Centre : Indian Institute of Vegetable Research
Name of the Protocol
Chitin synthase, a candidate gene for the molecular characterization of Fusarium isolates alternative to ITS region
Nature : Modification of existing protocol
Crop / Pathogen : Tomato and Chilli / Fusarium
Chitin synthase gene (CHS), which is responsible for virulence in Fusarium, was amplified in 19 isolates of F. solani and 22 isolates of F. oxysporum with specific primers CHS79-F / CHS354-R. PCR reactions were carried out in a 25µl volume consisting of 1X PCR buffer (Fermentas Inc., USA), 0.2mM of each dNTPs (Fermentas Inc., USA), 2mM MgCl2 (Fermentas Inc, USA), 0.5µM of each primers (Eurofins), 1U of Taq DNA polymerase (Genei, Bangalore), 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 95˚C for 10min followed by 35 cycles of 95˚C for 15s, 55˚C for 20s, and 72˚C for 60s with a 5min extension at 72˚C on the final cycle. In total 16 isolates of F. solani and seven isolates F. oxysporum were found positive and yielded 300bp fragment of CHS gene. The amplified products of CHS gene were eluted, purified and sequenced. In the phylogenetic analysis made using MEGA 5.0, all our 23 sequences from Fusarium solani and Fusarium oxysporum were grouped together with the fusarium sequences and form separate group from the other CHS gene.