The green alga Haematococcus pluvialis (Chlorophyta) is grown commercially for human and animal dietary supplementation and enhancing marketability of farm-raised salmon. Chytrid fungi are known pathogens of Haematococcus in monoculture, and JEL916 was recently isolated into pure culture from a farm in southwestern United States. This monocentric chytrid fungus initially grows epibiotically on individual host cells, later extending elongated rhizoids to other host cells; these rhizoidal extensions also occur in pure cultures on nutrient agar with no host present. Aside from a thickened initial rhizoidal axis, rhizoids are thin and isodiametric. Zoosporangia can mature within 3 days, producing 1–2 raised, inoperculate papillae; zoospores are spherical in motion and contain 1–3 lipid globules. Resting spores develop associated with host cells in senescent Haematococcus cultures but not on nutrient agar. In a phylogenomic analysis of currently available taxa, JEL916 was most closely affiliated with a member of the Lobulomycetales. Compared to lobulomycetalean chytrid fungi, however, zoosporic ultrastructure of JEL916 differs in its possession of (1) a small striated inclusion, (2) a rumposome over a lipid globule, (3) a variation of morphology of the flagellar plug, and (4) an array of microtubules densely populating a site proximal to the kinetosome. We will describe JEL916 as a new lineage within the Chytridiomycota; its importance lies both in increasing the known diversity of the Chytridiomycota and in its ability to disrupt agricultural production of an algal monoculture.

We describe a new culture collection resource focused on supporting research on zoosporic true fungi. This collection was founded by aggregating the two most important existing collections into a single resource. Drs. Peter M. Letcher, Martha J. Powell, and Joyce E. Longcore and their students have devoted a combined 100 years of research efforts into amassing culture collections with a focus on the zoosporic eufungi within the Chytridiomycota and Blastocladiomycota. In 2018, we coordinated the transfer of the JEL culture collection of Dr. Longcore (536 isolates representing 10 of 13 described orders including 13 ex-type cultures) from 4 liquid nitrogen Dewars needing weekly maintenance at the University of Maine, to the automated cryopreservation facility at the University of Michigan Research Museum Center. Of 409 non-Batrachochytrium frozen cultures, we have confirmed viability of 252, the majority of which are not identified by molecular methods. Additionally, we recently added 429 cultures (including 23 ex-type cultures) from the University of Alabama Chytrid Culture Collection (UACCC) to CZEUM, making it the largest aggregation of Chytridiomycota and Blastocladiomycota in the world.  We have extracted DNA from 249 JEL cultures and have used a MinION Oxford Nanopore Technologies device and R9.4.1 flow cells to sequence multiplexed rDNA amplifications of a 4–6kb region of the 18S-ITS1-5.8S-ITS2-LSU rDNA operon. Comprehensive metadata of the culture collection can be accessed on the website for the Collection of Zoosporic Eufungi at the University of Michigan (CZEUM), and cultures can be supplied for research for a modest recharge rate.

The clonal fungus Calonectria pseudonaviculata is the primary pathogen responsible for the global emergence of boxwood blight, a disease that threatens the production of ornamental plants and the health of native ecosystems. Commonly used genetic markers like microsatellites do not provide enough resolution to study the demographic history or migration of the fungus. The goal of this work was to overcome this limitation through the identification and analysis of genome-wide single nucleotide polymorphisms (SNPs) in global populations of C. pseudonaviculata. Fifty isolates of C. pseudonaviculata collected from four continents were sequenced using the Illumina MiSeq platform. Sequence data were aligned to a draft genome assembly and initial SNP calls were made using the Genome Analysis Toolkit. Further filtering of SNPs was performed using R packages and BCFtools. Following filtering, 1343 biallelic SNPs were identified across the 54 Mb genome of C. pseudonaviculata. Inference of a phylogenetic tree from these data found multiple well-supported genetic clades were present within this species. The majority of isolates from the United States formed a divergent genetic clade, distinct from European and Asian isolates. Only a few C. pseudonaviculata isolates from the west coast of the U.S. showed greater genetic similarity to isolates from Europe than other U.S. isolates. Overall, this work demonstrates the utility of using SNPs to study genetic diversity in clonal fungi and provides a means for future investigations into the historical and contemporary migration of C. pseudonaviculata.

Rhizopus fungi are ubiquitous, saprotrophic pin molds typically found in soil, decaying vegetable matter and dung.  Genome sequencing and phylogenomic efforts as part of the ZyGolife project have improved species level phylogenies within the paraphyletic Rhizopus and Mucor genera. Sporodiniella umbellata and Syzygites megalocarpus are animal and mycoparasitic fungi that are sister to the post harvest rot Rhizopus stolonifer. Comparison of these lineages provides an opportunity to examine how transitions in the substrate or host preference is reflected in genomic changes.  We sequenced an isolate of S. umbellata was cultured from a orb-weaver spider and a S. megalocarpus from a mushroom fruiting body infection. Our analysis found Pfam domain and CAZymes content differences between eight Rhizopus, two related Mucor species, S. umbellata and S. megalocarpus that could be attributed to differences in species ecology. S. umbellata have more copies of p450, LRR8, and HLH domains than R. stolonifer and encode fewer CAZymes for Glycohydrolases, Carbohydrate Binding Modules, Glycotransferases, and Carbohydrate Esterases. S. umbellata lack any pectinases which R. stolonifer uses to cause soft rot of fruits. S. umbellata also lack mannanases and encodes only one cellulase domain containing protein. These differences may be indicative of its ecology as an insect or arachnid parasite. Across the clade, relative number of protein domains vary greatly. These include Protein kinase, WD40 and the previously described CotH protein family implicated in host invasion. The diverse collection of undescribed CotH containing orthologs which may provide additional targets for understanding Mucoromyoctina fungi host interactions.

Fungi in the genus Beauveria (Cordycipitaceae: Hypocreales) occur worldwide as insect pathogens, plant endophytes, and/or soil-dwelling saprotrophs. Strains of several species are used as mycoinsecticides for the biological control of arthropod pests. The genomic basis of adaption to diverse plant, insect, and soil environments, including the production of an array of secondary metabolites is poorly understood. Moreover, the basis for the origins and maintenance of such diverse ecological capabilities, despite seemingly infrequent sexual reproduction, is unknown.  To investigate the genetic basis for this ecological flexibility, the genomes of 12 Beauveria species were sequenced, assembled, and annotated.  Each genome contained between 9,600 and 11,000 protein coding genes, including 4,984 single copy orthologs found in every genome.  The genus-level pangenome is composed of more than 18,000 protein coding genes, with an average of 600 unique proteins per genome. We describe the distributions of carbohydrate active enzymes, proteases, secreted proteins, secondary metabolite biosynthetic gene clusters, repetitive elements, and lineage specific regions of interest in these genomes.  The results provide a foundation from which to explore the genomic basis of their flexible life histories and a novel basis for the selection and improvement of applied strains.

Trichoderma (Hypocreales) is a widespread and diverse genus inhabiting many different environmental niches and is often found in soil and as plant endophytes. Multiple isolates of Trichoderma are commercial biocontrol agents, primarily due to strong mycoparasitic capabilities. Mycoparasitism is associated with increased speciation in Trichoderma but the evolutionary mechanisms for this are not known. For example, comparison of Trichoderma species that have apparently lost mycoparasitic abilities may therefore provide additional insight to the genetic basis of mycoparasitism. Here we report the genome sequencing and annotation of four Trichoderma endophyticum isolates obtained from living sapwood tissue of Hevea spp. We describe the re-annotation and comparison of 35 publicly available Trichoderma genomes from around the world representing a variety of non-exclusive lifestyles (soil-saprotrophy, mycotrophy, endophytism). Using this diverse Trichoderma genome dataset we identified genes and gene clusters associated with alternate Trichoderma lifestyles and investigated their origin, evolution, and putative functions. We further predicted genomic changes associated with the presumed loss of mycoparasitism in T. endophyticum in culture, as well as gene clusters predicted to degrade host defense compounds. This research enhances our understanding of the basis of endophytism and ecological transitions in the Trichoderma genus.

There have been thirteen species of Hypomyces identified that grow on gilled fungi, while two species of Hypomyces are known to parasitize mushrooms in the genus Russula, Hypomyces armeniacus and Hypomyces lactifluorum. Although studies have been done that focused on identifying Hypomyces species and their hosts using morphological data, no studies focus on building a comprehensive phylogeny that includes all Hypomyces species. In this study, I develop a robust phylogeny of the genus Hypomyces, focusing on the Lobster Mushroom to study cryptic species and host specificity. The Lobster Mushroom is believed to be Russula brevipes that has been parasitized by Hypomyces lactifluorum. When inspecting H. lactifluorum specimens located at the University of Michigan’s Herbarium, it was clear that the vast number of hosts had not been identified to species. Moreover, the hosts of H. lactifluorum have not been identified using molecular data. There are examples where the host has been identified to a morphologically distinct taxa other than R. brevipes. Using principal coordinate analysis, I determined if the Hypomyces phylogeny shows evidence of cophylogeny between host and parasite, and whether Hypomyces lactifluorum is host specific.

Downy mildew diseases cause significant losses to cereals and grains in many countries every year and represent a threat to crops in the United States. However, our knowledge of the morphological and genetic diversity of the oomycetes responsible for graminicolous downy mildews is limited, which negatively affects our ability to accurately identify, monitor, and quarantine these pathogens in the event of an outbreak. The U.S. National Fungus Collections contains ~300 specimens of globally collected graminicolous downy mildew specimens dating back 1800s that includes 14 species and at least five type specimens. Our primary goal is to use these specimens in combination with specimens from other herbaria to infer a robust phylogeny using mitochondrial (cox1, cox2, and rsp10) and nuclear loci (ITS and LSU).

Flag smut diseases are widespread on wild and cultivated grasses throughout the temperate and subtropical regions of the world. The pathogens (Urocystis spp., Urocystidales, Basidiomycota) cause a systemic infection on host plants, forming sori in the vegetative parts, most commonly in leaves, and present as narrow stripes between the leaf veins (Mordue & Walker, 1981). Most are believed to have a narrow host range on wild and cultivated grasses (Savchenko et al. 2017). Savchenko et al. (2017) clarified the taxonomy and phylogeny of flag smut pathogens of triticoid grasses using molecular and morphological analyses. 

The goal of the present study is to clarify the taxonomy and phylogeny of the flag smut pathogens on several additional grass hosts by analyzing morphological and molecular data. Specimens of Urocystis on several species of Elymus, Schizachne, Bromus, and Poa collected in the United States were examined and analyzed. The results of Bayesian analysis indicate that at least three undescribed species on Elymus, Poa, and Schizachne can be recognized and supported with morphological and host-specificity data. The new species on Schizachne is a first ever record of smut fungi on hosts from this genus. The new species on Elymus from New Mexico falls outside of the major clade of flag smut pathogens of grasses, representing a previously unknown lineage. Several other host-specific lineages were discovered in the phylogenetic analysis that will require additional sampling and further investigations. 

The USDA-ARS U. S. National Fungus Collections (BPI) contain approximately one million specimens representing all taxonomic groups with a special focus on phytopathogenic fungi. Of these, approximately 25,000 are listed as type specimens. Although label data in text form for more than 800,000 specimens are available online, the specimens, labels and annotations have not been imaged. Rust and smut fungi are particularly amenable to digital imaging for identification purposes, especially when the host plant is known, due to the obvious nature of their infections and the relatively large, ornamented spores that are produced.  In 2017, a project was initiated to identify and more completely document type specimens of rust and smut fungi housed at BPI. Following taxonomic assessment, type specimens were annotated and photographed. Images of specimens, packet contents and label information were photographed with a 35mm digital camera. Micrographs of diseased tissue were captured using a dissecting microscope while spores and other microscopic structures were captured using a compound microscope. Annotations will help researchers find type specimens at BPI and the micrographs can be used to identify pathogenic fungi without the need for additional sampling of original material. To date, 1517 type specimens, representing 704 species, have been identified. We present a summary of progress as well as examples of photo documentation generated in this project.

Two new species are described in the F. pinicola species complex in North America; Fomitopsis mounceae and F. schrenkii (Polyporales, Basidiomycota). Previous molecular phylogenetic analyses identified three well-delimited lineages that represent F. mounceae and F. ochracea from Canada, Appalachian Mountains, and northern USA and F. schrenkii from western and southwestern USA. Fomitopsis pinicola sensu stricto is restricted to Eurasia and does not occur in North America. Morphological characteristics of basidiocarps and cultures for F. mounceae, F. schrenkii, and F. ochracea are presented. The three species are readily differentiated by ITS1-5.8S-ITS2 (ITS) sequences, geographic distribution, and basidiospore size. Polyporus ponderosus H. Schrenk is an earlier illegitimate synonym of F. schrenkii. Both F. mounceae and F. schrenkii have a heterothallic, multi-allelic incompatibility system.

Whole genome duplication and gene-loss events leading to changes in ploidy levels are well-characterized in plants and animals. However, the frequency of ploidy changes and subsequent effects on diversification are poorly understood in mushroom-forming fungi. Previous research on the genus Hydnum (Cantharellales) has suggested that some species vary in ploidy level and differ by as much as a four-fold increase in chromosome sets. If so, ploidy level could correlate with changes in morphology observed across the phylogeny of Hydnum. Here, we present a framework to estimate ploidy levels in Hydnum and discuss hypotheses regarding the effect of polyploidy on morphology and diversification in the genus. To estimate ploidy levels in Hydnum, we collected dense basidiospore deposits from a range of species to compare relative gene content using flow cytometry. To study the phylogenetic distribution of ploidy level and other characters, we constructed a three-gene (ITS, rbp2, tef1) phylogeny from more than 50 samples of Hydnum, which provides a robust evolutionary framework for comparative analysis.

Tropical rain forests are the most species-rich biome on the earth. Studies point to the Russulales as the most abundant group of macrofungi in the tropics, but thus far, no member of the Hericiaceae has been described from Africa to our knowledge. Most known species of Hericium are from the Americas, Europe, and Asia. Here, we describe a new species of Hericium from the Dja Biosphere Reserve (DBR) in Cameroon. The DBR is a biodiversity hotspot with patches of monodominant ectomycorrhizal tree species (Gilbertiodendron dewevrei and Uapaca spp) scattered along the river banks.  Basidiomata were collected during the April–May early rainy season of 2018. The internal transcribed spacer (ITS) and large subunit rDNA (28S) regions were amplified using the primer combination ITS1F/ITS4B and LROR/LR6 respectively. The ITS and 28S sequences were BLASTn and available sequences of other described Hericium spp. were downloaded and analyzed for comparison. Laxitextum bicolor, Dentipellis fragrans and D. leptodon were used as the outgroup. Morphological examination and phylogenetic analysis support the Cameroonian collection as a new sister species to the H. coralloides complex. The new species differs from H. coralloides by having smaller spores (2.6–3.0 x 1.7–2.2 µm) and longer basidia (up to 27.0 µm). The Cameroon species possesses pleurocystidia, a feature that hasn’t been reported for other members of this genus. The new species like its temperate relatives, likely causes white rot to Congo Basin hardwoods. There is need to increase fungal sampling efforts of biodiversity hotspots such as the Congo Basin.

Marasmius Fr. (Marasmiaceae) is a highly diverse genus of approximately 600 species, with the greatest proportion of its diversity located in tropical ecosystems. Most Marasmius species are saprotrophic, meaning their distributions are rarely constrained by host plant distribution. Consequently, some Marasmius species are thought to have pantropical distributions. However, the distribution of many tropical fungal species is poorly understood due to fewer collecting efforts in these regions. To better understand the distribution of Marasmius species, we sequenced the recommended fungal barcode (the internal transcribed spacer region of the rDNA repeat, or ITS) from approximately 500 Marasmius collections from the Neotropics and the Caribbean collected over a 15 year period, and compared them to more recent collections from tropical Africa and Australia. From these data, we have: 1) expanded the known distributions of some Marasmius species, 2) provided a DNA barcode for species where one was not previously available, 3) described species that are new to science and 4) elucidated species complexes in this genus. This study provides further evidence that acquisition of vouchered fungal collections with corresponding DNA barcodes provides an invaluable tool not only for taxonomic studies, but for understanding the scope and distribution of fungal diversity. In particular, we found that many rhizomorph-forming species, thought to have a pantropical distribution, actually represent species complexes. These species, such as Marasmius crinis-equi and M. guyanensis, are character-poor, so species discrimination has been difficult. Ultimately, examination of molecular data from across the entire range of purported distributions is necessary to undertake revision of species complexes in character-poor groups. 

Species of Entolomataceae (Agaricales, Agaricomycetes, Basidiomycota) are found in most lowland tropical rainforests of the world. For tropical Africa, entolomatoid agarics have been comparatively well-documented from certain regions, such as parts of Ivory Coast, Gabon, Kenya, Tanzania, and Uganda, but are largely missing from mycofloristic studies of other areas, including the Congo Basin. Recent collecting activities in the Dja Biosphere Reserve of Cameroon have revealed a rich macrofungal mycota with numerous undescribed species across many agaric families and genera. Here we report on five new species of Entolomataceae and a new distribution record for E. semilanceatum (Romagn.) E. Horak, previously known only from the Congo. This work represents the first contemporary taxonomic study of the Entolomataceae from Cameroon. Macro- and micromorphological characters, habitat, and DNA sequence data of the new species will be presented.

Diversity of ectomycorrhizal (EM) fungi in the Neotropics is still relatively poorly known. Tropical rainforests dominate the region, but EM hosts constitute only a fraction of the tree diversity. However, in Guyana some forests are dominated by EM trees of the leguminous genus Dicymbe. Over 20+ years, macrofungal collecting in Dicymbe forests has yielded a great number of EM taxa. An important group of EM fungi in the Dicymbe system are the milkcap mushrooms of the genera Lactifluus and Lactarius. Molecular and morphological study of these milkcaps collections revealed a total of 17 species, of which nine are Lactifluus and eight Lactarius. The Lactifluus species are well distributed infragenerically, occurring in three out of four subgenera, and some exhibit unusual characteristics such as a partial veil or a pleurotoid habit. Only three of these Lactifluus species have been described so far. For Lactarius, eight species have been found in Guyana, a striking fact given that the main center of diversity for the genus is in Northern temperate regions. Seven of these newly discovered Lactarius species form an early diverging clade in Lactarius subgenus Plinthogalus. While this subgenus is predominantly known from the Northern hemisphere, it also contains early diverging African lineages. The discovery of Neotropical taxa in Lactarius subgen. Plinthogalus, previously thought to have originated in Africa, changes our ideas about the evolution and biogeography of the subgenus and even Lactarius as a whole. In addition, this illustrates the importance of including tropical taxa in the study of fungi. 

The ectomycorrhizal (EM) mutualism remains poorly characterized in tropical habitats compared to temperate and boreal ecosystems. Tropical EM fungi are notably understudied, particularly in the Guineo-Congolian forests of Central Africa. In this study, we report on an ongoing, intensive long-term sampling of the EM fungal community in a monodominant forest of the EM tree Gilbertiodendron dewevrei (Fabaceae subfam. Detarioideae) in southern Cameroon. Systematic, repeated-visit plot-based sampling for EM fungi was conducted yearly over the course of four years. During a season, in each of three 1-hectare permanent plots, fifty 100 m² subplots were exhaustively sampled over a 6-8 week period. Additional collections were made from outside the plots. To date, over 2000 sporocarp collections have been vouchered and ITS-barcoded. Morphological recognition of morphospecies along with operational taxonomic clustering of ITS sequences has revealed nearly 250 EM fungal species. Species accumulation curves indicate that saturation has not been reached, which is corroborated by the large number of unmatched EM OTUs recovered belowground in a separate study. This study highlights the importance of long-term sampling and vouchering of fresh collections within a given ecosystem to begin capturing the full breadth of macrofungal species within a guild. Therefore, we suggest that repeated sampling over multiple field seasons, in combination with DNA-based barcoding, is necessary in order to draw meaningful conclusions about the fungal diversity within a given ecosystem.

The diverse, primarily ectomycorrhizal (EM) mushroom genus Amanita (Amanitaceae, Agaricales, Basidiomycota) may have over 1000 species worldwide, with ~600 species formally described. Many Amanita species have broad distributions in higher latitude forests, while Amanita species in the Afrotropics are geographically restricted in lowland rainforests and woodlands dominated by EM trees of Fabaceae subfam. Detarioideae. Many African Amanita species described in the early twentieth century were based on a single type collection made by Mme Goossens-Fontana in Congolian forests of the EM detarioid Gilbertiodendron dewevrei. These species have been infrequently documented, if at all, in subsequent decades. Our collecting expeditions over 2014–2018 in Cameroonian Gilbertiodendron-dominated rainforests have yielded at least 40 distinct Amanita morphospecies. Some of these are new to science and others correspond to poorly-documented species based on the Goossens-Fontana collections. Here we present macro- and micromorphological characters and multi-locus DNA sequence data for two new species and four species to be epitypified. 

Amanita bisporigera is a pure white mushroom species that is deadly poisonous if consumed, hence its common name, the Destroying Angel. This mushroom species is native to North America and is responsible for human and pet fatalities every year. This mushroom contains lethal amounts of amatoxins and phallotoxins, which inhibit protein synthesis. The presence, composition and concentration of toxins varies across specimen. Recent phylogenetic studies indicate that A. bisporigera is a species-complex, likely consisting of multiple cryptic species. This hypothesis may help explain the wide toxin variability that has been found between mushroom specimen. However, this hypothesis has not been tested directly. To address this hypothesis, we are using multigene phylogenetics to resolve species in the Amanita bisporigera species-complex, and liquid chromatography and mass spectroscopy (LCMS) to test for variation in toxin presence and concentration within and between phylogenetic species. Fresh collections of white Amanita were collected in Michigan during the Fall of 2018 and the Summer of 2019. Each mushroom was flash-frozen and phenotyped for amatoxin and phallotoxin analysis with (LCMS). A multigene phylogeny was build with sequences of Internal Transcribed Spacer (ITS) rDNA, large subunit (LSU) rDNA, ß-tubulin, and RNA polymerase II (RPB2). Toxin profiles will be mapped onto the resulting phylogenetic tree.  Our preliminary analysis have identified at least one novel toxic species in this complex, which will be formally described.

Globally, many undescribed fungal taxa reside in the hyperdiverse, yet undersampled, tropics. These species are under increasing threat from habitat destruction by expanding extractive industry, in addition to climate change and other threats. Reserva Los Cedros is a primary cloud forest reserve of ~17,000 acres, and is among the last unlogged watersheds on the western slope of the Ecuadorian Andes. No fungal survey has been done there, presenting a rare opportunity to document fungi in an relatively undisturbed ecosystem in an underrepresented habitat and location. Six above-ground surveys from 2008–2019 resulted in ~1700 vouchered collections, mostly Agaricales sensu lato and Xylariales. We document diversity using a combination of ITS barcode sequencing, digital photography, and participation in online citizen science platforms, such as Mushroom Observer. Preliminary identifications indicate the presence of at least 5 phyla, 17 classes, 30 orders, 81 families, and 190 genera within the reserve. Two taxa at Los Cedros have recently been recommended to the IUCN Fungal Red List Initiative (Thamnomyces chocöensis and Callistodermatium aurantium), and we add occurrence data for two others already under consideration (Hygrocybe aphylla and Lamelloporus americanus). Plants and animals are known to exhibit exceptionally high rates of endemism in the Chocó bioregion, but endemism in fungi is poorly understood. Our collections contribute to understanding this important driver of biodiversity in the Neotropics. Mining expansion into protected forests, including Los Cedros, poses an existential threat to the fungi we have observed, and those that have escaped collection. 

Traditionally, Laetiporus genus was comprised of only two species: Laetiporus sulphureus (Bull.) Murrill and Laetiporus persicinus (Berk. & M.A. Curtis) Gilb. Those two were characterized as brown rot wood decomposers, producing large and soft basidiomata, dimitic hyphal system and white spore print. Both were easy to identify macroscopically with this delimitation. But recently, using molecular tools to test evolutionary trends within the genus, researchers found that L. sulphureus was a taxonomic complex hiding tens of species around the world regard their tree host and geographical location and some species that were synonymized to L. persicinus could be another genus until undescribed. We reviewed Laetiporus specimens in herbaria collections from Brazil and made new ones totalizing 52 basidiomata looking at macroscopic, microscopic and molecular level. All specimens that were in herbaria had the names L. sulphureus and L. persicinus. We found that those collection identified as L. persicinus were wrong interpreted and are from Polyporus talpae Cooke, that nowadays is considered a synonym of L. persicinus but it is more related to a genus described from Africa, Kusaghiporia usambarensis J. Hussein, S. Tibell & Tibuhwa, and need a new taxonomic combination. We found that those identified as L. sulphureus are Laetiporus gilbertsonii Burds. and that those could be jumping host to Melastomataceae tree, a host that was never described for Laetiporus species. Least, we found what seems to be a new species for science growing on Schinus sp. tree that´s been described.

White spruce growth at an arctic treeline may be limited by nutrient availability in cold soils. Ectomycorrhizal (ECM) fungi aid these trees in nutrient acquisition at a potentially high carbon cost. This study aims to examine changes in mycorrhizal dependence with elevation and to test the strength of the tree-fungal association in the face of experimental changes in soil nutrient availability. If fertilization diminishes spruce dependence on ECM fungi, will control trees display amino acid δ¹³C values, or isotopic fingerprinting, intermediate to a fungal fingerprint and the fertilized trees? Secondly, which taxon remain despite fertilization and is mantle development impaired? Roots and needles were collected from fertilized and control trees (n = 60) at three sites along an elevation gradient in northwestern Alaska. Root tips will be assessed for percentage of ECM fungal colonization and root tip architecture to determine mycorrhizal dependence. The 18S rRNA genes of pooled root tips from each tree will be sequenced with an Illumina platform. Needles will be analyzed for nitrogen and phosphorous concentrations to determine nutrient acquisition. Needles will also be analyzed for amino acid δ¹³C values to examine the viability of applying isotopic fingerprinting to mycorrhizal interactions.

Fungi in the ectomycorrhizal genus Suillus are widely recognized for their high specificity with tree hosts in the family Pinaceae, which consists of two clades: Pinoid and Abietoid. Although Suillus has only been documented to associate with the former clade, Suillus sporocarps have been found in Pinaceae forests in which no known Pinoid hosts (Pinus, Pseudotsuga, and Larix) are present. To investigate possible alternative host associations, we conducted a growth chamber bioassay using Suillus glandulosus, a species which commonly fruits in both Picea (Pinoid clade) and Abies (Abietoid clade) dominated Canadian forests. Seedlings of Larix laricina, Picea mariana, and Abies balsamea were grown in single- or two-host species pairings and inoculated with high densities of S. glandulosus spores to determine both host association and mechanism of colonization. We found that only L. laricina was colonized after 3 months, while both P. mariana and A. balsamea seedlings were lowly colonized in the two-host treatments with L. laricina after 6 months. These results suggest that while Larix is needed to trigger Suillus spore germination, both Picea and Abies can be colonized by mycelial extension. Results from the final part of the bioassay will also be presented in which colonized Picea and Abies seedlings have been isolated from co-planted Larix seedlings, to determine if alternative hosts can independently support the growth of Suillus. Collectively, these results indicate that Suillus fungi may be able to associate with Abietoid hosts in natural settings and that the classical conceptualization of Suillus host specificity may require revision.

The mutualism between temperate trees and ectomycorrhizal fungi (ECM) dominates much of the northern hemisphere. This association provides numerous benefits for the tree host such as nitrogen acquisition, drought tolerance, and defense. Recent manipulative research has only studied simplified systems with one ECM associate per host, however, ECM soil communities are highly diverse, and hundreds of species may colonize a single tree. Moreover, interspecific competition has been hypothesized to maintain this high alpha-diversity and may significantly alter ECM gene expression. To test these hypothesizes we are conducting a novel metatranscriptomic assay that seeks to elucidate the role of belowground competition and phylogenetic relatedness in gene expression of ECM fungi. Populus tremuloides seedlings will be grown in the presence of one to several ECM species (1-8) covering a vast array of phylogenetic diversity and ecological habits.  mRNA of each ECM will be extracted from ground whole root systems. We will compare the transcriptomes of each ECM species in and out of competitive environments to interspecific competition in ECM.  In addition, we are conducting a broad metabolomics survey using UPLC of the host leaves to identify changes in metabolite production due to belowground competition. We hypothesize that competition will have an additive effect on gene expression, where distantly related ECM up-regulate genes associated with nutrient acquisition and transport to become better mutualists, while closely related ECM shift expression to avoid niche competition. This study may lead to insights on the role of competition in ECM niche portioning and host phenotype.

Species distributions are expected to shift to higher elevations and latitudes in response to contemporary climate warming. However, because species appear to be shifting their distributions at different rates, some may reach environments that lack the species with which they usually interact. This may be a significant barrier to distribution shifts in species that depend on mutualistic interactions such as ectomycorrhizal fungi (EMF) and their plant hosts. To begin exploring this hypothesis, we describe the species composition of EMF communities of subalpine fir (Abies lasiocarpa) at their high elevation distribution limits. At our study site in Mount Rainier National Park (WA, USA), subalpine fir constitutes treeline and grows in small forest patches or as isolated individuals in subalpine meadows. The isolated trees in meadows are considered to represent the early stages of a climate-warming induced distribution shift for subalpine fir. We predicted that the roots of isolated subalpine fir would harbor only a subset of the EMF found in forest patches due to dispersal limitations and unfavorable abiotic conditions in the meadows. We used morphotyping and ITS sequence data to describe EMF communities in these two environments and quantified relevant soil variables (water holding capacity, C:N ratio of organic matter, inorganic P) at all sampling locations. The results of this study should provide insight into the potential distributional shifts of EMF in response to climate change.

Ectomycorrhization is important for early survival of seedlings of ectomycorrhizal (EM) tree species. In Guyanese forests dominated by EM canopy tree Dicymbe corymbosa (Fabaceae subfam. Detarioideae), conspecific seedlings experience high multi-year survivorship in crowded, often heavily-shaded environments. Ectomycorrhizal fungi may facilitate seedling survival under these challenging growth conditions. Little information exists, however, on the development of EM fungal communities in seedlings of EM tropical monodominant trees. This is especially true regarding how the extent of EM colonization or community assembly of EM fungi may influence early seedling survival. We sampled an even-aged cohort of seedlings of D. corymbosa at < 1, 6, and 12 months following a mast seeding event and determined their percent mycorrhization and mycobiont assemblage for each age class. Cross-generational sharing of EM mycobionts was assessed by comparing seedling EM fungal communities to those of nearby adult conspecifics. Percent mycorrhization did not increase over time, but rather was highest at 6 months, with no significant difference in colonization at < 1 and 12 months. However, species turnover in EM fungal communities over time was high, with only 20% overlap in EM fungal species between 6- and 12-month seedlings. Tomentella (Thelephorales) species were particularly abundant in each seedling age class and were also the most abundant in adult trees. This study suggests that the qualitative composition of EM fungal mutualists may play a larger role than the extent of mycorrhization in early seedling survival of tropical monodominant trees.

Invasive plants can leave lasting legacies on ecosystems, including changes to ectomycorrhizal fungi (EMF). Such legacies can make restoration difficult even after invaders have been removed. Previous research has identified suilloid fungi (Suillus and Rhizopogon) as important in early pine establishment, making them potentially useful in pine restoration. To test the hypothesis that Robinia pseudoacacia has a negative impact on resistant propagules of EMF associated with Pinus rigida we carried out soil bioassays with soil from sites where R. pseudoacacia had invaded but recently been removed and from non-invaded sites. Bioassay seedlings planted in R. pseudoacacia invaded soils had three EMF species compared to five species non-invaded sites. One EMF species was present in both. One suilloid species, Rhizopogon pseudoroseolus, was found on seedlings in non-invaded soils. To test the hypothesis that suilloid fungi can improve survival and that R. pseudoacacia has a legacy effect on P. rigida survival, P. rigida seedlings were inoculated with live or autoclaved locally collected suilloids spores and planted in a factorial field experiment (inoculation treatment x invasion history). There was no difference in survival of field seedlings between invaded or non-invaded sites after 8 months. However, 72% of seedlings inoculated with live spore inoculum survived, compared to 31% of seedlings inoculated with autoclaved spores (P < 0.001). These results suggest the legacy of R. pseudoacacia does not limit restoration of P. rigida at the Albany Pine Bush Preserve, but that establishment increases when pines are inoculated with locally adapted suilloid fungi.

Colonization by fungal endophytes have broad benefits for plants, including increased resistance against herbivores and pathogens. Endophytes in culture have been shown to produce secondary metabolites that can suppress herbivore and pathogen damage. It is unknown if these toxins are also synthesized during symbiosis within the host plant, or if endophytes stimulate plant chemical responses, thus affecting plant interactions with their enemies. We tested how endophyte colonization alters the production of defensive compounds in the toxic plant white snakeroot (Ageratina altissima), a native Indiana wildflower. We inoculated endophyte-free seedlings with one of three treatments: 1) inoculation by a single, dominant endophyte (Colletotrichum sp.), 2) inoculation with rainwater captured underneath wild snakeroot plants as a natural fungal spore source, or 3) application of sterile water as a control. After endophyte communities established in seedlings, we quantified colonization success using a culture-based approach, coupled with Sanger sequencing. Plants inoculated with rain water had the most diverse endophyte communities. To determine if endophyte community composition and diversity affected plant chemistry, we extracted phenolics from leaf tissue and performed liquid chromatography-mass spectrometry. Comparing the phenolic profiles across the three treatments revealed microbial colonizers alter the production of secondary metabolites in plants. Plants treated with Colletotrichum sp. and rain water had a significantly larger breadth of chemical compounds in their tissues than uninoculated seedlings. Additionally, abundance of individual phenolic compounds varied between treatments. Future work will examine if these chemicals were produced by endophytes or were produced by plant pathways triggered by fungal colonization. 

Prairies of the Pacific Northwest are highly threatened systems, with only ~2% of historic land area remaining.  The combined risk of global climate change and land use change make these systems a high conservation priority.  However, little attention has been paid to the microbiota of these systems.  Fungal endophytes are ubiquitous in plants and are important in ecosystem functioning and host dynamics.  While emphasis has largely been placed on single, economically relevant endophytes, there are often hundreds of species occupying a single leaf.  Despite our poor understanding of the functional role each fungal species plays within these complex systems, community composition may give insight into host-endophyte interactions.  Using high-throughput illumina sequencing, we investigated the diversity and composition of fungal foliar endophyte communities in two native, cool-season bunchgrasses along a natural latitudinal gradient.  We quantified the relative importance of host, host traits, climate, edaphic factors, and spatial distance in microbial community composition.  

We found markedly different communities between the southern and central-northern sites, suggesting a potential dispersal limitation in the Klamath Mountains (F_1,153 = 5.080, p < 0.001).  We also found that each host species was home to distinct fungal communities (F_1,153 = 5.965, p < 0.001).  Host species, spatial distance and climate were the strongest predictors of endophyte community, while host traits (e.g., plant size, reproductive status, density) were less important for community structure.  

Bark beetles have evolved symbioses with fungi and their tree hosts that range from highly specific, to loose associations, to asymmetrical dependence. Besides contributing to the advancement of symbiosis and evolutionary ecology theories, the beetle-fungus relationship has often been hugely destructive, with outbreaks and epidemics reaching record proportions in forests on every continent, costing billions of dollars per year and damaging important ecosystems. The scientific community working towards understanding and mitigating these emerging global threats is facing a critical shortage of expertise, comprehensive and curated public databases, updated research protocols and standards, and well-established knowledge flow systems that connect a global community of forest entomologists and pathologists. These challenges result in the use of incomplete or incorrect information by end-users who make policy decisions concerning international biosecurity, trade, and natural resources protection. This five-year project aims to coordinate research efforts of forest pathologists, entomologists and symbiologists throughout South Africa and the U.S. With over 22 individual researchers representing 17 institutions in five countries, our objective is to critically assess how bark beetles and their fungal associates are studied and interpreted and identify and recommend ways to improve current research approaches.

The emerald ash borer (EAB), a beetle endemic to northeastern Asia, has invaded eastern North America where it is causing a widespread die-off of ash (Fraxinus sp.). Adult beetles lay eggs on the tree bark and, after hatching, larvae feed on phloem tissue, destroying the tree’s ability to conduct water and nutrients. Microbial symbionts of other phytophagous insects have been shown to play important roles in lignocellulose degradation, breakdown of plant chemical defenses, or in nutrition and development of their insect hosts. There is some evidence to suggest an active fungal community within the EAB larval gut, but the taxonomic and functional diversity as well as drivers of community assembly of this gut microbiome are unknown. Larvae within EAB galleries may acquire a portion of their gut microbiome by consumption of fungi living as endophytes within ash phloem. In order to describe the relationships between EAB gut microbial species and ash endophytes, we conducted culture-dependent and -independent community profiling of EAB frass and phloem samples. We also aimed to determine whether any of the fungal taxa isolated from ash phloem or EAB frass potentially aid in EAB’s attack via lignocellulose degradation, detoxification of plant defense compounds, or other nutrient provision mechanisms. Preliminary lignocellulose and phenolic degradation assays will provide insight into the potential biochemical mechanisms within this microbial community to better assess the nutritional ecology of EAB.

With recent focus on both organismal and ecosystem microbiome composition, it is quickly becoming apparent that symbiotic fungal endophytes, those that asymptomatically infect plant tissues, are nearly ubiquitous throughout the plant kingdom providing a variety of services to their plant hosts. One such service is protection from herbivory. Nipponaclerda biwakoensis, an invasive scale insect recently discovered in high densities in the Mississippi River Delta (MRD), has been correlated with diebacks of a foundational plant species, Phragmites australis. This is of great concern because Louisiana’s coastal marshes are in danger of being lost to sea level rise and erosion and P. australisplays an integral role in the mitigation of these threats.Previous work has shown that there is considerable variation in scale infestation among Phragmites stands; specifically between two dominant Phragmites haplotypes, M and M1. In September 2018, I traveled to two sites in the MRD and collected samples of both haplotypes to assess infestation severity and fungal endophyte composition. My preliminary findings show that haplotype M1 is significantly more infested than haplotype M, and there is also substantial variation in infestation within each haplotype. I hypothesize thatdifferences in infestation between and within haplotypes can be attributed to the presence of mutualistic fungal endophytes which directly and indirectly confer resistance to N. biwakoensis. Overall, this work is an important first step in identifying key fungal players in plant-herbivore interactions in an important coastal system, which can eventually be leveraged to enhance restoration in the area.

            Ophiocordyceps camponoti-floridani, a species of “Zombie Ant Fungus,” infects and modifies the behavior of carpenter ants to further its own transmission at a lethal cost to the host. Manipulated ants perform a “death grip” biting and clinging behavior to attach themselves to plants. This novel behavior is understood as a fungal manipulation that benefits the parasite’s growth and transmission. Across the manipulative Ophiocordyceps, host modification is often observed at stereotypic times of day, on particular substrates, and in specific host species. The underlying mechanisms of how these fungi can dysregulate animal behavior in such a coordinated manner has yet to be described. Analysis of fungal gene activity across the entire transcriptome at the time of manipulation allows detailed investigations into the fundamental mechanisms used by these fungi. These explorations can then lead to functional testing of potentially key gene products and metabolites. Using a combination of Nanopore and Illumina sequencing technologies, we produced a hybrid assembly of the genome of this species. Subsequently, we performed RNAseq to characterize differential fungal gene expression across the course of infection and manipulation. To select a robust set of candidate “manipulation genes,” we will combine these data with a former gene expression study of O. kimflemingiae in a comparative transcriptomics analysis. Strong candidates will then be functionally tested for necessity in manipulated phenotypes, using controlled infections of transgenic knockout fungi with loss of function of the gene of interest.

Arthropods are an important source of nutrition for fungi, providing numerous niches for diversification of entomopathogens. Entomopathogenic fungi sometimes manipulate insect behavior as part of their spore dispersal phenotype, but the molecular and physiological mechanisms of manipulation are not well understood. Through analyses of clustering of related gene functions in multiple newly generated, Nanopore-Illumina genomes of Cordyceps s.l. species, we identified a putative neuroactive gene cluster. What we refer to as the GABA cluster is composed of 5 genes, including homologs of glutamate decarboxylase (GDC), Glutamine Phosphoribosylpyrophosphate Amidotransferase (GPA), and a Nitrate transporter (NRT2). The complete gene cluster appears to be ancestral in Cordyceps s.s., and is retained in Cordyceps militaris and Isaria fumosorosea, but partially lost in Beauveria bassiana. Here we present research whose ultimate goal is to characterize the function and evolution of the GABA gene cluster, and to investigate its possible role in behavior manipulation. We heterologously expressed GDC and GPA for use in enzymatic analyses, and monitored the production of GABA in vitro and in vivo using liquid chromatography-tandem mass spectrometry. This work represents a significant step in the understanding of neuroactive metabolite production by entomopathogenic fungi.

Endophytic insect pathogenic fungi are plant mutualists and insect antagonists and can therefore protect their plant hosts from detrimental insect pests. This study examined trade-offs between being a plant mutualist and an insect pathogen fungus. We assessed the endophytic and pathogenic capacities of 8 isolates of Beauveria and Metarhizium (B. pseudobassiana, B. bassiana, M. anisopliae, and M. pemphigi) to determine if single isolates are better at one strategy than the other. We performed two assays in order to test for the presence of trade-offs. In the insect pathogenicity assay, surface-sterilized wheat stem sawfly larvae were exposed to standardized spore solutions from the Beauveria and Metarhizium isolates. Infection and mortality of the larvae was tracked over a 7-day period. In the endophyte assay, wheat plants were cultivated from seed in a greenhouse and exposed to standardized conidial solutions of each fungal isolate and grown for four weeks. For each plant we measured chlorophyll content, height, and number of shoots, leaves, and inflorescence before and after the treatment. Our preliminary results show all isolates have high efficiency establishing as insect pathogens as we observed rapid and high mortality rates within larvae across all isolates. Wheat plants exposed to distinct isolates within the endophyte assay showed differential growth and physiological characteristics that may be due to endophyte establishment, suggesting a potential trade-off in endophytic pathogenic ability for some isolates. Our next steps include sequencing the fungal ITS region from plant tissue to ascertain endophyte presence and determine differential endophyte establishment and mutualistic ability.  

Fusarium is a genus associated with several economically important plant diseases and toxicoses. Some phytopathogenic fungi use mimicry to physically resemble plant structures that attract insects. Fieldwork conducted in western Guyana revealed an abundance of fungus-associated pseudoflowers on Xyris surinamensis and X. setigera (yellow-eyed grasses). An unusual new species of Fusarium was associated with these pseudoflowers, being the first report linking a Fusarium species to this phenomenon. Our study aims to 1) characterize the disease caused by this Fusarium sp., 2) determine the main dispersal mechanism for the fungus, 3) evaluate the role of pseudoflowers on insect attraction, and 4) determine if pseudoflower production is associated with the presence/absence of other microorganisms. Field observations will determine signs and symptoms produced by the disease. Greenhouse experiments will be used to evaluate infection mechanisms, while field samples will be tested for presence of the fungus on healthy plant tissue, seeds, and rhizosphere soil. Insect visitation studies will identify insects that come in contact with flowers and pseudoflowers on Xyris. A nested PCR method will be used to detect Fusarium DNA on collected insects. Volatile organic compound (VOCs) production will be compared between infected and uninfected plants to determine if fungal infection generates insect-attracting VOCs. The microbial community composition of pseudoflowers and inflorescences will be compared. Other pseudoflower-inducing fungi have been described, yet connections between disease-induced traits, transmission, and vector attraction require further research. Our study helps elucidate these plant-insect-pathogen interactions by providing understanding of their evolutionary ecology and transmission biology.

The serotonin-analog, psilocybin, and its chemical relatives, prevalent in Psilocybe species, have poorly understood ecological roles, as do fungal secondary compounds generally. Due to serotonin’s importance in animal nervous systems, these serotonergic compounds could have evolved for defensive purposes. This study investigated the defensive potential of Psilocybe cubensis alcoholic extracts on bacteria (Staphylococcus aureus [gram positive], Escherichia coli [gram negative]), Caenorhabditis elegans (Nematoda), soil microbes (undetermined), and brine shrimp (Arthropoda). Test organisms were introduced to P. cubensis extracts and toxic effects were measured after an allotted period of time. In the bacteria bioassay, methanol, P. cubensis extracts, and control fungus, Agaricus bisporus, extracts had no inhibitory effects with either bacterium at any concentrations tested. Similarly, results of the C. elegans bioassay demonstrated low toxicity across all treatments, and no correlation between P. cubensis extract concentration and mortality. In the soil microbe bioassay, P. cubensis extract exhibited significantly greater antimicrobial properties compared to untreated and methanol controls but did not differ from control fungus extract. In contrast to other bioassays, the brine shrimp bioassay demonstrated the most striking results, with near-universal mortality of all brine shrimp three hours after introduction of P. cubensis extract at all concentrations; results not seen in any control treatments, though control fungus extract concentration and mortality were positively correlated. Because P. cubensis has demonstrated high toxicity in an arthropod model and insect arthropods share decay niches with psilocybin-producing fungi and contain many fungivores, P. cubensis compounds may have evolved, in part, to defend against insects.

Fungi produce neuroactive metabolites that can influence animal behavior and fitness. Due to these effects, some of these metabolites and their synthetic derivatives are used medicinally and possess potential to treat recalcitrant psychiatric conditions, including addiction, depression, and anxiety. However, the ecological roles of these metabolites are poorly understood. Tryptamine-based metabolites are one class of neuroactive compounds that have independently evolved multiple times in Fungi. Multiple tryptamines have affinity to serotonin (5-hydroxytryptamine, 5-HT) receptors. The psychoactive tryptamine psilocybin was first described in the ‘magic mushroom’ genus Psilocybe, and primarily agonizes 5-HT₂ receptors. Psilocybin was recently found to be the product of a gene cluster with spotty distribution throughout Agaricales mushrooms, partly due to horizontal gene transfer (HGT). It was speculated that acquisition of the cluster by HGT increased fungal fitness under predation by mycophagous invertebrates. Notably, highly similar homologs of the five Agaricales psilocybin genes were also found unclustered with some gene family expansion in Atheliales. All of the genes were found in the termite symbiont Fibularhizoctonia sp. (teleomorph Athelia) whereas only psilocybin-associated transporter of unknown function was found in ectomycorrhizal Piloderma croceum. Here, we report the production of psilocybin in the sclerotia of Fibularhizoctonia sp. using multiple reaction monitoring (MRM) and product ion scan via tandem mass spectrometry. This provides additional evidence of psilocybin production in fungus-insect interactions.

Domesticated coral mushroom gardens (family Pterulaceae), farmed by Attine ants of the Apterostigma pilosum group, have a distinct evolutionary origin from ant-farmed gilled mushrooms, and convergently coevolved the same interactions with their Attine ant farmers, beneficial bacteria, and parasitic Escovopsis mold. Domestication of crops predicts stereotypical genetic changes that confer better crop phenotypes, such as lower toxicity through a loss of secondary metabolite gene clusters. Preliminary comparative genomic data from domesticated and free-living coral mushrooms suggest a reduction in secondary metabolite diversity in one of the coral mushroom cultivars. However, coral mushroom cultivars must still contend with attack from specialist parasitic Escovopsis fungi, possibly necessitating the retention of the ability to produce certain defensive compounds. This project will utilize comparative genomics to
 analyze differences in secondary 
metabolite-encoding gene clusters in
 domesticated and free-living Pterulaceae.


Seeds are an essential component of plant life histories, and seed endophytes have the potential to influence germination, seedling establishment and development. That said, seed endophytes are still a new area of study, both in the factors that influence which taxa are present and how these microbes alter plant function. The objectives of this study were to characterize the fungal endophytes present in native and introduced populations of Coastal Douglas-fir (Pseudotsuga menziesii var. menziesii) seeds, and to test whether some of these endophytes affect seedling drought response. Using culture-based techniques, endophytes were isolated from 8 native populations of Douglas-fir seeds in the United States and 3 introduced populations in New Zealand. Based on the ITS fungal barcode, the dominant taxa present in the sampled seed populations were Trichoderma spp. and Sydowia polyspora. Assessment of endophyte community composition in the United States populations indicates differences based on seed provenance, and future work could further investigate how these communities vary along environmental and plant genetic gradients. To test endophyte function, seedlings from one United States population were inoculated with two isolates of seed-borne Trichoderma spp. and grown under drought conditions. From this experiment, it is expected that inoculated seedlings will have longer survival and improved growth. If the expected results are observed, further study could be conducted on the role of seed endophytes in plant response to abiotic stresses, with applications in mitigating plant stress due to climate change. 

Old-growth and late-successional Pseudotsuga menziesii play important ecological roles in forests of the U.S Pacific Northwest. These conifers interact with the environment via their canopies, and how these interactions take place can differ within a single canopy along its vertical axis. This work seeks to identify whether canopy characteristics correlate with changes in endophytic fungal community compositions using an Illumina MiSeq metabarcoding approach. Eight trees in the HJ Andrews Experimental Research Forest (Oregon, USA) were climbed and accessible branches were collected along the length of the bole. A single age class of twigs and four age classes of needles were retained from each height. The ITS2 metabarcoding region was amplified directly from needle and twig DNA extractions and sequenced on a 2x300 Illumina MiSeq run. We found that twigs possess more diverse fungal communities than needles, and that the composition of twig and needle communities differed by source tree. Needle and twig communities both vary with vertical canopy position after accounting for source tree, and needle compositions differ according to needle age. Additionally, the relative abundance of Nothophaeocryptopus gaeumannii—the dominant endophyte of P. menziesii capable of causing Swiss needle cast—significantly affects the relatedness of needle communities across trees. These results suggest that spatiotemporality within Douglas-fir canopies is influential in the development of endophytic fungal communities. Determining the extent to which this spatiotemporality is due to microenvironmental properties and/or tree ontology requires further exploration.

Communities of animals, plants, and micro-organisms are commonly differentiated along precipitation gradients, yet it remains uncertain whether individuals within species are similarly ecotypically selected, particularly for microbes.  To better understand ecotypic adaptations of Ascomycota fungi to environmental conditions, we analyzed stress tolerance for six isolates of for five ascomycete species from sites that experienced up to two-fold differences in mean annual precipitation (MAP) across the central United States. We acquired fungal isolates from Sevilleta National Wildlife Refuge, ~250 mm MAP; Hays Agricultural Research Center, 450-660 mm MAP; and, Konza Prairie Biological Station, ~835 mm MAP.  First, utilizing quadrant Petri plates amended with sodium chloride (NaCl) at four concentrations (0–100g/L), we tested the halotolerance of the isolates. Second, we tested xerotolerance using liquid cultures amended with Poly-Ethylene Glycol (PEG) at four concentrations (representing approximate osmotic pressures from -1 to -15MPa). Analyses of estimated NaCl and PEG concentrations that reduced the fungal growth by 50% (ED₅₀) indicated that, although the fungal isolates varied in their halo- and xerotolerances, ecotypic adaptation to prevailing environmental conditions from their site of origin was limited. Our data suggest physiological plasticity in fungal populations adapted to environments with multiple potential stressors. Further experiments using conspecific isolates that vary in environmental tolerances could help to identify the underlying genomic and functional traits of stress tolerance in fungi. 

Plant species harbor important microbial symbionts that could help mediate plant response and adaptation to environmental factors. Endophytic Fungi (EF) represent one of the most important plant symbionts. However, processes governing EF community assembly and the effects of ecological context on the evolution of symbiotic interactions remains weakly understood. The objective of this study was to evaluate the effect of precipitation, host species, and tissue type on EF community diversity and richness. We collected leaves and roots samples from Schizachyrium scoparium (SCSC) and Zea mays (corn) in five locations along a rainfall gradient ranging from eastern South Dakota to south-eastern Minnesota . Fungi were isolated from plant tissues and sequenced using ITS rDNA region primers. EF community will be analyzed to evaluate the relative importance of yearly rainfall, hosts species, and tissue types to EF richness, diversity. We expect that the structure of EF communities will primarily vary with rainfall and geographic distance, and with host identity. Our preliminary results showed that EF colonization does not significantly varies across the rainfall gradient, but rather across host species and tissue types. In addition of their impacts on colonization and diversity, biotic and abiotic factors could have a significant impact on the interaction EF will maintain with their host in varying drought conditions.

All plants have a community of asymptomatic microbes inhabiting their tissue known as endophytes. Increasing evidence suggests that microbes are an extension of plant host phenotype and can ultimately help them adapt in response to stress, including drought (Compant et al. 2010). Additionally, stressful conditions may select for distinct endophyte taxa with specific functions (Lemanceau et al. 2017). Further understanding of how the structure of endophytes shift in response to drought is a potentially important avenue for identifying significant biotic interactions that may play a role in stress response to climate change and perhaps predicting species distribution shifts. The aim of this project is to examine changes in endophyte communities in plants suffering from drought. We ask, does drought alter microbiome composition, and if so, what part of the plant is changing and are there specific taxa that come into play? We sampled both roots and shoots of E. laciniata plants grown in native soil in laboratory 1) controlled and 2) drought conditions. Plant tissues were sampled at two time points in the plant life cycle to account for any shifts over time. All tissue was analyzed for bacterial and fungal taxa. Preliminary results indicate strong differences in endophyte between plant compartments (e.g. roots and shoots), suggesting that root communities are more impacted by the effects of drought than shoot communities. The diversity of endophytes was also greater in the root communities than in the shoot, suggesting transmission of endophytes from their native soil.

Current environmental change predictions forecast shifts in climatic means, greater climatic variability, and more frequent extremes including floods, droughts and heat waves. It is increasingly evident that plant communities are sensitive to drought and soil-inhabiting microbial communities vary along precipitation gradients. However, the drought sensitivity of the microbial communities remains unclear. Understanding microbial community responses to adverse environmental conditions is vital to elucidating their sensitivity to forecasted changes in precipitation regimes.  We sampled soils from an established drought experiment at two sites that vary in their mean annual precipitation (MAP) to assess fungal community responses to naturally existing and experimentally imposed precipitation regimes.  We hypothesized that fungal community composition and abundance of drought sensitive taxa would vary in response to MAP and drought treatment.  We analyzed our target communities using both culture-dependent and -independent tools to compare the conclusions derived from the two methods.  After sequencing communities from environmental DNA and from colony forming units on a drought simulating medium, we estimate that more than 10% of the fungal community and more than 20% of the ascomycetous community was culturable. Our data from the two approaches consistently indicate that while communities were distinct between the two sites differing in MAP, they did not differ between the experimental drought treatments. While recent research indicates that plant and bacterial communities respond to drought, fungal community responses are more elusive, particularly in experiments that impose chronic drought under field conditions.

Arbuscular mycorrhizal fungi play an important role in natural and agronomic settings, offering host plants increased uptake of mineral nutrients, pathogen resistance, and abiotic stress tolerance. Improved tolerance to drought stress of colonized plants is well-documented, but the molecular underpinnings of this benefit are poorly understood. To evaluate the impacts of drought on host and fungus, we inoculated carrots (Daucus carota cv. ‘Napoli’) with spores of Rhizophagus irregularis DAOM 197198. Carrots grew in a greenhouse. After carrots established, we imposed water restriction for ten days. Plants were flash frozen, and the fine roots of carrots were used for RNA-seq. Root staining revealed an average colonization percentage of 35% for inoculated plants and no evidence of fungi was found in mock-inoculated controls. Well-watered carrots had significantly higher rates of photosynthetic assimilation, transpiration, and stomatal conductance than those in the drought group, regardless of the inoculation status. Well-watered carrots grew taller shoots and outweighed their drought counterparts (p-value < 0.001). Within the drought treatment, mycorrhizal carrots grew 15% longer shoots than mock-inoculated carrots (p-value < 0.05). There were 12,087 differentially expressed transcripts of carrot and 3,224 for fungus (p-value < 0.05) between well-watered and drought treatments. Preliminary analyses revealed that transcripts associated with fungal aquaporins (AQPs) differed in their response to drought, with AQPF2 upregulated and both AQP1 and AQPF1 downregulated under drought conditions (p-values < 0.001, < 0.001, and < 0.05, respectively). Comparisons of transcriptomic differences between treatments will shed light on the mechanisms leading to improved drought-tolerance of mycorrhized plants.

Soybean (Glycine Max) is a globally significant crop in terms of nutritional and economic metrics. Soybean yields are expected to be at risk as global climate continues to become more erratic in the coming decades. In order to maintain yields, researchers must consider methods that supplement traditional breeding and genetics. One concept that is coming to the forefront in studying plant abiotic stress tolerance is the concept of the plant holobiont. 

In order to analyze the microbial component of the soybean holobiont, we performed amplicon sequencing of Fungi, Bacteria, and Oomycete communities associated with soybeans grown under organic, no-till and conventional management systems at Kellogg Biological Station. Sequencing was performed on rhizosphere soil, root, stem, and leaf samples taken at three different time points throughout the growing season. Analysis of Illumina sequencing results revealed significant differences between management systems using PCoA and PERMANOVA analyses in the fungal community associated with soil, but differences were obscured in roots. As expected, soil fungal communities were more diverse across all management systems with between 400 and 600 fungal taxa per sample while the roots contained on average between 150 and 250 taxa per sample. In the no-till management system, root-associated fungal communities were mostly dominated by taxa in Fusarium and Didymyella genera. In the soil, fungal communities were dominated by Fusarium and Mortierella. Network analysis performed on no-till communities showed several significant hub species with the most highly connected species being the yeast Sporidiobolus pararoseus. Efforts to manipulate the soybean microbiome are underway.

North American tallgrass prairies have been largely converted to row-crop agriculture and maybe only as little as 1% of the historic tallgrass prairies in North America remain intact.  Restoration efforts to re-establish prairie vegetation after long-term use in production agriculture is challenging and commonly burdened with questions about what are adequately desirable restoration outcomes.The evaluation of restoration largely relies on analyses of plant communities, whereas it is rarely evaluated whether or not microbial communities resemble those observed in prairie remnants without historic conversion to agriculture. We sampled two prairie sites in eastern Kansas, each with a remnant that had never been converted to row crop agriculture and a post-agricultural site that had been restored. We extracted genomic DNA from roots and leaves of the dominant grass, Schizachyrium scoparium, and MiSeq-analyzed the fungal community richness, diversity, and composition in plants collected from four replicate plots in each of two sites and two land use histories. Our data indicate that the root-associated fungal communities are indistinguishable between the plants from remnants and restored prairies. In contrast, the leaf-associated fungal communities are distinct and those from prairie remnants more diverse than those from restored prairies. While the restoration of the root-associated community composition seems to have been achieved, such outcomes were not obvious in the leaf-associated communities. As a result, we call for greater attention to evaluation of the restoration success of hidden biodiversity.

Restoration of highly disturbed ecosystems using mycorrhizal fungi to promote plant growth and survival has been used successfully in a variety of applications. However, there are several barriers that remain challenging for exceptionally degraded sites, such as those impacted by mining operations. Mine soils are typically characterized by low organic material, heavy metal contamination and low pH. The interactions between mycorrhizal fungi and soil amendments, such as biochar have the potential to increase soil pH, immobilize heavy metals while developing a stable store for carbon to promote native plant establishment in former mining sites. Although the possibility of synergism between mycorrhizal fungi and biochar is promising, few studies have tested this within the context of mining restoration. In a greenhouse experiment, I will evaluate the growth responses of two successionally distinct native plant species (Pseudotsuga menziesii, Elymus glaucus) in contaminated soil from the Formosa mine (Riddle, OR) amended with biochar, mycorrhizal fungi (from local inoculum), or a co-amendment of the two. I hypothesize that amending with biochar and mycorrhizal fungi individually will increase plant biomass compared to un-inoculated controls and that co-amended treatments will synergistically increase plant biomass compared to biochar or mycorrhizal fungi alone. Data on plant growth (e.g., height, leaf number, chlorophyll content) will be collected monthly for six months. Plants will be harvested for biomass and roots assessed for percent colonization by arbuscular and ectomycorrhizal fungi. Results from this study will be used to inform future field studies aimed at improving restoration efforts of abandoned mines.

Dead wood is an important component to the conservation of biodiversity in forests. When left to decompose on the forest floor, it protects soil from erosion, promotes nutrient cycling, and provides a unique ecological niche for decomposers such as fungi. The species richness of dead-wood inhabiting fungi can therefore serve as an indicator of the overall health of the forest. Most species of dead-wood inhabiting fungi are cryptic and do not always produce visible fruiting bodies for study. The goal of this project to evaluate new techniques that can be used to quickly assess the diversity of undetectable species present in decomposing wood. Samples of tissue from trees in varying stages of decay were collected near Elizabeth river, NJ and mixed, then DNA was extracted, PCR of ITS gene marker performed and sent for Next Generation Sequencing (NGS). Resulting DNA sequences were analyzed using SCATA pipeline and fungal species or OTU identified based on NCBI database. The results are to demonstrate how many dead wood trunks would require NGS assessment in order to collect information on dead wood fungi representative for the whole location.  

The focus of our research is to evaluate the potential of Next Generation Sequencing (NGS) method in identification of dead wood fungal species from a specific location, such as an urban park. In North America the research on biodiversity of dead wood fungi is still developing and there is no complete database so far. For our project, 37 fruiting bodies of various dead wood fungi were collected from Ocean County Park during October 2017. The species were identified morphologically where possible, with the conformation by DNA-barcoding. For the barcoding, DNA was isolated from each individual fungal body using DNeasy PowerSoil kit, then amplified by PCR using ITS specific primers to obtain a fragment of ITS gene, which serves as a barcode in fungal identification. This fragment was sequenced, the resulting sequence was compared to the database of fungal ITS sequences in NCBI portal using BLAST and species identified. As a result, a local database of the species found in Ocean County Park was created. NGS sequencing was used as a method allowing metabarcoding of a mixed DNA sample. This method allows to identify species all at once, without individual sequencing. The efficiency and reliability of this method in comparison to the individual species identification is discussed.

Despite the important role of saprotrophic fungi in decomposing organic matter and recycling nutrients, the development and diversity of wood decomposing communities remains poorly understood. In a 7-year field experiment in a boreal forest in the midwestern United States, we studied decay dynamics in red pine (Pinus resinosa) and birch (Betula papyrifera) logs using high-throughput sequencing and wood physiochemical analyses (i.e. density loss, ratio of lignin loss relative to density loss, alkali solubility). The effect of bark and decaying position (above- or on-ground contact) in wood decomposition was included in the treatment design. In our previous study, the optimization of environmental DNA extraction from wood samples was performed to obtain high quality DNA for high-throughput sequencing. We hypothesized more brown rot fungal taxa in above-ground wood, with fewer white rot or ectomycorrhizal fungal taxa than ground contact logs, resulting in different decay rate and wood characterization. Faster decay might also occur with bark on, due to high moisture content maintenance. In addition, we hypothesize different bacterial community structures between brown rot and white rot, given brown rot diffuse depolymerization mechanisms and the possible role of bacteria in assisting lignin degradation. Overall, our results, spanning a long-term field experiment and over 600 hundred samples, will provide a comprehensive view of wood decomposing community dynamics and interactions in complex natural ecosystems.

This study analyzes fungal diversity across varying stages of decay in dead aspen (Populus grandidtata) trees in a northern temperate forest. Populus grandidentata is a dominant early colonizing tree species in temperate forests across the Midwest since post deforestation due to extensive logging practices in the 19^th and 20^th centuries. Over the course of the coming decades P. grandidentata is beginning to naturally senesce as they are replaced by mid-successional tree species. This is particularly important as coarse woody debris (CWD) is one of the largest precursors to soil organic matter (SOM). Currently CWD contributes only a small portion of the total ecosystem C pool and respiration via the saprotrophs that are actively decomposing the wood. However, as succession in many forests progresses and as disturbances are predicted to increase, the effect of decomposing CWD on ecosystem C fluxes is expected to increase as well. Here we aim to answer two questions: 1) How do trends in fungal community structure differ along a decomposition gradient in Populus grandidenta, from standing dead to highly decomposed wood; and 2) how these differences, if any, are related to altered chemistry of the various stages of decomposing Populus logs.  

Anthropogenic activities such as fossil fuel combustion have increased the availability of reactive nitrogen (N) in terrestrial ecosystems. In many ecosystems, exogenous N addition has been shown to increase soil carbon (C) storage, and this response is sometimes associated with increased retention of lignin-derived compounds within the soil organic matter (SOM) and reductions in the abundance and activity of decomposer fungi in the phylum Basidiomycota. White- and brown-rot wood-degrading fungi within this phylum are important decomposers of lignocellulose, but use distinct metabolic mechanisms (synthesis of class II peroxidases within white-rot fungi vs. oxidative Fenton chemistry within brown-rot fungi) to gain energy from plant tissue. These processes may respond differently to N addition, in ways that depend on the lignin content of the substrate being decomposed. We present results from a laboratory decomposition experiment evaluating the influence of N fertilization on brown- and white-rot decomposition of Arabidopsis thaliana litter with high or low lignin. Decomposer activity of brown rot fungi was suppressed by low-lignin substrates. We also analyzed biomass, extracellular enzyme activity, and CO₂ respiration responses of five fungal taxa with variable metabolic strategies (Gloeophyllum traebum, Postia placenta, Schizophyllum commune, Trametes hirsute, and Pycnoporus cinnabarinus) to N addition treatments. The results provide context for understanding the functional mechanisms by which distinct microbial communities respond to N addition and provides opportunities to understand how these processes may confer changes in SOM composition and chemistry.

Over the past century, the frequency and intensity of hurricanes had increased on the Caribbean; resulting on deforestation and altering natural abiotic conditions. Therefore, it has challenged the stability of forest biogeochemical processes mediated by local microbial communities. Diverse bacteria and fungi driving resiliency and resistance of the forest ecosystem have disassembled by this biological disturbance. Uncovering how bacteria and fungi communities interact with ecosystems drivers under natural disturbances could help elucidate processes to help restore forest conditions. For this, a canopy trimming experiment, that simulated the pass of a hurricane, has been done at Luquillo experimental forest in Puerto Rico. It was designed to understand the effect of canopy opening and debris deposition at forest floor on microbial communities. Our objective is to determine temporal heterogeneity of bacterial and fungi communities in response to detritus deposition of simulated hurricane effect. Two treatments are considered: with and without detritus deposition trimmed from the local canopy. Soil samples were collected from plots, at various times for a period of two years. Total genomic DNA was extracted for the amplification of 16S rDNA and ITS to characterize bacterial and fungal communities using Terminal Restriction Fragment Length Polymorphisms. Bacteria was homogeneous over time for the same plot suggesting microbial succession in which rare microbiota became more prevalent over time. Two-Way PERMANOVA demonstrated significant differences through time and treatment (p=0.99) for the soil fungal and bacterial. Fungal and bacterial communities were heterogeneous among the treatments and through time. 

White rot fungi gained the capacity to degrade lignocellulose approximately 295 million years ago when they adapted oxidative enzymes to metabolize lignin. Since then, brown rot fungi have evolved a carbohydrate-selective, two-step mechanism, controlled through differentially expressed genes, that shortcuts lignin removal. In this mechanism, a reactive oxygen species (ROS) system is used to ‘loosen’ plant cell walls followed by the enzymatic hydrolysis of carbohydrates. It is still unclear how brown rot fungi regulate this seemingly ‘chaotic’ ROS system and avoid damaging their own enzymes and hyphae. Specifically, the process that turns ROS pathways on has not yet been identified, despite assumptions of an inducible mechanism. Many studies have suggested that the presence of either lignin or hemicellulose may initiate brown rot decay, but this has not been clearly shown experimentally. I propose to capture the earliest stages of brown rot decay and identify the ROS induction mechanism by using directional fungal growth on wood wafers to track decay progress – creating a space-for-time map of decay along the wafer – and analyzing the whole transcriptome at the incipient stage of decay. I also propose to study how wood components induce the expression of ROS-linked genes and enable brown rot decay by examining decay dynamics and ROS-linked gene expression on modified substrates, such as mutant strains of Arabidopsis thaliana. Understanding the decay mechanisms of brown rot fungi offers potential to harness those pathways for biotechnology applications as well as to make better predictions about the fate of carbon stored in wood.

Wetlands represent only ~7% of Earth’s surface but contain ~30% of global carbon (C) stores. However, wetland C storage capacity is sensitive to nutrient enrichment from anthropogenic sources such as excess fertilizer use and burning of fossil fuels. Whether a wetland is a C sink or source is primarily driven by rates of fungal and bacterial decomposition of soil organic matter. We can gain insights into mechanisms that determine C storage or loss by understanding how microbial metabolism changes in response to nutrient enrichment. Using a long-term fertilization and disturbance experiment, we examine how nutrient enrichment effects both bacterial and fungal composition and associated soil metabolic profiles. In a previous study, results revealed a distinct shift in bacterial community composition, an increase in bacterial diversity and copiotrophic species, and an increase in soil C in fertilized plots. To investigate how nutrient additions influence fungal and bacterial community composition, metabolic profiles, and microbial plant growth promotion, we compared microbial community structure-function relationships in nutrient enriched vs. ambient plots. We hypothesize a decrease in fungal species and increase in bacterial species diversity and higher metabolic diversity within fertilization treatments. Preliminary results suggest microbial communities from fertilized plots have greater substrate use diversity and faster substrate use rates. Over time, nutrient enrichment of historically low nutrient ecosystems alters C storage potential due to shifts in metabolic diversity of the microbial community. This work will identify substrates and microbial community members to target to further study the mechanisms driving C cycling in wetlands.

Despite their short duration, hurricanes can have strong impact on forests. Hurricane winds can damage trees and subsequently affect forest dynamics and associated ecosystem functions. Climate change is suspected to increase hurricane intensity. In this context, predicting hurricanes damage on trees populations could have a crucial economic importance as well as conservation value. In tropical forests, topography, tree species characteristics (i.e. diameter, growth rate, wood density and biogeographic origin) and tree health have been identified as potential predictors of trees resistance to hurricane. Tree health can be impacted by pathogenic microbes and notably stem wood-inhabiting fungi. Consequently, we speculate that trees infected by pathogenic fungi may be less resistant to hurricane winds by comparison to non-infected trees. To evaluate the potential role of wood-inhabiting fungal communities on the resistance of trees to hurricanes, we sampled logs of nineteen tree species damaged by the hurricane Maria in a Puerto Rican dry tropical forest in 2017. We categorized wood logs depending of two types of hurricane damage; uprooted trees and trees that in which stems were broken. We extracted fungal environmental DNA of logs corresponding to 109 samples. Fungal community composition were assessed using high-throughput DNA metabarcoding. We hypothesize that trees with the snapped stem break pattern will harbor fungal communities enriched in pathogenic fungi relatively to uprooted trees. Finally, we will discuss the utility of microbial communities as disturbance predictor in the light of the emergence of new sequencing technologies.

Due to the occurrence of hurricane Maria possible foreign pathogenic fungi could be affecting native tree species. Symptomatology has been observed for leaves, fruits, flowers and seeds, depending on the tree species.  We hypothesize the presence of pathogenic fungi are the main cause of the symptomatology. Species studied were Casearia arborea, Prestoea acuminate var. montana, Guarea guidonia and Dacryodes excelsa. For the leaves of Dacryodes excelsa, the symptoms found were the following: small necrotic circular lesion with a yellow halo, irregular lesion, and margin and apex necrosis on leaves. Also, fruits presented necrotic lesions, formation of pycnidia and shield softening. Furthermore, Guarea guidonia presented symptoms like partial necrosis with extended halo, regular and irregular necrotic circular lesions at the margin of the leave. Seeds and fruits were covered by mycelium. On the other hand, Casearia arborea presented flower necrosis and abortion and Prestoea acuminata var. montana, presented a pink mycelium in seeds collected from the soil. Using microscopy and morphological features, diverse pathogens were found, including: Phoma sp., Phomopsis sp., Pestalotiopsis sp., Colletrotrichum sp., Nigrospora sp. and Botryosphaeriaceae family. Thirteen anamorphs were observed to be pathogens. Koch’s postulates were performed to identify the causal agents for the symptoms. Future tests will include DNA extraction and PCR amplification with different gene regions depending on the pathogens to corroborate homology identity. Sequencing will be elaborate with Sanger sequencing method with Big Dye Terminator.

The edible wild mushrooms are cultivated and consumed worldwide. In Sri Lanka, only few such species have been accurately identified and domesticated. This study aims the molecular based identification of three edible mushroom species and development of protocols for their experimental domestication.  Mushrooms species collected were tentatively identified based on morphological characters of fruiting bodies and culture characteristics in four different media.  Genomic DNA were extracted and PCR amplifications of ribosomal Internal Transcribed Spacer (ITS) region were carried out. Initial molecular identification done by NCBI’s BLAST similarity search and phylogenetic analyses confirmed the isolates belong in Lentinus sajor-caju, L. tuber-regium, and L. squarrosulus. Out of rice, corn and corn grits with millet mixture that were tested as mother spawn media, corn showed the highest mycelial colonization density. Out of rubber saw dust (RSD), mango saw dust (MSD) and MSD with corn cobs (CC) mixture that were utilized as growth media, all three species showed the highest colonization rate on MSD. Successful fruiting body formations were observed after 88 and 74 days respectively for L. sajor-caju and L. tuber-regium on RSD.  Lentinus squarrosulus produced fruiting bodies on MSD after 49 days. L. squarrosulus fructified the most on MSD whereas L. tuber-regium showed the highest yields on RSD. All mushrooms tested show the antioxidant properties as assessed by a quantitative Ferric Reducing Antioxidant Power (FRAP) test. This is the first successful domestication effort of   L. sajor-caju and L. tuber-regium in Sri Lanka provided with freshly collected and DNA barcoded strains.

Having the designation of being biodegradable or compostable is a valuable marketing asset, especially given the push for ecofriendly and renewable products. Currently, some companies are utilizing natural organisms, such as fungi, to create more sustainable biobased materials. These materials lack the history of wood and other biomaterial testing, and they reflect novel substrates whose decomposition is of interest in other contexts (example: fungal necromass in carbon cycling). Here, we exposed a fungus-based biomaterial to a range of wood-degrading fungi and quantified various decay parameters, including mass loss, pH, and carbon fractions (lignin insolubles via gravimetric; acid-solubles via HPLC). To do this, we used a block of spent fungal biomass product developed in solid state and placed in a soil block jar with a pre-established hyphal lawn of a known decay fungi for 8 weeks. Nine fungi were tested, including white rot fungi, brown rot fungi, a 'grey rot' fungus (Schizophyllum commune), and one soil saprotroph. We found that the greatest mass loss was caused by the brown rot fungi, followed by white rot fungi, similar to rates and patterns typical in wood decay trials with these fungi. The wafer pH and carbon fractions are currently being assessed to track the losses of certain components in the fungal biomaterials, but preliminary acid-insolubles data indicate some disparities within rot types in removal efficiencies/patterns. 

Wood vinegar is a byproduct of wood pyrolysis, a process used to produce biochar, which has significant agricultural value. In its pure state, the wood vinegar is too acidic and concentrated for direct application as agricultural or compost amendments. It does, however, contain a rich diversity and high concentration of valuable organic compounds, which have proven useful and integral in a wide variety of applications (agricultural, industrial, medical, food-related, etc.). Because wood vinegar is generally around 30-40% of the average weight of charcoal/biochar produced during wood pyrolysis, it is valuable to develop more efficient methods of utilizing wood vinegar and making it more directly applicable. Dilutions of wood vinegar produced at Living Web Farms (Mills River, North Carolina) have already shown natural growth of fungi and bacteria species in mostly anaerobic conditions. These naturally occurring "SCOBY (Symbiotic Community of Bacteria and Yeast)-like" cultures were further propagated into media containing various dilutions of wood vinegar (1%, 5%, 20%, and 50%). These same dilutions of media containing wood vinegar were also inoculated with Pleurotus ostreatus, Trametes versicolor, Cordyceps militaris, Ganoderma lucidum, Aspergillus oryzae. Growth rates, metabolic efficiency, pH levels were recorded over a trial period of six weeks. The most successful species with the highest overall growth rates and metabolic efficiency was Aspergillus oryzae. These wood vinegar biological solutions can prospectively be used as an effect inoculant to improve crop vitality, soil health, and compost productivity.  

Mushrooms have been consumed by humans for many thousands of years. Fruiting bodies can be both aromatically enticing and pleasant to taste, but it has long been recognized that only select species of mushrooms are suitable for consumption. There are adverse health affects of consuming misidentified, inedible mushrooms ranging from mild illness to death. Many food products that have mushrooms as an ingredient use mushrooms that are commercially grown, avoiding these potential health hazards. There are many edible mushrooms that are unable or impractical to be grown commercially yet these mushrooms are still able to be harvested, sold and purchased as food. These products are often sold with the ingredient label of ‘wild mushrooms’, although in some cases the labels do have some level of specificity. For this study we used metagenomic analysis techniques to identify the species of mushrooms that were sold under the label of wild mushrooms in eight different food products. Some of the food products that were sold as porcini and bolete were shown to contain mushrooms that differed from their label, and in some cases the mushrooms present are ones that are not typically considered edible. This study demonstrates a lack of oversight in the labeling of wild collected mushrooms and shows a potential health hazard to consumers.

A P. ostreatus strain, appreciated as food and for the production of nutraceuticals, was grown on a commercial substrate, dried at low temperature (<40°C) and grinded in order to produce a mushroom powder. The bioactivity of the water extract conserved at 4°C in the dark was then assessed on F. graminearum, F. culmorum and F. musae at different time points from production (4 hrs, 40 days, 4 months). Moreover, the effect of the extracts on trichothecene type B production was measured exploiting a F. graminearum isolate expressing GFP-tagged trichodiene synthase. This allowed to monitor the first step of toxin production using a microplate fluorimeter. 

While mycelial growth of F. graminearum and F. culmorum was completely blocked at 3 mg/ml, mycelial growth of F. musae was inhibited at 90%. MIC50 was measured for F. graminearum and F. culmorum at 300 micrograms/mL. A loss of the bioactivity of P. ostreaus water extract on fungal growth was observed at 40 days (-30%) and of a further -30% at 4 months. A preliminary study on the biological activities of the extract identified a strong protease activity associated to low molecular weight proteins. Their bioactivity decreased over storage time in accordance with  a decreased proteolytic activity.

The P. ostreatus extract modulates trichothecene production independently from the protease activity, even at concentration where no mycelium inhibition was observed (down to 0.75 micrograms/mL).

Studies on the genetic determinants of the protease activity as well as the compounds able to modulate trichothecene production are ongoing.

Selenium (Se) is essential in moderate doses to most organisms for the production of selenoproteins, but elevated levels in the environment can cause detrimental biological repercussions. Selenium bioavailability is highly linked to its oxidation state in the environment, and the Se forms common in oxic surface environments are Se(VI) and Se(IV), which are highly water soluble and bioavailable. Current strategies for removing Se from wastewaters are expensive and inefficient, but some environmentally ubiquitous Ascomycetes remove Se from solution by aerobic reduction of dissolved Se(+IV/VI) to form solid Se(0) and volatile Se(-II). To this end, culture experiments with Se-reducing fungi, Alternaria alternata and Alternaria strain “F7”, an isolate from Se-contaminated soil, were performed to quantify total Se removed from solution in two Se-contaminated Minnesota wastewaters. In parallel, a second set of cultures were assembled with nutrient-lean culture media (“AY”) and 2000 µg/L or 25 µg/L Se(IV or VI) to reflect the wastewaters’ Se content. In AY, Se(IV) is quickly removed from solution by 7 days and concentrations remain low with time. Conversely, some Se(VI) is removed from solution by 20 days, then concentrations increase to near-initial values, suggesting that Se is recycled back to the media. In the wastewaters, total Se is also removed from solution by 20 days and is recycled back to the media ~8 days later. This experiment provides essential information about fungal mechanisms of Se reduction and information for engineering an efficient aerobic Se bioremediation strategy.

Mitotic recombination is a universal phenomenon in diploid genotypes. Regardless of being widespread and occurring at a higher rate than mutation, the impact of mitotic recombination on evolution is poorly understood. Previous research has shown that replicate populations of a highly heterozygous genotype of S. cerevisiae (cross between a European clinical strain and a wild Chinese strain) grown in a high salt environment have undergone parallel mitotic recombination events. Lines evolved from a heterozygous ancestor with a clinical and wildtype allele frequently become homozygous for the allele derived from the clinical parent, a loss of heterozygosity event. To test the effect of this event, CRISPR-Cas9 technology was utilized to create strains homozygous for the clinical or wildtype allele from the heterozygous strain using allele-specific guide RNAs. Strains homozygous for the clinical allele demonstrated a pronounced difference in growth rate compared to the homozygotes for the Chinese allele, indicating higher fitness in a high salt environment. Genotyping of all engineered strains has shown a variety of genotypes at linked loci, indicating the occurrence of complex crossing over events initiated by CRISPR double-strand DNA breaks. To introduce LOH specifically at a given locus, we are designing a CRISPR multiplexing protocol to create multiple chromosomal double-strand breaks. Multiplexing will be optimized to engineer either single locus or whole arm loss of heterozygosity events. With the development of this technique, it will become possible to study the specific fitness effects of mitotic recombination driven loss of heterozygosity events.