Gen 3 microplate system

The physiological and biochemical identification of XX2021 was performed as described previously (Yuan et al., 2021 (link)). The Biolog GenIII MicroPlate technique (protocol B, Biolog) was used for this experiment, and final results were recorded at 24 and 48 h of incubation. The physiological and biochemical characteristics of XX2021 could be identified by the phenotypic fingerprint shown on the Biolog GenIII MicroPlate system. The morphology of the bacteria was examined with Gram staining and observation under a scanning electron microscope with the method discribed by Aid et al. (2020) (link). The hemolytic activity of the bacteria was determined using the blood agar plate method. Briefly, the bacteria were inoculated on the medium containing 5% sheep blood. After 36 h of incubation at 28°C, hemolytic isolates were identified based on the presence of α- or β-hemolysis around the colonies.

A Biolog GEN III microplate system (Biolog Inc., Hayward, CA, United States) was used to analyze carbon source utilization and chemical sensitivity of strain QSB-6 as described in Hu et al. (2010) (link).

Carbon source utilization and tests for sensitivity to different chemicals were determined using the BIOLOG GEN III MicroPlate system (BIOLOG, Hayward, CA). Bacterial cells from nutrient agar were suspended in BIOLOG Inoculation Fluid and adjusted to 95% transmission (optical density = 0.02). Further, 100 μL of the suspension were added to each well of the GENIII MicroPlate and incubated for 48 h at 28°C. The results were recorded manually.

Unless stated otherwise, the strains were observed on TSA for their cultural, morphological, and physiological characteristics. Gram staining of strains T3-5-0-4, N1-5-1-14 and N5-1-1-5 was conducted with a Gram staining kit (Sigma-Aldrich, St. Louis, MO, USA). Cultures were conducted at 4, 10, 15, 20, 25, 30, 37, 42, and 50 °C to determine the optimal temperature range for growth. The optimal pH range for growth was carried out using trypticase soy broth (TSB, Difco), with pH adjustments ranging from 4.5 to 10.0 (at intervals of 0.5 units) [31 (link)]. The NaCl requirement and tolerance were investigated by NaCl-free TSB supplemented with 0–10% (w/v) NaCl at 1% intervals. The growth tests were performed on Reasoner’s 2A (R2A, Difco) agar, Marine Agar 2216 (MA, Difco), nutrient agar (NA, Difco), and TSA at 25 °C for 14 days. The morphology and motility of cells were observed after 48 h of incubation at 25 °C by transmission electron microscopy (JEM-1230; JEOL) as well as phase-contrast microscopy (HFX; Nikon, Tokyo, Japan). The activity of constitutive enzymes, utilization of carbon source, and other physiological properties were analyzed by the API ZYM and API 20NE strips (bioMérieux, Marcy l’Etoile, France), and BIOLOG GEN III MicroPlate system (BIOLOG, Hayward, CA, USA).

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 ).