A total of 63 strains of rhizobia isolated from Hainan Province, a tropical region of the People's Republic of China, and 27 representative strains belonging to the genera Rhizobium, Bradyrhizobium, and Agrobacterium were compared by performing numerical taxonomy, DNA hybridization, and DNA base composition analysis to determine the relationships among these rhizobia. The results indicated that the strains isolated from Hainan Province fell into two distinct phena, the slowly growing rhizobia and the fast-growing rhizobia. The slowly growing rhizobia, which formed three subphena that seemed to be three subspecies, are Bradyrhizobium japonicum strains. The fast-growing strains belong to the genus Rhizobium and might be further divided into three specific groups. Sometimes both slowly growing rhizobia and fast-growing rhizobia were isolated from host plants belonging to the same genus or species or even from the same nodule. There was no correlation between hosts and the distribution of rhizobia in the subphena. Isolates obtained from members of the same host genus or species fell into different groups or subgroups.The root nodule bacteria of tropical leguminous plants have been investigated by workers in many laboratories (1,(5)(6)(7)(8)10,23,25), but taxonomic studies of these bacteria have been sparse and have been limited to a few leguminous symbionts (16,19,24,27,28). Dreyfus and Dommergues (6) classified 13 Acacia species into three groups on the basis of effective nodulation with fast-growing and slowly growing tropical strains of rhizobia. The first group formed nodules effectively with slow growers; the second group formed effective nodules with fast growers; and the third group formed effective nodules with both fast and slow growers. Zhang et al. (27) obtained 12 clusters by using numerical taxonomy to study 97 strains isolated from root nodules of Acacia senagal, Prospopis chilensis, and other legumes. Thus, it is interesting to study the taxonomy of rhizobia isolated from a wide range of tropical legumes.In this work 63 rhizobial strains were isolated from various legumes, including trees, herbs, and vines, growing in different geographic regions of Hainan Province of the People's Republic of China, a tropical region south of latitude 20" N. These strains were compared with previously described species of rhizobia by performing numerical taxonomy, DNA composition, and DNA-DNA hybridization analysis. MATERIALS AND METHODSBacterial strains. A total of 90 strains were used in this study (Table 1); 27 of these strains were type and representative strains of Rhizobium, Bradyrhizobium, and Agrobacterium species, and 63 strains were isolated from root nodules collected in different geographic regions of Hainan Province. The purity of the cultures was assured by using single-colony isolates and checking colony morphology, bacterial morphology, and the Gram stain reaction. The identity of each strain was checked by performing a plant infection test with the original host plant.

Nine bacterial strains isolated from root nodules of Astragalus sinicus were compared with 41 reference strains, including the type strains of the type species of the genera Rhizobium, Bradyrhizobium, and Agrobacterium, by performing a numerical analysis of 200 phenotype features. Representative strains belonging to different clusters were further compared with similar bacteria by using data from gel electrophoresis of whole-cell proteins, DNA G + C content data, and DNA-DNA hybridization data. The rhizobial strains isolated from nodules of A . sinicus constitute a distinct homology group that is quite different from previously described Rhizobium, Bradyrhizobium, and Agrobacterium species and from strains isolated from other Astragalus species. We propose the name Rhizobium huakuii sp. nov. for the strains isolated from A . sinicus. Type strain CCBAU 2609 (= 103) has been deposited in the Culture Collection of Beijing Agricultural University, Beijing, People's Republic of China.Bacteria which form nitrogen-fixing nodules on leguminous plants are currently divided into the following four genera: Rhizobium, Bradyrhizobium, Azorhizobium, and Sinorhizobium (2, 5, 9). The genus Rhizobium comprises

Fifty-five Chinese isolates from nodules of Amorpha fruticosa were characterized and compared with the type strains of the species and genera of bacteria which form nitrogen-f ixing symbioses with leguminous host plants. A polyphasic approach, which included RFLP of PCR-amplif ied 165 rRNA genes, multilocus enzyme electrophoresis (MLEE), DNA-DNA hybridization, 165 rRNA gene sequencing, electrophoretic plasmid profiles, cross-nodulation and a phenotypic study, was used in the comparative analysis. The isolates originated from several different sites in China and they varied in their phenotypic and genetic characteristics. The majority of the isolates had moderate to slow growth rates, produced acid on YMA and harboured a 930 kb symbiotic plasmid (pSym). Five different RFLP patterns were identified among the 16s rRNA genes of all the isolates. Isolates grouped by PCR-RFLP of the 165 rRNA genes were also separated into groups by variation in MLEE profiles and by DNA-DNA hybridization. A representative isolate from each of these DNA homology groups had a separate position in a phylogenetic tree as determined from sequencing analysis of the 165 rRNA genes. A new species, Mesorhizobium amorphae, is proposed for the majority of the isolates, which belonged to a moderately slow-to slow-growing, acid-producing group based upon their distinct phylogenetic position, their unique electrophoretic type, their low DNA homology with reference strains representing the species within the genus Mesorhizobium and their distinct phenotypic features. Strain ACCC 19665 was chosen as the type strain for M. amorphae sp. nov.

The nitrogen-fixing rhizobial symbionts of Sesbania herbacea growing in the nature reserve a t the Sierra de Huautla, Mexico, were isolated and characterized. All 104 isolates together with the type strain for Rhizobium galegae, HAMBI 540T, had similar 165 rRNA genes as revealed by PCR-RFLP analysis. Similarity in the sequences of the 16s rRNA genes placed the isolates on a phylogenetic branch shared with R. galegae. Among 66 randomly selected isolates, three closely related electrophoretic alloenzyme types (ETs) were identified, which were distinct from 10 ETs distinguished among 23 strains of R. galegae. A new species Rhizobium huautlense, represented by the Sesbania isolate SOZT, is proposed based upon low estimates of DNA relatedness between our chosen type strain and the type strains for the other species, the dissimilarity of the nucleotide sequence of the 165 rRNA genes, and their distinct ETs compared with R. galegae. The description of R. huaut/ense is significant because in the reconstruction of the phylogeny of R. huautlense there was a shift in the node of the branch of Agrobacterium vitis relative to that of R. galegae. The revised phylogenetic tree would tend to indicate common ancestry between R. galegae and Rhizobium leguminosarum .