Coupled shifts in phosphorus fractions and P-cycling genes driven by long-term cattle manure amendment across the 0–40 cm profile of a Mollisol

Sustainable intensification of agriculture is dependent on higher fertiliser inputs and balanced nutrient management to increase crop productivity while simultaneously maintaining soil health. Since the long-term use of higher doses of inorganic fertilisers can negatively affect soil ecosystem stability, manure inputs may be a viable alternative that also alters microbial phosphorus (P) metabolism. Using metagenomics and genome binning, we evaluated the shifts in microbial communities and P-cycling genes in a Mollisol after substitution of mineral fertilisers with manure additions. Soil samples were collected at different depths (0–10, 10–20, 20–30, and 30–40 cm) from a field that had undergone 30 years of mineral fertiliser application before transitioning to the following four treatments, each of which was applied for 12 years: no fertilisers (CK), continued mineral fertilisers (CF), CF with 15 Mg ha −1 of cattle manure per year (FM1), and CF with 30 Mg ha −1 of cattle manure per year (FM2). In comparison with CF, CK decreased the concentrations of most P fractions, whereas manure addition had positive effects on P fractions. Different fertilisers yielded distinct P-related microbial communities and networks across various soil depths. At the functional gene level, manure application increased the relative abundance of inorganic P solubilisation genes, particularly in the 0–10 cm and 30–40 cm layers, while reducing the abundance of P-starvation-response genes across all layers. Both mineral fertilisers and manure enhanced the abundance of organic P mineralisation genes in the 10–20 cm and 30–40 cm layers. The abundance of the gcd gene showed a positive relationship with inorganic P solubilisation in the surface layer, whereas those of the ppk1 , ppa , and ppx genes were positively correlated with inorganic P solubilisation in the subsoil layers. The construction of metagenome-assembled genomes (MAGs) showed that deeper soil layers offer greater potential for harbouring novel and undescribed microbial lineages and functional genes involved in the P cycle. Overall, supplementation of mineral fertilisers with manure additions significantly affected not only the P fractions but also the P metabolic capacities of the soil microbiome, influencing the soil P cycle in fertilised ecosystems.