This study highlights the potential of grass–legume intercropping systems in enhancing soil carbon sequestration and cycling nutrientes, providing ecological support for sustainable agriculture. This study evaluated the chemical composition and root biomass of signal grass (Urochloa decumbens cv. Basilisk) pasture grown in monoculture or intercropped with forage peanut (Arachis pintoi cv. Belmonte) under different row spacings (40, 50, 60, 70, and 80 cm), 4–5 years after establishment. The experiment was conducted in a randomized block design with four replicates under intermittent sheep grazing. Results (p < 0.05) indicated that root biomass was higher in the intercropped pastures (6752 to 11,167 kg OM ha−1) than in the monoculture (3696 kg OM ha−1). Carbon (C) and N stored in root biomass were also influenced by the row spacings, with narrower spacings, particularly 40 cm (1282 kg C ha−1) and 50 cm (1424 kg C ha−1), showing higher C storage than in the monoculture (509 kg C ha−1). Similarly, the 40 cm (122 kg N ha−1) and 50 cm (138 kg N ha−1) spacings showed higher N accumulation in root biomass than the monoculture (38.9 kg N ha−1). These spacing also reduced C/N and lignin/ADIN ratios, which enhanced nitrogen cycling efficiency. Based on these results, 40 and 50 cm spacings are recommended for intercropped pastures, as they optimize C and N accumulation and nutrient cycling, and support sustainable pasture management.
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