Centre : ICAR Research Complex For NEH Region
Name of the Protocol
A new approach for PCR based rapid detection of Ralstonia solanacearum from infected plants
Nature : Modification of existing protocol
Crop / Pathogen : Solanaceous crops/Ralstonia solanacearum
A presumptive ooze test was performed following standard protocol for initial identification of bacterial wilt infected plant materials, samples were cut into small pieces aseptically and were placed in 1ml sterile nuclease and protease free water. Then those were kept for incubation at room temperature for 5 min or until the water became turbid followed by a final incubation at 960C for 6 min in thermal cycler. Finally, the suspension was directly used for PCR amplification using previously reported Rsol-fliC primers designed to amplify a ~400bp fragment of fliC gene specific for R. solanacearum (The PCR has 1X PCR buffer, 3 mM MgCl2, 6% DMSO, 50µM of each dNTPs, 10 pmol of each primer, 1 U of Taq DNA polymerase and 2µl of bacterial ooze suspension. A known and identified bacterial strain of R. solanacearum was used as positive control and sterile nuclease free water as negative control. The PCR cycling conditions are: initial denaturation at 960C for 9 min, followed by 34 cycles of 950C for 30 sec, 640C for 1 min, 720C for 2min and final extension of 720C for 10 min. Successful amplification of the expected fragment was confirmed by gel electrophoresis.