| Available phosphorus deficiency in agricultural soils has become one of the limited factors for crop production. P-solubilizing bacteria(PSB) are known to play vital roles in biogeochemical P cycling in natural and agricultural ecosystems, in respect that can solubilize P from inorganic and organic P compounds in soil to available forms for plants by secreting organic acid and relevant enzymes.In this study, six most efficient isolates were successfully screened from the soil samples that obtained from paddy fields, compost, and hog farm septic tank at urban areas of Wuhan. The strain A5-4 was selected with phosphate-solubilizing efficiency of 204.70mg/L and 58.3 mg/L for Ca3(PO4)2 and calcium phytate, respectively. In the modified PVK liquid medium amended calcium phytate as sole P, phytase and alkaline phosphatase activity of A5-4 reached 0.397 U/mL and 1.897 U/mL. In the pot experiments, rice inoculated with selected isolates exhibited increased plant height, root length, fresh weight, dry weight and P content of leaf to various extent with respect to control. Among the bacterial inoculations after 49 days growth. The best treatment was the application of A5-4, which increased plant height, root length, fresh weight, dry weight P content of leaf by37.3%,72.2%,64.7%,76.4% and 38.1% respectively. The results of plant growth regulators(PGRs) tests revealed the ability of the strain with the excretion of indole-acetic acid(IAA) and siderophores. Based on Morphological, physiological and biochemical characteristics and partial sequencing analysis of their 16S rDNA, the strain A5-4 was identified as Clostridium butyricums (98% sequence homology).The P-solubilizing characteristics of C.butyricums A5-4 in PVK liquid medium(amended calcium phytate or Ca3(PO4)2 as the single P source) and pot experiment soil were investigated. The results suggested:(Ⅰ) There was a positive correlation between the concentration of biomass and solubilized P in the culture; no matter which kind of P source. (Ⅱ) The secretion of phytase and phosphatase was mainly in exponential phase of C.butyricums A5-4, which was induced by organic P substrate.(Ⅲ) No significant correlation was found between the culture pH and the P solubilized from calcium phytate, whereas a significantly negative correlation (p<0.05) was found between the culture pH and the P solubilized from Ca3(PO4)2. (Ⅳ) Six kinds of organic acid have been detected in the culture filtrates by GC, the amount of organic acids was accumulated to 2.659g/L. (Ⅴ) The available P contents, phosphate and phophatase activities of rhizosphere soil(RS) were significantly (P≤0.05) increased by C.butyricum A5-4 during the entire period(0～49d).The screening experiments, Plackett-Burman design, Steepest ascent experiments, Central composite experimental design and response surface methodology were adopted to derive a statistical model for optimizing the composition of the medium and the fermentation conditions of C.butyricum A5-4. The optimum medium and conditions that describe as following (g/L):glucose 23.41, yeast extract 4.42, peptone 7.23, K2HPO4·3H2O 2.90, MgSO4·7H2O 0.6, NaCl 0, KCl 0.30, FeSO4·7H2O 0.03, MnSO4·H2O 0.015, initial pH 7.5, temperature 37℃, culture time 36 h, seed age 12 h, inoculum level 3%. The populations of the viable organisms could reach 1.12±0.11×109colony forming units CFU/mL. |
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