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Chronic synthetic nitrogen (N) application can result in a significant accumulation of nitrate in the subsoil, which could alter subsoil N cycle and subsequently affect subsoil N levels. To understand how elemental interactions affect the cycle and storage of subsoil N, we examined the soils receiving no fertilizer control (CK), 30-year applications of synthetic fertilizer (CF), and CF plus organic manure (CF + OM). The N cycling microbial groups and activity were investigated through analyzing abundance of bacteria, nitrifiers and denitrifiers, potential nitrification (PNA) and denitrification (DEA) rates in the topsoil (0–20 cm) and subsoil depths (20–80 cm). Compared with the CK, the CF application increased subsoil nitrate but reduced or did not change subsoil microbial biomass N and total N. Corresponding to the increased nitrate, the abundances of denitrifiers increased in the CF subsoils. By contrast, the abundances of nitrifiers increased in the CF topsoil. Significant correlation between the abundances of nitrifiers and soil PNA was found in the topsoil, while significant correlation was also found in the subsoil between the abundances of nirS- and/or nirK-type denitrifiers and DEA. These results suggest that the depleted or less changed subsoil total N by CF application might be partly related to the enriched denitrifiers groups and the related potential activity. The contrasting responses of nitrifiers and denitrifiers in the CF subsoil indicate a decoupling of both processes. Our findings highlight that the leached nitrate by synthetic fertilizer addition not only occurs as an environmental risk causing groundwater contamination but may also alter the subsoil N cycle through the denitrifier groups.

Fertilizer application has contributed substantially to increasing crop yield. Despite the important role of soil fungi in agricultural production, we still have limited understanding of the complex responses of fungal taxonomic and functional groups to organic and mineral fertilization in long term. Here we report the responses of the fungal communities in an alkaline soil to 30-year application of mineral fertilizer (NP), organic manure (M) and combined fertilizer (NPM) by the Illumina HiSeq sequencing and quantitative real-time PCR to target fungal internal transcribed spacer (ITS) genes. The results show: (1) compared to the unfertilized soil, fertilizer application increased fungal diversity and ITS gene copy numbers, and shifted fungal community structure. Such changes were more pronounced in the M and NPM soils than in the NP soil (except for fungal diversity), which can be largely attributed to the manure induced greater increases in soil total organic C, total N and available P. (2) Compared to the unfertilized soil, the NP and NPM soils reduced the proportion of saprotrophs by 40%, the predominant taxa of which may potentially affect cellulose decomposition. (3) Indicator species analysis suggested that the indicator operational taxonomic units (OTUs) in the M soil occupied 25.6% of its total community, but that only accounted for 0.9% in the NP soil. Our findings suggest that fertilization-induced changes of total fungal community were more responsive to organic manure than mineral fertilizer. The reduced proportion of cellulose decomposition-related saprotrophs in mineral fertilizer treatments may potentially contribute to increasing their soil C stocks.