Fungal Communities Associated with Siricid Wood Wasps: Focus on Sirex juvencus, Urocerus gigas, and Tremex fuscicornis.

We investigated the diversity and occurrence of wood wasps in Lithuania and determined communities of associated fungi. Trapping of wood wasps resulted in three different species, including Sirex juvencus, Urocerus gigas, and Tremex fuscicornis. Fungal culturing from adult females of T. fuscicornis mainly resulted in fungi from the genera Penicillium and Trichoderma. High-throughput sequencing of ITS2 rDNA resulted in 59,797 high-quality fungal sequences, representing 127 fungal OTUs. There were 93 fungal OTUs detected in U. gigas, 66 in S. juvencus, and 10 in T. fuscicornis. The most common fungi were Fusarium sporotrichioides (63.1% of all fungal sequences), Amylostereum chailletii (14.9%), Penicillium crustosum (7.8%), Microascus sp. 2261_4 (5.0%), and Pithoascus ater (2.1%). Among these, only A. chailletii was found in all three insect species with the highest relative abundance in U. gigas (15.2%), followed by S. juvencus (7.7%), and the lowest in T. fuscicornis (0.3%) (p < 0.0003). Correspondence analysis of fungal communities showed a distant placement of different species of wood wasps, indicating that fungal communities in each of these were largely different. In conclusion, the study showed that the economically important tree pathogen A. chailletii was among the most common fungal OTUs vectored by siricid wood wasps.

Long-term Dynamics of Fungal Communities Inhabiting Decaying Stumps of Quercus robur.

We studied the diversity, composition, and long-term dynamics of wood-inhabiting fungi in Quercus robur stumps left after commercial tree harvesting in Lithuania. Sampling of wood was carried out at three sites and from stumps, which were 10-, 20-, 30-, 40-, and 50-year-old. DNA was isolated from wood samples and fungal communities analyzed using high-throughput sequencing. Results showed that stump age had a limited effect on fungal diversity. The development of fungal communities in oak stums was found to be a slow process as fungal communities remained similar for decades, while larger changes were only detected in older stumps. The most common fungi were Eupezizella sp. (18.4%), Hyphodontia pallidula (12.9%), Mycena galericulata (8.3%), and Lenzites betulinus (7.1%). Fistulina hepatica, which is a red-listed wood-decay oak fungus, was also detected at a low relative abundance in stump wood. In the shortage of suitable substrate, oak stumps may provide habitats for long-term survival of different fungal species, including red-listed and oak-related fungi.

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.

An Overview of Phytophthora Species on Woody Plants in Sweden and Other Nordic Countries.

The genus Phytophthora, with 326 species in 12 phylogenetic clades currently known, includes many economically important pathogens of woody plants. Different Phytophthora species often possess a hemibiotrophic or necrotrophic lifestyle, have either a broad or narrow host range, can cause a variety of disease symptoms (root rot, damping-off, bleeding stem cankers, or blight of foliage), and occur in different growing environments (nurseries, urban and agricultural areas, or forests). Here, we summarize the available knowledge on the occurrence, host range, symptoms of damage, and aggressiveness of different Phytophthora species associated with woody plants in Nordic countries with a special emphasis on Sweden. We evaluate the potential risks of Phytophthora species to different woody plants in this geographical area and emphasize the increasing threats associated with continued introduction of invasive Phytophthora species.

Fungal Communities in Leaves and Roots of Healthy-Looking and Diseased Ulmusglabra.

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.

Fungal Communities in Re-Emerging Fraxinus excelsior Sites in Lithuania and Their Antagonistic Potential against Hymenoscyphus fraxineus.

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.

Principal Drivers of Fungal Communities Associated with Needles, Shoots, Roots and Adjacent Soil of Pinus sylvestris.

The plant- and soil-associated microbial communities are critical to plant health and their resilience to stressors, such as drought, pathogens, and pest outbreaks. A better understanding of the structure of microbial communities and how they are affected by different environmental factors is needed to predict and manage ecosystem responses to climate change. In this study, we carried out a country-wide analysis of fungal communities associated with Pinus sylvestris growing under different environmental conditions. Needle, shoot, root, mineral, and organic soil samples were collected at 30 sites. By interconnecting the high-throughput sequencing data, environmental variables, and soil chemical properties, we were able to identify key factors that drive the diversity and composition of fungal communities associated with P. sylvestris. The fungal species richness and community composition were also found to be highly dependent on the site and the substrate they colonize. The results demonstrated that different functional tissues and the rhizosphere soil of P. sylvestris are associated with diverse fungal communities, which are driven by a combination of climatic (temperature and precipitation) and edaphic factors (soil pH), and stand characteristics.

Fungal Communities Vectored by Ips sexdentatus in Declining Pinus sylvestris in Ukraine: Focus on Occurrence and Pathogenicity of Ophiostomatoid Species.

Drought-induced stress and attacks by bark beetle Ips sexdentatus currently result in a massive dieback of Pinus sylvestris in eastern Ukraine. Limited and fragmented knowledge is available on fungi vectored by the beetle and their roles in tree dieback. The aim was to investigate the fungal community vectored by I. sexdentatus and to test the pathogenicity of potentially aggressive species to P. sylvestris. Analysis of the fungal community was accomplished by combining different methods using insect, plant, and fungal material. The material consisted of 576 beetles and 96 infested wood samples collected from six sample plots within a 300 km radius in eastern Ukraine and subjected to fungal isolations and (beetles only) direct sequencing of ITS rDNA. Pathogenicity tests were undertaken by artificially inoculating three-to-four-year-old pine saplings with fungi. For the vector test, pine logs were exposed to pre-inoculated beetles. In all, 56 fungal taxa were detected, 8 exclusively by isolation, and 13 exclusively by direct sequencing. Those included nine ophiostomatoids, five of which are newly reported as I. sexdentatus associates. Two ophiostomatoid fungi, which exhibited the highest pathogenicity, causing 100% dieback and mortality, represented genera Graphium and Leptographium. Exposure of logs to beetles resulted in ophiostomatoid infections. In conclusion, the study revealed numerous I. sexdentatus-vectored fungi, several of which include aggressive tree pathogens.

First Report of the Larch Longhorn (Tetropium gabrieli Weise, Coleoptera: Cerambycidae: Spondylidinae) on Larix spp. in Lithuania.

Tetropium gabrieli is known to be native to the Alps in Europe where it breeds in European larch (Larix decidua), but it has spread to other areas and was reported in Poland, Sweden, Denmark and Belorussia. Although T. gabrieli is considered an important secondary pest of Larix spp., it can be particularly harmful to trees subjected to abiotic stress. Here we report that in Lithuania, T. gabrieli was for the first time captured in 2019 using sticky traps attached to Larix spp. trees. Two adult beetles were trapped at two different sites in central Lithuania, and this was in the period between 10th of May and 5th of June. Regarding potential threats caused by this insect pest, this new finding requires special attention, particularly on its biology, ecology, and local distribution.

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%), andPythium 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.

In vitro evidence of root colonization suggests ecological versatility in the genus Mycena.

The root-associated habit has evolved on numerous occasions in different fungal lineages, suggesting a strong evolutionary pressure for saprotrophic fungi to switch to symbiotic associations with plants. Species within the ubiquitous, saprotrophic genus Mycena are frequently major components in molecular studies of root-associated fungal communities, suggesting that an evaluation of their trophic status is warranted. Here, we report on interactions between a range of Mycena species and the plant Betula pendula. In all, 17 Mycena species were inoculated onto B. pendula seedlings. Physical interactions between hyphae and fine roots were examined using differential staining and fluorescence microscopy. Physiological interactions were investigated using 14 C and 32 P to show potential transfer between symbionts. All Mycena species associated closely with fine roots, showing hyphal penetration into the roots, which in some cases were intracellular. Seven species formed mantle-like structures around root tips, but none formed a Hartig net. Mycena pura and Mycena galopus both enhanced seedling growth, with M. pura showing significant transfer of 32 P to the seedlings. Our results support the view that several Mycena species can associate closely with plant roots and some may potentially occupy a transitional state between saprotrophy and biotrophy.

Occurrence and pathogenicity of Corinectria spp. - an emerging canker disease of Abies sibirica in Central Siberia.

During recent years, a new disease of Siberian fir (A. sibirica) emerged in Central Siberia, exhibiting symptoms of stem/branch deformation, cambium necrosis, and dieback of branches and twigs, the causal agent remaining unknown. The aim was to identify agent of the disease and to investigate its pathogenicity to A. sibirica and Norway spruce (Picea abies). Symptomatic tissues of fir were subjected to pure culture isolation of anticipated pathogen(s). Obtained isolates were subjected to molecular identification, phylogenetic analyses, and pathogenicity tests with A. sibirica saplings, and seeds and seedlings of A. sibirica and P. abies. The study demonstrated that, (i) most commonly isolated fungus from canker wounds of A. sibirica exhibited Acremonium-like anamorphs; (ii) phylogeny demonstrated that investigated fungi belong to genus Corinectria, but are genetically well separated from other worldwide known Corinectria spp.; (iii) one species of isolated fungi has the capacity to cause the disease and kill A. sibirica saplings and seedlings, but also seedlings of P. abies. Guidelines for future research were defined in order to generate needed information on species description, its origin and ecology, and estimation of potential risks upon the eventual invasion of the pathogen to new geographic areas, in particular of Europe.

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.

Secondary Metabolites from the Root Rot Biocontrol Fungus Phlebiopsis gigantea.

Three cyclopentanoids (phlebiopsin A⁻C), one glycosylated p-terphenyl (methyl-terfestatin A), and o-orsellinaldehyde were isolated from the biocontrol fungus Phlebiopsis gigantea, and their structures were elucidated by 1D and 2D NMR spectroscopic analysis, as well as by LC-HRMS. The biological activity of the compounds against the root rot fungus Heterobasidion occidentale, as well as against Fusarium oxysporum and Penicillium canescens, was also investigated, but only o-orsellinaldehyde was found to have any antifungal activity in the concentration range tested.

Growing evidence for facultative biotrophy in saprotrophic fungi: data from microcosm tests with 201 species of wood-decay basidiomycetes.

Ectomycorrhizal (ECM) symbioses have evolved a minimum of 78 times independently from saprotrophic lineages, indicating the potential for functional overlap between ECM and saprotrophic fungi. ECM fungi have the capacity to decompose organic matter, and although there is increasing evidence that some saprotrophic fungi exhibit the capacity to enter into facultative biotrophic relationships with plant roots without causing disease symptoms, this subject is still not well studied. In order to determine the extent of biotrophic capacity in saprotrophic wood-decay fungi and which systems may be useful models, we investigated the colonization of conifer seedling roots in vitro using an array of 201 basidiomycete wood-decay fungi. Microtome sectioning, differential staining and fluorescence microscopy were used to visualize patterns of root colonization in microcosm systems containing Picea abies or Pinus sylvestris seedlings and each saprotrophic fungus. Thirty-four (16.9%) of the tested fungal species colonized the roots of at least one tree species. Two fungal species showed formation of a mantle and one showed Hartig net-like structures. These features suggest the possibility of an active functional symbiosis between fungus and plant. The data indicate that the capacity for facultative biotrophic relationships in free-living saprotrophic basidiomycetes may be greater than previously supposed.

Impact of Heterobasidion root-rot on fine root morphology and associated fungi in Picea abies stands on peat soils.

We examined differences in fine root morphology, mycorrhizal colonisation and root-inhabiting fungal communities between Picea abies individuals infected by Heterobasidion root-rot compared with healthy individuals in four stands on peat soils in Latvia. We hypothesised that decreased tree vitality and alteration in supply of photosynthates belowground due to root-rot infection might lead to changes in fungal communities of tree roots. Plots were established in places where trees were infected and in places where they were healthy. Within each stand, five replicate soil cores with roots were taken to 20 cm depth in each root-rot infected and uninfected plot. Root morphological parameters, mycorrhizal colonisation and associated fungal communities, and soil chemical properties were analysed. In three stands root morphological parameters and in all stands root mycorrhizal colonisation were similar between root-rot infected and uninfected plots. In one stand, there were significant differences in root morphological parameters between root-rot infected versus uninfected plots, but these were likely due to significant differences in soil chemical properties between the plots. Sequencing of the internal transcribed spacer of fungal nuclear rDNA from ectomycorrhizal (ECM) root morphotypes of P. abies revealed the presence of 42 fungal species, among which ECM basidiomycetes Tylospora asterophora (24.6 % of fine roots examined), Amphinema byssoides (14.5 %) and Russula sapinea (9.7 %) were most common. Within each stand, the richness of fungal species and the composition of fungal communities in root-rot infected versus uninfected plots were similar. In conclusion, Heterobasidion root-rot had little or no effect on fine root morphology, mycorrhizal colonisation and composition of fungal communities in fine roots of P. abies growing on peat soils.

The ectomycorrhizal community of conifer stands on peat soils 12 years after fertilization with wood ash.

We studied long-term effects of fertilization with wood ash on biomass, vitality and mycorrhizal colonization of fine roots in three conifer forest stands growing in Vacciniosa turf. mel. (V), Myrtillosa turf. mel. (M) and Myrtillosa turf. mel./Caricoso-phragmitosa (MC) forest types on peat soils. Fertilization trials amounting 5 kg/m(2) of wood ash were established 12 years prior to this study. A total of 63 soil samples with roots were collected and analysed. Ectomycorrhizal (ECM) fungi in roots were identified by morphotyping and sequencing of the fungal internal transcribed spacer (ITS) region. In all forest types, fine root biomass was higher in fertilized plots than in control plots. In M forest type, proportion of living fine roots was greater in fertilized plots than in control plots, while in V and MC, the result was opposite. Fifty ECM species were identified, of which eight were common to both fertilized and control plots. Species richness and Shannon diversity index were generally higher in fertilized plots than in control plots. The most common species in fertilized plots were Amphinema byssoides (17.8%) and Tuber cf. anniae (12.2%), while in control plots, it was Tylospora asterophora (18.5%) and Lactarius tabidus (20.3%). Our results showed that forest fertilization with wood ash has long-lasting effect on diversity and composition of ECM fungal communities.

Cytotoxic Illudane Sesquiterpenes from the Fungus Granulobasidium vellereum (Ellis and Cragin) Jülich.

Eight illudane sesquiterpenes were obtained from the wood-decomposing fungus Granulobasidium vellereum (Ellis and Cragin) Jülich; among them were the enantiomers of the known compounds illudin M (1) and dihydroilludin M (4) and the diastereomers of illudin M (2) and illudin S (3), as well as two previously undescribed illudanes (5, 6). The cytotoxicity of compounds 1-4 and 6 was evaluated against two tumor cell lines (Huh7 and MT4), which showed that compounds 1-3 had potent cytotoxic activity, whereas compounds 4 and 6 had no or only moderate effects at concentrations up to 400 µM. Surprisingly, both compounds 2 and 3 were about 10 times more potent than 1. When the chemical reactivity of 1 and 2 was tested, compound 2 was shown to have a substantially higher reaction rate when reacted both with 2 M HCl and with cysteine, indicating that the difference in cytotoxicity is probably due to chemical reactivity and not to enzymatic affinity.

High-Throughput Sequencing Reveals Drastic Changes in Fungal Communities in the Phyllosphere of Norway Spruce (Picea abies) Following Invasion of the Spruce Bud Scale (Physokermes piceae).

The aim of this study was to assess the diversity and composition of fungal communities in damaged and undamaged shoots of Norway spruce (Picea abies) following recent invasion of the spruce bud scale (Physokermes piceae) in Lithuania. Sampling was done in July 2013 and included 50 random lateral shoots from ten random trees in each of five visually undamaged and five damaged 40-50-year-old pure stands of P. abies. DNA was isolated from 500 individual shoots, subjected to amplification of the internal transcribed spacer of fungal ribosomal DNA (ITS rDNA), barcoded and sequenced. Clustering of 149,426 high-quality sequences resulted in 1193 non-singleton contigs of which 1039 (87.1 %) were fungal. In total, there were 893 fungal taxa in damaged shoots and 608 taxa in undamaged shoots (p < 0.0001). Furthermore, 431 (41.5 %) fungal taxa were exclusively in damaged shoots, 146 (14.0 %) were exclusively in undamaged shoots, and 462 (44.5 %) were common to both types of samples. Correspondence analysis showed that study sites representing damaged and undamaged shoots were separated from each other, indicating that in these fungal communities, these were largely different and, therefore, heavily affected by P. piceae. In conclusion, the results demonstrated that invasive alien tree pests may have a profound effect on fungal mycobiota associated with the phyllosphere of P. abies, and therefore, in addition to their direct negative effect owing physical damage of the tissue, they may also indirectly determine health, sustainability and, ultimately, distribution of the forest tree species.

Archaeorhizomyces borealis sp. nov. and a sequence-based classification of related soil fungal species.

The class Archaeorhizomycetes (Taphrinomycotina, Ascomycota) was introduced to accommodate an ancient lineage of soil-inhabiting fungi found in association with plant roots. Based on environmental sequencing data Archaeorhizomycetes may comprise a significant proportion of the total fungal community in soils. Yet the only species described and cultivated in this class is Archaeorhizomyces finlayi. In this paper, we describe a second species from a pure culture, Archaeorhizomyces borealis NS99-600(T) (=CBS138755(ExT)) based on morphological, physiological, and multi-locus molecular characterization. Archaeorhizomyces borealis was isolated from a root tip of a Pinus sylvestris seedling grown in a forest nursery in Lithuania. Analysis of Archaeorhizomycete species from environmental samples shows that it has a Eurasian distribution and is the most commonly observed species. Archaeorhizomyces borealis shows slow growth in culture and forms yellowish creamy colonies, characteristics that distinguish A. borealis from its closest relative A. finlayi. Here we also propose a sequence-based taxonomic classification of Archaeorhizomycetes and predict that approximately 500 species in this class remain to be isolated and described.