Title : Molecular phylogenetic analysis of Rhizobium sullae isolated from Algerian Hedysarum flexuosum
Hedysarum flexuosum, annual species, commonly called Sulla flexuosa. It originates from the western Mediterranean region and North Africa, disappeared in Tunisia and only isolated populations persist in Algeria and Morocco. H. flexuosum has the capacity to produce high levels of quality green matter (500 q/ha). Rhizobium is nitrogen-fixing bacteria nodulating legumes. The 16S rRNA, recA, nifH and nodD are genes markers for Rhizobium Species identification. Currently, there is no information about the phylogeny of the microsymbionts of H. flexuosum in Algeria. Hence, the aim of this research was to investigate the phylogenetic affiliations of the strains isolated from root nodules of H. flexuosum growing in this country using sequence analysis of the 16S rRNA gene, housekeeping gene (recA) and the symbiotic genes (nifH and nodD). Additionally, they were tested for their tolerance to NaCl, pH, temperatures, antibiotics and heavy metals resistance. Interestingly, the isolate Hf_04N appeared resistant to ZnCl2 (50 μg/mL) and grew at high saline concentration up to 9%. The phylogenetic positions of five isolates were studied by comparative sequence analysis of 16S rRNA, recA, nifH and nodD genes. There were grouped close to the Rhizobium sullae type strain in relation to their 16S rRNA, recA and nifH genes-based phylogenies. By contrast, the tree of nodD gene was not congruent with ribosomal, housekeeping and nitrogen fixation genes. We suggest that our strains have a novel nodD gene. The detection of conserved domains of NodD protein and nitrogenase reductase enzyme, confirm their ability to nodulate and fix nitrogen. This report is, to our knowledge, the first study carried out with H. flexuosum microsymbionts in Algeria.