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Identification of Erwinia amylovora, the Fireblight Pathogen, by Colony Hybridization with DNA from Plasmid pEA29
H Falkenstein1, P Bellemann, S Walter
1Max-Planck-Institut für medizinische Forschung, Abteilung Molekulare Biologie, Jahnstrasse 29, D-6900 Heidelberg, and Biologische Bundesanstalt für Land-und Forstwirtschaft, Institut für Pflanzenschutz im Obstbau, D-6915 Dossenheim, Federal Republic of Germany.
Abstract:
All strains of Erwinia amylovora characterized carry a medium-size plasmid of 29 kilobases (pEA29). We mapped this plasmid with various restriction enzymes, cloned the whole DNA into an Escherichia coli plasmid, and subcloned restriction fragments. These DNA species were used for identification of E. amylovora after handling of strains in the laboratory and also in field isolates. About 70 strains of E. amylovora and 24 strains from nine other species, mainly found in plant habitats, were checked in a colony hybridization test. Virulent and avirulent E. amylovora strains reacted positively, whereas the other species were negative. Apart from the hybridization assay, the positive strains were additionally tested for ooze production on rich agar with 5% sucrose and on immature-pear slices. Unspecific background hybridization of non-E. amylovora strains found for hybridization with the whole E. amylovora plasmid was almost eliminated when a 5-kilobase SalI fragment from pEA29 was used as a probe and when the washes after the hybridization procedure were done with high stringency. Under these conditions, E. amylovora could be readily identified from field isolates.
Insights
A specific plasmid, pEA29, from Erwinia amylovora can reliably identify this plant pathogen. This method distinguishes Erwinia amylovora from other species, aiding in accurate disease diagnosis in field samples.
Area of Science:
- Plant Pathology
- Microbial Genetics
- Molecular Biology
Background:
- Erwinia amylovora is a significant plant pathogen causing fire blight disease.
- Accurate identification of Erwinia amylovora is crucial for disease management.
- Existing identification methods may lack specificity or require extensive laboratory procedures.
Purpose of the Study:
- To develop a reliable method for identifying Erwinia amylovora using its characteristic plasmid.
- To assess the specificity and sensitivity of a DNA-based probe for E. amylovora detection.
- To enable rapid and accurate identification of field isolates.
Main Methods:
- Characterization and mapping of the 29-kilobase plasmid (pEA29) from Erwinia amylovora.
- Cloning of plasmid DNA and subcloning of restriction fragments.
- Colony hybridization tests using plasmid DNA fragments as probes against various bacterial strains, including field isolates.
Main Results:
- All characterized Erwinia amylovora strains harbored the pEA29 plasmid.
- A 5-kilobase SalI fragment of pEA29, used as a probe under high stringency conditions, specifically detected E. amylovora.
- Non-E. amylovora strains showed no significant hybridization, indicating high specificity.
Conclusions:
- The pEA29 plasmid, particularly a specific fragment, serves as an effective DNA marker for identifying Erwinia amylovora.
- This molecular method allows for rapid and accurate differentiation of E. amylovora from other plant-associated bacteria.
- The developed hybridization assay is suitable for identifying E. amylovora in laboratory and field settings.
