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


RESULTS
The molecular assay was based on TaqMan 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 TaqMan assay was 78.6%, compared with only 14.3% of positive isolations on growth media for two sites.


CONCLUSIONS
In shoots, the quantitative Rt PCR assay detected down to 8.0-fg fungal DNA per microgram of total DNA extracted.


CONCLUSIONS
The assay developed in quantitative Rt PCR, by using TaqMan chemistry, revealed a rapid and sensitive method useful for the early detection of B. nummularia in symptomless European beech twigs.
Luchi
N
N
Dipartimento di Biotecnologie Agrarie, Sezione di Patologia vegetale, Università degli Studi di Firenze, Piazzale delle Cascine, 28 I-50144 Florence, Italy.
Capretti
P
P
Vettraino
A M
AM
Vannini
A
A
Pinzani
P
P
Pazzagli
M
M
D017931:DNA Primers; D004271:DNA, Fungal / Q000032:analysis / Q000302:isolation & purification; D029964:Fagus / Q000382:microbiology*; D010935:Plant Diseases / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase; D014197:Trees / Q000382:microbiology*; D013098:Xylariales / Q000235:genetics / Q000302:isolation & purification*
D017931:DNA Primers; D004271:DNA, Fungal; D019914:Taq Polymerase