植物的叶际存在数量巨大且多种多样的微生物种群，这些叶际微生物种群间的相互作用以及它们的代谢物影响着植物的健康。微生物可以借助化学交流的方式，利用群体感应调控自身的行为，通过种群内和种群间的相互作用发挥种群的生态学功能。尽管群体感应对于实现微生物种群的生态学功能具有重要的作用，然而对基于群体感应的叶际微生物种群行为的研究甚少。本论文以烟草叶际微生物为研究对象，综合运用了微生物学和分子生态学的研究手段和方法，调查了烟草叶际群体感应信号分子酰基高丝氨酸内脂（AHL）相关的微生物，同时也研究了产AHL的重组烟草对整个叶际微生物种群的影响，并探讨了不同AHL的浓度对叶际微生物群落的影响以及AHL相关的典型菌在叶际的个体行为，研究获得了以下结果：

1、烟草叶际存在大量的AHL产生菌以及AHL降解菌，这些细菌在叶际具有一定的普遍性，其中AHL产生菌在可培养微生物中的比例达7.9～11.7%；AHL降解菌在可培养微生物中所占的比例约为14%。烟草叶际的AHL产生菌以及AHL降解菌也具有一定的多样性，AHL产生菌分布在Pseudomonas、Acinetobacter、Citrobacter、Enterobacter、Pantoea、Serratia和Rhizobium 这7个不同的属中；而叶际AHL降解菌主要是利用AHL内酯酶降解AHL导致信号分子失活，这些产AHL内酯酶的叶际AHL降解菌主要分布在Myroides、Serratia、Acinetobacter、Pseudominas、Lysinibacillus、Bacillus 等6个不同的属，不同AHL降解菌的AHL内酯酶的酶活差异较大，其中酶活相对较高的菌株主要分布在Bacillus属中。

2、γ-变形菌纲、α-变形菌纲和厚壁菌门的细菌是烟草叶际主要菌群，β-变形菌纲和放线菌门相对比例随生长期有下降趋势。其中γ-变形菌的相对比例最大(70～90%)是叶际主要细菌，α-变形菌和厚壁菌相对比例较为稳定。聚类分析结果表明产AHL重组烟草和野生型烟草的叶际微生物群落结构差异显著。AHL信号分子影响了烟碱降解菌，产AHL重组烟草叶际没有发现烟碱降解菌，而野生型烟草叶际其相对丰度随生长期而增加。假单胞菌为叶际优势菌群，在产AHL重组烟草主要生长期内其相对比例要大于野生型。产AHL重组烟草影响了常见叶际致病菌泛菌和塔特姆菌的相对比例，对塔特姆菌的抑制尤为明显。

3、1 µM 和10 µM两个浓度的信号分子均不同程度影响了烟草叶际相关细菌群落结构。经1 µM的信号分子处理后革兰氏阳性菌相对比例明显增加，革兰氏阴性细菌则略有减少；10 µM的信号分子处理下对革兰氏阳性菌具有一定的抑制，革兰氏阴性细菌则较好的生存。

4、标记细菌NTL223/pBQGFP可以在烟草叶际短时间内定殖，在野生型烟草叶片的气孔、纤毛和叶脉都随着时间延长最初有增加的趋势，后期在不同的时间点开始减少，标记细菌第3天后在纤毛上开始减少，第5天后在叶脉处开始减少。标记细菌在产AHL重组烟草叶际气孔周围分布略有变化；纤毛、叶脉处标记细菌随着时间而增加，分别在第4天和第7天达到顶值。

The phyllosphere harbors large and diverse naturally occuring epiphytic microbial populations, of which bacteria, are the numerous. Bacterial populations coexisting in the phyllosphere niche have important effects on plant health. Quorum sensing (QS) grants bacterial communication via diffusible signal molecules, but QS-dependent behaviors in phyllosphere bacterial populations are poorly understood. In this research, we investigate AHL-producing phyllosphere bacteria and quorum-quenching microbes using modern biotechnological methods. The objective of our study was to assess the AHL-mediated influence on the composition of the leaf-associated bacterial community and individual behavior of the AHL -producing bacteria in the phyllosphere of tobacco. The major contents and finding of the dissertation are as follows:

1.        Our results indicated that approximately 7.9-11.7% of the culturable leaf-associated bacteria have the ability to produce AHL based on the assays with whole-cell biosensors. While the occurrence of 3-oxo-C₆-HSL-degrading strains isolated from the phyllosphere upto 14%. 16S rRNA gene sequencing revealed that AHL-active strains were assigned to two phylogenetic groups with Gammaproteobacteria (93%) as the predominant group followed by Alphaproteobacteria. All the AHL-producing Alphaproteobacteria affiliated to the genus Rhizobium, whereas the AHL-producing bacteria belonging to the Gammaproteobacteria mainly fell within the genera Pseudomonas, Acinetobacter, Citrobacter, Enterobacter, Pantoea and Serratia. 16S rRNA gene sequencing showed that AHL-degrading bacteria mainly fell within the genera Myroides, Serratia, Acinetobacter, Pseudominas, Lysinibacillus and Bacillus. AHL-lactoase of Bacillus genus had the highest enzyme activity.

2.        As shown by the 16S rRNA gene sequencing analysis, the tobacco phyllosphere microbial populations predominantly belonged to Gammaproteobacteria following with Alphaproteobacteria and Phylum Firmicutes. As well as the relative abundance of Beltaproteobacteria and Actinomycetes declined at the late growth stage of tobacco. The Ochrobactrum strains were found in the phyllophsere of wild lines increasing with the growth of tobacco. The relative proportion of Pseudomonas of transgenic tobacco was greater than that of the wild line at 11, 14 weeks. AHL had a significant effect on the phytopathogen of Pantoea and Tatumella. Cluster analysis showed that phylloshpere bacterial community structure of AHL-producing transgenic line was significantly different with that of the control.

3.        The changes in the bacterial community composition in tobacco phyllosphere were also obatained after treatment with different concentration of 3-oxo-C₆-HSL. Notably, the ratio of Gram-positive (GP) bacteria increased in response to 1 μM AHL treatment but decreased incipiently when treated with 10 μM AHL. These observations provide insight into the composition of the leaf-colonizing epiphyte community, particularly GP bacteria for they do not use the AHL as a signaling molecule for QS, responsible for the AHLs.

Pseudomonas sp. NTL223 was tagged with green fluorescent protein (gfp), and inoculation was carried out through phyllosphere. Observations by CLSM and SEM showed that the gfp marker is stably expressed in tobacco phyllosphere allowing for easy visualization of Pseudomonas sp. NTL223. Pseudomonas sp. NTL223 could distribute at different sites on leaves, such as stomatal, cilia and leaf vein. Time course of the settlement of Pseudomonas sp. NTL223 showed that higher numbers of GFP-markered strains in stomatal and leaf vein appeared at 3-day and 5-day respectively. Moreover, there was no noticeable difference of the colonization of strain NTL223 around stomatal in transgenic line throughout the time-course experiment. While Pseudomonas sp. NTL223 switched at the sites of cilia and leaf vein assembling together at 4-day and 7-day respectively.