DETECTION OF RALSTONIA SOLANACEARUM IN POTATO TUBERS, SOLANUM DULCAMARA AND ASSOCIATED IRRIGATION WATER.
Elphinstone J. G., Stanford H., Stead D. E.
Central Science Laboratory, Sand Hutton, York, YO4 1LZ, UK
A number of rapid detection methods were compared for the testing of potato tubers, Solanum dulcamara and surface irrigation water for infection by Ralstonia solanacearum biovar 2-A. Methods using immunofluorescence (IF) microscopy, ELISA, polymerase chain reaction (PCR), dilution plating on semi-selective medium and bioassay in tomato seedlings were all sufficiently sensitive for reliable detection of the pathogen in samples of 200 healthy tubers to which a single tuber with brown rot symptoms had been added. Lower pathogen populations equivalent to those found in latently infected potatoes were most reliably detected by culture on semi-selective medium followed by identification of presumptive isolates by fatty acid analysis or REP-PCR. The sensitivity of ELISA and PCR methods were improved by pre-incubation of tuber extracts in semi-selective broth. A scheme of methods was devised in order to maximise the probability of detection of low pathogen populations. For pathogen detection in aquatic roots of Solanum dulcamara, ELISA was the least sensitive method, whereas PCR could be inhibited by plant and soil components and cultures could be overgrown or inhibited by commonly-occurring saprophytic or antagonistic organisms. Reliable detection could therefore be achieved only when all three methods were used simultaneously. Detection of R. solanacearum in surface water was possible by direct plating onto semi-selective medium but only during warm summer months. Sensitivity of detection in water samples was increased when the bacterial fraction was concentrated by centrifugation or ultra-filtration.
