To investigate the effects and mechanism of prolonged inorganic nitrogen (N) fertilization on the N-use efficiency of spring wheat (Triticum aestivum L.), a long-term study initiated in 2003 was conducted. The study analyzed how N fertilization affects dry matter translocation, N translocation, soil NO3-N, and N-use efficiency. Five different N-fertilizer rate treatments were tested: N0, N52.5, N105, N157.5, and N210, corresponding to annual N fertilizer doses of 0, 52.5, 105.0, 157.5, and 210.0 kg N ha−1, respectively. Results showed that increasing N-fertilizer rates significantly enhanced the two-year average dry matter accumulation amount (DMA) at maturity by 22.97–56.25% and pre-flowering crop growth rate (CGR) by 17.11–92.85%, with no significant increase beyond 105 kg N ha−1. However, no significant correlation was observed between the dry matter translocation efficiency (DTE) and wheat grain yield. Both insufficient and excessive N applications resulted in an imbalanced N distribution favoring vegetative growth over reproductive growth, thus negatively impacting N-use efficiency. At maturity, the N-fertilized treatments significantly increased the two-year average N accumulation amount (NAA) by 52.04–129.98%, with no further increase beyond 105 kg N ha−1. N fertilization also improved the two-year average N translocation efficiency (NTE) by 56.89–63.80% and the N contribution proportion (NCP) of wheat vegetative organs by 27.79–57.83%, peaking in the lower-N treatment (N52.5). However, high-N treatment (N210) led to an increase in NO3-N accumulation in the 0–100 cm soil layer, with an increase of 26.27% in 2018 and 122.44% in 2019. This higher soil NO3-N accumulation in the 0–100 cm layer decreased NHI, NUE, NAE, NPFP, and NMB. Additionally, N fertilization significantly reduced the two-year average N harvest index (NHI) by 9.89–12.85% and N utilization efficiency (NUE) by 11.14–20.79%, both decreasing with higher N application rates. The NAA followed the trend of anthesis > maturity > jointing. At the 105 kg N ha−1 rate, the highest N agronomic efficiency (NAE) (9.31 kg kg−1), N recovery efficiency (NRE) (38.32%), and N marginal benefit (NMB) (10.67 kg kg−1) were observed. Higher dry matter translocation amount (DTA) and N translocation amount (NTA) reduced NHI and NUE, whereas higher NTE improved NHI, NUE, and N partial factor productivity (NPFP). Overall, N fertilization enhanced N-use efficiency in spring wheat by improving N translocation rather than dry matter translocation.
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