具促生功能拟蕈状芽孢杆菌(Bacillus paramycoides) SH-1464发酵条件优化及其活性

Optimization of fermentation conditions of Bacillus paramycoides SH-1464 with growth-promoting activity

摘要:

【背景】植物根际促生菌在促进植物生长、提高植物系统抗性及改善土壤养分状况等方面具有极大的应用潜力。【目的】为微生物菌肥的开发利用提供高效微生物资源。【方法】采用稀释涂布平板法分离烟草根际土壤可培养细菌，利用PVK培养基筛选具强溶无机磷活性的菌株，结合钼锑抗比色法测定菌株溶磷活性；利用Salkowski’s比色液检测菌株产吲哚乙酸(indole acetic acid, IAA)活性；以菌株溶磷量和IAA产量为指标，采用单因素试验设计进行发酵条件优化，以得到菌株溶磷活性和产IAA的最佳发酵方案；基于种子促发芽及幼苗促生长试验验证菌株的促生效果；结合形态学、生理生化和16S rRNA基因序列对菌株进行分类学鉴定。【结果】于烟草根际土壤分离得到127个不同分类的菌株，10个菌株具溶无机磷的能力，其中菌株SH-1464溶磷活性最强，溶磷圈直径为16.11 mm，溶磷量为119.22 mg/L；Salkowski’s比色结果表明，菌株具有极强的产IAA活性，为39.88μg/mL。单因素试验结果表明，菌株SH-1464溶磷的最佳碳源为10 g/L葡萄糖，最佳氮源为0.5 g/L蛋白胨，最佳无机磷源及其添加量为2.5 g/L磷酸三钙，最佳初始pH值为7.5，温度30 ℃，转速200 r/min；菌株SH-1464产IAA最佳培养条件为：麦芽糖10 g/L，酵母粉5 g/L，NH₄NO₃ 10 g/L，氯化钾2.5 g/L，l-色氨酸0.1g/L，pH 6.5，温度30 ℃，转速180 r/min。基于单因素试验结果进行优化前后菌株溶磷量及IAA产量对比试验可知，优化后菌株的溶磷量为225.9 mg/L，提升84.5%；IAA产量为75.3μg/mL，提升88.8%。促生试验结果表明：菌株SH-1464处理下番茄种子的发芽率提高84.5%，株高提高55.1%，茎围提高32.5%，根长、根重分别提高16.4%和149%，地上部分鲜重和干重分别提高172.2%和203.6%。结合形态学和分子生物学鉴定菌株SH-1464为拟蕈状芽孢杆菌(Bacillus paramycoides，Genbank登录号为ON417365，保藏号为CCTCCM 2022924)；此外，菌株还具有产蛋白酶、产纤维素酶、固氮、分泌铁载体的能力。【结论】菌株SH-1464具有强溶无机磷、固氮、分泌IAA、产蛋白酶、产纤维素酶及分泌铁载体等能力，可显著提高番茄种子的发芽率，对番茄幼苗株高、茎围、根长、根重、地上部分鲜重和干重等农艺性状具有显著促进作用，可作为高效微生物资源在微生物菌剂的开发利用等方面展现广泛应用前景。

Abstract:

[Background] Plant growth-promoting rhizobacteria (PGPR) has great potential in improving plant resistance and regulating soil nutrient status. [Objective] To provide efficient microbial resources for the development and utilization of microbial fertilizer, we isolated and screened out the strains with strong growth-promoting properties from the rhizosphere soil of tobacco. [Methods] The culturable bacteria in the rhizosphere soil of tobacco were isolated by the dilution-plate coating method. The strains with strong phosphorus-solubilizing activity were screened by PVK medium, and the phosphorus-solubilizing activity of each strain was determined by molybdenum-antimony resistance colorimetry. Salkowski’s colorimetric solution was used to determine the production of indole acetic acid (IAA). With the phosphorus-solubilizing amount and IAA yield as indicators, we optimized the fermentation conditions by single factor tests to obtain the optimal fermentation scheme for phosphorus solubilization and IAA production. The growth-promoting activity of the strain was verified by seed germination and seedling growth experiments. The strain was identified based on the morphological, physiological, and biochemical characteristics and 16S rRNA gene sequence. [Results] A total of 127 strains were isolated from the rhizosphere soil of tobacco. Ten of the strains had the ability to solubilize inorganic phosphorus, among which SH-1464 had the highest phosphorus-solubilizing activity with a diameter of 16.11 mm and an amount of 119.22 mg/L. Strain SH-1464 had strong IAA production of 39.88 μg/mL. The optimal fermentation conditions of SH-1464 for solubilizing phosphorus was 10 g/L glucose as the carbon source, 0.5 g/L peptone as the nitrogen source, 2.5 g/L Ca₃(PO₄)₂ as the inorganic phosphorus source, initial pH 7.5, temperature of 30 ℃, and rotation speed of 200 r/min. The optimum conditions of strain SH-1464 for producing IAA were 10 g/L maltose, 5 g/L yeast powder, 10 g/L NH₄NO₃, 2.5 g/L KCl, 0.1 g/L l-tryptophan, pH 6.5, 30 ℃, and 180 r/min. The phosphorus-solubilizing amount and IAA yield of the strain were compared before and after optimization. After optimization, the phosphorus-solubilizing amount and IAA yield of the strain was 225.9 mg/L and 75.3 μg/mL, which increased by 84.5% and 88.8%, respectively, compared with those before optimization. The inoculation of SH-1464 increased the germination rate of tomato seeds by 84.5%, plant height by 55.1%, stem circumference by 32.5%, root length by 16.4%, root weight by 149%, aboveground part fresh weight by 172.2%, and aboveground part dry weight by 203.6%. Strain SH-1464 was identified as Bacillus paramycoides (GenBank accession number: ON417365, deposit number: CCTCCM 2022924). In addition, the strain had the abilities of producing protease and cellulase, fixing nitrogen, and secreting siderophore. [Conclusion] Strain SH-1464 had the abilities of solubilizing inorganic phosphorus, fixing nitrogen, and producing IAA, protease, cellulase, and siderophore. It significantly improved the germination rate of tomato seeds and the seedling agronomic traits such as plant height, stem circumference, root length, root weight, and fresh and dry weight of aboveground part. This strain has a wide application prospect in the development and utilization of microbial agents.