Screening and biodiversity analysis of cultivable inorganic phosphate–solubilizing bacteria in the rhizosphere of Hydrilla verticillata

Materials and methods Source of experimental samples Cultivable IPB were isolated and screened from the roots and rhizosphere sediments of Hydrilla verticillata collected from an urban landscape water in Chuangzhi Park, Pidu District, Chengdu, Sichuan Province (E103˚540, N30˚480). Culture medium The inorganic phosphorus medium (NBRIP) [17]: glucose, 10 g; Ca3(PO4)2, 5 g; MgCl2�6H2O, 5 g; MgSO4�7H2O, 0.25 g; KCl, 0.2 g; (NH4)2SO4, 0.1 g; distilled water, 1 L; with a pH 7.0–7.5. Ca3(PO4)2 was required to be sterilized separately; NBRIP solid medium was added with 20 g agar. The Luria-Bertani (LB) medium: NaCl, 10 g; yeast paste, 5 g; peptone, 10 g; agar, 20 g; and distilled water, 1 L; with a pH 7.0–7.2. The phosphorus-deficient medium [18]: CH3COONa�3H2O, 3.23 g; Na2HPO4�2H2O, 23 mg; NH4Cl, 152.8 mg; MgSO4�7H2O, 81.12 mg; K2SO4, 17.83 mg; CaCl2�2H2O, 11 mg; 4- (2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 7 g; trace element solution, 2 mL; and distilled water, 1000 mL; with a pH = 7. Isolation and screening of IPB The roots with good growth conditions and mud were selected from the collected Hydrilla ver- ticillata and cut up with scissors. Firstly, 10 g cut mud-bearing roots were added to a conical flask containing 100 mL of sterile water and glass beads. The conical flask was placed in a shak- ing incubator at 600 rpm and 28˚C for two hours to form a sediment suspension. Then, 1 mL of the suspension was taken and 9 mL of sterile distilled water was added to it to form a 10−1

bacterial suspension. The suspension was taken and diluted step by step to 10−2–10−9. Then, 0.1 mL of each was taken and spread on the NBRIP solid plate medium, with three replicates of each concentration. Next, colony with obvious phosphate-solubilizing ring on the NBRIP solid medium was picked for lineation and separation by repeated lineation for more than four times. After the single colony was confirmed as pure culture by morphological character- istic and microscopic examination, the LB slant medium was inoculated, cultured and stored in a 4˚C refrigerator for further use. Detection of phosphate-solubilizing characteristic of IPB The strains were inoculated in a phosphorus-deficient medium and incubated at 28˚C for 24 h. The culture solution was poured into a centrifuge tube and centrifuged at 4000 rpm for five minutes. After centrifugation, the supernatant was discarded, and distilled water was added and mixed with a vortex to prepare a bacterial suspension with an OD600 (Optical Density) value of about 0.2. Then, 2 mL of the suspension was added to 100 mL of freshly sterilized NBRIP liquid medium and incubated at 28˚C for seven days. Each treatment was replicated three times. The uninoculated medium was used as the control. The concentration of soluble reactive phosphate (SRP) in the culture solution from three to seven days was determined by the molybdenum antimony anti-colorimetric method [19], the pH of the culture solution was measured using a pHS-3C acidity meter, the concentration of dissolved inorganic phosphorus

(DIP) was calculated from the standard curve, with three replicates per group, phosphate-solu- bilizing efficiency was calculated by the ratio of the concentration of dissolved inorganic phos- phorus in the culture solution to the original phosphorus concentration, and the PLOS ONE Screening and biodiversity analysis of cultivable inorganic phosphate–solubilizing bacteria PLOS ONE | https://doi.org/10.1371/journal.pone.0297047 January 19, 2024 3 / 11