Pathogenicity test

Bacterial isolates were evaluated for their pathogenicity on pepper seedlings (Capsicum annuum var M Southern Star Seeds S. de R.L. de C.V., Mexico City). Seedlings were grown in 10-cm diameter pots with sterile expanded perlite particles, under a 16 h light at 28 °C and 8 h dark at 18 °C. Pathogenicity tests were conducted on 30-d-old pepper seedlings. Before and after testing, seedlings were conditioned two days in darkness, with relative humidity above 90% in a closed 27-gallon plastic chamber (Plastic Trends, Jalisco, Mexico). Bacterial suspensions were prepared from a 24-h-old culture grown on nutrient broth medium at a concentration of 1 × 10⁸ colony forming units (CFU) per milliliter, corresponding to an optical density (OD) of 0.01 at 600 nm by UV-visible spectrophotometry (Evolution 60 S; Thermo Fisher Scientific Inc., Waltham, MA, USA) (Mbega et al., 2012). Next, 10 µL of each bacterial suspension were infiltrated into leaves using Plastipak SFP 1 mL syringes (Becton-Dickinson, Brooklyn, NY, USA), and sterile distilled water as a control. Three leaves were infiltrated with two dots in five seedlings for each bacterial isolate. Disease severity was determined after 5 and 10 d, calculating the leaf damage percentage, using the Canopeo app (https://canopeoapp.com). Canopeo is an application that uses a red, green, and blue (RGB) system. It produces pixels analysis according to R/G, B/G ratios, and the green index excess. Results are interpreted in binary images where white pixels represent the pixels that satisfied the selection criteria (green canopy), whereas black pixels represent the pixels that did not comply with the selection criteria (not green canopy) (Patrignani & Ochsner, 2015). Severity was evaluated considering green percentage present in leaves. A 100% value was associated with healthy leaves, whereas lower percentages were considered as Xanthomonas damaged leaves.