Nitrogen Fertilizer Reduction in Rice–Eel Co-Culture System Improves the Soil Microbial Diversity and Its Functional Stability

The ecological rice–eel co-culture system is not only beneficial for enhancing productivity and sustainability in agriculture but also plays a crucial role in promoting environmental health. In the present study, based on the long-term positioning trial of the rice–eel co-culture system that began in 2016 and was sampled in 2023, the effects of reduced nitrogen fertilizer application on soil physico-chemical properties and the bacterial community were investigated. Treatments included a conventional regular fertilization treatment (RT), rice–eel co-culture system regular fertilization (IT), and nitrogen-reduction 10%, 30%, and 50% fertilization treatments (IT90, IT70, and IT50). Our research demonstrated the following: (1) Compared to RT, IT significantly increased soil water-stable macroaggregates (R0.25), mean weight diameter (MWD), geometric mean diameter (GMD), and available phosphorus content, with the increases of 15.66%, 25.49%, 36.00%, and 18.42%, respectively. Among the nitrogen-reduction fertilization treatments, IT90 showed the most significant effect. Compared to IT, IT90 significantly increased R0.25, MWD, GMD, and available nitrogen content, with increases of 4.4%, 7.81%, 8.82%, and 28.89%, respectively. (2) Compared to RT, at the phylum level, the diversity of Chloroflexi was significantly increased under IT and IT50, and the diversity of Gemmatimonadota was significantly increased under IT90, IT70, and IT50. The diversity of Acidobacteriota was significantly higher in IT90 and IT70 compared to IT. It was shown that the rice–eel co-culture system and nitrogen fertilizer reduction could effectively improve the degradation capacity of organic matter and promote soil nitrogen cycling. In addition, redundancy analysis (RDA) identified total phosphorus, total nitrogen, and available nitrogen (<i>p</i> = 0.007) as the three most important environmental factors driving changes in the bacterial community. (3) The functional prediction analysis of soil microbiota showed that, compared to RT, the diversity of pathways related to biosynthesis (carbohydrate biosynthesis and cell structure biosynthesis) and metabolism (L-glutamate and L-glutamine biosynthesis) was significantly higher under IT70, IT90, IT, and IT50 (in descending order). However, the diversity of pathways associated with degradation/utilization/assimilation (secondary metabolite degradation and amine and polyamine degradation) was significantly lower under all the rice–eel co-culture treatments. In conclusion, the rice–eel co-culture system improved soil physicochemical properties and the soil microbial environment compared with conventional planting, and the best soil improvement was achieved with 10% less N fertilizer application.