Untargeted profiling of secondary metabolites and phytotoxins associated with stemphylium blight of lentil.

Stemphylium botryosum alters lentil secondary metabolism and differentially affects resistant and susceptible genotypes. Untargeted metabolomics identifies metabolites and their potential biosynthetic pathways that play a crucial role in resistance to S. botryosum. The molecular and metabolic processes that mediate resistance to stemphylium blight caused by Stemphylium botryosum Wallr. in lentil are largely unknown. Identifying metabolites and pathways associated with Stemphylium infection may provide valuable insights and novel targets to breed for enhanced resistance. The metabolic changes following infection of four lentil genotypes by S. botryosum were investigated by comprehensive untargeted metabolic profiling employing reversed-phase or hydrophilic interaction liquid chromatography (HILIC) coupled to a Q-Exactive mass spectrometer. At the pre-flowering stage, plants were inoculated with S. botryosum isolate SB19 spore suspension and leaf samples were collected at 24, 96 and 144 h post-inoculation (hpi). Mock-inoculated plants were used as negative controls. After analyte separation, high-resolution mass spectrometry data was acquired in positive and negative ionization modes. Multivariate modeling revealed significant treatment, genotype and hpi effects on metabolic profile changes that reflect lentil response to Stemphylium infection. In addition, univariate analyses highlighted numerous differentially accumulated metabolites. By contrasting the metabolic profiles of SB19-inoculated and mock-inoculated plants and among lentil genotypes, 840 pathogenesis-related metabolites were detected including seven S. botryosum phytotoxins. These metabolites included amino acids, sugars, fatty acids and flavonoids in primary and secondary metabolism. Metabolic pathway analysis revealed 11 significant pathways including flavonoid and phenylpropanoid biosynthesis, which were affected upon S. botryosum infection. This research contributes to ongoing efforts toward a comprehensive understanding of the regulation and reprogramming of lentil metabolism under biotic stress, which will provide targets for potential applications in breeding for enhanced disease resistance.