GENOTYPIC CHARACTERIZATION OF BRADYRHIZOBIUM STRAINS NODULATING ENDEMIC WOODY LEGUMES OF THE CANARY-ISLANDS BY PCR-RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF GENES ENCODING 16S RIBOSOMAL-RNA (16S RDNA) AND 16S-23S RDNA INTERGENIC SPACERS, REPETITIVE EXTRAGENIC PALINDROMIC PCR GENOMIC FINGERPRINTING, AND PARTIAL 16S RDNA SEQUENCING

We present a phylogenetic analysis of nine strains of symbiotic nitrog en-fixing bacteria. isolated from nodules of tagasaste (Chamaecytisus proliferus) and other endemic woody legumes of the Canary Islands, Spa in. These and several reference strains mere characterized genotypical ly at different levels of taxonomic resolution by computer-assisted an alysis of 16S ribosomal DNA (rDNA) PCR-restriction fragment length pol ymorphisms (PCR-RFLPs), 165-23S rDNA intergenic spacer (IGS) RFLPs, an d repetitive extragenic palindromic PCR (rep-PCR) genomic fingerprints with BOX, ERIC, and REP primers. Cluster analysis of 16S rDNA restric tion patterns with four tetrameric endonucleases grouped the Canarian isolates with the two reference strains, Bradyrhizabium japonicum USDA 110spc4 and Bradyrhizobium sp. strain (Centrosema) CIAT 3101, resolvi ng three genotypes within these bradyrhizobia. In the analysis of IGS RFLPs with three enzymes, six groups were found, whereas rep-PCR finge rprinting revealed an even greater genotypic diversity, with only two of the Canarian strains having similar fingerprints. Furthermore, we s how that IGS RFLPs and even very dissimilar rep-PCR fingerprints can b e clustered into phylogenetically sound groupings by combining them wi th 16S rDNA RFLPs in computer-assisted cluster analysis of electrophor etic patterns. The DNA sequence analysis of a highly variable 264-bp s egment of the 16S rRNA genes of these strains was found to be consiste nt with the fingerprint-based classification. Three different DNA sequ ences were obtained, one of which was not previously described, and al l belonged to the B. japoaicum/Rhodopseudomonas rDNA cluster. Nodulati on assays revealed that none of the Canarian isolates nodulated Glycin e max or Leucaena leucocephala, but all nodulated Acacia pendula, C. p roliferus, Macroptilium atropurpureum, and Vigna unguiculata.