The efficiency of carbon and nitrogen uptake in apple trees is co-regulated by plant genotype and rhizosphere microbial communities. However, the mechanisms by which different scion varieties modulate microbial structure and function under varying nitrogen levels remain poorly understood. In this study, Malus sieversii was used as the rootstock, onto which three scion cultivars (M. sieversii, Malus domestica cv. Hanfu, and Malus domestica cv. Red Fuji) were grafted under two nitrogen regimes. A combination of 13C/15N isotope labeling, Illumina MiSeq amplicon sequencing, and metagenomic analysis was employed to elucidate how scion–rootstock interactions and nitrogen availability affect carbon and nitrogen acquisition. Under nitrogen-deficient conditions, Red Fuji exhibited stronger root activity and larger root surface area, indicating enhanced nutrient foraging capacity. Conversely, under nitrogen application, Hanfu showed significantly greater 13C and 15N uptake, with 5.7-fold and 1.6-fold higher 13C accumulation in roots and stems, respectively, and markedly higher 15N utilization efficiency in roots and leaves compared with M. sieversii. In parallel, Hanfu under nitrogen input showed enrichment of beneficial microbial taxa and more complex microbial co-occurrence networks. Metagenomic analysis and random forest analyses revealed that the relative abundance of specific functional genes related to carbon and nitrogen transformation (rbcL, abfA, napB/C, nasA) was significantly higher under specific scion–nitrogen combinations, contributing to enhanced microbial carbon fixation and nitrogen reduction. Collectively, these results demonstrate that scion genotype modulates rhizosphere microbial structure, physiological root traits, and carbon–nitrogen distribution patterns, thereby improving nutrient uptake efficiency under different nitrogen inputs.

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