Time-resolved multi-omics reveals stage-specific root defense associated with pepper resistance to southern blight

Following infection by Sclerotium rolfsii , pepper root defense responses are time-dependent, yet evidence for timing differences across defense layers between resistant and susceptible cultivars remains limited. Here, we integrated physiological and biochemical measurements, transcriptomics, and LC–MS-based untargeted metabolomics to compare root responses of the resistant cultivar Hongtai 666 and susceptible cultivar Layan 101 from 0 to 48 h after S. rolfsii inoculation, resolving the temporal basis of resistance. Hongtai 666 showed a smaller transcriptional response than Layan 101: at 48 hpi, the number of differentially expressed genes was 49% of that in Layan 101, and the set of genes responsive across multiple post-inoculation time points was also smaller. Hongtai 666 exhibited a higher basal relative salicylic acid abundance, whereas SA abundance decreased after inoculation in both cultivars, with a larger decline in Layan 101. The TCA/glyoxylate-related metabolite index showed distinct temporal changes in the two cultivars, with a higher value in Hongtai 666 at 24 hpi. At 24–48 hpi, Hongtai 666 also maintained a higher CHI–β-1,3-GA composite activity index, while the phenylpropanoid/flavonoid-associated metabolite profiles differed temporally between the two cultivars. By contrast, Layan 101 underwent broader transcriptional reprogramming and showed distinct late-stage metabolic changes, including stronger representation of glutathione- and cyanoamino-acid-associated pathways. Weighted gene co-expression network analysis identified candidate nodes within resistance-associated modules, providing targets for subsequent functional validation. These results indicate that pepper root resistance differs not only in response magnitude but also in the temporal organization of defense, providing a basis for evaluating candidate time-series resistance-associated features.