Bacterial Volatiles Induce ISR in Arabidopsis

To determine if exposure of Arabidopsis seedlings to bacterial volatiles elicited ISR against Pseudomonas syringae pv. maculicola ES4326, we developed a 24-well microtitre-based disease assay system (Fig. 1A). The 24-multimicrotitre plate of bacterial suspensions of PGPR strains B. subtilis GB03 and P. polymyxa E681 at OD₆₀₀ = 1 (10^8–9 cfu/ml) was inoculated in an empty well (without plant). For assessing ISR by bacterial volatile and its derivatives, the I-plate system was employed. After application of 30 µl of 10 mM and 100 µM decane, undecane, and dodecane, the I-plate was tightly sealed with parafilm. Seven days after inoculation with PGPR or VOCs, 2.5 ml of a bacterial suspension (at OD₆₀₀ = 1) of P. syringae pv. maculicola ES4326 grown on King's B medium was added to each well. A whole seedling in each well was then soaked in the bacterial suspension for 5 min, and the suspension was removed. The plant was rinsed with sterile distilled water three times. The 24-multimicroliter plate with challenged Arabidopsis seedlings was then placed in a growth chamber that was maintained at 21°C in 12/12 day and night condition. Disease severity was measured four to seven days after pathogen challenge.