Aims: Real‐time PCR, based on TaqMan^® chemistry, was used to detect Biscogniauxia mediterranea, a fungal pathogen that after a long endophytic phase may cause charcoal disease in oak trees.

Methods and Results: Specific primers and probe were designed and tested on axenic cultures of B. mediterranea and other fungi commonly colonizing oaks. Twig samples were collected in Tuscany from apparently healthy oaks (Quercus cerris, Quercus ilex and Quercus pubescens) growing near trees infected with the fungus. Twigs were divided into two groups: one for isolation in agar plates, and one for real‐time PCR after DNA extraction. The detection limit of the assay was 0·01 pg/DNA, whereas the amounts of fungal DNA detected in asymptomatic tissue were >0·5 pg μg⁻¹ total DNA extracted. In the apparently healthy twigs the frequency of isolation found on agar was 25·0%, much lower than that with real‐time PCR (96·4%).

Conclusions: Real‐time PCR is a sensitive and fast technique able to specifically detect and quantify the DNA of B. mediterranea in oak tissue.

Significance and Impact of the Study: This diagnostic method is a precise tool to localize fungi in symptomless plant tissues and promises to advance our understanding of fungal infection during their latent phase.