Suillus: an emerging model for the study of ectomycorrhizal ecology and evolution.

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.

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

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.IMPORTANCEUsing 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.

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

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.

The pan-genome of Aspergillus fumigatus provides a high-resolution view of its population structure revealing high levels of lineage-specific diversity driven by recombination.

Aspergillus fumigatus is a deadly agent of human fungal disease where virulence heterogeneity is thought to be at least partially structured by genetic variation between strains. While population genomic analyses based on reference genome alignments offer valuable insights into how gene variants are distributed across populations, these approaches fail to capture intraspecific variation in genes absent from the reference genome. Pan-genomic analyses based on de novo assemblies offer a promising alternative to reference-based genomics with the potential to address the full genetic repertoire of a species. Here, we evaluate 260 genome sequences of A. fumigatus including 62 newly sequenced strains, using a combination of population genomics, phylogenomics, and pan-genomics. Our results offer a high-resolution assessment of population structure and recombination frequency, phylogenetically structured gene presence-absence variation, evidence for metabolic specificity, and the distribution of putative antifungal resistance genes. Although A. fumigatus disperses primarily via asexual conidia, we identified extraordinarily high levels of recombination with the lowest linkage disequilibrium decay value reported for any fungal species to date. We provide evidence for 3 primary populations of A. fumigatus, with recombination occurring only rarely between populations and often within them. These 3 populations are structured by both gene variation and distinct patterns of gene presence-absence with unique suites of accessory genes present exclusively in each clade. Accessory genes displayed functional enrichment for nitrogen and carbohydrate metabolism suggesting that populations may be stratified by environmental niche specialization. Similarly, the distribution of antifungal resistance genes and resistance alleles were often structured by phylogeny. Altogether, the pan-genome of A. fumigatus represents one of the largest fungal pan-genomes reported to date including many genes unrepresented in the Af293 reference genome. These results highlight the inadequacy of relying on a single-reference genome-based approach for evaluating intraspecific variation and the power of combined genomic approaches to elucidate population structure, genetic diversity, and putative ecological drivers of clinically relevant fungi.

Avian-associated Aspergillus fumigatus displays broad phylogenetic distribution, no evidence for host specificity, and multiple genotypes within epizootic events.

Birds are highly susceptible to aspergillosis, which can manifest as a primary infection in both domestic and wild birds. Aspergillosis in wild birds causes mortalities ranging in scale from single animals to large-scale epizootic events. However, pathogenicity factors associated with aspergillosis in wild birds have not been examined. Specifically, it is unknown whether wild bird-infecting strains are host-adapted (i.e. phylogenetically related). Similarly, it is unknown whether epizootics are driven by contact with clonal strains that possess unique pathogenic or virulence properties, or by distinct and equally pathogenic strains. Here, we use a diverse collection of Aspergillus fumigatus isolates taken from aspergillosis-associated avian carcasses, representing 24 bird species from a wide geographic range, and representing individual bird mortalities as well as epizootic events. These isolates were sequenced and analyzed along with 130 phylogenetically diverse human clinical isolates to investigate the genetic diversity and phylogenetic placement of avian-associated A. fumigatus, the geographic and host distribution of avian isolates, evidence for clonal outbreaks among wild birds, and the frequency of azole resistance in avian isolates. We found that avian isolates were phylogenetically diverse, with no clear distinction from human clinical isolates, and no sign of host or geographic specificity. Avian isolates from the same epizootic events were diverse and phylogenetically distant, suggesting that avian aspergillosis is not contagious among wild birds and that outbreaks are likely driven by environmental spore loads or host comorbidities. Finally, all avian isolates were susceptible to Voriconazole and none contained the canonical azole resistance gene variants.

Host Lung Environment Limits Aspergillus fumigatus Germination through an SskA-Dependent Signaling Response.

Aspergillus fumigatus isolates display significant heterogeneity in growth, virulence, pathology, and inflammatory potential in multiple murine models of invasive aspergillosis. Previous studies have linked the initial germination of a fungal isolate in the airways to the inflammatory and pathological potential, but the mechanism(s) regulating A. fumigatus germination in the airways is unresolved. To explore the genetic basis for divergent germination phenotypes, we utilized a serial passaging strategy in which we cultured a slow germinating strain (AF293) in a murine-lung-based medium for multiple generations. Through this serial passaging approach, a strain emerged with an increased germination rate that induces more inflammation than the parental strain (herein named LH-EVOL for lung homogenate evolved). We identified a potential loss-of-function allele of Afu5g08390 (sskA) in the LH-EVOL strain. The LH-EVOL strain had a decreased ability to induce the SakA-dependent stress pathway, similar to AF293 ΔsskA and CEA10. In support of the whole-genome variant analyses, sskA, sakA, or mpkC loss-of-function strains in the AF293 parental strain increased germination both in vitro and in vivo. Since the airway surface liquid of the lungs contains low glucose levels, the relationship of low glucose concentration on germination of these mutant AF293 strains was examined; interestingly, in low glucose conditions, the sakA pathway mutants exhibited an enhanced germination rate. In conclusion, A. fumigatus germination in the airways is regulated by SskA through the SakA mitogen-activated protein kinase (MAPK) pathway and drives enhanced disease initiation and inflammation in the lungs. IMPORTANCE Aspergillus fumigatus is an important human fungal pathogen particularly in immunocompromised individuals. Initiation of growth by A. fumigatus in the lung is important for its pathogenicity in murine models. However, our understanding of what regulates fungal germination in the lung environment is lacking. Through a serial passage experiment using lung-based medium, we identified a new strain of A. fumigatus that has increased germination potential and inflammation in the lungs. Using this serially passaged strain, we found it had a decreased ability to mediate signaling through the osmotic stress response pathway. This finding was confirmed using genetic null mutants demonstrating that the osmotic stress response pathway is critical for regulating growth in the murine lungs. Our results contribute to the understanding of A. fumigatus adaptation and growth in the host lung environment.

Fungal biodiversity and conservation mycology in light of new technology, big data, and changing attitudes.

Fungi have successfully established themselves across seemingly every possible niche, substrate, and biome. They are fundamental to biogeochemical cycling, interspecies interactions, food production, and drug bioprocessing, as well as playing less heroic roles as difficult to treat human infections and devastating plant pathogens. Despite community efforts to estimate and catalog fungal diversity, we have only named and described a minute fraction of the fungal world. The identification, characterization, and conservation of fungal diversity is paramount to preserving fungal bioresources, and to understanding and predicting ecosystem cycling and the evolution and epidemiology of fungal disease. Although species and ecosystem conservation are necessarily the foundation of preserving this diversity, there is value in expanding our definition of conservation to include the protection of biological collections, ecological metadata, genetic and genomic data, and the methods and code used for our analyses. These definitions of conservation are interdependent. For example, we need metadata on host specificity and biogeography to understand rarity and set priorities for conservation. To aid in these efforts, we need to draw expertise from diverse fields to tie traditional taxonomic knowledge to data obtained from modern -omics-based approaches, and support the advancement of diverse research perspectives. We also need new tools, including an updated framework for describing and tracking species known only from DNA, and the continued integration of functional predictions to link genetic diversity to functional and ecological diversity. Here, we review the state of fungal diversity research as shaped by recent technological advancements, and how changing viewpoints in taxonomy, -omics, and systematics can be integrated to advance mycological research and preserve fungal biodiversity.

Aspergillus fumigatus In-Host HOG Pathway Mutation for Cystic Fibrosis Lung Microenvironment Persistence.

The prevalence of Aspergillus fumigatus colonization in individuals with cystic fibrosis (CF) and subsequent fungal persistence in the lung is increasingly recognized. However, there is no consensus for clinical management of A. fumigatus in CF individuals, due largely to uncertainty surrounding A. fumigatus CF pathogenesis and virulence mechanisms. To address this gap in knowledge, a longitudinal series of A. fumigatus isolates from an individual with CF were collected over 4.5 years. Isolate genotypes were defined with whole-genome sequencing that revealed both transitory and persistent A. fumigatus in the lung. Persistent lineage isolates grew most readily in a low-oxygen culture environment, and conidia were more sensitive to oxidative stress-inducing conditions than those from nonpersistent isolates. Closely related persistent isolates harbored a unique allele of the high-osmolarity glycerol (HOG) pathway mitogen-activated protein kinase kinase, Pbs2 (pbs2C2). Data suggest this novel pbs2C2 allele arose in vivo and is necessary for the fungal response to osmotic stress in a low-oxygen environment through hyperactivation of the HOG (SakA) signaling pathway. Hyperactivation of the HOG pathway through pbs2C2 comes at the cost of decreased conidial stress resistance in the presence of atmospheric oxygen levels. These novel findings shed light on pathoadaptive mechanisms of A. fumigatus in CF, lay the foundation for identifying persistent A. fumigatus isolates that may require antifungal therapy, and highlight considerations for successful culture of persistent Aspergillus CF isolates. IMPORTANCE Aspergillus fumigatus infection causes a spectrum of clinical manifestations. For individuals with cystic fibrosis (CF), allergic bronchopulmonary aspergillosis (ABPA) is an established complication, but there is a growing appreciation for A. fumigatus airway persistence in CF disease progression. There currently is little consensus for clinical management of A. fumigatus long-term culture positivity in CF. A better understanding of A. fumigatus pathogenesis mechanisms in CF is expected to yield insights into when antifungal therapies are warranted. Here, a 4.5-year longitudinal collection of A. fumigatus isolates from a patient with CF identified a persistent lineage that harbors a unique allele of the Pbs2 mitogen-activated protein kinase kinase (MAPKK) necessary for unique CF-relevant stress phenotypes. Importantly for A. fumigatus CF patient diagnostics, this allele provides increased fitness under CF lung-like conditions at a cost of reduced in vitro growth under standard laboratory conditions. These data illustrate a molecular mechanism for A. fumigatus CF lung persistence with implications for diagnostics and antifungal therapy.

Aspergillus fumigatus Strain-Specific Conidia Lung Persistence Causes an Allergic Broncho-Pulmonary Aspergillosis-Like Disease Phenotype.

Aspergillus fumigatus is a filamentous fungus which can cause multiple diseases in humans. Allergic broncho-pulmonary aspergillosis (ABPA) is a disease diagnosed primarily in cystic fibrosis patients caused by a severe allergic response often to long-term A. fumigatus colonization in the lungs. Mice develop an allergic response to repeated inhalation of A. fumigatus spores; however, no strains have been identified that can survive long-term in the mouse lung and cause ABPA-like disease. We characterized A. fumigatus strain W72310, which was isolated from the expectorated sputum of an ABPA patient, by whole-genome sequencing and in vitro and in vivo viability assays in comparison to a common reference strain, CEA10. W72310 was resistant to leukocyte-mediated killing and persisted in the mouse lung longer than CEA10, a phenotype that correlated with greater resistance to oxidative stressors, hydrogen peroxide, and menadione, in vitro In animals both sensitized and challenged with W72310, conidia, but not hyphae, were viable in the lungs for up to 21 days in association with eosinophilic airway inflammation, airway leakage, serum IgE, and mucus production. W72310-sensitized mice that were recall challenged with conidia had increased inflammation, Th1 and Th2 cytokines, and airway leakage compared to controls. Collectively, our studies demonstrate that a unique strain of A. fumigatus resistant to leukocyte killing can persist in the mouse lung in conidial form and elicit features of ABPA-like disease.IMPORTANCE Allergic broncho-pulmonary aspergillosis (ABPA) patients often present with long-term colonization of Aspergillus fumigatus Current understanding of ABPA pathogenesis has been complicated by a lack of long-term in vivo fungal persistence models. We have identified a clinical isolate of A. fumigatus, W72310, which persists in the murine lung and causes an ABPA-like disease phenotype. Surprisingly, while viable, W72310 showed little to no growth beyond the conidial stage in the lung. This indicates that it is possible that A. fumigatus can cause allergic disease in the lung without any significant hyphal growth. The identification of this strain of A. fumigatus can be used not only to better understand disease pathogenesis of ABPA and potential antifungal treatments but also to identify features of fungal strains that drive long-term fungal persistence in the lung. Consequently, these observations are a step toward helping resolve the long-standing question of when to utilize antifungal therapies in patients with ABPA and fungal allergic-type diseases.

Comparative genomics reveals dynamic genome evolution in host specialist ectomycorrhizal fungi.

While there has been significant progress characterizing the 'symbiotic toolkit' of ectomycorrhizal (ECM) fungi, how host specificity may be encoded into ECM fungal genomes remains poorly understood. We conducted a comparative genomic analysis of ECM fungal host specialists and generalists, focusing on the specialist genus Suillus. Global analyses of genome dynamics across 46 species were assessed, along with targeted analyses of three classes of molecules previously identified as important determinants of host specificity: small secreted proteins (SSPs), secondary metabolites (SMs) and G-protein coupled receptors (GPCRs). Relative to other ECM fungi, including other host specialists, Suillus had highly dynamic genomes including numerous rapidly evolving gene families and many domain expansions and contractions. Targeted analyses supported a role for SMs but not SSPs or GPCRs in Suillus host specificity. Phylogenomic-based ancestral state reconstruction identified Larix as the ancestral host of Suillus, with multiple independent switches between white and red pine hosts. These results suggest that like other defining characteristics of the ECM lifestyle, host specificity is a dynamic process at the genome level. In the case of Suillus, both SMs and pathways involved in the deactivation of reactive oxygen species appear to be strongly associated with enhanced host specificity.

Does fungal competitive ability explain host specificity or rarity in ectomycorrhizal symbioses?

Two common ecological assumptions are that host generalist and rare species are poorer competitors relative to host specialist and more abundant counterparts. While these assumptions have received considerable study in both plant and animals, how they apply to ectomycorrhizal fungi remains largely unknown. To investigate how interspecific competition may influence the anomalous host associations of the rare ectomycorrhizal generalist fungus, Suillus subaureus, we conducted a seedling bioassay. Pinus strobus seedlings were inoculated in single- or two-species treatments of three Suillus species: S. subaureus, S. americanus, and S. spraguei. After 4 and 8 months of growth, seedlings were harvested and scored for mycorrhizal colonization as well as dry biomass. At both time points, we found a clear competitive hierarchy among the three ectomycorrhizal fungal species: S. americanus > S. subaureus > S. spraguei, with the competitive inferior, S. spraguei, having significantly delayed colonization relative to S. americanus and S. subaureus. In the single-species treatments, we found no significant differences in the dry biomasses of P. strobus seedlings colonized by each Suillus species, suggesting none was a more effective plant symbiont. Taken together, these results indicate that the rarity and anomalous host associations exhibited by S. subaureus in natural settings are not driven by inherently poor competitive ability or host growth promotion, but that the timing of colonization is a key factor determining the outcome of ectomycorrhizal fungal competitive interactions.

Genome-based estimates of fungal rDNA copy number variation across phylogenetic scales and ecological lifestyles.

Ribosomal DNA (rDNA) copy number variation (CNV) has major physiological implications for all organisms, but how it varies for fungi, an ecologically ubiquitous and important group of microorganisms, has yet to be systemically investigated. Here, we examine rDNA CNV using an in silico read depth approach for 91 fungal taxa with sequenced genomes and assess copy number conservation across phylogenetic scales and ecological lifestyles. rDNA copy number varied considerably across fungi, ranging from an estimated 14 to 1,442 copies (mean = 113, median = 82), and copy number similarity was inversely correlated with phylogenetic distance. No correlations were found between rDNA CNV and fungal trophic mode, ecological guild or genome size. Taken together, these results show that like other microorganisms, fungi exhibit substantial variation in rDNA copy number, which is linked to their phylogeny in a scale-dependent manner.

Ectomycorrhizal host specificity in a changing world: can legacy effects explain anomalous current associations?

Despite the importance of ectomycorrhizal (ECM) fungi in forest ecosystems, knowledge about the ecological and co-evolutionary mechanisms underlying ECM host associations remains limited. Using a widely distributed group of ECM fungi known to form tight associations with trees in the family Pinaceae, we characterized host specificity among three unique Suillus-host species pairs using a combination of field root tip sampling and experimental bioassays. We demonstrate that the ECM fungus S. subaureus can successfully colonize Quercus hosts in both field and glasshouse settings, making this species unique in an otherwise Pinaceae-specific clade. Importantly, however, we found that the colonization of Quercus by S. subaureus required co-planting with a Pinaceae host. While our experimental results indicate that gymnosperms are required for the establishment of new S. subaureus colonies, Pineaceae hosts are locally absent at both our field sites. Given the historical presence of Pineaceae hosts before human alteration, it appears the current S. subaureus-Quercus associations represent carryover from past host presence. Collectively, our results suggest that patterns of ECM specificity should be viewed not only in light of current forest community composition, but also as a legacy effect of host community change over time.

Fusarium graminearum: pathogen or endophyte of North American grasses?

Mycotoxin-producing Fusarium graminearum and related species cause Fusarium head blight on cultivated grasses, such as wheat and barley. However, these Fusarium species may have had a longer evolutionary history with North American grasses than with cultivated crops and may interact with the ancestral hosts in ways which are biochemically distinct. We assayed 25 species of asymptomatic native grasses for the presence of Fusarium species and confirmed infected grasses as hosts using re-inoculation tests. We examined seed from native grasses for the presence of mycotoxin-producing Fusarium species and evaluated the ability of these fungi to produce mycotoxins in both native grass and wheat hosts using biochemical analysis. Mycotoxin-producing Fusarium species were shown to be prevalent in phylogenetically diverse native grasses, colonizing multiple tissue types, including seeds, leaves and inflorescence structures. Artificially inoculated grasses accumulated trichothecenes to a much lesser extent than wheat, and naturally infected grasses showed little to no accumulation. Native North American grasses are commonly inhabited by Fusarium species, but appear to accommodate these toxigenic fungi differently from cultivated crops. This finding highlights how host identity and evolutionary history may influence the outcome of plant-fungal interactions and may inform future efforts in crop improvement.

Population Subdivision of Fusarium graminearum from Barley and Wheat in the Upper Midwestern United States at the Turn of the Century.

Fusarium graminearum, the causal agent of Fusarium head blight (FHB) in wheat and barley, is one of the most economically destructive pathogens of these grains worldwide. Recent population genetic studies of the pathogen obtained from wheat in North America supported population subdivision in part correlated with the spectrum of trichothecene mycotoxins (chemotype) produced by individuals within each population. In contrast, a recent study of F. graminearum obtained from diseased barley in the upper Midwestern United States concluded that only a single population was present, consisting of individuals with various chemotypes. To test whether strains derived from different hosts potentially have different population dynamics, we obtained the barley strains used in the previous study and compared them with wheat strains isolated at a similar time and geographic origin. A total of 247 F. graminearum isolates from barley were assigned firmly into two clusters using a Bayesian clustering method. Subdivision within the barley population corresponded to the previously described NA1 (correlated with the 15ADON chemotype) and NA2 (correlated with the 3ADON chemotype) populations from wheat. However, in both sampling periods the barley population exhibited a higher level of genetic differentiation between NA1 and NA2 populations, fewer admixed individuals and evidence of unidirectional gene introgression (15ADON strains with NA2 genetic backgrounds). These results suggest less recombination between NA1 and NA2 populations on barley compared with wheat. The frequency of 3ADON chemotype strains in the most recently surveyed barley population suggests a latitudinal cline from the northern (49%), central (40%) to the southern (29%) sampling area. The potential to produce a novel trichothecene, 3α-acetoxy,7α,15-dihydroxy-12,13-epoxytrichothe-9-ene (NX-2), was not detected in the barley population but occurred at a low rate (2.4%) in the wheat population.