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Detection of Chalara fraxinea in common ash (Fraxinus excelsior) using real time PCR



A real time PCR assay was developed for the detection of Chalara fraxinea in common ash. PCR primers and Taqman probes, based on the internal transcribed spacer region of the multi-copy gene rDNA, were tested for specificity and sensitivity. The primers amplified an 81 bp fragment for C. fraxinea but did not amplify DNA from other Chalara species or from other fungi isolated from ash, whether pathogenic or saprophytic. The limit of detection was 5 pg of genomic DNA per PCR. Moreover, naturally-infected samples were correctly diagnosed. A procedure for DNA extraction from woody tissues using an electric drill yielded DNA of an appropriate quality for real time PCR. This molecular method could be useful for routine analysis of this emergent pathogen and for epidemiological studies.