根际环境对超累积植物东南景天内生细菌的多样性和定殖的影响

The characteristics of endophytic bacterial diversity and distribution are conditioned by biotic and abiotic factors. Under heavy pollution environment, plants might select endophytic bacteria which can assist their host plants in overcoming phytotoxic effects and promoting plant growth. Our previous study proved that the ability of uptake and accumulation of Zn/Cd differed significantly between hyperaccumulator ecotype and non-hyperaccumulator ecotype of Sedum alfredii. Furthermore, the cultureable endophytic bacteria number and community structure were affected by soil environment. However, we know little about the biodiversity, infection and colonization of endophytic bacteria in hyperaccumulator plant S. alfredii. In this study, the differences of genetic diversity and community of endophytic bacteria between hyperaccumulator ecotype and non-hyperaccumulator ecotype of S. alfredii were assessed using a combination of dilution plating of plant macerates followed by isolation and characterization of isolates and the restriction enzyme 16S fragment rDNA polymorphism (16SrDNA-RFLP), denaturing gradient gel electrophoresis (DGGE)and 454 pyrosequencing on the basis of DNA extracted from plants. The influence of soil environment, soil Zn and Cd pollution level and endophytic bacteria of seedling on the community structures and biological roles of endophytic bacterial in the root, stem and leaf tissues of two ecotypes of S. alfredii at different growth and development stage were also investigated. Colonization dynamics of plant growth promoting endopytic bacteria Pseudomonas fluorescens labeled with GFP (pDSK-GFPuv) in rhizosphere soils and the root, stem and lef tissues of S. alfredii plants were investigated by the use of GFP-tagged strains in combination with spread plate method，fluorescence microscopy and confocal laser scanning microscopy (CLSM). The results will reveal the endophyte communities of S. alfredii , clarify the relationships among S. alfredii, soil environment and the endophytic bacterial diversity and community structures and discover new endophytic bacteria resouces, in order to lay a good scientific foundation for developing technogy of endophytic bacteria assisting phytoremediation of heavy metal contaminated soils.

在长期的选择和进化过程中，内生细菌与宿主植物形成一种和谐联合的关系，共同抵抗不利环境条件。当土壤重金属浓度增加时，超积累植物是否会选择性让土壤中某些金属抗性和（或）促植物生长细菌进入体内，或植物体内原有的内生菌大量增殖，进而协同超积累植物完成对重金属吸收、积累、解毒过程呢？本项目以典型的锌镉超积累植物东南景天为研究对象，通过培养方法和非培养方法（PCR-DGGE和高通量测序）分析其体内内生菌多样性及其随土壤环境条件变化；采用pDSK-GFPuv基因标记技术、荧光显微镜技术和共聚焦激光扫描显微镜技术跟踪特定内生菌对东南景天的侵染、定殖和分布特征，深入认识东南景天与内生细菌在超积累重金属过程中的相互关系，从"植物与内生细菌相互作用"的角度来补充解释东南景天对锌、镉的吸收、积累及解毒的生理生态机制。

本项目研究了土壤环境对东南景天内生菌群落结构的影响及其侵染特征。主要结果如下：（1）非超积累和超积累生态型东南景天的根际和内生菌的群落结构基本相似，但超积累生态型东南景天的根际细菌、根系和叶片内生菌更丰富，且存在大量金属抗性和促植物生长特征的菌株。（2）随着东南景天的生长和对锌、镉、铅的积累，其内生菌的多样性和群落结构发生了变化，主要受到其体内锌或镉含量、气温和降雨量的影响。（3）随着土壤Cd浓度的增加，除了超积累生态型东南景天茎中可培养内生菌数量和多样性增加外，两种生态型东南景天根系、茎和叶片可培养内生菌数量和内生细菌的多样性均减少。鞘氨醇单胞菌属、假单胞菌属、肠杆菌属、叶杆菌属、甲基杆菌属、慢生根瘤菌属的相对丰度与土壤、植物Cd浓度正相关。（4）随着土壤Zn浓度增加，东南景天根际和茎内生菌的丰度和多样性减少，尤其是变形菌门的相对丰度减少；根系和叶片内生菌的丰度和多样性增加，尤其是变形菌门与放线菌门。贪铜菌属、假诺卡氏菌属、黄杆菌属、食酸菌属、鞘氨醇单胞菌、无色杆属、黄杆菌属、伯克氏菌属、根瘤菌属等与土壤和植物Zn含量正相关。（5）开瓶3天后，东南景天组培苗转移到以泥炭、蛭石和椰糠混配的基质上，保持60~65%基质含水率、20~25℃下50%~60%遮阴培养7天，自然环境下继续培养20~30天即可移栽到土壤（成活率≥95%）。（6）根际土壤接种Ⅱ8L4-GFP后，其从根表或根毛侵入根内后，迁移到根维管束，进一步迁移到茎维管组织和表皮层、叶片维管束髓、木质部和韧皮部。在亚细胞水平上，Ⅱ8L4-GFP主要分布在细胞间隙，少量在细胞内。（7）溶磷内生菌通过酸化和分泌为萄糖酸、α-酮戊二酸、丁二酸和丙酸溶解Ca3(PO4)2、FePO4和AlPO4，从而显著降低土壤中有效铅含量降低，促进油麦菜的生长和减少对铅的吸收。上述研究成果阐明了根际环境影响东南景天内生菌群落结构，筛选出的溶磷内生菌可以原位钝化土壤铅，东南景天组培苗可为今后利用东南景天大规模修复土壤提供种苗。