Isolation，Identification and Phylogenetic Analysis of Endophytic Diazotrophs in Tengxian Medical-use Oryza officinalis

doi:10.13560/j.cnki.biotech.bull.1985.2016.06.016

Abstract

Abstract: This research aims to select the endophytic diazotrophs from wild medical-use Oryza officinalis growing on Teng County of Guangxi for collecting the strain resources contributing to the production of microbial fertilizer. Endophytic diazotrophs were isolated from O. officinalis using three selective mediums of free nitrogen and plate streaking method. Acetylene reduction assay was used to determine the activity of nitrogenase in the endophytic diazotrophs. The SDS-PAGE(dodecyl sulfate sodium salt-polyacrylamide gel electrophoresis)patterns of whole-cell proteins and IS-PCR DNA fingerprinting patterns were employed for the clustering analysis of the isolated endophytic diazotrophs. The 16S rRNA gene sequences of the representative strains of each group were analyzed to construct the phylogenetic tree. The abilities of producing auxin，producing siderophores，dissolving phosphorus and the activity of ACC deaminase were tested by colorimetry of spectrophotometer method. Thirty-four strains of endophytic diazotrophs were isolated from O. officinalis and assigned to 5 groups and their nitrogenase activity was between 5.0 to 1 036.7 nmol/(mL·h). Group I and II each had 8 strains. Group III，IV，V had 2，5，11 strains respectively. The homology of 16S rRNA gene was 97.13% between Group I and Azospirillum amazonense ATCC 35119^T，99.71% between Group II and Klebsiella variicola DSM 15968^T，99.86% between Group III and Pseudomonas monteilii ATCC 700476^T，100% between Group IV and Xanthobacter flavus ATCC 35867^T，and 99.86% between Group V and Burkholderia vietnamiensis LMG 10929^T. In conclusion，some strains have the ability of producing auxin，producing siderophores，ACC deaminase activity，and dissolving phosphorus. The results suggest that endophytic diazotrophs in medical-use Oryza officinalis present genetic diversity and potential applications in agricultural practices；and group I probably is new.

Key words: medical-use Oryza officinalis, endophytic diazotrophs, phylogenetic analysis, whole-cell protein electrophoresis, insertion sequence-based PCR

HU Wen-zhe, TAN Ze-wen, WANG Yong, XU Xian-wei, TAN Zhi-yuan. Isolation，Identification and Phylogenetic Analysis of Endophytic Diazotrophs in Tengxian Medical-use Oryza officinalis[J]. Biotechnology Bulletin, 2016, 32(6): 111-119.

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