Fifteen bacterial strains isolated from molasses grass (Melinis minutiflora Beauv.) were identified as nitrogen-fixers by using the acetylene-reduction assay and PCR amplification of nifH gene fragments. These strains were classified as a unique group by insertion sequence-PCR fingerprinting, SDS-PAGE protein patterns, DNA–DNA hybridization, 16S rRNA gene sequencing and morphological characterization. Phylogenetic analysis of the 16S rRNA gene indicated that these diazotrophic strains belonged to the genus Azospirillum and were closely related to Azospirillum lipoferum (with 97?5% similarity). In all the analyses, including in addition phenotypic characterization using Biolog MicroPlates and comparison of cellular fatty acids, this novel group was found to be different from the most closely related species, Azospirillum lipoferum. Based on these data, a novel species, Azospirillum melinis sp. nov., is proposed for these endophytic diazotrophs of M. minutiflora, with TMCY 0552T (=CCBAU 5106001T=LMG 23364T=CGMCC 1.5340T) as the type strain.

In addition to forming symbiotic nodules on legumes, rhizobial strains are members of soil or rhizosphere communities or occur as endophytes, e.g., in rice. Two rhizobial strains which have been isolated from root nodules of the aquatic legumes Aeschynomene fluminensis (IRBG271) and Sesbania aculeata (IRBG74) were previously found to promote rice growth. In addition to analyzing their phylogenetic positions, we assessed the suitability of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (IGS) sequences for the differentiation of closely related rhizobial taxa and for the development of PCR protocols allowing the specific detection of strains in the environment. 16S rDNA sequence analysis (sequence identity, 99%) and phylogenetic analysis of IGS sequences showed that strain IRBG271 was related to but distinct from Bradyrhizobium elkanii. Rhizobium sp. (Sesbania) strain IRBG74 was located in the Rhizobium-Agrobacterium cluster as a novel lineage according to phylogenetic 16S rDNA analysis (96.8 to 98.9% sequence identity with Agrobacterium tumefaciens; emended name, Rhizobium radiobacter). Strain IRBG74 harbored four copies of rRNA operons whose IGS sequences varied only slightly (2 to 9 nucleotides). The IGS sequence analyses allowed intraspecies differentiation, especially in the genus Bradyrhizobium, as illustrated here for strains of Bradyrhizobium japonicum, B. elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium sp. (Chamaecytisus) strain BTA-1. It also clearly differentiated fast-growing rhizobial species and strains, albeit with lower statistical significance. Moreover, the high sequence variability allowed the development of highly specific IGS-targeted nested-PCR assays. Strains IRBG74 and IRBG271 were specifically detected in complex DNA mixtures of numerous related bacteria and in the DNA of roots of gnotobiotically cultured or even of soil-grown rice plants after inoculation. Thus, IGS sequence analysis is an attractive technique for both microbial ecology and systematics.

A novel rhizobial group, cluster 9, defined in previous research [Tan, Z. Y., Wang, E. T., Peng, G. X., Zhu, M. E., Martı!nez-Romero, E. & Chen, W. X. (1999). Int J Syst Bacteriol 49, 1457–1469], was further characterized by determination of DNA base composition, whole-cell protein SDS-PAGE analysis, DNA–DNA hybridization, 16S rRNA gene sequencing and host specificity. These isolates were collected from the wild legumes Amphicarpaea trisperma, Coronilla varia and Gueldenstaedtia multiflora growing in arid and semi-arid regions in northwestern China. Isolates within cluster 9 grouped into a single cluster above a similarity level of 90<6% in a cluster analysis based on protein SDS-PAGE, and they were differentiated from defined rhizobial species. Comparative analysis of 16S rRNA gene sequences showed that isolate CCBAU 71623T, representing cluster 9, was most related to Rhizobium gallicum and Rhizobium mongolense. The DNA–DNA homologies were lower than 42<4% among cluster 9 and defined species, including R. gallicum and R. mongolense. These data indicated that cluster 9 was a unique genomic species. Isolates within this cluster could share their host plants. They could not nodulate Galega orientalis and Leucaena leucocephala and formed ineffective nodules on Phaseolus vulgaris. This group could also be differentiated from defined species by phenotypic characteristics. It is therefore proposed as a new species, Rhizobium yanglingense, with isolate CCBAU 71623 as the type strain.

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDSPAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens (more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens (≥86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.

Nodule isolates from 11 species of wild legumes in north-western China were characterized by numerical taxonomy, PCR-based 165 rRNA gene RFLP and sequence analyses, DNA-DNA hybridization, restriction patterns of nodDA5 and nifH genes, and symbiotic propetties. Based on the results of numerical taxonomy, most of the 35 new isolates were grouped into five clusters (clusters 7, 9, 12, 14 and 15). Clusters 7 and 12 were identified as Mesorhizobium amorphae and Agrobacterium tumefaciens, respectively, based on their high DNA homologies with the reference strains for these species, their 165 rRNA gene analysis and their phenotypic features. Results of 16s rDNA PCR-RFLP analysis showed that cluster 9 belonged to Rhizobium. Clusters 14 and 15 were identified as Mesorhizobium based on their moderately slowgrowing, acid-producing characters and the high similarity of their 165 rDNA PCR-RFLP patterns to those of Mesorhizobium species. These two clusters were genomic species distinct from all described species based on analysis of DNA relatedness within this genus. The isolates in cluster 12 (Agrobacterium tumefaciens) failed to nodulate their original host and other selected hosts and they did not hybridize to nif or nod gene probes. The possibility of opportunistic nodulation of these isolates is discussed. Identical restriction patterns were obtained in the nif or nod gene hybridization studies from the three isolates within cluster 15, which were isolated from the same host species. The isolates from different host plants in each of clusters 9 and 14 produced different nodDA5 RFLP patterns, but similar nifH RFLP patterns appeared (one band for each isolate). Different patterns were observed among different clusters from both the nod and nif gene hybridization studies. Crossnodulation was recorded among the isolates and the host plants in the same cluster and promiscuous properties were found among some of the hosts tested.

The genetic and phylogenetic relationships for strains of Mesorhizobium tianshanense and its relatives were compared by an analysis of the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) of whole-cell proteins, DNA-DNA hybridization, and full 16s rRNA gene sequencing. The strains of M. tianshanense formed a cluster which was distinct from those of other rhizobium species in the clustering analysis of SDS-PAGE. DNA-DNA relatedness between A-1BS (type strain of M. tianshanense) and the type or eference strains for Mesorhizobium loti, M. huakuii, M. ciceri, M. mediterraneum, and cluster U, an unnamed hizobial group, ranged from 4.4 to 43.8%. The phylogenetic analysis based on the 16s rRNA gene sequences howed that M. tianshunense was closely related to the Mesorhizobium phylogenetic branch and could be istinguished from the other four species in this branch. These results further confirmed that these bacteria onstitute a distinct rhizobial species.

Twenty-nine rhizobial isolates from root nodules of Astragalus and Lespedeza spp. growing in the Loess Plateau of China were characterized by numerical taxonomy, RFLP and sequencing of PCR-amplified 16S rRNA genes, measurement of DNA G+C content, DNA–DNA relatedness and cross-nodulation with selected legume species. Based on the results of numerical taxonomy, the isolates formed two clusters (1 and 2) with some single isolates at a similarity level of 82 %. Cluster 1 contained six isolates from Astragalus and Lespedeza spp. Cluster 2 consisted of nine isolates from Astragalus spp. DNA relatedness was greater than 80% among isolates within cluster 2. Phylogenetic analysis based on 16S rRNA gene sequences showed that CCBAU 7190BT, representing cluster 2, was closely related to Rhizobium galegae and Rhizobium huautlense. DNA–DNA relatedness between CCBAU 7190BT and reference strains of R. galegae, R. huautlense and other related species ranged from 0 to 48?6 %. The cluster 2 isolates could also be differentiated phenotypically from related species. Based on these data, a novel species, Rhizobium loessense sp. nov., is proposed for cluster 2, with the type strain CCBAU 7190BT (=AS1.3401T=LMG 21975T).

Sinorhizobium morelense sp. nov. is described to designate a group of bacteria isolated from root nodules of Leucaena leucocephala. S. morelense shows 98% 16S rRNA gene sequence similarity to some Sinorhizobium species and to Ensifer adhaerens. This novel species is distinguished from other Sinorhizobium species and from E. adhaerens by DNA–DNA hybridization, 16S rRNA gene restriction fragments and sequence and some distinctive phenotypic features. Strains of this species are highly resistant to some antibiotics, such as carbenicillin (1 mg mlN1), kanamycin (500 lgmlN1) and erythromycin (300 lgmlN1). They do not form nodules, but a nodulating strain, Lc57, is closely related to the novel species. Strain Lc04T (=LMG 21331T= CFN E1007T) is designated as the type strain of this novel species.

Eight fast-growing rhizobial isolates from Xinjiang soils were identified as Sinorhizobium xinjiangense by analyses of 16S rRNA gene sequences, SDSPAGE of proteins, intergenic spacer sequences and DNA–DNA hybridization. Based on all of the results, these isolates and the reference strains for S. xinjiangense were a distinct genomic species, although the 16S rRNA genes were closely related to that of Sinorhizobium fredii.

Forty-eight rhizobial isolates from root nodules of Indigofera and Kummerowia, two genera of annual or perennial wild legumes growing in the Loess Plateau in north-western China, were characterized by a polyphasic approach. Two main groups, cluster 1 and cluster 2, were defined based upon the results of numerical taxonomy, SDS-PAGE of whole-cell proteins and DNA relatedness. All the isolates within cluster 1 were isolated from Indigofera and they were identified as Rhizobium strains by 16S rRNA gene analysis. DNA relatedness of 29<5–48<9% was obtained among the cluster 1 isolates and the reference strains for defined Rhizobium species. Cluster 2 consisted of isolates from Kummerowia stipulacea and was identified as belonging to Sinorhizobium by 16S rRNA gene analyses. DNA relatedness varied from 5<2 to 41<7% among the isolates of cluster 2 and reference strains for Sinorhizobium species. Considering the existence of distinctive features among these two groups and related species within the genera Rhizobium and Sinorhizobium, we propose two novel species, Rhizobium indigoferae sp. nov. for cluster 1, with isolate CCBAU 71714T (=AS 1.3046T) as the type strain, and Sinorhizobium kummerowiae sp. nov. for cluster 2, with isolate CCBAU 71042T (=AS 1.3045T) as the type strain.