Isolation and identification of Azospirillum bacteria of wheat and maize rhizosphere and determination of some plant growth promoting characteristics

Azospirillum bacteria are one of the most important plant growth promoting rhizobacteria which exist in the rhizosphere of many crop plants. In this research, some soil and plant roots were collected from wheat and maize rhizosphere of the fields in Isfahan and were cultured in the tubes containing Nitrogen Free Bromothymol (NFB) medium. In order to isolate Azospirillum strains, the grown bacteria pellicles were streaked on NFB and Rojo Congo (RC) specific and semi-specific media. The bacteria were isolated and purified using morphological characteristics on the medium. After gram staining, catalase, oxidase and oxidative-fermentative tests, the detection and identification of Azospirillum isolates was done by molecular tests using PCR test with Az16S-A and Azo494-F/Azo756-R specific primers and 16S rRNA gene sequencing. Plant growth promoting characteristics (PGP) of Azospirillum isolates were determined by investigating the presence of NifH and NifD genes of nitrogenase enzyme by PolF/PolR and FdB260/FdB261 specific primers respectively. The properties of phosphate solubilization and production of chitinase, HCN, siderophores and indole acetic acid of isolates were performed by using specific tests. The replication of 646, 263, 360, and 390 bp fragments by Az16S-A, Azo494-F/Azo756-R, PolF/PolR and FdB260/FdB261 primers respectively, confirmed the presence of Azospirilum bacteria and the presence of NifH and NifD genes. Specific tests also revealed the ability of indole acetic acid production, insoluble phosphate solubilizationwith average value of 15 and 18.8 mg/l respectively and siderophore production in isolates. The results showed that some of the isolates possessed several PGP properties, while some of them had one or no characteristic. Finally, the sequencing of the 16S rRNA gene in some isolates determined and three species of A. brasilense, A. lipoferum and A. zeae identified in rhizosphere of wheat and maize plants.