To address the low nitrogen use efficiency caused by long-term dependence on chemical nitrogen fertilizer in maize-soybean rotation systems in the black soil region of Northeast China, a two-year field experiment was conducted during 2023-2024 at the Xiangyang demonstration base of Northeast Agricultural University, Harbin, to clarify how partial substitution of nitrogen fertilizer with bio-organic fertilizer regulated soil nitrogen transformation and crop nitrogen utilization across the rotation. A randomized block design with three replicates was adopted, in which bio-organic fertilizer containing humus and Bacillus subtilis (OB), sterilized bio-organic fertilizer (O), or Bacillus subtilis inoculant alone (B) replaced 10%, 20%, or 30% of the synthetic nitrogen fertilizer, with an unfertilized treatment as the control. Soybean (Dongnong 252) and maize (Dongnong 2004) were grown in rotation under rain-fed conditions. At harvest, crop yield, nitrogen concentration and accumulation in individual organs were determined, and nitrogen use efficiency, agronomic efficiency, yield response index, and yield sustainability were calculated. Topsoil samples (0-20 cm) were collected at key growth stages of both crops: microbial biomass nitrogen was determined by the chloroform fumigation-extraction method, inorganic nitrogen fractions were measured with a continuous flow analyzer, net nitrogen mineralization and nitrification rates were estimated by laboratory incubation, and urease and protease activities were assayed colorimetrically. Soybean nodule number and dry weight were recorded, nitrogenase activity was measured by the acetylene reduction assay, and superoxide dismutase, peroxidase, and leghemoglobin contents were determined to evaluate symbiotic nitrogen fixation capacity. One-way analysis of variance followed by the least significant difference test, redundancy analysis, correlation and interaction analysis, and structural equation modeling were integrated to dissect the pathways linking soil nitrogen transformation to crop nitrogen use and yield formation.

The results showed that substituting 20% of the nitrogen fertilizer with bio-organic fertilizer (OB2) was the optimal treatment. Compared with the control, soybean yield increased by 25.97% and nitrogen use efficiency rose from 11.11% to 44.77%; in the following maize season, yield increased by 25.60% and nitrogen use efficiency rose from 32.37% to 64.37%. However, none of these indicators increased linearly with the substitution rate, indicating an optimal input threshold. The OB2 treatment significantly and persistently enhanced soil nitrogen transformation activity: the soil net mineralization rate increased by 12.16% and 8.49%, and the nitrification rate by 20.70% and 29.12%, at the maturity stages of soybean and maize, respectively, demonstrating a clear carry-over effect across seasons. At soybean maturity, soil inorganic nitrogen content increased by 19.12% and organic nitrogen by 24.69%. Meanwhile, OB2 markedly promoted soybean nodulation and nitrogen fixation, with nodule number, nodule dry weight, and nitrogenase activity increasing by 70.02%, 84.19%, and 26.25%, respectively. Redundancy analysis indicated that during the nutrient-demanding stages, soil nitrogen transformation was most significantly and positively associated with inorganic nitrogen content, whereas at maturity it was closely linked to the microbial biomass nitrogen pool (P ≤ 0.005). Structural equation modeling further revealed that nitrogen was efficiently transferred to grains mainly through a cascade in which elevated soil mineralization and nitrification rates raised ammonium and organic nitrogen availability, then crop total nitrogen accumulation, and finally grain yield, with key standardized path coefficients of 0.656, 0.996, 0.767, and 0.307. These findings demonstrated that bio-organic fertilizer, through the synergistic action of humus and functional Bacillus subtilis, fostered an efficient nodule system, enlarged the soil nitrogen pool, and regulated microbial transformation processes, thereby synchronously improving nitrogen use efficiency and yield in the maize-soybean rotation system. These results highlight the central role of soil nitrogen transformation in coordinating crop nitrogen supply within rotation systems, providing a theoretical basis and quantitative support for precise bio-organic fertilizer application in cold-region black soils.