Search

The ability of Bradyrhizobium spp. to nodulate and fix atmospheric nitrogen in soybean root nodules is critical to meeting humanity's nutritional needs. The intricacies of soybean bradyrhizobia-plant interactions have been studied extensively; however, bradyrhizobial ecology as influenced by phages has received somewhat less attention, even though these interactions may significantly impact soybean yield. In batch culture, four soybean bradyrhizobia strains, Bradyrhizobium japonicum S06B (S06B-Bj), B. japonicum S10J (S10J-Bj), Bradyrhizobium diazoefficiens USDA 122 (USDA 122-Bd), and Bradyrhizobium elkanii USDA 76 T (USDA 76-Be), spontaneously (without apparent exogenous chemical or physical induction) produced tailed phages throughout the growth cycle; for three strains, phage concentrations exceeded cell numbers by ~3-fold after 48 h of incubation. Phage terminase large-subunit protein phylogeny revealed possible differences in phage packaging and replication mechanisms. Bioinformatic analyses predicted multiple prophage regions within each soybean bradyrhizobia genome, preventing accurate identification of spontaneously produced prophage (SPP) genomes. A DNA sequencing and mapping approach accurately delineated the boundaries of four SPP genomes within three of the soybean bradyrhizobia chromosomes and suggested that the SPPs were capable of transduction. In addition to the phages, S06B-Bj and USDA 76-Be contained three to four times more insertion sequences (IS) and large, conjugable, broad host range plasmids, both of which are known drivers of horizontal gene transfer (HGT) in soybean bradyrhizobia. These factors indicate that SPP along with IS and plasmids participate in HGT, drive bradyrhizobia evolution, and play an outsized role in bradyrhizobia ecology. IMPORTANCE Previous studies have shown that IS and plasmids mediate HGT of symbiotic nodulation ( nod ) genes in soybean bradyrhizobia; however, these events require close cell-to-cell contact, which could be limited in soil environments. Bacteriophage-assisted gene transduction through spontaneously produced prophages provides a stable means of HGT not limited by the constraints of proximal cell-to-cell contact. These phage-mediated HGT events may shape soybean bradyrhizobia population ecology, with concomitant impacts on soybean agriculture.

The internally transcribed spacer (ITS) sequences of several members within each of 17 soybean bradyrhizobial serogroups were determined to establish whether the regions within all members of each serogroup were identical. The rationale was to provide a sequence-based alternative to serology. The objective also was to link the extensive older literature on soybean symbiosis based on serology with ITS sequence data for more recent isolates from both soybean and other legumes nodulated by rhizobia within the genus Bradyrhizobium. With the exception of serogroup 31 and 110 strains, sequence identity was established within each serogroup. Variation ranged from 0 to 23 nucleotides among serogroup 31 strains, and the regions in the type strains USDA 31 (serogroup 31) and USDA 130 (serogroup 130) were identical. Sequence identity was established among most strains within serogroup 110. The exceptions were USDA 452 and USDA 456, which had ITS sequences that were identical with those of the serotype 124 strain, USDA 124. Perhaps this would imply that USDA 452, USDA 456, and serogroup 31 strains are members of rhizobial lineages resulting from genetic exchange and homologous recombination events. This conclusion would be supported by the construction of a phylogenetic network from the ITS sequence alignment implying that the genomes of extant members of the genus Bradyrhizobium are likely the products of reticulate evolutionary events. A pairwise homoplasy index (phi or Phi(w)) test was used to obtain further evidence for recombination. The ITS sequences of USDA 110 and USDA 124 were more divergent (53 nucleotides) than this region between the type strain Bradyrhizobium japonicum USDA 6(T) and the proposed species Bradyrhizobium yuanmingense (28 nucleotides) and Bradyrhizobium liaoningense (48 nucleotides). Therefore, support for assigning discrete species boundaries among these three proposed species appears limited, considering the evidence for recombination, the narrow divergence of the ITS sequence, and their relative placement on the phylogenetic network.

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.Key words: bradyrhizobium, phylogeny, soybean, Glycine max, FAME, AFLP.Le USDA, la Collection Nationale de germoplasmes de Rhizobium de l'ARS contient 143 accessions de souches de soya à croissance lente parmi lesquels on retrouve 17 groupes sérologiques distincts. Cependant, 11 souches semblent n'avoir aucune affinité sérologique avec ces 17 sérogroupes. Ainsi, nous avons déterminé si ces souches étaient diversifiées et examiné leur localisation phylogénétique. Neuf souches ont formé des symbioses de fixation de l'azote avec des plants de soya, indiquant que ces accessions n'étaient pas des contaminants. Nous avons conclu à partir des résultats d'AFLP (polymorphisme de longueur des fragments amplifiés), en utilisant trois amorces sélectives avec 8 souches, qu'elles étaient génétiquement dissemblables. La composition en acides gras de neuf souches a été étudié en utilisant des dérivés d'esters méthyliques d'acides gras (FAME). Les résultats de FAME avec 5 souches et souches sérotypiques de Bradyrhizobium elkanii étaient semblables, tandis que les résultats avec les deux autres paires restantes étaient soit semblables à la souche type de Bradyrhizobium japonicum (USDA 6), soit semblables à USDA 110. L'évolution de 9 souches a été reconstruite à partir de divergences de séquences issues de la combinaison du gène complet de l'ARNr 16S, de la région de l'espace traduit interne (ITS) et d'environ 400 bases de l'extrémité 5' du gène de l'ARNr 23S. Cinq souches furent localisées à l'intérieur de B. elkanii, 2 autres étaient incluses dans USDA 110, et les deux autres dans USDA 6. Nous avons donc conclu que les isolats de soya qui ne peuvent être placés à l'intérieur de l'un des 17 sérogroupes établis sont aussi diversifiés phénotypiquement et génétiquement que les souches sérotypées.Mots clés : bradyrhizobuim, phylogénie, soya, Glycine max, FAME, AFLP.[Traduit par la Rédaction] [ABSTRACT FROM AUTHOR]