Biofilm Formation by Rice Rhizosphere Nitrogen-Fixing Microorganisms and Its Effect on Rice Growth Promotion

Background and Aims: Excessive nitrogen fertilizer use contributes to environmental pollution and undermines agricultural sustainability. Enhancing symbiotic interactions between rice and nitrogen-fixing microorganisms offers a promising strategy to improve nitrogen use efficiency (NUE). This study investigates the role of rice root exudates in promoting biofilm formation by nitrogen-fixing microbes to enhance nitrogen fixation. Methods: Nine nitrogen-fixing microbial strains were evaluated for biofilm formation in response to flavone and apigenin treatments, with Gluconacetobacter diazotrophicus (KACC 12358) serving as the reference strain. The most responsive strain was selected, and a li-brary of 1,597 natural compounds was screened to identify those that promote biofilm formation in both the selected and reference strains. Results: Azoarcus indigens (KACC 11682) exhibited the highest biofilm-forming capacity. Screening identified 68 compounds that enhanced biofilm formation by more than 500% compared to the control. Among them, eight compounds induced strong biofilm for-mation (O.D. > 2.0) in Azoarcus indigens. Cardamomin, a flavonoid biosynthesized by rice, emerged as the most effective compound. Growth promotion assays showed that Azoarcus indigens increased rice fresh weight by approximately 128% compared to untreated con-trols. Conclusion: This study demonstrates the potential of rice root exudate-derived com-pounds to promote beneficial symbiosis with nitrogen-fixing microbes. These findings of-fer a novel approach to enhancing rice NUE. Future research will focus on developing rice cultivars with increased production of biofilm-inducing metabolites.