6-year nitrogen addition alters fungal network structure and extracellular enzyme activities in alpine grasslands: divergent responses between rhizosphere and bulk soils

Abstract

Background

Nitrogen deposition significantly alters carbon and nitrogen cycling in terrestrial ecosystems, yet how microbial network restructuring drives extracellular enzyme activity changes under continuous nitrogen input remains elusive. Traditional studies frequently overlook rhizosphere microenvironments, treating soil as a homogeneous system. To bridge this gap, we leveraged a 6-year nitrogen addition gradient experiment (0, 10, 15, and 20 kg nitrogen ha⁻¹ yr⁻¹) in an alpine grassland of the Sejila Mountains. Integrating co-occurrence network, Mantel, and redundancy analyses, we systematically compared the divergent responses of fungal communities, enzyme activities, and soil carbon and nitrogen fractions between rhizosphere and bulk soils.

Results

In the bulk, nitrogen inputs exceeding 10 kg ha⁻¹ yr⁻¹ induced significant acidification and substrate depletion, structurally simplifying Saprotroph-dominated fungal networks. Consequently, substrate scarcity downregulated enzyme synthesis, driving an overall decline in carbon- and nitrogen-cycling enzyme activities. Conversely, the rhizosphere—bolstered by continuously enriched microbial biomass carbon and nitrogen—maintained highly modular fungal networks and stable Symbiotrophs. Synergistic substrate–community–enzyme interactions sustained high activities of key metabolizing enzymes, including β-glucosidase and urease.

Conclusions

Overall, the structural and functional differentiation between rhizosphere and bulk compartments under 6-year nitrogen addition constitutes a crucial complementary mechanism underpinning ecosystem stability. Future research should prioritize microhabitat scales, coupling in situ isotope tracing with functional gene validation to enhance carbon and nitrogen pool stability and persistence assessments in alpine ecosystems.

Graphical abstract