Introduction
Climate change is increasing the number of natural disasters (e.g., storms, droughts, high temperatures) that are able to damage the shallow root system of Norway spruce (Picea abies L. Karst.) (Klavina et al., Reference Klavina, Menkis, Gaitnieks, Velmala, Lazdins, Rajala and Pennanen2015). Consequently, trees are weakened and exposed to attacks by bark beetles, which are among the most common damaging agents to these trees (Christiansen & Bakke, Reference Christiansen, Bakke and Berryman1988). The great spruce bark beetle (Dendroctonus micans Kug.) is among the largest sized bark beetles and the only representative of this genus in Europe. It often attacks older stands and clonal seed orchards of P. abies, especially following natural disasters or when trees have been weakened by other insects. During outbreaks, D. micans is able to destroy entire stands. The trees are killed mostly by girdling when several broods develop in the same tree (Evans et al., Reference Evans, King and Wainhouse1984). The life cycle of many bark beetles is dependent on many microorganisms, especially fungi. These beetles are anatomically and morphologically adapted to vector and transmit these microorganisms from one generation to the next that can infect the attacked trees (Vega & Blackwell, Reference Vega and Blackwell2005). Among the European bark beetles, the interaction between the European spruce bark beetle (Ips typographus L.) and Ophiostoma (Ascomycota) fungi is a well-known example. Depending on the Ophiostoma species that are vectored by the beetle, there may be variable effects, including antagonism, commensalism or mutualism (Vega & Blackwell, Reference Vega and Blackwell2005). In contrast, symbioses of D. micans and fungi are less well studied, revealing the need for better understanding of associated fungal communities and their potential effects on insect and attacked tree species. For example, Lieutier et al. (Reference Lieutier, Vouland, Pettinetti, Garcia, Romary and Yart1992) showed that only O. canum was cultured from D. micans or from its galleries with a significant frequency of association, while other species, including O. penicillatum, O. minus and other unidentified Ophiostoma species were found only very occasionally.
The aim of the present study was to assess occurrence, phenology and morphology of D. micans in Lithuania and the fungi associated at different developmental stages.
Material and methods
Assessing the occurrence of D. micans
The assessment was carried out in July 2013. The study sites were selected based on the information provided by the Lithuanian State Forest Service and included 19 seed orchards of P. abies, which were at least 40 years old and as such were suitable for D. micans reproduction (table 1, fig. 1). The occurrence of D. micans in each seed orchard was evaluated by visual inspection of 100 randomly selected trees of P. abies. Research Randomizer (available at https://www.randomizer.org/) was used to select the trees in each seed orchard. As all trees in each seed orchard are numbered, the random numbers generated by Research Randomizer were used to select the trees. Figure 2 shows a schematic map of a seed orchard (Site 15, table 1) with a random distribution of selected trees. Characteristic symptoms of damage by D. micans (Francke-Grosmann & Ruhm, Reference Francke-Grosmann and Ruhm1954; Forestry Commision, 2015) were assessed on the stems (from the ground level and up to a height of 2 m) and the damage symptoms were divided into three categories: fresh with living beetles, fresh without living beetles (buried in resin) and old (of the previous or earlier years).
Fig. 1. Map of Lithuania showing visited seed orchards of P. abies and detected (filled circles) and undetected (open circles) incidences of D. micans. Seed orchards are numbered as in table 1. The P. pungens plantation is noted with a triangle.
Table 1. Seed orchards of P. abies used to evaluate occurrence of D. micans in different parts of Lithuania.
Determining the phenology of D. micans
The phenology of D. micans was investigated during April–September 2014. This was done at two sites and included two tree species: a P. abies seed orchard (Site 15, table 1) and a plantation of Picea pungens that was located at the Ukmergė forest nursery (N55.24, E24.88) (fig. 1). The distance between the sites was ca. 110 km. Average temperatures for each site and observation period were obtained from the local meteorological stations of the Lithuanian Hydrometeorological Service (Vilnius, Lithuania) and are presented in fig. 3. In total, five trees of P. abies and five of P. pungens were selected. The selected trees were characterized by a high colonization and activity of D. micans in the lower part of their stems, which were easily accessible for regular observations. The relatively low number of selected trees was due to low incidences of D. micans in 2014. The observations were performed regularly at 2-week intervals. During each observation, the bark was carefully removed from the investigated trees and between 10 and 20 eggs and/or individual D. micans at different developmental stages were extracted each time from the nests. Special care was taken not to damage extracted individuals and not to disturb the nests. After each extraction of D. micans, the bark was carefully returned and fastened to the trees in order to preserve the remaining eggs/individuals of D. micans and to minimize the negative effects of disturbance that may interfere with their further development. The collected D. micans eggs/individuals were placed separately in 1.5 ml centrifugation tubes in 70% ethanol solution and transported to the laboratory for morphological assessment. The length and width of collected eggs, larvae, pupae and adults of D. micans, as well as width of larval heads was measured at the accuracy of 0.01 mm using a dissection microscope (Stemi 2000, Carl Zeiss, Oberkochen, Germany). It is known that heads of larvae at different developmental stages differ in width and this feature can be used for their classification (Voolma, Reference Voolma1980).
Fig. 2. A schematic map of the Šakiai seed orchard (Site 15, table 1) of P. abies showing random distribution of selected trees, which were used to assess incidents of D. micans. The D. micans-infested trees are shown as triangles, non-infested trees are shown as filled circles and non-assessed trees are shown as open circles. Gaps in the seed orchard indicate missing trees.
Fig. 3. Average temperatures for April–September 2014 recorded at the nearest hydrometeorological station for each P. abies seed orchard and P. pungens plantation, which were used for phenology observations of D. micans. Measurements are averaged and presented for every 10 days of each month.
Culturing of fungi from D. micans
Fungal culturing was carried out from the 25 larvae, ten pupae and ten adults of D. micans sampled at each site of phenology observations. Eggs were not used for culturing. Two nutrient media were used to culture the fungi associated with D. micans, malt extract agar medium and yeast extract agar medium (Atlas, Reference Atlas1997) supplemented with 0.5% of each antibiotic chloramphenicol and streptomycin. Before the beetles were placed in 9 cm diameter Petri dishes, these were surface sterilized for 2 min in 5% hypochlorite and rinsed twice in sterile distilled water. One individual was placed per Petri dish. Prepared dishes with insects were stored for 1 month at room temperature (ca. 21°C) in the dark. The Petri dishes were inspected every 2–3 days and any outgrowing mycelium was immediately transferred to new Petri dishes with malt agar medium. Fully developed fungal colonies were divided into morphological groups and representative cultures per each group were taxonomically identified based on morphological characteristics of fungal mycelia and reproductive structures, which were examined using a light microscope (Axiostar plus, Carl Zeiss, Oberkochen, Germany).
Direct sequencing of fungi from D. micans
For the identification of fungal communities, larvae and adults of D. micans were sampled during field surveys in the P. abies seed orchards (table 1) and in a plantation of P. pungens. In trees with signs of beetle attack, the bark was carefully removed using a knife, and adults and larvae were placed into 1.5 ml centrifugation tubes and transported to the laboratory. Collected insects were stored at −20°C until used for DNA extraction and sequencing of fungi. Genomic DNA was isolated from 125 larvae (25 larvae were collected in each of five sites: 2, 7, 14, 15 and 16 as in table 1) and from 25 adults (collected in Ukmergė forest nursery during phenology observations) of D. micans. Prior to isolation of DNA, larvae and adults were placed separately in 2-ml screw-cap centrifugation tubes and freeze-dried at −60°C for 2 days. No surface sterilization was carried out. Isolation of DNA, amplification and sequencing was carried out as described by Menkis et al. (Reference Menkis, Marčiulynas, Gedminas, Lynikienė and Povilaitienė2015). Briefly, material from larvae and adults were homogenized separately using a FastPrep shaker (Precellys 24; Bertin Technologies, Rockville, MD, USA) and subjected to isolation of genomic DNA using the CTAB method (Menkis et al., Reference Menkis, Marčiulynas, Gedminas, Lynikienė and Povilaitienė2015). Following DNA isolation, concentration of genomic DNA was determined using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Diluted (1–10 ng µl−1) genomic DNA samples were amplified separately by PCR using the primer pair ITS9 (5′-GAACGCAGCRAAIIGYGA-3′) (Ihrmark et al., Reference Ihrmark, Bodeker, Cruz-Martinez, Friberg, Kubartova, Schenck, Strid, Stenlid, Brandstrom-Durling, Clemmensen and Lindahl2012) and ITS4 (5′-xxxxxxxxTCCTCCGCTTATTGATATGC-3′) (White et al., Reference White, Bruns, Lee, Taylor, Innis, Gelfand, Sninsky and White1990) containing 8 base pairs (bp) sample identification barcodes denoted by ×. The concentration of purified PCR products was determined using a ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and an equimolar mix of all PCR products was used for Ion Torrent sequencing. The sequencing library construction and sequencing using a 316 chip as a part of the larger sample were carried out by SciLifeLab (Uppsala, Sweden).
Bioinformatics
The sequences generated were subjected to quality control and clustering in the SCATA NGS sequencing pipeline (http://scata.mykopat.slu.se). Quality filtering of the sequences included the removal of short sequences (<200 bp), sequences with low read quality, primer dimers and homopolymers, which were collapsed to 3 bp before clustering. Sequences that were missing a tag or primer were excluded. The primer and sample tags were then removed from the sequence, but information on the sequence association with the sample was stored as meta-data. The sequences were then clustered into different taxa using single-linkage clustering based on 98.5% similarity. The most common genotype (real read) for each cluster was used to represent each taxon. For clusters containing two sequences, a consensus sequence was produced. Fungal taxa were taxonomically identified using RDP pipeline classifier available at https://pyro.cme.msu.edu/index.jsp (Center for Microbial Ecology, Michigan State University, Michigan, USA). Sequences showing <80% similarity to phylum level were considered to be of non-fungal origin and were excluded from further analyses. Taxonomic names identified using RDP pipeline classifier are listed in online Supplementary table 1 and represent the closest match as compared to the reference sequence at a particular level of similarity. The following similarity levels should be used for reliable identification of fungal taxa: taxon level 98–100%, genus level 94–97% and unidentified <94% (online Supplementary table 1). In addition, the 20 most common fungal taxa were identified using GenBank (NCBI) database and the Blastn algorithm. The criteria used for identification were: sequence coverage >80%; similarity to taxon level 98–100%, similarity to genus level 94–97%. Sequences not matching these criteria were considered unidentified and were given unique names as shown in table 2.
Table 2. Relative abundance (%) of the 20 most common fungal taxa directly sequenced from larvae and adult beetles of D. micans. The number of individuals (larvae and adult beetles) sequenced is shown in the parenthesis. Sites are numbered as in table 1.
1 The sequence similarity column shows the base pair comparison between the query sequence and the reference sequence at NCBI database, with the percentage of sequence similarity in the parenthesis.
Statistical analyses
Differences in relative abundance (estimated from the total number of sequences for each sample) of dominant fungal taxa detected in larvae and in adults of D. micans was compared by non-parametric chi-square (χ2) test (Magurran, Reference Magurran1988). Shannon diversity index and quantitative Sorensen similarity index were used to characterize diversity and composition of fungal communities in larvae and adults of D. micans (Shannon, Reference Shannon1948; Magurran, Reference Magurran1988). Clark–Evans nearest neighbour method (Clark & Evans, Reference Clark and Evans1954) was used to estimate whether the spatial distribution of attacked trees in the Šakių seed orchard (Site 15) of P. abies and in a plantation of P. pungens (both used for phenology observations) were random (R = 1), even (R > 1) or clustered (R < 1). The statistical test of significance of the nearest neighbour statistic provides the degree to which the distribution of individuals on a given area differs from that of a random distribution, and is estimated as the ratio of expected and observed mean value of the nearest neighbour distances.
Results
Occurrence of D. micans
Inspection of different seed orchards of P. abies showed that the occurrence D. micans was relatively rare and in many cases was not detected in western Lithuania (fig. 1). Recent and characteristic signs of damage, as well as, viable individuals of D. micans were detected in seed orchards of Dubravos (Site 2), Marijampolės (Site 7), Rokiškio (Site 14), Šakių (Site 15) and Šilutės (Site 16) forest enterprises (table 1). In the remaining seed orchards, the majority of D. micans occurrences were from the previous year or earlier years. In these sites, there were a number of adult females that were buried in the resin, and thereby failed to colonize the trees. In addition to the seed orchards of P. abies, D. micans was also detected in a plantation of P. pungens situated in mid-eastern Lithuania (fig. 2). Analysis of the spatial distribution of attacked trees in the Šakių seed orchard (Site 15) of P. abies and in the plantation of P. pungens using Clark–Evans nearest-neighbour method revealed that in both sites, there was clustered distribution of attacked trees (R < 0.35 and R < 0.47, respectively).
Phenology of D. micans
D. micans overwintered under the bark as larvae of the II and III developmental stages. Following overwintering, larvae were actively feeding and at the end of April under the bark of P. pungens there were larvae of both III and V developmental stages, and pupae. Their abundance was 26.0, 57.0 and 17.0%, respectively. At the same time, under the bark of P. abies there were larvae of II, III and IV developmental stages abundance was 14.8, 37.0 and 48.2%, respectively. In the middle of May, in P. pungens there were 79.0% of the V developmental stage larvae, 4.3% of pupae and 16.7% of young adults. For P. abies there were only larvae of III (6.0%), IV (67.0%) and V (27.0%) developmental stages. In the beginning of June, in P. pungens there were 24.2% of larvae of the V developmental stage, 24.2% of pupae, 30.3% of young adults and 21.3% of mature adults, while in P. abies we detected only larvae of the V developmental stage. By mid-June, in P. pungens we observed the beginning of the flying season of mature adults and new entry holes on the trees, while under the bark there were 34.5% of young adults and 65.5% of mature adults of D. micans. At this time, in P. abies there were only larvae of the V developmental stage. At the end of June, under the bark of P. pungens we detected a new generation of eggs of D. micans, while in P. abies there were larvae of the V developmental stage (44.4%) and pupae (55.6%). In the beginning of July, in P. pungens we recorded a period of intensive flying of mature adults of D. micans, numerous new entry holes in the same trees and in the neighbouring trees, and extensive egg laying under the bark. Eggs were laid in the cambium and each female produced up to 300 eggs, which were laid in groups of 50–80 in interconnected chambers. At the same time, in P. abies under the bark there were still larvae of the V developmental stage and pupae, but all of them were dead as these were destroyed by microbial parasites and/or predating insects. Thus, further observations in P. abies were not possible. Observations in P. pungens were continued and in the beginning of August there were 56.9% of eggs and 20.0% of larvae of the I developmental stage and 23.1% of the II stage under the bark. In September, we detected larvae of both II (60.0%) and III (40.0%) developmental stages, and later observations showed that larvae of these two stages remained under the bark for overwintering. In both sites, the average temperatures were similar during the entire period of phenology observations (fig. 3).
Morphology of D. micans
Morphological assessment showed that the head width of larvae of the I developmental stage was 0.48 ± 0.02 mm, and the body length was 2.20 ± 0.01 mm; of the II stage it was 0.68 ± 0.03 and 2.90 ± 0.04 mm, respectively; of the III stage it was 0.98 ± 0.03 and 4.79 ± 0.11 mm, respectively; of the IV stage it was 1.26 ± 0.02 and 5.39 ± 0.03 mm, respectively; and of the V stage it was 1.71 ± 0.01 and 8.18 ± 0.15 mm, respectively. The body length of pupae was 7.06 ± 0.24 mm. The body length of young adults was 7.10 ± 0.25 mm and width was 2.84 ± 0.06 mm, and remained similar at maturity. The young adults were light brown in colour, and as they matured, the colour darkened to brown and black. Eggs of D. micans were white, oval and 0.61 ± 0.01 mm in width and 1.10 ± 0.03 mm in length.
Fungi cultured from D. micans
Fungal culturing resulted in 115 pure cultures, and based on mycelial morphology were divided into 10 groups potentially representing different taxa. Morphological identification showed that in a majority of the cases, fungal culturing resulted in yeasts of the genus Saccharomyces, which were growing out from 70.0% of larvae but not from pupae or adult beetles. In contrast, fungi from the genus Penicillium, that represented 6.1% of cultures, were cultured from different developmental stages of D. micans. Leptographium sp. represented 22.0% of the fungal cultures. This species was also cultured from different developmental stages of D. micans. A single culture of Graphium sp. was cultured from an adult beetle.
Fungi directly sequenced from D. micans
Ion Torrent sequencing from 125 larvae and 25 adults of D. micans and subsequent quality filtering showed the presence of 139,097 high-quality ITS rDNA sequences (mean length of 347 bp). Clustering of these sequences resulted in 376 non-singleton contigs (at 98.5% similarity representing different taxa) and in 1167 singleton contigs, which were excluded from the further analyses. Taxonomic identification using the RDP pipeline classifier showed that 319 taxa were of fungal origin (all non-singleton fungal sequences are provided in online Supplementary table 1) and 57 of non-fungal origin, which were later excluded from further analyses. In this study, the detected fungi were 82.3% Ascomycota, 16.8% Basidiomycota and 0.9% Mucoromycotina. The absolute richness of fungal taxa was higher in larvae (277 taxa out of 86,784 sequences) than in adults (95 out of 50,188), and the χ2 test showed that taxa richness between these datasets differed significantly (P < 0.0001). Information on the 20 most common fungal taxa representing 98.4% of all fungal sequences is shown in table 2.
Among these, however, five taxa representing 7.2% of fungal sequences could not be identified to taxon or genus level and remained unidentified (table 2). The most common fungal taxa were Peterozyma toletana (37.5%), Yamadazyma scolyti (30.0%) and Kuraishia capsulate (17.7%). The plant pathogen Leptographium lundbergii (0.2%) was the fourteenth most common fungus, though at low abundance, was detected in both larvae and beetles (table 2). The χ2 test showed that among the 20 most common fungal taxa, the relative abundance of P. toletana, K. capsulate, Unidentified sp. 2183_3, Nakazawaea holstii, Candida sp. 2183_5, Rhodotorula sp. 2183_7, Unidentified sp. 2183_11, Phoma herbarum, Zalerion arboricola and Aureobasidium pullulans was significantly higher in larvae than in beetles. On the other hand, the relative abundance of Y. scolyti, Cladosporium herbarum, Candida sp. 2183_8, L. lundbergii and Unidentified sp. 2183_22 was significantly higher in beetles than in larvae. The remaining 299 fungal taxa were very rare and their relative abundances varied between 0.001 and 0.04% (online Supplementary table 1). The Sorensen index of similarity of fungal communities was low (0.23) when these were compared between larvae and beetles. The Shannon diversity index of fungal communities was 2.1 in larvae and 1.3 in beetles.
Discussion
During outbreaks, D. micans can cause severe damage to stands of Picea spp. (Bevan & King, Reference Bevan and King1983). At the time of this study, the population density of D. micans appeared to be at the minimum, and the beetles caused relatively mild damage to trees of P. abies in central and eastern parts of Lithuania. Clark–Evans nearest-neighbour analysis of spatial distribution showed that D. micans likely disperses over relatively short distances and attacks primarily the neighbouring trees, and thus resulting in the clustered distribution of damaged trees (fig. 2). The surveys showed that at the same time point within a site there were individuals of D. micans of different developmental stages. Moreover, it appeared that the development of D. micans was greatly dependent on the host tree species as temperatures in both sites were similar during the period of observations (fig. 3). However, the possibility should not be excluded that multiple factors have influenced the development of D. micans, suggesting that further studies might be needed to address this issue. Moreover that changing environments may have a major effect on the population dynamics and phenology of insects (Chaves et al., Reference Chaves, Morrison, Kitron and Scott2012). Nevertheless, in the present study, development of D. micans was quicker and D. micans had larger nests under the bark of P. pungens than of P. abies. This is further supported by Wainhouse & Beechgarwood (Reference Wainhouse and Beechgarwood1994) who showed that development of D. micans larvae differed in different conifer trees species. Additionally, the association of D. micans with P. pungens and its local outbreaks were also reported from Czech Republic (Lukasova et al., Reference Lukasova, Holusa and Knizek2014). Lukasova et al. (Reference Lukasova, Holusa and Knizek2014) concluded that it may constitute a new threat to urban and forest trees of P. pungens, and that special attention should be given to these trees while monitoring population dynamics of D. micans. In the present study, the development of D. micans under the bark of P. abies was prolonged and often unsuccessful due to activity of microbial parasites and/or predating insects. Sevim et al. (Reference Sevim, Demir, Tanyeli and Demirbag2010) showed that pathogenic efficacy of entomopathogenic fungi against D. micans was 83–100% for larvae and 23–100% for beetles. Rhizophagus grandis, the specific predator of D. micans, was shown to have up to 84% effectiveness against D. micans when co-occurring in the galleries (Alkan Akinci et al., Reference Alkan Akinci, Özcan and Eroglu2003). Furthermore, our study confirmed that in Lithuania, D. micans exhibits the following developmental stages: adult, egg, I–V developmental stage larvae and pupa. These developmental stages and morphological characteristics were similar to those reported from other European countries (e.g., Forestry Commision, 2015) indicating that information acquired in the present study has broader application elsewhere with similar environmental conditions.
Although the bacterial flora associated with D. micans has been relatively well investigated (Yilmaz et al., Reference Yilmaz, Sezen, Kati and Demirbag2006), the fungal communities have received less attention. The fungal studies have been mainly focused on the isolation and testing of fungal pathogens for biocontrol (Sevim et al., Reference Sevim, Demir, Tanyeli and Demirbag2010). The present study, by using both fungal culturing methods and high-throughput sequencing, revealed that D. micans is associated with species-rich fungal communities that was dominated by the yeasts (table 2). Among these, P. toletana has been mainly isolated from insect frass in fir and spruce trees or from wood products. This suggests that the primary habitat of, P. toletana is in association with wood-boring insects and their larvae (Kurtzman et al., Reference Kurtzman, Fell and Boekhout2011). The second most common fungus of this study, Y. scolyti, was also commonly isolated from tree-boring insect frass (Kurtzman et al., Reference Kurtzman, Fell and Boekhout2011). Interestingly, Boone et al. (Reference Boone, Six, Zheng and Raffa2008) reported that parasites and dipteran predators may exploit volatiles of fungal symbionts including that of Y. scolyti to locate bark beetles. K. capsulata, the third most common fungus, has similar ecology to that of P. toletana and Y. scolyti. However, K. capsulata produces extracellular polysaccharides that cause fungal cells to adhere to the bark beetles, suggesting that the fungus has likely co-evolved with its beetle host, and thereby has specifically adapted to dispersal by the beetles (Wicherham et al., Reference Wicherham, Hansenula and Sydow1970). Similar to what has been shown for I. typographus (Persson et al., Reference Persson, Vasaitis, Langstrom, Ohrn, Ihrmark and Stenlid2009), the present study reports the occasional occurrence of several basidomycetes wood-decay fungi including Fomitopsis pinicola, Sistotrema brinkmannii and Ganoderma applanatum (online Supplementary table 1). Among these, F. pinicola was previously detected on several Dendroctonus species in North America including: D. pseudotsugae, D. brevicomis, D. ponderosae and D. valens (Castello et al., Reference Castello, Shaw and Furniss1976; Pettey & Shaw, Reference Pettey and Shaw1986; Lim et al., Reference Lim, Kim, Lu and Breuil2005). Our study also revealed the presence of several plant pathogens including L. lundbergii (table 2). The genus Leptographium include important forest pathogens and blue stain fungi (Jacobs et al., Reference Jacobs, Solheim, Wingfield and Wingfield2005), which are commonly found in association with bark beetles (Linnakoski et al., Reference Linnakoski, de Beer, Duong, Niemela, Pappinen and Wingfield2012). Although Lieutier et al. (Reference Lieutier, Vouland, Pettinetti, Garcia, Romary and Yart1992) were able to culture several ophiostomatoid fungi, with O. canum being most common, in the present study only O. bicolor was detected occasionally in the larvae of D. micans by the sequencing method (online Supplementary table 1). For I. typographus, Persson et al. (Reference Persson, Vasaitis, Langstrom, Ohrn, Ihrmark and Stenlid2009) have also detected a number of ophiostomatoid fungi including O. bicolor using both fungal culturing and molecular methods. Together, this demonstrates that O. bicolor is associated with different species of beetles. Furthermore, these studies highlight that fungal culturing and sequencing methods are both necessary and required to obtain a more complete picture of fungal communities associated with beetles (Lim et al., Reference Lim, Kim, Lu and Breuil2005; Giordano et al., Reference Giordano, Garbelotto, Nicolotti and Gonthier2012). Taken together, our study demonstrated that D. micans associates with different functional groups of fungi, including pathogens, and that some of these fungi may have negative effects on both the health of forest trees and timber quality.
Conclusions
At the time of the study, the population of D. micans was at its minimum with individuals mainly detected in central and eastern parts of Lithuania. D. micans is an annual univoltine species but development of individuals is largely dependent on the host tree species and/or the specific environmental factors of a site. D. micans is associated with a species-rich community of fungi, whereby the majority belong to yeasts from a class Saccharomycetes.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0007485316001048.
Acknowledgements
We thank Dr Diem Nguyen at the Department of Forest Mycology and Plant Pathology, SLU for language revision. This research was funded by the European Regional Development Fund under the Global Grant measure, project no. VP1-3.1-ŠMM-07-K-02-001.
