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Article

Fungal and Bacterial Communities of the Red Turpentine Beetle (Dendroctonus valens LeConte) in the Great Lakes Region, USA

by
Andrew J. Mann
1,*,
Rin M. Barnum
1,
Benjamin W. Held
1,
Kathryn E. Bushley
2,
Brian H. Aukema
3 and
Robert A. Blanchette
1
1
Department of Plant Pathology, University of Minnesota, Saint Paul, MN 55108, USA
2
Emerging Pests and Pathogens Unit, USDA-ARS, Ithaca, NY 14853, USA
3
Department of Entomology, University of Minnesota, Saint Paul, MN 55108, USA
*
Author to whom correspondence should be addressed.
Forests 2025, 16(10), 1604; https://doi.org/10.3390/f16101604
Submission received: 12 August 2025 / Revised: 11 October 2025 / Accepted: 17 October 2025 / Published: 19 October 2025
(This article belongs to the Special Issue Diagnosing and Managing Forest and Tree Diseases: Advances, Challenges and Strategies)
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Abstract

Fungi and bacteria associated with bark beetles can facilitate successful tree colonization, and, in some cases, these fungi act as pathogens of trees. The red turpentine beetle (RTB, Dendroctonus valens) is a bark beetle native to North America that colonizes stressed pines, rarely killing healthy trees. The fungal communities associated with RTB adults, larval galleries, and control tree phloem from red pine (Pinus resinosa) and white pine (P. strobus) forests in the Great Lakes region of the United States were characterized using both culture-independent and culture-dependent methods. Similarly, the bacterial communities associated with RTB adults in the same region were characterized using a culture-independent method. There were significant differences between the adult beetle fungal communities and the tree-based fungal communities. Culture-independent sequencing of RTB adults showed high abundances of the fungal order Filobasidiales (red pine: 28.71% relative abundance, white pine: 6.91% relative abundance), as well as the bacterial orders Enterobacterales (red pine: 53.72%, white pine: 22.15%) and Pseudomonadales (red pine: 15.86%, white pine: 12.91%). In contrast, we isolated high amounts of fungi in the orders Pleosporales (red pine: 21.79%, white pine: 15.90%) and Eurotiales (red pine: 15.38%, white pine: 16.51%) from the adult beetles by culturing. Culture-independent sequencing of beetle galleries yielded high abundances of fungi in the orders Helotiales (red pine: 22.23%, white pine: 23.21%), whereas culture-based isolation from the same galleries yielded high amounts of Eurotiales (red pine: 17.91%, white pine: 17.91%), Hypocreales (red pine: 16.42%, white pine: 16.42%), and Ophiostomatales (red pine: 23.39%, white pine: 23.39%). This contrasts with the culture-independent method, where, likely due to limitations in the sequencing method, the Ophiostomatales accounted for only around 2% of the fungi from RTB galleries in both pine species. We observed a high species-level diversity of Ophiostomatales associated with RTB, isolating 14 species from the Great Lakes region. Leptographium terebrantis, a species that has been described in association with RTB throughout the United States, was the most common species (e.g., >35% of the Ophiostomatales relative abundance in red pine environments and >14% of the Ophiostomatales relative abundance in the white pine environment). This study enhances our understanding of RTB-associated fungi and bacteria in the beetle’s native range at both the community and species levels.

1. Introduction

Many bark beetles (Coleoptera: Curculionidae: Scolytinae) maintain close associations with fungal and bacterial communities, collectively acting as a ‘holobiont’ [1,2,3]. While some microorganisms may disperse via wind or already exist within the tree as endophytes, many are transported from one tree to another by adult beetles carrying the microbes externally as ectosymbionts or internally in mycangia [4]. Some of the associated microorganisms may facilitate key processes for the beetles. For example, fungi and bacteria associated with bark beetles may aid with chemical signaling for mass aggregation [5,6,7], digestion of wood lignocellulose [8,9], nutrient acquisition from wood [10,11,12], and defense against insect pathogens [11,12,13]. Some symbiotic microorganisms may also degrade tree defenses, allowing beetles to colonize new hosts [12,14,15]. Other microorganisms may be detrimental to insect populations by outcompeting nutritionally beneficial microorganisms [10,16] or by directly killing their insect host as pathogens [17].
The red turpentine beetle (RTB), Dendroctonus valens LeConte (Coleoptera: Curculionidae: Scolytinae), is a bark beetle native to North America that rarely kills healthy trees [18]. It colonizes the lower portion of pines, typically as a secondary pest of trees stressed by fire, drought, or after a more aggressive bark beetle has attacked the tree [18]. RTB has received considerable attention in recent decades because it has caused substantial damage to Pinus tabuliformis and P. bungeana as an invasive species in Asia [19]. Between the early 1980s and the 2010s, RTB is estimated to have killed more than 10 million pine trees on over 500,000 ha in China [19].
There are several distinct subpopulations of RTB throughout its native range in North America. The beetles that are found in Minnesota and Wisconsin are genetically distinct from the RTB subpopulations in western North America and China [20]. RTB does not have a mycangium and, in North America, is said to mainly associate with several Ophiostomatales (Fungi: Ascomycota) species, including Leptographium procerum, L. terebrantis, and Ophiostoma gilletteae [21,22,23], although there are records of at least 29 Ophiostomatales species that have been isolated from RTB environments, including many recently described new species [21]. The genomic divide across different geographical areas throughout North America appears to extend to the fungi, where eastern North America RTB, which includes the Great Lakes region, associates with a different collection of Ophiostomatales than the western North America RTB [23].
Additionally, while RTB and its fungi may not be aggressive tree-killers in North America, RTB is part of a complex of root- and lower-bole-feeding beetles that are documented in association with ‘red pine pocket decline and mortality’ (RPPDM) on red pine (Pinus resinosa) plantations in the Great Lakes region of North America [24,25]. RPPDM is an understudied forest health issue. There has been some uncertainty regarding the exact species identities of the Ophiostomatales due to a lack of easily identifiable species-specific morphological traits and a lack of species-level clarity provided by sequencing some common fungal barcoding regions [3,26].
The non-Ophiostomatales fungi associated with RTB in the Great Lakes region are largely unknown. The most common yeasts isolated from RTB elsewhere in North America are in the genera Candida, Cyberlindnera, and Ogataea, with Cyberlindnera americana being among the most isolated species [27]. A metabarcoding study has also revealed the high association of RTB with yeasts, especially in the subphylum Saccharomycotina, orders Phaffomycetales and Serinales, and the genera Cyberlindnera and Candida [28].
The bacteria associated with RTB in the Great Lakes region have been documented through culture-independent [29] and culture-dependent [30] methods. The culture-dependent study isolated Enterobacter, Pseudomonas, Frigoribacterium, and Paenibacillus from RTB adults in red pine stumps [30]. A cross-country comparison also found geographical differences in the bacterial communities of RTB adults, where the Great Lakes bacterial community differed from the western USA RTB bacterial communities [29]. The bacterial richness in bark beetle systems is known to be relatively low compared to other wood-feeding insects, comprising only around a dozen species in several genera [1,31,32].
In this study, we characterize the fungal and bacterial communities associated with the red turpentine beetle using culture-based and non-culture-based approaches at sites in Minnesota and Wisconsin within the Great Lakes region of the United States. We aim to address three research questions: (1) Are there differences in alpha diversity or community composition of fungal communities associated with RTB adults, larval galleries, and non-infested phloem? (2) Are there differences in alpha diversity and community composition of fungi associated with these same sample types or bacteria associated with RTB adults between red versus white pine (Pinus strobus) hosts? (3) What species of Ophiostomales fungi are found in association with RTB adults and galleries in Minnesota and Wisconsin, USA? The results of this study will advance our understanding of the microbial communities associated with this ubiquitous bark beetle.

2. Materials and Methods

2.1. Field Collection

Between April and July in 2021–2023, RTB adults were collected in Lindgren funnel traps at three sites in Minnesota and one site in Wisconsin (Table 1). Each site was selected due to its high likelihood of having RTB present. RTB is found in pine forests across North America, but it is not extremely abundant unless there is a predisposing stressor. The diameter at breast height (DBH, 1.37 m) was measured on one hundred and fifty trees in a transect to determine an average ± 1 SD DBH at each site. The Anoka, Minnesota site is an old-growth white pine stand where trees measured 27.25 ± 22.89 cm. The Sherburne, Minnesota site is a red pine stand adjacent to a recently logged stand, with trees measuring 22.33 ± 12.55 cm in diameter at breast height. The Carlton, Minnesota site is in a red pine stand that had recently been prescribed a low-intensity controlled burn, where the trees measured 19.81 ± 10.16 cm in DBH. The stand is also adjacent to an old-growth red pine stand. The Jackson, Wisconsin site was chosen because it is an area with documented RPPDM. Its trees measured 19.23 ± 9.88 cm in DBH.
Nine funnel traps were spaced at least 90 m apart at each site and baited with 3-carene and frontalin (Dendroctonus valens combo lure, Synergy Semiochemicals, Burnaby, BC, Canada). Beetles were collected from traps at least once per week. Each collection cup contained one-quarter of a Hot Shot No-Pest Strip (18.6% dichlorvos, Spectrum Brands, Madison, WI, USA) and plastic Easter grass (Amscan, Elmsford, NY, USA) to reduce predation.
When collecting beetles from the traps, RTB adults were placed into sterile Whirl-Pak bags (Madison, WI, USA) using sterile forceps. Bycatch was separated from RTB adults and placed into separate plastic bags. RTB adults were identified by their reddish-brown color, large (greater than 5 mm) body length, and shiny declivity [33]. After all the insects were removed from the trap at each collection, the entire contents of the collection cup, including the Hot Shot No-Pest Strip and plastic Easter grass, were sterilized with 70% ethanol (EtOH).
Trees with RTB larval galleries were located by red pitch tubes in the lower 1.5 m of the tree [34]. A 1.9 cm arch punch (General Tools, Secaucus, NJ, USA) was taken at the site of the RTB gallery and placed into a sterile Whirl-Pak bag. Non-infested control phloem, referred to as ‘phloem’ throughout this paper, was collected by taking a punch at the base of a nearby tree of the same size without pitch tubes present, and each sample was placed into separate sterile Whirl-Pak bags. The arch punch was sterilized with 70% EtOH prior to taking each sample. A single gallery or control phloem sample was collected from each tree. Ten gallery samples and ten control phloem samples were collected from each site at the end of each flight season. Adult beetles, larval galleries, and control phloem samples were transported from field sites to the laboratory on ice and stored at −20 °C until processing.

2.2. DNA Extraction for Culture-Independent Sequencing

Total DNA was extracted from 72 adult beetles (48 from red pine, 24 from white pine), 14 larval galleries (seven from red pine, seven from white pine), and 10 control phloem samples (six from red pine, four from white pine) collected in Minnesota during 2021 using the Qiagen PowerPlant Kit (Qiagen N.V., Hilden, Germany). Entire adult beetles were crushed whole during the tissue disruption step. The outer edge of the larval gallery and control phloem samples was removed to reduce transport or lab contamination. For the tissue disruption step, a Roche MagNA Lyser (Roche, Penzberg, Germany) was used for one 30 s cycle at 5500 rpm.

2.3. PCR and Culture-Independent Sequencing

The ITS1 (ITS1F-ITS2) and 16S V3-V4 (357F-806R) regions were sequenced for fungi and bacteria, respectively. Illumina sequencing was performed at the University of Minnesota Genomics Center (Oakdale, MN, USA) according to the methods in [35], using one 2 × 300 v3 Illumina MiSeq flow cell (Illumina, San Diego, CA, USA).

2.4. Culturing of Fungal Communities

Three types of media with prior success in culturing diverse fungal communities from wood environments [36,37,38] were used to culture the RTB fungal communities from the adult beetles, galleries, and control phloem samples. A malt extract agar plus antibiotics medium (M+) containing 1.5% Bacto malt extract (Thermo Fisher, Waltham, MA, USA), 1.5% granulated agar (Apex Bioresearch Products, El Cajon, CA, USA), and amended with 100 ppm streptomycin sulfate salt dissolved in sterile water after autoclaving and cooling (Sigma-Aldrich, Saint Louis, MO, USA) was used as a non-selective fungal medium. A cycloheximide medium [39] was used to select for Ophiostomatales, which tend to be resistant to cycloheximide [40,41]. The cycloheximide medium was prepared by adding 100 ppm cycloheximide (Sigma-Aldrich) dissolved in 95% ethanol to 2% M+ (2.0% Bacto malt extract, 1.5% granulated agar) after autoclaving and cooling. Finally, a semi-selective Basidiomycota medium [42] was prepared by adding Difco yeast extract at 0.2% (Sigma-Aldrich) and benomyl pre-dissolved in 95% ethanol at 0.006% (Santa Cruz Biotechnology, Dallas, TX, USA) to 1.5% M+ prior to autoclaving. After autoclaving and cooling, 85% lactic acid at 0.2% v/v (Aqua Solutions, Deer Park, TX, USA) and 100 ppm streptomycin salt dissolved in water were added.
Culturing took place as soon as possible, within one week of collection. The external and internal fungi were cultured together to capture the full culturable fungal community from adult beetles. This was achieved by rolling each adult beetle in a zigzag pattern across the media to create individual colonies and crushing the adult at the end of the path. A total of 30 adult beetles were cultured at each site and year (10 adult beetles per media type, per site, and year). To culture from tree samples, four pieces of either the RTB galleries or control phloem were placed into the three types of media to induce fungal growth. Petri dishes were checked every 3 days for growth and sub-cultured onto the same type of media from which they originated.

2.5. DNA Extraction from Pure Cultures

DNA was extracted from each unique morphotype after two weeks of growth. The number of plates from each unique morphotype was recorded and used to calculate abundances. At least one of every four plates per morphotype was extracted to reduce the potential of missing cryptic diversity.
Initially, DNA was extracted from each morphotype using a NaOH extraction, modified from [43]. Briefly, mycelia were scraped from actively growing Petri dishes into a microcentrifuge tube containing four glass beads (BioSpec Products, Bartlesville, OK, USA) and 200 µL of 0.5 M NaOH solution (MCB Reagents, Cincinnati, OH, USA). After vortexing for one min and centrifuging for 10 s, 5 µL of the supernatant was transferred to a microcentrifuge tube containing 495 µL Tris-HCl (Thermo Fisher). For isolates where the NaOH extraction was not successful, and for Ophiostomatales, another extraction following the methods described in [44] was performed with slight modifications. Briefly, mycelia from actively growing Petri dishes were transferred to a microcentrifuge tube containing three glass beads and an extraction buffer made up of NaCl (IBI Scientific, Dubuque, IA, USA), Tris-HCl (pH 8.0), ethylenediaminetetraacetic acid (EDTA, Research Products International, Mt. Prospect, IL, USA), and polyvinylpyrrolidone (PVP, Sigma-Aldrich). The tube containing the mycelia and extraction buffer was briefly vortexed and incubated for 15 min at 65 °C, with a one min vortex midway through the incubation period. The tube was then centrifuged (Eppendorf, Hamburg, Germany) at 8000 rpm for 10 min. 500 µL of the wash buffer containing sodium dodecyl sulfate (Sigma-Aldrich) and 0.5 M KCl (Thermo Fisher) was added to the supernatant, vortexed for one min, and centrifuged at 15,000 rpm for 10 min. The supernatant was washed with 99.5% isopropanol (Thermo Fisher) by gentle inversion and centrifugation at 15,000 rpm for 10 min. The pellet was washed a third time with 70% ethanol (Thermo Fisher) by centrifuging for five min at 15,000 rpm. Finally, the pellet was resuspended in sterile molecular-grade water (Thermo Fisher).

2.6. PCR and Sanger Sequencing of Pure Cultures

For every isolate, the full internal transcribed spacer region (ITS) was sequenced using the ITS1F [45] and ITS4 [46] primers. For the Ophiostomatales, an additional four loci were sequenced: translation elongation factor 1α (EF-1α; EF2F [47] and EF2R [48]), nuclear large subunit (LSU; LR0R and LR5 [49]), RNA polymerase II (RPBII; Oph-RPB2F1 and Oph-RPB2R1 [50]), and β-tubulin (βt; T10 [51] and BT2B [52]).
The forward and reverse primers of the ITS polymerase chain reaction (PCR) mixture were prepared to a final primer concentration of 0.2 µM. The EF-1α, LSU, and βt primers had a final concentration of 0.4 µM, and the RPBII primers were prepared to a final concentration of 1.0 µM. All PCR amplifications for Sanger sequencing were performed in a Bio-Rad T100 Thermo Cycler (Hercules, CA, USA). The PCR conditions for each gene region are listed in Table 2. Gel electrophoresis and Sanger sequencing were completed according to [53].

2.7. Community Analysis of Culture-Independent Sequences

Cutadapt version 4.1 [54] was used to remove primers and adapters. The remaining analytical steps were completed in R version 4.4.2 [55]. Forward and reverse sequences were paired, filtered, and chimeras were removed before assigning amplicon sequence variants (ASVs) in DADA2 version 1.31.0 [56]. To maintain a quality score above 30, the fungal sequences were truncated at 277 bp for the forward reads and 279 bp for the reverse reads, and the bacterial sequences were truncated at 256 bp for the forward reads and 260 bp for the reverse reads. The fungal taxonomy was assigned to ITS ASVs with UNITE version 21.04.2024 [57], and the bacterial taxonomy was assigned to 16S ASVs with SILVA version 138.1 [58]. The UNITE-assigned taxonomic names were updated according to the September 2025 classifications in MycoBank [59].
Singleton ASVs, as well as non-fungal and non-bacterial taxa, were removed from the ITS and 16S datasets, respectively. Samples were rarefied at 1005 reads for the fungal dataset and 941 reads for the bacterial dataset. For the fungal dataset, this removed one control phloem sample, one adult beetle sample, and two gallery samples from the Anoka, Minnesota white pine site, plus one control phloem and one adult beetle from the Sherburne, Minnesota red pine site. Due to the low sequencing depth of the bacterial dataset, only adult beetles were included in the bacterial analysis. Despite the poor sequencing quality for the tree-based samples, only six of the adult beetle samples were removed. The removed samples included five of the adult beetles from the Anoka, Minnesota, white pine site and one adult beetle from the Carlton, Minnesota, red pine site. A total of 3,865 fungal ASVs and 2545 bacterial ASVs remained for analysis.
The iNEXT package version 3.0.2 [60,61] was used to create rarefaction curves by sample type (Figures S1 and S2). Plots were visualized with ggplot2 version 3.5.2 [62] and arranged using the cowplot package version 1.2.0 [63]. Alpha diversity indices (species richness, Pielou’s evenness index, Shannon’s index, and Simpson’s index) were calculated using the vegan version 2.7.1 [64] and phyloseq version 1.50.0 [65] packages. Linear mixed-effects models were run using the lmer function in lme4 [66] with site as a random effect for the red pine samples and the lm function for the white pine stand.
Microbial community beta diversity differences among sample type, tree species, and site were evaluated using permutational multivariate analysis of variances (PERMANOVA) with the adonis2 function and 9999 permutations on a Bray–Curtis dissimilarity matrix computed from total-sum-scaled data. We calculated the PERMANOVA pseudo-R2 for the model terms using the default sequential partitioning. The sample type and tree species differences were visualized with non-metric multidimensional scaling (NMDS) plots (metaMDS function, k = 2) on the same Bray–Curtis matrix. The final NMDS stress values were 0.18 for the fungal and 0.216 for the bacterial dataset. Differences in fungal and bacterial orders were represented by sample type and by tree species using 100% stacked bar plots in ggplot2.
Differences in fungal and bacterial orders by sample type and tree species were also calculated using linear mixed-effects models with the lmer function to include site as the random effect, as described above for red pine, and the lm function for the one white pine site. Adjusted p-values using the false discovery rate (FDR) are reported for all multiple comparison analyses in this study.

2.8. Community Analysis of Culture-Dependent Sequences

Sequences were trimmed in Geneious Prime 11.1.5 (Biomatters Ltd., Auckland, New Zealand) and compared to published sequences using BLASTn version 2.17.0 [67]. Taxonomy was assigned to each BLAST result using the September 2025 classification in MycoBank to match the taxonomy from the culture-independent sequences. Sample completeness curves by sample type and tree species were created in ggplot2 and the iNEXT packages. Individual sites in a single year were considered one ‘sample’ for the culture-dependent analysis because many individual specimens only included one fungal species. Stacked bar plots were created in ggplot2 as described above, and linear mixed-effects models were applied to fungal order differences by sample type and tree species using the lmer function with the terms for ‘site’ and ‘year’ as random effects for the red pine sites and the term ‘year’ as a random effect for the white pine site.

2.9. Phylogenetic Placement of Ophiostomatales

Ophiostomatales sequences were aligned to reference sequences using the default settings on the MAFFT v7 online tool [68]. Aligned sequences were then fed through Gblocks 0.91b with the less stringent options selected [69,70]. Each individual locus was then concatenated in R using the ape package version 5.8.1 [71], and maximum likelihood trees were created in the online version of IQ-Tree with 1000 bootstrap replicates [72]. The reference sequences used in the phylogenetic tree included all known RTB-associated fungi and close BLAST matches of the isolates in the present study [21,50]. Afroraffaelea ambrosiae isolate CBS 141678 was used as an outgroup.

3. Results

3.1. Alpha Diversity of Culture-Independent Samples

Individual sites explained 7.00% of the variance in fungal richness, and none of the fungal variance in Pielou’s evenness, Shannon’s, or Simpson’s diversity indices. Additionally, there were no significant differences in fungal alpha diversity among sample types or between forest types (Figure 1).
The random effect of site explained 18% of the total variance in bacterial species richness and none of the total variance in the Pielou’s evenness, Shannon’s, or Simpson’s diversity indices. Similar to the fungal results, there were no significant differences in the alpha diversity indices between bacterial communities of red pine and white pine RTB adult beetles (Figure 2).

3.2. Community Composition of Culture-Independent Samples

The fungal communities differed overall by tree species (F1, 88 = 3.17; p < 0.001) and sample type (F2, 87 = 3.77; p < 0.001). The interaction among site × sample type × tree species explained 14% of the variation in fungal community composition (F4, 85 = 3.48; p < 0.001). In the fungal interaction, there were significant differences between the white pine gallery and adult beetle communities (F1, 14 = 2.91; p = 0.02), between the white pine control phloem and adult beetle communities (F1, 14 = 2.23; p = 0.02), between the red pine gallery and adult beetle communities (F1, 14 = 3.74; p = 0.02), between the red pine control phloem and adult beetle communities (F1, 14 = 3.27; p = 0.02), and between the white and red pine adult beetles (F1, 14 = 4.28; p = 0.02) (Figure 3a). The site × tree species interaction explained 8% of the variation in bacterial community composition (F2, 62 = 2.63; p < 0.001). There was a significant difference between the red pine and white pine adult beetle bacterial communities (F1, 63 = 3.20; p < 0.001) (Figure 3b).

3.3. Taxonomic Composition of Culture-Independent Samples

For the red pine culture-independent dataset, there were many orders that differed significantly among the sample types. Notably, Filobasidiales (most common genera Naganishia and Filobasidium) were more abundant on the red pine adult beetles (28.71% relative abundance) than in the red pine galleries (0.10%; t55.02 = 3.61; p = 0.006) or red pine control phloem samples (1.63%; t55.02 = 3.27; p = 0.008). Helotiales (most common genera: Pezicula and Proliferodiscus) were more abundant in the red pine galleries (22.23%; t55.11 = −4.30; p < 0.001), white pine galleries (23.21%; t28 = −2.94; p = 0.03), red pine control phloem (22.20%; t55.09 = −4.22; p < 0.001), and white pine control phloem (35.31%; t28 = −3.97; p = 0.004) than on the adult beetles from red (2.38%) and white pine (3.22%) forests. Finally, Polyporales (most common genera: Cryptoporus, Hyphoderma, Cabalodontia, Phlebiopsis) were more abundant in the red pine galleries (7.44%) than on adult beetles captured in red pine forests (0.94%; t55.12 = −2.87; p = 0.02). Two orders in the subphylum Saccharomycotina, such as Phaffomycetales (most common genera: Cyberlindnera, Wickerhamomyces) and Serinales (most common genus: Candida) each had >6% relative abundance in both the red and white pine adult beetles, >0.40% relative abundance in the galleries of both tree species, and were absent from the phloem samples in each tree species, but the differences were not significant. There was a similar trend for the Ophiostomatales (most common genera: Ceratocystiopsis and Ophiostoma), where the differences were not significant despite making up 2.9% of the fungi from red pine adult beetles, 11.38% of the white pine adult beetle fungi, and >2% of the gallery fungi of both tree species, versus less than 0.6% of both the red and white pine control phloem (Figure 4a and Table S1).
There were no significant differences in the bacterial order abundances between the red pine and white pine adult beetles; however, insignificant differences were present. Xanthomonadales (most common genera: Frateuria and Rhodanobacter) made up 0.68% of the red pine adult beetle bacteria and 6.48% of the white pine adult beetle communities. Rickettsiales (most common genera: Wolbachia and Rickettsia) were absent from the red pine adult beetle samples but made up 3.29% of the white pine adult beetle communities. Pseudomonadales (genus: Pseudomonas) were abundant in the adults of both red pine (15.86%) and white pine (12.91%). The Enterobacterales (most common genera: Arsenophonus, Enterobacter, Erwinia, Klebsiella, Lelliottia, Pantoea, Rahnella, Raoultella, and Serratia) were also abundant in red pine (53.72%) and white pine (22.15%) adult beetles. Finally, the Betaproteobacteriales (genus: Massilia) were also common in association with the adult beetles of red pine (11.26%) and white pine (9.84%) forests (Figure 4b).

3.4. Taxonomic Composition of Culture-Dependent Samples

A total of 205 species from four phyla and 37 orders were cultured from adult beetles, galleries, and control phloem of red and white pine forests in the Great Lakes region. There were no statistically significant differences among sample types or between tree species. Like with the culture-independent bacterial samples, some insignificant trends did emerge. Pleosporales (most common genera: Alternaria and Epicoccum) were very abundant in the red pine adult beetles (21.79%), white pine adult beetles (15.90%), red pine control phloem (16.00%), and white pine control phloem (16.07%) samples. Eurotiales (most common genera: Penicillium and Talaromyces) were also commonly isolated across sample types, accounting for at least 15% of the total relative abundance in every sample type and 40% of the red pine control phloem. For the Phaffomycetales (genus: Cyberlindnera) and Serinales (genera: Candida, Danielia), the Phaffomycetales made up >1% of the adult fungal communities for beetles captured in both forest types, and 0.75% of the red pine gallery fungi. The Serinales accounted for >2.5% of the fungi isolated from adults in both red and white pine forests, and were 1.5% of the fungi from red pine galleries. Neither order was isolated from the red or white pine control phloem samples. Finally, the Ophiostomatales (genera: Ceratocystiopsis, Ophiostoma, Graphilbum, Leptographium, and Sporothrix) made up 22.39% of the fungi isolated from red pine galleries, 22.39% of the fungi isolated from white pine galleries, and 11.86% of the fungi isolated from adults captured in red pine forests. However, the Ophiostomatales comprised only 2.14% of the fungi isolated from white pine adults. (Figure 4c and Table 3).

3.5. Ophiostomatales Species Isolated in the Culture-Dependent Samples

A total of 14 Ophiostomatales species were isolated from RTB environments in the Great Lakes region. The most common species isolated was Leptographium terebrantis, which made up >30% of the Ophiostomatales isolated from red pine galleries, white pine galleries, and red pine adult beetles. Graphilbum sp. A is likely a new species related to Graphilbum ipis-grandicollis, matching Gra. ipis-grandicollis at 96.77% (Figure 5 and Table 3).

4. Discussion and Conclusions

In this study, we used culture-independent and culture-dependent methods to examine the fungal and bacterial communities associated with the red turpentine beetle adults, galleries, and the phloem of nearby control trees. There were no significant differences in the alpha diversity metrics, such as species richness and evenness, among different sample types and between red and white pine forests for the fungal or bacterial datasets. We also measured community-level differences in the fungal and bacterial communities between red and white pine stands. The fungal communities associated with adult RTB differed significantly from those on the tree-based fungal communities; however, there were no overall differences between the gallery and phloem communities. We also observed taxonomic differences in the fungi among sample types using culture-independent techniques. The Helotiales and Polyporales were more abundant in the galleries than on adults, while the Filobasidiales were more abundant on the adults than in the galleries. For the bacteria, advances in sequencing technologies did not reveal a novel diversity of bacteria that were not present in previous studies. A high species richness of Ophiostomatales species was isolated in this study, contributing to the substantial number of Ophiostomatales that RTB associates with in its native range.
The adult beetles in our study carried a high diversity of fungi that were not detected in the galleries. This phenomenon has been noted before [73] and may be a consequence of a trap-based study where the beetles picked up more wind-disseminated fungi than they would have flying directly between trees, a lack of surface sterilization, or potentially storing at freezing temperatures. Although we attempted to minimize the transfer of fungal spores from trap bycatch to RTB adults, it is possible that the predators captured in the traps contributed to the fungal diversity recovered from RTB adults. Additionally, it is possible that the dichlorvos in the Hot Shot No-Pest Strip acted as a fungicide, altering the adult fungal communities, but more testing on this potential limitation is needed.
We observed differences between the red and white pine fungal and bacterial communities. The red turpentine beetle has an expansive range, inhabiting many pine species across most of North America [18]. Previous studies have found differences in the genomics of the beetles [18], as well as the associated fungal communities [23] and associated bacterial communities [29] across large geographical areas. These differences may be due, in part, to variation in host tree species, as the main differences observed in these studies are between eastern and western RTB subpopulations, where there is no overlap in pine host species. In our study, we demonstrate that there are differences in RTB microbial communities within the same geographic region.
There were also differences between the RTB adult fungal communities and the tree-based (gallery and phloem) fungal communities, but not between the gallery and phloem communities, in our study. This is similar to findings from the emerald ash borer system in its native range in Asia [74]. The emerald ash borer and RTB have similar life histories in their respective native ranges, where each beetle acts as a secondary colonizer of stressed trees and rarely kills healthy trees [19,75]. It is possible that the chemical defenses of healthy trees do not allow significantly different microbial communities to establish in association with these beetles. Another alternative hypothesis for the lack of community-wide differences between the RTB galleries and control phloem is that the galleries in our study were sampled too soon after colonization, before the microbial communities in the galleries had a chance to develop.
We recovered a significantly higher abundance of Polyporales in the red pine galleries than were associated with the red pine adult beetles using the culture-independent extraction. This finding contrasts with the culturing study, where we recovered the same amount of Polyporales on red pine adult beetles as in red pine galleries and a relatively high rate of Polyporales on the white pine adults, versus none in the white pine galleries. Cryptoporus was the most common Polyporales genus recovered with both the culture-independent and culture-dependent methods. Cryptoporus volvatus is a white rot fungus and often fruits around bark beetle galleries, but bark beetles reportedly do not pick up C. volvatus from their galleries [73]. Rather, previous studies have demonstrated that the spores are mainly wind-dispersed, and the high abundance cultured from the adults in our study may be a result of the trap-based study [73].
Yeast volatiles seem to play important roles in insect signaling, including for bark beetles [27,76]. Despite the relatively high abundance of budding yeasts (subphylum Saccharomycotina, orders Alaninales, Dipodascales, Phaffomycetales, Pichiales, Saccharomycetales, and Serinales) measured from adults (>18%) and galleries (>6%) of both tree species using the culture-independent method, and from adults (>6%) and from galleries (>3%) using the culturing method, we found much lower rates of yeasts using both culture-independent and culture-dependent methods than the >90% relative abundance that had previously been reported for RTB in North America [28]. The low rates of Saccharomycotina isolation may be because we did not use a yeast-selective medium, unlike other studies which focused on RTB-associated yeasts such as [77]. We isolated several isolates that closely match Candida piceae (>98% match), Danielia oregonensis (syn Candida oregonensis) (100% match), and Cyberlindnera americana (100% match). Cyberlindnera americana and Candida piceae have been reported as the main yeast associates of RTB in both North America [27] and Asia [27,77]. The high abundance of Cyberlindnera americana is supported by the culture-independent results in the present study, where Cyberlindnera was the most abundant genus in the entire fungal dataset. Previous studies have hypothesized that Cyberlindnera may play important roles for bark beetles, such as potentially degrading tree defensive chemicals [78] or contributing to producing volatiles that are attractive to beetles [79]. Further testing is needed to determine whether Cyberlindnera contributes to RTB’s ability to kill trees in Asia.
Despite advances in sequencing technology, our bacterial findings were consistent with the Dendroctonus bacterial biodiversity surveys that took place more than a decade ago [1]. The most common bacteria associated with RTB adults in our study were Pseudomonas and Enterobacterales such as Enterobacter, Rahnella, Raoultella, and Serratia. The bacteria isolated from RTB systems seem to have interesting interactions with pine monoterpenes, where RTB-associated Enterobacter can grow on limonene without inhibition, RTB-associated Pseudomonas can grow on 3-carene and alpha pinene without inhibition, and both genera can grow on myrcene without inhibition [30]. Additionally, Rahnella and Serratia have been shown to reduce alpha pinene and abietic acid, respectively [15]. Wolbachia was also relatively common on RTB adults in the white pine stand. This genus was present in the red pine samples, but at low enough levels that it was excluded from the analysis during the rarefaction step. Wolbachia is found abundantly across insects, including other bark beetles, and may play a role in selecting for more females in the population [80].
The most studied associates of bark beetles are members of the Ophiostomatales. These fungi are well-adapted for dispersal by beetles due to their sticky spores at the tips of long sexual or asexual structures [50]. Most of the Ophiostomatales cause staining in the sapwood of trees, which does not impact the structural integrity of the wood. Overall, the Ophiostomatales did not make up a large portion of the culture-independent fungal communities. Although we recovered a higher relative abundance of Ophiostomatales by culturing, the culture-based approach still recovered relatively low amounts of Ophiostomatales compared to other bark beetle studies [81]. This low recovery rate may be partially because we did not surface sterilize our samples, and due to limitations of ITS as a metabarcoding region for Ophiostomatales [3,9]. Future RTB microbiome studies should test primers for other gene regions, such as the 28S ribosomal large subunit (LSU), which may more reliably allow researchers to detect Ophiostomatales at the levels that are present in nature [9].
In China, RTB commonly associates with Leptographium procerum and Ophiostoma minus [19]; however, in North America, over 20 Ophiostomatales species have been isolated from RTB, and the beetle seems to be without a clear symbiont [21,22]. In the present study, we isolated several species previously isolated from RTB environments in North America, including Leptographium terebrantis, L. gordonii, L. procerum, Ophiostoma gilletteae, O. minus, and O. ips. The most isolated Ophiostomatales species in our study was L. terebrantis, which has previously been described in association with RTB in both eastern and western North America [21,23]. We also isolated two recently described species, O. gilletteae and L. gordonii [21]. Ophiostoma gilletteae is a species that had previously only been isolated from western RTB subpopulations, and L. gordonii has only been found in eastern RTB subpopulations [21,23], indicating that the Great Lakes region may be an area where ‘eastern’ and ‘western’ Ophiostomatales mix, although further testing is needed. Furthermore, this study contributes sequences for O. gilletteae using the recently created Ophiostomatales-specific RPBII primers, which will help future researchers separate O. gilletteae from the phylogenetically related O. adjuncti (Table S2).
We also isolated L. procerum and L. terebrantis, two of the fungi that have been described in association with RPPDM in the Great Lakes region [24,25]. This study also provides the first RPBII sequence of L. terebrantis, which will help field practitioners, disease diagnosticians, and researchers identify the Ophiostomatales in stands with RPPDM. Additionally, L. procerum is the main associate of RTB in China [19]. Leptographium procerum has not been isolated from western North America [23]; however, based on the genomics of RTB, the subpopulation causing damage in China likely originated in the western United States [20]. Researchers have hypothesized that the L. procerum associated with RTB in China may have arrived independently of the beetle and may have its origins in Europe [82].
Finally, we isolated eight species that had not been previously described in association with RTB, namely Ceratocystiopsis brevicomis, C. minima, C. yantaiensis, Graphilbum fragrans, Gra. pusillum, Sporothrix lunata, S. variecibatus, and a species similar to Gra. ipis-grandicollis. All the new associates came from genera that have not been isolated very frequently in association with RTB [21]. RTB associates with a high diversity of Ophiostomatales in low abundances, and future studies will likely continue to isolate many additional incidental associates as researchers move away from ITS-only Ophiostomatales identification.
Collectively, the results of this study provide more insight into the fungal and bacterial communities associated with RTB in its native range. Future studies should expand upon this study by evaluating RTB culture-independent communities using gene regions in addition to ITS for potentially better recovery of the Ophiostomatales. Additionally, the fungal and bacterial communities associated with RTB in the western United States should be studied in greater depth to determine how the microbiota of RTB in its native range directly compares to that of RTB in its invaded range in Asia. Finally, although most of the Ophiostomatales associated with RTB appear to be incidental associates, occurring sparingly and in low densities, studies on the potential functions of these species would add to the understanding of bark beetle-fungal interactions.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f16101604/s1, Figure S1: Fungal rarefaction curves; Figure S2: Bacterial rarefaction curves; Figure S3: Sample completeness curves; Table S1: Comparisons of the abundances of microbial orders; Table S2: Accession numbers of fungi included in this study.

Author Contributions

Conceptualization, A.J.M., K.E.B. and R.A.B.; methodology, A.J.M., B.W.H., K.E.B., B.H.A. and R.A.B.; formal analysis, A.J.M.; investigation, A.J.M. and R.M.B.; resources, B.W.H., B.H.A. and R.A.B.; data curation, A.J.M.; writing—original draft preparation, A.J.M.; writing—review and editing, A.J.M., R.M.B., B.W.H., K.E.B., B.H.A. and R.A.B.; visualization, A.J.M.; supervision, R.A.B.; project administration, K.E.B. and R.A.B.; funding acquisition, K.E.B., B.W.H., B.H.A. and R.A.B. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the U.S. National Science Foundation Dimensions of Biodiversity, grant number 2030036.

Data Availability Statement

The Accession numbers for the Sanger sequences are found in Table S2. The Illumina sequences have been deposited in BioProject: PRJNA1308577. The aligned sequences for Figure 5 can be downloaded at https://github.com/andrewmann1/Red-turpentine-beetle-fungi-and-bacteria (accessed on 12 August 2025).

Acknowledgments

We thank Lou McCarthy and Tessa Kothlow for assistance in extracting fungal DNA, Brian Schwingle of the Minnesota Department of Natural Resources, and Alex Hornung of the Wisconsin Department of Natural Resources for assistance in locating field sites with RTB. We appreciate Kyle Gill and all the staff at the University of Minnesota Cloquet Forestry Center for their help in selecting trapping locations and collecting beetles. Thanks also to Nickolas Rajtar for help collecting beetles. We also thank Rob Venette and the Minnesota Invasive Terrestrial Plant and Pest Center at the University of Minnesota for their collaboration, discussions, and advice.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
RTBRed turpentine beetle (Dendroctonus valens)
RPPDMRed pine pocket decline and mortality

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Figure 1. The alpha diversity of culture-independent fungal communities associated with red turpentine beetle adults, galleries, and control phloem in Minnesota, USA. Panels show (a) fungal richness; (b) fungal evenness; (c) Shannon’s diversity; (d) Simpson’s diversity. Each sample was rarefied to 1005 reads. The large points indicate model-estimated marginal means, error bars represent ±1 SD, and smaller points represent all the sample points.
Figure 1. The alpha diversity of culture-independent fungal communities associated with red turpentine beetle adults, galleries, and control phloem in Minnesota, USA. Panels show (a) fungal richness; (b) fungal evenness; (c) Shannon’s diversity; (d) Simpson’s diversity. Each sample was rarefied to 1005 reads. The large points indicate model-estimated marginal means, error bars represent ±1 SD, and smaller points represent all the sample points.
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Figure 2. The alpha diversity for culture-independent bacterial samples from red turpentine beetle adults in Minnesota, USA. Panels show (a) bacterial richness; (b) bacterial evenness; (c) Shannon’s diversity; (d) Simpson’s diversity. Each sample was rarefied to 941 reads. The large points indicate model-estimated marginal means, error bars represent ±1 SD, and smaller points represent all the sample points.
Figure 2. The alpha diversity for culture-independent bacterial samples from red turpentine beetle adults in Minnesota, USA. Panels show (a) bacterial richness; (b) bacterial evenness; (c) Shannon’s diversity; (d) Simpson’s diversity. Each sample was rarefied to 941 reads. The large points indicate model-estimated marginal means, error bars represent ±1 SD, and smaller points represent all the sample points.
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Figure 3. NMDS plots for (a) culture-independent fungi and (b) bacteria. The green points represent samples from adult red turpentine beetles, orange points represent samples from red turpentine beetle galleries, and the violet points represent samples from control phloem samples. The samples from red pine forests are represented by circles, and triangles represent the samples from white pine forests. For (a) ellipses represent differences among sample types for both red and white pine fungal samples. For (b) ellipses represent differences between red and white pine adult beetle bacterial communities.
Figure 3. NMDS plots for (a) culture-independent fungi and (b) bacteria. The green points represent samples from adult red turpentine beetles, orange points represent samples from red turpentine beetle galleries, and the violet points represent samples from control phloem samples. The samples from red pine forests are represented by circles, and triangles represent the samples from white pine forests. For (a) ellipses represent differences among sample types for both red and white pine fungal samples. For (b) ellipses represent differences between red and white pine adult beetle bacterial communities.
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Figure 4. 100% stacked bar plots by (a) culture-independent fungi, (b) culture-independent bacteria, and (c) culture-dependent fungi. For (a) each sample was rarefied to 1,005 reads, for (b) each sample was rarefied to 941 reads, for (c) the number of fungi isolated from each sample type differed: red pine adult n = 312; red pine gallery n = 134; red pine phloem n = 75; white pine adult n = 327; white pine gallery n = 67; white pine phloem n = 56. The most abundant orders are displayed according to the corresponding color in the legends. Less abundant orders are classified into the “Other” category (gray).
Figure 4. 100% stacked bar plots by (a) culture-independent fungi, (b) culture-independent bacteria, and (c) culture-dependent fungi. For (a) each sample was rarefied to 1,005 reads, for (b) each sample was rarefied to 941 reads, for (c) the number of fungi isolated from each sample type differed: red pine adult n = 312; red pine gallery n = 134; red pine phloem n = 75; white pine adult n = 327; white pine gallery n = 67; white pine phloem n = 56. The most abundant orders are displayed according to the corresponding color in the legends. Less abundant orders are classified into the “Other” category (gray).
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Figure 5. Ophiostomatales phylogenetic tree based on the concatenated sequences of five loci, including ITS, LSU, βt, EF-1α, and RPBII. Afroraffaelea ambrosiae isolate CBS 141678 is designated as the outgroup. Species in bold were isolated in the present study. Graphilbum sp. A did not align with any described species. Node supports are presented as ML bootstrap (>60).
Figure 5. Ophiostomatales phylogenetic tree based on the concatenated sequences of five loci, including ITS, LSU, βt, EF-1α, and RPBII. Afroraffaelea ambrosiae isolate CBS 141678 is designated as the outgroup. Species in bold were isolated in the present study. Graphilbum sp. A did not align with any described species. Node supports are presented as ML bootstrap (>60).
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Table 1. Collection sites of red turpentine beetle (RTB) adults, galleries, and control phloem. In each sampling year, larval galleries were sampled from ten infested trees and control phloem was collected from ten trees without RTB infestation.
Table 1. Collection sites of red turpentine beetle (RTB) adults, galleries, and control phloem. In each sampling year, larval galleries were sampled from ten infested trees and control phloem was collected from ten trees without RTB infestation.
SiteStateLongitudeLatitudeElevation (m)Tree SpeciesYears Sampled
AnokaMinnesota45.33113−93.12880277White pine2021
2022
SherburneMinnesota45.44212−93.69684298Red pine2021
2022
CarltonMinnesota46.69949−92.52472386Red pine2021
2022
2023
JacksonWisconsin44.349704−90.695295282Red pine2023
Table 2. PCR conditions used in this study.
Table 2. PCR conditions used in this study.
Gene RegionConditions
ITS94 °C for 5 min. 35 cycles of 94 °C for 60 s, 52 °C for 60 s, 72 °C for 60 s. 72 °C for 5 min.
LSU95 °C for 3 min. 30 cycles of 94 °C for 60 s, 50 °C for 45 s, 72 °C for 2 min. 75 °C for 5 min.
EF-1α95 °C for 5 min. 31 cycles of 94 °C for 30 s, 55 °C for 45 s, 72 °C for 90 s. 72 °C for 10 min.
RPBII94 °C for 3 min. 40 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 60 s. 72 °C for 5 min.
βt94 °C for 3 min. 31 cycles of 94 °C for 30 s, 58 °C for 30 s, 72 °C for 60 s. 72 °C for 7 min.
Table 3. Red turpentine beetle Ophiostomatales species isolated in the Great Lakes region, USA, in this study. The relative abundance value is the percent of that species per Ophiostomatales species within that tree species and sample type.
Table 3. Red turpentine beetle Ophiostomatales species isolated in the Great Lakes region, USA, in this study. The relative abundance value is the percent of that species per Ophiostomatales species within that tree species and sample type.
Tree SpeciesSample TypeSpeciesRelative Abundance (%)
Red PineAdultCeratocystiopsis brevicomis2.70
Graphilbum sp. A16.22
Graphilbum pusillum5.41
Leptographium gordonii10.81
Leptographium terebrantis35.14
Ophiostoma minus10.81
Sporothrix lunata18.92
GalleryCeratocystiopsis yantaiensis3.13
Graphilbum fragrans6.25
Graphilbum sp. A6.25
Graphilbum pusillum18.75
Leptographium procerum6.25
Leptographium terebrantis50.00
Ophiostoma gilletteae3.13
Ophiostoma ips3.13
Ophiostoma minus3.13
White PineAdultGraphilbum sp. A42.86
Leptographium terebrantis14.29
Ophiostoma gilletteae28.57
Ophiostoma minus14.29
GalleryCeratocystiopsis brevicomis6.67
Ceratocystiopsis minima26.67
Leptographium procerum6.67
Leptographium terebrantis40.00
Sporothrix lunata6.67
Sporothrix variecibatus13.33
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Mann, A.J.; Barnum, R.M.; Held, B.W.; Bushley, K.E.; Aukema, B.H.; Blanchette, R.A. Fungal and Bacterial Communities of the Red Turpentine Beetle (Dendroctonus valens LeConte) in the Great Lakes Region, USA. Forests 2025, 16, 1604. https://doi.org/10.3390/f16101604

AMA Style

Mann AJ, Barnum RM, Held BW, Bushley KE, Aukema BH, Blanchette RA. Fungal and Bacterial Communities of the Red Turpentine Beetle (Dendroctonus valens LeConte) in the Great Lakes Region, USA. Forests. 2025; 16(10):1604. https://doi.org/10.3390/f16101604

Chicago/Turabian Style

Mann, Andrew J., Rin M. Barnum, Benjamin W. Held, Kathryn E. Bushley, Brian H. Aukema, and Robert A. Blanchette. 2025. "Fungal and Bacterial Communities of the Red Turpentine Beetle (Dendroctonus valens LeConte) in the Great Lakes Region, USA" Forests 16, no. 10: 1604. https://doi.org/10.3390/f16101604

APA Style

Mann, A. J., Barnum, R. M., Held, B. W., Bushley, K. E., Aukema, B. H., & Blanchette, R. A. (2025). Fungal and Bacterial Communities of the Red Turpentine Beetle (Dendroctonus valens LeConte) in the Great Lakes Region, USA. Forests, 16(10), 1604. https://doi.org/10.3390/f16101604

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