Objective Long-term fertilization regimes and apple growth stages exert important regulatory effects on soil fungal communities in orchard soils. To elucidate the mechanisms by which different long-term fertilization regimes and growth stages influence the structure and activity of soil fungal communities, this study systematically analyzed the response characteristics of total and active fungal communities in orchard soils under different long-term fertilization regimes across key apple growth stages.

Methods This study was based on a 28-year long-term positioning experiment conducted at the Baishui Apple Experimental Station of Northwest A&F University. Four fertilization treatments were selected: no fertilizer (CK), organic manure alone (M), chemical fertilizer alone (NPK), and combined organic and chemical fertilizer (MNPK). Surface soil samples were collected at the fruit setting, fruit swelling, and fruit maturity stages. High-throughput sequencing of 18S rDNA and 18S rRNA was employed to simultaneously characterize the composition and diversity of total and active fungal communities. Combined with soil nutrient measurements, the regulatory mechanisms of different fertilization regimes and growth stages on soil fungal communities were explored through diversity analysis, community structure comparison, and co-occurrence network construction.

Results 1) The α-diversity of total fungal communities was significantly reduced under NPK and MNPK treatments compared to CK, with the Shannon index decreasing by 20.1% under MNPK at the fruit setting stage, which was significantly negatively correlated with soil available phosphorus content (r=−0.415). 2) The α-diversity of active fungal communities was most sensitive to fertilization at the fruit swelling stage, with Shannon and Chao1 indices under NPK and MNPK treatments being 69.1%, 58.2% and 53.9%, 75.9% higher than those under M, respectively, while the relative abundance of Basidiomycota reached 62.7% under M treatment, representing a significant enrichment. 3) Fertilization significantly altered both total and active fungal community structures. PERMANOVA analysis revealed that the explanatory power of fertilization treatment for total fungal community structure increased with growth stage progression, from 44.08% at the fruit setting stage to 56.47% at the fruit maturity stage. 4) At the fruit maturity stage, MNPK significantly increased the relative abundance of arbuscular mycorrhizal fungi (Glomeromycetes) in the active fungal community (13.1%), which was positively correlated with soil nitrate nitrogen content. 5) The relative abundances of multiple dominant classes in both total and active fungal communities were significantly correlated with soil carbon (SOC, DOC, MBC), nitrogen (TN, NO₃⁻-N), and phosphorus (AP, TP) fractions, with Agaricomycetes showing positive correlations with SOC and NO₃⁻-N, Sordariomycetes showing negative correlations with SOC and MBC, and Glomeromycetes showing positive correlations with NO₃⁻-N, indicating that fertilization-driven fungal community succession is closely associated with soil nutrient transformation. 6) The active fungal co-occurrence network under MNPK exhibited relatively high modularity (0.819) and average clustering coefficient (0.998), suggesting the formation of a more stable and functionally organized interaction system.

Conclusion Long-term balanced application of NPK chemical fertilizer, alone or in combination with organic manure, significantly increased soil available phosphorus and nitrate nitrogen contents, which in turn reduced the α-diversity of total fungal communities, but significantly increased the α-diversity of active fungal communities at the fruit swelling stage, while also significantly altering the β-diversity of both total and active fungal communities. NPK application increased the relative abundance of Ascomycota (Sordariomycetes) in the total fungal community, while organic manure or combined organic-inorganic fertilization increased the proportion of Basidiomycota. Notably, combined organic-inorganic fertilization substantially increased the abundance of arbuscular mycorrhizal fungi (Glomeromycetes) in active fungal communities at the fruit maturity stage. In summary, combined organic-inorganic fertilization shaped the active fungal co-occurrence network with the highest modularity and clustering coefficients, indicating the most stable structure, whereas chemical fertilization alone resulted in relatively lower stability of the total fungal network. Therefore, the adoption of combined organic-inorganic fertilization in orchards contributes to the establishment of a stable and functionally differentiated fungal interaction network, which activates beneficial functional microbial groups during critical stages such as fruit swelling, thereby enhancing soil ecological functions.