En Tao Wang

Rhizobium huakuii sp. nov. Isolated from the Root Nodules of Astragalus sinicus

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, Peoples Republic of China.

Characterization of rhizobia that nodulate legume species of the genus Lespedeza and description of Bradyrhizobium yuanmingense sp. nov.

Legume species belonging to the genus Lespedeza are annual or perennial herb or shrub plants that grow in the northern hemisphere. They are known for the formation of root nodules, but little information is available about their microsymbionts. In this study, 58 root-nodule isolates from Lespedeza spp., obtained from China and the USA, were characterized using numerical taxonomic analysis of phenotypic features, SDS-PAGE analysis of whole-cell proteins, DNA-DNA hybridization, 16S rRNA gene sequence analysis and cross-nodulation with selected legume species. From the results generated using these approaches, it was concluded that Lespedeza spp. were promiscuous hosts for rhizobia. Four main clusters of bacteria, which included 35 of the strains isolated from Lespedeza spp., were defined upon numerical taxonomic analysis; these groups corresponded to those determined from analyses of protein electrophoretic and DNA-DNA hybridization data. The four clusters were found to define strains belonging to one of four species, Sinorhizobium saheli, Bradyrhizobium japonicum, Bradyrhizobium elkanii or a novel species of the genus Bradyrhizobium. The strains of B. japonicum and B. elkanii were all from the USA soil samples, and their representative strains could not nodulate soybean. The seven strains found to represent the novel Bradyrhizobium sp. were from China. These were differentiated from recognized species of the genus Bradyrhizobium by all of the taxonomic methods used here; hence, it is proposed that the novel strains isolated from Lespedeza spp. represent a novel species of the genus Bradyrhizobium, Bradyrhizobium yuanmingense. The type strain of the novel species, CCBAU 10071(T) (= CFNEB 101(T)), formed ineffective nodules on Medicago sativa and Melilotus albus but did not nodulate soybean. The other 23 bacterial strains isolated from Lespedeza spp. were found to form single branches or small groups (two to three strains) that were related to Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium spp. on the basis of numerical taxonomic analysis, indicating the possibility that other rhizobial species are also associated with Lespedeza spp.

Novel nitrogen-fixing acetic acid bacteria, Gluconacetobacter johannae sp. nov. and Gluconacetobacter azotocaptans sp. nov., associated with coffee plants

Diazotrophic bacteria were isolated, in two different years, from the rhizosphere and rhizoplane of coffee (Coffea arabica L.) plants cultivated in Mexico; they were designated as type DOR and type SAd isolates. They showed characteristics of the family Acetobacteraceae, having some features in common with Gluconacetobacter (formerly Acetobacter) diazotrophicus, the only known N2-fixing species of the acetic acid bacteria, but they differed from this species with regard to several characteristics. Type DOR isolates can be differentiated phenotypically from type SAd isolates. Type DOR isolates and type SAd isolates can both be differentiated from Gluconacetobacter diazotrophicus by their growth features on culture media, their use of amino acids as nitrogen sources and their carbon-source usage. These results, together with the electrophoretic mobility patterns of metabolic enzymes and amplified rDNA restriction analysis, suggested that the type DOR and type SAd isolates represent two novel N2-fixing species. Comparative analysis of the 16S rRNA sequences revealed that strains CFN-Cf55T (type DOR isolate) and CFN-Ca54T (type SAd isolate) were closer to Gluconacetobacter diazotrophicus (both strains had sequence similarities of 98.3%) than to Gluconacetobacter liquefaciens, Gluconacetobacter sacchari (similarities < 98%) or any other acetobacteria. Strain CFN-Cf55T exhibited low levels of DNA-DNA reassociation with type SAd isolates (mean 42%) and strain CFN-Ca54T exhibited mean DNA-DNA reassociation of 39.5% with type DOR isolates. Strains CFN-Cf55T and CFN-Ca54T exhibited very low DNA reassociation levels, 7-21%, with other closely related acetobacterial species. On the basis of these results, two novel N2-fixing species are proposed for the family Acetobacteraceae, Gluconacetobacter johannae sp. nov. (for the type DOR isolates), with strain CFN-Cf55T (= ATCC 700987T = DSM 13595T) as the type strain, and Gluconacetobacter azotocaptans sp. nov. (for the type SAd isolates), with strain CFN-Ca54T (= ATCC 70098ST = DSM 13594T) as the type strain.

Rhizobium indigoferae sp. nov. and Sinorhizobium kummerowiae sp. nov., respectively isolated from Indigofera spp. and Kummerowia stipulacea.

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 71714(T) (= AS 1.3046(T)) as the type strain, and Sinorhizobium kummerowiae sp. nov. for cluster 2, with isolate CCBAU 71042(T) (= AS 1.3045(T)) as the type strain.

Biodiversity and Biogeography of Rhizobia Associated with Soybean Plants Grown in the North China Plain

ABSTRACT As the putative center of origin for soybean and the second largest region of soybean production in China, the North China Plain covers temperate and subtropical regions with diverse soil characteristics. However, the soybean rhizobia in this plain have not been sufficiently studied. To investigate the biodiversity and biogeography of soybean rhizobia in this plain, a total of 309 isolates of symbiotic bacteria from the soybean nodules collected from 16 sampling sites were studied by molecular characterization. These isolates were classified into 10 genospecies belonging to the genera Sinorhizobium and Bradyrhizobium, including four novel groups, with S. fredii (68.28%) as the dominant group. The phylogeny of symbiotic genes nodC and nifH defined four lineages among the isolates associated with Sinorhizobium fredii, Bradyrhizobium elkanii, B. japonicum, and B. yuanmingense, demonstrating the different origins of symbiotic genes and their coevolution with the chromosome. The possible lateral transfer of symbiotic genes was detected in several cases. The association between soil factors (available N, P, and K and pH) and the distribution of genospecies suggest clear biogeographic patterns: Sinorhizobium spp. were superdominant in sampling sites with alkaline-saline soils, while Bradyrhizobium spp. were more abundant in neutral soils. This study clarified the biodiversity and biogeography of soybean rhizobia in the North China Plain.

Characterization of symbiotic and endophytic bacteria isolated from root nodules of herbaceous legumes grown in Qinghai-Tibet plateau and in other zones of China

Qinghai–Tibet plateau is the highest place in the world and the environment in that plateau is hard for animals and plants, with low temperature, low concentration of oxygen and high solar radiation. In this study, 61 root nodule isolates from Vicia, Oxytropis, Medicago, Melilotus and Onobrychis species grown in Qinghai–Tibet plateau and in loess plateau were comparatively characterized. Based upon the results of numerical taxonomy, ARDRA, AFLP, DNA–DNA hybridization and 16S rDNA sequencing, the isolates were classified as Rhizobium leguminosarum, Sinorhizobium meliloti, Sinorhizobium fredii, Mesorhizobium sp., Phyllobacterium sp., Stenotrophomonas sp. and two non-symbiotic groups related to Agrobacterium and Enterobacteriaceae. The strains isolated from Qinghai–Tibet plateau and from the loess plateau were mixed in these species or groups. Oxytropis spp. and Medicagoarchiducis-nicolai grown in Qinghai–Tibet plateau were recorded as new hosts for R. leguminosarum, as well as Oxytropis glabra and Medicago lupulina for S. fredii. In addition, strains resistant to high alkaline (pH 11) and high concentration of NaCl (3–5%, w/v) were found in each of the rhizobial species. This was the first systematic study of rhizobia isolated from Qinghai–Tibet plateau

Endophytic Occupation of Root Nodules and Roots of Melilotus dentatus by Agrobacterium tumefaciens

Agrobacterium strains have been frequently isolated from the root nodules of different legumes. Various possible mechanisms have been proposed to explain the existence of these bacteria in nodules, but there is no sufficient experimental evidence to support the estimations. In this work, we proved that the Agrobacterium strain CCBAU 81181, which was originally isolated from the root nodules of Onobrychis viciaefolia, and a symbiotic strain of Sinorhizobium meliloti CCBAU 10062 could coinhabit the root nodules of Melilotus dentatus. Analyses were performed by using a fluorescence marker, reisolation of bacteria from nodules, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) of whole cellular proteins, and polymerase chain reaction amplification of symbiotic genes. The inoculation of A. tumefaciens CCBAU 81181 did not affect the growth and nodulation of plants. CCBAU 81181 and 24 other Agrobacterium strains isolated from nodules were incapable of nodulating on their original or alternative host and 22 strains of these strains were endophytes in the roots and stems of their hosts. Also, the tumor-inducing A. tumefaciens strains IAM 13129T and C58 were found capable of entering the roots of Glycyrrhiza pallidiflora, but did not cause pathogenic symptoms. With these results, we conclude that A. tumefaciens strains could be endophytic bacteria in the roots, stems, and root nodules. This finding partially explains why Agrobacterium strains were frequently isolated from the surface-sterilized nodules.

Unique community structure and biogeography of soybean rhizobia in the saline-alkaline soils of Xinjiang, China

To investigate the community composition and biogeography of soybean rhizobia in Xinjiang, a total of 151 strains were investigated with RFLP and phylogenetic analyses of 16S rRNA gene, 16S–23S intergenic spacer, three housekeeping genes (atpD, glnII and recA), and two symbiotic genes (nifH and nodC), as well as cross-nodulation. Two rhizobial species, Bradyrhizobium liaoningense and Sinorhizobium fredii, were found as dominant groups in communities of soybean rhizobia in Xinjiang, whereas three Rhizobium genomic species, B. yuanmingense and B. japonicum, were minor groups. These genomic species showed clear correlations with eco-regions, and their symbiotic genes were identical or very similar to those of the reference strains for the corresponding species. Conclusively, the dominant soybean rhizobia S. fredii and B. liaoningense in Xinjiang might be introduced from other Chinese regions, but they have been selected as the rhizobia adapted to the saline-alkaline soils. The high pH, salinity, and phosphate concentration in soil might be the environmental factors determining the biogeography of these bacteria. It is worth mentioning that a novel Rhizobium species that may have acquired the symbiotic genes from a Bradyrhizobium lineage was identified.

Shinella kummerowiae sp. nov., a symbiotic bacterium isolated from root nodules of the herbal legume Kummerowia stipulacea.

Bacterial strain CCBAU 25048(T) was isolated from root nodules of Kummerowia stipulacea grown in Shandong province of China. Cells of the strain were Gram-negative, strictly aerobic, non-spore-forming, motile short rods. Phylogeny of 16S rRNA gene sequences revealed that the strain belonged to the genus Shinella, a member of family Rhizobiaceae. Its closest phylogenetic relatives were Shinella granuli Ch06(T) and Shinella zoogloeoides IAM 12669(T), respectively showing 98.3 and 98.9 % 16S rRNA gene sequence similarity. Strain CCBAU 25048(T) had DNA-DNA relatedness of 43.5 and 34.8 %, respectively, with S. zoogloeoides JCM 20728(T) and S. granuli JCM 13254(T). In addition, in TP-RAPD analysis, different patterns were obtained for these three strains and some rhizobial strains. The nifH, nodC and nodD sequences of CCBAU 25048(T) were identical or very similar to those of bean-nodulating Rhizobium tropici strains. Several phenotypic characteristics, including the use of citrate and d-ribose as carbon sources and growth at pH 11.0, as well as the fatty acid composition, could differentiate CCBAU 25048(T) from the two defined Shinella species. Therefore, a novel species Shinella kummerowiae sp. nov. is proposed, with strain CCBAU 25048(T) (=JCM 14778(T) =LMG 24136(T)) as the type strain.

Identification of isolates from soybean nodules in Xinjiang Region as Sinorhizobium xinjiangense and genetic differentiation of S. xinjiangense from Sinorhizobium fredii.

Eight fast-growing rhizobial isolates from Xinjiang soils were identified as Sinorhizobium xinjiangense by analyses of 16S rRNA gene sequences, SDS-PAGE 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.