Genetic diversity of native bradyrhizobia isolated from soybeans (Glycine max L.) in different agricultural-ecological-climatic regions of India

Abstract

Fifty isolates from root nodules of soybean plants sampled in five agricultural-ecological-climatic regions of India were analyzed by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene, the intergenic spacer region between the 16S and 23S rRNA genes (IGS), and the nifH and nodC genes. Eight haplotypes assigned to the Bradyrhizobium genus were identified, and the genetic diversity was conserved across regions. Sequence analyses of the IGS and the dnaK, glnII, recA, and nifH genes revealed three groups. One of them (26% of isolates) was assigned to Bradyrhizobium liaoningense. A second group (36% of isolates) was identified as B. yuanmingense but likely forms a new biovar able to nodulate soybean plants. The third lineage (38% of isolates) was different from all described Bradyrhizobium species but showed the same symbiotic genotype as B. liaoningense and B. japonicum bv. glycinearum.

FIG. 1.

Distribution of bradyrhizobial genotypes (I to VIII) defined by the combined data from PCR-RFLP analysis of the 16S rRNA gene, IGS, nifH, and nodC among agricultural-ecological-climatic regions of India.

FIG. 2.

Phylogenetic ML tree based on 938-bp alignment of nucleotide sequences of the IGS between the 16S and 23S rRNA genes. Only bootstrap probability values of ≥70% (over 100 replicates) are indicated at the branching points. The scale bar indicates the number of substitutions per site. Soybean isolates are shown in bold, with their IGS haplotypes indicated. The letter “T” indicates the type strain of the species. Accession numbers of sequences extracted from GenBank are as follows: PAC53, AY628088; type XIX, AY493859; LMG R16434^T, AJ534605; MSDJ G17, AF338851; PAC40, AY628087; LMG 6138^T, AJ279264; BC-P22, AY386706; LMG 21987^T, AJ631967; USDA 110, BA000040; LMG 18230^T, AJ279301; USDA 135, AF208511; LMG 11944, AJ279287; LMG 10726, AJ279281; EC550-1, AY628085; PAC43, AY628089; PAC18, AY628093; LMG 10677, AJ534597; LMG 10671, AJ279272; LMG 10705, AJ534592; LMG 6134^T, AJ279308; LMG 11951, AJ534594; and LMG 8293, AJ279311.

FIG. 3.

Phylogenetic ML tree based on 1,493-bp alignment of concatenated nucleotide sequences of dnaK (489 bp), glnII (519 bp), and recA (482 bp). Only bootstrap probability values of ≥70% (over 100 replicates) are indicated at the branching points. The scale bar indicates the number of substitutions per site. Soybean isolates are shown in bold, with their IGS haplotypes indicated. The letter “T” indicates the type strain of the species. Accession numbers of sequences extracted from GenBank are as follows: CCBAU 10071^T, AY923039 (dnaK), AY386780 (glnII), and AY591566 (recA); LMG 18230^T, AY923041 (dnaK), AY386775 (glnII), and AY591564 (recA); USDA 6^T, AM168362 (dnaK), AF169582 (glnII), and AM168341 (recA); USDA 110, BA000040 (complete genome); LMG 21987^T, AY923046 (dnaK), AB353733 (glnII), and AB353734 (recA); BTA-1^T, AY923047 (dnaK), AY386765 (glnII), and AY591553 (recA); USDA 76^T, AM168363 (dnaK), AY599117 (glnII), and AM168342 (recA); ORS 278, CU234118 (complete genome); and BTAI1, CP000495 (complete genome).

FIG. 4.

Phylogenetic ML tree based on 612-bp alignment of nucleotide sequences of the nifH gene. Only bootstrap probability values of ≥70% (over 100 replicates) are indicated at the branching points. The scale bar indicates the number of substitutions per site. Soybean isolates are shown in bold, with their nifH haplotypes indicated. The letter “T” indicates the type strain of the species. Accession numbers of sequences extracted from GenBank are as follows: CCBAU 83698, EU146010; CCBAU 23230, AY934871; CCBAU 61071, EU113228; USDA 110, BA000040; S 127, DQ485701; IFO 14791^T, AB094963; CPAC 402, DQ485714; MAFF303099, BA000012; 3841, AM236084; and 1021, AE007235.