Nitrogen fertilizer form and application rate jointly govern the ecological stoichiometric characteristics of plants, soils, and microorganisms in perennial cultivated grasslands.

To elucidate the regulatory mechanisms by which nitrogen fertilizer form and application rate affect the carbon, nitrogen, and phosphorus (C:N:P) stoichiometric characteristics of the plant-soil-microbe system in perennial cultivated grasslands, this study used a mixed sward of Festuca sinensis Keng ex E.B.Alexeev cv. Qinghai + Poa pratensis L. cv. Qinghai in Qinghai as the experimental system. A no-fertilization treatment served as the control, while three N forms, namely amide-N, ammonium-N, and nitrate-N, were applied at three N addition rates (22.5, 45.0, and 67.5 kg ha-1·yr-1) using a randomized block design. Combined with analysis of variance, a homeostasis model, redundancy analysis (RDA), and structural equation modeling (SEM), the study explored the stoichiometric characteristics of the system, homeostatic responses, and the interaction pathways among its components. The results showed that N form, N application rate, and their interaction all had significant effects on the C:N:P stoichiometric characteristics of the system, and that the regulatory effects of N form were distinct. amide-N was most effective in increasing N content in plants and soil, whereas ammonium-N was more conducive to soil phosphorus accumulation and to increases in microbial biomass carbon and nitrogen. Significant differences in stoichiometric homeostasis were observed among different plant organs: stems were the most sensitive to changes in the soil environment, while some indicators exhibited absolute homeostasis. Significant coordinated variation in stoichiometric characteristics was observed among plants, soil, and microorganisms. Nitrogen form directly and negatively regulated soil stoichiometric ratios, whereas N application rate indirectly regulated system stoichiometric characteristics through soil nutrients. This study demonstrated that N fertilizer form and N application rate jointly regulate the stoichiometric characteristics of perennial sown grassland ecosystems, and that the application of amide-N at 45 kg ha-1·yr-1 is more conducive to optimizing nutrient balance within the ecosystem. These findings provide important theoretical support for the formulation of precision fertilization strategies and the ecologically sustainable management of alpine perennial cultivated grasslands on the Qinghai-Tibet Plateau.