Evaluation of transgenic plants resistance to citrus canker

The disease resistance assay for transgenic plants against citrus canker was performed according to Peng et al. [6]. A Xcc strain, XccYN1 [6], was used to investigate plant disease resistance. Three mature healthy leaves per plant were tested. In total, 24 punctures were made per leaf with a needle containing the bacterial suspension (0.5 × 10⁵ CFU ml⁻¹). The inoculated leaves were maintained at 28°C under a 16-h light/8-h dark photoperiod with 45 μmol m⁻² s⁻¹ illumination and 60% relative humidity. Photographs were taken at 10 dpi. The area of all diseased spots was assessed with ImageJ 2.0 software (National Institutes of Health, Bethesda, MD, USA). The disease intensity of an individual line was based on the mean values of the diseased areas surrounding the 24 punctures on three leaves using the rating index described by Peng et al. [6]. The test was repeated three times.

The Xcc population levels in transgenic plants were determined as described by Peng et al. [32]. At 1, 3, 5, 7 and 9 dpi, bacterial colonies were counted and used to estimate the number of bacterial cells per unit leaf area (cm²).