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Abstract:
Monitoring of fireblight can be done by plating on semi-selective agar, by immunofluorescence or by hybridization of bacterial colonies with DNA from a 29 kb plasmid common to Erwinia amylovora. The specific but labour intensive procedure of DNA hybridization can be circumvented by PCR-detection of a 0.9 kb Pstl-fragment from the 29 kb plasmid, which was partially sequenced. Two oligonucleotides derived from sequence information of both ends of the fragment were used as specific primers for amplification of bacterial DNA and produced a 0.9 kb band, which was not observed from DNA isolated from other plant pathogenic bacteria. The signal was also detected when the bacteria were added directly to the PCR reaction mixture and the method was more sensitive, when a detergent was present in the amplification assay.
Detection was still possible for about 50 E. amylovora-cells, which were added directly to the PCR reaction mixture with detergent. Samples from infected plant tissue also produced a clear signal when subjected to PCR-analysis in this system. The specific signal was obtained with E. amylovora-strains isolated at various geographic regions and at different times. A few E. amylovora cells could be detected in an excess of other plant-associated bacteria which did not change the signal strength. As an alternative method for confirmation of strains preliminary classified as E. amylovora one primer amplification (RAPD-PCR) can be applied to develop signals characteristic for the fireblight pathogen.
