噬菌体协同生防细菌抑制土传青枯菌入侵番茄根际的机制研究

The increasing demand for food supply requires more efficient control of plant diseases. Biocontrol, the use of natural antagonists or competitors, has recently re-emerged as a promising alternative to chemical pesticides. However, it is well know that pathogen quickly evolves to adapt the changing surrounding environment, especially the abiotic (e. g., chemical pesticides) and biotic (e. g., interference competition and parasitism) pressures, making it difficult to consistently reproduce the beneficial effects of biocontrol. Previous study shows that the incidence of bacterial wilt disease of tomato caused by the pathogen Ralstonia solanacearum (a soilborne bacterial pathogen) is significantly reduced by combination of R. solanacearum-antagonistic bacterium (interference competition) and R. solanacearum-specific phages (parasitism), compared with interference competition or parasitism treatment alone or the control treatment. Here we will use real-time experimental evolution to study these complex dynamics in laboratory and tomato plant rhizosphere. We will concentrate on devastating plant pathogen, R. solanacearum bacterium and three different biocontrol agents: pathogen-specific viruses (phages) and bacterial competitors, Bacillus amyloliquefaciens T-5 that show direct antagonism towards the pathogen, Ralstonia pickettii QL-A6 that could not produce antibacterial substrates and only compete fiercely from plant-derived nutrients in the rhizosphere. We will first study the effect of application of bacterial competitors alone or combining with phage on pathogen invasion both in the lab and greenhouse conditions. On the basis of the trade-offs theory, we expect the pathogen resistance evolution will be constrained by applying different biocontrol agents in combination. The evolved pathogen colonies will be isolated from different biocontrol agents’ combination treatment, and their phenotypic, physiological and genetic changes will be evaluated and compared with those of the ancestral pathogen. We expect that when facing the pressures from competitive biocontrol bacteria and parasitic phage, the pathogen R. solanacearum 1) will loss the virulence to maintain its survival in certain environment; 2) will loss the ability to be resistant to one pressure if it evolves to be resistant to this other pressure; and finally loss its ability to survive and infect host plant. We will also compare which biotic pressures (interference competition, nutrition competition and parasitism) drive pathogen evolution process more and which direction to evolve by using the transcriptome study. Finally lab, greenhouse and field experiments will be carried out to test how abiotic environment (resource level and temperature) affect the success of biocontrol outcomes. Proposed research will help to understand eco-evolutionary dynamics in complex microbial communities and offers a novel and predictive framework for applied agriculture to manipulate plant root communities in evolutionary stable manner.

土传植物病原青枯菌 Ralstonia solanacearum （RS）的环境胁迫进化适应能力强，其对生防菌产生适应性进化可能是生防产品难以稳定控病的重要原因之一。前期研究发现相对于单一措施，寄生侵染型噬菌体和竞争型生防细菌配合能有效提高土传青枯病防控效率。本研究拟从噬菌体协同生防细菌阻控RS进化适应性的角度，运用Trade-off理论，阐明两者协同高效抑病的作用机制。我们推测当RS面对双重生物胁迫时，可能1）通过减少 对致病力的投入，来维持其生存；2）对一种生物胁迫产生进化抵抗时，会降低其对另一种生物胁迫的抵抗能力；导致其整体生存或致病能力下降。拟以拮抗竞争型生防细菌、营养竞争型生防细菌和寄生侵染型噬菌体为材料，应用微生物生态学、实验进化学和转录组学等研究方法，从生理和基因水平上揭示不同生物胁迫下RS进化适应的Trade-off机制，为开发高效稳定的生防产品提供理论和技术支撑。

土传病原青枯菌引起的作物减产问题日益严重，传统化学防治方法短期见效快，但存在二次侵染的风险。前期研究表明根际噬菌体在土传病害生态防控中有巨大潜力。本项目应用微生物生态学、实验进化学和转录组学等研究方法，从生理和基因水平上探究噬菌体单独或与生防菌协同抑制青枯菌入侵的效果和机制。主要研究结果如下： .1. 从不同地区采集植株根际土壤样品，分离病原青枯菌及其专性噬菌体，进一步通过测定噬菌体-青枯菌的交互侵染探究根际噬菌体与青枯菌的互作规律。结果表明，植株根际普遍存在能够侵染青枯菌的噬菌体，噬菌体的形态呈现高度多样性，且噬菌体对不同来源青枯菌的侵染性存在显著差异。.2. 噬菌体组合显著提高了青枯病的防控效率，进一步探究其作用机制，结果发现噬菌体能够直接猎杀青枯菌，增加了病原菌对噬菌体抗性成本，降低其竞争力。同时噬菌体通过靶定宿主青枯菌，间接修复青枯菌入侵对细菌群落的影响，提高根际拮抗型有益菌的丰度。 .3. 室内和温室试验均发现噬菌体与生防菌组合显著提高青枯病的防控效果。进一步分析发现，噬菌体诱导青枯菌抗性的同时增加了青枯菌对生防菌产生的拮抗物质的敏感性。说明二者通过驱动不同抗性间的进化权衡而协同抑制青枯菌。转录组结果表明青枯菌对不同胁迫响应不同：噬菌体处理趋向于下调青枯菌相关基因的表达，生防菌的正负向调控作用相当，二者组合对青枯菌基因表达的影响由有益菌主导，具体机制还有待进一步验证。.4. 不同温度和资源均会影响噬菌体对青枯菌的抑菌抑制结果。低温、低资源条件下青枯菌表型转换率高；高温、高资源能够增强噬菌体对青枯菌表型转换的驱动。表型转换后的青枯菌生物量降低，致病能力减弱。.综上所述，根际噬菌体能够直接降低病原青枯菌数量，削弱病原菌的竞争能力，间接调控土著微生物群落；噬菌体与拮抗型有益菌通过权衡机制协同抑制病原菌；且噬菌体的抑菌效果受环境因素的影响。本项目的研究成果为土传病害的微生态防控提供了新策略和新机制。