer. Forty eight chemical carbons previously reported to be existing in tomato tissue will be selected and assumed as components of tomato root exudates. First, the carbon (tomato root exudate and chemical carbons) utilization characteristics (carbon utilization spectra, carbon utilization rate and antibiotics production) of PGPRs will be analyzed by using microplates- ELIASA system.With these data we can further analyze the relationship between which characteristics contribute more to community resistance to Rs invasion. Second, the effects of culture temperature, pH, carbon source amount and diversity and Rs inoculation rate on the stability of community resistance to Rs invasion will be evaluated by using microplates- ELIASA system.Third, we will gnotobiotic microcosm system to assess the effects of diverse PGPR communities on the biomass and morbidity of tomato invaded by Rs. Quantitative PCR and PCR-DGGE technique will be employed to evaluate the dynamic changes of Rs population and bacterial communities in tomato rhizosphere. Finally, based on the results of the above 3 experiments, we will choose a group of potential PGPR consortia who perform better in resistance to Rs invasion. Then we will carry out field experiments to test the stabilities of PGPR consortia in controlling BWT at different growth crop seasons. The results of this study will clarify the interation (root exudates competition) between PGPR communities and Rs in tomato rhizosphere and supply a technique support for construction of PGPR consortia which can efficiently control BWT under field condition.

研究发现PGPR群落多样性有利于提高防控土传病害的效率，但PGPR菌群稳定抵御病原菌入侵的机制尚不清楚。本研究以从番茄根际土壤获得的8株具有拮抗土传青枯病致病菌（RS）能力的PGPR为材料，用断棒模型设计不同丰富度水平的PGPR群落，（1）通过微孔板系统研究PGPR菌群利用番茄根系分泌物（基质培养收集和化学碳源模拟）的特征（碳源利用谱、碳源利用效率、产拮抗物质能力），分析这些特征与群落入侵抵抗力之间的关系；（2）通过微孔板系统研究温度、pH、碳源总量、碳源种类及RS种群数量的变化对群落入侵抵抗力的影响；（3）通过限菌微宇宙系统研究PGPR群落对番茄生长和发病率的影响，用定量PCR和PCR-DGGE技术研究RS入侵对PGPR群落演替的影响；（4）筛选出高效的PGPR菌群，田间条件下研究其防控土传青枯病的效果和作用机制。为构建高效PGPR菌群及阐明其抵御RS入侵番茄根系的机制提供理论依据。

根际土著有益微生物的多样性以及它们之间的互作关系以各种方式影响病原菌入侵的结果和植物健康，且这些互作关系易受环境因素影响，从而影响有益菌群落功能。阐明根际有益微生物间互作关系，以及不同互作关系和不同多样性的群落抑制病原菌入侵对于设计高效抑制植物病害的微生物群落十分重要。本研究主要围绕构建高效抑制青枯菌的有益菌群落这一主题，从有益菌群落多样性与抑病、有益菌间互作关系与抑病和有益菌群落功能的稳定性三个方面开展工作。研究了有益菌群落的不同多样性（1，2，4，8株菌）和有益菌间不同类型的相互作用（便利型和对抗型）对病原青枯菌入侵的影响，以及资源浓度对有益菌群落抵抗青枯菌入侵的调控。研究结果发现：.1）随着有益菌群（8株荧光假单胞菌）丰富度的增加，有益菌群在植物根际的定殖能力和对植株青枯病的抑制能力都有所增加。项目构建的最佳有益菌群在资源利用上具有互补效应，在拮抗物质合成过程中具有协同效应。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）与促进型土著微生物群落相比，对抗型土著微生物群落抑制了病原菌的入侵和病害的发生。而且利用简单群落成对相互作用能很好预测多物种复杂群落的互作关系以及对病原菌入侵的抵抗能力。这一结果对筛选有益微生物，开发高效提升根际健康的复合益生菌群产品有重要参考意义。.3）资源浓度能通过改变有益菌和青枯菌的竞争关系调节有益菌群落多样性——抗入侵关系（非致病性青枯菌同属菌群）。在资源浓度较低时，与病原菌具有高分解代谢相似性的有益菌有效抑制入侵，而在资源浓度高时，生长速度快的有益菌有效抑制入侵。这一发现对于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具有指导意义。.4）增加接种荧光假单胞菌群落的丰富度明显增加了植物生物量和植物组织对各种重要营养物质的吸收同化。这一研究结果为将来环境友好型农业和作物生产的发展提供了基础。