Isolation, identification and characterization of associative nitrogen-fixing endophytic bacterium Kosakonia radicincitans GXGL-4A in maize

Abstract:

[Objective] Nitrogen-fixing microorganisms are the main component of biological nitrogen fixation agent, and the breeding of nitrogen-fixing-bacterium provides a solid foundation for the investigation and application in biological nitrogen fixation field. A nitrogen-fixing bacterium was identified and furthermore its nitrogenase activities were determined. [Methods] Ashby medium is used for the isolation and purification of nitrogen-fixing-bacterium. Morphological, physiological and biochemical analyses were conducted combining with 16S rRNA gene sequencing and genomic scanning to identify the bacteria. The ammonia-secreting capacity of bacterial cells was measured by indophenol blue spectrophotometric method and the nitrogenase activity was determined using the acetylene reduction assay (ARA). [Results] Kosakonia radicincitans GXGL-4A, a gram-negative nitrogen-fixing bacterium with the size of 1.5 μm×0.5 μm was finally isolated from maize root tissues. The strain were observed to show single cell distribution and joint of two tandem bacterial cells under a microscope. Phylogenic analysis based on 16S rRNA gene sequence revealed that GXGL-4A shares 95% of DNA homology with the Kosakonia oryzae strain Fo8A1d. Eventually, the isolate was identified as Kosakonia radicincitans and the stain was named as K. radicincitans GXGL-4A. The nitrogen-fixing gene, nifH was successfully amplified by PCR technique. The content of ammonia nitrogen secreted by GXGL-4A bacterial cells was assayed at 2.5 mg/L through indophenol blue spectrophotometric method. The results showed that this strain has a good capacity in ammonium secretion. The nitrogen-fixing bacterium GXGL-4A grown on nitrogen-free media can effectively restore acetylene, and its nitrogenase activity expressed in ethylene production capacity was evaluated at 232.94 nmol C2H4/(mL·h). [Conclusion] K. radicincitans GXGL-4A will benefit for the research of microbial nitrogen fixation.