Synergism of Bacillus subtilis and Azospirillum brasilense for enhanced N‐use efficiency and maize growth: Evidence from 15 N isotopic and physiological responses

Abstract We explored the impact of inoculating maize ( Zea mays L.) seeds with plant growth‐promoting rhizobacteria (PGPR ‐ Bacillus subtilis and Azospirillum brasilense ), either individually or in combination, under different nitrogen input levels – control, low, average and high N levels (0, 30, 60, 90 and 120 mg L ‐1 equivalent to between 0 and 240 kg N ha ‐1 ) in a greenhouse setting. Leaf‐ and plant‐level biometrics, nutritional, biochemical, and physiological evaluations were supplemented by isotopic methods ( 15 N isotope dilution and natural abundance ‐ δ 15 N‰) along with root scanning to investigate N acquisition and distribution. Dual inoculation not only enhanced the recovery of applied N but also bolstered nitrogenase activity, leading to increased biological N fixation (BNF) even at an average level of N (120 kg N ha ‐1 ). In instances where dual inoculated plants were grown at low (60 kg N ha ‐1 ) and average N levels, a cascade effect was observed, such as encompassing root growth stimulation, enhanced fertilizer and soil exploitation, and increased biomass production. Increasing N application to 240 kg N ha ‐1 reduced plant biomass by 19–47% compared to 120 kg N ha ‐1 , depending on inoculation strategy. This led to downregulation of nitrogenase activity, diminished PGPR efficiency, and a significant decline in BNF. This study shows how a change in the rhizosphere microbial population can influence a myriad of responses inside the plants. Our research provides valuable insights for studies using 15 N isotopic and dual inoculation techniques, offering strong potential for practical implementation.