Distinct metabolic architectures differentiate HLB-tolerant lemon and susceptible sweet orange.

Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CLas), is a devastating citrus disease that severely threatens global citrus production, yet the metabolic mechanisms underlying citrus tolerance to HLB remain elusive. Citrus genotypes exhibit differential tolerance to HLB, with lemon (Citrus limon) showing greater resilience compared to susceptible sweet orange (Citrus sinensis). In this study, we integrated untargeted liquid chromatography-high resolution mass spectrometry metabolomics with a bootstrap-enhanced LASSO approach to characterize the metabolic responses of HLB-tolerant C. limon and susceptible C. sinensis to CLas infection. A total of 1,294 metabolites were identified. Bootstrap-enhanced LASSO identified stable discriminant metabolites (DMs) across four comparative frameworks, with phenylpropanoids (flavonols, furanocoumarins, and hydroxycinnamic acids) and limonoids being the most prominent DM classes. Lemon exhibited constitutively higher levels of kaempferol/quercetin-derived flavonols, linear furanocoumarins, monolignol glycosides, and limonoids compared to orange. While CLas infection induced quantitative changes in specific metabolic branches, the overall metabolic organization remained primarily determined by genotype. In the two cultivars examined, these results suggest that pre-existing metabolic configurations distinguish the tolerant lemon from the susceptible sweet orange and are associated with their contrasting HLB responses. This study provides robust metabolic signatures and candidate pathways for improving HLB tolerance through citrus breeding programs.