Real‐time PCR assays for the detection of Heterobasidion irregulare,H. occidentale,H. annosum sensu stricto and the Heterobasidion annosum complex

A set of quantitative hierarchical real‐time PCR assays was developed for the detection of Heterobasidion irregulare, H. occidentale, H. annosum sensu stricto and of the entire Heterobasidion annosum complex. These assays enable specific and accurate detection and quantification of the target species from DNA extracted on airborne collected spores. Heterobasidion‐specific TaqMan? real‐time PCR detection assays were designed to function under homogeneous conditions so that they may be used in 96‐ or 384‐well plate format arrays for high‐throughput testing of large numbers of samples against multiple targets. Assays were validated for (i) specificity, (ii) sensitivity and (iii) repeatability. All assays were highly specific when evaluated against a panel of pure cultures of target and phylogenetically closely related species. Sensitivity, evaluated by assessing the limit of detection (with a threshold of 95% of positive samples), was found to be between one and a hundred ITS gene region copies or one conidia count equivalent. Precision or repeatability of each assay revealed a mean coefficient of variation of 5.9%. These molecular tools are now available for rapid and reliable monitoring of one of the most significant pathogen species complex of temperate northern coniferous forest around the world.