Fungi isolated from shoots showing ash dieback in the Wolica Nature Reserve in Poland and artificially inoculated seedlings with Hymenoscyphus fraxineus

Ash dieback caused by an alien, invasive fungus Hymenoscyphus fraxineus is a serious disease of European ash species in many parts in Europe. In Poland, the disease was recorded in the beginning of the 1990s. This study was performed in 2016–2017 with the aim to identify fungi isolated from ash shoots showing dieback symptoms in the Wolica Nature Reserve in Poland, as well as from shoots of two years-old ash seedlings inoculated with H. fraxineus in the greenhouse. The most frequently isolated fungi from shoots of common ash (associated with the pathogenic fungus H. fraxineus) were identified on the basis of sequencing of the internal transcribed spacer region (ITS1) of fungal rDNA. In total, 19 fungal taxa were identified for ash shoots as follows: H. fraxineus, Fusarium avenaceum, Alternaria spp., Phomopsis oblonga, Diplodia mutila and other Phomopsis spp. The pathogen H. fraxineus was not found for all the shoots samples; one year after inoculation the aforementioned fungi and other species as: Alternaria alternata, Bionectria ochroleuca, Epicoccum nigrum, F. acuminatum, F. avenaceum, and Paraphaesphaeria neglecta were identified in inoculation point, as well as H. fraxineus. The same quantitative and qualitative changes of organisms were observed in the case of artificially colonised ash seedlings in the greenhouse, as well as in the shoots of adult ash trees in the forest.