Polyphasic analysis reveals correlation between phenotypic and genotypic analysis in soybean bradyrhizobia (Bradyrhizobium spp.).

Soybean bradyrhizobia (Bradyrhizobium spp.) are bacteria that fix atmospheric nitrogen within the root nodules of soybean, a crop critical for meeting global nutritional protein demand. Members of this group differ in symbiotic effectiveness, and historically both phenotypic and genotypic approaches have been used to assess bradyrhizobial diversity. However, agreement between various approaches of assessment is poorly known. A collection (n=382) of soybean bradyrhizobia (Bradyrhizobium japonicum, B. diazoefficiens, and B. elkanii) were characterized by Internal Transcribed Spacer - Restriction Fragment Length Polymorphism (ITS-RFLP), cellular fatty acid composition (fatty acid methyl esters, FAME), and serological reactions to assess agreement between phenotypic and genotypic methods. Overall, 76% of the accessions demonstrated identical clustering with each of these techniques. FAME was able to identify all 382 accessions, whereas 14% were non-reactive serologically. One ITS-RFLP group, containing 36 Delaware isolates, produced multiple ITS amplicons indicating they possess multiple ribosomal RNA (rrn) operons. Cloning and sequencing revealed that these strains contained as many as three heterogenous rrn operons, a trait previously unknown in bradyrhizobia. A representative subset of 96 isolates was further characterized using 16S rRNA and Internal Transcribed Spacer (ITS) amplicon sequencing. ITS sequences showed better inter- and intra-species discrimination (65-99% identity) than 16S sequences (96-99% identity). This study shows that phenotypic and genotypic approaches are strongly correlated at the species level but should be approached with caution. We also suggest using combined 16S and ITS genotyping data to obtain better inter- and intra-species resolution in bradyrhizobia classification.

Microbial community responses to atrazine exposure and nutrient availability: linking degradation capacity to community structure.

Repeated pesticide exposure may enhance biodegradation through selective enrichment of pesticide-metabolizing microorganisms, particularly when the compound is used as a C and energy source. The relationship between pesticide application history and degradation rate is unclear when the chemical is utilized as a nutrient source other than C. Atrazine, a poor source of C and energy, was chosen as a model compound because it can serve as an N source for some microorganisms. Soils with (H-soil) and without (NH-soil) prior s-triazine treatment history were repeatedly exposed to atrazine and a variety of C and N source amendments. Exposure to atrazine and inorganic-N availability were the dominant factors leading to the development of microbial communities with an enhanced capacity to degrade atrazine. The density of the atrazine-degrading microorganisms increased immediately, up to 1000-fold, with atrazine exposure in the H-soil, but comparable increases were not observed in the NH-soil until 12 weeks following laboratory acclimation, despite high rates of atrazine mineralization in these soils immediately following the acclimation period. Whole-soil fatty acid methyl ester (FAME) analysis showed that the application of alternative C and N sources in addition to atrazine resulted in a microbial community composition that was distinctly different from that in either the atrazinealone treatment or water controls for both the H- and NH-soils. These data suggest that the microbial communities in both soils were altered differently in response to the treatments but developed a similar enhanced capacity to mineralize atrazine.

Characterization of soybean bradyrhizobia for which serogroup affinities have not been identified.

The USDA, ARS National Rhizobium Germplasm Collection contains 143 accessions of slow-growing soybean strains among which there are 17 distinct serological groups. However, 11 strains appear to have no serological affinity with the 17 serogroups. Therefore, we determined whether these strains were diverse and examined their phylogenetic placement. Nine strains formed nitrogen-fixing symbioses with soybean indicating that these accessions were not contaminants. We concluded from results of amplified fragment length polymorphism (AFLP) analysis, using 3 selective primers with 8 strains, that they were genetically dissimilar. Nine strains were examined for their fatty acid composition using fatty acid methyl ester (FAME) derivatives. The FAME results with 5 strains and serotype strains of Bradyrhizobium elkanii were similar, while results with each of the remaining 2 pairs were either similar to the type strain of Bradyrhizobium japonicum (USDA 6) or to USDA 110. Evolutionary history of 9 strains was reconstructed from sequence divergence of a combination of the complete 16S rRNA gene, the internally transcribed spacer region, and about 400 bases of the 5' end of the 23S rRNA gene. Placement of 5 strains was nested within B. elkanii, 2 with USDA 110, and the other 2 with USDA 6. We concluded that soybean isolates that cannot be placed within one of the 17 established serogroups are phenotypically and genetically as diverse as the serotype strains.

Evolutionary relationships among the soybean bradyrhizobia reconstructed from 16S rRNA gene and internally transcribed spacer region sequence divergence.

From sequence divergence of 16S rRNA genes and the internally transcribed spacer (ITS) region it is reported that variation in phylogenetic placement exists among the 17 different serotype strains of Bradyrhizobium that have been isolated from nodules of soybean. Evolutionary relationships among the bradyrhizobia were more resolved using reconstructions derived from ITS than from 16S rRNA gene sequence divergence. Strain USDA 129 was placed together with USDA 62, 110, 122 and 126, but did not cluster with USDA 123 and 127, with which it shares antigenic determinants. The results from the phylogenetic analysis were supported with data from determinations of genetic diversity among additional strains within each of these serogroups using amplified fragment length polymorphism analysis. From these results it was concluded that strains of serogroup 129 were more similar to strains of serogroups 62, 110 and 122 than they were to strains of serogroups 123 and 127. The serotype strain of Bradyrhizobiumjaponicum USDA 135 and the type strain for Bradyrhizobium liaoningense possessed identical 16S rRNA gene and ITS region sequences. Also, the type strain for B. liaoningense cross-reacted with antisera prepared against somatic antigens of USDA 135. Therefore, it was not possible to distinguish B. liaoningense from serogroup 135 in our analysis of B. japonicum and Bradyrhizobium elkanii.