Standardization of methods for detection of Ralstonia solanacearum in potato

In support of EU Council Directive 98/57/EC on the control of potato brown rot disease, caused by Ralstonia solanacearum, a ring test was conducted to evaluate and standardize currently recommended methods for official testing of potato tubers. Test tubers were either naturally infected (symptomatic) or vacuum-infiltrated with different R. solanacearum suspensions or water. Testing was performed independently in 19 EU plant health laboratories using standardized protocols based on those annexed to the Directive. With high pathogen concentrations, fully reliable results were obtained with dilution plating on a selective medium, immunofluorescent antibody staining (IFAS), enzyme-linked immunosorbent assay (ELISA), fluorescent in situ hybridization and a tomato bioassay. The reliability of diagnoses across all laboratories decreased with inoculum concentration in asymptomatic tubers, irrespective of the methods used. Comparable results were obtained with 3 commercial antisera in IF AS or with 2 antisera in ELISA but the IFAS method was slightly more reliable. In several laboratories, diagnoses obtained using polymerase chain reaction (PCR) protocols were less reliable than those using the other methods. Independent optimization of PCR conditions in each laboratory and the introduction of internal positive PCR controls are recommended.