Centre : Indian Institute of Vegetable Research
Name of the Protocol
Molecular characterization of Fusarium oxysporum f.sp. lycopersici isolates infecting tomato using polygalacturonase gene
Nature : Modification of existing protocol
Crop / Pathogen : Tomato/Fusarium oxysporum F.sp. lycopersici
Polygalacturonase gene (pgl), virulence gene in Fusarium, was amplified in 13 isolates of F. oxysporum f.sp. lycopersici (FOL) out of 17 isolates with specific primers PGLENDO F/R. PCR reaction was performed in Thermal cycler (BIORAD, USA). PCR reactions were carried out in a 25µl volume consisting of 1X PCR buffer (Fermentas Inc. Maryland, USA), 0.2 mM of each dNTPs (Fermentas Inc. Maryland, USA), 2 mM MgCl2 (Fermentas Inc. Maryland, USA), 0.5 µM of each primers (Eurofins), 1 U of Taq DNA polymerase (Genei, Banagalore), 2.5 µl of template DNA and sterile distilled water was added to make up the final volume. The thermal cycling conditions used were: initial denaturation at 94°C for 4 min, followed by 40 cycles of denaturation, annealing and elongation steps respectively at 94°C for 45 sec, 59ºC for 60 sec and 72ºC for 45 sec and a final extension step of 72ºC for 10 min. Thirteen isolates of FOL were found positive yielded an amplicon size of ~1.5kb gene fragment.