Five-needle pines, which belong to the subgenus Strobus (section Quinquefoliae), are unique in their ecology and distribution. There are 24 Pinus spp. in this subgenus, which are native to Europe, North and Central America and Asia (Gernandt et al. 2005). The majority of these species are characterized by a narrow distribution and high-altitude habitats i.e. growing under harsh environmental conditions. Information about fungal diversity associated with those Pinus spp. is still limited, while the available knowledge is mainly on the above ground sporocarps or on ECM fungi. For example, ECM communities of P. cembra were described from European Alps (Rainer et al. 2015; Bacher et al. 2010), of P. albicaulus (Mohat et al. 2008; Cripps and Antibus 2011; Jenkins et al. 2018), P. flexilis (Cripps and Antibus 2011) and P. monticola from the North America, of P. walchiniana from Himalaya (Sagar and Lakhanpal 2005; Tyub et al. 2018) and of Pinus amamiana from Japan (Murata et al. 2017). These studies emphasise the importance of ECM fungi for natural regeneration and survival of those tree species, which all have limited distribution and represent important tree species in montane ecosystems. These studies have also revealed the prevailing ECM species, which could potentially be used for seedling mycorrhization owing the conservation of these pine species. Apart from ECM fungi, data on fungi associated with needles and soil of five-needle pines is scarce, but could provide important knowledge on potential pathogens and endophytic fungi.
Survey of fungal fruitbodies in native forests in North Macedonia and Bulgaria have showed that P. peuce is associated with nearly 400 fungal species (Kalucka et al. 2013), while only six fungal species have been recorded in Montenegro (Perić and Perić 2004; Kasom and Karadelev 2012). Heterobasidion annosum and Phaeolus schweinitzii were reported as decay fungi of mature P. peuce trees (Papazov 1969; Rosnev 1985; Tomanić et al. 1998). Cenangium ferugginosum, Cenangium abietis and Ungulina marginata where frequently recorded on weakened trees (Papazov 1969; Tomanić et al. 1998). Previous studies on needle pathogens in native forests revealed the presence of a potentially invasive species Dothistroma septosporum in Montenegro (Lazarević et al. 2017) and Cytospora pinastri in Bulgaria (Georgieva and Marković 2018). Lophodermium fungi were shown to be present in forests and in forest plantations (Georgieva and Marković 2018; Tomanić et al. 1998).
The results of the present study have expanded the available knowledge on endemic P. peuce, demonstrating that needles, rootlets and the rhizosphere soil are inhabited by taxa-rich communities of fungi (Table 3). The detected fungal communities were largely specific to each particular substrate (Fig. 3–5), showing their adaptation and substrate preferences. In support, the qualitative Sørensen similarity index was very low when compared between the above- (needles) and belowground (rootlets or soil) substrates, repeatedly demonstrating the potential importance and functional preferences of associated fungi. Interestingly, the site conditions had only limited effect on associated fungal communities as in different sites these were similar (Fig. 4–5). The latter may suggest that associated fungal communities were largely determined by the host tree species, substrate properties and functional capabilities of associated fungi. This may also suggest that fungi detected at one particular site could be used in different habitats (e.g. for ECM inoculation, or for biocontrol of pests or diseases) i.e. within the distribution of P. peuce.
Among the principal fungi identified in the soil and rootlets, there were taxa from genera Suillus and Rhizopogon, which are closely related and almost exclusively restricted to Pinaceae (Bruns et al. 2002). A long co-evolutionary history between Suillus and Pinus species (Wu et al. 2000) represents an example of host specificity and adaptation (Jenkis 2018). Moreover, a limited number of Suillus fungi appear to be specific to five-needle pines (Klofac 2013), and this symbiosis can be essential for the survival under harsh environmental conditions. For example, Suillus fungi could be regarded as the most important and widespread symbionts of P. cembra in the Alps (Rainer et al. 2015) and of P. albicaulus in the North America (Mohat et al. 2008). Suillus sibiricus readily forms an ECM symbiosis with five-needle pines (Liao et al. 2016), which are found in different regions worldwide (Reiner 2015; Mohat 2008). It is a protected fungus in many European countries, including North Macedonia (Karadelev 1998), Bulgaria (Boev 2011) and Montenegro (Kasom and Karadelev 2012), where it was recorded in native forest stands of P. peuce. In agreement, the results of the present study provided the evidence that S. sibiricus is a common and an important symbiont of P. peuce as it was detected in both the rootlets and the soil (Table 4, Supplementary Table 1). Surveys on the aboveground sporocarp production and analyses of fine roots have shown that Suillus granulatus is the dominant fungus in P. heldreichii forests in Montenegro (Lazarević et al. 2011; Lazarević and Menkis 2018). In the present study, S. granulatus was only at the Visitor site, where P. heldreichii forest is in near proximity (see above). Liao et al (2016) have shown that S. granulatus can also readily form ECMs with pines from the subgenus Strobus. Suillus luteus and S. variegatus were also detected in rootlet and soil samples (Table 4, Supplementary Table 1), showing that P. peuce is associated with different suilloid fungi, which can colonise tree roots in high-altitude habitats. Fungi from the genus Rhizopogon are also primarily associated with Pinacea, but are not strictly host specific (Bruns et al 2002) and are also known to form ECM symbioses with trees in high-altitude coniferous forests (Kjøller and Bruns 2003; Mohatt et al. 2008; Lazarević and Menkis 2018). Contrary to Suillus spp., Rhizopogon fungi produce hypogeous sporocarps and their spores are mainly dispersed by animals (Grubisha et al. 2007; Mohatt et al. 2008). This makes the gene flow of Rhizopogon species more restricted (Grubisha et al. 2007), leading to genetic differentiation among isolated populations (Grubisha et al. 2007; Murata et al. 2017) and eventually to the evolution of different Rhizopogon species. Rhizopogon mohelensis was one of the most commonly detected fungi in this study, particularly in pure P. peuce forests, but less abundantly found in mixed forests. By contrast, R. falax was commonly detected in all study sites (Table 4). Rhizopogon mohelensis was reported from many countries in Europe (Holec et al. 2013). It belongs to R. roseolus group and sometimes can be confused with R. rubescens. For example, R. salebrosus is exclusively associated with P. strobus (Kohout et al. 2011), what limits such misidentification.
It appears that both Suillus and Rhyzopogon fungi possess specific ecological adaptations important for the establishment of Pinus spp. on marginal habitats and after forest disturbance (Kjøller and Bruns 2003; Mohatt et al. 2008). Such host specialists may often represent the dominant ECM species in high-altitude habitats characterised by extreme conditions (Bruns et al. 2002; Antibus and Cripps 2010). Certain suilloid fungi can be of special importance to five-needle pines due to host specialisation, high efficiency of nutrient and water uptake and transfer between the symbiosis partners, and an exclusion of mycoheterotrophy (Cripps and Antibus 2011). The common occurrence of suilloid fungi was particularly notable at the Visitor site containing newly regenerated P. peuce trees, but their abundance was lower in old-growth stands (Tables 1 and 3).
Among other fungi commonly detected in rootlets, there were Thelephora terrestris and Phialocephla fortinii (Table 4). Thelephora terestris is a typical ECM fungus of long-distance exploration type. This property allows efficient transportation of nutrients and water over long distances, which appears to be especially suitable for undisturbed, but very stony habitat with high soil heterogeneity (Reiner at al. 2015). Phialocephala fortinii belongs to a complex of dark septate endophytes, which forms non-specific associations with many plant hosts. The complex includes ECM-forming fungi as well as numerous pathogenic fungi (Tedersoo et al. 2008). Interestingly, P. fortinii was also commonly detected in rootlets of old growth P. heldreichii in high-altitude habitats, but not in its pioneer forests (Lazarević and Menkis 2017).
Tomentella brasodellea and Tylospora asterophora were the other two ECM fungi found in common association with P. peuce rootlets (Table 4). Tomentella fungi have been shown to be among dominant species in older coniferous forests worldwide (Lilleskov and Bruns 2005; Mrak et al. 2020). Tylospora asterophora is known as one of the most consistent and abundant ECM fungus associated with P. abies (Eberhard et al. 1999). Melinomyces bicolor (Piceirhiza bicolorata) was also commonly recorded (Table 4) and it is known to colonise roots of Pinus, Picea and Betula trees, but also forms ericoid mycorrhiza with shrubs from the Ericaceae family (Grelet et al. 2009). A high presence of M. bicolor in the present study could be influenced by the occurrence of Vaccinium myrtilus in the ground vegetation. According to Horton et al. (1999), the sharing of ECM fungi between coniferous trees and plants from Ericaceae may play a major role in plant community dynamics. Soil microorganisms associated with arbutoid members of Ericaceae may enhance growth, survival, mycorrhizal root formation, and nitrogenase activity of conifer tree seedlings (Amaranthus et al. 1990). Fungal networks shared between those hosts (Pinaceae-Ericoid) remain even after disturbance events as these fungi are able to form associations with different plants (Perry et al. 1989). Ericaceous plants was shown to play an important role in the formation of ECM communities associated with P. strobus (Kohout et al. 2011). Besides, ericoid plants are very important in Mediterranean forests as these support ECM and ericoid fungi after fire events (Bergero et al. 2003).
Although a number of fungi were shared between the rootlet and soil samples as indicated by a high value of Sørensen similarity index, soil samples were characterised by a higher abundance of saprotrophic fungi as compared to the rootlet samples (dominated by ECM fungi) (Tables 4 and 5), which led to the differentiation of fungal communities in these two substrates (Fig. 4). Among the dominant fungi in the soil, there was P. gigantea, which is known as a common saprophytic fungus that causes white rot in conifer logs and stumps (Copenhaver et al. 2014), thereby playing an important role in the decomposition of conifer wood debris. Its common occurrence at the Bogićevica site was likely associated with the vast availability of dead wood, which was absent at the other two sites. Solicoccozyma terricola was another commonly detected saprotrophic fungus known from soils of temperate forests (Mašínová et al. 2016). Hygrocybe intermedia and Neohygrocybe ingrata were also among dominant fungi, which are known to be common in montane grasslands in Montenegro. These fungi, while being red-listed, are often associated with habitats of P. heldreichii (Perić and Perić 2004), which are nutrient-poor, but support high diversity of fungi (Lazarević and Menkis 2018). Interestingly, recently described ubiquitous soil fungi of the genus Archaeorhizomyces (Rosling et al. 2011) were also detected (Table 5). Although functional properties, reproduction structures and dispersal strategy of these fungi are largely unknown, the current observation expands available knowledge on the host tree species and geographical distribution.
A number of ECM fungi were also commonly detected in the soil, including Tylospora asterophora, Russula vesca, Inocybe whitei, Laccaria laccata and Cenococcum geophilum (Table 5), showing that these may be important symbionts of forest trees grown in high-altitude habitats. However, as these ECM fungi were mainly detected at the Zeletin site (Table 5), the possibility should not be excluded that their occurrence was also affected by the other three species (A. alba or F. sylvatica) present there.
Living needles are known to be associated with diverse trophic groups of fungi, including endophytes, epiphytes and pathogens. Many of these can be functionally important and metabolically active taxa, which respond to changes in the environment (Nguyem et al. 2016). They appear to be able to colonise different tree species, but factors driving their distribution remains largely unclear (Tehronen et al. 2019). Among the dominant fungi associated with the needles of P. peuce, there were Dothideomycetes sp. 3360_7, Dothideomycetes sp. 3360_10 and Leotiomycetes sp. 3360_16 (Table 6), which could not be identified to the species or genus level, thereby not only posing a challenge to fungal taxonomy, but also limiting the identification of their ecology and functional roles. Further, Sydowia polyspora was detected in P. peuce needles (Table 6), which is the fungus with a wide geographical distribution and common occurrence in Europe (Botella and Diez 2010). The pathogenic behaviour of S. polyspora to young conifers (genera Thuja, Abies, Tsuga, Larix, Picea and Pinus) was previously reported (Talgo et al. 2018). Besides, it has been recently reported as one of the most abundant needle pathogens in high-altitude P. heldreichii forests in Montenegro (Lazarević and Menkis 2020), showing that it is not restricted by harsh environmental conditions prevailing in these habitats. Sydowia symptoms include needle discoloration, necrosis and shoot dieback. However, the fungus is favoured by a warm climate, especially if the host is stressed by drought or insect attack (Munoz – Adalia et al. 2017). Celosporium larixicola was another commonly detected fungus (Table 6), which was recently described from needles of Larix lyallii in Canada (Tsuneda et al. 2010) and needles of P. abies in Sweden (Ngyen et al. 2016). Fungi from the genus Celosporium was shown to be commonly recovered from alpine habitats and may be biotrophic or necrotrophic (Brown et al. 2015). Lophodermium pinastri was also common in needles of P. peuce (Table 6). It has global distribution and is commonly associated with pines (Millberg et al. 2015; Reignoux et al. 2014). It was shown recently that L. pinastri colonises healthy needles latently as an endophyte, initiates active growth at the beginning of needle senescence and sporulates after the needle fall. It is a dominant coloniser of dying needles and a saprotroph contributing to the decomposition of the needles (Reignoux et al. 2014). Besides, L. pinastri was one of the most commonly detected fungi on P. heldreichii needles in Montenegro (Lazarević and Menkis 2020) and frequently reported from P. nigra and P. sylvestris grown in forests, forest nurseries and plantations in the Balkan region (Karadžić and Miijašević 2008; Dobrova et al. 2016).
In summary, P. peuce in high altitude mountain habitats harbour diverse communities of fungi, composition of which appears to be largely determined by the host tree species, substrate properties and functional capabilities of these fungi.