Rhizobium miluonense sp. nov., a symbiotic bacterium isolated from Lespedeza root nodules

Seventeen strains of lespedeza rhizobia, a previously defined novel group, were further characterized by comparison with phylogenetically related species using a polyphasic approach, including SDS-PAGE of whole-cell proteins, PCR-based restriction fragment length polymorphism analysis of ribosomal intergenic spacer sequences, determination of DNA G+C contents, DNA–DNA hybridization and analysis of phenotypic features. Based on data obtained in the present and previous studies, a novel species, Rhizobium miluonense sp. nov., is proposed for these strains. The type strain of R. miluonense is CCBAU 41251^T (=LMG 24208^T=HAMBI 2971^T). Strain CCBAU 41251^T could form ineffective nodules on Phaseolus vulgaris in addition to its original host Lespedeza chinensis.

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Abbreviations: IGS, intergenic spacer; RFLP, restriction fragment length polymorphism

The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Rhizobium miluonense CCBAU 41251^T is EF061096.

Phenotypic characteristics of strains of Rhizobium miluonense sp. nov. are given in Supplementary Table S1, which is available with the online version of this paper.

Rhizobia are soil bacteria that can form nodules in symbiosis with leguminous plants. Currently, about 50 rhizobial species within more than 10 genera of the alpha- and betaproteobacteria have been described. Among these genera, Rhizobium was the first described species, named 100 years ago. After a series of taxonomic changes since the 1980s, the genus Rhizobium now contains about 20 species, including three recently described species: Rhizobium daejeonense (Quan et al., 2005), Rhizobium lusitanum (Valverde et al., 2006) and Rhizobium cellulosilyticum (García-Fraile et al., 2007).

In our previous study (Gu et al., 2007), diverse rhizobia associated with Lespedeza spp. in temperate and subtropical regions of China were characterized and two novel Rhizobium groups (6 and 7) were identified based on 16S rRNA gene sequence analysis and DNA–DNA hybridization. In the present study, the main novel group, group 7, was further studied to clarify its taxonomic status.

Seventeen test strains in group 7 (Gu et al., 2007) and six reference strains were used in this study (Table 1). These test strains were isolated from root nodules of nine Lespedeza species grown in Hunan province, China. Nodulation on the original host plant of each isolate has been confirmed previously (Gu et al., 2007). All strains were maintained on yeast-mannitol agar (YMA) medium at 4 °C during the study.

Table 1. Bacterial strains used in this study

Previously, these 17 strains were defined as group 7 since they formed a cluster using SDS-PAGE of proteins that was distinct from reference strains and other lespedeza rhizobia at a similarity of 88 %. These 17 strains also clustered in PCR-based ribosomal intergenic spacer restriction fragment length polymorphism (IGS-RFLP) analysis, showing 99.5–100 % similarity within the group and 70 % similarity with other rhizobia (Gu et al., 2007). 16S rRNA gene sequencing and phylogenetic analysis revealed that three representative strains of group 7 had identical sequences and were closely related to Rhizobium tropici type B CIAT 899^T, R. tropici type A LMG 9517, R. lusitanum P1-7^T and Rhizobium rhizogenes IFO 13257^T, with 16S rRNA gene sequence similarities of 99.3, 98.8, 99.6 and 99.4 %, respectively (Gu et al., 2007).

Since the reference strains for the Rhizobium species most related to group 7 were not included in SDS-PAGE of whole-cell proteins and IGS-RFLP analysis, these two methods were used in this study to compare the group 7 strains and the related species. For SDS-PAGE, all strains were incubated in yeast-mannitol (YM) broth at 28 °C for 2 days. The preparation of protein samples, SDS-PAGE and silver-staining of the gels were performed as described previously (Tan et al., 1997). For IGS-RFLP, the ribosomal IGS between the 16S and 23S rRNA genes was amplified using primers FGPS6 and 23S-38 and the protocol of Rasolomampianina et al. (2005). The PCR products were digested separately with MspI, CfoI and HaeIII as described by Rasolomampianina et al. (2005). Restriction fragments were separated by electrophoresis in 3 % agarose gels and photographed under UV light after ethidium bromide staining. Cluster analyses of the protein and RFLP patterns were performed as described previously (Gu et al., 2007).

In both SDS-PAGE of proteins and ribosomal IGS-RFLP analysis, the 17 strains of group 7 showed almost identical patterns (99–100 % similarity). Similarities between the group 7 strains and R. tropici type B LMG 9503^T, R. tropici type A CFN 299, R. lusitanum LMG 22705^T and R. rhizogenes LMG 150^T were ≤64.5 % in SDS-PAGE of whole-cell proteins (Fig. 1) and ≤81 % in ribosomal IGS-RFLP analysis (Fig. 2). These data demonstrated that members of group 7 differed from phylogenetically related Rhizobium species.

Table 1) were almost identical to that of strain CCBAU 41251T.

(67K): Fig. 2. Ribosomal IGS-RFLP patterns showing the genomic relationships between strains of R. miluonense sp. nov. and phylogenetically related reference strains.

Previously, DNA–DNA hybridization has been performed between group 7 strain CCBAU 41251^T and the reference strains of several defined species. To determine the relationships between group 7 strains and all the most related species, DNA G+C contents were determined and further DNA–DNA hybridizations were performed. The DNA was prepared according to the method of Marmur (1961). DNA G+C contents were measured using the thermal melting protocol of De Ley (1970) with Escherichia coli K-12 as the standard. DNA relatedness was determined by the initial renaturation rate method (De Ley et al., 1970). The DNA G+C contents of the three group 7 strains CCBAU 41251^T, CCBAU 41029 and CCBAU 41128 were 58.4, 58.8 and 58.2 mol%, respectively. The DNA–DNA relatedness data are presented in Table 2. Low to medium DNA–DNA relatedness (15–45 %) was obtained between strain CCBAU 41251^T and six reference strains of R. tropici type B, R. tropici type A, R. lusitanum and R. rhizogenes (Table 2). DNA–DNA relatedness of 96–100 % was obtained between strain CCBAU 41251^T and two other strains that were randomly selected as representatives of group 7 (Table 2). The large gap between the DNA–DNA relatedness values within the group and with the reference strains obviously indicated that the group 7 strains formed a genomic species that differed from related Rhizobium species.

Table 2. DNA–DNA relatedness between strain CCBAU 41251T and phylogenetically related reference strains Data are mean values of at least three repetitions.

To clarify whether the group 7 strains could be defined as a species, phenotypic features that differentiated group 7 from related species were analysed according to the current criterion of bacterial species definition (Stackebrandt et al., 2002). In the present study, phenotypic features, including the utilization of sole carbon and nitrogen sources, resistance to antibiotics, tolerance to NaCl and pH and temperature ranges for growth (see Supplementary Table S1, available in IJSEM Online) were analysed as described previously (Gao et al., 1994). Among the 96 tested features, 68 were the same (positive or negative) for all the strains. In the cluster analysis (Fig. 3) using the S_SM coefficient (S_SM=ΣI/96, where ΣI is the number of features identical for the compared strain pair; 96 is the total number of tested features) and unweighted pair-group method with arithmetic averages (UPGMA) (Sneath & Sokal, 1973), the 17 strains in group 7 showed 98.5 % or greater similarity and clearly differed from the reference strains of recognized species (Fig. 3). Distinctive features of group 7 are shown in Table 3 and more features are presented in the species description. In this analysis, colony morphology (i.e. wet and translucent) could distinguish group 7 strains from R. tropici type A and R. rhizogenes, whereas no growth in Luria–Bertani (LB) medium and sensitivity to 1 % NaCl could differentiate group 7 strains from R. tropici type B and R. lusitanum.

(16K): Fig. 3. Dendrogram showing the phylogenetic relationships among strains of R. miluonense sp. nov. and phylogenetically related reference strains. The dendrogram was constructed based upon 96 phenotypic features using SSM coefficients and the UPGMA method (Sneath & Sokal, 1973).

Table 3. Distinctive features of R. miluonense sp. nov. and phylogenetically related species Strains: 1, R. miluonense CCBAU 41251T sp. nov.; 2, R. tropici A CFN 299; 3, R. tropici B LMG 9503T; 4, R. lusitanum LMG 22705T; 5, R. rhizogenes LMG 150T. –, No growth; +, growth;±, light growth.

To check the host range, seeds of Phaseolus vulgaris, Medicago sativa, Pisum sativum and Leucaena leucocephala were surface-sterilized, germinated and inoculated with the representative strain CCBAU 41251^T according to standard protocols (Vincent, 1970). The growth conditions of the plants were as described previously (Yao et al., 2002). After 7 weeks of growth, inefficient (white) nodules were observed on Phaseolus vulgaris inoculated with strain CCBAU 41251^T, but no nodules were found on Medicago sativa, Pisum sativum or Leucaena leucocephala. This failure to nodulate P. vulgaris effectively might be an additional phenotypic feature to enable the differentiation of group 7 members from related species since R. tropici, R. rhizogenes and R. lusitanum strains could form effective root nodules with this plant (Valverde et al., 2006).

In conclusion, the 17 strains in group 7 were very similar in all the genomic and phenotypic analyses. They were identified as members of the genus Rhizobium and were related to R. tropici, R. rhizogenes and R. lusitanum by 16S rRNA gene sequencing (Gu et al., 2007), but formed a group that differed from the recognized species of the genus Rhizobium in SDS-PAGE of proteins (Fig. 1), ribosomal IGS-RFLP (Fig. 2), DNA–DNA hybridization (Table 2) and numerical taxonomy (Fig. 3). Taking the recently reported Rhizobium species as references (García-Fraile et al., 2007; Quan et al., 2005; Valverde et al., 2006), the strains in group 7 could be defined as a novel species of the genus Rhizobium based on the results presented in this study and in our previous work (Gu et al., 2007); the name proposed for this species is Rhizobium miluonense sp. nov.

Description of Rhizobium miluonense sp. nov.
Rhizobium miluonense (mi.lu.o.nen'se. N.L. fem. adj. miluonense pertaining to the Miluo River, a famous river located in Hunan Province, where the bacterium was isolated).

Aerobic, Gram-negative, non-spore-forming rods, 0.6–0.8x1.8–3.2 µm. Colonies on YMA are circular, convex, translucent and usually 2–3 mm in diameter after 3 days incubation at 28 °C. Amygdalin, D-arabinose, calcium gluconate, calcium malonate, meso-erythritol, D-fructose, D-galactose, D-glucose, inositol, lactose, sodium DL-malate, maltose, D-mannose, turanose, raffinose, L-rhamnose, salicin, sodium citrate, sodium D-gluconate, sodium succinate, sorbose, sucrose, trehalose, D-xylose, L-arginine, L-proline and L-aspartic acid are used as sole carbon sources for growth, but adipic acid, dextrin, inulin, melezitose, sodium acetate, sodium formate, sodium hippurate, soluble starch, syringic acid, potassium sodium tartrate, vanillic acid, glycine, L-threonine and L-methionine are not utilized. DL-α-Aminopropionic acid, L-arginine, L-aspartic acid, L-cystine, D-glutamic acid, L-glutamic acid, hypoxanthine, L-isoleucine, L-lysine, L-phenylalanine, L-valine, glycine, L-threonine and L-hydroxyproline are used as sole nitrogen sources for growth, but D-threonine is not utilized. Resistant to 100 µg ampicillin ml^–1 and 5 µg ml^–1 each of chloramphenicol, neomycin sulfate and streptomycin sulfate. Sensitive to 300 µg ampicillin ml^–1, 5 µg ml^–1 each of gentamicin sulfate, erythromycin and kanamycin sulfate, and 50 µg ml^–1 each of chloramphenicol, neomycin sulfate and streptomycin sulfate. Optimum growth temperature is 25–30 °C; can grow at 37 °C, but not 4 °C. Optimum pH is 6–8. Sensitive to 1 % (w/v) NaCl in YMA. Does not grow in Luria–Bertani medium.

The type strain is CCBAU 41251^T (=LMG 24208^T=HAMBI 2971^T), isolated from root nodules of Lespedeza species grown in Hunan province, China. The DNA G+C content of strain CCBAU 41251^T is 58.4 mol%.