Sensitivity of different methods for the detection of Ralstonia solanacearum in potato tuber extracts

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Abstract
The sensitivities of various methods for the detection of Ralstonia solanacearum following dilution in healthy potato tuber tissue macerate were compared. Estimated pathogen populations in undiluted macerates, from samples of 200 heel‐end vascular cores each containing a single diseased and 199 healthy tubers, ranged from 1.2 × 106–7.4 × 107 colony‐forming units per ml. Following concentration by high‐speed centrifugation and resuspension in phosphate buffer, the pathogen was detected by all methods studied, including culture on semi‐selective media, enzyme‐linked immunosorbent assay (ELISA), indirect immunofluorescent‐antibody staining (IFAS) of fixed cells, immunofluorescent colony staining (IFCS), detection of specific DNA sequences following amplification by the polymerase chain reaction (PCR) and bioassay in tomato seedlings. Both ELISA and PCR methods were improved by pre‐enrichment of samples in semi‐selective broth prior to testing. A nested PCR method was evaluated which could detect fewer than 10 cells per ml in the potato extracts. Of the other methods only dilution plating on semi‐selective medium and tomato bioassay could detect fewer than 104 cells per ml. In order to combine ease and speed of use with sensitive detection, it was recommended that a series of methods be used for routine screening of potato tuber stocks for infection by R. solanacearum.