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

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.