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Article

Exploring Fungal Communities in the Needles of Marginal Conifer Tree Populations

by
Jelena Lazarević
1,* and
Audrius Menkis
2,*
1
Biotechnical Faculty, University of Montenegro, Mihaila Lalića 1, 81000 Podgorica, Montenegro
2
Department of Forest Mycology and Plant Pathology, Uppsala BioCenter, Swedish University of Agricultural Sciences, 75007 Uppsala, Sweden
*
Authors to whom correspondence should be addressed.
Forests 2025, 16(6), 968; https://doi.org/10.3390/f16060968
Submission received: 26 April 2025 / Revised: 26 May 2025 / Accepted: 4 June 2025 / Published: 7 June 2025
(This article belongs to the Special Issue Recent Scientific Developments in Forest Pathology)
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Abstract

:
In Montenegro, coniferous forests play a key ecological role in maintaining ecosystem stability. Root-associated mycorrhizal fungi and saprotrophic fungi inhabiting forest soils are well known for their roles in nutrient cycling, organic matter decomposition, and supporting host tree health. In contrast, the fungal communities residing within conifer needles, despite potentially important ecological functions, remain largely underexplored, particularly in natural and marginal forest ecosystems such as those in the Balkans. This study aimed to investigate the diversity and community composition of needle-associated fungi in three native conifers: Picea abies and Abies alba (at the edge of their native range), and the endemic Pinus heldreichii, from different mountainous regions in Montenegro. High-throughput sequencing was conducted to assess fungal diversity and community composition. Dothideomycetes dominated fungal communities in all three tree species, followed by Leotiomycetes and Tremellomycetes. Multivariate analysis revealed distinct fungal communities in P. heldreichii, whereas fungal communities in A. alba and P. abies were partially overlapping. Functional classification showed a dominance of saprotrophic, pathogenic, and endophytic fungi, with P. heldreichii exhibiting the highest proportion of saprotrophs, while A. alba and P. abies showed a considerable proportion of pathogens. The findings highlight strong host specificity, biogeographical influences, and the ecological importance of fungal communities in coniferous forests. This study provides new insights into the diversity and functional roles of needle-associated fungi, emphasizing the need for conservation efforts to maintain microbial biodiversity in native forests of Montenegro.

1. Introduction

Forests are vital ecosystems that play a key role in maintaining global biodiversity, supporting ecosystem services such as soil stabilization, including erosion control, carbon sequestration, water regulation, and nutrient cycling. Montenegro is a predominantly mountainous country in Southeastern Europe (41°52′ and 43°32′ latitude North and 18°26′ and 19°32′ longitude East), with forest covering more than 60% of the land area. Forests are predominantly natural and forest types vary across altitudes, hosting a rich biodiversity shaped by geographical and climatic diversity in the country [1].
The conifer forest zone in Montenegro is limited to mountain–subalpine regions. Norway spruce (Picea abies Karst) and silver fir (Abies alba Mill.) are keystone European conifers with important ecological and economic values [2,3]. On the Dinaric Mountains in Montenegro, both P. abies and A. alba reach the southern edge of their natural distribution. P. abies is a key component in the boreal forest ecosystems [4], which is well adapted to the continental mountain climates of northern and central regions of Montenegro, where it grows in mixed stands with A. alba. Bosnian pine (Pinus heldreichii H. Christ) is a tertiary relict and subendemic to the Balkans, occurring in fragmented forest stands at high altitudes (1200–2000 m) on karst terrain in the Oro-Mediterranean zone and often under harsh environmental conditions [5,6]. The coniferous forest of P. abies and A. alba, and those of P. heldreichii, occupy distinct ecological niches that reflect their evolutionary adaptations to the diverse climatic and geographical conditions such as boreal and the Mediterranean, respectively (Figure 1). These mountain forests are hotspots of unique biodiversity and provide important ecosystem services. However, they represent vulnerable ecosystems, facing the effects of climate change and over-exploitation.
Needle-associated fungi may have a significant effect on host tree health, ecosystem functioning, and forest resilience [7,8], particularly in regions with unique ecological conditions such as the Balkan region [9]. However, studies investigating needle-associated fungal communities in conifers from the Balkan Peninsula are scarce, leaving a significant knowledge gap regarding fungal diversity and its ecological implications.
The aim of this study was to explore the diversity and community composition of fungi associated with healthy-looking needles of P. abies and A. alba at the edge of their native range, as well as the endemic P. heldreichii, across different mountainous regions in Montenegro. We hypothesized that fungal communities associated with conifer needles would exhibit significant differences between tree species due to host specificity. We have implemented an extensive and wide-scale sampling with the intention to both reveal the diversity of fungal taxa with important ecological functions and to uncover the unique biodiversity of these forests by employing high-throughput sequencing techniques.

2. Materials and Methods

2.1. Study Sites and Sampling

Sampling was conducted across geographically distinct regions in Montenegro to represent the diverse climatic zones and major coniferous forest types of the studied coniferous forests in the region (Figure 2). In northwestern and central Montenegro, the Mt. Durmitor site comprises mixed coniferous forests of P. abies and A. alba (Ass. Abieti-Piceetum abietis [10] ) at 1350–1450 m alt., developed on kalkomelanosols [11] (Table 1). Associated vegetation includes species such as Vaccinium myrtilis, Galium rotundifolum, Luzula sp., and Oxalis acetosella. The Mt. Bjelasica site comprises high-altitude (1700 m) mixed P. abies and A. alba (Abietinum albae subalpinum) forests of low density, developed on ranker [11]. Associated vegetation includes species such as Juniperus nana, Hypericum alpigenum, H. maculatum, Vaccinium sp., and Rosa alpina. Both the Mt.Durmitor and Mt.Bjelasica sites are characterized by a humid boreal mountain climate (Dfc-Dfbx‘’, with mean temperatures during the growing season (April–October) averaging 14.3 °C, extreme temperatures ranging from −30.4 °C to 29.6 °C, and annual precipitation of 1100 mm, of which 460 mm falls during the growing season [12].
In southeastern Montenegro, the Mt. Prokletije (Bogićevica) site was dominated by P. abies forests with admixture of A. alba (Abieti-Piceetum abietis) at 1400–1700 m alt. at Dystrict cambisol [10,11] (Table 1). The Mt. Visitor site comprises mixed coniferous forests of P. abies and A. alba at high altitudes of 1700–1900 m, on kalkocambisol [11]. In this region, the forest understory includes many endemic and relict species such as Wulfenia blecicci, Daphne blagayana, Pinus peuce, and Acer heldreichii [10]. These forests experience a perhumid sub-Mediterranean mountain climate (Cfs’’b), with a mean growing season temperature of 13.1 °C, extremes of −31 °C to 27 °C, annual precipitation of 880 mm, and growing season precipitation of 294 mm [12].
Relict pine forests dominated by P. heldreichii at the Mt. Kuči site are composed of Pinetum heldreichii bertisceum forest, across a wide zone, up to the tree line (1250–1700 m), while on the Mt. Prekornica and Mt. Orjen sites, they were shown at a lower altitude (1200–1300 m), where the bigger forest populations (Ass. Pinetum heldreichii mediteraneo-montanum) are present [10] (Table 1). Forests on Mt. Kuči support a diverse understory including Daphne oleoides, Daphne blagayana, Wulfenia blecicci, Thymus albanicus, Acinos alpinus spp. Dinaricus, and Achillea abrotanoides. Sites at Mt. Orjen and Mt. Prekornica had the following understory vegetation: Juniperus sibirica, Senecio visianianus, Dianthus petraceus, Festuca heterophylla, and Gentiana amblyphylla. These forests represent Mediterranean and sub-Mediterranean mountain climates (Cfsb/Cfws’’bx’’), with annual temperatures averaging 2–4 °C, extreme seasonal temperatures ranging from −37 °C to 40 °C, and annual precipitation between 2500 and 5000 mm, with only 8%–10% falling in the summer, and all three were developed on soils of varied development, i.e., with different depths between leptosol and molic leptosol [10,11,12].
At each sampling site, the forest stands were healthy-looking. Sampling took place during the autumn/winter months (November–December). Sampling was conducted by randomly selecting 1–2-year-old shoots with needles from 50 trees of each tree species per site (Table 1), growing at a minimal distance of 50 m between individual trees. Samples were placed in individual plastic bags and transported to the laboratory, where healthy-looking current-year needles were selected for DNA extraction, and stored at −20 °C before further processing. Taken together, the sampling resulted in 150 needle samples of P. abies from three sites, 200 needle samples of A. alba from four sites, and 150 of P. heldreichii from three forest sites.

2.2. Laboratory Work

Individual needle samples containing current-year healthy-looking needles were freeze-dried at −60 °C for 48 h (Alpha 1–4 LD, Martin Christ, Osterode Am Harz, Germany). After freeze-drying, about 200 mg of freeze-dried needle material was homogenized in a Tissue Lyser II (Qiagen, Hilden, Germany) and used for isolation of genomic DNA, using a DNeasy 96 Plant kit (Qiagen, Hilden, Germany) according to the protocol of the producer. The DNA concentration of each sample was determined using a NanoDropTM One spectrophotometer (Thermo Scientific, Rodchester, NY, USA) and adjusted to 10 ng/µL. Following DNA isolation, the fungal-specific ITS2 rDNA region was amplified by PCR using the barcoded primers gITS7 (5′-GTGARTCATCGARTCTTTG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) as described by Ihrmark et al. [14]. All DNA samples of the same tree species and site (50 samples) were amplified using primers with the same barcode, resulting in a total of 10 different barcodes. Each PCR reaction included template DNA (10 ng/µL), primers with unique barcodes, and Taq polymerase under the manufacturer’s recommended conditions. PCR controls were also included. Amplifications were performed using the Applied Biosystems 2720 thermal cycler (Foster City, CA, USA). An initial denaturation step started at 95 °C for 2 min, followed by 27 amplification cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 60 s. The thermal cycling was ended by a final extension step at 72 °C for 7 min. Amplified products were verified using 1% agarose gel electrophoresis stained with Nancy-520 (Sigma-Aldrich, Stockholm, Sweden) and purified using a sodium acetate and ethanol mixture (1:15). Purified PCR products were quantified using a Qubit fluorometer 4.0 (Thermo Fisher Scientific, Waltham, MA, USA), pooled in equimolar mix, and subjected to high-throughput sequencing using the PacBio RSII platform at SciLifeLab, Uppsala, Sweden.

2.3. Bioinformatics

The bioinformatics methodology followed the principles outlined by Mischerikova et al. [15]. Sequence processing was conducted using the SCATA NGS sequencing pipeline, accessible at https://scata.mykopat.slu.se/ (accessed on 10 February 2025). The process began with quality control, which involved filtering out sequences shorter than 200 bp, low-quality reads, primer dimers, and homopolymers, which were collapsed to 3 bp prior to clustering. Only sequences containing both a barcode and primer were retained. Barcodes and primers were subsequently removed from the sequences, but metadata linking samples to sequences was preserved. Sequences were clustered into taxa using single-linkage clustering with a 98% similarity threshold. For each cluster, the most common genotype sequence was selected for taxonomic identification. In cases where clusters contained only two sequences, a consensus sequence was generated. Taxonomic identification was performed using the GenBank database and the Blastn algorithm [16]. The reliability of reference sequences was manually verified for each taxon by examining the metadata of top BLASTn hits in GenBank, prioritizing sequences from type specimens, well-annotated voucher specimens or peer-reviewed studies. Identification criteria included sequence coverage > 80%, 94%–97% similarity for genus-level identification, and ≥98% similarity for species-level identification. Sequences not meeting these criteria were assigned to higher taxonomic ranks and given unique identifiers. Assembled representative sequences of fungal non-singleton OTUs have been submitted to GenBank under accession numbers PV529837–PV530182.

2.4. Statistical Analyses

Fungal community diversity and composition were analyzed using the Shannon diversity index (to assess alpha diversity), the qualitative Sørensen similarity index (to evaluate beta diversity based on presence/absence data), and detrended correspondence analysis (DCA) (to visualize compositional differences among sites and host tree species) using Canoco 5 [17,18,19]. Fungal richness (total number of OTUs) among different tree species was compared using non-parametric chi-square tests. Differences in Shannon diversity index values between sites and tree species were assessed using the non-parametric Mann–Whitney U test. Fungal functional guilds were assigned using the FUNGuild online database available at https://www.funguild.org/(accessed on 15 March 2025). To assess the association between tree species and fungal OTUs, we performed an indicator species analysis using the Kruskal–Wallis test on the relative abundance of each OTU to identify those showing significant differences among host species. Only OTUs with a significance level of p < 0.05 were considered. These statistical analyses were conducted using Python (version 3.12.0) and the SciPy package [20]. All analyses were performed on non-transformed data.

3. Results

High-throughput sequencing and quality filtering yielded 15,269 high-quality reads. Clustering these reads at a 98% similarity threshold identified 428 OTUs. Of these, 346 OTUs, represented by 9549 reads (1364 reads per sample on average), were confirmed as fungal (Table 2) and retained for further analysis (Table S1), while 82 non-fungal OTUs were excluded. Observed OTU richness was highest in needles of P. abies (229), followed by A. alba (218) and P. heldreichii (111) (Table 2). When comparing OTU richness among tree species, significant differences were observed between A. alba and P. abies (p < 0.0001) and between A. alba and P. heldreichii (p < 0.0001), while no significant difference was detected between P. abies and P. heldreichii (p = 0.48).
The analysis of fungal OTU richness in needles among different tree species revealed that A. alba had 55 unique OTUs, P. abies had 67 unique OTUs, and P. heldreichii had 53 unique OTUs. A total of 113 OTUs were shared between A. alba and P. abies, 12 OTUs were shared between A. alba and P. heldreichii, and 8 OTUs were shared between P. abies and P. heldreichii. In total, 38 fungal OTUs were found to be common across all three species (A. alba, P. abies, and P. heldreichii) (Figure 3).
The fungal community composition varied among the three tree species, with A. alba, P. abies, and P. heldreichii showing distinct distributions of fungal classes (Figure 4). Dothideomycetes was the most dominant fungal class across all tree species, representing 33.1% of the community in A. alba, 63.7% in P. abies, and 54.7% in P. heldreichii. In A. alba, the second most abundant class was Leotiomycetes (23.3%), followed by Pucciniomycetes (10.8%) and Tremellomycetes (9.5%). Similarly, P. abies exhibited Leotiomycetes as the second most dominant class (10.5%), but with a greater relative abundance of Tremellomycetes (7.0%) and Eurotiomycetes (6.2%). For P. heldreichii, Leotiomycetes accounted for 21.2% of the fungal community, while Pucciniomycetes (12.0%) and Tremellomycetes (5.6%) were also common. Less abundant classes, including Pezizomycotina, Microbotryomycetes, Exobasidiomycetes, Lecanoromycetes, Sordariomycetes, and Taphrinomycetes, contributed to the overall diversity, but their relative proportions varied among tree species.
The 20 most abundant fungal OTUs across all tree species are shown in Table 3. Among these, the most abundant was the unidentified fungus ID 3598_3 with a total relative abundance of 10.7%. It was particularly dominant in needles of P. heldreichii (25.2%) but was less frequent in P. abies (6.4%) and A. alba (3.7%). Another unidentified species (ID 3598_2) was very common in P. abies (15.7%) but less abundant in A. alba (2.2%) and absent in P. heldreichii. Similarly, the yeast-like fungus Vishniacozyma victoriae showed a higher presence in P. abies (5.4%) but was less frequent in A. alba (1.3%) and absent in P. heldreichii. Some taxa showed a relatively balanced distribution across all species, including Pseudeurotiaceae sp., which was present in all three hosts with similar abundances (2.0% in A. alba, 4.5% in P. abies, and 2.2% in P. heldreichii). Similarly, Cladosporium sp. exhibited comparable abundance across all tree species (1.3%–2.7%). A few taxa demonstrated host specificity. The rust fungus Chrysomyxa nagodhii, known to be associated with needle infections, was exclusively detected in P. abies (12.6%), while unidentified fungus (ID 3598_24) was only detected in P. heldreichii (11.6%) (Table 3).
The DCA ordination plot shows the distribution of fungal communities associated with three tree species across different mountain sites (Figure 5). The presence of distinct clustering patterns indicated clear differences in fungal communities among the tree species. Pinus heldreichii was positioned to the left in the ordination, exhibiting a unique fungal community structure compared to A. alba and P. abies, which were more closely associated and showed a degree of overlap. The mountain sites demonstrated geographic variation in fungal communities. The fungal communities from Mt. Kuči (K), Mt. Orjen (O), and Mt. Prekornica (Pr) were clustered within the P. heldreichii group, whereas sites from Mt. Bjelasica (B), Mt. Prokletije (P), and Mt. Visitor (V) were within the overlapping A. alba and P. abies regions. Notably, the fungal communities from Mt. Durmitor (D) and Mt. Bjelasica (B) appeared at distinct positions within the ordination, reflecting their unique communities (Figure 5).
The Shannon diversity index at different sites ranged from 2.74 to 3.97 for A. alba, 3.14 to 4.45 for P. abies, and 2.46 to 3.24 for P. heldreichii. The Mann–Whitney test showed no significant differences in Shannon diversity index values among the three tree species (p > 0.05). The Sørensen qualitative similarity index of fungal communities was high (0.68) between A. alba and P. abies, but low (0.30) between A. alba and P. heldreichii, and low (0.27) between P. abies and P. heldreichii.
The composition of fungal trophic modes varied among A. alba, P. abies, and P. heldreichii, with differences in the relative abundance of unknown, saprotrophic, pathogenic, and endophytic fungi (Figure 6). For A. alba, fungi with an unknown trophic mode accounted for 42.5% of the total community, followed by saprotrophs (28.5%), pathogens (23.0%), and endophytes (6.0%). P. abies showed a higher proportion of fungi with an unknown trophic mode (59.8%), while saprotrophs represented 23.8%, pathogens 14.4%, and endophytes 2.0%. In P. heldreichii, unknown fungi comprised 49.2%, saprotrophs 36.2%, pathogens 8.9%, endophytes 5.4%, and mycorrhizal fungi 0.2%. The distribution patterns showed that fungi with an unknown trophic role dominated across all three tree species, particularly in P. abies. P. heldreichii exhibited the highest proportion of saprotrophs, while A. alba had a more even distribution of fungal functional groups (Figure 6).
The indicator species analysis identified fungal OTUs that were significantly associated with specific tree species (p < 0.05). OTU 3598_24 and 3598_53 were found to be significantly associated with P. heldreichii, while OTUs 3598_15 and 3598_50 were linked to both A. alba and P. abies. OTU 3598_78 exhibited a broader association, being significantly linked to A. alba, P. abies, and P. heldreichii (Table 1 and Table S1).

4. Discussion

The results of this study provided new insights into the fungal communities associated with the needles of three native conifer species (P. abies, A. alba, and P. heldreichii) in the Balkan region. High-throughput sequencing revealed high fungal diversity and specificity in community composition among these tree species, hence contributing to the general knowledge on forest microbiomes by providing a detailed information of needle-associated fungal diversity in a relatively understudied region. However, a high proportion of fungal OUTs remained unidentified or were classified only at higher taxonomic levels, showing limitations in the available reference databases. The recent rapid increase in fungal barcoding studies has revealed vast fungal diversity across a broad spectrum of ecological niches. However, it is estimated that only about 15% of fungal diversity, or only about 100,000 of the 740,000 to 6,000,000 predicted fungal species, has been formally described [21]. Moreover, among the fungal species described, around 50% still lack any DNA sequence information in public databases [21]. This suggests that many of our unidentified OUTs may represent species yet to be discovered or described, emphasizing the need for future taxonomic and functional studies of phyllosphere communities in native forests [22]. In addition, many unidentified fungi also have unknown ecological functions (in terms of behavior and/or resource use), which limits the resolution of trophic mode analyses.
The diversity of fungal communities associated with tree needles was shown to vary according to host species, environmental conditions, and geographical location [15]. These fungal communities are adapted to needle-specific environments [23] and belong to diverse trophic groups, which can be functionally important and respond to changes in the environment [24].
Considering that P. abies represents an important commercial conifer species in Europe [2], several studies have been carried out in central and northern Europe [15,24,25]. Those studies investigated whether and how fungal communities associated with P. abies needles correlate with variation in tree species richness along the tree species diversity gradient [24], along the latitudinal gradient of P. abies distribution in Europe [15], or under different edaphic and management practices in the southern Baltics [25]. Also, other studies revealed that a plant genotype, and even different clones, affect the composition of fungal communities on assimilative plant parts [7,26]. Moreover, fungal communities evolve together with host plants [27], suggesting that outside the principal areal of distribution, such as on marginal habitats or on the edges of species distribution, P. abies (and A. abies) can be associated with a specific fungal community, what was also confirmed in the present study. In the past, fungal communities of A. alba needles were rarely studied, and these studies were largely based on cultivation methods [28,29]. Regarding P. heldreichii, our previous study [30] examined fungal communities present in older needles (2–4 years old), comparing trees growing under high environmental stress with those growing under milder conditions. In the presented study, sampling was performed in larger P. heldreichii populations, and under the conditions of species ecological optimum. These favorable environmental conditions may support a more stable or diverse fungal community, thereby contributing to a more comprehensive understanding of the fungal diversity associated with this relict conifer.
A key finding of this study is the host tree specificity of fungal communities as shown by the DCA. P. heldreichii exhibited a distinct fungal community compared to A. alba and P. abies, which shared a greater degree of overlap in fungal taxa. This pattern suggests that host species exert selective pressures on their associated fungal communities, potentially due to differences in needle chemistry, nutrient availability, and microhabitat conditions [31,32,33]. Furthermore, in the DCA, the separation along Axis 1 showed that the fungal communities of P. heldreichii were compositionally distinct, potentially due to environmental factors specific to its sites (Table 1), where this species was the dominant or sole conifer, unlike P. abies and A. alba, which occurred in mixed stands. This distinctiveness likely also reflects the relict status of this tree species and its long-term adaptation to harsh montane environments (e.g., temperature extremes, soil composition, and precipitation patterns) in southern Europe [8,34].
The dominance of Dothideomycetes across all tree species aligns with global fungal diversity patterns in phyllosphere habitats, and is also consistent with the results of Nguyen et al. [24] and Mischerikova et al. [15]. The relative abundance of Leotiomycetes and Tremellomycetes suggests that these also play important roles in coniferous ecosystems.
The fungal communities were dominated by several fungal taxa (Table 3). Understanding the roles of these taxa is important for assessing their contributions to forest health, tree resilience, ecosystem stability, and nutrient cycling in ecosystems. For example, the predominant presence of C. nagodhii, a recently described rust pathogen [35], suggests its ecological role as a potential plant pathogen of P. abies. Rust fungi from the Chrysomixa complex (C. ledi) were identified as abundant species on current-year needles in Finland [24]. Rust fungi from the genus Crysomixa (abietis) are known to infect P. abies needles in Montenegro [36]. Similarly, the occurrence of S. polyspora, a well-known endophyte and opportunistic pathogen, aligns with its documented association with conifer needles under stress conditions. Only S. polyspora was more abundantly represented on all three hosts. In published research, it was the most common fungus in 12 P. abies sampling sites along a latitudinal gradient in Europe [15]; on P. abies, it was abundantly represented in mixed coniferous–deciduous forests in Romania and Germany, as well as in coniferous semiboreal and boreal forests in Poland and Finland [24]. Regarding A. alba, S. polyspora was abundantly detected on symptomatic and asymptomatic needles from Tatra mountains [29], and studied as a causal agent of current-season needle necrosis of Abies spp. [37] across Europe. On older needles of P. heldreichii, S. polyspora was among the most dominant fungi [30]. Sydowia polyspora has a wide geographical range [38] and is common in Europe. The high representation of this fungus on current-year needles highlights that it can be an important component in these habitats and not merely a latent pathogen of coniferous forests in this region. Its importance may increase in the future due to climate change and abiotic stress affecting trees. Yeast-like fungi, such as V. victoriae, are often found in extreme environments and have been tested in biocontrol and antifungal interactions [39]. It was detected in association with P. abies and A. alba needles. Their presence in conifer needles could indicate a role in microbial competition and plant defense mechanisms. Additionally, the presence of Cladosporium sp. across all three species suggests its role as a generalist species, commonly detected in plant tissues and known for its antifungal properties [40]. It is also abundantly detected on P. abies needles across different forests in Europe, including the southern Baltics [24,25].
In the present study, fungi from the genii Ceramothyrium and Trichomerium dioscoreae were detected on P. abies and A. alba needles at a relatively high abundance (Table 3). Many species of Ceramothyrium are found on tropical broadleaves [41,42] and Trichomerium dioscoreae is known to be associated with monocotyledonous flowering plants from the family Discoreaceae [43], primarily outside Europe. Interestingly, Nguyen et al. [24] have also found both Ceramothyrium fungi and Trichomerium dioscoreae [24,25] in similar abundance on P. abies from coniferous, hemi boreal, and temperate beech–spruce forests. The latter may suggest that some Ceramothyrium, as well as Trichomerium dioscoreae, can be specific to P. abies and A. alba as a host species. Similarly, the Exobasidium taxa detected on all three hosts in this study are already known from P. abies needles in boreal and semiboreal forests [24,25]. It is known that temperate Ericales, commonly found in coniferous forest understories, are preferred hosts for parasitic fungi of the genus Exobasidium [44].
The trophic mode analysis revealed that fungi with an unknown ecological function accounted for a significant proportion of the fungal communities in all three tree species, showing the need for continued research on the fungal communities associated with these key European conifer tree species. The presence of pathogenic, endophytic, and saprotrophic fungi provided valuable ecological insights. The great proportion of detected potential pathogens in A. alba and P. abies may reflect increased susceptibility to fungal infections, particularly in marginal habitats, and under high environmental stresses such as drought [4]. The relatively high abundance of saprotrophic fungi in P. heldreichii needles may indicate latent establishment in healthy-looking tissues but also the capacity for nutrient recycling in high-altitude pine forests, where decomposition rates are generally slower [45].
These findings have important implications for forest conservation and management in Montenegro and in similar temperate and montane ecosystems. The high fungal diversity in these native conifers shows the importance of preserving natural forests, which stabilize marginal mountain environments, but also serve as reservoirs of microbial biodiversity. Future research should focus on the functional roles of dominant fungal taxa in coniferous forests, particularly in the context of climate change and forest disturbance [32].
In conclusion, this study highlights the relationships between native conifers and their associated fungi, emphasizing the ecological importance of needle-associated fungi in forest ecosystems. By advancing our understanding of fungal biodiversity and host specificity, these findings lay the foundation for future research and conservation efforts aimed at preserving the microbial diversity and ecosystem functions at the edges of tree populations in the Balkan region, particularly in Montenegrin forests.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f16060968/s1, Table S1: Relative abundance of fungal taxa associated with needles of Abies alba, Picea abies and Pinus heldreichii in Montenegro

Author Contributions

Conceptualization, J.L. and A.M.; methodology, J.L. and A.M.; formal analysis, J.L. and A.M.; investigation, J.L. and A.M.; resources, J.L.; data curation, J.L. and A.M.; writing—original draft preparation, J.L. and A.M.; writing—review and editing, J.L. and A.M.; funding acquisition, J.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Ministry of Science of Montenegro through the grants INVO HERIC No: 01-646 and 01-001 and FunGforest project No 04-082/23-2532/1. The collaboration within this project was also supported by STSM grants from COST FP 1401 and COST FP 1202.

Data Availability Statement

The original contributions presented in the study are included in the article and Supplementary Material; further inquiries can be directed to the corresponding author.

Acknowledgments

Special thanks go to Ivana Stojanović, and the Laboratory at the CNR–Institute for Biosciences and BioResources in Florence, Italy, for working on DNA extractions from all sampled material in this study.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Forests 16 00968 g001
Figure 1. Forests of Picea abies and Abies alba (Abieti-Piceatum abietis) on Mt. Durmitor (left), photo J. Lazarević; forest of Pinus heldreichii (Pinetum heldreichii mediteraneo-montanum) on Mt. Orjen (right), photo Ž. Starčević.
Figure 1. Forests of Picea abies and Abies alba (Abieti-Piceatum abietis) on Mt. Durmitor (left), photo J. Lazarević; forest of Pinus heldreichii (Pinetum heldreichii mediteraneo-montanum) on Mt. Orjen (right), photo Ž. Starčević.
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Forests 16 00968 g002
Figure 2. Map of Montenegro showing the distribution of tree vegetation. Sampling sites are numbered as follows: (1) Mt. Durmitor, (2) Mt. Bjelasica, (3) Mt. Prokletije, (4) Mt. Visitor, (5) Mt. Kuči, (6) Mt. Prekornica, and (7) Mt. Orjen. The map is adapted from [13]. Coniferous Picea abies and Abies alba forest (blue); coniferous forest of Pinus heldreichii (burgundy); termophillus broadleaf forest, mainly oaks (green); mesophillous broadleaf forests, mainly beech (orange); high mountain pastures and shrubs (gray); Skadar lake (pink).
Figure 2. Map of Montenegro showing the distribution of tree vegetation. Sampling sites are numbered as follows: (1) Mt. Durmitor, (2) Mt. Bjelasica, (3) Mt. Prokletije, (4) Mt. Visitor, (5) Mt. Kuči, (6) Mt. Prekornica, and (7) Mt. Orjen. The map is adapted from [13]. Coniferous Picea abies and Abies alba forest (blue); coniferous forest of Pinus heldreichii (burgundy); termophillus broadleaf forest, mainly oaks (green); mesophillous broadleaf forests, mainly beech (orange); high mountain pastures and shrubs (gray); Skadar lake (pink).
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Forests 16 00968 g003
Figure 3. Venn diagram showing unique and shared OTUs among different groups of samples. Within each group, data from different sites are combined. The size of each circle represents the number of unique OTUs. Pinus heldreichii (blue), Picea abies (green), and Abies alba (red).
Figure 3. Venn diagram showing unique and shared OTUs among different groups of samples. Within each group, data from different sites are combined. The size of each circle represents the number of unique OTUs. Pinus heldreichii (blue), Picea abies (green), and Abies alba (red).
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Figure 4. Relative abundance (% of sequences) of fungal classes associated with needles of Abies alba, Picea abies, and Pinus heldreichii. Data from different sites are combined. Other includes fungal classes with a relative abundance < 1%.
Figure 4. Relative abundance (% of sequences) of fungal classes associated with needles of Abies alba, Picea abies, and Pinus heldreichii. Data from different sites are combined. Other includes fungal classes with a relative abundance < 1%.
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Figure 5. Ordination diagram based on detrended correspondence analysis of fungal communities in needles of Picea abies (points—squares; polygon—green), Abies alba (points—circles; polygon—red), and Pinus heldreichii (points—diamonds; polygon—blue). Sites of the same tree species are enclosed in color-shaded areas, illustrating the spatial distribution of fungal communities and their compositional differences and overlaps among tree species. The size of each plot (square, circle, or diamond) shows the relative richness of fungal OTUs at each site. Site names are represented as B—Mt. Bjelasica, D—Mt. Durmitor, K—Mt. Kuči, O—Mt. Orjen, Pr—Mt. Prekornica, P—Mt. Prokletije, and V—Mt. Visitor (Table 1). In the ordination, 23.9% of the variation was explained on the x-axis, and 14.8% on the y-axis.
Figure 5. Ordination diagram based on detrended correspondence analysis of fungal communities in needles of Picea abies (points—squares; polygon—green), Abies alba (points—circles; polygon—red), and Pinus heldreichii (points—diamonds; polygon—blue). Sites of the same tree species are enclosed in color-shaded areas, illustrating the spatial distribution of fungal communities and their compositional differences and overlaps among tree species. The size of each plot (square, circle, or diamond) shows the relative richness of fungal OTUs at each site. Site names are represented as B—Mt. Bjelasica, D—Mt. Durmitor, K—Mt. Kuči, O—Mt. Orjen, Pr—Mt. Prekornica, P—Mt. Prokletije, and V—Mt. Visitor (Table 1). In the ordination, 23.9% of the variation was explained on the x-axis, and 14.8% on the y-axis.
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Forests 16 00968 g006
Figure 6. Relative abundance (% of sequences) of fungal trophic modes associated with needles of Abies alba, Picea abies, and Pinus heldreichii in Montenegro. Data from different sites are combined.
Figure 6. Relative abundance (% of sequences) of fungal trophic modes associated with needles of Abies alba, Picea abies, and Pinus heldreichii in Montenegro. Data from different sites are combined.
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Table 1. Characteristics of the study sites and tree species sampled at each location.
Table 1. Characteristics of the study sites and tree species sampled at each location.
Forest Vegetation [10]SiteGeographical Position Altitude (m)Climate [12]Soil Type [11]Sampled
Abieti-Piceetum abietisMt. DurmitorN 43.1455
E 19.0981		1350–1450Dfbx’’KalkomelanosolP. abies, A. alba
Abietinum albae subalpinumMt. BjelasicaN 42.8748
E 19.6969		1700Dfbx’’Ranker P. abies, A. alba
Abieti-Piceetum abietisMt. ProkletijeN 42.5964
E 20.0306		1400–1700Cfs’’bDystrict cambisolP. abies, A. alba
Abieti-Piceetum abietisMt. VisitorN 42.6146
E 19.8821		1700–1900-Cfs’’bKakocambisol A. alba
Pinetum heldreichii bertisceumMt. KučiN 44.8150
E 20.4347		1250–1700Cfws’’bx’’Leptosol/Molic leptosol P. heldreichi
Pinetum heldreichii mediteraneo-montanumMt. PrekornicaN 42.6240
E 19.1995		1300Cfsb/Cfsb’’Leptosol/Molic leptosolP. heldreichi
Pinetum heldreichii mediteraneo-montanumMt. OrjenN 42.5436
E 18.5240		1200–1300CfsbLeptosol/Molic leptosolP. heldreichii
Table 2. The number of high-quality fungal reads and OTUs in needles of Abies alba, Picea abies and Pinus heldreichii from different sites in Montenegro.
Table 2. The number of high-quality fungal reads and OTUs in needles of Abies alba, Picea abies and Pinus heldreichii from different sites in Montenegro.
SitePicea abiesAbies albaPinus heldreichiiAll
ReadsOTUsReadsOTUsReadsOTUsReadsOTUs
Mt. Durmitor529801341134--1870158
Mt. Bjelasica737666121--79878
Mt. Prokletije3138168982137--4120211
Mt. Visitor--23259--23259
Mt. Kuči----1032410324
Mt. Prekornica----109171109171
Mt. Orjen----134174134174
Total4404226261021825351119549346
Table 3. Relative abundance (%) of fungal taxa in needles of Abies alba, Picea abies, and Pinus heldreichii from Montenegro. Data from different sites were aggregated by combining all sequencing reads per tree species across all sites, without calculating averages.
Table 3. Relative abundance (%) of fungal taxa in needles of Abies alba, Picea abies, and Pinus heldreichii from Montenegro. Data from different sites were aggregated by combining all sequencing reads per tree species across all sites, without calculating averages.
OUT ID.TaxonPhylum *Genbank ReferenceSequence Length, bpCompared, bpSimilarity, %Abies alba, %Picea abies, %Pinus heldreichii, %All, %
3598_3Unidentified sp.AKX220267257257/257100%3.86.425.210.7
3598_2Unidentified sp.AMN902478240240/240100%2.215.7-7.8
3598_4Chrysomyxa nagodhiiBGU049432317312/31798%-12.6-5.8
3598_7Pseudeurotiaceae sp.AON865419240239/24099%2.04.52.23.2
3598_24Unidentified sp.AMT241934259259/259100%--11.63.1
3598_12Herpotrichiellaceae sp.AJF449676258257/25899%5.03.3-2.9
3598_10Vishniacozyma victoriaeBMF927673234234/234100%1.35.4-2.8
3598_20Cladosporium sp.ALC317546243243/243100%1.32.72.72.3
3598_28Unidentified sp.AMZ983694242242/242100%4.10.73.42.3
3598_38Mycosphaerellaceae sp.AKJ406801240217/22397%0.80.27.12.2
3598_41Unidentified sp.AMN903736246246/246100%4.30.62.42.1
3598_27Botryosphaeriales sp.APP759557244244/244100%4.71.5-2.0
3598_15Phaeosphaeria sp.AKR909136249244/25098%7.10.0-2.0
3598_45Ceramothyrium sp.AKC978733252246/25397%3.12.4-1.9
3598_35Leotiomycetes sp.AOQ066890240240/240100%0.53.60.01.8
3598_37Sydowia polysporaAKY659505256256/256100%1.81.02.71.7
3598_39Trichomerium dioscoreaeANR_137946265261/26598%3.61.1-1.5
3598_58Pseudeurotiaceae sp.AKR267039243243/243100%1.32.3-1.4
3598_59Exobasidium bisporumBAB180368281266/26899%2.10.51.31.2
3598_49Unidentified sp.AMN902540240235/24098%3.10.6-1.1
Total of the 20 taxa 62.956.556.059.9
* A = Ascomycota, B = Basidiomycota.
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Lazarević, J.; Menkis, A. Exploring Fungal Communities in the Needles of Marginal Conifer Tree Populations. Forests 2025, 16, 968. https://doi.org/10.3390/f16060968

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Lazarević J, Menkis A. Exploring Fungal Communities in the Needles of Marginal Conifer Tree Populations. Forests. 2025; 16(6):968. https://doi.org/10.3390/f16060968

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Lazarević, Jelena, and Audrius Menkis. 2025. "Exploring Fungal Communities in the Needles of Marginal Conifer Tree Populations" Forests 16, no. 6: 968. https://doi.org/10.3390/f16060968

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Lazarević, J., & Menkis, A. (2025). Exploring Fungal Communities in the Needles of Marginal Conifer Tree Populations. Forests, 16(6), 968. https://doi.org/10.3390/f16060968

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