Table 1.
Gene’s candidates and their primers sequencing for transcript analysis after the elicitors treatment on potato plants.
Fig 1.
Désirée metabolic profiles of non-supervised PCA on (A, C) ESI- and (B, D) ESI+ ionization modes in two independent trials. Grouping of individual samples obtained with the various treatments were based on normalized abundances of the identified features from UPLC-qTOF-MSe. Each point represents one sample and each color is associated with one treatment: control (blue), CCF (green), BABA (red) and Ulva extract (purple). Colored ellipses represent the 95% confidence regions for each treatment.
Fig 2.
Representation of identified (“Level 2”) metabolites belonging to secondary metabolism pathways.
Normalized abundance heatmap of 178 putative secondary metabolites (“Level 2” identification) found in Désirée samples treated with BABA, CCF, Ulva extract or water and revealed under ESI- and ESI+ ionization modes: (A) 42 phenylpropanoids; (B) 34 flavonoids; (C) 46 alkaloids; and (D) 56 terpenoids. Each row represents one metabolite and the columns represent the average abundance per treatment. The hierarchical clustering tree brings together the metabolites according to the correlation-based distance calculated with Pearson correlation coefficient and Ward linkage. Clusters make it possible to visually identify metabolic profiles specific to each treatment by showing up-regulation (red), neutral effect (black) and down-regulation (green) by treatments.
Fig 3.
Effect of elicitors on selected metabolite regulations.
Semi-quantitative analysis of (A) phenylpropanoid, (B) flavonoid and (C) alkaloid/terpenoid metabolites based on its normalized abundances. Different letters indicate significant differences based on Tukey HSD test (p-value<0.05).
Fig 4.
Secondary metabolism pathways and main genes involved in the biosynthesis of the metabolites.
Schematic representation of (A) phenylpropanoid and flavonoid pathways; (B) terpenoid and alkaloid pathways. All pathways were based on KEGG maps. The target genes (orange bold) regulated by the elicitors were analyzed by RT-qPCR. Rutin, caffeoyl quinic acid and α-chaconine (black bold metabolites) were quantified by UPLC-qTOF-MSe. Solid arrows indicate a direct link between the metabolites and dotted arrows indicate non-direct link with the mentioned metabolite. The dataset was analyzed by linear mixed-effects model fit by REML and significant differences based on same test (p-value <0.05). The transcript expression relative to the water control for each gene is indicated as colored circles: red circles mean significant induction, green circles significant repression and black circles non-significant differences.
Fig 5.
Quantification of phytohormones from secondary metabolism and PR gene expression (A) SA (p-value 0.3870), (B) OPDA (0.1371), (C) DHJA (0.9737), (D) JA (<0.0001), (E) JA-ILE (<0.0001) and (F) ACC (0.0032). The target metabolites regulated by the elicitors were analyzed by UHPLC-MS/MS. The concentration in ng/mg of lyophilized material are illustrated by histograms. The target genes related with the phytohormones correspond to either the SA (G) or JA (H) pathways. The transcript expression relative to the water control for each gene is indicated as colored circles: red circles mean a significant induction, green circles significant repression and black circles non-significant differences. The dataset was analyzed by linear mixed-effects model fit by REML. Different letters indicate significant differences based on same test (p-value <0.05).
Fig 6.
Quantification of secondary antimicrobial metabolites and test in vitro against P. infestans.
(A) caffeoyl quinic acid, (B) rutin and (C) α-chaconine, regulated by the elicitors were analyzed by UPLC-qTOF-MSe in ESI- ionization mode. The concentrations in ng/mg are illustrated by histograms. The dataset was analyzed by linear mixed-effects model fit by REML. Different letters indicate significant differences based on same test (p-value <0.05). (D) Mycelial growth of P. infestans was measured for 8 days after deposition of pathogen in Petri dish on pea agar with respectively, α-chaconine (26 μg/ml), rutin (4 μg/ml) or caffeoyl quinic acid (24 μg/ml) or water. ImageJ software was used to this analysis. Different letters indicate significant differences based Tuckey test (p-value <0.05). (E) The sporangia production were counted twice by each Petri dish with a Malassez cell.