Objectives This study aims to explore the mechanisms by which the addition of sheep manure and biochar drives enzyme activities and their spatial distribution characteristics in the maize rhizosphere, providing a theoretical basis for the efficient application of organic fertilizers.

Methods A rhizobox-based maize cultivation experiment was conducted with four treatments: no fertilization (CK), sole application of sheep manure (SM), sole application of biochar (BC), and combined application of sheep manure and biochar (SM+BC). The maize was harvested after six weeks of growth. Using in situ soil zymography and high-throughput sequencing techniques, we systematically analyzed the physicochemical properties, microbial community composition, and spatial patterns of enzyme activities in the rhizosphere soil of maize.

Results 1) Compared with CK, SM and SM+BC significantly increased soil pH, total nitrogen (TN), alkali-hydrolyzable nitrogen (AN), available phosphorus (AP), and total phosphorus (TP), promoting maize aboveground and root growth; while BC had no significant effect on plant growth. 2) SM and SM+BC significantly increased the activities of alkaline phosphatase (ALP), β-1,4-N-acetylglucosaminidase (NAG), and β-glucosidase (BG) in the rhizosphere soil, with increases ranging from 200.8%−589.0% for ALP and 146.7%−400.8% for BG and NAG, respectively. Both treatments expanded the enzyme activity hotspot area and extended the rhizosphere extent. BC had no significant effect on hydrolytic enzyme activity. 3) Sheep manure enriched copiotrophic bacteria such as Proteobacteria, Bacteroidota, Actinobacteriota, and Firmicutes, as well as fungal groups such as Ascomycota, while inhibiting oligotrophic taxa such as Acidobacteriota. Microbial community composition was significantly associated with soil nutrients and enzyme activity. The structural equation model indicated that exogenous organic matter primarily improved the soil chemical environment, regulated plant traits and bacterial community structure, thereby synergistically enhancing rhizosphere enzymatic activity and expanding functional zones.

Conclusions Sheep manure significantly increased rhizosphere soil pH and nutrient levels (e.g., nitrogen and phosphorus), enriched copiotrophic bacterial taxa including Proteobacteria, Bacteroidota, Actinobacteriota, and Firmicutes, as well as fungal groups such as Ascomycota, while suppressing oligotrophic taxa such as Acidobacteriota. It enhanced the activities of C, N, and P cycling-related enzymes (BG, NAG, and ALP), expanded enzyme activity hotspot areas, and extended the rhizosphere extent, thereby substantially promoting maize aboveground and root growth. In contrast, biochar was less effective than sheep manure in directly improving nutrient availability, exerted a lower impact on rhizosphere microorganisms and N and P metabolism-related enzyme activities, and did not significantly promote maize growth. The long-term effects of sheep manure and biochar on rhizosphere physicochemical and biological properties still require long-term experimental validation.