The legacy of long-term fertilization reshapes functional partitioning of the rhizosphere and hyphosphere through a plant-mediated cascade effect

The rhizosphere and hyphosphere are critical interfaces for plant-microbe interactions. However, the regulatory impact of long-term fertilization on the functional niche partitioning between these two compartments remains poorly understood. To address this, we conducted a pot experiment with wheat grown in preconditioned soils from a century-old fertilization trial. A 41-μm nylon mesh was used to physically separate the rhizosphere from the hyphosphere, enabling independent measurements of enzyme activities, microbial biomass, and available nutrient concentrations in each compartment. Our results showed that nitrogen (N) availability was the dominant factor among the fertilization regimes influencing plant performance, belowground C, nutrient dynamics, and prokaryote communities. Under N-limited conditions, plant–fungus cooperation was intensified, leading to a larger amount (24-41%) of dissolved organic C than in N-rich treatments. The dissolved organic C enrichment induced in the hyphosphere was 12-16% higher than that induced in the rhizosphere. This is evidenced by the strong positive correlation between arbuscular mycorrhizal fungal colonisation and hyphosphere dissolved organic C enrichment. In the fully mineral-fertilized NPK treatment, C-, N- and P-acquiring enzyme activities were 43-102% higher in the rhizosphere compared to the hyphosphere. Under combined manure and mineral fertilization, the highest overall levels of enzyme activities, nutrient availability, dissolved organic carbon, and microbial biomass carbon were observed in both compartments, but no differentiation between rhizosphere and hyphosphere was evident, reflecting the homogeneity of the microhabitats in these microbial functional traits. Linear discriminant analysis revealed that fertilization regimes significantly shaped microbial community composition, with combined manure and mineral fertilization consistently enriching Nitrososphaeria in both compartments. However, niche differentiation was evident between the two microhabitats: the rhizosphere uniquely recruited Planctomycetota under PK fertilization, whereas the hyphosphere was characterized by an enrichment of Chloroflexi under PK. This suggests that while fertilization drives broad taxonomic shifts, the rhizosphere imposes specific selective filters distinct from the hyphosphere. Together, these findings demonstrate that distinct fertilization regimes restructure the spatial partitioning of dissolved organic C dynamics and microbial functioning in the rhizosphere–hyphosphere by plant mediated cascading effect. Our results underscore the necessity of evaluating the rhizosphere and hyphosphere as distinct compartments to elucidate belowground C–N interactions under varying fertilization regimes. Accordingly, future research should examine these compartments separately to accurately capture fertilization-induced shifts in belowground C–N dynamics.