Spatial distribution of nitrification and denitrification genes in Isohumosols soil profiles

Soil microorganisms are pivotal drivers of nitrogen (N) cycling and profoundly influence crop productivity and ecosystem stability. Although there has been extensive research into N transformations in surface soils, the mechanisms governing deep-soil N cycling, particularly across Isohumosol (Chernozem) diagnostic horizons, remain poorly understood. This study elucidated the vertical distribution patterns for N-cycling functional genes in Ustic (semi-arid) and Udic (humid) Isohumosols and identified the key environmental drivers regulating these processes. Soil samples were collected from four profiles that contained three diagnostic horizons: humus (A), illuvium (B), and parent material (C). Real-time qPCR was employed to quantify key functional gene types: nitrification markers (Comammox, AOA amoA, and AOB amoA) and denitrification markers (nirK, nirS, and nosZ). Potential nitrification (PNR) and denitrification rates (PDR) were measured and their relationships with soil physicochemical properties were analyzed using redundancy analysis (RDA) and structural equation modeling (SEM). The results revealed a pronounced depth-dependent decline in gene abundance, with 41.8%−96.8% reductions across diagnostic horizons (P<0.05). Notably, Udic Isohumosol exhibited distinct N-cycling gene profiles compared to Ustic Isohumosol counterparts. In horizon A, the nosZ, nirS, nirK, and Comammox genes were 49.7%, 89.7%, 30.4%, and 18.8% more abundant in the Udic Isohumosol, respectively (P<0.05), but the AOA amoA and AOB amoA abundances were 64.2% and 20.1% lower, compared to those in Ustic Isohumosol. These differences diminished in horizons B and C, except for Comammox and AOA amoA. Correspondingly, the PNR and PDR were averagely 53.7% and 25.5% lower in horizons B compared to horizon A (P<0.05), respectively. The PNRs in Udic Isohumosol were 26.6%−71.9% lower across all horizons, but the PDR decreased by only 7.9% in the A layer, compared to those in Ustic Isohumosol. There were strong positive correlations between PNR and the nitrification genes (R²=0.82, P<0.05) and between PDR and the denitrification genes (R²=0.34, P<0.05), which confirmed that functional gene abundance is a robust predictor of N-cycling potential. This study identified available phosphorus (AP) and pH as the primary regulators of N-cycling processes. There were significant positive correlations between AP and Comammox, AOB amoA, nirK, nirS, and nosZ abundances (P<0.001); whereas pH negatively influenced Comammox (P<0.001) and nirS (P<0.05 >, but was positively correlated with AOB amoA (P<0.05). The RDA and SEM results demonstrated that AP and pH collectively explained 61% and 21% of the variation in PNR and PDR, respectively, and that AP indirectly regulated nitrification by modulating gene abundance (path coefficient=−0.21, P<0.05), whereas pH exerted direct effects on both nitrifiers and denitrifiers. The SEM further highlighted that the total AP effect on PDR surpassed that of the denitrification genes, which underscored its pivotal role in N metabolism. Additional factors, including soil organic carbon (SOC), C∶N ratio, clay content, and ammonium/nitrate levels, contributed to microhabitat differentiation. Two key innovations emerge from this study. The first is the first comprehensive evidence of contrasting N-cycling strategies between Ustic and Udic Isohumosols and these strategies are linked to moisture-driven redox conditions. Udic Isohumosol favored Comammox and denitrifiers in surface layers, which is probably due to chronic waterlogging and NH₄⁺ accumulation, while Ustic Isohumosol supported ammonia oxidizers under drier, higher-pH conditions. Second, the identification of AP (rather than traditional N substrates) as a key variable redefines the understanding of nutrient coupling in Isohumosols and suggests that phosphorus availability critically constrains microbial N transformation across soil profiles. These findings advance the mechanistic framework for predicting N cycling dynamics in deep soils and offer actionable insights for optimizing fertilization strategies. By highlighting AP management and pH adjustment as potential strategies to mitigate N loss and enhance N retention, this study establishes a scientific basis for sustainable soil management in Isohumosol-dominated agroecosystems. Future research should explore how long-term phosphorus amendment interacts with moisture regimes to shape N-cycling microbial networks at the genome-level.