Long-term green manuring increases phosphorus availability in soil by modifying microbial functional genes and enzyme activities

Substitution of mineral fertilizers with milk vetch (Astragalus sinicus L.) organic fertilization strongly influences the dynamics and availability of phosphorus (P) in soil. How milk vetch organic fertilization affects the specific roles of P cycling functional genes in soil and, in turn, the mechanisms of altered P turnover remain unclear. Soil samples were taken from a 10-year field experiment that included mineral fertilizer and partial substitution of mineral fertilizer with milk vetch, together with an unfertilized control. The P-cycling functional genes in soil determined by metagenomics were related to phosphatase enzyme activities and P fractions with various availability. Milk vetch substitution decreased soil total P and NaHCO3-OP (8 and 65 %) but maintain similar available P and high phosphatase activities compared with mineral fertilizer, indicating a net solubilization/mineralization of soil P occurring in response to milk vetch substitution. The Proteobacteria, followed by Chloroflexi, Acidobacteria, Actinobacteria, Nitrospirae, Verrucomicrobia, and Gemmatimonadetes were the most abundant bacteria phyla involved in soil P-cycling. Shifts in P-cycling functional gene community were more related to soil total N and the ratio of SOC to total P. The relative abundance of P-cycling functional genes, increased the production of PQQ-GDH (gcd, pqqE), fumaric acid (fumC), acetic acid (aldh), acid phosphatase (phoN), alkaline phosphatase (phoD), the catabolism of organophosphonates (phnJ, phnN, and phnW) and the dissimilation of glycerol-3-phosphate (glpB, glpF, and glpR) by 4 %–80 %. Phosphatase activities increased with milk vetch substitution, because of raised abundance of related genes and gene-harboring communities (especially Chloroflexi). The phoN gene was the key predictor of organic P mineralization and thus available P. Overall, milk vetch substitution increased the P-acquiring enzyme activities and boosted the involvement of the functional genes in P cycling, which in turn stimulated P bioavailability. These results highlight the connections between milk vetch substitution and P cycling functional genes, which also have implications for offsetting the future demand for P mineral fertilizer by milk vetch substitution in agricultural systems. © 2025 Elsevier B.V., All rights reserved.