*4.6. Molecular Characterization*

The actinobacterial strains CBQ-EA-2 and CBQ-B-8 were grown in tryptone-soya broth (BioCen) at 30 ◦C for three days, and centrifuged at 16,000 rpm. DNA was extracted from the resulting pellet using the PureLink™ Genomic DNA Mini Kit reagent (Invitrogen, Waltham, MA, USA), following the manufacturer's instructions. The universal primers 27f

and 1492r [48] for eubacteria were used to amplify the 16S rRNA gene via Polymerase Chain Reaction (PCR). Each reaction mixture contained each primer at 20 μM, dNTPs at 10 μM, 5 μL of 10X MgSO4 and buffer, dimethyl sulfoxide (5%), 1 μg of genomic DNA and 1 unit of taq DNA polymerase, for a final volume of 50 μL. PCR steps included an initial denaturation at 94 ◦C for 3 min, followed by 30 cycles at 94 ◦C for 30 s, 47 ◦C for 33 s and 72 ◦C for 90 s and a final extension step at 72 ◦C for 7 min. PCR products were run through 1% agarose gel electrophoresis stained with RedSafe™ dye (iNtRONBiotechnology), followed by purification using the PureLink™ kit (Invitrogen, Waltham, MA, USA) and determination of amplicon quality by spectrophotometry (NanoDrop 2000, ThermoScientific; Waltham, MA, USA). Sequencing was carried out on the ABI310 Prism automated sequencer (Applied Biosystems; Waltham, MA, USA), and the resulting sequences were compared with those in the GenBank database using the BLAST (Basic Local Alignment Search Tool) algorithm to identify closely related sequences [49,50]. The consensus sequences were uploaded to GenBank data base (Table 6).
