The hidden role of rhizospheric viruses in promoting nitrogen fixation in soils

Biological nitrogen fixation is a cornerstone of terrestrial nitrogen cycling, traditionally attributed to bacterial nitrogenase activity. However, the potential contribution of rhizospheric viruses remains largely unexplored. Here, we reveal the global distribution of nitrogen-fixing genes, with widespread detection of nifA, nifL, nifU, and nifH in both bacteria and viruses, and identify nifU as a viral auxiliary metabolic gene (AMG). Analysis of viral communities in rhizosphere and bulk soils cultivated with cowpea showed that viral nifU expression was significantly upregulated in rhizosphere soils. Using ¹⁵N₂ stable-isotope tracing and virus transplantation experiments, we demonstrate that virus-encoded nitrogen-fixing AMGs, horizontally transferred from bacteria such as Azospirillum thermophilum (70-99% homology), increased nitrogenase activity from 1.79 to 3.14 nmol C₂H₄ g⁻¹ dry soil h⁻¹. This enhancement was accompanied by shifts in bacterial community composition, with the relative abundance of the nitrogen-fixing genus Azotobacter reaching 90.8%. These results uncover a previously hidden role of rhizospheric viruses in promoting bacterial nitrogen fixation, suggesting that viral-mediated gene transfer could be leveraged to enhance nitrogen cycling in soils and inform sustainable soil management strategies.