2.13. Pst DC3000 Inoculation and Bacterial Growth in Tomato Plants

To test whether AY001 induces resistance against Pst DC000, AY001 was pre-treated either on tomato leaves or on roots. For leaf treatment, AY001 suspension (OD₆₀₀ = 1.0; 2 × 10⁷ cfu/mL) or sterilized tap water (STW) was sprayed on three-week-old tomato leaves at 24 h before inoculation with Pst DC3000. For root treatment, three-week-old tomato roots were uprooted and submerged either into AY001 suspension (OD₆₀₀ = 1.0) or STW for 30 min, then planted into a new plastic pot (10 × 9 cm). After 24 h, Pst DC3000 was inoculated by the spraying method. For Pst DC3000 inoculation, Pst DC3000 was cultured in KB medium (MBcell, Seoul, Korea) containing 50 μg/mL rifampicin (Rif) at 28 °C for 24 h. Bacterial cells were collected by centrifugation, resuspended in 10 mM MgCl₂ solution (1 × 10⁶ cfu/mL) with 0.05% Tween 20, and evenly sprayed over the entire leaves. Each treatment was performed with 8 replicates. To measure the Pst DC3000 growth, 6 discs of tomato leaves were collected by using a cork borer (diameter = 6.5 mm). Samples were surface sterilized with 1% NaOCl for 30 s, rinsed three times with SDW, then grounded in 300 µL SDW using a silamat S6 and glass beads. Serial dilutions were placed on a KB agar plate containing 50 μg/mL Rif by the dot-plating method. Experiments were repeated 3 times with similar results.