Synergy of FeNPs and PGPR Strain Enhances Nitrogen Fixation by Linking Root Metabolites and Rhizosphere Microbiome Assembly in Alfalfa

Increasing evidences show plant growth-promoting rhizobacteria (PGPR) benefit legume-rhizobium symbiosis, and iron-based nanoparticles (FeNPs) act as rhizobia microenvironment stabilizers. However, few studies explored if their combination exerts synergistic effects on the symbiosis in legume. Here, we compared the effects of FeNPs, Pseudomonas rhizovicinus M30-35, and their co-application (Fe + M) on alfalfa growth, nitrogen fixation, root metabolites, and rhizosphere microbiome. Compared with FeNPs and M30-35, Fe + M increased shoot height, root length, root activity, chlorophyll content, and net photosynthetic rate (Pn) by 63.2% and 45.4%, 61.1% and 70.6%, 56.2% and 47.1%, 20.1% and 18.6%, and 41.1% and 30.6%, respectively; the nodule number, nitrogenase activity, ureide content, and leghemoglobin content rose by 29.6% and 31.4%, 58.5% and 78.7%, 20.4% and 15.1%, and 9.7% and 12.4%, respectively. Metabolomic analysis showed that Fe + M enhanced the accumulation of benzenoid compounds in roots, while microbial co-occurrence network analysis indicated reduced complexity and connectivity of rhizosphere bacterial and fungal communities. Importantly, core microbes, such as Hydrogenophaga, Nocardioides, unidentified_Mitochondria, and Scedosporium, were positively associated with benzenoid compounds, which contribute to nutrient cycling in the rhizosphere. Our findings demonstrate that FeNPs and PGPR strain together achieve synergistic effects on the nitrogen fixation in alfalfa.