Early detection of Biscogniauxia nummularia in symptomless European beech (Fagus sylvatica L.) by TaqMan quantitative real-time PCR

To develop a quantitative real-time PCR (Rt PCR) assay for the early detection of Biscogniauxia nummularia, a xylariaceous fungus that causes strip-canker and wood decay on European beech (Fagus sylvatica L.). The molecular assay was based on TaqManTM chemistry using species-specific primers and a fluorogenic probe designed on the ITS1 sequence of rRNA gene clusters. The specificity of the oligonucleotides and the probe were tested using the DNA of B. nummularia isolates from different geographic areas, of phylogenetically related species, and of some fungi commonly colonizing European beech bark and wood. A total of 31 symptomless and symptomatic shoots of European beech were collected from three forest sites in the Apennine Mountains of Italy. The percentage of positive detections of B. nummularia with the TaqManTM assay was 78·6%, compared with only 14·3% of positive isolations on growth media for two sites. In shoots, the quantitative Rt PCR assay detected down to 8·0-fg fungal DNA per microgram of total DNA extracted. The assay developed in quantitative Rt PCR, by using TaqManTM chemistry, revealed a rapid and sensitive method useful for the early detection of B. nummularia in symptomless European beech twigs.