Population Structure of Geosmithia morbida,the Causal Agent of Thousand Cankers Disease of Walnut Trees in the United States

The ascomycete Geosmithia morbida and the walnut twig beetle Pityophthorus juglandis are associated with thousand cankers disease of Juglans (walnut) and Pterocarya (wingnut). The disease was first reported in the western United States (USA) on several Juglans species, but has been found more recently in the eastern USA in the native range of the highly susceptible Juglans nigra. We performed a comprehensive population genetic study of 209 G. morbida isolates collected from Juglans and Pterocarya from 17 geographic regions distributed across 12 U.S. states. The study was based on sequence typing of 27 single nucleotide polymorphisms from three genomic regions and genotyping with ten microsatellite primer pairs. Using multilocus sequence-typing data, 197 G. morbida isolates were placed into one of 57 haplotypes. In some instances, multiple haplotypes were recovered from isolates collected on the same tree. Twenty-four of the haplotypes (42%) were recovered from more than one isolate; the two most frequently occurring haplotypes (H02 and H03) represented 36% of all isolates. These two haplotypes were abundant in California, but were not recovered from Arizona or New Mexico. G. morbida population structure was best explained by four genetically distinct groups that clustered into three geographic regions. Most of the haplotypes isolated from the native range of J. major (Arizona and New Mexico) were found in those states only or present in distinct genetic clusters. There was no evidence of sexual reproduction or genetic recombination in any population. The scattered distribution of the genetic clusters indicated that G. morbida was likely disseminated to different regions at several times and from several sources. The large number of haplotypes observed and the genetic complexity of G. morbida indicate that it evolved in association with at least one Juglans spp. and the walnut twig beetle long before the first reports of the disease.     

Geosmithia species in southeastern USA and their affinity to beetle vectors and tree hosts

The fungal genus Geosmithia is best known due to one species, G. morbida, which is vectored by the walnut twig beetle (WTB) and contributes to Thousand Cankers Disease (TCD) on walnut and wingnut trees. However, the genus is globally very diverse and abundant, and dominates a ubiquitous but understudied niche – the twig-infesting, phloem-feeding bark beetle mycobiome. The Geosmithia community in North America is only now beginning to be described. Very limited information is available for the South East, despite the region's potential to be a Geosmithia diversity hotspot. To survey the Geosmithia community in the subtropical USA, to assess their beetle and tree associations, and to test for the presence of G. morbida, we systematically deployed branch sections of nine tree species, including three Juglandaceae, in North Florida. We recovered 55 Geosmithia isolates from 195 beetle specimens from 45 exposed branch units. Neither G. morbida nor its beetle vector were detected. We identified 14 Geosmithia species; those in the G. pallida species complex were the most prevalent. Four undescribed phylogenetic species were recovered, indicating that the Geosmithia diversity in North America remains under-documented. Analysis of the association of Geosmithia with beetles and trees suggested that most species are generalists, five display preference for certain tree species, and none is specific to any beetle species.     

Fungal,host and non-host volatiles modify attraction of the walnut twig beetle,Pityophthorus juglandis,to pheromone lures

1. Thousand Cankers Disease (TCD) of walnut trees is caused by the pathogenic fungus Geosmithia morbida vectored by the walnut twig beetle (WTB) Pityophthorus juglandis. Monitoring efforts for WTB rely on pheromone-baited traps, but lures are likely effective at attracting beetles only over short distances. Fungal-derived kairomones may increase the efficacy of current lures, while additional volatiles may repel beetles from valuable trees.
2. The objective of this study was to determine the extent to which fungal, host and non-host volatiles modify the attraction of WTB to pheromone-baited traps. A trapping study that combined fungal, host-associated and non-host compounds with WTB-pheromone lures was conducted over three years in black walnut plantations experiencing a TCD outbreak in Walla Walla, WA.
3. Traps baited with pheromone and G. morbida volatiles (i.e., isoamyl and isobutyl alcohol) consistently attracted more WTB, while other fungal volatiles inconsistently increased attraction compared to those baited with pheromone lure alone. This is the first field study that demonstrates fungal volatiles can increase the attraction of a bark beetle to its pheromone in a hardwood system.
4. One fungal (benzyl alcohol) and two additional volatiles (limonene, piperitone) repelled WTB from pheromone-baited traps. Although limonene is known to repel WTB, this is the first demonstration that benzyl alcohol and piperitone repel a bark beetle.
5. Fungal volatiles may increase the efficacy of monitoring efforts and may play an important role in management tactics for WTB, especially in detecting the introduction and establishment of nascent populations and protecting trees from colonizing beetles.

     

Inhibition of virulent and hypovirulent Cryphonectria parasitica growth in dual culture by fungi commonly isolated from chestnut blight cankers

Chestnut blight cankers, caused by the fungus Cryphonectria parasitica, are prone to invasion by other microorganisms as the canker ages. This microbial community has the potential to alter canker expansion, which may influence the probability that the canker girdles the infected stem. Hypoviruses infect the pathogen mycelium directly and are known to decrease pathogen virulence (i.e. hypovirulent). These viral infections can slow pathogen growth, decreasing the rate of canker expansion and lowering the probability of girdling. Saprophytic fungi also invade the expanding canker and may antagonize C. parasitica leading to reduced pathogen growth. The combined effects of fungal antagonism and a hypovirulent pathogen could work in combination to reduce the probability of girdling the infected stem. We assessed the ability of different fungal taxa, isolated from low severity cankers, to inhibit the growth of virulent and hypovirulent forms of C. parasitica in dual culture tests on two cultural media. Percent growth inhibition of virulent C. parasitica by potentially antagonistic fungi ranged from 2 % to 34 %, while inhibition of hypovirulent C. parasitica ranged from 18 % to 54 %. Only one isolate, identified as Umbelopsis isabellina (UmbelopsisWS) inhibited the virulent form of the pathogen more than the hypovirulent form. All three Trichoderma isolates caused the greatest growth inhibition of virulent C. parasitica, but they, like all other fungal isolates tested, inhibited the hypovirulent form of the pathogen more than the virulent form. These results suggest that commonly occurring fungi in chestnut blight cankers, including Trichoderma, may inhibit the hypovirulent C. parasitica more than virulent C. parasitica. Thus, the presence of other fungi in cankers may not enhance the effect of hypovirulent C. parasitica to delay cankers from girdling a stem but instead intensify canker development.     

Phylogeography of the Walnut Twig Beetle,Pityophthorus juglandis,the Vector of Thousand Cankers Disease in North American Walnut Trees

Thousand cankers disease (TCD) of walnut trees (Juglans spp.) results from aggressive feeding in the phloem by the walnut twig beetle (WTB), Pityophthorus juglandis, accompanied by inoculation of its galleries with a pathogenic fungus, Geosmithia morbida. In 1960, WTB was only known from four U.S. counties (in Arizona, California, and New Mexico), but the species has now (2014) invaded over 115 counties, representing much of the western USA, and at least six states in the eastern USA. The eastern expansion places TCD in direct proximity to highly valuable (> $500 billion) native timber stands of eastern black walnut, Juglans nigra. Using mitochondrial DNA sequences, from nearly 1100 individuals, we examined variation among 77 samples of WTB populations across its extended range in the USA, revealing high levels of polymorphism and evidence of two divergent lineages. The highest level of genetic diversity for the different lineages was found in the neighboring Madrean Sky Island and Western New Mexico regions, respectively. Despite their proximity, there was little evidence of mixing between these regions, with only a single migrant detected among 179 beetles tested. Indeed, geographic overlap of the two lineages was only common in parts of Colorado and Utah. Just two haplotypes, from the same lineage, predominated over the vast majority of the recently expanded range. Tests for Wolbachia proved negative suggesting it plays no role in "driving" the spread of particular haplotypes, or in maintaining deep levels of intraspecific divergence in WTB. Genotyping of ribosomal RNA corroborated the mitochondrial lineages, but also revealed evidence of hybridization between them. Hybridization was particularly prevalent in the sympatric areas, also apparent in all invaded areas, but absent from the most haplotype-rich area of each mitochondrial lineage. Hypotheses about the specific status of WTB, its recent expansion, and potential evolutionary origins of TCD are discussed.     

High diversity of root-associated fungi isolated from three epiphytic orchids in southern Ecuador

Knowledge of fungal root-associates is essential for effective conservation of tropical epiphytic orchids. We investigated the diversity of root-associated fungi of Cyrtochilum myanthum, Scaphyglottis punctulata and Stelis superbiens from a tropical mountain rainforest in southern Ecuador, using a culture dependent approach. We identified 115 fungal isolates, corresponding to 49 fungal OTUs, based on sequences of the nrDNA ITS and partial 28S region. Members of Ascomycota were unambiguously dominant (37 OTUs), including Trichoderma sp. as the most frequent taxon. Members of Basidiomycota (Agaricales and Polyporales) and Mucoromycota (Umbelopsidales and Mortierellales) were also identified. Four potential mycorrhizal OTUs of Tulasnellaceae and Ceratobasidiaceae were isolated from C. myanthum and S. superbiens. Fungal community composition was examined using Sørensen and Jaccard indices of similarity. Alfa diversity was significantly different between C. myanthum and S. superbiens. No difference in beta diversity of the fungal communities between the 3 orchid species and the collecting sites was detected. The study revealed a high diversity of fungi associated with orchid roots. Our results contribute to a better understanding of specific relationships between epiphytic orchids and their root-associated fungi.     

Detection of a root-associated group of Hyaloscyphaceae (Helotiales) species that commonly colonizes Fagaceae roots and description of three new species in genus Glutinomyces

We used a culture-dependent approach to investigate root-associated fungal communities in Fagaceae roots at four fagaceous species-dominant forests in Japan. In total 1029 isolates were collected and classified, based on colony morphological features and molecular information. Species of order Helotiales (Ascomycota) were dominantly isolated at all four sites, in which a globally-distributed putative endophytic group in Hyaloscyphaceae predominated. This group of fungi was morphologically and phylogenetically investigated using these isolates as well as additional isolates collected from 8 different sites in Japan. Among the Hyaloscyphaceae, Glutinomyces species were frequently detected, and three novel species, G. inflatus, G. vulgaris, and G. takaragaikensis spp. nov. were identified and described according to their morphology and genealogical concordance.     

Genetic variability among populations of Fusicladium species from different host trees and geographic locations in the USA

Peach and almond scab caused by Venturia carpophila and pecan scab caused by Fusicladium effusum result in yield loss, downgrading of fruit, defoliation and subsequent decline of an orchard. To understand the levels of genetic diversity and divergence of pathogens from different hosts and locations 51 isolates were genotyped and analyzed using 10 RAPD and 5 UP-PCR markers, including 18 isolates of V. carpophila from peach trees in the southeastern United States, 12 isolates of V. carpophila from almond trees in California, and 21 isolates of F. effusum (a related species) from pecan trees in the southeastern United States. The combined marker results showed a low incidence of polymorphisms among the peach isolates (4.2 % of markers), but a higher incidence of polymorphisms among the almond isolates (42.0 %) and the pecan isolates (61.0 %). The Dice coefficient of similarity ranged from 0.932 to 1.000 for the peach V. carpophila isolates, 0.214 to 0.976 for the almond V. carpophila isolates, and 0.528 to 0.920 for the pecan F. effusum isolates. UPGMA bootstrap values indicated that UP-PCR data were slightly more robust and, based on the combined data, the UPGMA bootstrap analysis (1,000 runs) gave a high node value (100 %) differentiating all the isolates of V. carpophila from F. effusum and a moderate node value differentiating the peach and almond isolates of V. carpophila (68 %). The results suggest some divergence between the V. carpophila populations of almond trees in California and peach tree populations in the southeastern United States, and different levels of genetic diversity within the two populations.     

Comparative analysis of fungal communities in colonies of two leaf-cutting ant species with different substratum preferences

Fungus gardens of leaf-cutting ants harbor diverse alien fungi in addition to their fungal cultivar. Previous work suggested that alien microorganisms are likely derived from the substrata foraged by ant workers and incorporated into the fungus gardens. To test this hypothesis, we sampled 1014 garden fragments from 16 field colonies of Atta sexdens rubropilosa (a dicot-cutting ant) and Atta capiguara (a grass-cutting ant) in Brazil. From a total of 615 fungal isolates recovered, we observed similar diversity of fungi between colonies of both ant species. However, fungal communities differed in composition of taxa between ant colonies. Trichoderma spirale, Trichosporon chiarellii and Penicillium citrinum were prevalent accounting for 18.5%, 12.2% and 11.7% of the total isolates, respectively. As expected, fungal communities clustered in two major groups supporting the hypothesis that plant substratum has an impact on the composition of the alien fungi found in leaf-cutting ant gardens.     

Psychrophilic endophytic fungi with biological activity inhabit Cupressaceae plant family

Psychrophilic microorganisms are cold-adapted organisms that have an optimum growth temperature below 15 °C, and often below 5 °C. Endophytic microorganisms live inside healthy plants and biosynthesize an array of secondary metabolites which confer major ecological benefits to their host. We provide information, for the first time, on an endophytic association between bioactive psychrophilic fungi and trees in Cupressaceae plant family living in temperate to cold, semi-arid habitats. We have recovered psychrophilic endophytic fungi (PEF) from healthy foliar tissues of Cupressus arizonica, Cupressus sempervirens and Thuja orientalis (Cupressaceae, Coniferales). In total, 23 such fungi were found out of 110 endophytic fungal isolates. They were identified as ascomycetous fungi, more specifically Phoma herbarum, Phoma sp. and Dothideomycetes spp., all from Dothideomycetes. The optimal growth temperature for all these 23 fungal isolates was 4 °C, and the PEF isolates were able to biosynthesize secondary metabolite at this temperature. Extracted metabolites from PEF showed significant antiproliferative/cytotoxic, antifungal and antibacterial effects against phytopathogenic fungi and bacteria. Of special interest was their antibacterial activity against the ice-nucleation active bacterium Pseudomonas syringae. Accordingly, we suggest that evergreen Cupressaceae plants may benefit from their psychrophilic endophytic fungi during cold stress. Whether such endosymbionts confer any ecological and evolutionary benefits to their host plants remains to be investigated in vivo.     

Fungi at a Small Scale: Spatial Zonation of Fungal Assemblages around Single Trees

Biological communities are often structured by environmental factors even at small spatial scales. Fungi are no exception, though the patterns and mechanisms underlying their community structure are usually unknown. Previous work documented zonation in fungi under tree canopies primarily through their fruiting patterns. Here we investigate the existence of zonation patterns in fungal communities around isolated Pinus muricata trees of different ages in northern coastal California. Using a combination of ingrowth bags and pyrosequencing to target underground mycelium we found highly diverse soil fungal communities associated with single trees. Both ectomycorrhizal and non-ectomycorrhizal fungi were present in all samples, but the latter were more species rich, dominated the samples by sequence read abundance, and showed partitioning by canopy-defined zones and tree age. Soil chemistry was correlated with fungal zonation, but host root density was not. Our results indicate different guilds of fungi partition space differently and are driven by distinct environmental parameters.     

Fungal communities associated with acorn woodpeckers and their excavations

Wood-decay fungi soften wood, putatively providing opportunities for woodpeckers to excavate an otherwise hard substrate, yet the fungal community composition in tree cavities and the specificity of these relationships is largely unknown. We used high-throughput amplicon sequencing of the fungal ITS2 region to examine the fungal communities associated with acorn woodpeckers (Melanerpes formicivorus) and their cavities in mature valley oak (Quercus lobata) and blue oak (Q. douglasii) trees in an oak savannah of central coastal California, USA. Acorn woodpeckers and their excavations harbored over 1500 fungal taxa, including more than 100 putative wood-decay fungi. The fungal communities found on the birds were more similar to those found in excavated cavities than those found in trees without excavated holes. These results suggest that symbiotic associations between acorn woodpeckers and fungi are highly diverse, with low specificity. Symbiotic associations between cavity-excavators and fungi are likely more common and widespread than previously thought.     

In situ seed baiting to isolate germination-enhancing fungi for an epiphytic orchid,Dendrobium aphyllum (Orchidaceae)

Orchid conservation efforts, using seeds and species-specific fungi that support seed germination, require the isolation, identification, and germination enhancement testing of symbiotic fungi. However, few studies have focused on developing such techniques for the epiphytes that constitute the majority of orchids. In this study, conducted in Xishuangbanna Tropical Botanical Garden, Yunnan, China, we used seeds of Dendrobium aphyllum, a locally endangered and medicinally valuable epiphytic orchid, to attract germination promoting fungi. Of the two fungi isolated from seed baiting, Tulasnella spp. and Trichoderma spp., Tulasnella, enhanced seed germination by 13.6 %, protocorm formation by 85.7 %, and seedling development by 45.2 % (all P?Epulorhiza, another seed germination promoting fungi isolated from Cymbidium mannii, also enhanced seed germination (6.5 %; P?P?Trichoderma suppressed seed germination by 26.4 % (P?Tulasnella was the only treatment that produced seedlings. Light increased seed imbibition, protocorm formation, and two-leaved seed development of Tulasnella inoculated seeds (P?Tulasnella be introduced for facilitating D. aphyllum seed germination at the protocorm formation stage and that light be provided for increasing germination as well as further seedling development. Our findings suggest that in situ seed baiting can be used to isolate seed germination-enhancing fungi for the development of seedling production for conservation and reintroduction efforts of epiphytic orchids such as D. aphyllum.     

Bacterial community composition and chitinase gene diversity of vermicompost with antifungal activity

Bacterial communities and chitinase gene diversity of vermicompost (VC) were investigated to clarify the influence of earthworms on the inhibition of plant pathogenic fungi in VC. The spore germination of Fusarium moniliforme was reduced in VC aqueous extracts prepared from paper sludge and dairy sludge (fresh sludge, FS). The bacterial communities were examined by culture-dependent and -independent analyses. Unique clones selected from 16S rRNA libraries of FS and VC on the basis of restriction fragment length polymorphism (RFLP) fell into the major lineages of the domain bacteria Proteobacteria, Bacteroidetes, Verrucomicrobia, Actinobacteria and Firmicutes. Among culture isolates, Actinobacteria dominated in VC, while almost equal numbers of Actinobacteria and Proteobacteria were present in FS. Analysis of chitinolytic isolates and chitinase gene diversity revealed that chitinolytic bacterial communities were enriched in VC. Populations of bacteria that inhibited plant fungal pathogens were higher in VC than in FS and particularly chitinolytic isolates were most active against the target fungi.     

Monitoring of the bacterial and fungal biodiversity and dynamics during Massa Medicata Fermentata fermentation

The microbial community dynamics play an important role during Massa Medicata Fermentata (MMF) fermentation. In this study, bacterial and fungal communities were investigated based on the culture-dependent method and polymerase chain reaction-denaturing gradient gel electrophoresis analysis. Meanwhile the dynamic changes of digestive enzyme activities were also examined. Plating results showed that MMF fermentation comprised two stages: pre-fermentation stage (0–4 days) was dominated by bacterial community and post-fermentation stage (5–9 days) was dominated by fungal community. The amount of bacteria reached the highest copy number 1.2?×?10¹⁰ CFU/g at day 2, but the fungi counts reached 6.3?×?10⁵ CFU/g at day 9. A total of 170 isolates were closely related to genera Enterobacter, Klebsiella, Acinetobacter, Pseudomonas, Mucor, Saccharomyces, Rhodotorula, and Amylomyces. DGGE analysis showed a clear reduction of bacterial and fungal diversity during fermentation, and the dominant microbes belonged to genera Enterobacter, Pediococcus, Pseudomonas, Mucor, and Saccharomyces. Digestive enzyme assay showed filter paper activity; the activities of amylase, carboxymethyl cellulase, and lipase reached a peak at day 4; and the protease activity constantly increased until the end of the fermentation. In this study, we carried out a detailed and comprehensive analysis of microbial communities as well as four digestive enzymes' activities during MMF fermentation process. The monitoring of bacterial and fungal biodiversity and dynamics during MMF fermentation has significant potential for controlling the fermentation process.     

Isolation and Characterization of Trichoderma spp. for Antagonistic Activity Against Root Rot and Foliar Pathogens

Trichoderma, soil-borne filamentous fungi, are capable of parasitising several plant pathogenic fungi. Twelve isolates of Trichoderma spp. isolated from different locations of South Andaman were characterized for their cultural, morphological and antagonistic activity against soil borne and foliar borne pathogens. The sequencing of these isolates showed seven different species. The isolates revealed differential reaction patterns against the test pathogens viz., Sclerotium rolfsii, Colletotrichum gloeosporioides and C. capsici. However, the isolates, TND1, TWN1, TWC1, TGD1 and TSD1 were most effective in percentage inhibition of mycelial growth of test pathogens. Significant chitinase and β-1,3-glucanase activities of all Trichoderma isolates has been recorded in growth medium. T. viride was found with highest chitinase whereas T. harzianum was recorded with highest β-1,3-glucanase activities.     

Soil fungal diversity in three nature reserves of Jiuzhaigou County,Sichuan Province,China

Jiuzhaigou County is located at the southern transition zone of Sichuan Basin and the Qinghai–Tibet Plateau and is the site of three famous nature reserves, namely, the Jiuzhaigou Nature Reserve (JZNR), Baihe Nature Reserve (BHNR) and Wujiao Nature Reserve (WJNR). The soil fungal diversity in this region has not yet been investigated. In this study, we collected 25 soil samples from these three nature reserves. Soil fungi were isolated using the soil dilution plate technique and Rose Bengal agar medium. The culturable soil fungal density based on analysis of the 25 samples ranged from 2.18 log to 4.38 log CFU g^?1 dry weight soil, with the fungal density being highest in samples from JZNR and lowest in those from BHNR. Based on morphological characters and the results of phylogenetic analysis of the internal transcribed spacer (ITS) of the rDNA operon, we identified 38 genera (two genera could not be identified) belonging to Ascomycota, Zygomycota and Basidiomycota. The dominant genera were Penicillium, Humicola, Aspergillus and Trichoderma. The species richness index S, biodiversity index H′ and evenness index E of the 25 sampling sites were in the range 10–29, 1.96–3.05 and 0.74–0.95, respectively. The highest mean values of the S, H′ and E indices were in soil samples from BHNR, where the values of these indices were 20.00, 2.66 and 0.90, respectively. These results indicate that the diversity of culturable fungi in these three nature reserves was high. Furthermore, a total 14 Trichoderma isolates were tested for their antagonism activity against mycelium growth of three pathogens: Bipolaris maydis, Curvularia lunata, Rhizoctonia solani. The results showed that six Trichoderma isolates had good antagonistic effects on the three pathogenic fungi.     

Effect of larch (Larix gmelini Rupr.) root exudates on Manchurian walnut (Juglans mandshurica Maxim.) growth and soil juglone in a mixed-species plantation

The establishment and productivity of a Manchurian walnut (Juglans mandshurica Maxim.) plantation can be improved by inter-planting with larch (Larix gmelini Rupr.) in Northeast China, but the potential mechanism remains obscure. We carried out a series of experiments in a 20-year-old mixed-species plantation, as well as in Manchurian walnut and larch plantations. Manchurian walnut seedlings had difficulty surviving in the Manchurian walnut plantation because their growth was inhibited by their own soil and root exudates. In sharp contrast, Manchurian walnut seedlings grew well in larch and mixed-species plantations. Larch soil and root exudates greatly stimulated the growth of Manchurian walnut seedlings in controlled conditions. In particular, larch root exudates can increase the soil microbial populations, including bacteria, actinomycetes, azotobacter and cellulose-decomposing microorganisms; larch root exudates can also increase the enzyme activities of saccharase, urease, proteinase and polyphenol oxidase. Significant results led to a rapid degradation of the root-exuded phytotoxic juglone from Manchurian walnut. Manchurian walnut root exudates contained juglone at a high concentration of 121.3?±?6.6 mg g^?1, while juglone concentrations in the soil beneath Manchurian walnut trees ranged from 2.9–6.2 µg g^?1 soil. It appears from the results that juglone may be released from Manchurian walnut roots into the soil in a sufficient quantity but rapidly degrades due to interactions with soil factors. Furthermore, juglone was more resistant toward degradation in the Manchurian walnut soil (t _1/2 ?=?7.36?±?0.63 h) when compared to the larch soil (t _1/2 ?=?4.66?±?0.82 h). The results suggest that larch may improve the establishment and productivity of Manchurian walnut in a mixed-species plantation through the release of root exudates.     

Fungi Vectored by the Bark Beetle Ips typographus Following Hibernation Under the Bark of Standing Trees and in the Forest Litter

The bark beetle Ips typographus has different hibernation environments, under the bark of standing trees or in the forest litter, which is likely to affect the beetle-associated fungal flora. We isolated fungi from beetles, standing I. typographus-attacked trees, and forest litter below the attacked trees. Fungal identification was done using cultural and molecular methods. The results of the two methods in detecting fungal species were compared. Fungal communities associated with I. typographus differed considerably depending on the hibernation environment. In addition to seven taxa of known ophiostomoid I. typographus-associated fungi, we detected 18 ascomycetes and anamorphic fungi, five wood-decaying basidomycetes, 11 yeasts, and four zygomycetes. Of those, 14 fungal taxa were detected exclusively from beetles that hibernated under bark, and six taxa were detected exclusively from beetles hibernating in forest litter. The spruce pathogen, Ceratocystis polonica, was detected occasionally in bark, while another spruce pathogen, Grosmannia europhioides, was detected more often from beetles hibernating under the bark as compared to litter. The identification method had a significant impact on which taxa were detected. Rapidly growing fungal taxa, e.g. Penicillium, Trichoderma, and Ophiostoma, dominated pure culture isolations; while yeasts dominated the communities detected using molecular methods. The study also demonstrated low frequencies of tree pathogenic fungi carried by I. typographus during its outbreaks and that the beetle does not require them to successfully attack and kill trees.