Sreening, stress toleranceexamination, and phosphate-solubilizing condition optimization of phosphate-solubilizing bacteria

Abstract: [Background] Phosphorus in the soil mainly exists in the form of insolublephosphate, which is inaccessible for crops. Phosphate-solubilizing microorganisms can convertinsoluble phosphorus into available phosphorus, significantly improving soil phosphorusbioavailability and regulating phosphorus transformation. [Objective] To screen the efficientphosphate-solubilizing microorganisms so as to provide the strain resources for developingmicrobial fertilizers. [Methods] The strain was identified based on the morphological, physiological,and biochemical properties and 16S rDNA gene sequence. The tolerance of the strains to salt, pH,temperature, and drought was examined. The phosphate-solubilizing and plant growth-promotingcharacteristics of the strain were investigated in terms of siderophore production, ammoniageneration, and plant hormone secretion. The organic acids were qualitatively analyzed by ultra-highperformance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-TOF-MS).[Results] Strain 4-4-4 capable of efficiently solubilizing phosphate was isolated from the rhizospheresoil of wheat in Xinjiang and identified as Enterobacter. Enterobacter sp. 4-4-4 could grow at pH4.5−9.5, 12−45 °C, and tolerate 10% NaCl and 80 g/L PEG6000, thereby demonstrating strong stresstolerance. This strain was capable of simultaneously solubilizing insoluble inorganic phosphorus(Ca3(PO4)2) and insoluble organic phosphorus (lecithin). It could secrete 28.07 mg/L ofindole-3-acetic acid (IAA) and fix 23.18 mg/L nitrogen, thus promoting the growth of tomato plants,which was evidenced by the plant height, root length, stem diameter, total dry weight, and total freshweight increases of 66.67%, 21.14%, 34.93%, 233.30%, and 211.70%, respectively. Under the cultureconditions optimized by response surface methodology, the strain achieved the soluble phosphoruscontent of 902.1 mg/L from Ca3(PO4)2. This result represents an increase of 30.1% compared withthat before optimization and is 1.25–16.20 times higher than that of most reportedphosphate-solubilizing Enterobacter strains. UPLC-TOF-MS results verified that gluconic acid wasthe key metabolite responsible for phosphate solubilization. [Conclusion] Enterobacter sp. 4-4-4shows an excellent phosphate-solubilizing capability, certain plant growth-promoting potential, andstrong stress tolerance, serving as a candidate strain for developing microbial fertilizers.

摘要：[背景] 土壤中的磷多以难溶性磷酸盐形式存在，难以被作物吸收利用。溶磷微生物可将难溶性磷转化为有效磷，显著提升土壤磷生物有效性并调控磷素转化。[目的] 筛选高效溶磷微生物，为微生物肥料开发提供菌株资源。[方法] 结合形态学、生理生化特性及16S rDNA基因序列对菌株进行鉴定；检测菌株对盐、pH、温度及干旱的耐受性；通过产铁载体、产氨及植物激素分泌等维度，探究菌株的溶磷与促生特性；采用超高效液相色谱-四极杆飞行时间质谱（ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry, UPLC-TOF-MS）对有机酸进行定性分析。[结果] 从新疆小麦根际土壤中分离得到一株高效溶磷菌株4-4-4，鉴定为肠杆菌属（Enterobacter）。肠杆菌属菌株4-4-4可在pH 4.5~9.5、12~45 ℃条件下生长，耐受10% NaCl及80 g/L聚乙二醇6000（PEG6000），表现出较强的胁迫耐受性。该菌株可同时溶解难溶性无机磷（磷酸三钙，Ca₃(PO₄)₂）与难溶性有机磷（卵磷脂，lecithin）；可分泌28.07 mg/L吲哚-3-乙酸（indole-3-acetic acid, IAA），固氮量达23.18 mg/L，能够促进番茄植株生长：番茄株高、根长、茎粗、总干重及总鲜重分别提升66.67%、21.14%、34.93%、233.30%及211.70%。在响应面法优化后的培养条件下，该菌株对磷酸三钙的溶磷量可达902.1 mg/L，较优化前提升30.1%，较多数已报道的溶磷肠杆菌菌株高出1.25~16.20倍。UPLC-TOF-MS结果证实，葡萄糖酸为介导溶磷过程的关键代谢产物。[结论] 肠杆菌属菌株4-4-4具有优异的溶磷能力、一定的促生潜力及较强的胁迫耐受性，可作为开发微生物肥料的候选菌株。