Chemidoc mp photodocumentation system

Phylotype was determined by multiplex PCR using a set of phylotype-specific primers Nmult:21:1F, Nmult:21:2F, Nmult:22:InF, Nmult:23:AF, and Nmult21:RR (Fegan and Prior, 2005 ). Amplification was carried out in a total volume of 15 μl containing 1 X PCR buffer, 2.5 mM MgCl2, 0.2 mM of each dNTP, 0.2 μM of each primer, 0.3 U of GoTaq G2 Flexi DNA polymerase (PROMEGA) and 1 μl DNA template. Amplifications were performed in an Applied Biosystem Veriti thermocycler as follows: an initial denaturation step at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 23 s, and a final extension step at 72°C for 5 min. PCR products (10 μl) were analyzed by electrophoresis through 1% (w/v) agarose gels with 0.01 μl /ml GelRed^TM 10,000X (Biotium) and photographed under UV light in The Chemidoc^TM MP Photodocumentation System (BIO-RAD). DNA template from strains CIP-277 (phylotype I), CIP-435 (phylotype II), and CIP-358 (phylotype III) were used as positive amplification controls. The size of the amplified fragments was estimated by comparison with a 1 Kb Plus marker ladder.

For DNA extraction, the bacteria were streaked on MKM without TZC and the plates incubated at 30°C for 2 days. Then one colony was suspended in 100 μl of sterile NFW, boiled for 10 min and kept at -20°C prior to use.
The taxonomic identity of R. solanacearum was verified with primers 759/760 (Opina et al., 1997 ). PCR amplification was performed in a total volume of 15 μl containing 1X of PCR Buffer, 2.5 mM MgCl2, 0.2 μM each dNTP, 0.2 μM of each primer, 0.3 U of GoTaqG2 Flexi DNA polymerase (PROMEGA) and 1 μl DNA template. Amplification was performed in an Applied Biosystem Veriti thermocycler as follows: an initial denaturation step at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 17 s, and a final extension step at 72°C for 5 min. PCR products (10 μl) were analyzed by electrophoresis through 1% (w/v) agarose gels with 0.01 μl /ml GelRed^TM 10,000X (Biotium) and photographed under UV light in a The Chemidoc^TM MP Photodocumentation System (BIO-RAD). Fragments were compared with a 1 Kb Plus marker ladder. A positive identification was based on the presence of a 282 bp amplicon.