Identification of the isolates as E. amylovora was carried out by PCR following the standard diagnostic protocols for E. amylovora as recommended by EPPO [59], using three pairs of chromosomal specific [6062], and one pair of pEA29 specific primers [63] (Table 2). PCR reactions were carried out using a reaction mixture containing 1x DreamTaq Buffer with 2.0 mM MgCl2 (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 0.2 mM of dNTPs (GRiSP, Porto, Portugal), 0.2 μM of each primer, 1U of DreamTaq DNA Polymerase (Thermo Fisher Scientific, Waltham, Massachusetts, USA), and 25 ng of DNA template. PCR cycling conditions for the chromosomal specific primers (G1-F+G2-R, FER1-F+FER1-R, and FER1-F+rgER2R) and for the plasmid specific primers (PEANT1+PEANT2) were the same as detailed by the EPPO standard diagnostic protocol [59]. PCR products were separated by electrophoresis in a 0.8% agarose gel stained with GreenSafe Premium (NZYTech, Lisbon, Portugal), with constant voltage (90V) in 1x Tris-EDTA (TE) buffer. The agarose gel was observed with a GelDoc™ (Bio-Rad Laboratories, California, USA).

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