2.4.2. Double-Antibody Sandwich Enzyme-Linked Immunosorbent Assay (DAS-ELISA)

The direct double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA Complete kits, Bioreba, Switzerland) was used to determine the PVX, PVY, PVM, PVS and potato leaf roll virus (PLRV) content, as described previously [24]. ELISA was performed on both challenged inoculated and non-inoculated leaves (to check systemic virus movement) of five tomato plants per replicate for each treatment at 14 dpi for the growth chamber experiments and 10 weeks after viral infection for the greenhouse experiments. In the potato plants on which the PVX-19AA and PVY-19KV isolates were maintained, ELISA was performed directly on the leaves from which the inoculum was obtained (Figure S1, see Supplementary Materials). Samples were considered positive for the presence of viruses if the absorbance value A₄₀₅ exceeded three times a threshold value equal to the mean of the absorbance value of the healthy control samples. Three independent biological replicates were performed for each experiment.