Nickel oxide nanoparticles (NiO-NPs) pose potential threats to agricultural production. Bacillus subtilis has emerged as a stress-mitigating microbe that alleviates the phytotoxicity caused by NiO-NPs. However, the mechanisms underlying its effectiveness, particularly in root-nodule symbiosis and biological N2-fixation (BNF), remain unclear. Here, we tested the combined exposure of NiO-NPs (50 mg kg-1) and B. subtilis on soybean (Glycine max L.) growth and BNF. Combined exposure increased root length, shoot length, root biomass, and shoot biomass by 19% to 26%, while Ni (200 mg kg-1) reduced them by 38% to 53% compared to the control. NiO-NPs at 100 and 200 mg kg-1 significantly (P < 0.05) reduced nodule formation by 16% and 58% and Nitrogen assimilation enzyme activities levels (urease, nitrate reductase, glutamine synthetase, and glutamate synthetase) by 13% to 57%. However, co-exposure with B. subtilis improved nodule formation by 22% to 44%. Co-exposure of NiO-NPs (200 mg kg-1) with B. subtilis increased peroxidase, catalase, and glutathione peroxidase activity levels by 20%, 16%, and 14% while reducing malondialdehyde (14%) and hydrogen peroxide (12%) levels compared to NiO-NPs alone. Additionally, co-exposure of NiO-NPs (100 and 200 mg kg-1) with B. subtilis enhanced the relative abundance of Stenotrophomonas, Gemmatimonas, and B. subtilis, is associated with N2-cycling and N2-fixation potential. This study confirms that B. subtilis effectively mitigates NiO-NP toxicity in soybean, offering a sustainable method to enhance BNF and crop growth and contribute to addressing global food insecurity.
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