Oomycete Soil Diversity Associated with Betula and Alnus in Forests and Urban Settings in the Nordic-Baltic Region

For about 25 years, forest managers in Sweden have been treating stumps following harvesting with Phlebiopsis gigantea, retailed as Rotstop®S gel, against spore infections of Heterobasidion, which cause root rot in order to minimize losses in timber production. However, not all forest managers trust the efficacy of stump treatment and this fact has hindered widespread adoption of stump treatment using P. gigantea. In this study, we evaluated stump treatment in the field during commercial thinning operations across 15 sites, by assessing the degree of stump coverage and subsequent infection levels in stump discs. In total, 45 % of all stumps were infected with Heterobasidion spp.. Nineteen percent of all stumps were considered to have full (100 %) coverage by Rotstop®S but contractors failed to achieve the manufacturers stipulated minimum coverage (85 %) in approximately 1/3 of all stumps. Using PCR, we could only detect the presence of P. gigantea in 73 % of the tested stumps. Large variation occurred between stump coverage and the recovery of P. gigantea in wood chip samples collected from stump discs across sites. In the worst case, we detected P. gigantea in only three out of ten treated stumps at one site. Despite this discrepancy we saw a clear reduction of the size of Heterobasidion infections on stumps where stump treatment coverage was more than 85 % of the stump surface.Our results suggest that forest operators in Sweden repeatedly fail to either apply a spore solution of P. gigantea or cover enough of the stumps to provide the desired protection. The outcome of such sub-standard application, could further undermine the usage of biological control agents in forestry and limit any potential control against Heterobasidion spp.

Seasonal and Site-Specific Patterns of Airborne Fungal Diversity Revealed Using Passive Spore Traps and High-Throughput DNA Sequencing

The aim of the present work was to study seasonal and site-specific patterns of airborne fungal diversity, focusing on plant pathogens. The sampling of fungal spores was carried out for twelve months, i.e., between September 2017 and August 2018, using passive spore traps that were placed at three different sites in western (Lenkimai), central (Dubrava), and eastern (Labanoras) Lithuania. Samples were collected every 7-10 days, resulting in 146 samples altogether. Following DNA isolation, samples were individually amplified using ITS2 rRNA as a marker and subjected to high-throughput sequencing. Clustering and taxonomic classification of 283,006 high-quality reads showed the presence of 805 non-singleton fungal taxa. The detected fungi were 53.4% Ascomycota, 46.5% Basidiomycota, and 0.1% Mucoromycota. The most common fungal taxon at Labanoras and Lenkimai was Hannaella coprosmae (23.2% and 24.3% of all high-quality fungal sequences, respectively), while at Dubrava it was Cladosporium macrocarpum (16.0%). In different sites, plant pathogenic fungi constituted between 1.6% and 14.6% of all fungal taxa and among these the most common were Protomyces inouyei (4.6%) and Sydowia polyspora (1.9%). The results demonstrated that the diversity of airborne fungi was mainly determined by the surrounding vegetation and climatic factors, while the occurrence of pathogenic fungi was affected by the availability of their hosts

Can Larix sp. Mill. Provide Suitable Habitats for Insects and Lichens Associated with Stems of Picea abies (L.) H. Karst. in Northern Europe?

Recent observations suggest that climate change affects the growth conditions and range of tree species distribution in Europe. This may also have a major effect on communities of different organisms associated with these tree species. We aimed to determine whether Larix sp. could provide suitable habitats to insects and lichens associated with P. abies to conserve their biodiversity under climate change. The study sites were 10 Larix sp. and 10 P. abies forest stands in Lithuania. Both living and dead trees were included. Sticky traps, bark sheets, and exit hole methods were used for the assessment of insects. Independent plots on tree stems were established for the assessment of lichens. There were 76 and 67 different insect species on dead and living P. abies, respectively, using sticky traps. Similarly, there were 64 and 68 on dead and living Larix sp., respectively. The overall community of xylophagous insects consisted of nine and eight species, which were detected using the bark sheet and exit hole methods, respectively. The bark area colonized by lichens was 34.3% on dead P. abies and 63.2% on dead Larix sp., and 40.4% on living P. abies and 78.0% on living Larix sp. Taken together, the results demonstrate that native P. abies and introduced Larix sp. support similar diversity of stem-associated insect and lichen species

DNA-Metabarcoding of Belowground Fungal Communities in Bare-Root Forest Nurseries: Focus on Different Tree Species

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

Fungi and Oomycetes in the Irrigation Water of Forest Nurseries

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 Anykiai, 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. Gueho, 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 & Levesque (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

High Variability of Fungal Communities Associated with the Functional Tissues and Rhizosphere Soil of Picea abies in the Southern Baltics

Climate change, which leads to higher temperatures, droughts, and storms, is expected to have a strong effect on both health of forest trees and associated biodiversity. The aim of this study was to investigate the diversity and composition of fungal communities associated with the functional tissues and rhizosphere soil of healthy-looking Picea abies to better understand these fungal communities and their potential effect on tree health in the process of climate change. The study sites included 30 P. abies stands, where needles, shoots, roots, and the rhizosphere soil was 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 232,547 high-quality reads, which following clustering were found to represent 2701 non-singleton fungal OTUs. The highest absolute richness of fungal OTUs was in the soil (1895), then in the needles (1049) and shoots (1002), and the lowest was in the roots (641). The overall fungal community was composed of Ascomycota (58.3%), Basidiomycota (37.2%), Zygomycota (2.5%), Chytridiomycota (1.6%), and Glomeromycota (0.4%). The most common fungi based on sequence read abundance were Aspergillus pseudoglaucus (7.9%), Archaeorhizomyces sp. (3.6%), and Rhinocladiella sp. (2.0%). Pathogens were relatively rare, among which the most common were Phacidium lacerum (1.7%), Cyphellophora sessilis (1.4%), and Rhizosphaera kalkhoffii (1.4%). The results showed that the detected diversity of fungal OTUs was generally high, but their relative abundance varied greatly among different study sites, thereby highlighting the complexity of interactions among the host trees, fungi, and local environmental conditions

Mycobiota Associated with Symptomatic and Asymptomatic Fraxinus excelsior in Post-Dieback Forest Stands

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

The potential of biological control against Heterobasidion root rot is not realized in practical forestry

For about 25 years, forest managers in Sweden have been treating stumps following harvesting with Phlebiopsis gigantea, retailed as Rotstop (R) S gel, against spore infections of Heterobasidion, which cause root rot in order to minimize losses in timber production. However, not all forest managers trust the efficacy of stump treatment and this fact has hindered widespread adoption of stump treatment using P. gigantea. In this study, we evaluated stump treatment in the field during commercial thinning operations across 15 sites, by assessing the degree of stump coverage and subsequent infection levels in stump discs. In total, 45 % of all stumps were infected with Heterobasidion spp.. Nineteen percent of all stumps were considered to have full (100 %) coverage by Rotstop (R) S but contractors failed to achieve the manufacturers stipulated minimum coverage (85 %) in approximately 1/3 of all stumps. Using PCR, we could only detect the presence of P. gigantea in 73 % of the tested stumps. Large variation occurred between stump coverage and the recovery of P. gigantea in wood chip samples collected from stump discs across sites. In the worst case, we detected P. gigantea in only three out of ten treated stumps at one site. Despite this discrepancy we saw a clear reduction of the size of Heterobasidion infections on stumps where stump treatment coverage was more than 85 % of the stump surface.Our results suggest that forest operators in Sweden repeatedly fail to either apply a spore solution of P. gigantea or cover enough of the stumps to provide the desired protection. The outcome of such sub-standard application, could further undermine the usage of biological control agents in forestry and limit any potential control against Heterobasidion spp.

Managed and Unmanaged Pinus sylvestris Forest Stands Harbour Similar Diversity and Composition of the Phyllosphere and Soil Fungi

The aim was to assess fungal communities associated with living needles and soil of Pinus sylvestris in managed and unmanaged forest stands to get a better understanding of whether and how different intensities of forest management affects fungal diversity and community composition under the north temperate forest zone conditions. The study was carried out in three national parks in Lithuania. Each included five study sites in managed stands and five in unmanaged stands. At each site, three random soil cores and five random last-year needle samples were collected. Following DNA isolation, a DNA fragment of the ITS2 rRNA gene region of each sample was individually amplified and subjected to high-throughput sequencing. Analysis of 195,808 high-quality reads showed the presence of 1909 fungal taxa. Richness and composition of fungal taxa were similar in each substrate (needles and soil) in managed vs. unmanaged sites. The most common fungi in needles were Coleosporium campanulae (12.4% of all fungal sequences), Unidentified sp. 3980_1 (12.4%), Unidentified sp. 3980_4 (4.1%) and Sydowia polyspora (3.1%). In soil: Unidentified sp. 3980_21 (8.6%), Umbelopsis nana (8.2%), Archaeorhizomyces sp. 3980_5 (8.1%) and Penicillium spinulosum (6.3%). The results demonstrated that managed and unmanaged P. sylvestris stands support similar diversity and composition of fungal communities associated with living needles and soil

Biogeography of Fungal Communities Associated with Pinus sylvestris L. and Picea abies (L.) H. Karst. along the Latitudinal Gradient in Europe

We assessed the diversity and composition of fungal communities in different functional tissues and the rhizosphere soil of Pinus sylvestris and Picea abies stands along the latitudinal gradient of these tree species distributions in Europe to model possible changes in fungal communities imposed by climate change. For each tree species, living needles, shoots, roots, and the rhizosphere soil were sampled and subjected to high-throughput sequencing. Results showed that the latitude and the host tree species had a limited effect on the diversity and composition of fungal communities, which were largely explained by the environmental variables of each site and the substrate they colonize. The mean annual temperature and mean annual precipitation had a strong effect on root fungal communities, isothermality on needle fungal communities, mean temperature of the warmest quarter and precipitation of the driest month on shoot fungal communities, and precipitation seasonality on soil fungal communities. Fungal communities of both tree species are predicted to shift to habitats with a lower annual temperature amplitude and with increasing precipitation during the driest month, but the suitability of these habitats as compared to the present conditions is predicted to decrease in the future