Bradyrhizobium sp. nodulating the Mediterranean shrub Spanish broom (Spartium junceum L.) P. Quatrini, G. Scaglione, M. Cardinale, F. Caradonna and A.M. Puglia Dipartimento di Biologia Cellulare e dello Sviluppo, Universita Á di Palermo, Palermo, Italy 2001/11: received 13 March 2001, revised 13 June 2001 and accepted 2 July 2001 P. QUATRINI, G. SCAGLIONE, M. CARDINALE, F. CARADONNA AND A.M. PUGLIA. 2002. 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 con®rmed 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 con®rm 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. Signi®cance and Impact of the Study: The present paper is, to our knowledge, the ®rst report on rhizobia nodulating a Mediterranean wild woody legume. INTRODUCTION Rhizobia are widespread soil bacteria able to induce the formation of root nodules and to ®x nitrogen on cultivated and wild legumes. The family Leguminosae (Fabaceae) is represented mainly by herbaceous and shrubby legumes in temperate regions while tree species are dominant in tropical regions (Pignatti 1982; Crepet and Taylor 1985; Paiero et al. 1993). In the Mediterranean area members of the subfamily Faboideae are dominant; they are mainly represented by herbaceous species and also by a minor group of woody genera (Genista, Spartium, Cytisus, Anthyllis, etc.) able to colonize poor and arid soils, up to 2000 m above sea level, such as the volcanic lavas of Mount Etna in Sicily (Italy). Both deserti®cation and ecosystem degradation problems are common in Mediterranean and subtropical regions, partic- ularly in Sicily and in the rest of southern Italy, where low precipitation and human activities enhance the erosion and deserti®cation processes. It is widely recognized that indigenous rhizobia play an important role in the dominance of Fabaceae in poor and arid soils (Zaharan 1999) but, so far, only a few wild Correspondence to: A.M. Puglia, Dipartimento di Biologia Cellulare e dello Sviluppo, Sez. Genetica, Universita Á di Palermo, Viale delle Scienze Parco D'Orleans II 90128 Palermo, Italy (e-mail: ampuglia@unipa.it). ã 2002 The Society for Applied Microbiology Journal of Applied Microbiology 2002, 92, 13±21