Identification Of Endophytic Diazotrophs Isolated From Tengxian Oryza Officinalis

The collection and digging of nitrogen fixing bacteria resources would help to the development and research of microbial fertilizer. The rational combination of microbial fertilizer and chemical fertilizer can improve the utilization of chemical fertilizer,reduce the cost and pollution,but keep high crop yields.The Oryza officinalis which were collected at the valley of Natang village, Mengjiang town, Teng county, Wuzhou City, Guangxi Autonomous Regionwere used for experimental materials. The endophytic diazotrophs were collected on three nitrogen-free selective medium by plate streak separation; The activity of nitrogenasewas tested by Acetylene Reduction Assay and verified by isotopic abundance method; The SDS-PAGE(Sodium Dodecyl Sulfate Polyacrylamide Gel Electropheresis) fingerprinting patterns of whole-cell proteins and IS-PCR(Insertion Sequence PCR) DNA fingerprinting patterns were used for clustering analysis; The 16 S r RNA gene sequences were analyzed of the representative strains of each group and constructed the phylogenetic tree; The phosphorus-releasing silicon capacity qualitatively was tested by dissolution transparent circle method; The auxin production ability was detected quantitatively by salkowski colorimetry; The phosphorus solubilizing capability was tested quantitatively by Mo-Sb colorimetry; The activity of ACC deaminase was tested quantitatively by 2,4-dinitrophenylhydrazine colorimetry; The siderophores production ability was tested by complex and cyanide method(CAS).Thebelow are main conclusions of the study:1. Thirty-four endophytic diazotrophswere isolated from Oryza officinalis and their nitrogenase activity were between 5.0 to 1036.7 nmol/m L?h. The isolatesassigned to 5 groups by IS-PCR DNA fingerprinting patterns and SDS-PAGE fingerprinting patterns of whole-cell proteins. Group I and II each had 8 strains. Group III, IV, V had 2, 5, 11 strains respectively. The strains named HU31 had the highest nitrogenase activity, and the 15 N isotopic abundance of it could reached 25.15% tested by isotopic abundance method, far more than the natural isotopic abundance 0.36%.2. The 16 S r RNA gene sequence analysis of the representative strains of different types indicated that: the homology of 16 S r RNA 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, 99.86% between group V and Burkholderia vietnamiensis LMG 10929 T.3. Both the representative strains HU31 and CM05 had the ability that could dissolve phosphorus anddesilication, and the ability of dissolving phosphorus were reached 105.4±6.9 ?g/m L and 71.8±9.0 ?g/m L. The representative strains HU31, HU17 and CM05 can product siderophore. The strains of the 5 groups all can produce auxin. When used tryptophan, the ability ofauxinproducing of the trains HU13, HU17, and HU31 is better than without tryptophan,so it indicated that,these 3 strains rely on tryptophan during the process they're producing auxin.Meanwhile, the representative strains of these 5 groups all are tested to have the activity of the ACC deaminase.4. The representative strains HU31 made the seal plastic bottle with full of N2 depressed when put in culture medium containing low nitrogen.Endophytic diazotrophs isolated from Oryza officinalis have biodiversity in this study, and have protencial in agriculture production and application, meanwhile, provide the resources of strains. The representative strains of group I maybe mean a new groups, and it is necessary to be further verified by DNA hybridization and observation by electron microscope. The representative strains HU31 of group II can fix nitrogen efficiently under the low nitrogen in liquid culture medium, it maybe indicate a new nitrogen fixation system.