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Zheleznyakov S.V., Kalinina T.V., Deeva V.K., Laktionov Yu.V., Jacobi L.M. The study of Agrobacterium radiobac- ter 10 and Pseudomonas fluorescens PG7 phosphate-mobilizing abilities in vitro. Sel'skokhozyaistvennaya Biologiya [Agricultural Biology], 2022, Vol. 57, № 1, p. 158-170.
(how to cite this article)

doi: 10.15389/agrobiology.2022.1.158eng

UDC: 579.64:631.811.2

Acknowledgements:
Supported financially from the Russian Foundation for Basic Research (the project No. 19-016-00197)

 

THE STUDY OF Agrobacterium radiobacter 10 AND Pseudomonas fluorescens PG7 PHOSPHATE-MOBILIZING ABILITIES in vitro

S.V. Zheleznyakov, T.V. Kalinina, V.K. Deeva, Yu.V. Laktionov,
L.M. Jacobi ✉

All-Russian Research Institute for Agricultural Microbiology, 3, sh. Podbel’skogo, St. Petersburg, 196608 Russia, e-mail krosh15_02@mail.ru, sotvk@yandex.ru, vdvalerie21@mail.ru, laktionov@list.ru, lidija-jacobi@yandex.ru (✉ corresponding author)

ORCID:
Zheleznyakov S.V. orcid.org/0000-0001-5321-4051
Laktionov Y.V. orcid.org/0000-0001-6241-0273
Kalinina T.V. orcid.org/0000-0003-1255-6498
Jacobi L.M. orcid.org/0000-0003-0387-5024
Deeva V.K. orcid.org/0000-0001-8387-2602

November 15, 2021

 

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, MgSO4·7H2O — 0.1, NaCl — 0.1, CaCl2·2H2O — 0.1; FeSO4·7H2O — 0.01, MnSO4·7H2O — 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, MgCl2·6H2O — 5.0, MgSO4·7H2O — 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, MgSO4·7H2O — 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 cultured in the liquid PSM, both strains actively grew and multiplied, obviously using glucose as a source of carbon and energy. Under these conditions, a significant amount of immobilized phosphorus accumulated in the bacterial cell sediment, while the content of free orthophosphate in the medium remained at the control level. In addition, bacterial growth led to significant acidification of the medium, which contributed to the non-enzymatic hydrolysis of sodium phytate. Therefore, the research data could not drive to an unambiguous conclusion about the ability of strains to enzymatic hydrolysis of sodium phytate when cultured in the liquid PSM with two carbon sources. The halos around the colonies of P. chlororaphis PG7 on solid media indicated its ability to dissolve inorganic phosphates and phytin by solubilization. Unlike the Pseudomonas strain, the A. radiobacter 10 showed no solubilizing ability. This indicates its individual physiological features, since, as follows from special publication, many representatives of the genus Rhizobium are potential solubilizers. Thus, the ability of strains to solubilize mineral phosphates should be tested on solid nutrient media, where the formation of halos around colonies is a criterion for evaluating phosphate dephosphorization. The ability of strains to mobilize phosphorus from phytates should be assessed in liquid media in order to avoid false positive or false negative results. The main indicators of the enzymatic hydrolysis of phytates are the accumulation of immobilized phosphorus in the sediment of bacterial cells and free orthophosphate in the medium.

Keywords: phosphate-mobilizing ability, Agrobacterium radiobacter, Pseudomonas fluorescens, phytate, tricalcium phosphate, hydroxyapatite, selective nutrient media, immobilized phosphorus, orthophosphate.

 

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