Isolation and identification of phosphate solubilizer Azospirillum, Bacillus and Enterobacter strains by 16SrRNA sequence analysis and their effect on growth of wheat (Triticum aestivum L.)

The aim of the present study was to isolate phosphate solubilizing bacteria from wheat rhizosphere and investigate their potential for plant growth promotion. Three phosphate solubilizing bacterial strains were isolated by serial dilution method from the rhizosphere of wheat grown under wheat-cotton and wheat-rice crop rotation. 16S rRNA gene sequence of the isolates WS-1, T-34 and T-41 showed 98% similarity to those of Azospirillum, Bacillus and Enterobacter sequences in NCBI data base, respectively. The bacterial strain WS-1 clustered with Azospirillum brasilense strains in the phylogenetic tree constructed using NCBI data base sequences of the genus Azospirillum. The bacterial isolate T-34 formed cluster with Bacillus licheniformis strains in the phylogenetic tree constructed using 16S rRNA sequences of the genera Bacillus and Paenibacillus. In the phylogenetic tree constructed using 16S rRNA gene sequences of genus Enterobacter, the isolate T-41 clustered with Enterobacter amnigenus strains. Phosphate solubilizing activity of the bacterial strains in Pikovskaya medium was investigated by molybdate blue color method using spectrophotometer while organic acids produced in the medium were detected on HPLC. In a growth medium containing insoluble tri-calcium phosphate and supplemented with sucrose, maltose, glucose or galactose as single C-source, all the three isolates produced organic acids like acetic acid, citric acid and gluconic acid for phosphate solubilization. Among the organic acids detected, acetic acid was produced in highest amounts by all strains tested in the media containing different sugars. Maximum acetic acid (56.7 mug/ml) was produced by Bacillus strain T-34 in medium supplemented with sucrose and citric acid (36.2 mug/ml) by the same strain on glucose supplemented medium. Oxalic acid was produced by Bacillus strain T-34 (2.89 mug/ml) and Enterobacter strain T-41 (5.93 mug/ml) only in the medium containing galactose as C-source. Maximum amount of gluconic acid (25.4 mug/ml) was produced by Azospirillum strain WS-1 in the medium containing glucose as C-source. Highest P solubilization activity (298.3 mug/ml) was observed in Bacillus strain T-34 grown on sucrose supplemented medium. Maximum P solubilization was detected in Enterobacter strain T-41 (292.2 mug/ml) in medium containing maltose. Azospirillum strain WS-1 showed maximum P solubilization (218.1 mug/ml) in medium containing glucose as single C-source. All the three bacterial strains produced growth hormone IAA in the growth medium supplemented with tryptophan. Quantification on HPLC indicated maximum IAA production (31.15 mug/ml) by Bacillus strain T-34. A pot experiment conducted to study the effect of bacterial isolates on growth of wheat showed that inoculation with Azospirillum, Bacillus and Enterobacter strains increased the grain yield of wheat by 9.3%, 14.8%, 13.1%, respectively over non-inoculated control. Under field conditions, increase in grain yield of plants inoculated with Azospirillum, Bacillus and Enterobacter was 11.2%, 15.7% and 5.6%, respectively compared with non-inoculated plants.These results indicated that bacterial isolates having plant-beneficial traits like phosphate solubilization and IAA production and capable of improving growth of wheat when used as inoculants qualify for production of biofertilizer for wheat crop