Hymenoscyphus fraxineus, an introduced ascomycete fungus and primary causal agent of European ash dieback, was investigated onFraxinus mandshuricatrees in its native range in Primorye region of Far East Russia. This evidence is the first report ofH. fraxineuson healthy, asymptomaticF. mandshuricatrees. High-throughput sequencing revealed 49 distinct fungal taxa associated with leaves ofF. mandshurica, 12 of which were identified to species level. Phyllosphere fungal assemblages were similar among sites despite being largely geographically distant. Many organisms comprising the foliar fungal community onF. mandshuricain Far East Russia have similarity to those reported inhabitingF. excelsiorin Europe based on previous studies. However,Mycosphaerellasp., the most dominant species in this study and detected in nearly all samples, was associated only withF. mandshurica. Genetic diversity ofH. fraxineuswas significantly higher in the Far East Russian population than in Europe. In contrast to its aggressive behaviour onFraxinus excelsiorin Europe,H. fraxineusappears to be a benign associate of indigenousF. mandshuricathat initially induces quiescent and asymptomatic infections in healthy trees prior to active host colonization normally associated with modification of host tissue during senescence.

Natural and urban forests worldwide are increasingly threatened by global change resulting from human-mediated factors, including invasions by lethal exotic pathogens. Ash dieback (ADB), incited by the alien invasive fungus Hymenoscyphus fraxineus, has caused large-scale population decline of European ash (Fraxinus excelsior) across Europe, and is threatening to functionally extirpate this tree species. Genetically controlled host resistance is a key element to ensure European ash survival and to restore this keystone species where it has been decimated. We know that a low proportion of the natural population of European ash expresses heritable, quantitative resistance that is stable across environments. To exploit this resource for breeding and restoration efforts, tools that allow for effective and efficient, rapid identification and deployment of superior genotypes are now sorely needed. Here we show that Fourier-transform infrared (FT-IR) spectroscopy of phenolic extracts from uninfected bark tissue, coupled with a model based on soft independent modelling of class analogy (SIMCA), can robustly discriminate between ADB-resistant and susceptible European ash. The model was validated with populations of European ash grown across six European countries. Our work demonstrates that this approach can efficiently advance the effort to save such fundamental forest resource in Europe and elsewhere.

Ten juvenile Fraxinus excelsior half-sib families from two Lithuanian populations have been tested in the controlled environment for their response to ash dieback disease caused by Hymenoscyphus fraxineus, detecting changes of genetic variation and heritability, as well as estimating genotype by environment (G×E) interaction and phenotypic plasticity following artificial spring frost and summer drought treatments. In 2014, a batch of 200 four-year-old ash seedlings was used for each treatment and control (no treatment). Health condition, bud flushing phenology and height were assessed for each seedling, and disease incidence and survival ratios were assessed for each family both before (at the beginning of the vegetation season) and after the treatments (at the end of the vegetation season). Disease incidence ratio increased from 0.77-0.80 up to 0.90-0.95. Tree mortality rates during one vegetation season were significantly lower in the frost treatment (21%) than in the drought treatment (25%) or control (31%). None of the tested F. excelsior families were completely resistant to ash dieback, although significant among-family differences in disease incidence and damage rates suggest an additive mode of gene action and thus a quantitative resistance to the disease. Neither disease incidence rates, nor tree health condition scores differed significantly among the applied treatments (including control) indicating in general a negligible effect of the simulated adverse conditions on health status of the ash seedlings. However, G×E interaction was found to be significant (at P > 0.05) for disease incidence, length of necrotic shoots and tree survival, implying that susceptibility of ash families to the dieback disease unequally depends on environmental conditions, and indicating a presence of genetic variation in plasticity and reaction norms of the tested families across environments (treatments). Substantially increased coefficients of additive genetic variation and heritability in health condition following both frost and drought treatments and compared to control showed that simulated stress conditions may noticeably contribute to expression of differences among the tested F. excelsior families in their resistance traits, thus enabling a better evaluation of performance of different families, an effective family selection for resistance, and achievement of a marked genetic gain.

The production of tree seedlings in forest nurseries and their use in the replanting of clear-cut forest sites is a common practice in the temperate and boreal forests of Europe. Although conifers dominate on replanted sites, in recent years, deciduous tree species have received more attention due to their often-higher resilience to abiotic and biotic stress factors. The aim of the present study was to assess the belowground fungal communities of bare-root cultivated seedlings of Alnus glutinosa, Betula pendula, Pinus sylvestris, Picea abies and Quercus robur in order to gain a better understanding of the associated fungi and oomycetes, and their potential effects on the seedling performance in forest nurseries and after outplanting. The study sites were at the seven largest bare-root forest nurseries in Lithuania. The sampling included the roots and adjacent soil of 2–3 year old healthy-looking seedlings. Following the isolation of the DNA from the individual root and soil samples, these were amplified using ITS rRNA as a marker, and subjected to high-throughput PacBio sequencing. The results showed the presence of 161,302 high-quality sequences, representing 2003 fungal and oomycete taxa. The most common fungi were Malassezia restricta (6.7% of all of the high-quality sequences), Wilcoxina mikolae (5.0%), Pustularia sp. 3993_4 (4.6%), and Fusarium oxysporum (3.5%). The most common oomycetes were Pythium ultimum var. ultimum (0.6%), Pythium heterothallicum (0.3%), Pythium spiculum (0.3%), and Pythium sylvaticum (0.2%). The coniferous tree species (P. abies and P. sylvestris) generally showed a higher richness of fungal taxa and a rather distinct fungal community composition compared to the deciduous tree species (A. glutinosa, B. pendula, and Q. robur). The results demonstrated that the seedling roots and the rhizosphere soil in forest nurseries support a high richness of fungal taxa. The seedling roots were primarily inhabited by saprotrophic and mycorrhizal fungi, while fungal pathogens and oomycetes were less abundant, showing that the cultivation practices used in forest nurseries secured both the production of high-quality planting stock and disease control.

The aim of the present study was to assess fungal and oomycete communities in the irrigation water of forest nurseries, focusing on plant pathogens in the hope of getting a better understanding of potential pathogenic microorganisms and spreading routes in forest nurseries. The study sites were at Anykščiai, Dubrava, Kretinga and Trakai state forest nurseries in Lithuania. For the collection of microbial samples, at each nursery five 100-L water samples were collected from the irrigation ponds and filtered. Following DNA isolation from the irrigation water filtrate samples, these were individually amplified using ITS rDNA as a marker and subjected to PacBio high-throughput sequencing. Clustering in the SCATA pipeline and the taxonomic classification of 24,006 high-quality reads showed the presence of 1286 non-singleton taxa. Among those, 895 were representing fungi and oomycetes. The detected fungi were 57.3% Ascomycota, 38.1% Basidiomycota, 3.1% Chytridiomycota, 0.8% Mucoromycota and 0.7% Oomycota. The most common fungi were Malassezia restricta E. Guého, J. Guillot & Midgley (20.1% of all high-quality fungal sequences), Pezizella discreta (P. Karst.) Dennis (10.8%) and Epicoccum nigrum Link (4.9%). The most common oomycetes were Phytopythium cf. citrinum (B. Paul) Abad, de Cock, Bala, Robideau, Lodhi & Lévesque (0.4%), Phytophthora gallica T. Jung & J. Nechwatal (0.05%) and Peronospora sp. 4248_322 (0.05%). The results demonstrated that the irrigation water used by forest nurseries was inhabited by a species-rich but largely site-specific communities of fungi. Plant pathogens were relatively rare, but, under suitable conditions, these can develop rapidly, spread efficiently through the irrigation system and be a threat to the production of high-quality tree seedlings.