Integrated microbiological and metabolomic analysis reveals the mechanisms by which biochar and wood vinegar enhance the cold tolerance of alfalfa

Freezing stress limits the sustainable production of alfalfa in northern China. While biochar and wood vinegar enhance crop stress tolerance, their potential to improve alfalfa cold resistance remains unknown. This field study compared the effects of a control group (CK), biochar (B, 10 t/ha), wood vinegar (W, diluted 200-fold), and a combined treatment (BW). Results indicate that B, W, and BW significantly enhanced cell membrane stability and alleviated oxidative stress, as evidenced by increases in root soluble protein (SP), soluble sugar (SS), and proline (Pro) content by 7.74 %-11.21 %, 12.81 %-51.54 %, and 3.50 %-43.00 %, respectively (p < 0.05). Superoxide dismutase (SOD) and catalase (CAT) activities increased by 20.13 %-46.18 % and 163.54 %-235.16 %, respectively (P < 0.05); Maldodialdehyde (MDA) content and relative electrical conductivity in BW were significantly reduced by 22.55 % and 16.56 %, respectively, compared to CK (p < 0.05). Biochar and wood vinegar increased the contents and availability of nutrients such as nitrogen, phosphorus, and potassium in the soil and improved the structure of the rhizosphere bacterial community (for example, bacteria of the phyla Bacteroidetes, Actinobacteria, and Acidobacteria were significantly enriched in B, W, and BW, and the community structure became more complex). They also activated metabolic pathways such as flavonoid and isoflavone biosynthesis in the roots, thereby enhancing the cold tolerance of alfalfa and increasing the yield of alfalfa after overwintering by 13.63 %–19.91 %. The findings of this study suggest that the concurrent utilisation of wood vinegar and biocharcan effectively augment the cold tolerance of alfalfa, thereby facilitating the sustainable production of this crop. • Biochar and wood vinegar can enhance the cold resistance of alfalfa. • Biochar and wood vinegar enhanced isoflavonoid and flavonoid biosynthesis in roots. • Biochar and wood vinegar significantly improved soil fertility. • Biochar and wood vinegar improved the structure and function of rhizosphere bacteria.