Biological nitrogen fixation in maize (Zea mays L.) by 15N isotope-dilution and identification of associated culturable diazotrophs

The nitrogen-fixing capacity of a range of commercial cultivars of maize (Zea mays L.) was evaluated by the ¹⁵N isotope-dilution method. Biological nitrogen fixation (BNF) expressed as percent nitrogen derived from air (Ndfa) ranged from 12 to 33 regardless of nitrogen fertilization. BNF was not affected by mineral nitrogen fertilization except on cultivar Topacio and PAU-871 cultivars. Subsequently, culturable bacterial diazotrophs were isolated from endophytic tissue of maize: seed, root, stem, and leaf. All isolates were able to grow on N-free semisolid medium. Eleven bacteria isolates showed nitrogen-fixing capacity by the reduction of acetylene to ethylene and confirmed by PCR the presence of nifH gene in their genome. Identification of the 11 isolates was performed by bacteriological methods, 16S rRNA gene sequences, and phylogenetic analysis, which indicated that the bacteria isolated were closely related to Pantoea, Pseudomonas, Rhanella, Herbaspirillum, Azospirillum, Rhizobium (Agrobacterium), and Brevundimonas. This study demonstrated that maize cultivars obtain significant nitrogen from BNF, varying by maize cultivar and nitrogen fertilization level. The endophytic diazotrophic bacteria isolated from root, stem, and leaf tissues of maize cultivars may contribute to BNF in these plants.