Characterization Of Mineral Phosphate-Solubilizing Bacterium K₃ And Colonization Under Different Soil

Phosphate solubilizing microorganisms (PSMs) are ubiquitous in soil and could play an important role in phosphorous cycling, converting insoluble phosphate into soluble forms which are available to plants. Pseudomonas sp. K3 was previously identified with a high ability to solubilize tricalcium phosphate (TCP) and to promote the growth of maize seedlings. This study investigated the mechanism of phosphate solubilization and assessed the influence of different factors (e.g., carbon source, nitrogen forms, phosphate source and buffer capacity) on the phosphate solubilizing ability of K3 in vitro. Furthermore, pot experiments were carried out to study the behavior of GFP-labelled strain K3 in different types of soils and rhizosphere. Different organic materials were also tested with K3 to screen the best organic carrier. The relationships among the number of GFPK3, dynamic distributing in the rhizosphere, and phosphorus accumulation in plant were systemically investigated. The results were as follows:1. After seven days of K3 growth in a liquid medium NBRIP, the P concentration in the solution increased from 6.54μg/mL to 655.23μg/mL, while the pH value decreased from 7.00 to 3.99. Results from HPLC analysis showed that the solubilization of TCP was mainly caused by the release of 47.39 mmol/L malic acid,25.67 mmol/L lactic acid and 1.89 mmol/L oxalic acid. At these concentrations, malic acid, lactic acid and oxalic acid could solubilize P about 535.31μg/mL,279.06μg/mL and 46.58μg/mL from TCP, respectively, suggesting that malic acid excretion by K3 was the main mechanism for P solubilization.2. The effects of carbon and nitrogen forms and carbon to nitrogen ratios (C/N) on solubilization of TCP by K3 were assessed. The results showed that carbon sources had strong influence on phosphate solubilization, and K3 had the highest capacity to dissolve TCP when glucose was added. Release of P reached maximum under glucose at 653.24μg/mL and minimum under soluble starch at 4.83μg/mL. Ammonium significant promoted P solubilization when compared with nitrate and urea. The ability of K3 to dissolve TCP went down to 32.13μg/mL when ammonium and nitrate were added together. The P solubilization activity was strongly associated with C/N ratio, i.e., the lower C/N ratio, the higher activity of P solubilization. The highest dissolved P concentration was attained with the C/N ratio at 8:1. Results also showed that addition of soluble P in the medium could repress the ability of K3 to dissolve TCP. In addition, strain K3 was resistant to Zn, Pb and Cr, but sensitive to Cu.3. Greenhouse experiment was conducted by using GFP labeled strain K3 (GFPK3). The results showed that the wheat yield, shoot height and dry weight were increased by 14.75%, 19.37% and 2.8% respectively, after supplied bio-organic fertilizer of GFPK3 when compared with the common organic manure treatment. The population of GFPK3 was gradually decreasing in soil with the wheat growing. The total population of GFPK3 decreased from 107 CFU/g of soil to 105 CFU/g of soil when growing from sowing to wheat seedling stage. GFPK3 was further decreased to about 103 CFU/g of soil at harvest time. Bands of DGGE and similarity coefficient of soil microbial populations showed that the microbe diversity of treatments of the bio-organic fertilizer with GFPK3 (M+GFPK3) and the bio-organic fertilizer with GFPK3 plus P addition (M+1/3P+GFPK3) were more plentiful than the control, revealing that the phosphate solubilizing bacterium had remarkable effect on the microbial community in soil.4. Pot experiments were conducted with maize growing in three types of soil to investigate the colonization of GFPK3 on rhizosphere soil in greenhouse. The results showed that the population of GFPK3 in the sandy soil was more than that in the silt soil and the sand-silt mixed soil. The number of GFPK3 in the silt soil was dramatically decreased to 104 CFU/g of soil 42 days after sowing. However, in the sandy and the sand-silt mixed soil, the population of GFPK3 was about 105 CFU/g of soil at the same time, then declined to 104 CFU/g of soil. The results suggested that manure inoculation with GFPK3 could enhance the available phosphate content in soil and significantly promote the maize biomass and phosphorus content in plant.5. Experiment about the effect of different organic manure on colonization of GFPK3 in soil was conducted in greenhouse. The results showed that GFPK3 was able to grow and colonize under various organic manure addition. The population of GFPK3 in amino manure was highest than the other treatments. The total population of GFPK3 strain decreased from 106 CFU/g of soil at 7th day to 105 CFU/g of soil at 56th day after sowing. Results from root scanning showed that inoculation of K3 could enhance the total root length and root surface area, which improves plant nutrition and plant growth.