Evidence of horizontal transfer of symbiotic genes from a Bradyrhizobium japonicum inoculant strain to indigenous diazotrophs Sinorhizobium (Ensifer) fredii and Bradyrhizobium elkanii in a Brazilian Savannah soil

Abstract

The importance of horizontal gene transfer (HGT) in the evolution and speciation of bacteria has been emphasized; however, most studies have focused on genes clustered in pathogenesis and very few on symbiosis islands. Both soybean (Glycine max [L.] Merrill) and compatible Bradyrhizobium japonicum and Bradyrhizobium elkanii strains are exotic to Brazil and have been massively introduced in the country since the early 1960s, occupying today about 45% of the cropped land. For the past 10 years, our group has obtained several isolates showing high diversity in morphological, physiological, genetic, and symbiotic properties in relation to the putative parental inoculant strains. In this study, parental strains and putative natural variants isolated from field-grown soybean nodules were genetically characterized in relation to conserved genes (by repetitive extragenic palindromic PCR using REP and BOX A1R primers, PCR-restriction fragment length polymorphism, and sequencing of the 16SrRNA genes), nodulation, and N(2)-fixation genes (PCR-RFLP and sequencing of nodY-nodA, nodC, and nifH genes). Both genetic variability due to adaptation to the stressful environmental conditions of the Brazilian Cerrados and HGT events were confirmed. One strain (S 127) was identified as an indigenous B. elkanii strain that acquired a nodC gene from the inoculant B. japonicum. Another one (CPAC 402) was identified as an indigenous Sinorhizobium (Ensifer) fredii strain that received the whole symbiotic island from the B. japonicum inoculant strain and maintained an extra copy of the original nifH gene. The results highlight the strategies that bacteria may commonly use to obtain ecological advantages, such as the acquisition of genes to establish effective symbioses with an exotic host legume.

FIG. 1.

Cluster analysis (UPGMA with the coefficient of Jaccard) of the DNA amplification products obtained in the analyses of: rep-PCR (primers BOX A1R and REP) (a) and PCR-RFLP of the 16S rRNA genes (b) of 17 soybean rhizobia. Strains belonged to two serogroups, one comprised of the parental B. japonicum strain SEMIA 586 (also CB1809) and the commercial variant CPAC 7 and the other comprised of the B. japonicum parental strain SEMIA 566 and the commercial variant CPAC 15.

FIG. 2.

Phylogenetic tree based on the 16S rRNA sequences of B. japonicum strains USDA 6^T, SEMIA 566, SEMIA 586, CPAC 7, and CPAC 15; S. fredii USDA 205^T; B. elkanii USDA 76^T; and the variant strains S 127 and CPAC 402. The numbers in the main branches indicate bootstrap values obtained with 2,000 replicates.

FIG. 3.

Cluster analysis (UPGMA with the coefficient of Jaccard) of the DNA fragments obtained in the analysis of PCR-RFLP of the nodC (a) and nodY-nodA (b) regions of 17 strains of soybean rhizobia. Strains belonged to two serogroups, one comprised the parental B. japonicum SEMIA 586 (also CB 1809) and the commercial variant CPAC 7 and the other comprised of the parental B. japonicum SEMIA 566 and the commercial variant CPAC 15.

FIG. 4.

Phylogenetic tree based on the nodC partial sequences of B. japonicum strains USDA 110, SEMIA 586, SEMIA 566, CPAC 7, and CPAC 15; B. elkanii CCBAU23174; and S. fredii HH103. The numbers in the main branches indicate bootstrap values obtained with 2,000 replicates.

FIG. 5.

Phylogenetic tree based on the nodY-nodA partial sequences of B. japonicum strains USDA 110, SEMIA 566, SEMIA 586, CPAC 7, and CPAC 15; B. elkanii USDA 94; and the variant strains CPAC 390, CPAC 403, S 370, CPAC 404, S 340, S 478, CPAC 394, CPAC 402, CPAC 392, S 516, S 372, and S 127. The numbers in the main branches indicate bootstrap values obtained with 2,000 replicates.

FIG. 6.

Cluster analysis (UPGMA with the coefficient of Jaccard) of the DNA fragments obtained in the analysis of PCR-RFLP of the nifH genes of 17 soybean rhizobia. Strains belonged to two serogroups, one comprised of the parental B. japonicum SEMIA 586 (also CB 1809) and the commercial variant CPAC 7 and the other comprised of the parental B. japonicum SEMIA 566 and the commercial variant CPAC 15.

FIG. 7.

Phylogenetic tree based on the nifH partial sequences of B. japonicum strain USDA 110, B. elkanii USDA 76, S. fredii USDA 191, and the variant strains S 127 and CPAC 402. The two nifH sequences from CPAC 402 are discriminated. The numbers in the main branches indicate bootstrap values obtained with 2,000 replicates.