ACE protein as a candidate proxy for microbial functional processes in contrasting Australian cropping soils

Abstract Soil microbial processes underpin carbon and nutrient cycling, soil resilience and plant health outcomes, making them pertinent to soil health assessments. Despite this, there remains little consensus on which biological indicators to measure, how to interpret them and how to integrate results. Here, we evaluated 82 microbially relevant indicators measured at pre-sowing, in-crop and post-harvest across four field experiments in Australian broadacre cropping systems spanning contrasting soils, climates and management regimes. Multivariate profiles of six enzymes involved in carbon (C), nitrogen (N), phosphorus (P) and sulfur (S) cycling varied significantly among sampling times and sites (R² = 0.72, P < 0.001). Soil pH was the best single BIOENV model predictor (Spearman ρ = 0.598), consistent with its role as a master regulator of microbial communities and extracellular enzyme systems, while total C (TC) and soil autoclavable citrate-extractable (ACE) protein constituted the most parsimonious model (ρ = 0.705). Co-occurrence network inference identified soil ACE protein as the central node of consistently high importance, linking soil physicochemical properties and enzyme activities involved in nutrient cycling. Soil ACE protein showed strong and consistent correlations with chitinase and phosphatase activities across all sites and sampling times (r = 0.62–0.76, P < 0.001). While ACE protein has previously been proposed as an indicator of labile organic N and general soil health, our results show that it also tracks variation in enzyme-mediated nutrient cycling across contrasting soils and sampling times. These findings position soil ACE protein as a candidate low-cost, robust proxy for microbial functional processes.