Isolation and identification of Rhizobacteria in a symbiotic relation with some non-cultivated legumes of Alborz province

Rhizobacteria that benefit plants by stimulating growth and suppressing disease are called plant growth promoting rhizobacteria (PGPR) which includes rhizobia. In addition to nitrogen supply, rhizobia promote the growth of plant root system and improve nutritional uptake. In this research, for isolation and identification of rhizobacteria especially rhizobia in a symbiotic relation with non-cultivated legumes, 21 plant samples from 12 non-agricultural sites of Alborz province were collected. In order to isolate bacteria, serial dilutions were prepared from nodules, rhizosphere soil and roots. Bacteria were cultured and purified on YMA medium and morphological and biochemical characteristics of isolates were studied. In total, 16 purified strains were obtained. Bacterial DNA of all strains were extracted and 16S rRNA gene was amplified with specific primers using polymerase chain reaction (PCR). Strains were identified by sequences of this gene and using EzTaxon database. All isolated strains in this research belonged to 12 species. PCR reactions were also done for atpD, nodA and nifH genes by specific primers. Phylogenetic trees were constructed by maximum-likelihood statistical method and evolutionary distances were computed by using the Tamura-Nei model. Based on the obtained results from four strains of Ensifer meliloti which were isolated in this study, only Tal31 strain showed a higher growth with the increace of NaCl from one percent to two percent (w/v), therefore this strain is probably salt tolerant. This is the first time that Rhizobium laguerreae (Tal71) and Neorhizobium alkalisoli (Krj1) were isolated from root nodules of Trifolium pretense and Colutea buhsei respectively. These findings show that the host range of rhizobial strains are broader than it was estimated before.