Compost Substrate Formulations Reshape Soil Physicochemical Properties and Rhizosphere Microbiota: A PLS-SEM Analysis

Abstract

To optimize and evaluate multi-source ultra-high-temperature compost formulations derived from livestock manure, municipal sludge, and spent mushroom substrate. The goal is to identify an optimal formulation that balances nutrient supply with environmental safety, and to elucidate how these formulations influence soil–root–microbe interactions regulating Medicago sativa (alfalfa) growth. Using Medicagosativa as a model plant, soil physicochemical properties and rhizosphere bacteria (16S rRNA) and fungi (ITS) were profiled before and after cultivation. Data were analyzed using partial least squares structural equation modeling (PLS-SEM) and parallel mediation models to quantify soil–root–microbe–plant linkages. The SMS-enriched formulation (FSM) reduced electrical conductivity (EC) by 64.00 µS·cm− 1 and maintained NH4+-N stability (p > 0.05). It also resulted in the largest increase in total nitrogen (TN, + 2.1 g·kg− 1) and significantly increased total phosphorus (TP, p < 0.001). Bacterial communities responded rapidly to shifts in salinity and nutrients, whereas fungal changes were delayed and more closely associated with soil physical structure. FSM increased Mortierella from 1% to 35%. PLS-SEM revealed a negative path from soil chemistry to root exudates (β = −1.035, p < 0.001), with positive paths from root to bacteria (f² = 0.56) and fungi (f² = 0.52), indicating a greater fungal impact on plant performance. Parallel mediation analysis confirmed the consistent directionality of these effects. FSM simultaneously improved soil physical and chemical conditions and strengthened positive, root-mediated fungal feedback to alfalfa, providing a controllable framework for optimizing multi-source compost substrates in legume-based systems.