Co-Culture Duration Reshapes the Rhizosphere Microbial Functional Potential for Nitrous Oxide Production and Consumption in a Traditional Rice–Fish System

Rice–fish co-culture is widely promoted for mitigating nitrous oxide (N2O) emissions from paddy soils, yet how the duration of co-culture reshapes the underlying nitrogen-cycling microbial community under low-nitrogen input remains poorly understood. This study aimed to (i) characterize how co-culture duration alters the rhizosphere microbial functional potential for N2O production and consumption, and (ii) identify the water and soil variables linking fish activity to that response. The experiment was conducted during the 2024 rice growing season in the Qingtian rice–fish system (Zhejiang Province, China), a traditional agricultural heritage system managed without chemical fertilizer or supplementary feed. Three treatments (i.e., rice monoculture, first-year co-culture, and long-established (~10-year) co-culture) were compared using six independently bunded replicate plots each. Rhizosphere soils were collected at the tillering, heading and maturity stages for shotgun metagenomic profiling of nitrogen-cycling functional genes, with concurrent measurement of N2O flux and water and soil physicochemical properties. Fluxes were uniformly low and did not differ among treatments (p > 0.05), defining a substrate-limited baseline. Against this baseline, first-year co-culture induced a coordinated shift toward complete denitrification (nosZ increased by 25–33% across all stages; nosZ/(nirK + nirS) rose to 0.99 at heading), associated with a transient water organic carbon pulse and dissolved-oxygen availability. The long-established system resembled monoculture, indicating a non-monotonic, duration-dependent response.