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

Abstract

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.

Invasive legumes can associate with many mutualists of native legumes, but usually do not

“…2010) and one with S. junceum in its native range in Sicily (Sj4‐ ITS only; Genbank Accession Number ; Quatrini et al. ).…”

Molecular analysis of lichen-associated bacterial communities

“…After external sterilization by immersion in H 2 O 2 (9%) for 4 min, total DNA was extracted from approximately 0.3–0.5 g of lichen thalli using the CTAB/chloroform‐isoamyl alcohol method as previously described (Cubero et al , 1999). The regions of DNA including the 16S ribosomal gene, the internal transcribed ribosomal spacer and approximately 100 nucleotides of the 23S ribosomal gene were amplified using 5 μL of crude extracted DNA as template, 0.4 μM of each fD1 and FGPL 132–38 primers (Quatrini et al , 2002), 0.2 mM of each dNTP, 2.5 units of Taq DNA polymerase (Sigma‐Aldrich) and 2 mM MgCl 2 , according to the PCR‐protocol previously described for 16S rRNA gene and ITS 16S–23S amplifications. A second nested amplification was performed to amplify the ITS 16S–23S region of all bacterial communities using 10 μL of the first PCR product as template and the primers ITSF and ITSReub.…”

Root reinforcement and slope bioengineering stabilization by Spanish Broom (<i>Spartium junceum</i> L.)

“…Site A was next to where the root-ball Spanish Broom plants were planted; site B was 30 m away on the same contour line, isolated from the other Spanish Broom plants. This distribution was chosen in order to verify a possible correlation between the soil that was already colonized by Bradyrhizobium spp., bacteria and Glomus fungi and the rooting ability only for the first test) (Quatrini et al, 2002). In turn, the two sites were divided into two sections: stem cuttings with or without use of plant hormone for rooting.…”

“…This protocol allows a high rate of young plants production in a short period of time. Quatrini et al (2002) proposed using plants that were inoculated with nitrogen-fixing bacteria.…”