大豆根际溶无机磷细菌Klebsiella sp.wj6的分离鉴定与溶磷特性分析

Screening,identification and solubilizing properties of the inorganic phosphate-solubilizing bacteria Klebsiella sp. wj6 isolated from soybean rhizosphere soil

[目的]为了明确黑土种植的大豆根际溶磷菌的种类及不同溶磷菌溶无机磷的特性,并有效利用溶磷菌,增加黑土的可持续利用能力。[方法]以NBRIP为分离培养基从大豆根际土壤中分离到1株溶磷菌(命名为wj6),采用Vitek 2自动分析系统和16S rDNA方法对其进行鉴定;以NBRIP分离培养基为基础,利用L25(53)正交设计试验,从碳源、氮源及pH值3个方面对菌株wj6溶磷培养基进行优化;利用优化后的NBRIP培养基测定wj6溶解Ca3(PO4)2、FePO4和AlPO4的特点以及溶磷过程中磷酸酶活性和葡萄糖脱氢酶活性。[结果]经Vitek 2自动分析系统和16S rDNA方法鉴定菌株wj6为Klebsiella sp.。菌株wj6以葡萄糖和蛋白胨为碳源和氮源,pH值为8时溶磷量最高;对Ca3(PO4)2、FePO4、AlPO4均有较强的溶解能力,最高溶磷量分别为409.28、60.59和32.90μg·m L-1。菌株wj6在溶解FePO4和AlPO4时受环境pH值的影响较大,当接种到pH值为8的溶磷培养基中,能溶解FePO4和AlPO4,而在培养基原液(pH3)中不具有溶磷能力;以Ca3(PO4)2为唯一磷源,在溶磷过程中酸性磷酸酶、碱性磷酸酶和葡萄糖脱氢酶活性先增加后减小,36 h内葡萄糖脱氢酶活性较大,与其他时间内酶活差异显著。[结论]从大豆根际分离到的溶磷菌Klebsiella sp.wj6在不同难溶性磷酸盐中溶磷能力差异较大,且溶磷受环境pH值的影响。磷酸酶和葡萄糖脱氢酶在溶磷过程中起重要作用。

[ Objectives] In order to clear and define the species of phosphate-solubilizing bacteria（PSB）strains from the black soil of soybean rhizosphcre, the characteristics of dissolving inorganic phosphorus of different PSB and maintain the sustainable utilization ability of the black soil by using PSB. [ Methods] Strain bacterium wj6 was isolated from rhizosphere soil of soybean by NBRIP medium. The Vitek 2 automatic analysis system and 16S rDNA sequences technology were used to identify the bacterium;on the basis of NBRIP medium, the orthogonal design L25（ 53） was employed in decision medium optimization from the carbon source, nitrogen source and pH value for strain w j6 phosphorus solubilization;the dissolving characteristics of Ca3 （PO4 ）2, FePO4, AlPO4 and phosphatase activity and glucose dehydrogenase activity in optimized NBRIP medium were determined. [ Results] The results indicated that the effective PSB wj6 was screened and identified as Klebsiella sp.. The highest capacity of solubilizing phosphate appeared when strain wj6 was supplied with glucose and peptone as carbon source and nitrogen source and the medium pH value was 8. The strain wj6 exhibited higher salubilizing abilities in Ca3 （PO4）2, FePO4, AlPO4 as sole source of phosphorus, and maximum amount of phosphorus solubilization were 409.28,60.59,and 32.90 μg·mL^-1, respectively. Furthermore, the solubilizing phosphorus ability of strain wj6 was strongly influenced by environmental pH value when FePO4 and AlPO4 were the phosphorus source. The strain wj6 could dissolve FePO4 and AlPO4 when it was inoculated to soluble phosphorus medium（ pH 8）. However,no phosphorus solubilizing was found in the medium（ pH 3）. With the use of C% （PO4 ）2 as the sole source of phosphorus, the activity of acid phosphatase, alkaline phosphatase, and glucose dehydrogenase showed the reduction after the first increase in the process of solubilizing phosphorus. Glucose dehydrogenase activity was larger in 36 h than that of other times and showed the significant differences. [ Conclusions ] Klebsiella so. wi6 isolated from soybean rhizosphere showed the significant difference in so｜ubilizing Ca3 （PO4 ）2, FePO4 and ALP04, and phosphorus solubilization was influenced by environmental pH value. Phosphatase and glucose dehydrogenase played an important role in the process of solubilizing phosphorus.