In present study, we report our extensive survey on the diversity and biogeography of rhizobia associated with Sophora flavescens, a sophocarpidine (matrine)-containing medicinal legume. We additionally investigated the cross nodulation, infection pattern, light and electron microscopies of root nodule sections of S. flavescens infected by various rhizobia. Seventeen genospecies of rhizobia belonging to five genera with seven types of symbiotic nodC genes were found to nodulate S. flavescens in natural soils. In the cross-nodulation tests, most representative rhizobia in class α-Proteobacteria, whose host plants belong to different cross-nodulation groups, form effective indeterminate nodules, while representative rhizobia in class β-Proteobacteria form ineffective nodules on S. flavescens. Highly host-specific biovars of Rhizobium leguminosarum (bv. trifolii and bv. viciae) and Rhizobium etli bv. phaseoli could establish symbioses with S. flavescens, providing further evidence that S. flavescens is an extremely promiscuous legume and it does not have strict selectivity on either the symbiotic genes or the species-determining housekeeping genes of rhizobia. Root-hair infection is found as the pattern that rhizobia have gained entry into the curled root hairs. Electron microscopies of ultra-thin sections of S. flavescens root nodules formed by different rhizobia show that the bacteroids are regular or irregular rod shape and nonswollen types. Some bacteroids contain poly-β-hydroxybutyrate (PHB), while others do not, indicating the synthesis of PHB in bacteroids is rhizobia-dependent. The extremely promiscuous symbiosis between S. flavescens and different rhizobia provide us a basis for future studies aimed at understanding the molecular interactions of rhizobia and legumes.

Five bacterial strains representing 45 isolates originated from root nodules of the medicinal legume Sophora flavescens were defined as two novel groups in the genus Rhizobium based on their phylogenetic relationships estimated from 16S rRNA genes and the housekeeping genes recA, glnII and atpD. These groups were distantly related to Rhizobium leguminosarum USDA 2370 T (95.6 % similarity for group I) and Rhizobium phaseoli ATCC 14482 T (93.4 % similarity for group II) in multilocus sequence analysis. In DNA-DNA hybridization experiments, the reference strains CCBAU 03386 T (group I) and CCBAU 03470 T (group II) showed levels of relatedness of 17.9-57.8 and 11.0-42.9 %, respectively, with the type strains of related species. Both strains CCBAU 03386 T and CCBAU 03470 T contained ubiquinone 10 (Q-10) as the major respiratory quinone and possessed 16 : 0, 18 : 0, 19 : 0 cyclo v8c, summed feature 8 and summed feature 2 as major fatty acids, but did not contain 20 : 3 v6,8,12c. Phenotypic features distinguishing both groups from all closely related species of the genus Rhizobium were found. Therefore, two novel species, Rhizobium sophorae sp. nov. for group I (type strain CCBAU 03386 T 5E5 T 5LMG 27901 T 5HAMBI 3615 T) and Rhizobium sophoriradicis sp. nov. for group II (type strain CCBAU 03470 T 5C-5-1 T 5LMG 27898 T 5HAMBI 3510 T), are proposed. Both groups were able to nodulate Phaseolus vulgaris and their hosts of origin (Sophora flavescens) effectively and their nodulation gene nodC was phylogenetically located in the symbiovar phaseoli. Rhizobia isolated from the medicinal legume Sophora flavescens have not been surveyed previously, although the genetic diversity of rhizobia associated with another wild species in the same genus, Sophora alopecuroides, have been extensively studied. Rhizobia isolated from Sophora alopecuroides were classified into five genera and nine genospecies, with Mesorhizobium alhagi and Mesorhizobium gobiense as the main groups, and Agrobacterium tumefaciens, Mesorhizobium amorphae, Phyllobacterium trifolii, Rhizobium giardinii, Rhizobium indigoferae, Ensifer fredii (former Sinorhizobium fredii) and Ensifer meliloti (former Sinorhizobium meliloti) as the minor groups (Zhao et al., 2010). At the time of writing, 76 species have been classified and described in the genus Rhizobium (Euzéby, 1997; Parte, 2014), including recently described species

We explored the genetic basis of the promiscuous symbiosis of Sophora flavescens with diverse rhizobia. To determine the impact of Nod factors (NFs) on the symbiosis of S. flavescens, nodulation-related gene mutants of representative rhizobial strains were generated. Strains with mutations in common nodulation genes (nodC, nodM, and nodE) failed to nodulate S. flavescens, indicating that the promiscuous nodulation of this plant is strictly dependent on the basic NF structure. Mutations of the NF decoration genes nodH, nodS, nodZ, and noeI did not affect the nodulation of S. flavescens, but these mutations affected the nitrogen-fixation efficiency of nodules. Wild-type Bradyrhizobium diazoefficiens USDA110 cannot nodulate S. flavescens, but we obtained 14 Tn5 mutants of B. diazoefficiens that nodulated S. flavescens. This suggested that the mutations had disrupted a negative regulator that prevents nodulation of S. flavescens, leading to nonspecific nodulation. For Ensifer fredii CCBAU 45436 mutants, the minimal NF structure was sufficient for nodulation of soybean and S. flavescens. In summary, the mechanism of promiscuous symbiosis of S. flavescens with rhizobia might be related to its nonspecific recognition of NF structures, and the host specificity of rhizobia may also be controlled by currently unknown nodulation-related genes.

Phyllobacterium sophorae sp. nov., a symbiotic bacterium isolated from root nodules of Sophora flavescens Two novel Gram-stain-negative strains (CCBAU 03422 T and CCBAU 03415) isolated from root nodules of Sophora flavescens were classified phylogenetically into the genus Phyllobacterium based on the comparative analysis of 16S rRNA and atpD genes. They showed 99.8 % rRNA gene sequence similarities to Phyllobacterium brassicacearum LMG 22836 T , and strain CCBAU 03422 T showed 91.2 and 88.6 % atpD gene sequence similarities to strains Phyllobacterium endophyticum LMG 26470 T and Phyllobacterium brassicacearum LMG 22836 T , respectively. Strain CCBAU 03422 T contained Q-10 as its major quinone and showed a cellular fatty acid profile, carbon source utilization and other phenotypic characteristics differing from type strains of related species. DNA-DNA relatedness (lower than 48.8 %) further confirmed the differences between the novel strains and the type strains of related species. Strain CCBAU 03422 T could nodulate and fix nitrogen effectively on its original host plant, Sophora flavescens. Based upon the results mentioned above, a novel species named Phyllobacterium sophorae is proposed and the type strain is CCBAU 03422 T (5A-6-3 T 5LMG 27899 T 5HAMBI 3508 T ).The genus Phyllobacterium was originally proposed by Knösel (1962) Table 1). It is obvious that root nodules are the preferred habitats for species of the genus Phyllobacterium more so than leaf nodules (Mergaert & Swings, 2001).In studies on genetic diversity of nodule bacteria from Sophora flavescens, a legume traditionally used as herbal medicine in China, two strains (CCBAU 03422 T and CCBAU 03415) were found to be distinct from the rhizobial species in the genera Mesorhizobium, Ensifer and Rhizobium based on the sequence analysis of the recA (recombinase A)Abbreviations: DDH, DNA-DNA hybridization ; ML, maximum-likelihood.The GenBank/EMBL/DDBJ accession number for the 16S rRNA, atpD, nodC and nifH gene sequences of strain CCBAU 03422 T are KJ685937, KJ685941, KJ685943 and KJ685942, respectively.

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