Co-existence of Rhizobia and Diverse Non-rhizobial Bacteria in the Rhizosphere and Nodules of Dalbergia odorifera Seedlings Inoculated with Bradyrhizobium elkanii, Rhizobium multihospitium–Like and Burkholderia pyrrocinia–Like Strains

Rhizobia induce root nodules and fix atmospheric N₂ for most legume species in exchange for carbon. However, the diverse endophytic non-rhizobial bacteria in legume nodules that co-exist with rhizobia are often ignored because they are difficult to cultivate using routine cultivation approaches. To enhance our understanding of the incidence and diversity of legume-bacteria associations, a high-throughput sequencing analysis of bacterial 16S rRNA genes was used to examine the bacterial community in the rhizospheres and root nodules of <i>Dalbergia odorifera</i> seedlings that were uninoculated or inoculated with <i>Bradyrhizobium elkanii</i> H255, <i>Rhizobium multihospitium-</i>like HT221, or <i>Burkholderia pyrrocinia-</i>like H022238, in two growth media (nitrogen [N]-supplied soil or N-omitted potting mix). Seedlings inoculated with <i>Bradyrhizobium</i> had significantly more nodules than seedlings in the other inoculation conditions, regardless of growth media. Using the ¹⁵N natural abundance method, it was shown that the inoculated plants had significantly higher N₂ fixation efficiency (48-57%) and specific nodule activity [269-313 μg N mg^-1 of dry weight (dwt) nodule] compared to the uninoculated plants (203 μg N mg^-1 dwt nodule). The 16S rRNA gene analysis showed that there was generally a higher bacterial diversity in the rhizosphere than in the nodules in the corresponding condition. Both rhizobial inoculation and media status significantly altered the bacterial communities in the rhizospheres and nodules (<i>P</i> < 0.05), with the exception of the inoculated soil rhizospheres. Regarding non-rhizobial bacteria, three genera, i.e., <i>Lactococcus, Bacillus</i>, and <i>Pseudomonas</i>, were consistently enriched in the rhizosphere and <i>Bradyrhizobium, Chloroplast</i> norank (which belongs to <i>Cyanobacteria</i>), and <i>Lactococcus</i> were commonly found in the nodules. In contrast, common rhizobial genera (including <i>Rhizobium, Mesorhizobium</i>, and <i>Burkholderia</i>) were only present in the nodules at low relative abundances (0.01-3.41%). Regarding non-rhizobial bacteria, 32 genera were found in the nodules, with non-rhizobial bacteria being predominant in the N omitted potting mix (with a relative abundance of 56-87%). This study suggests that legume nodules are inhabited by a high diversity of non-rhizobial bacteria, which may play a vital role in nodulation and N₂ fixation in the host plants.