Genotypic characterization of Bradyrhizobium strains nodulating endemic woody legumes of the Canary Islands by PCR-restriction fragment length polymorphism analysis of genes encoding 16S rRNA (16S rDNA) and 16S-23S rDNA intergenic spacers, repetitive extragenic palindromic PCR genomic fingerprinting, and partial 16S rDNA sequencing

Abstract

We present a phylogenetic analysis of nine strains of symbiotic nitrogen-fixing bacteria isolated from nodules of tagasaste (Chamaecytisus proliferus) and other endemic woody legumes of the Canary Islands, Spain. These and several reference strains were characterized genotypically at different levels of taxonomic resolution by computer-assisted analysis of 16S ribosomal DNA (rDNA) PCR-restriction fragment length polymorphisms (PCR-RFLPs), 16S-23S rDNA intergenic spacer (IGS) RFLPs, and repetitive extragenic palindromic PCR (rep-PCR) genomic fingerprints with BOX, ERIC, and REP primers. Cluster analysis of 16S rDNA restriction patterns with four tetrameric endonucleases grouped the Canarian isolates with the two reference strains, Bradyrhizobium japonicum USDA 110spc4 and Bradyrhizobium sp. strain (Centrosema) CIAT 3101, resolving three genotypes within these bradyrhizobia. In the analysis of IGS RFLPs with three enzymes, six groups were found, whereas rep-PCR fingerprinting revealed an even greater genotypic diversity, with only two of the Canarian strains having similar fingerprints. Furthermore, we show that IGS RFLPs and even very dissimilar rep-PCR fingerprints can be clustered into phylogenetically sound groupings by combining them with 16S rDNA RFLPs in computer-assisted cluster analysis of electrophoretic patterns. The DNA sequence analysis of a highly variable 264-bp segment of the 16S rRNA genes of these strains was found to be consistent with the fingerprint-based classification. Three different DNA sequences were obtained, one of which was not previously described, and all belonged to the B. japonicum/Rhodopseudomonas rDNA cluster. Nodulation assays revealed that none of the Canarian isolates nodulated Glycine max or Leucaena leucocephala, but all nodulated Acacia pendula, C. proliferus, Macroptilium atropurpureum, and Vigna unguiculata.

FIG. 1

Dice/UPGMA cluster analysis of combined CfoI, DdeI, MboI, and MspI restriction patterns of amplified 16S rDNA of nodule isolates of endemic woody legumes of the Canary Islands and reference strains (see Table 1). The individual RFLPs are shown as bands defined on the actual restriction fragments. The sizes of the restriction fragments for each type of pattern are presented in Table 2. Clusters A, B, and C contain all the Canarian isolates and the two reference Bradyrhizobium strains. R. etli, Rhizobium etli; R. tropici, Rhizobium tropici; A. tumefaciens, Agrobacterium tumefaciens; P. fluorescens, Pseudomonas fluorescens.

FIG. 2

Dice/UPGMA cluster analysis of combined DdeI, HaeIII, and MspI restriction patterns of amplified 16S-23S rDNA IGSs of nodule isolates of endemic woody legumes of the Canary Islands and reference Bradyrhizobium strains (see Table 1). The individual RFLPs are shown as bands defined on the actual restriction fragments. Six genotypes were resolved. Not all groupings are consistent with those obtained by ARDRA.

FIG. 3

Dice/UPGMA dendrogram based on the combined CfoI, DdeI, MboI, and MspI 16S rDNA RFLPs and 16S-23S rDNA IGS RFLPs of nodule isolates of endemic woody legumes of the Canary Islands and reference Bradyrhizobium strains (see Table 1). The groupings revealed in this cluster analysis are consistent with both the ARDRA and the IGS PCR-RFLP analyses.

FIG. 4

Product-moment/UPGMA cluster analysis of linearly combined BOX, ERIC, and REP PCR genomic fingerprints of nodule isolates of endemic woody legumes of the Canary Islands and reference strains (see Table 1). The only significant cluster is denoted by I. See the text for explanations. On the scale, r values are expressed as percentages. R. etli, Rhizobium etli; R. trop., Rhizobium tropici; A. tum., Agrobacterium tumefaciens; P. fluor., Pseudomonas fluorescens; B. jap., Bradyrhizobium japonicum.

FIG. 5

Product-moment/UPGMA cluster analysis of linearly combined BOX, ERIC, and REP PCR genomic fingerprints and CfoI, DdeI, MboI, and MspI restriction patterns of amplified 16S rDNA of nodule isolates of endemic woody legumes of the Canary Islands and reference strains (see Table 1). Clusters A, B, and C contain all the Canarian isolates and the two reference Bradyrhizobium strains. On the scale, r values are expressed as percentages. For species abbreviations, see the legend for Fig. 4.

FIG. 6

Alignment of sequence stretches containing variable positions within the 264-bp 16S rDNA segment amplified by primer pair Y1/Y2 from Canarian and reference bradyrhizobia (see Table 1). GenBank accession numbers of the complete sequences are listed in Materials and Methods.