Detection and Molecular Characterization of some Egyptian Isolates of Ralstonia solanacearum by Nested-PCR and PCR-RFLP Analyses

Abstract

Brown rot disease of potato caused by the bacterium Ralstonia solanacearum (smith) is a destructive disease limiting potato production in several areas of the world. Nineteen isolates of the bacterium were recovered from infected potato tubers, showing typical symptoms of the disease that had been collected from Alexandria, Behera, Ismailia and Menofia Governorates. Isolates were identified as R. solanacearum biovar 2 (race 3), based on nutritional biochemical and enzymatic features as well as, standard biovar determination tests. All isolates were identified at the molecular level through Polymerase Chain Reaction (PCR) using a pair of specific primers (OLI-1 and Y2) that produced a 288 bp specific PCR product. Nested-PCR with the primer pair OLI-1/OLI-2 followed by JE2/Y2 proved to be highly specific and sensitive, producing the expected 220 bp band with all isolates tested beside a number of naturally infected potato tuber samples. The PCR-restriction fragment length polymorphism (PCR-RFLP) technique, based on the hypersensitive response and pathogenicity (hrp) gene region was used to determine race and biovar of nine R. solanacearum isolates. The RFLP banding patterns produced specific restriction profiles corresponded to biovar 2. The PCR-RFLP can, thus be used as a reliable sensitive technique to rapidly identify potential R. solanacearum isolates.