Unravelling the metabolic reconfiguration of the post-challenge primed state in sorghum bicolor responding to colletotrichum sublineolum infection

Priming is a natural phenomenon that pre-conditions plants for enhanced defence against a wide range of pathogens. It represents a complementary strategy, or sustainable alternative that can provide protection against disease. However, a comprehensive functional and mechanistic understanding of the various layers of priming events is still limited. A non-targeted metabolomics approach was used to investigate metabolic changes in plant growth-promoting rhizobacteria (PGPR)-primed Sorghum bicolor seedlings infected with the anthracnose-causing fungal pathogen, Colletotrichum sublineolum, with a focus on the post-challenge primed state phase. At the 4-leaf growth stage, the plants were treated with a strain of Paenibacillus alvei at 10⁸ cfu mL^-1. Following a 24 h PGPR application, the plants were inoculated with a C. sublineolum spore suspension (10⁶ spores mL^-1), and the infection monitored over time: 1, 3, 5, 7 and 9 days post-inoculation. Non-infected plants served as negative controls. Intracellular metabolites from both inoculated and non-inoculated plants were extracted with 80% methanol-water. The extracts were chromatographically and spectrometrically analysed on an ultra-high performance liquid chromatography (UHPLC) system coupled to high-definition mass spectrometry. The acquired multidimensional data were processed to create data matrices for chemometric modelling. The computed models indicated time-related metabolic perturbations that reflect primed responses to the fungal infection. Evaluation of orthogonal projection to latent structure-discriminant analysis (OPLS-DA) loading shared and unique structures (SUS)-plots uncovered the differential stronger defence responses against the fungal infection observed in primed plants. These involved enhanced levels of amino acids (tyrosine, tryptophan), phytohormones (jasmonic acid and salicylic acid conjugates, and zeatin), and defence-related components of the lipidome. Furthermore, other defence responses in both naïve and primed plants were characterised by a complex mobilisation of phenolic compounds and de novo biosynthesis of the flavones, apigenin and luteolin and the 3-deoxyanthocyanidin phytoalexins, apigeninidin and luteolinidin, as well as some related conjugates.