Figure 2
Pathogen defense-inducing activity of SAGs. (A) Superoxide radical production in leaves of plants after 0, 2, 4 and 6 hours post-treatment with SAGs (10 µg/ml) or water (control) measured by quantification of formazan produced after NBT histochemical staining. (B) Aniline blue staining to detect callose accumulation in leaves of SAGs- (10 µg/ml) or water-treated (control) plants later inoculated or not inoculated (Mock) with Pseudomonas viridiflava. Bright blue leaf areas (mm2) were measured from digital images of the stained leaves. Four leaves per plant and four plants per treatment were evaluated in three independent experiments for NBT and Aniline blue staining. (*) Denote values statistically different using the DGC test (p < 0.05). (C) Relative expression of PR1 and PDF1.2 genes calculated as 2∆CT in water-treated (control) and SAGs-treated (10 µg/ml) Arabidopsis plants at 1, 3, 6 and 24 hours post treatment (hpt). (*) Denote statistically significant differences in RNA expression between control and SAGs-treated plants using the ∆CT method. Three technical and three biological replicates were analyzed for each qPCR reaction. (D) Protection assay on Arabidopsis plants inoculated with P. viridiflava. Bacterial population in plants pretreated with SAGs (10 µg/ml), BION 500 or water (infection control) were expressed as the logarithm of colony forming units per leaf fresh weight (log CFU/g FW). (E) Protection assay on Arabidopsis plants inoculated with Botrytis cinerea. Disease severity was measured as average of leaf lesion diameters in plants pretreated with SAGs (10 µg/ml), BION 500, or water (infection control). Non-inoculated plants were denoted as Mock. (F) Protection assay on soybean plants inoculated with Corynespora cassiicola. The area under the disease progress curve (AUDPC) 4, 7 and 10 days post pathogen inoculation was determined in soybean plants pre-treated with SAGs (100 µg/ml), BION 500 or water (infection control). Eight and six plants per treatment were used in the protection assays on Arabidopsis and soybean, respectively. Both experiments were repeated four times. Different letters indicate significant difference among treatments using the DGC test (p < 0.05). Images of symptoms in leaves from plants corresponding to each treatment are shown under each graph bar.
