The aim of this study was to investigate fungal communities associated with leaves and roots of healthy-looking and declining U. glabra trees. The study was expected to demonstrate whether and how the diversity and composition of fungal communities change in these functional tissues following the infection by Dutch elm disease-causing fungi. The study sites included six U. glabra sites in Lithuania, where leaves and roots were sampled. DNA was isolated from individual samples, amplified using ITS2 rRNA as a marker, and subjected to high-throughput sequencing. The sequence analysis showed the presence of 32,699 high-quality reads, which following clustering, were found to represent 520 non-singleton fungal taxa. In leaves, the fungal species richness was significantly higher in healthy-looking trees than in diseased ones (p < 0.05). In roots, a similar comparison showed that the difference was insignificant (p > 0.05). The most common fungi in all samples of roots were Trichocladium griseum (32.9%), Penicillium restrictum (21.2%), and Unidentified sp. 5238_7 (12.6%). The most common fungi in all samples of leaves were Trichomerium sp. 5238_8 (12.30%), Aureobasidium pullulans (12.03%), Cladosporium sp. 5238_5 (11.73%), and Vishniacozyma carnescens (9.86%). The results showed that the detected richness of fungal taxa was higher in samples collected from healthy-looking trees than from diseased ones, thereby highlighting the negative impact of the Dutch elm disease on the overall fungal diversity.

In Lithuania, the dieback of European ash (Fraxinus excelsior L.), caused by alien ascomycete Hymenoscyphus fraxineus, started in the mid-1990s, resulting in a large-scale decline of F. excelsior and its dominated forest habitats. Nevertheless, the recent inventories show the presence of several hundred hectares of naturally regenerated F. excelsior stands. We used seven naturally regenerated sites and three planted progeny trials of F. excelsior to collect leaves, shoots, roots, and the surrounding soil to study ash-associated fungal communities based on high-throughput sequencing. Results showed that fungal communities associated with F. excelsior in re-emerging stands in post-dieback areas were composed of 1487 fungal taxa. Among these, 60.5% were Ascomycota, 37.5%—Basidiomycota, 1.7%—Zygomycota, and 0.2% were Chytridiomycota. Revealed mycobiota was largely composed of endophytic fungal communities as these were dominated by Cladosporium sp., Fraxinicola fraxini (syn. Venturia fraxini) and Vishniacozyma foliicola. Identified mycobiota also included a range of ash-specific fungal taxa. Hymenoscyphus fraxineus occurred in all stands but was not frequent. Cladosporium sp. showed strongest negative correlation with the presence of H. fraxineus. This ascomycete, given its dominance in leaves, shoots and in the organic soil layer, might be the limiting factor for the infection rate or spread of H. fraxineus. Although fungal communities in asymptomatic and symptomatic samples of F. excelsior differed significantly from each other, the majority of the most frequently found fungal taxa were not host-specific, suggesting that these were negligibly affected by ash dieback. Investigated stands in natural F. excelsior habitats exhibited larger diversity of fungal taxa (especially ash-specific), than progeny trials planted on former grasslands, indicating the importance of natural habitats in F. excelsior restoration programs.

Fifty-nine fungal taxa, isolated from re-emerging Fraxinus excelsior sites in Lithuania, were in vitro tested against three strains of Hymenoscyphus fraxineus on agar media to establish their biocontrol properties. All tested fungi were isolated from leaves and shoots of relatively healthy Fraxinus excelsior trees (<30% defoliation), which were affected by ash dieback but their phytosanitary condition has not worsened during the last decade. The inhibition of H. fraxineus growth by tested fungal taxa ranged between 16–87%. Occasionally isolated fungal taxa such as Neonectria coccinea, Nothophorma quercina, and Phaeosphaeria caricis were among the most effective fungi inhibiting the growth of H. fraxineus cultures. Among the more commonly isolated fungal taxa, Cladosporium sp., Fusarium sp., Malassezia sp., and Aureobasidium pullulans showed a strong growth inhibition of H. fraxineus.