Screening and molecular identification of phosphate- solubilizing bacteria in rhizosphere soils in Hainan ecosystem

Abstract:

[Objective] To know the activities of dissolving Ca3(PO4)2 and FePO4 of phosphate-solubilizing bacteria (PSB) isolated from acidic soil in Hainan province in China and to screen PSB with high and stable activity (PSBHS). [Methods] Soil samples of 21 plant species were isolated with nutrient agar, crystal violet-nutrient agar, and yeast extract-mantol agar with dilution-plate method. Bacterial representative of the predominant morphologically distinct colonies present on plates were selected randomly and purified on minimal nutrient agar. First screening was done on solid medium with tricalcium phosphate (Ca-P) as the sole P source, and after 5 days of cultivation the colonies with phosphate-solubilizing zones were selected for further screening. Secondary screening was conducted by cultivating the selected colonies in Ca-P liquid medium for 6 days at 32℃ with shaking of 200 r/min, and PSB capable of solubilizing phosphorus up to more than 200 mg/L were selected. The third screening—6-day shaking cultivation in Ca-P liquid media—was conducted after 4 successive cycles of transfer-culture plus 15 d-storage at 4℃. PSB capable of solubilizing phosphorus up to more than 200 mg/L was selected and termed as PSBHS. Then PSBHS were cultivated in FePO4 liquid media for 6 days, and the activity of dissolved phosphorus was determined. The 16S rDNA gene sequencing and blast search against the public database were used to identified PSBHS. [Results] In total 363 bacterial representatives were isolated from soil samples, and the following three screenings reduced the number of representatives to 126, 45, and 14 respectively. The amounts of dissolved phosphorus by 14 PSBHS after 6 days of cultivation in Ca-P liquid media were up to 201.0 mg/L~623.3 mg/L, the pH values of media decreased to 3.82~4.34 from initial 7.0, and the final pH values were found to be significantly negatively correlated to the amounts of dissolved phosphorus. After 6-day of cultivation in FePO4 liquid media, only a small amount of phosphorus (1.6 mg/L ~34.2 mg/L) were solubilized by PSBHS, and the final pH values of media decreased to 2.87~5.67— also significantly negatively correlated to the amounts of dissolved phosphorus. The 16S rDNA gene sequencing analysis identified 6 PSBHS to be Acinetobacter, 3 to be Pseudomonas, 3 to be Serratia, 2 to be Enterobacter.