SAR assay in tomato

Two bottom leaflets (closest to main stem) of third or fourth compound leaves of 4- to 5-week-old S. lycopersicum VF36 plants were infiltrated with 10 mM MgCl₂ (mock) or 10 mM MgCl₂ containing 1 mM NHP. After chemical incubation for 24 hours, three top leaflets (distal to main stem) on the same branch were inoculated with a suspension of Pst (1 × 10⁵ CFU/ml). Plants were incubated in greenhouse (16-hour light/8-hour dark, 25° to 28°C). The titer of Pst in the bottom leaflets was quantified at 4 dpi by homogenizing leaves discs in 1 ml of 10 mM MgCl₂, plating appropriate dilutions on NYGA medium with rifampicin (100 μg/ml), incubating plates at 28°C for 2 days, and then counting bacterial colonies. Symptoms of infected leaves were photographed at 4 dpi. Three biological repeats were performed per treatment in two independent experiments.