Herbaria are a promising but still poorly applied information source for retrospective microbiological studies. In order to find any evidence of the virulent European origin of ash dieback agent Hymenoscyphus fraxineus and other fungal pathogens, we analyzed 109 leaf samples from three different Estonian botanical herbaria, sampled during 171 years from 20 ash species and cultivars, using a PacBio third-generation sequencing of the fungal internal transcribed spacer ITS1-5.8S-ITS2 ribosomal DNA region. We identified a large amount of saprotrophic fungi naturally colonizing ash leaves. Hymenoscyphus fraxineus colonized a Fraxinus chinensis subsp. rhynchophylla specimen and a F. chinensis specimen collected from Tallinn Botanic Garden in July 1978 and July 1992, respectively. The samples originated from trees grown in this garden from seeds collected from Shamora, Far-East Russia, in 1961 and from a Beijing botanical garden in eastern China in 1985, respectively. Repeated subsequent DNA extraction, real-time quantitative PCR, and Sanger and Illumina sequencing confirmed our findings of these apparently oldest cases of the ash dieback agent in Europe. These results show that H. fraxineus evidently was present in Estonia 19 years earlier than our previous data from fungal herbaria documented and 14 years before the first visible damage of ash trees was registered in Poland. Because we found no evidence of the saprotrophic H. albidus from earlier mycological and botanical herbarium specimens, the presence of H. albidus in Estonia remains questionable.
Keywords: Hymenoscyphus albidus; Illumina and PacBio sequencing; ash dieback; botanical herbarium specimens; invasive pathogen; mycobiome.