Supplementation with Mo, Co, and Ni Enhances the Effectiveness of Co-Inoculation with the Rhizobacteria Azospirillum brasilense and Bradyrhizobium diazoefficiens in Soybean

Efficient biological nitrogen fixation (BNF) is crucial for sustainable soybean productivity. Current strategies involve the use of <i>Bradyrhizobium diazoefficiens</i> and co-inoculation with plant growth-promoting bacteria like <i>Azospirillum brasilense</i>. To further optimize BNF and plant performance, we investigated the effect of co-inoculation with <i>A. brasilense</i> and <i>B. diazoefficiens</i> combined with the strategic application of the micronutrients Molybdenum (Mo), Cobalt (Co), and Nickel (Ni) on soybean grown under greenhouse conditions. We evaluated plant growth, photosynthetic parameters, accumulation of N, nitrate reductase activity, and <i>nifH</i> gene expression at the R1 reproductive stage. Our main finding was that the co-inoculation combined with the simultaneous application of Mo, Co, and Ni significantly maximized vegetative growth, photochemical efficiency, and BNF. Specifically, this triple supplementation increased <i>nifH</i> gene expression (0.22) compared to the inoculated control (0.003), leading to a substantial enhancement of photosynthetic parameters, including photosystem II (PSII) efficiency and net carbon assimilation (<i>A</i>). For example, the total dry mass was 14.36 g in the Mo + Co + Ni + AZO + BRADY combination and 6.50 g in the non-inoculated and non-micronutrient-treated plants. The total N content was also higher in the plants treated with Mo + Co + Ni + AZO + BRADY (73.20 g kg^-1). Crucially, the data also demonstrated that excessive levels of Co impaired the symbiosis, underscoring the necessity of precise dose management. These results confirm the strong synergistic potential of combining microbial co-inoculation with targeted mineral nutrition as a high-impact, sustainable strategy for boosting soybean productivity.