*4.3. Molecular Identification*

The bacterial isolates that demonstrated potentially antagonistic effects against the tested target microorganisms were previously morphologically identified under a light microscope (60×) and then by the molecular method based on the analysis of genomic DNA (gDNA) sequences. The gDNA of each studied isolate was extracted using a Qiagen Genomic DNA Kit (Qiagen, Heidelberg, Germany). The extracted gDNA was amplified using the universal primers for bacteria Y1/Y2 (Table 5). The PCR reaction was carried out in a final volume of 25 μL containing: 200 ng DNA, 0.2 μL of 1 U *Taq* DNA polymerase, 2.5 μL *Taq* buffer (20 mM MgCl2), 5 μL of each primer (2.5 μM), 5 μL of dNTPs (4 mM) and ultrapure dH2O for a final volume of 25 μL. Both the concentration and purity of the total DNA extracted from each sample were measured using a Nano-drop (Thermo Fisher Scientific, Waltham, MA USA). Each DNA sample was subjected to PCR amplification following the cycling profile: 94 ◦C for 5 min (initial denaturation), followed by 34 cycles of 94 ◦C for 30 c (denaturation), 57 ◦C for 30 s (annealing), and 72 ◦C for 1 min (extension), with a final extension step of 5 min at 72 ◦C. The amplified DNA, stained by Bromophenol blue (3 μL/10 μL), was applied for agarose gel electrophoresis (1.2%) stained by SYBR green dye (4 μL/100 gel). The obtained amplicons were directly sequenced and compared with those available in the GenBank nucleotide archive using BLAST software [47].
