A Simple and Efficient Method for Extracting S. Rolfsii DNA For PCR Based Diversity Studies

Materials and Methods Plant sample collection, pathogen isolation and purificationInfected bean plants showing symptoms of Sclerotium root rots were collected from the fields during the cropping season of February -April 2018 at Kawanda, Uganda.Isolation of the fungus was conducted at the CIAT-Uganda laboratory at NARL-Kawanda by washing the infected roots under running tap water to remove soil and debris and blotted dry using sterile tissue (Kleen ® , Kampala SITI Industries, Uganda).Roots were then cut into small pieces ~5 mm long and placed on water agar (WA) media (13.2 g/L, Biolife Italiana, Milano, Italy) and incubated for 24 h in darkness.Emerging fungal colonies were then transferred from the WA media to potato dextrose agar (PDA), [19.5 g/L, Sigma-Aldrich Chemistry GmbH, Steinheim, Germany] supplemented with 0.03g/L Rifamycin (Sigma-Aldrich Chemistry GmbH, Steinheim, Germany)].Actively growing agar plugs of ~3 mm of suspected Sclerotium spp.were again grown on WA for 24 h in darkness following single hyphal isolation.Hyphal tipping was done using a sterile needle under a stereo microscope (Leica, Wild M3B, Leica systems, Germany), and cultures grown on PDA containing 0.03 g/L of rifamycin.The resulting pure cultures were incubated in darkness at room temperature for 10 days.Nine S. rolfsii plugs were recovered from the PDA and inoculated in 30 ml of sterilized clarified V8 broth (200 ml of V8 juice; 3 g of calcium carbonate and 800 ml of distilled water) for 10 days at room temperature.Thereafter, 0.1 g of the mycelia were harvested using some sterile forceps and blotted on a napkin and paced in a 1.5 ml tube.