Objective To alleviate the economic and and environmental pollution caused by excessive nitrogen fertilizer application in Guangxi's citrus industry, culturable nitrogen-fixing strains in the roots and rhizosphere soil of Citrus sinensis 'Lane Late' were isolated,identified phylogenetic positions and evaluated their comprehensive plant growth-promoting traits, aiming to provide strain resources for the development of citrus-specific microbial fertilizers.

Method Diazotrophs were isolated and purified from the roots and rhizosphere of C. sinensis Lane Late 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 (Sphingobium) exhibited the greatest nitrogen-fixing enzyme activity with the ability to produce indole and gelatinase. Inoculation experiments with Chinese cabbage seedlings demonstrated that treatment with strains SN-1-4 and SG-3-2 (Rhizobium) promoted both plant height and fresh weight to a certain extent.

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 exhibited the highest nitrogenase activity and multiple growth-promoting properties on cabbages and was considered for potential application as a microbe for citrus orchard fertilization.