Development of a multiplex PCR assay to discriminate native Hymenoscyphus albidus and introduced Hymenoscyphus fraxineus in Britain and assess their distribution

Two Hymenoscyphus species are associated with ash (Fraxinus) in Europe; the recently arrived ash dieback pathogen Hymenoscyphus fraxineus and apparently native Hymenoscyphus albidus. However, in parts of Europe invasive H. fraxineus appears to be replacing H. albidus which may, therefore, face extinction. We designed primers based on calmodulin sequences for use in singleplex and multiplex PCR assays to detect and discriminate between these two morphologically very similar species. The multiplex assay was tested against 50 isolates and 26 apothecial field samples of H. albidus/H. fraxineus from Europe and Asia, plus isolates of Hymenoscyphus fructigenus, Hymenoscyphus koreanus and Hymenoscyphus occultus. Use of the assay confirmed both H. albidus and H. fraxineus were present across southern Britain between 2014 and 2015, with H. albidus detected at eight geographically distinct sites. The sensitivity, specificity and potential applications of the multiplex assay are discussed.