Biochemical Characterization of E. amylovora

The E. amylovora strains were characterized through their biochemical pattern resorting to Biolog GEN III Microplate™ system (Biolog™, USA), according to manufacturer’s instructions. Briefly, the strains were first grown on solid YNA medium (4 g of meat extract; 5 g of peptone; 2.5 g yeast extract; 5 g of NaCl; 15 g of agar; distilled water up to 1 L; pH 7.0) for 48 h at 28 °C. Fresh colonies were transferred, with a cotton-tipped swab, to new vials containing Inoculating Fluid A. The inoculum density was adjusted to a transmittance of 95–98% resorting to a turbidimeter. After that, 100 µL of the inoculum was dispensed into each well of the Biolog MicroPlate. MicroPlates were then incubated at 30 °C during 24 h, and then the plates were read in a microplate photometer (Mulstiskan™ FC; Thermo Fisher Scientific, Waltham, MA, USA). For all strains, two independent replicates were performed in different dates. Results were considered positive if the OD at 595 nm (OD₅₉₅) was higher than 50% of the positive control, whilst they were considered negative if the OD₅₉₅ was below 25% of the positive control. Results between these two parameters were considered borderlines (Flores et al., 2018 (link)). The dendrogram (UPGMA, bootstrap of 1,000) was obtained resorting to RStudio (RStudio Team, 2020 ).