Bradyrhizobium sp. nodulating the Mediterranean shrub Spanish broom (Spartium junceum L.)

Aims: The molecular diversity of 25 strains of rhizobia, isolated in Sicily from root nodules of the Mediterranean shrubby legume Spanish broom (Spartium junceum L.), is presented in relation to the known rhizobial reference strains. Methods and Results: Our approach to the study of the S. junceum rhizobial diversity combined the information given by the 16S and the intergenic spacer (IGS) 16S–23S rDNA polymorphic region by obtaining them in a single polymerase chain reaction (PCR) step. The PCR fragment size of the S. junceum isolates was 2400–2500 bp and that of the reference strains varied from 2400 in Bradyrhizobium strains to 2800 in Sinorhizobium strains. Inter- and intrageneric length variability was found among the reference strains. Restriction fragment length polymorphisms (RFLP) analysis allowed us to identify eight genotypes among the S. junceum rhizobia that were clustered into two groups, both related to the Bradyrhizobium lineage. Sequencing of representative strains of the two clusters confirmed these data. The 16S–IGS PCR–RFLP approach, when applied to rhizobial reference strains, allowed very close species (i.e. Rhizobium leguminosarum/R. tropici) to be separated with any of the three enzymes used; however, cluster analysis revealed inconsistencies with the 16S-based phylogenesis of rhizobia. Conclusions: Rhizobia nodulating S. junceum in the Mediterranean region belong to the Bradyrhizobium lineage. Our results confirm the resolution power of the 16S–23S rDNA in distinguishing among rhizobia genera and species, as well as the usefulness of the PCR–RFLP method applied to the entire 16S–IGS region for a rapid tracking of the known relatives of new isolates. Significance and Impact of the Study: The present paper is, to our knowledge, the first report on rhizobia nodulating a Mediterranean wild woody legume.