Supplementary file 1_Soil acidification drives a massive accumulation of nitrite as a mechanistic bottleneck to inhibit maize growth and productivity.pdf

Crucially, nitrite (NO₂^–-N) underwent a 5.85-fold hyper-accumulation, reaching up to 28.52 mg kg^–1. At the molecular level, this accumulation was driven by a transcriptional and functional decoupling between nitrifying cohorts: while ammonia oxidation genes (amoA/amoB in AOA and AOB) were upregulated or sustained under acidity, nitrite oxidoreductase (NxrC) expression and gaseous denitrification pathways were almost completely suppressed.

To decouple nitrite phytotoxicity from direct proton (H⁺) stress, exogenous sodium nitrite (NaNO₂) was spiked into neutral soil (pH 7.0) matching the acidification gradient (1.75–28.52 mg kg^–1; M7–M2). Dose-response validation revealed that NO₂^–-N exceeding 13.21 mg kg^–1 exerted severe phytotoxicity, significantly inhibiting maize germination, leaf area, and height, with total plant mortality occurring at ≥27.78 mg kg^–1 NO₂^–-N. Collectively, this study establishes that acid-induced functional decoupling of the nitrifying community causes a massive NO₂^–-N accumulation, acting as an relatively overlooked, independent phytotoxic driver of crop failure in acidified soils.

These insights demonstrate that restoring acidified agroecosystems requires shifting from simple pH neutralization to integrated microbiome management aimed at relieving N-transformation bottlenecks.</p>