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

作　　者：申云鑫 赵江源[3] 王楠 李铭刚[3] 施竹凤 冯路遥 李者芬 陈齐斌[1] 杨佩文[2] SHEN Yunxin;ZHAO Jiangyuan;WANG Nan;LI Minggang;SHI Zhufeng;FENG Luyao;LI Zhefen;CHEN Qibin;YANG Peiwen(College of Plant Protection,Yunnan Agricultural University,Kunming 650000,Yunnan,China;Agricultural Environment and Resources Institute,Yunnan Academy of Agricultural Sciences,Kunming 650205,Yunnan,China;Yunnan University,Kunming 650091,Yunnan,China)

机构地区：[1]云南农业大学植物保护学院,云南昆明650000 [2]云南省农业科学院农业环境资源研究所,云南昆明650205 [3]云南大学,云南昆明650091

出　　处：《微生物学通报》2023年第6期2436-2451,共16页Microbiology China

基　　金：云南省科技计划重大科技专项(202202AE090015,202202AE090010);国家自然科学基金(32060624)。

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16.11mm,溶磷量为119.22mg/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,NH4NO310 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�[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 39.88μg/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-so

关 键 词：拟蕈状芽孢杆菌 无机磷 吲哚乙酸(IAA) 发酵条件优化 微生物肥料 

分 类 号：TQ920.1[轻工技术与工程—发酵工程] TQ446