Objective Culturable diazotrophs in the roots and rhizosphere soil of Citrus sinensis navel orange were isolated, identified, and characterized for potential application as a microbial fertilizer to reduce the use of chemicals and improve ecology for the orchards.

Method Diazotrophs were isolated and purified from the roots and rhizosphere of C. sinensis Lane Late navel orange at flowering stage on Ashby nitrogen-free medium. Molecular phylogenetic analysis was conducted by 16S rRNA sequencing, physiological and biochemical characteristics of the isolated strains determined using microbial biochemical tubes and nitrogen-fixing enzyme ELISA kits, and growth-promoting effects observed by inoculation on cabbage seedings.

Result From the rhizosphere, 3 strains of nitrogen-fixing bacteria belonging to Rhizobium, Burkholderia and Pantoea, and in the roots, two strains of Sphingobium and Agrobacterium, were isolated and identified. The colonies of the rod or elliptical shaped diazotrophic microbes significantly differed morphologically and in color. But they all were Gram-negative and contained the nifH gene encoding nitrogen-fixing enzymes. Most of them secreted indole and gelatinase. Among them, SN-1-4 exhibited the greatest nitrogen-fixing enzyme activity with the ability to produce indole and gelatinase. In addition, it, as well as SG-3-2, promoted the plant height and fresh weight of the Chinese cabbage seedings.

Conclusion Five strains of nitrogen-fixing bacteria in 5 genera were isolated and identified from the roots and rhizosphere soil of C. sinensis Lane Late. Of the identified diazotrophs, Sphingobium SN-1-4 displayed the greatest nitrogenase activity with comprehensive growth-promoting properties on cabbages and was considered for potential application as a microbe for citrus orchard fertilization.