几株高效溶磷解钾药用稻内生固氮菌的筛选与鉴定

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摘要从药用稻(Oryza officinalis)中筛选具有高效溶磷解钾的内生固氮菌，在农业生产上具有很大的潜在应用价值。本研究以药用稻为材料，采用两种选择性无氮培养基结合乙炔还原法进行内生固氮菌的分离，利用平板筛选法从所分离的内生固氮菌中筛选出高效溶磷解钾菌，应用全细胞蛋白SDS-聚丙烯酰氨凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)对所筛选到的菌株进行快速聚类。选取每个类群的代表菌株进行16S rRNA基因序列测定及生理生化鉴定。通过钼锑抗比色法和四苯硼钠法测定其溶磷解钾量，同时通过接种水稻对其促生作用进行分析。结果表明，从药用稻中筛选到了4株具有高效溶磷解钾能力的内生固氮菌，其固氮酶活性在8.78~8.88 μmol C2H4/(mL·h)之间。SDS-PAGE全细胞蛋白电泳将4株菌聚为1个类群。16S rRNA基因序列相似性分析及理化鉴定表明，代表菌株yy01为久留里伯克霍尔德菌(Burkholderia kururiensis)，菌株yy01发酵培养5 d后，溶无机磷量达116.28 mg/L，是参比菌株变栖克雷伯氏菌(Klebsiella variicola)的2.73倍；解钾量达268.31 mg/L，是参比菌株变栖克雷伯氏菌的4.67倍。此外，yy01还具有分泌生长素等特性，接种水稻之后能明显的促进水稻的生长, 其中水稻叶长增加了25.90%，根增长了42.30%，分蘖数增加了79.64%，鲜重增加了166.90%，氮、磷、钾的含量分别增加了61.54%、41.18%和54.05%。因此，从药用稻中筛选到的4株具有高效溶磷解钾能力的内生固氮菌是一类对农业生产有着重要意义的菌株。

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关键词 ：伯克霍尔德菌, 固氮, 溶磷, 解钾, 筛选, 鉴定, 促生长作用

Abstract：The study aimed to screen endophytic diazotrophs with high capability of phosphate solubilizing and potassium decomposing from Oryza officinalis. The endophytic diazotrophs with high capability of phosphate solubilizing and potassium decomposing properties potentially supplied the foundation for the future agricultural application. The endophytic diazotrophs isolated from the O. officinalis were evaluated by using 2 nitrogen-free selective media in combination with the acetylene reduction method, the capability of phosphate solubilizing and potassium decomposing. The isolated endophytic diazotrophs with high efficient capability of phosphate solubilizing and potassium decomposing were grouped by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns of the whole-cell protein electrophoresis. The representative strains were further studied by physiological and biochemical tests, and 16S rRNA gene sequencing analysis. The soluble phosphorus and decomposing potassium content in the fermentation liquid were determined by molybdenum antimony colorimetric method and 4 benzene boron sodium assay, respectively. The possible promotion of rice growth was also carried out. The nitrogenase activity of 4 isolated endophytic diazotrophs ranged from 8.78~8.88 μmol C2H4/(mL·h). The 4 strains were assigned to one group by analysis of SDS-PAGE patterns of the whole-cell protein electrophoresis. 16S rRNA gene sequencing analysis and physiological and biochemical tests of the representative strain yy01 showed that the strain was closely related to Burkholderia kururiensis. The inorganic phosphorus solubilizing ability (116.28 mg/L) of strain yy01 was 2.73 times as much as the reference strain Klebsiella variicola, and potassium decomposing activity (268.31 mg/L) was 4.67 times as much as the the reference strain K. variicola within 5 d fermentation in the liquid medium. In addition, the strain yy01 could also secrete indole-3-acetic acid. Strain yy01 inoculated with rice showed significant promotion rice growth, the length of rice leaf increased of 25.9%, the length of root increased of 42.30%, tiller number increased of 79.60%, fresh weight increased of 166.90%; The N, P and K content of rice increased of 61.54%, 41.18% and 54.05%, respectively. Based on above results, these 4 endophytic diazotrophs with high capability of phosphate solubilizing and potassium decomposing isolated from the O. officinalis have the potential application in agriculture production.

Key words： Burkholderia kururiensis Nitrogen fixation Phosphate solubilizing Potassium decomposing Screening Identification Growth-promoting properties

收稿日期: 2015-06-13 出版日期: 2015-12-29

基金资助:国家自然科学基金;广东省科技项目;广东省科技项目

通讯作者: 谭志远 E-mail: zytan@scau.edu.cn;houwei1020@126.com

引用本文:

阳洁江院王晓甜尹坤秦莹溪谭志远. 几株高效溶磷解钾药用稻内生固氮菌的筛选与鉴定[J]. , 2016, 24(2): 186-195.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2016/V24/I2/186

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