Legacy Effects of 32 Years of Tillage and Crop Diversification on Soil Biological Activity in Paraguay

Soil biological activity integrates microbial processes involved in organic matter decomposition and nutrient cycling, yet its long-term response under agricultural systems in Paraguay remains poorly documented. This study evaluated soil biological activity in a 32-year field experiment in the Eastern Region of Paraguay, comparing cropping systems differing in tillage intensity and crop rotation diversification. Soil samples from the 0–20 cm layer were analyzed for microbial biomass carbon (MBC), β-glucosidase (BG), urease (URE), acid phosphatase (AP), arylsulfatase (ARS), soil organic carbon (SOC), total nitrogen (TN), available phosphorus (P), sulfur (S), and pH. Our results revealed that BG, URE, and AP increased under no-tillage, particularly in the most diversified no-tillage rotation, with 71%, 90%, and 51% higher activities, respectively, than conventional tillage. MBC and ARS were not significantly affected by cropping systems. Principal component analysis, Spearman correlations, and Mantel analysis indicated that enzymatic responses were associated with SOC, TN, P, S, and pH, linking soil biological activity with chemical attributes related to nutrient cycling. These findings show that diversified no-tillage strengthens soil biological functioning under representative Paraguayan grain-production conditions, providing long-term local evidence to guide soil-health management, crop diversification strategies, and more sustainable agricultural systems in the region.