The Bacterial Diversity Of Paris Polyphylla Var. Yunanensis From Rhizome And Phylogenimics Of Rhizobium

The further improvement of artificial cultivation of Paris polyphylla var. yunnanensis is the only route for its industrial development. Besides the optimization of germplasm resources and field management, the endophytic resources are of great advantage in the sustainable utilization of P'. polyphylla var. yunnanensis. The objective of this study is to analyse the bacterial community structure in the rhizome of P. polyphylla var. yunnanensis. By using selective pure culture and high-throughput sequencing strategies, the endophytic resources of P. polyphylla var. yunnanensis will be analysed and exploited. Furthermore, bacterial diversity investigation showed that Rhizobiales was one of predominant group and a batch of isolates belonging to this group were obtained, consequently, the phylogenomics of Rhizobium and related groups were performed.By using high-throughput sequencing technique, the biodiversities of rhizosphere soil and rhizome endophytic habitats of P. polyphylla var. yunnanensis from four sampling sites of Yunnan province had been studied. In addition, we compared the population function amongst different habitats. Although there were more diverse bacteria in the rhizosphere habitats, they had the same predominant groups belonging to phyla Actinobacteria, Firmicutes and Proteobacteria, comparing with endophytic environments. The bacterial community structure of rhizosphere habitat was totally different with that of endophytic one. On order level, there were similarities and differences on the aspect of predominant groups. By population function predication and comparison, we found that the population functions between rhizosphere and endophytic microbial communities were also different. Furthermore, we found that the bacterial diversities of endophytic habitats from Zhongdian's samples were different with others. However, they had similar population function. It indicated their predominated groups were consistent.In study of pure culture, seven isolation media were used to isolated endophytic bacteria from different samples of Yunnan province. Totally,289 bacterial isolates were obtained and identified. They distributed in three phyla, five classes,14 orders,25 families and 83 genera. Meanwhile, steroidal saponins compounds were detected from the fermentation products of 35 isolates; and 25 isolates could grow on the nitrogen free medium. There were 22 potential novel taxa. And through polyphasic identification, four isolates were identified and proposed. Rhizobium radicis sp. nov. and Rhizobium gengmaense sp. nov. are proposed respectively with py1111t and py1134T as type strains. Oceanobacillus endoradicis sp. nov. is proposed with py1294T as type strain, and Yimella radicis sp. nov. is proposed with py12921 as type strain.The draft genomic sequences of 41 type species of Rhizobium were determined. The phylogenomics study found that Rhizobium and related groups formed four main phylogenetic clades. Most of Rhizobium species with nitrogen fixation function belonged to the latest originated branch. The further analysis focusing on the symbiotic nitrogen fixation related proteins demonstrated that the homologous proteins of symbiotic plasmid originated earlier among Rhizobium and related groups, and gene loss might be the main cause leading to the deficiency of nitrogen fixation in some Rhizobium species. The average similarities of NifH, FixC and NodC that are key proteins in nitrogen fixation had apparent different. That indicated they underwent different selective pressures. Alternatively, some individual genes had been affected by horizontal gene transfers. This work analysed the phylogenetic relationships among Rhizobium and relative species, and revealed the main evolutionary mechanism of symbiotic nitrogen fixation in this group.The result of this study will contribute to understand the relationships between bacterial community structures of rhizosphere and rhizome endophytic habitats of P. polyphylla var. yunnanensis, and will be underpinned for the accumulation of P. polyphylla var. yunnanensis endophytic bacterial resources. It will provide more theoretical guidance for the optimization of artificial cultivation. A batch of endophytic bacterial resource were obtained, they will be the valuable materials for the development of strain resources. The phylogenomic analysis addressed the taxonomic question about Rhizobium and related groups, and provided new insights into the evolution and symbiotic nitrogen fixation in this group. Microbial communities associated with plant host and microbial resources are important research objectives for the development of microbial function, population ecology, and systematic evolution. Along with the breakthrough of high-throughput sequencing and pure-culture techniques, endophytic microbiology will keep on promoting the development and utilization of microbial resources.