The study of Agrobacterium radiobacter 10 and Pseudomonas fluorescens PG7 phosphate-mobilizing abilities in vitro

There is a need to improve the phosphorus nutrition of agricultural plants due to the mobilization of phosphorus from hard-to-reach soil compounds and fertilizers by useful rhizosphere microorganisms (PGPB). For this purpose, phosphate-mobilizing bacteria are being selected to create biologicals with a fertilizing action. Here, our research data for the first time show that the strain Agrobacterium radiobacter 10 can metabolize phytate to utilize it as a source of carbon and energy in the absence of other sources, and the strain Pseudomonas chlororaphis PG7 can solubilize inorganic phosphates (tricalcium phosphate, hydroxyapatite) and organic phosphates (calcium phytate). The aim of the work is to investigate the potential of phosphate-mobilizing ability of two strains, A. radiobacter 10 and P. chlororaphis PG7. The stock cultures were propagated on pea agar (according to Khotyanovich). The phosphate mobilizing ability of the strains was assessed in vitro on selective nutrient media at 28 °С. Dephosphorylation of sodium phytate was examined in two liquid media. Medium II had the following composition (g/l distilled water): (NH4)2SO4 - 1.0, K2SO4 - 0.2, Na phytate (Sigma-Aldrich, USA) - 10, corn extract - 0.2, pH 6.8. PSM (phytase screening medium) composition was as follows (g/l distilled water): D-glucose - 15.0, (NH4)2SO4 - 5.0, KCl - 0.5, MgSO4ʺ7H2O - 0.1, NaCl - 0.1, CaCl2ʺ2H2O - 0.1; FeSO4ʺ7H2O - 0.01, MnSO4ʺ7H2O - 0.01; Na phytate (Sigma-Aldrich, USA) - 5, pH 6.5. The content of total phosphorus added to media with Na phytate was determined by the method of E. Truog and A.H. Meyer modified by J.B. Rodriguez et al. (1994) after ashing as per N.E. Ginsburg and G.M. Shcheglova (1960). The growth of strains in liquid media was estimated by the bacteria abundance (CFU/ml of suspension) during incubation. The ability of the strains to solubilize inorganic phosphates (tricalcium phosphate, hydroxyapatite) and organic phosphate (calcium phytate) was carried out on three solid nutrient media, the NBRIP, glucose-aspartic medium (according to G.S. Muromtsev) and PSM. NBRIP (National Botanical Research Institute's phosphate growth medium) composition was as followed (g/l of distilled water): D-glucose - 10, Ca3(PO4)2 - 5.0, MgCl2ʺ6H2O - 5.0, MgSO4ʺ7H2O - 0.25, KCl - 2.0, (NH4)2SO4 - 0.1, agar-agar - 20, pH 6.8. The glucose-aspartic medium with hydroxyapatite (according to G.S. Muromtsev) (43) contained (g/l of distilled water) D-glucose - 10, asparagine - 1, K2SO4 - 0.2, MgSO4ʺ7H2O - 0.2, corn extract - 0.2, Ca5(PO4)3O5 - 4, agar-agar - 20, pH 6.8. The PSM composition is as hereinabove, added with agar-agar 20 g/l, pH 6.5 adjusted to by adding a 1 0% aqueous solution of Ca(OH)2 to convert soluble sodium phytate into insoluble calcium phytate. The formation of halos around the colonies was recorded. The research revealed that A. radiobacter 10 cultured in the liquid medium uses phytate as a source of carbon and phosphorus for growth and enzymatically dephosphorylates phytate. This was evidenced by a significant increase in abundance of the bacteria during 4-day growth, a relatively small decrease in pH of the liquid broth compared to the control without inoculation, and the accumulation of immobilized phosphorus in the bacterial cell sediment and free orthophosphate in the liquid medium. P. chlororaphis PG7 could not mobilize phytate in the medium II. In particular, despite an increase in the P. chlororaphis PG7 aundance, there was no noticeable accumulation of bacterial cell sediment and free orthophosphate in the liquid medium. It was shown that when cul