Bark beetles (Coleoptera: Scolytinae) play a dual ecological role in forest ecosystems as disturbance agents and vectors of symbiotic fungi, including blue-stain taxa that affect wood quality and tree health. This study assessed fungal communities specific to four bark beetle species—Ips acuminatus (Gyllenhal, 1827), Ips sexdentatus (Börner, 1776), Ips typographus (Linnaeus, 1758), and Pityogenes chalcographus (Linnaeus, 1761)—colonizing Scots pine (Pinus sylvestris L.) in Slovakia. Fungal DNA was extracted from beetle surfaces and analyzed using ITS2 metabarcoding on the Illumina MiSeq platform to characterize the diversity and structure of associated mycobiota. Alpha- and beta-diversity analyses revealed a taxonomically and functionally rich fungal assemblage dominated by Ascomycota, comprising over one thousand operational taxonomic units. Fungal richness and diversity varied among beetle species: I. typographus and P. chalcographus supported the most diverse communities, I. sexdentatus harbored the least diverse assemblages, and I. acuminatus showed contrasting patterns depending on the index used. Beta-diversity analysis indicated that community composition was primarily structured by beetle species identity, with weaker effects of locality and sampling method. Ophiostomatoid fungi, particularly Geosmithia pallida (G. Sm.) M. Kolařík, Kubátová & Pažoutová, Ophiostoma distortum (R.W. Davidson) de Hoog & R.J. Scheff., and Ophiostoma minus (Hedgc.) Syd. & P. Syd., were consistently prevalent and formed the core mycobiome. Random forest classification and differential abundance analyses confirmed host-specific enrichment of several ophiostomatoid and yeast taxa. Yeasts (e.g., Kuraishia, Candida, Yamadazyma), saprotrophic molds (e.g., Penicillium, Davidiella), and the entomopathogen Beauveria bassiana (Bals.-Criv.) Vuill. also occurred frequently. These findings provide the first DNA-based evidence of host-specific fungal assemblages in Scots pine bark beetles in Slovakia and emphasize their ecological significance for beetle–fungus symbioses and pine forest health.

Bark beetle-fungal associations are critical drivers of forest health, yet the factors structuring these communities in Central European pine forests remain poorly understood. This study characterises and compares the culturable filamentous fungal assemblages associated with the epicuticle of two sympatric bark beetles, Ips acuminatus and Ips sexdentatus , infesting Pinus sylvestris in western Slovakia. Using culture-based methods and DNA barcoding, we identified distinct fungal assemblages associated with each beetle species. A total of 153 fungal isolates representing 15 species were obtained from 118 individuals of I. acuminatus , whereas 482 isolates representing 28 species were recovered from 209 individuals of I. sexdentatus . Ophiostomatoid fungi dominated the assemblages of both beetle species; however, their dominance structure differed markedly. Communities associated with I. acuminatus were strongly dominated by Graphilbum acuminatum , resulting in a comparatively uniform fungal assemblage. In contrast, I. sexdentatus harboured a significantly more diverse and heterogeneous community characterised by several co-dominant ophiostomatoid taxa. Both beetle species were also associated with entomopathogenic fungi and diverse saprotrophic taxa. Individual beetles of I. sexdentatus harboured significantly higher fungal alpha diversity than those of I. acuminatus , while beta-diversity analyses revealed pronounced differences in fungal community composition between beetle species, along with temporal variation within each host species. Notably, Graphilbum species ( G. acuminatum , G. furuicola , and G. sexdentatum ) were recorded for the first time in Slovakia, while Sporothrix pseudoabietina represents the first record of an Ips -associated occurrence in Slovakia. These results indicate that bark beetle identity is associated with differences in culturable filamentous fungal assemblages, although temporal variation and sampling context may also contribute to community structure.

Ophiostomatoid fungi are important associates of bark beetles (Scolytinae) and cause sapstain and tracheomycosis in trees. We examined the growth and fructification of seven species from the genera Ophiostoma, Graphilbum, Leptographium , and Grosmannia , isolated from bark beetle species of the genera Ips, Orthotomicus , and Pityogenes collected from Scots pine ( Pinus sylvestris ). Ten isolates were cultivated on water agar (WA), malt extract agar (MEA), MEA with tyrosol (T), and MEA with vitamins (biotin, thiamine, pyridoxine) to determine the optimal medium for growth and fructification. Radial growth varied significantly among media and isolates. The fastest radial growth was observed on MEA supplemented with tyrosol at 0.5 µL per 250 mL (T0.5). Fructification of Ophiostoma minus and Leptographium piceaperdum was significantly higher on the nutrient-poor WA, MEA with tyrosol, and vitamin-enriched MEA than on plain MEA. The results highlight the importance of medium composition for fungal fructification and provide a basis for optimizing fungal development under different conditions.