In addition to the rhizobia, other non-rhizobial endophytes (NREs) have been simultaneously isolated from the root nodules. The existence of NREs in leguminous root nodules is a universal phenomenon, and they have the potential to enhance legume survival, especially under conditions of environmental stress. However, the diversity and biogeographic patterns of microbial communities inhabiting root nodules are not well studied or understood. Here, we explored and characterized the diversity of NRE bacteria by using 16S rRNA gene high-throughput amplicon sequencing. Additionally, we compared the biogeography and co-occurrence patterns in review of the bacterial microbiota inhabiting the rhizosphere, the bulk soil and the root nodule bacterial communities associated with Sophora davidii, a native N-fixing wild leguminous shrub in hilly and gully regions of the Loess Plateau of China. The results showed the presence of a large diversity of bacteria belonging to 81 phyla, 154 classes, 333 orders, 463 families, and 732 genera inside the nodules. Proteobacteria were dominant in the nodule and rhizosphere soil samples, and Actinomycetes were dominant in the bulk soil samples. Mesorhizobium was the dominant genus in the nodules, accounting for between 60.15 and 83.74% of the bacteria. The microbial community composition of the NRE in the root nodules differed from that in the rhizosphere soil and the bulk soil of S. davidii. Moreover, we found that the biogeographic patterns and assembly process of the rhizobia and non-rhizobia communities differed in the root nodule, the rhizosphere soil and the bulk soil. Furthermore, the correlation analysis between the soil’s physical and chemical properties and the bacteria showed that available phosphorus was the predominant factor affecting the bacterial diversity within the rhizosphere soil. Finally, our results revealed that the microbial network diagram of co-occurrence patterns showed more complexes in the soil than in the root nodules. This indicates that only specific microorganisms could colonize and thrive in the rhizosphere through the selection and filtering effects of roots. In conclusion, there are significant differences in bacterial community composition in the nodules, rhizosphere and bulk soil in the hilly and gully region of the Loess Plateau, which is the result of environmental filtration. Our study improves the understanding of the biogeographic patterns and diversity of bacterial microbiota inhabiting root nodules and can help quantify and define the root nodule assemblage process of S. davidii.

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An opaque, pink-coloured, gram-positive, aerobic bacteria (designated as FBM22-1T), was isolated from microbial fermentation bed material from a pig farm in northwestern China. Optimal growth occurred at 30–37 °C, pH 7.0 and with 0.5 % NaCl (w/v). The strain had nitrification and denitrification functions. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the novel isolate belonged to the genus Rhodococcus . Strain FBM22-1T was closely related to Rhodococcus zopfii NBRC 100606T and Rhodococcus rhodochrous NBRC 16069T, with 16S rRNA gene sequence similarities of 97.9 and 97.7 %, respectively. The predominant menaquinone in strain FBM22-1T was MK-8(H2). The cellular fatty acids consisted primarily of C16 : 1ω7c and/or C16 : 1 ω6c, C16 : 0 and 10-methyl C18 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and glycolipid. The G+C content of strain FBM22-1T was 68.64 mol%. Based on the phenotypic, phylogenetic and chemotaxonomic characterization results, in combination with low values of digital DNA–DNA hybridization between strain FBM22-1T and its closest neighbours, FBM22-1T represents a novel species of the genus Rhodococcus , for which the name Rhodococcus yananensis sp. nov. is proposed; the type strain is FBM22-1T (=KCTC 49502T=CCTCC AB2020275T).

Nodule-associated nitrogen-fixing microorganisms (diazotrophs) residing in legume root nodules, and they have the potential to enhance legume survival. However, the succession characteristics and mechanisms of leguminous diazotrophic communities remain largely unexplored. We performed a high-throughput nifH amplicon sequencing with samples of root nodules and soil in the three developmental phases (young nodules, active nodules and senescent nodules) of the Sophora davidii (Franch.) Skeels root nodules, aiming to investigate the dynamics of nodule-endophytic diazotrophs during three developmental phases of root nodules. The results demonstrated the presence of diverse diazotrophic bacteria and successional community shifting dominated by Mesorhizobium and Bradyrhizobium inside the nodule according to the nodule development. The relative abundance decreased for Mesorhizobium, while decreased first and then increased for Bradyrhizobium in nodule development from young to active to senescent. Additionally, strains M. amorphae BT-30 and B. diazoefficiens B-26 were isolated and selected to test the interaction between them in co-cultured conditions. Under co-culture conditions: B. diazoefficiens B-26 significantly inhibited the growth of M. amorphae BT-30. Intriguingly, growth of B. diazoefficiens B-26 was significantly promoted by co’culture with M. amorphae BT-30 and could utilize some carbon and nitrogen sources that M. amorphae BT-30 could not. Additionally, the composition of microbial community varied in root nodules, in rhizosphere and in bulk soil. Collectively, our study highlights that developmental phases of nodules and the host microhabitat were the key driving factors for the succession of nodule-associated diazotrophic community.