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.