Isolation, Identification, and Characterization of Phosphate-Solubilizing Bacteria from Tunisian Soils

Abstract

Soil microorganisms play an important role in maintaining natural ecological balance through active participation in carbon, nitrogen, sulfur, and phosphorous cycles. Phosphate-solubilizing bacteria (PSB) are of high importance in the rhizosphere, enhancing the solubilization of inorganic phosphorus complexes into soluble forms available for plant nutrition. The investigation of this species of bacteria is of major interest in agriculture, as they can be used as biofertilizers for crops. In the present study, 28 isolates of PSB were obtained after the phosphate enrichment of soil samples from five Tunisian regions. Five PSB species were identified by 16S rRNA gene sequencing including Pseudomonas fluorescens, P. putida, and P. taiwanensis, Stenotrophomonas maltophilia, and Pantoea agglomerans. Solid and liquid Pikovskaya's (PVK) and National Botanical Research Institute's (NBRIP) media containing insoluble tricalcium phosphate were used for the evaluation of the phosphate solubilization ability of the bacterial isolates by two methods: visual evaluation of the solubilization zone around colonies (halo) and determination of solubilized phosphates in liquid medium by the colorimetric method of the vanado-molybdate yellow. Based on the results of the halo method, the isolate of each species that showed the higher phosphate solubilization index was selected for evaluation of phosphate solubilization by the colorimetric method. In the liquid media, the bacterial isolates showed phosphate solubilization ranging from 535.70 to 618.57 µg mL^-1 in the NBRIP medium, and 374.20 to 544.28 µg mL^-1 in the PVK medium, with the highest values produced by P. fluorescens. The best phosphate solubilization ability and higher reduction in broth pH, which indicates higher organic acid production, were achieved in NBRIP broth for most of the PSB. Strong correlations were observed between the average capability of PSB to solubilize phosphates and both the pH and total phosphorous content in the soil. The production of the hormone indole acetic acid (IAA), which can promote plant growth, was observed for all five PSB species. Among them, P. fluorescens obtained from the forest soil of northern Tunisia showed the highest production of IAA (50.4 ± 0.9 µg mL^-1).

Keywords: indole acetic acid; phosphate solubilization index; phosphate-solubilizing bacteria; tricalcium phosphate.

Figure 1

Partial sequence 16SrRNA gene phylogenetic tree of Pseudomonas (A–C), Pantoea agglomerans (D), and Stenotrophomonas (E) species. (Species described in this study are indicated with a black square).

Figure 2

Relative abundance of the different species of PSB in the studied Tunisian soils.

Figure 3

Phosphate solubilization by bacterial isolates grown in NBRIP and PVK media at 28 ± 2 °C. Vertical bars represent confidence intervals at a confidence level of 95% (n = 3). Same letters indicate that differences among the means is not statistically significant (Tukey’s HSD post hoc test).

Figure 4

Variation of pH in NBRIP culture medium. Vertical bars represent confidence intervals at a confidence level of 95% (n = 3). Same letters indicate that differences among the means is not statistically significant (Tukey’s HSD post hoc test).

Figure 5

Variation of pH in PVK culture medium. Vertical bars represent confidence intervals at a confidence level of 95% (n = 3). Same letters indicate that differences among the means is not statistically significant (Tukey’s HSD post hoc test).

Figure 6

IAA production (µg/mL) by bacterial isolates. Vertical bars represent confidence intervals at a confidence level of 95% (n = 3). Same letters indicate that differences among the means is not statistically significant (Tukey’s HSD post hoc test).