Isolation and Characterization of Phosphate Solubilizing Bacteria from Paddy Field Soils in Japan

Abstract

Phosphorus (P) is abundant in soil and is essential for plant growth and development; however, it is easily rendered insoluble in complexes of different types of phosphates, which may lead to P deficiency. Therefore, increases in the amount of P released from phosphate minerals using microbial inoculants is an important aspect of agriculture. The present study used inorganic phosphate solubilizing bacteria (iPSB) in paddy field soils to develop microbial inoculants. Soils planted with rice were collected from different regions of Japan. Soil P was sequentially fractionated using the Hedley method. iPSB were isolated using selective media supplemented with tricalcium phosphate (Ca-P), aluminum phosphate (Al-P), or iron phosphate (Fe-P). Representative isolates were selected based on the P solubilization index and soil sampling site. Identification was performed using 16S rRNA and rpoB gene sequencing. Effectiveness was screened based on rice cultivar Koshihikari growth supplemented with Ca-P, Al-P, or Fe-P as the sole P source. Despite the relatively homogenous soil pH of paddy field sources, three sets of iPSB were isolated, suggesting the influence of fertilizer management and soil types. Most isolates were categorized as β-Proteobacteria (43%). To the best of our knowledge, this is the first study to describe the genera Pleomorphomonas, Rhodanobacter, and Trinickia as iPSB. Acidovorax sp. JC5, Pseudomonas sp. JC11, Burkholderia sp. JA6 and JA10, Sphingomonas sp. JA11, Mycolicibacterium sp. JF5, and Variovorax sp. JF6 promoted plant growth in rice supplemented with an insoluble P source. The iPSBs obtained may be developed as microbial inoculants for various soil types with different P fixation capacities.

Keywords: Japan; phosphate solubilizing bacteria; rice; soil phosphorus fractionation.

Fig. 1.

Geographical locations of soil samples used in the isolation of phosphate solubilizing bacteria.

Fig. 2.

Percentage of fractionated phosphorus to total phosphorus in soil samples. AVG, average value of soil samples; FS1, Fukushima site 1; FS2, Fukushima site 2; HK, Hokkaido; HN, Honmachi; KG1, Kagawa site 1; KG2, Kagawa site 2; NG, Nagano; and SG, Saga. The significance of differences between the overall average and soil samples was assessed by Dunnett’s test (*** P<0.001, ** P<0.01, * P<0.05). Error bars indicate the standard deviation of three replicates.

Fig. 3.

Quantitative estimation of phosphate solubilization by representative isolates on P growth media amended with tricalcium phosphate (A), aluminum phosphate (B), or iron phosphate (C). Values represent the net soluble P by deducting the value of uninoculated samples. NBRC 15308 served as the positive control. The significance of differences between NBRC 15308 and isolates was assessed by Dunnett’s test (*** P<0.001, ** P<0.01, * P<0.05). Means and standard deviations (n=3) are shown.

Fig. 4.

Composition of representative phosphate solubilizing bacteria based on 16S rRNA gene sequencing. Numbers at nodes indicate the level of bootstrap support (%) based on the 1,500-bp DNA fragment and neighbor-joining ana­lysis with 1,000 replications. The scale bar indicates 0.05 changes per site.

Fig. 5.

Venn diagram for isolated iPSB genera Unique and/or shared genera among the types of inorganic P used to isolate bacteria (A), between fertilizer management regimes (B), and soil types (C) in paddy field sources. Al-P: AlPO₄, Ca-P: Ca₃(PO₄)₂, Fe-P: FePO₄