The development of a species‐specific test to detect H ymenoschyphus pseudoalbidus in ash tissues

Ash dieback, caused by the pathogen H ymenoscyphus pseudoalbidus , is an emerging lethal disease of F raxinus excelsior in large parts of E urope. To develop a method for the early detection of H . pseudoalbidus, we designed primers for 46 microsatellites (simple sequence repeats, SSR s) of the pathogen. Seven pairs of primers ( SSR 38, SSR 58, SSR 114, SSR 198, SSR 206, SSR 211 and SSR 212) were found to bind only to the genome of H . pseudoalbidus , but not to the genome of H . albidus or to 52 different fungal endophytes isolated from F . excelsior and F . angustifolia . Using these seven primer pairs, H . pseudoalbidus was identified in fruiting bodies and different types of ash tissues including dead leaves, dead petioles and discoloured or non‐discoloured wood. Along one twig, H . pseudoalbidus was detected at different levels of intensity, which depended on the distance from symptomatic tissue. The detection limit was 0.9–1.8 pg of genomic DNA per PCR . Of 50 analysed commercially available seedlings, six were infected with H . pseudoalbidus . Two SSR loci ( SSR 198 and SSR 211) showed fragment length polymorphism. Our results showed that the new primers not only provide an easy and inexpensive means of detecting H . pseudoalbidus in ash tissues, but can also provide information on the genetic heterogeneity of the species.