Nutrients cause consolidation of soil carbon flux to small proportion of bacterial community.

Nutrient amendment diminished bacterial functional diversity, consolidating carbon flow through fewer bacterial taxa. Here, we show strong differences in the bacterial taxa responsible for respiration from four ecosystems, indicating the potential for taxon-specific control over soil carbon cycling. Trends in functional diversity, defined as the richness of bacteria contributing to carbon flux and their equitability of carbon use, paralleled trends in taxonomic diversity although functional diversity was lower overall. Among genera common to all ecosystems, Bradyrhizobium, the Acidobacteria genus RB41, and Streptomyces together composed 45-57% of carbon flow through bacterial productivity and respiration. Bacteria that utilized the most carbon amendment (glucose) were also those that utilized the most native soil carbon, suggesting that the behavior of key soil taxa may influence carbon balance. Mapping carbon flow through different microbial taxa as demonstrated here is crucial in developing taxon-sensitive soil carbon models that may reduce the uncertainty in climate change projections.

Endophytic Bradyrhizobium spp. isolates from sugarcane obtained through different culture strategies.

Brazilian sugarcane has been shown to obtain part of its nitrogen via biological nitrogen fixation (BNF). Recent reports, based on the culture independent sequencing of bacterial nifH complementary DNA (cDNA) from sugarcane tissues, have suggested that members of the Bradyrhizobium genus could play a role in sugarcane-associated BNF. Here we report on the isolation of Bradyrhizobium spp. isolates and a few other species from roots of sugarcane cultivar RB867515 by two cultivation strategies: direct isolation on culture media and capture of Bradyrhizobium spp. using the promiscuous legume Vigna unguiculata as trap-plant. Both strategies permitted the isolation of genetically diverse Bradyrhizobium spp. isolates, as concluded from enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR) fingerprinting and 16S ribosomal RNA, nifH and nodC sequence analyses. Several isolates presented nifH phylotypes highly similar to nifH cDNA phylotypes detected in field-grown sugarcane by a culture-independent approach. Four isolates obtained by direct plate cultivation were unable to nodulate V. unguiculata and, based on PCR analysis, lacked a nodC gene homologue. Acetylene reduction assay showed in vitro nitrogenase activity for some Bradyrhizobium spp. isolates, suggesting that these bacteria do not require a nodule environment for BNF. Therefore, this study brings further evidence that Bradyrhizobium spp. may play a role in sugarcane-associated BNF under field conditions.

Diversity of rhizobia associated with leguminous trees growing in South Korea.

This study was carried out to examine the diversity of 34 isolates collected from 11 species of leguminous trees growing in South Korea. Phylogenetic relationships between these 34 isolates and reference strains of the Azorhizobium, Bradyrhizobium, Mesorhizobium, Rhizobium and Ensifer/Sinorhizobium were analysed by using 16S rRNA gene sequences. Twenty-one isolates were related to Mesorhizobium, four isolates to Rhizobium, and nine isolates to Bradyrhizobium. But none of isolates were related to Sinorhizobium/Ensifer and Azorhizobium. Robinia pseudoacacia and Amorpha fruticosa were nodulated by various genotypes of rhizobia out of them, most of the isolates belonged to the genus Mesorhizobium. The isolates from Lespedeza bicolar belonged to diverse genera of Mesorhizobium, Rhizobium, and Bradyrhizobium. The isolates from Maackia amurensis and Lespedeza maximowiezii var. tomentella were phylogenetically related to the genera of Bradyrhizobium. PCR-based RAPD method and phylogenetic analysis of the 16S rRNA results revealed a high phylogenetic diversity of rhizobial strains nodulating leguminous trees in South Korea. Also, the relationships between host and bacterial phylogenies showed that only Robinia pseudoacacia, and Wisteria floribunda have significantly unique branch length than expected by chance based on phylogenetic tree.

Oxidation of elemental sulfur by Fusarium solani strain THIF01 harboring endobacterium Bradyrhizobium sp.

Nineteen fungal strains having an ability to oxidize elemental sulfur in mineral salts medium were isolated from deteriorated sandstones of Angkor monuments. These fungi formed clearing zone on agar medium supplemented with powder sulfur due to the dissolution of sulfur. Representative of the isolates, strain THIF01, was identified as Fusarium solani on the basis of morphological characteristics and phylogenetic analyses. PCR amplification targeting 16S rRNA gene and analyses of full 16S rRNA gene sequence indicated strain THIF01 harbors an endobacterium Bradyrhizobium sp.; however, involvement of the bacterium in the sulfur oxidation is still unclear. Strain THIF01 oxidized elemental sulfur to thiosulfate and then sulfate. Germination of the spores of strain THIF01 was observed in a liquid medium containing mineral salts supplemented with elemental sulfur (rate of germinated spores against total spores was 60.2%), and the culture pH decreased from pH 4.8 to 4.0. On the contrary, neither germination (rate of germinated spores against total spores was 1.0%) nor pH decrease was observed without the supplement of elemental sulfur. Strain THIF01 could also degrade 30 ppmv and ambient level (approximate 500 pptv) of carbonyl sulfide.

Phenotypic, genomic and phylogenetic characteristics of rhizobia isolated from root nodules of Robinia pseudoacacia (black locust) growing in Poland and Japan.

Rhizobial strains, rescued from the root nodules of Robinia pseudoacacia growing in Japan and Poland, were characterized for the phenotypic properties, genomic diversity as well as phylogeny and compared with the reference strains representing different species and genera of nodule bacteria. They had a moderately slow growth rate, a low tolerance to antibiotics, a moderate resistance to NaCl and produced acid in yeast mannitol agar. Cluster analysis based on the phenotypic features divided all bacteria involved in this study into four phena, comprising: (1) Rhizobium sp. + Sinorhizobium sp., (2) Bradyrhizobium sp., (3) R. pseudoacacia microsymbionts + Mesorhizobium sp., and (4) Rhizobium galegae strains at similarity coefficient of 74%. R. pseudoacacia nodule isolates and Mesorhizobium species were placed on a single branch clearly distinct from other rhizobium genera lineages. Strains representing R. pseudoacacia microsymbionts shared 98-99% 16S rDNA sequence identity with Mesorhizobium species and in 16S rDNA phylogenetic tree all these bacteria formed common cluster. The rhizobia tested are genomically heterogeneous as indicated by the AFLP (Amplified Fragment Length Polymorphism) method. The bacteria studied exhibited high degree of specificity for nodulation. Nitrogenase structural genes in these strains were located on 771-961 kb megaplasmids.

Bradyrhizobium betae sp. nov., isolated from roots of Beta vulgaris affected by tumour-like deformations.

Some varieties of sugar beet, Beta vulgaris, cultivated in northern Spain have large deformations that resemble the tumours produced by Agrobacterium species. In an attempt to isolate the agent responsible for these deformations, several endophytic slow-growing bacterial strains were isolated, the macroscopic morphology of which resembled that of Bradyrhizobium species. These strains were not able to produce tumours in Nicotiana tabacum plants and, based on phylogenetic analysis of their 16S rRNA, they are closely related to the genus Bradyrhizobium. Phenotypic and molecular characteristics of these strains revealed that they represent a species different from all Bradyrhizobium species previously described. Sequence analysis of the 16S-23S rDNA intergenic spacer region indicated that these novel strains form a homogeneous group, related to Bradyrhizobium japonicum, Bradyrhizobium liaoningense and Bradyrhizobium yuanmingense. DNA-DNA hybridization confirmed that these strains represent a novel species of the genus Bradyrhizobium, for which the name Bradyrhizobium betae sp. nov. is proposed. The type strain is PL7HG1T (=LMG 21987T=CECT 5829T).

Small-subunit rRNA genotyping of rhizobia nodulating Australian Acacia spp.

The structure of rhizobial communities nodulating Acacia in southeastern Australia from south Queensland to Tasmania was investigated by a molecular approach. A total of 118 isolates from nodule samples from 13 different Acacia species collected at 44 sites were characterized by small-subunit (SSU) ribosomal DNA (rDNA) PCR-restriction fragment length polymorphism analysis. Nine rhizobial genomospecies were identified, and these taxa corresponded to previously described genomospecies (B. Lafay and J. J. Burdon, Appl. Environ. Microbiol. 64:3989-3997, 1998). Eight of these genomospecies belonged to the Bradyrhizobium lineage and accounted for 96.6% of the isolates. The remaining genomospecies corresponded to Rhizobium tropici. For analysis of geographic patterns, results were grouped into five latitudinal regions regardless of host origin. In each region, as observed previously for rhizobial isolates taken from non-Acacia legumes (Lafay and Burdon, Appl. Environ. Microbiol. 64:3989-3997, 1998), rhizobial communities were dominated by one or two genomospecies, the identities of which varied from place to place. Despite this similarity in patterns, the most abundant genomospecies for Acacia isolates differed from the genomospecies found in the non-Acacia-derived rhizobial collection, suggesting that there is a difference in nodulation patterns of the Mimosoideae and the Papilionoideae. Only two genomospecies were both widespread and relatively abundant across the range of sites sampled. Genomospecies A was found in all regions except the most northern sites located in Queensland, whereas genomospecies B was not detected in Tasmania. This suggests that genomospecies A might be restricted to the more temperate regions of Australia, whereas in contrast, genomospecies B occurs in different climatic and edaphic conditions across the whole continent. The latter hypothesis is supported by the presence of genomospecies B in southwestern Australia, based on partial SSU rDNA sequence data (N. D. S. Marsudi, A. R. Glenn, and M. J. Dilworth, Soil Biol. Biochem. 31:1229-1238, 1998).

Numerical taxonomy of Sarothamnus scoparius rhizobia.

Twenty nodule isolates from Sarothamnus scoparius (broom) growing in Poland and nine strains from plants growing in Japan were studied for phenotypic properties, plasmid presence, phage sensitivity, and host plant specificity. By numerical analysis of phenotypic properties, it was found that the studied nodule bacteria, originating from geographically different countries, constitute two separate groups affiliated to the bradyrhizobium cluster. The membership of S. scoparius rhizobia in the Bradyrhizobium genus was also supported by their long generation time, alkaline reaction in YEM medium with mannitol, lack of plasmids, and wide host plant range.

AFLP fingerprint analysis of Bradyrhizobium strains isolated from Faidherbia albida and Aeschynomene species.

The diversity of Bradyrhizobium isolates from Faidherbia albida and Aeschynomenee species was assessed using AFLP analysis, a high-resolution genomic fingerprinting technique. Reference strains from Bradyrhizobium japonicum, Bradyrhizobium elkanii and Bradyrhizobium liaoningense were included for comparison. At a similarity level of 50%, a total of 34 different groups were obtained by cluster analysis of the genomic fingerprints. Four of these clusters correspond to the three reference species, demonstrating the large diversity of the isolates studied. Comparison with other data demonstrates that AFLP has a higher resolution than restriction analysis of 16S rRNA genes, SDS-PAGE analysis of proteins and phenotypic analysis. Results of the latter two methods showed little correspondence with the genotypic data.

Relationships of bradyrhizobia from Platypodium and Machaerium (Papilionoideae: tribe Dalbergieae) on Barro Colorado Island, Panama.

Enzyme electrophoresis and rRNA sequencing indicated that root nodule bacteria from the canopy tree Platypodium elegans and the lianas Machaerium milleflorum and Machaerium arboreum on Barro Colorado Island, Panama, were highly diverse on a local scale. A total of 11 distinct multilocus genotypes [ETs (electrophoretic types)] was found among the 33 isolates analysed. On average, ETs differed from one another at 74% of the 11 enzyme loci assayed, and separate nodules on a single host individual were often occupied by genetically divergent ETs. Certain ETs were sampled multiple times from both Platypodium and Machaerium, suggesting a lack of specificity toward the two legume genera. Within the intervening sequence (IVS) region in the 5' end of 23S rRNA, seven ETs had a length variant similar to that of Bradyrhizobium japonicum USDA 110, and the other four ETs had an IVS region 26-28 bp shorter. Parsimony analysis of both partial 23S rRNA and nearly full-length 16S rRNA sequences indicated that all Platypodium and Machaerium isolates were related to B. japonicum rather than Bradyrhizobium elkanii. The 16S rRNA sequence of one isolate was >99% similar to that of B. japonicum USDA 110, and the closest known relatives for other isolates were Philippine bradyrhizobia from the legumes Stylosanthes and Samanea.

Divergent Bradyrhizobium symbionts on Tachigali versicolor from Barro Colorado Island, Panama.

Relationships of root-nodule bacteria from the tree Tachigali versicolor (legume subfamily Caesalpinioideae) were analyzed for 20 isolates sampled from juvenile plants growing on Barro Colorado Island (BCI), Panama. Bacterial genetic diversity appeared to be low. In the highly polymorphic 5' intervening sequence region of 23S rRNA, all isolates had the same length variant. A 472 bp segment spanning this region was sequenced in four isolates, and all proved to be identical at every nucleotide position. RFLP analysis of a 868 bp fragment of the nitrogenase alpha-subunit gene likewise indicated that all 20 isolates shared an identical set of restriction sites. Phylogenetic analysis of both partial 23S rRNA and nearly full-length 16S rRNA sequences showed that bacterial symbionts of T. versicolor fall into the genus Bradyrhizobium. However, they are divergent from the bradyrhizobia associated with other BCI legumes, as well as from other currently known bacteria in this genus. Inoculation tests with two promiscuously-nodulating legumes showed that bacteria from T. versicolor were unable to form nodules on Vigna unguiculata, but did nodulate Macroptilium atropurpureum, although the nodules lacked nitrogen fixation activity. The association of Tachigali with a divergent lineage of Bradyrhizobium is noteworthy in view of this plant's position within a clade of the mostly non-nodulating "primitive" legume subfamily Caesalpinioideae that gave rise to the predominantly nodulating subfamily Mimosoideae.

Relationships of bradyrhizobia from the legumes Apios americana and Desmodium glutinosum.

Multilocus enzyme electrophoresis, partial 23S rRNA sequences, and nearly full-length 16S rRNA sequences all indicated high genetic similarity among root-nodule bacteria associated with Apios americana, Desmodium glutinosum, and Amphicarpaea bracteata, three common herbaceous legumes whose native geographic ranges in eastern North America overlap extensively. A total of 19 distinct multilocus genotypes (electrophoretic types [ETs]) were found among the 35 A. americana and 33 D. glutinosum isolates analyzed. Twelve of these ETs (representing 78% of all isolates) were either identical to ETs previously observed in A. bracteata populations, or differed at only one locus. Within both 23S and 16S rRNA genes, several isolates from A. americana and D. glutinosum were either identical to A. bracteata isolates or showed only single nucleotide differences. Growth rates and nitrogenase activities of A. bracteata plants inoculated with isolates from D. glutinosum were equivalent to levels found with native A. bracteata bacterial isolates, but none of the three A. americana isolates tested had high symbiotic effectiveness on A. bracteata. Phylogenetic analysis of both 23S and 16S rRNA sequences indicated that both A. americana and D. glutinosum harbored rare bacterial genotypes similar to Bradyrhizobium japonicum USDA 110. However, the predominant root nodule bacteria on both legumes were closely related to Bradyrhizobium elkanii.