R. Vilgalys’s research while affiliated with Duke University and other places

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Publications (903)


Mycorrhiza formation rate in response to organic matter and inorganic nitrogen (N) treatments. (a) Mycorrhiza formation rate of Pinus taeda seedlings only grown under iron (Fe)‐coated sand. Variations across treatments were evaluated by a two‐way ANOVA followed by HSD post hoc test. Uppercase and lowercase letters represent significant differences in mycorrhiza formation rate across different N treatments under conditions with and without organic matter addition, respectively. (b) Pearson's correlation analysis was performed to assess the correlation between treated N levels and mycorrhiza formation rate under conditions with and without organic matter addition, respectively. Boxes indicate interquartile range; horizontal lines within the boxes indicate the medians of the data; whiskers above and below the boxes indicate the highest and lowest values. +Fe, with Fe‐coated sand addition; −OM, without organic matter addition; +OM, with organic matter addition.
Synchrotron X‐ray microfluorescence analysis identifying iron (Fe) concentrations in ectomycorrhizas in response to organic matter and inorganic nitrogen (N) treatments. Only Pinus taeda seedlings grown under Fe‐coated sand were analyzed. The Fe concentration of different mycorrhizal compartments, including whole mycorrhizas (a, e), mantle (b, f), cortex (c, g), and vascular compartments (d, h), was quantified. In (a–d), the effect of organic matter and N treatments on the Fe concentration in each compartment was evaluated by a two‐way ANOVA and followed by HSD post hoc test. Uppercase and lowercase letters indicate significant differences among different added inorganic N levels under conditions with and without organic matter addition, respectively. In (e–h), Pearson's correlation analysis was performed to identify the linear correlation between the added inorganic N levels and Fe concentration in each ectomycorrhizal compartment under conditions with and without organic matter addition, respectively. Boxes indicate interquartile range; horizontal lines within the boxes indicate the medians of the data; whiskers above and below the boxes indicate the highest and lowest values; dots in each box indicate replicates within each treatment. +Fe, with Fe‐coated sand addition; −OM, without organic matter addition; +OM, with organic matter addition. ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Synchrotron X‐ray microfluorescence analysis illustrating the distribution of iron (Fe), zinc (Zn), calcium (Ca), and copper (Cu) across ectomycorrhizal compartments. The Pinus taeda–Suillus cothurnatus grown under conditions with Fe‐coated (+Fe) sand and organic matter (+OM) were selected to compare the element distribution along a gradient of NH4NO3 treatments (0, 4.2, or 42 mg NH4NO3–N kg⁻¹ sand). Spatial distribution analyses of elements were performed on ectomycorrhizas. In (a–c), the fluorescence intensity of Fe (red), Zn (green), and Ca (blue), Cu (yellow) were collected based upon fluorescence counts per second, and the color wheel shows composite intensities of elements Fe (red), Zn (green), and Ca (blue). The cellular‐scale Fe distribution (μg g⁻¹) within a thin section of each mycorrhiza (30 μm thickness) was further quantified, as indicated by a white box with an arrowhead in (a–c). In (d–f), histogram plots show the distribution of Fe concentration in each compartment under different N treatments. Each column in the histograms represents mean values of Fe intensity at each pixel position (4 μm per pixel) within the marked area, with colors red, green, and blue corresponding to Fe extracted from the fungal mantle, cortex, and vascular compartments, respectively. Grey bars represent the SE associated with each measurement. The same analysis was performed for the other elements (Zn, Ca, and Cu) and the results were shown in Supporting Information Fig. S3.
Synchrotron X‐ray microfluorescence analysis identifying the concentration of calcium (Ca) (a–d), zinc (Zn) (e–h), and copper (Cu) (i–l) in ectomycorrhizas in response to organic matter and nitrogen (N) treatments. Only Pinus taeda seedlings grown under Fe‐coated sand were analyzed. The concentration of elements across different mycorrhizal compartments, including whole mycorrhizas (a, e, i), mantle (b, f, j), cortex (c, g, k), and vascular (d, h, l), was quantified. The effect of organic matter and N treatments on the element concentration in each compartment was evaluated by a two‐way ANOVA and followed by HSD post hoc test. Uppercase and lowercase letters indicate the significant differences among different added inorganic N levels under conditions with and without organic matter addition, respectively. Boxes indicate interquartile range; horizontal lines within the boxes indicate the medians of the data; whiskers above and below the boxes indicate the highest and lowest values. +Fe, with Fe‐coated sand addition; −OM, without organic matter addition; +OM, with organic matter addition.
Relationships between iron (Fe) concentration and those of calcium (Ca), zinc (Zn), and copper (Cu) in ectomycorrhizas. Only the Pinus taeda seedlings grown under Fe‐coated sand (+Fe) and organic matter addition (+OM) were analyzed. Element concentrations were averaged across the entire mycorrhizas. Dot colors represent the gradient of inorganic N levels: black, 0 mg NH4NO3–N kg⁻¹ sand; blue, 4.2 mg NH4NO3–N kg⁻¹ sand; red, 42 mg NH4NO3–N kg⁻¹ sand.
Inorganic nitrogen and organic matter jointly regulate ectomycorrhizal fungi‐mediated iron acquisition
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January 2025

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Ectomycorrhizal fungi (EMF) play a crucial role in facilitating plant nutrient uptake from the soil although inorganic nitrogen (N) can potentially diminish this role. However, the effect of inorganic N availability and organic matter on shaping EMF‐mediated plant iron (Fe) uptake remains unclear. To explore this, we performed a microcosm study on Pinus taeda roots inoculated with Suillus cothurnatus treated with +/−Fe‐coated sand, +/−organic matter, and a gradient of NH4NO3 concentrations. Mycorrhiza formation was most favorable under conditions with organic matter, without inorganic N. Synchrotron X‐ray microfluorescence imaging on ectomycorrhizal cross‐sections suggested that the effect of inorganic N on mycorrhizal Fe acquisition largely depended on organic matter supply. With organic matter, mycorrhizal Fe concentration was significantly decreased as inorganic N levels increased. Conversely, an opposite trend was observed when organic matter was absent. Spatial distribution analysis showed that Fe, zinc, calcium, and copper predominantly accumulated in the fungal mantle across all conditions, highlighting the mantle's critical role in nutrient accumulation and regulation of nutrient transfer to internal compartments. Our work illustrated that the liberation of soil mineral Fe and the EMF‐mediated plant Fe acquisition are jointly regulated by inorganic N and organic matter in the soil.

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Biosynthetic Gene Cluster Diversity Across Native and Introduced Populations of an Ectomycorrhizal Fungus, Suillus luteus

September 2024

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9 Reads

Non-pathogenic fungi are being increasingly recognized for their involvement in biological invasions, where they have the potential to cause ecological and economical harm. One notable example is the ectomycorrhizal (EcM) fungus, Suillus luteus, which is commonly found co-invading with non-native pines that have escaped from commercial pine plantations across the Southern Hemisphere. In such invasions, selection pressures imposed by novel plants, soil microbes, and environmental conditions, as well as impoverished assemblages of co-invading EcM fungi, may exert particularly strong selective effects and contribute to driving evolutionary divergence in traits. We investigated the potential impact of this global co-introduction on biosynthetic gene cluster (BGC) diversity in native and introduced populations of S. luteus. We found that native populations demonstrated higher BGC diversity at both the gene and clan levels compared to introduced populations. Additionally, we identified a collection of unique BGCs as well as 24 highly conserved clans, including two featuring known pathways, within both native and introduced populations. Furthermore, our study confirmed the presence of three previously identified clades of S. luteus with distinct gene cluster family (GCF) diversity. Thus, our study concludes that the introduction of S. luteus has driven evolutionary changes in its BGCs, underscoring the broader implications of global co-introductions on fungal adaptation in new environments.


FIGURE 5
First rotation and second rotation site designs and descriptions.
Generalized linear regression results from fungal biomass response to increases in P carryover rate by the site (Alfisol & Spodosol), bag treatment (C & T), and burial period.
Fungal biomass and ectomycorrhizal community assessment of phosphorus responsive Pinus taeda plantations

May 2024

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51 Reads

Frontiers in Fungal Biology

Ectomycorrhizal fungi and non-ectomycorrhizal fungi are responsive to changes in environmental and nutrient availabilities. Although many species of ectomycorrhizas are known to enhance the uptake of phosphorus and other nutrients for Pinus taeda , it is not understood how to optimize these communities to have tangible effects on plantation silviculture and P use efficiency. The first step of this process is the identification of native fungi present in the system that are associated with P. taeda and influence P uptake efficiency. We used sand-filled mesh bags baited with finely ground apatite to sample ectomycorrhizal and non-ectomycorrhizal fungi associated with the rhizosphere of P-responsive P. taeda under several field conditions. Mesh bags were assessed for biomass accumulation over three years using a single three-month burial period pre-harvest and three six-month burial periods post-planting. Amplicon sequencing assessed ectomycorrhizal and non-ectomycorrhizal communities between phosphorus treatments, sites, mesh bags, and the rhizosphere of actively growing P. taeda in the field. We found biomass accumulation within the mesh bags was inversely related to increasing phosphorus fertilization (carryover) rates from pre-harvest to post-planting. Up to 25% increases in total biomass within the bags were observed for bags baited with P. Taxonomic richness was highest in Alfisol soils treated with phosphorus from the previous rotation and lowest in the Spodosol regardless of phosphorus treatment.


Morphological and phenotypic variation across Suillus. (a–h) Representative members of the major clades within the Suillus phylogeny: Larix‐associated species (a) Suillus ampliporus displaying the radiating pores characteristic of most members of the genus. (b) Suillus clintonianus‐displaying the copious viscus slime often produced by Suillus fruit bodies. (c) Suillus grisellus highlighting morphological variation present within Larix‐associated species. (d) Pseudotsuga‐associated S. lakei (Rocky Mountain form) highlighting the significant carbon allocation needed to form Suillus fruit bodies, which here exceed 15 cm in diameter. Pinus‐associated species: (e) S. subalutaceus representing species with longer stipes. (f) Suillus luteus, the type species of the genus, displaying a prominent veil, a diagnostic feature for some species. (g) Suillus spraguei, displaying distinctive red scales on the cap cuticle. (h) Suillus tomentosus with robust fruiting bodies and variable bluing reactions of the trama tissue. (i–p) Other diagnostic and morphologically important characteristics of Suillus (i) A young specimen of S. weaverae showing milky droplets and punctate stipe as a result of glandular cells. (j) An old specimen of S. caerulescens, strongly stained blue, with insect larvae tunnels. (k) Insect larvae quickly consume Suillus fruit bodies and can ruin an attempt at making spore prints. (l) A spore print of S. lakei on aluminum foil for spore collection. (m) A portion of an S. lakei fairy ring with large fruit bodies, which require substantial carbon allocations from the host. (n) A mycorrhizal root of S. paluster with rhizomorphs at the base, typically found in Suillus mycorrhizas. (o) The mycoparasitic Chroogomphus vinicolor (left) fruiting next to its host, a senescing S. pungens (right). (p) Hyphae of C. vinicolor, stained dark violet with Melzer's reagent, within a mycorrhizal root of S. pungens.
Representatives of the Suillus Genome Strain Culture Collection on four media types. Cultures were grown in 9 cm Petri plates on four media types including (a, e, i, m) Modified Melin‐Norkrans (MMN), (b, f, j, n) Modified Fries Media (Fries). (c, g, k, o) Modified Hagem's Agar (Hagem's), and (d, h, l, p) Pachlewski's Media (Pachlewski's). See Supporting Information Notes S3 for Suillus‐optimized media recipes. All media types were prepared at their full respective carbon concentrations and adjusted to pH 6 before autoclaving. Cultures were grown for 28 d, at room temperature, in the dark, before being photographed. Strains represented here are (a–d) S. weaverae FC27, (e–h) S. pungens FC27, (i–l) S. fuscotomentosus FC203, and (m–p) the monokaryotic strain S. hirtellus EM16. These four strains are part of the Suillus Genome Strain Culture Collection, and the Suillus phenotype database, SuilluScope. To access phenotype information and NRRL accession numbers for the full strain collection, please visit www.SuilluScope.com.
Total colony area over time for representatives of the Suillus Genome Strain Culture Collection. Cultures grown in 9 cm Petri plates and started by placing 3‐mm agar plugs on Modified Melin‐Norkrans (MMN) media, adjusted to pH 6 before autoclaving. Cultures were grown in temperature‐adjustable incubators, in the dark, for a total of 33 d (at n = 4 replicates per species per temperature treatment). Starting on Day 8, colony area was recorded twice per week over the course of the assay by marking the colony margin on the back of each Petri dish with a fine‐tip sharpie. After 33 d of growth, the back of the Petri plates were imaged using a flatbed scanner, and colony area was calculated for each time point using the program imagej. The data presented here are for replicates grown at room temperature (20°C). To access growth data at other temperatures, and for the full Suillus Genome Strain Culture Collection, please visit www.SuilluScope.com.
Associations between Suillus and their Pinaceae hosts. Time‐calibrated phylogenies of proposed Suillus subgenera and Pinaceae genera and subgenera, displaying distinct topologies and no indication of cospeciation. Molecular clock dating supports the earliest divergence of Suillus between 40.2 and 71.1 million years ago depending on the method of fossil calibration used. Dashed lines represent host associations, and ancestral host‐switching events, highlighting ancestral associations with Larix and the host switch of Suillus subgenus Suillus from the host subgenera Strobus to the subgenus Pinus, which is thought to have occurred at least four times independently. Figure redrawn with permission from Zhang et al. (2022).
Biological introduction records. Records from the Global Database of Alien Macrofungi (Monteiro et al., 2020) were pruned to exclude taxa with uncertain species‐level assignments, and those annotated as having established from uncertain geographic origins. Guild was assigned across the database using FUNGuild retaining only species that could be confidently identified as Ectomycorrhizal (ECM), resulting in 946 observations. (a) Out of 55 genera of ECM fungi with introduction records, Suillus accounted for the majority of observed introductions, totaling 193 records. (b) In total, 24 species of Suillus have introduction records, with the most common species identified as S. luteus (44 observations) and S. granulatus (34 observations).
Suillus: an emerging model for the study of ectomycorrhizal ecology and evolution

April 2024

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385 Reads

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13 Citations

Research on mycorrhizal symbiosis has been slowed by a lack of established study systems. To address this challenge, we have been developing Suillus, a widespread ecologically and economically relevant fungal genus primarily associated with the plant family Pinaceae, into a model system for studying ectomycorrhizal (ECM) associations. Over the last decade, we have compiled extensive genomic resources, culture libraries, a phenotype database, and protocols for manipulating Suillus fungi with and without their tree partners. Our efforts have already resulted in a large number of publicly available genomes, transcriptomes, and respective annotations, as well as advances in our understanding of mycorrhizal partner specificity and host communication, fungal and plant nutrition, environmental adaptation, soil nutrient cycling, interspecific competition, and biological invasions. Here, we highlight the most significant recent findings enabled by Suillus, present a suite of protocols for working with the genus, and discuss how Suillus is emerging as an important model to elucidate the ecology and evolution of ECM interactions.


Genome mining revealed diversity of compounds encoded in three species of Suillus. (A) Genome size, number of genes, and predicted BGCs vary across S. cothurnatus VC 1858, S. hirtellus EM16, and S. decipiens EM49. (B) The tool antiSMASH v.6.0.1 detected and characterized the number of BGCs in the genome of S. cothurnatus VC1858, S. hirtellus EM16, and S. decipiens EM49. The tool BiG-SCAPE showed low grouping between architecture and sequence similarity for (C) NRPS-like and (D) terpene-classified BGCs.
Principal component analysis (PCA) plot and UpSet plot showed metabolite variation among different culture conditions. PCA for metabolites observed in (A) ethylacetate fraction and (B) aqueous fraction showed clear separation among culture conditions. The replicates are closer together, while culture groups are well separated along the PCA space. UpSet plot showing the number of putative metabolites that are present in either multiple-culture condition or present in only a specific-culture group for (C) ethylacetate and (D) aqueous fractions. Only a subset of the UpSet plot is shown to show the number of metabolites shared among different sample groups.
Compound classification highlighted chemical diversity of the putatively identified metabolites. The tool ClassyFire classified the chemical taxonomy of compounds for the non-redundant list of 487 compounds identified using Compound Discoverer workflow. This analysis showed carboxylic acids and derivatives, benzene and substituted derivatives, and prenol lipids are the most abundant chemical classes observed in LC-MS/MS analysis for the three species of Suillus used in this study.
Prenol lipids relative abundance varied among culture conditions. (A) A heatmap illustrates the relative abundances of selected prenol lipids metabolites that varied in their abundance among culture conditions in ethylacetate fraction. These putative identifications were the result of an untargeted metabolomics analysis using Compound Discoverer. For each compound, the average intensity across biological replicates was Log10 transformed into a scaled value. Mirror match images for (B) sandaracopimaric acid, (C) abietic acid, and (D) isopimaric acid. The experimentally observed MS/MS spectrum is shown at the top, and a representative MS/MS spectrum from a pure standard is shown in blue color at the bottom.
Classical molecular network uncovered predominant annotated and unannotated MS/MS spectra. (A) The GNPS-derived molecular network was visualized by the Cytoscape software. Each node represents an MS/MS spectrum from this study. Nodes colored white represent unannotated MS/MS spectrum, and colored nodes represent an MS/MS spectrum associated with a putative metabolite annotation with a relatively high spectral similarity score (correlation >0.7). Nodes with putative annotations were annotated by the MolNetEnhancer workflow in GNPS to illustrate the different superclass annotations. Only a subset of the molecular network is shown by removing subnetworks with fewer than six nodes. (B) A bar plot shows the number of metabolites belonging to a chemical class reported by the MolNetEnhancer workflow in GNPS. The full molecular network and associated chemical classes are illustrated in Fig. S4.
Decoding the chemical language of Suillus fungi: genome mining and untargeted metabolomics uncover terpene chemical diversity

March 2024

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154 Reads

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3 Citations

Ectomycorrhizal fungi establish mutually beneficial relationships with trees, trading nutrients for carbon. Suillus are ectomycorrhizal fungi that are critical to the health of boreal and temperate forest ecosystems. Comparative genomics has identified a high number of non-ribosomal peptide synthetase and terpene biosynthetic gene clusters (BGC) potentially involved in fungal competition and communication. However, the functionality of these BGCs is not known. This study employed co-culture techniques to activate BGC expression and then used metabolomics to investigate the diversity of metabolic products produced by three Suillus species (Suillus hirtellus EM16, Suillus decipiens EM49, and Suillus cothurnatus VC1858), core members of the pine microbiome. After 28 days of growth on solid media, liquid chromatography–tandem mass spectrometry identified a diverse range of extracellular metabolites (exometabolites) along the interaction zone between Suillus co-cultures. Prenol lipids were among the most abundant chemical classes. Out of the 62 unique terpene BGCs predicted by genome mining, 41 putative prenol lipids (includes 37 putative terpenes) were identified across the three Suillus species using metabolomics. Notably, some terpenes were significantly more abundant in co-culture conditions. For example, we identified a metabolite matching to isomers isopimaric acid, sandaracopimaric acid, and abietic acid, which can be found in pine resin and play important roles in host defense mechanisms and Suillus spore germination. This research highlights the importance of combining genomics and metabolomics to advance our understanding of the chemical diversity underpinning fungal signaling and communication. IMPORTANCE Using a combination of genomics and metabolomics, this study’s findings offer new insights into the chemical diversity of Suillus fungi, which serve a critical role in forest ecosystems.


Populus MYC2 orchestrates root transcriptional reprogramming of defence pathway to impair Laccaria bicolor ectomycorrhizal development

February 2024

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184 Reads

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5 Citations

The jasmonic acid (JA) signalling pathway plays an important role in the establishment of the ectomycorrhizal symbiosis. The Laccaria bicolor effector MiSSP7 stabilizes JA corepressor JAZ6, thereby inhibiting the activity of Populus MYC2 transcription factors. Although the role of MYC2 in orchestrating plant defences against pathogens is well established, its exact contribution to ECM symbiosis remains unclear. This information is crucial for understanding the balance between plant immunity and symbiotic relationships. Transgenic poplars overexpressing or silencing for the two paralogues of MYC2 transcription factor (MYC2s) were produced, and their ability to establish ectomycorrhiza was assessed. Transcriptomics and DNA affinity purification sequencing were performed. MYC2s overexpression led to a decrease in fungal colonization, whereas its silencing increased it. The enrichment of terpene synthase genes in the MYC2‐regulated gene set suggests a complex interplay between the host monoterpenes and fungal growth. Several root monoterpenes have been identified as inhibitors of fungal growth and ECM symbiosis. Our results highlight the significance of poplar MYC2s and terpenes in mutualistic symbiosis by controlling root fungal colonization. We identified poplar genes which direct or indirect control by MYC2 is required for ECM establishment. These findings deepen our understanding of the molecular mechanisms underlying ECM symbiosis.


MicroFisher: Fungal taxonomic classification for metatranscriptomic and metagenomic data using multiple short hypervariable markers

January 2024

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75 Reads

Profiling the taxonomic and functional composition of microbes using metagenomic (MG) and metatranscriptomic (MT) sequencing is advancing our understanding of microbial functions. However, the sensitivity and accuracy of microbial classification using genome- or core protein-based approaches, especially the classification of eukaryotic organisms, is limited by the availability of genomes and the resolution of sequence databases. To address this, we propose the MicroFisher, a novel approach that applies multiple hypervariable marker genes to profile fungal communities from MGs and MTs. This approach utilizes the hypervariable regions of ITS and large subunit (LSU) rRNA genes for fungal identification with high sensitivity and resolution. Simultaneously, we propose a computational pipeline (MicroFisher) to optimize and integrate the results from classifications using multiple hypervariable markers. To test the performance of our method, we applied MicroFisher to the synthetic community profiling and found high performance in fungal prediction and abundance estimation. In addition, we also used MGs from forest soil and MTs of root eukaryotic microbes to test our method and the results showed that MicroFisher provided more accurate profiling of environmental microbiomes compared to other classification tools. Overall, MicroFisher serves as a novel pipeline for classification of fungal communities from MGs and MTs.


Effect of ectomycorrhizal fungi (EMF) treatment and Fe addition on mycorrhiza formation rate, Pinus contorta biomass, and Fe uptake. (a, b) A two‐way ANOVA was used to determine the interaction of EMF treatment and Fe addition, and the significant differences among EMF treatments or treatments with and without Fe were labeled with letters and asterisks, respectively. Lowercase letters above the boxes indicate significant differences among EMF treatments with Fe, determined by a HSD test (P < 0.05). Asterisks under lines indicate the significant differences between treatments with and without Fe within each EMF treatment using a t‐test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Statistical analysis for mycorrhiza formation rate and P. contorta biomass is shown in Supporting Information Table S1. (c) Microscopic imaging of P. contorta–Suillus rhizoboxes with Fe. (d) Synchrotron X‐ray fluorescence (μ‐XRF) imaging of one of representative mycorrhizae/root tips. Fe intensity in both rhizosphere and roots represents the actual Fe counts that are proportional to the number of Fe atoms. The degree of brightness can be referred to as the number of Fe atoms. Arrows in different colors mark the mycorrhizae (red), Fe‐coated sand (yellow), and dissolved Fe sand (gray). (e, f) Box plots show the Fe distribution within the whole imaged root (including root branches) (e) and root tip (non‐EMF treatment)/mycorrhizae (EMF‐inoculated treatments) (f) in response to Fe addition. Boxes indicate interquartile range; horizontal lines within the boxes indicate the medians of the data; whiskers above and below the boxes indicate the highest and lowest values; circles in each box indicate replicates within each EMF treatment. Fe intensity was transformed by log10. Lowercase letters indicate significant differences among EMF treatments, determined by a one‐way ANOVA followed by a HSD test for post hoc comparison. Ctrl, non‐EMF inoculation; EM44, S. spraguei; SB120, S. brevipes.
Gene expression of Pinus roots in response to ectomycorrhizal fungi (EMF) treatment and Fe addition. (a) Principal components analysis (PCA) shows the normalized gene expression of Pinus contorta genes across different treatments. Different shapes represent Fe treatments (circles for Fe addition; triangles for non‐Fe addition), and different colors represent EMF treatments (black for non‐EMF (Ctrl); red for S. spraguei EM44; blue for S. brevipes SB120; orange for S. brevipes SB120/S. spraguei EM44). (b) Volcano plots visualize the counts and expression rate of P. contorta genes that were up‐ (red dots) and downregulated (blue dots) following the Fe addition at each EMF treatment, with the cutoff values of log2FoldChange > 1 and P < 0.01. (c) Heatmap shows the significant differential expressed genes regulating Fe processes under different EMF treatments. Red and blue cells represent enrichment and depletion of functions.
Comparison of Pinus contorta gene expression between non‐ectomycorrhizal fungi (EMF) (Ctrl) and EMF‐ inoculated treatments in the presence of Fe. (a) Volcano plots visualize the counts and expression rate of P. contorta genes that were up‐ (red dots) and downregulated (blue dots) in response to EMF inoculation, with the cutoff values of |log2FC| > 1 and P < 0.01. (b) Venn plot shows the counts of shared and unique upregulated pine genes among different EMF‐inoculated treatments. (c) Soft clustering uncovers the relative expression of differential expressed genes across different EMF treatments with Fe addition. (d) Line plots show the gene expression patterns among EMF treatments in each cluster. (e, f) Bubble plots show the most enriched GO: biological process (GO‐BP; e) and molecular function (GO‐MF; f) across these five clusters, respectively. The size and color of bubbles are proportional to the number of genes and gene expression rate represented by −log10 (P‐value). (g) Treeplot displays the hierarchical clustering of the most enriched GO‐based gene sets (Top10) shared across five clusters using the GSEA. Ctrl, non‐EMF inoculation; EM44, S. spraguei; SB120, S. brevipes.
Effects of ectomycorrhizal fungi (EMF) treatment and Fe addition on symbiosis‐related metabolites derived from NMR. (a–f) Uppercase letters indicate significant differences among EMF treatments irrespective of Fe addition, determined by a two‐way ANOVA followed by a HSD test (P < 0.05). Lowercase letters indicate significant differences among EMF treatments within Fe addition (subfigures inside b and d), determined by a one‐way ANOVA followed by HSD test (P < 0.05). †, P < 0.1; *, P < 0.05; **, P < 0.01. Statistical analysis for symbiosis‐related metabolites is shown in Supporting Information Table S1. Ctrl, non‐EMF inoculation; EM44, S. spraguei; SB120, S. brevipes.
Interconnections between plant responses and symbiotic performance under the condition with (a) and without (b) Fe addition. Line width is proportional to the partial Mantel's r statistic, and line color denotes the statistical significance based on 999 permutations (orange: 0.01 < P < 0.05; green: P < 0.01). Pairwise comparisons of plant nutrients and metabolites are also shown, with a gradient of color and size of circles denoting Pearson's correlation coefficient. P‐values were adjusted for multiple testing with the Bonferroni–Holm method. Al, aluminum; B, boron; C, carbon; Ca, calcium; Fe, iron; K, potassium; Mg, magnesium; Mn, manganese; N, nitrogen; P, phosphorus; Zn, zinc.
Ectomycorrhizal fungi enhance pine growth by stimulating iron‐dependent mechanisms with trade‐offs in symbiotic performance

December 2023

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263 Reads

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6 Citations

Iron (Fe) is crucial for metabolic functions of living organisms. Plants access occluded Fe through interactions with rhizosphere microorganisms and symbionts. Yet, the interplay between Fe addition and plant–mycorrhizal interactions, especially the molecular mechanisms underlying mycorrhiza‐assisted Fe processing in plants, remains largely unexplored. We conducted mesocosms in Pinus plants inoculated with different ectomycorrhizal fungi (EMF) Suillus species under conditions with and without Fe coatings. Meta‐transcriptomic, biogeochemical, and X‐ray fluorescence imaging analyses were applied to investigate early‐stage mycorrhizal roots. While Fe addition promoted Pinus growth, it concurrently reduced mycorrhiza formation rate, symbiosis‐related metabolites in plant roots, and aboveground plant carbon and macronutrient content. This suggested potential trade‐offs between Fe‐enhanced plant growth and symbiotic performance. However, the extent of this trade‐off may depend on interactions between host plants and EMF species. Interestingly, dual EMF species were more effective at facilitating plant Fe uptake by inducing diverse Fe‐related functions than single‐EMF species. This subsequently triggered various Fe‐dependent physiological and biochemical processes in Pinus roots, significantly contributing to Pinus growth. However, this resulted in a greater carbon allocation to roots, relatively reducing the aboveground plant carbon content. Our study offers critical insights into how EMF communities rebalance benefits of Fe‐induced effects on symbiotic partners.


Decoding the chemical language of Suillus fungi: genome mining and untargeted metabolomics uncover terpene chemical diversity

November 2023

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48 Reads

Ectomycorrhizal fungi establish mutually beneficial relationships with trees, trading nutrients for carbon. Suillus are ectomycorrhizal fungi that are critical to the health of boreal and temperate forest ecosystems. Comparative genomics has identified a high number of non-ribosomal peptide synthetase and terpene biosynthetic gene clusters (BGC) potentially involved in fungal competition and communication. However, the functionality of these BGCs is not known. This study employed co-culture techniques to activate BGC expression and then used metabolomics to investigate the diversity of metabolic products produced by three Suillus species ( S. hirtellus EM16, S. decipiens EM49, and S. cothurnatus VC1858), core members of the Pine microbiome. After 28 days of growth on solid media, liquid chromatography–tandem mass spectrometry identified a diverse range of extracellular metabolites (exometabolites) along the interaction zone between Suillus co-cultures. Prenol lipids were among the most abundant chemical classes. Out of the 62 unique terpene BGCs predicted by genome mining, 116 putative terpenes were identified across the three Suillus species using metabolomics. Notably, some terpenes were significantly more abundant in co-culture conditions. For example, we identified a metabolite matching to isomers isopimaric acid, sandaracopimaric acid, and abietic acid, which can be found in pine resin and play important roles in host defense mechanisms and Suillus spore germination. This research highlights the importance of combining genomics and metabolomics to advance our understanding of the chemical diversity underpinning fungal signaling and communication. Importance Using a combination of genomics and metabolomics, this study’s findings offer new insights into the signaling and communication of Suillus fungi, which serve a critical role in forest ecosystems.


PacBio high‐throughput multi‐locus sequencing reveals high genetic diversity in mushroom‐forming fungi

October 2023

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145 Reads

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7 Citations

Molecular Ecology Resources

Multi‐locus sequence data are widely used in fungal systematic and taxonomic studies to delimit species and infer evolutionary relationships. We developed and assessed the efficacy of a multi‐locus pooled sequencing method using PacBio long‐read high‐throughput sequencing. Samples included fresh and dried voucher specimens, cultures and archival DNA extracts of Agaricomycetes with an emphasis on the order Cantharellales. Of the 283 specimens sequenced, 93.6% successfully amplified at one or more loci with a mean of 3.3 loci amplified. Our method recovered multiple sequence variants representing alleles of rDNA loci and single copy protein‐coding genes rpb 1, rpb 2 and tef 1. Within‐sample genetic variation differed by locus and taxonomic group, with the greatest genetic divergence observed among sequence variants of rpb 2 and tef 1 from corticioid Cantharellales. Our method is a cost‐effective approach for generating accurate multi‐locus sequence data coupled with recovery of alleles from polymorphic samples and multi‐organism specimens. These results have important implications for understanding intra‐individual genomic variation among genetic loci commonly used in species delimitation of fungi.


Citations (47)


... The set of sporocarps was predominantly composed by fungi with macroscopic characteristics similar to Suillus placidus, S. luteus (L.) Roussel and S. granulatus (L.) Roussel, and Amanita caesarea-complex. The fungal species were chosen depending upon field availability of sporocarps and their know capacity to form mycorrhizal associations with Pinus species (Endo et al. 2013;Lofgren et al. 2024). Collected sporocarps were shipped to the multipurpose laboratory at the Campo Experimental Valle del Guadiana of the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (23°59′21.82″ ...

Reference:

Improving ectomycorrhizal colonization and morpho-physiological traits of Pinus cooperi seedlings through organic nitrogen fertilization
Suillus: an emerging model for the study of ectomycorrhizal ecology and evolution

... The diterpene isopimaric acid has a wide range of applications in agriculture and is of great value in research into seed germination and seedling growth [31]. This study investigated the biological activity of isopimaric acid on rice seedlings. ...

Decoding the chemical language of Suillus fungi: genome mining and untargeted metabolomics uncover terpene chemical diversity

... The MYC2 transcription factor, central to JA signaling, is also responsive in Populus trichocarpa under drought, indicating a conserved role in stress adaptation. Moreover, comparative transcriptomic studies have revealed that JA-responsive genes are differentially expressed in drought-tolerant vs. susceptible Populus genotypes, underscoring the hormone's role in species-specific drought resilience [117]. ...

Populus MYC2 orchestrates root transcriptional reprogramming of defence pathway to impair Laccaria bicolor ectomycorrhizal development

... (Ca), from deeper soil layers (Jobb agy & Jackson, 2001;H€ ogberg & H€ ogberg, 2002;Agnan et al., 2019;Sokol et al., 2019). This ability allows them to aid their plant partners in the acquisition of micro-and macronutrients from the soil, in exchange for carbon (C) sources derived from plant photosynthesis (Smith & Read, 2010;K. Zhang et al., 2021K. Zhang et al., , 2023. EMF may also participate in the decomposition of soil organic matter (SOM), whereby organic matter is broken down into smaller N-bearing organic molecules (Read & Perez-Moreno, 2003;Averill et al., 2014;Lindahl & Tunlid, 2015;Shah et al., 2016;Zak et al., 2019), which are then mined by extramatrical hyphae of EMF (Nicol as et al., 2019 ...

Ectomycorrhizal fungi enhance pine growth by stimulating iron‐dependent mechanisms with trade‐offs in symbiotic performance

... To simplify sequence identification among different barcoding regions, it will be important to link fungal ITS data with other barcoding regions in such databases as UNITE (Abarenkov et al. 2024) and GlobalFungi (Větrovský et al. 2020). Different species recovered in our study by different barcode markers confirmed the goodpractice recommendation to combine widely used molecular markers with an additional one for higher taxonomic resolution within individual groups of organisms (Lekberg et al. 2018;van der Loos and Nijland 2021;Ogier et al. 2019;Swenie et al. 2024). This study showcases the advantage of the combined metabarcoding markers approach on the example of three Basidiomycota lineages which cover a significant phylogenetic variation within Agaricomycotina, but future testing on a broader representation of other fungal lineages could strengthen the conclusions. ...

PacBio high‐throughput multi‐locus sequencing reveals high genetic diversity in mushroom‐forming fungi
  • Citing Article
  • October 2023

Molecular Ecology Resources

... Although BUSCO analysis yielded a high, 95 % genome completeness result, CheckM2 contamination analysis showed 8.63 % contamination. Further analysis using BlobTools2 proved that the contamination was detected most likely due to the incompleteness and incorrectly annotated fungal sequences in the reference databases as fungal genomes sequencing lags significantly behind other organism groups [60]. ...

Sequencing the Genomes of the First Terrestrial Fungal Lineages: What Have We Learned?

... And, although propagule pressure is a major driver of Vol.: (0123456789) biological invasions, biotic interactions could become more relevant when nonnative species represent a novel life-form in the native community (Taylor et al. 2016b). Furthermore, in previous studies only aboveground interactions were taken into account, while belowground interactions were ignored (Policelli et al. 2023). Until now, belowground interactions between native and nonnative species had not been evaluated in terms of the distribution of root biomass across different soil layers, as a proxy for belowground competition. ...

Global pine tree invasions are linked to invasive root symbionts

... However, this fungal species is considered questionable by Keller (1991) and Bałazy (1993), and any potential synonymous species are not known in North America, and so it has not been included in this checklist. One isolate of Entomophaga conglomerata is listed in the ARSEF culture collection (ARSEF 2020), but a recent publication demonstrated that this isolate instead belongs in the genus Batkoa (Gryganskyi et al. 2022b), and therefore this example was not included. Tipulids in NHand NC (US), collected by Thaxter, harbored a fungus named Entomophthora thaxteri (MacLeod and Müller-Kögler 1973), but this species was not adequately described. ...

The Early Terrestrial Fungal Lineage of Conidiobolus —Transition from Saprotroph to Parasitic Lifestyle

... Specifically, the abundance proportion of Wilcoxina was significantly higher in seedlings grown with the soil microbial legacy of trees than in seedlings inoculated with the soil microbial legacies of herbs and shrubs (Fig. 4a, b). Wilcoxina is known to be an excellent and rapid root colonizer on Pinaceae seedlings, as it has little host specificity and lower carbohydrate requirements than other EcM fungi (Jones et al. 2010;Wen et al. 2018;Milani et al. 2022). Wilcoxina have also been suggested to arise from a potential capacity for facultative saprotrophy and to increase the resistance of seedlings to pathogens (Yu et al. 2001;Rosenstock et al. 2019). ...

Co-invading ectomycorrhizal fungal succession in pine-invaded mountain grasslands
  • Citing Article
  • December 2022

Fungal Ecology

... Of the twelve taxa shared between the rhizosphere and P-treated mesh bags, all genera found except Sistotrema, Chloridium, and Xerocomus are known early successional colonizers of P. taeda in North Carolina (Hackman et al., 2022). BLAST results of the sequences from the three genera against the UNITE database (Nilsson et al., 2019) could not discern any further details on possible species. ...

NPK fertilizer use in loblolly pine plantations: Who are we really feeding?
  • Citing Article
  • September 2022

Forest Ecology and Management