The transition from monoculture to mixed-species agroforestry systems affects soil organic matter and microbial activity. However, the specific dynamics of these changes, particularly within medicinal plant-based agroforestry, remain underexplored. This study investigates the impact of monoculture Pine (Pinus massoniana) forests and four agroforestry models: (M1) Pinus massoniana and Alpina oxyphylla, (M2) Pinus massoniana and Ficus simplicissima, (M3) Pinus massoniana and Amomum villosum, and (M4) Pinus massoniana and Curcuma longa on soil properties and microbial activity in rhizosphere and non-rhizosphere environments. Our results showed significantly higher pH (4.80) and total nitrogen (N) content (1.77 g kg−1) in the rhizosphere of model (M4) compared to (CK). Total organic carbon (TOC) and carbon fractions (POC, DOC, and MBC) also differed significantly across monoculture and agroforestry models, with highest TOC concentrations (31.70 g kg⁻1) in rhizosphere of CK. Exchangeable cations, including Ca2⁺, and Mg2⁺ were significantly higher in the rhizosphere of agroforestry models compared to CK, particularly in M4, where Ca2⁺ was recorded at 12.03 cmol kg−1 with the highest percent base saturation (PBS) at 90.17%. Enzymes leucine aminopeptidase and polyphenol oxidase varied significantly, with higher activity in the rhizosphere of agroforestry models and greater activity in non-rhizosphere of monoculture. Soil microbial respiration (MRes) revealed substantial differences, with an average 17% decrease in rhizosphere soil for models M2 and M4 and a 20.83% reduction in non-rhizosphere soil for model M1 compared to CK. Generalized Linear Model (GLM) demonstrated a significant positive correlation between TOC and MRes (R2 = 0.885, p < 0.01), indicating that higher TOC levels are linked with increased MRes. In conclusion, model M4 most effectively enhanced soil fertility and nutrient availability followed by the other agroforestry models tested. This suggests that integrating medicinal plants into agroforestry systems is a viable strategy for improving ecosystem functioning compared to monoculture practices.
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