Pathogenicity potential of the 36 E. amylovora isolates were evaluated by dot blot hybridization of three virulence markers designated by hrpNM, hrpLM, and amsGM, targeting respectively the hrpL gene which is a transcriptional switch of the T3SS hrp operon [38, 65]; the hrpN gene coding for a translocator protein [66, 67]; and amsG, a gene coding the amylovoran biosynthesis protein AmsG, involved in the ams gene cluster [38, 68].

DNA probes for dot blot hybridization were prepared by PCR amplification of the three virulence specific markers on E. amylovora type strain LMG 2024, using primers previously described by Pester et al. [64] for each gene, and by designing their respective complementary primers (Table 2), resorting to the full genome of the type strain LMG 2024 (AN: CAPB00000000.1), to obtain DNA probes with the following sizes: 368bp for amsGM, 378bp for hrpLM, and 410bp for hrpNM. A 50 μL PCR reaction mix consisted of 1x DreamTaq Buffer with 2.0 mM MgCl2 (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 0.2 mM of dNTPs (GRiSP, Porto, Portugal), 0.5 μM of each forward and reverse primer, 1.25U of DreamTaq DNA Polymerase (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and 10 ng of DNA template. For negative control, H2O was added to the reaction mix instead of DNA template. PCR cycle parameters were carried out with a first amplification cycle of 3 min at 95°C, followed by 30 cycles at 95°C for 30 s, 50°C for 30 s, and 72°C for 60 s, and a final extension at 72°C for 5 min. The obtained PCR products were purified using the illustra GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, Chicago, Illinois, USA), and sequenced (STAB Vida, Caparica, Lisbon, Portugal) to confirm the identity of each amplicon used as probe. Probes were labelled with digoxigenin using the DIG-High Prime kit, according to the manufacturer’s instructions (Roche Diagnostics, Basel, Switzerland). The dot blot hybridization with labelled probes was carried out using 100 ng bacterial DNA from the 36 E. amylovora isolates, that were spotted onto nylon membranes (Roche Diagnostics, Basel, Switzerland) using a Bio-Dot apparatus (Bio-Rad, Hercules, California, USA). Each membrane was hybridized overnight at 68°C to ensure high stringency with a final probe concentration of 100 ng/mL, and stringency washes were performed following manufacturers recommendation. Probe-target hybrids were detected with chemiluminescent alkaline phosphatase substrate (CDP-Star®) reagent (Roche Diagnostics, Basel, Switzerland) and the images were acquired using a Molecular Imager ChemiDoc™ system (Bio-Rad, Hercules, California, USA). Three dot blot hybridization replicates for each probe were carried out.

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