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

Compost substrate formulations are critical for improving the performance of organic fertilizers, yet the pathways through which plant roots modulate their effects remain unclear. In this study, Medicago sativa was used as a model legume to evaluate three treatments: CK (control), FSM (2.5% (w/w), dry weight; F:S:M = 1:1:3), and FS (5% (w/w), dry weight; F:S = 1:1), where F represents livestock manure, S denotes municipal sludge, and M refers to spent mushroom substrate (SMS). Soil physicochemical properties and rhizosphere microbiota (16S rRNA and ITS regions) were analyzed before and after planting. The data were integrated using partial least squares structural equation modeling (PLS-SEM) and parallel mediation models. Results showed that FSM treatment reduced EC (−64.00 μS·cm⁻¹), stabilized NH₄⁺-N (p > 0.05), and produced the most significant increases in TN (+2.1 g·kg⁻¹) and TP (p < 0.001). Bacterial communities responded rapidly to changes in salinity and nutrient status, whereas fungal communities showed delayed responses that were more closely associated with shifts in soil physical structure. Under FSM, the relative abundance of Mortierella increased from 1% to 35%, indicating strong enrichment of saprotrophic fungi. PLS-SEM revealed a strong negative path from soil chemistry to root exudates (β = −1.035, p < 0.001), and indicated that fungi contributed more to plant performance than bacteria. Collectively, the FSM formulation simultaneously improved soil physical and chemical conditions and strengthened positive, root-mediated fungal feedback to Medicago sativa. The study provided an integrated framework linking compost substrate formulation, soil physicochemistry, rhizosphere microbiota and plant growth. It offered guidance for the design and optimization of functional substrates in legume-based systems.