Soilborne diseases are important problems in modern agricultural production. Fusarium oxysporum f. sp. cucumerinum (FOC) is one of the predominant soilborne pathogens threatening cucumber cultivation, especially in Hainan, China. This study assessed FOC-resistant rootstocks using incidence rate, disease severity index (DSI), and area under the disease severity index curve (AUDRC), revealing “JinJiaZhen (Mc-4)” as resistant and “JinGangZhuan 1901 (Mc-18)” as susceptible. Comprehensive transcriptome and metabolome analyses were conducted to investigate the defense mechanisms of these rootstocks, revealing key pathways, such as the mitogen-activated protein kinase (MAPK) signaling pathway, starch and sucrose metabolism, and phenylpropanoid biosynthesis, which are crucial for plant disease resistance. Additionally, the study compared the resistance mechanisms of two other rootstocks, Mc-4 and Mc-18, against FOC infection through transcriptomic and metabolomic analyses. Mc-4 exhibited a higher number of differentially expressed genes (DEGs) related to phenylpropanoid biosynthesis compared to Mc-18. Untargeted metabolomics identified 4093 metabolites, with phenylpropanoid biosynthesis, isoquinoline alkaloid biosynthesis, and porphyrin metabolism as primary annotated pathways. On the sixth day post-inoculation, when the number of DEGs and differentially accumulated metabolites (DAMs) was highest, phenylpropanoid biosynthesis emerged as a key pathway in Mc-4, with 37 DEGs and 8 DAMs identified. Notably, Mc-4 showed upregulated expression of genes encoding enzymes involved in phenylpropanoid biosynthesis and increased accumulation of related metabolites, such as coniferyl-aldehyde, coniferyl alcohol, and coniferyl acetate. These findings highlight the differential defense mechanisms between resistant and sensitive rootstocks and provide insights into plant–pathogen interactions. This study’s results will contribute to the development of better and disease-free cucumber varieties, promoting sustainable agriculture.

Cucumbers (Cucumis sativus L.) are highly sensitive to cold, but grafting onto cold-tolerant rootstocks can enhance their low-temperature resilience. This study investigates the physiological and molecular mechanisms by which exogenous vitamin C (Vc) mitigates cold stress in grafted cucumber seedlings. Using cucumber ‘Chiyu 505’ as the scion and pumpkin ‘Chuangfan No.1’ as the rootstock, seedlings were grafted using the whip grafting method. In the third true leaf expansion stage, seedlings were foliar sprayed with Vc at concentrations of 50, 100, 150, and 200 mg L⁻¹. Three days after initial spraying, seedlings were subjected to cold stress (8 °C) for 3 days, with continued spraying. After that, morphological and physiological parameters were assessed. Results showed that 150 mg L⁻¹ Vc treatment was most impactive, significantly reducing the cold damage index while increasing the root-to-shoot ratio, root vitality, chlorophyll content, and activities of antioxidant enzymes (SOD, POD, CAT). Moreover, this treatment enhanced levels of soluble sugars, soluble proteins, and proline compared to control. However, 200 mg L⁻¹ treatment elevated malondialdehyde (MDA) content, indicating potential oxidative stress. For transcriptomic analysis, leaves from the 150 mg L⁻¹ Vc and CK treatments were sampled at 0, 1, 2, and 3 days of cold stress. Differential gene expression revealed that genes associated with photosynthesis (LHCA1), stress signal transduction (MYC2-1, MYC2-2, WRKY22, WRKY2), and antioxidant defense (SOD-1, SOD-2) were initially up-regulated and subsequently down-regulated, as validated by qRT-PCR. Overall, we found that the application of 150 mg L⁻¹ Vc enhanced cold tolerance in grafted cucumber seedlings by modulating gene expression networks related to photosynthesis, stress response, and the antioxidant defense system. This study provides a way for developing Vc biostimulants to enhance cold tolerance in grafted cucumbers, improving sustainable cultivation in low-temperature regions.