阴沟肠杆菌AA4与极小短小杆菌AA3稳定生防菌群结构的分子互作机制研究

The biocontrol microbial consortia are microbial communities capable of controlling crop diseases. They have a few advantages: consistent efficacy, high adaptability, diverse biocontrol mechanisms, etc. The co-existence of biocontrol bacterial strains is the key for the biocontrol bacterial consortia to fully function. Currently, the knowledge about the mechanisms of the assembly of the biocontrol bacterial consortia of stable structure is still very limited. In a previous study, on maize rhizoplane the proposer assembled a consortium, composed of seven biocontrol bacterial strains of different species, possessing the function of controlling maize seedling blight disease. The interspecies interaction between Enterobacter cloacae AA4 and Curtobacterium pusillum AA3 is suggested to be crucial for maintaining the stable co-existence of all the strains in this consortium. Based on these results, by using Phenotype Microarray analysis, fluorescence in situ hybridization, genome sequencing analysis, random transposon mutagenesis, gene knockout mutagenesis and transcriptomics analysis, we are planning to study on E. cloacae AA4 and C. pusillum AA3 to reveal the molecular mechanisms of the interaction between the two strains for stabilizing the composition of the consortium through inhibiting root colonization of strain AA3 by AA4 and to build a model of interspecies interactions in biocontrol bacterial consortia. Our findings will contribute to elucidate the mechanisms of the assembly of the biocontrol bacterial consortia of stable compositions and serve as guidance on the construction of the biocontrol bacterial consortia and on the development and efficient application of the biological control agents of multiple bacterial strains.

生防菌群是由多个生防菌株组成的具有防治作物病害功能的微生物群落，具有稳定、适应性强及防病机制多样等优点。各菌株稳定共存，是生防菌群充分发挥功能的关键。目前，生防菌株形成结构稳定的菌群的分子互作机制尚不明确。在前期研究中，申请人在玉米根表构建了一个由7种细菌组成的能够防治玉米苗枯病的生防菌群，阴沟肠杆菌AA4与极小短小杆菌AA3的种间互作是维持该菌群中各菌株稳定共存的关键，该互作的分子机制尚不明确。本项目拟以菌株AA4与AA3为研究对象，利用表型微阵列分析、荧光原位杂交、基因组-转录组测序分析、转座子随机突变及基因敲除突变等技术，揭示菌株AA4抑制AA3在玉米根表定殖，稳定生防菌群结构的分子互作机制，并建立生防菌群种间互作模型。预期的研究结果将为阐明生防细菌在作物上形成（结构）稳定菌群的机制奠定理论基础，为生防菌群的构建及多菌株混合生防微生物制剂的开发与高效应用提供理论指导。

生防菌群充分发挥功能的关键是群落中各菌株的稳定共存。在前期研究中，课题负责人在玉米根表构建了一个由7种细菌组成的生防菌群，菌群基石物种路德维希肠杆菌AA4的稳定定殖和菌株AA4与极小短小杆菌AA3的种间互作是维持该菌群稳定的关键。本项目以菌株AA4与AA3为研究对象，利用全基因组测序分析、表型微阵列分析、荧光原位杂交、转座子随机突变体库测序分析和基因敲除等技术，对菌株AA4与AA3稳定生防菌群结构的分子互作机制进行了研究。. 通过全基因组测序分析，菌株AA4被进一步鉴定为路德维希肠杆菌（Enterobacter ludwigii）；利用表型微阵列技术结合细菌纯培养，确定了菌株AA4与AA3能够竞争利用玉米根系分泌物中的柠檬酸、天冬氨酸、果糖等碳源；分析发现，菌株AA4初始含量越低，群落的稳定性越差；采用荧光原位杂交结合激光共聚焦显微镜观察，明确了菌株AA4与AA3能够稳定定殖于玉米根表皮细胞间隙、根毛及根表皮细胞表面。以上结果表明菌株AA4与AA3对根系分泌物中碳源的竞争以及菌株AA4在玉米根表的稳定定殖是维持群落稳定的关键。. 通过构建菌株AA4转座子随机突变体库，并对文库进行测序分析，筛选获得了46个定殖相关基因，其中44个基因与菌株AA4的定殖能力呈正相关，2个基因呈负相关。利用Eggnog数据库对定殖相关基因进行了功能分析，结果显示这些基因可被归入运动性、代谢、调节基因等类别，表明上述生物学功能与菌株AA4的定殖能力密切相关。对5个定殖相关基因的突变体菌株进行了定殖相关表型检测，发现这些基因的突变均造成了菌株定殖能力的降低，同时表现出某些定殖相关生物学性状的改变。. 研究团队提出了基于宏基因组测序技术与培养组学技术相结合的合成生防菌群构建策略，明确了菌群中各菌株的兼容性、多样性、根际定殖能力以及病害防控作用方式等因子是影响合成生防菌群构建的关键因素。