Viral regulation of N₂O emissions depends on soil acidity

Acidification generally enhances N₂O emissions from soils. Yet, the adaptive responses of denitrifiers and the regulatory roles of soil viruses in denitrifier-mediated N₂O emissions under persistent acidity remain poorly understood. Here, we combined D₂O-labeled single-cell Raman spectroscopy, ¹⁵N tracing, metagenomics, machine learning, and virus-addition experiments to examine virus-denitrifier interactions across soils with contrasting long-term acidity conditions maintained for > 17 years. Acidic soils showed the highest N₂O emissions despite the lowest potential denitrification rate and highest microbial anabolic activity. Denitrifiers and viral populations in acidic soils exhibited reduced microdiversity, stronger purifying selection, and distinct functional specialization, while viruses showed a lower virulent-to-temperate ratio and greater genetic potential for host interaction and persistence. Host antiviral defense capacity was the strongest genomic predictor of abundance responses to active virus. In acidic soils, active viruses increased N₂O reductase level by 33.2% without altering nosZ abundance and reduced N₂O accumulation by up to 62.5%, indicating an efficiency-optimized response. Neutral soils showed a quantity-driven response, whereas alkaline soils showed no significant reduction in N₂O accumulation. These findings reveal acidity-dependent viral regulation of N₂O emissions, strongest in long-term acidic soils.