Abstract
Soil acidification poses a critical threat to global agricultural sustainability, particularly in high-buffering Mollisol (black soil) ecosystems. However, the microbially mediated biochemical mechanisms underlying nitrogen (N) transformation blockages and subsequent phytotoxicity under acid stress remain poorly understood. Here, using a controlled soil acidification gradient (pH 2.0–7.0) combined with 16S rRNA gene sequencing, functional profiling (FAPROTAX), quantitative PCR (qPCR), and chemical N speciation, we unravel a key microbially driven mechanism governing acid-induced crop failure. Our findings demonstrate that soil acidification imposes strong deterministic environmental filtering (βNTI approaching +2) on the rhizosphere microbiome, driving a drastic collapse in microbial phylogenetic diversity (Chao1 index). This structural transition severely alters N-cycling functional guilds, inducing a profound functional bottleneck. In strong acid treatments (pH 2.0–4.0), ammonium (NH
4
+
) and nitrate (NO
3
–
) concentrations plummeted to 0.25-fold and 0.20-fold of those in circumneutral soils, respectively. Crucially, nitrite (NO
2
–
-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 (NaNO
2
) 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 NO
2
–
-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
NO
2
–
-N. Collectively, this study establishes that acid-induced functional decoupling of the nitrifying community causes a massive NO
2
–
-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.