<em>Azorhizobium caulinodans </em> ORS 571–<em>Aspergillus spp.</em> biofilm in the presence of flavonoid naringenin: An extremely effective association for rice root colonization with a definite future as a nitrogen bio-fertilizer

Abstract

Use of diazotrophs to provide biologically fixed nitrogen to rice is a challenging but an important alternative to reduce the ailments of excessive use of nitrogen fertilizer on environment, health and economy of a country. The present study focuses on finding an effective strategy for induced colonization of rice roots by <em>Azorhizobium caulinodans </em> leading to significantly beneficial nitrogen providing the plant-microbe association with a very high potential to be developed as a nitrogen bio-fertilizer. <em>Azorhizobium caulinodans</em> was labelled with a green fluorescent protein (GFP) for detection <em>in</em>-<em>vivo</em>. Three separate rice rhizosphere fungi were tested for biofilm formation with the GFP-labelled bacterium <em>in-vivo </em> and successful formation of a biofilm was observed with <em>A. caulinodans-Aspergillus</em> spp. 1 (AAB). <em>A. caulinodans</em> in the presence of flavonoid naringenin, AAB and <em>A. caulinodans</em> in water limiting conditions were tested for induced Azorhizobial colonization of three local rice varieties. The degree of colonization and its effectiveness were screened through epifluorescent microscopy, the degree of endophytic colonization and acetylene reduction assay. Green fluorescence intensity measurements revealed that the treatments, biofilm and naringenin have induced colonization at significantly higher values. Treatment biofilm resulted in the highest nitrogenase activity. Combination of the treatments biofilm, and naringenin, reflected significantly much higher values in fluorescence intensities, acetylene reduction assay and endophytic colonizations. This study reveals that the combination of the biofilm and naringenin promote extremely efficient rice root colonization, with a definite future as a nitrogen bio-fertilizer for rice.