A new isolation methodology for phosphate-solubilizing bacteria using a step-wise enrichment process

Phosphate-solubilizing bacteria (PSB) screened for their ability to solubilize Ca3(PO4)2 in a laboratory may not be effective in solubilizing phosphorus (P) in soils that contain different forms of P. We developed an efficient isolation method to obtain PSB from soils collected from diverse soil types that vary in P forms.

PSB isolated through enrichment in media containing AlO4P, Ca3(PO4)2, or FeO4P as sole P sources were compared with a previous collection of Pseudomonas- and Bacillus-like bacteria that solubilized P. There was a significant positive association (P < .05) between the number of culturable PSB and several properties from the original soils, including calcium, magnesium, potassium, total N, P, carbon concentrations, electrical conductivity, and silt content. The number of culturable PSB was inversely related to the sand content of the soil. Additionally, the most efficient PSB were obtained from sites with a high aridity index and from alkaline soils. After enrichment, there was a significant increase in PSB among all culturable bacteria. Isolates obtained after step-wise enrichment solubilized 1.8-2.2, 1.4-2.9, and 2.2-3.3 times more P from Ca3(PO4)2, AlO4P, and FeO4P, respectively, compared with strains selected using a taxonomic method. Isolates enriched using FeO4P produced predominantly tri- and dicarboxylates in vitro. Additionally, we identified an efficient P solubilizer that is closely related to Pseudomonas granadensis, a species not previously reported as a P solubilizer.

Using a step-wise enrichment method in culture media with sparingly soluble P increased the likelihood of isolating high-efficiency PSB from soil.