2.7. Detached leaf assay

Detached leaves assay was performed using five weeks old tomato leaves without any wounds and selected leaves were surface sterilized by soaking in a 1% sodium hypochlorite solution for 2 min. These leaves were then thoroughly rinsed with sterile distilled H₂O, leaves were dried for 5 min. For treatments, the overnight grown P. fluorescens VSMKU3054 and R. solanacearum cell density was adjusted to 1x10⁸ CFU/ml. Each treatment consisted of one detached leaf. As soon as the surface of detached leaves dried, a 50 µl of both bacterial cell suspensions was dropped for the inoculation in detached tomato leaves. Similar volumes of 30 µg/ml of partially purified DAPG and commercial tetracycline was injected in detached leaves of tomato. Distilled H₂O was used as negative control for the comparison of other treatments. A total of three replications were maintained for all the experiments. All the treated detached leaves of tomato were placed on the 0.5% agar surface in Petri dishes. All plates were incubated at 28 ± 2 °C for 1, 3, and 7 days in light/dark conditions (16/8 h) and humidity was maintained. After seven days of incubation, disease severity was assessed (Afroz et al., 2009). The disease scale was calculated and the relative AUDPC (Area under the disease progress curve) percentage was determined as described by Lee et al., (2012).