Transcriptomics of Temporal- versus Substrate-Specific Wood Decay in the Brown-Rot Fungus Fibroporia radiculosa.

Brown-rot fungi lack many enzymes associated with complete wood degradation, such as lignin-attacking peroxidases, and have developed alternative mechanisms for rapid wood breakdown. To identify the effects of culture conditions and wood substrates on gene expression, we grew Fibroporia radiculosa in submerged cultures containing Wiley milled wood (5 days) and solid wood wafers (30 days), using aspen, pine, and spruce as a substrate. The comparative analysis revealed that wood species had a limited effect on the transcriptome: <3% of genes were differentially expressed between different wood species substrates. The comparison between gene expression during growth on milled wood and wood wafer conditions, however, indicated that the genes encoding plant cell wall-degrading enzymes, such as glycoside hydrolases and peptidases, were activated during growth on wood wafers, confirming previous reports. On the other hand, it was shown for the first time that the genes encoding Fenton chemistry enzymes, such as hydroquinone biosynthesis enzymes and oxidoreductases, were activated during submerged growth on ground wood. This illustrates the diversity of wood-decay reactions encoded in fungi and activated at different stages of this process.

Diverse Xylaria in the Ecuadorian Amazon and their mode of wood degradation.

BACKGROUND: Xylaria is a diverse and ecologically important genus in the Ascomycota. This paper describes the xylariaceous fungi present in an Ecuadorian Amazon Rainforest and investigates the decay potential of selected Xylaria species. Fungi were collected at Yasuní National Park, Ecuador during two collection trips to a single hectare plot divided into a 10-m by 10-m grid, providing 121 collection points. All Xylaria fruiting bodies found within a 1.2-m radius of each grid point were collected. Dried fruiting bodies were used for culturing and the internal transcribed spacer region was sequenced to identify Xylaria samples to species level. Agar microcosms were used to assess the decay potential of three selected species, two unknown species referred to as Xylaria 1 and Xylaria 2 and Xylaria curta, on four different types of wood from trees growing in Ecuador including balsa (Ochroma pyramidale), melina (Gmelina arborea), saman (Samanea saman), and moral (Chlorophora tinctoria). ANOVA and post-hoc comparisons were used to test for differences in biomass lost between wood blocks inoculated with Xylaria and uninoculated control blocks. Scanning electron micrographs of transverse sections of each wood and assay fungus were used to assess the type of degradation present. RESULTS: 210 Xylaria collections were sequenced, with 106 collections belonging to 60 taxa that were unknown species, all with less than 97% match to NCBI reference sequences. Xylaria with sequence matches of 97% or greater included X. aff. comosa (28 isolates), X. cuneata (9 isolates) X. curta and X. oligotoma (7 isolates), and X. apiculta (6 isolates)., All Xylaria species tested were able to cause type 1 or type 2 soft rot degradation in the four wood types and significant biomass loss was observed compared to the uninoculated controls. Balsa and melina woods had the greatest amount of biomass loss, with as much as 60% and 25% lost, respectively, compared to the controls. CONCLUSIONS: Xylaria species were found in extraordinary abundance in the Ecuadorian rainforest studied. Our study demonstrated that the Xylaria species tested can cause a soft rot type of wood decay and with the significant amount of biomass loss that occurred within a short incubation time, it indicates these fungi likely play a significant role in nutrient cycling in the Amazonian rainforest.

Proteome of the Wood Decay Fungus Fomitopsis pinicola Is Altered by Substrate.

The brown rot fungus Fomitopsis pinicola efficiently depolymerizes wood cellulose via the combined activities of oxidative and hydrolytic enzymes. Mass spectrometric analyses of culture filtrates identified specific proteins, many of which were differentially regulated in response to substrate composition.

Grapevine trunk diseases of cold-hardy varieties grown in Northern Midwest vineyards coincide with canker fungi and winter injury.

Grapevine trunk diseases make up a disease complex associated with several vascular fungal pathogenic species. Surveys to characterize the composition of grapevine trunk diseases have been conducted for most major grape growing regions of the world. This study presents a similar survey characterizing the fungi associated with grapevine trunk diseases of cold-hardy interspecific hybrid grape varieties grown nearly exclusively in the atypical harsh winter climate of Northern Midwestern United states vineyards. From the 172 samples collected in 2019, 640 isolates obtained by culturing were identified by ITS sequencing and represent 420 sample-unique taxa. From the 420 representative taxa, opportunistic fungi of the order Diaporthales including species of Cytospora and Diaporthe were most frequently identified. Species of Phaeoacremonium, Paraconiothyrium, and Cadophora were also prevalent. In other milder Mediterranean growing climates, species of Xylariales and Botryosphaeriales are often frequently isolated but in this study they were isolated in small numbers. No Phaeomoniellales taxa were isolated. We discuss the possible compounding effects of winter injury, the pathogens isolated, and management strategies. Additionally, difficulties in researching and understanding the grapevine trunk disease complex are discussed.

New Findings on the Biology and Ecology of the Ecuadorian Amazon Fungus Polyporus leprieurii var. yasuniensis.

Polyporus leprieurii var. yasuniensis is a prolific wood-decay fungus inhabiting the forest floor of one of the most biodiverse places on earth, the Yasuní National Park in Ecuador. Basidiocarps and aerial rhizomorphs are commonly found growing on woody debris distributed along the floor of this forest ecosystem. Because of the extraordinary abundance of this fungus in the tropical rainforest, we carried out investigations to better understand the biological and ecological aspects contributing to its prolific distribution. Data on growth inhibition in paired competition studies with sixteen fungal isolates exemplifies defense mechanisms used to defend its territory, including pseudosclerotial plates and the development of a melanized rhizomorphic mat. Results of biomass loss on eleven types of tropical wood in microcosm experiments demonstrated the broad decay capacity of the fungus. In and ex situ observations provided information on how long rhizomorphs can prevail in highly competitive ecosystems as well as stressful conditions in the laboratory. Finally, high concentrations of metal ions occur on rhizomorphs as compared to colonized wood. Sequestration of metal ions from the environment by the melanized rhizomorphs may offer protection against competitors. The development of melanized rhizomorphs is key to find and colonize new substrates and resist changing environmental conditions.

Taxonomy of the major rhizomorphic species of the "Melanopus group" within Polyporaceae in Yasuní National Park, Ecuador.

Yasuní National Park in Ecuador is one of the most biodiverse places on earth. The fungi in this tropical rainforest are also diverse but have received little research attention. This research paper focuses on an important group of fungi in the family Polyporaceae and examines the genera Polyporus, Atroporus, and Neodictyopus that form aerial melanized cord-like structures called rhizomorphs. Phylogenetic analyses, macro and micromorphological descriptions of basidiomata and rhizomorphs, as well as cultural characterization were completed to better understand these ecologically important fungi. Here we describe four new species: Atroporus yasuniensis, Atroporus tagaeri, Neodictyopus sylvaticus, and Polyporus taromenane, and a new variety Polyporus leprieurii var. yasuniensis. The information presented in this study adds important new knowledge about the unusual rhizomorph producing fungi found in Yasuní National Park, Ecuador and other tropical rainforests.

Retracted and Republished from: "Substrate-Specific Differential Gene Expression and RNA Editing in the Brown Rot Fungus Fomitopsis pinicola"

Wood-decaying fungi tend to have characteristic substrate ranges that partly define their ecological niche. Fomitopsis pinicola is a brown rot species of Polyporales that is reported on 82 species of softwoods and 42 species of hardwoods. We analyzed gene expression levels of F. pinicola from submerged cultures with ground wood powder (sampled at 5 days) or solid wood wafers (sampled at 10 and 30 days), using aspen, pine, and spruce substrates (aspen was used only in submerged cultures). Fomitopsis pinicola expressed similar sets of wood-degrading enzymes typical of brown rot fungi across all culture conditions and time points. Nevertheless, differential gene expression was observed across all pairwise comparisons of substrates and time points. Genes exhibiting differential expression encode diverse enzymes with known or potential function in brown rot decay, including laccase, benzoquinone reductase, aryl alcohol oxidase, cytochrome P450s, and various glycoside hydrolases. Comparing transcriptomes from submerged cultures and wood wafers, we found that culture conditions had a greater impact on global expression profiles than substrate wood species. These findings highlight the need for standardization of culture conditions in studies of gene expression in wood-decaying fungi. IMPORTANCE All species of wood-decaying fungi occur on a characteristic range of substrates (host plants), which may be broad or narrow. Understanding the mechanisms that allow fungi to grow on particular substrates is important for both fungal ecology and applied uses of different feedstocks in industrial processes. We grew the wood-decaying polypore Fomitopsis pinicola on three different wood species­aspen, pine, and spruce­under various culture conditions. We found that F. pinicola is able to modify gene expression (transcription levels) across different substrate species and culture conditions. Many of the genes involved encode enzymes with known or predicted functions in wood decay. This study provides clues to how wood-decaying fungi may adjust their arsenal of decay enzymes to accommodate different host substrates.

Fungi associated with galleries of the emerald ash borer.

The emerald ash borer (EAB) is an exotic forest pest that has killed millions of ash trees in the United States and Canada, resulting in an ecological disaster and billions of dollars in economic losses of urban landscape and forest trees. The beetle was first detected in Michigan in 2002 and has spread through much of the Eastern and Midwestern U.S., reaching Minnesota in 2009. Since then, it has spread across the state and poses a great risk to the more than 1 billion ash trees in Minnesota. The larval stage of EAB creates wounds on trees as they feed on the inner bark, causing disruption of water and sap flow that results in tree death. The fungal community associated with EAB larval galleries is poorly understood and the role these fungi may play in tree death is not known. This study describes fungi isolated from EAB larval galleries sampled throughout the main geographic areas of Minnesota where ash is affected by EAB. Fungal cultures were identified by extracting genomic DNA and sequencing the ITS region of the rDNA. Results from 1126 isolates reveal a diverse assemblage of fungi and three functional guilds comprised of canker pathogens, wood decay, and entomopathogenic fungi. The most common canker-associated genera were Cytospora followed by Phaeoacremonium, Paraconiothyrium, Coniothyrium, Nectria, Diplodia, and Botryosphaeria. Fungi in the Basidiomycota were nearly all wood decay causing fungi and many were species of pioneer colonizing genera including Sistotrema, Irpex, Peniophora, Phlebia and Ganoderma. Some of these fungi seriously affect urban trees, having the potential to cause rapid wood decay resulting in hazardous tree situations. Several entomopathogenic genera with the potential for biological control of EAB were also isolated from galleries. Purpureocillium was the most commonly isolated genus, followed by Beauveria, Clonostachys, Lecanicillium, Akanthomyces, Cordyceps, Microcera, Tolypocladium, and Pochonia. The results identify important fungal functional guilds that are occupying a new niche in ash trees resulting from EAB and include fungi that may accelerate decline in tree health, increase hazard tree situations, or may provide options for biological control of this destructive invasive insect.

Fungal mycelial mats used as textile by indigenous people of North America.

The indigenous people of the United States and Canada long have used forest fungi for food, tinder, medicine, paint, and many other cultural uses. New information about historical uses of fungi continues to be discovered from museums as accessions of fungi and objects made from fungi collected over the last 150+ years are examined and identified. Two textiles thought to be made from fungal mats are located in the Hood Museum of Art, Dartmouth College, and the Oakland Museum of California. Scanning electron microscopy and DNA sequencing were used to attempt to identify the fungus that produced the mats. Although DNA sequencing failed to yield a taxonomic identification, microscopy and characteristics of the mycelial mats suggest that the mats were produced by Laricifomes officinalis. This first report of fungal mats used for textile by indigenous people of North America will help to alert museum curators and conservators as well as mycological researchers to their existence and hopefully lead to more items being discovered that have been made from fungal fabric.

Fungi attacking historic wood of Fort Conger and the Peary Huts in the High Arctic.

Historic wooden structures in Polar Regions are being adversely affected by decay fungi and a warming climate will likely accelerate degradation. Fort Conger and the Peary Huts at Lady Franklin Bay in northern Ellesmere Island are important international heritage sites associated with early exploration in the High Arctic. Fort Conger, built by Adolphus Greely and expedition members during the First International Polar Year in 1881, was dismantled and used by Robert Peary and his expedition crew in the early 1900's to build several smaller shelters. These historic structures remain at the site but are deteriorating. This investigation examines the fungi associated with wood decay in the historic woods. Soft rot was observed in all 125 wood samples obtained from the site. The major taxa found associated with the decayed wood were Coniochaeta (18%), Phoma (13%) Cadophora (12%), Graphium (9%), and Penicillium (9%) as well as many other Ascomycota that are known to cause soft rot in wood. Micromorphological observations using scanning electron microscopy of historic wooden timbers that were in ground contact revealed advanced stages of type I soft rot. No wood destroying Basidiomycota were found. Identification of the fungi associated with decay in these historic woods is a first step to better understand the unusual decomposition processes underway in this extreme environment and will aid future research to help control decay and preserve this important cultural heritage.

Fungal Diversity in Multiple Post-harvest Aged Red Pine Stumps and Their Potential Influence on Heterobasidion Root Rot in Managed Stands Across Minnesota.

Thinning operations that occur in managed red pine (Pinus resinosa) stands, create tree stumps that can serve as a habitat for fungi, especially Heterobasidion irregulare, the cause of a serious root disease. Different fungi can colonize stumps early and the community of fungi can change over time as initial fungal species become replaced. Samples were collected from both the native and non-native range of red pine from stumps that were cut at different time periods. Stumps that were harvested at 0-1, 2-3, 5-6, and 10-12 years before sampling were used to provide data on the diversity of fungi that colonize tree stumps and how these communities can change over time as well as how they influence colonization of H. irregulare. Traditional culturing methods and Illumina MiSeq sequencing were used to identify the fungi in the samples. Of particular interest was Phlebiopsis gigantea, which can colonize cut stumps and prevent H. irregulare from becoming established. Overall, P. gigantea was the most abundant fungus isolated and sequenced via Illumina MiSeq. Results show that Phlebiopsis gigantea was isolated from 90% of all stumps sampled for sites harvested within 3 years of sampling in the native range of red pine compared to 33% in the non-native range. For Illumina MiSeq, 5,940 total amplicon sequence variants (ASVs) were detected. P. gigantea represented 14% of the total reads and composed 19% of the reads in the native range and 8% in non-native range of red pine. Furthermore, P. gigantea represented 38% of the reads for stumps that were harvested within 3 years of sampling in the native range of red pine compared to 14% in the non-native range. These results help demonstrate that a higher amount of P. gigantea is present in the native range of red pine and could be acting as a native biological control agent. Additional fungi, including Resinicium bicolor, Hypochnicium cremicolor, Leptographium spp., and others identified at different cutting times are also discussed. Finally, different diversity indices revealed similar, but slightly higher diversity for southern sites via Shannon and Simpson Diversity indices. Beta diversity demonstrated a similar species composition in stumps harvested at different times with these stumps being grouped together based on harvesting years.

Fungal attack on archaeological wooden artefacts in the Arctic-implications in a changing climate.

Climate change is expected to accelerate the microbial degradation of the many extraordinary well-preserved organic archaeological deposits found in the Arctic. This could potentially lead to a major loss of wooden artefacts that are still buried within the region. Here, we carry out the first large-scale investigation of wood degradation within archaeological deposits in the Arctic. This is done based on wooden samples from 11 archaeological sites that are located along a climatic gradient in Western Greenland. Our results show that Ascomycota fungi are causing extensive soft rot decay at all sites regardless of climate and local environment, but the group is diverse and many of the species were only found once. Cadophora species known to cause soft rot in polar environments were the most abundant Ascomycota found and their occurrence in native wood samples underlines that they are present locally. Basidiomycota fungi were also present at all sites. In the majority of samples, however, these aggressive and potentially very damaging wood degraders have caused limited decay so far, probably due to unfavorable growth conditions. The presence of these wood degrading fungi suggests that archaeological wooden artefacts may become further endangered if climate change leads to more favorable growth conditions.

Using Wood Rot Phenotypes to Illuminate the "Gray" Among Decomposer Fungi.

Wood-decomposing fungi use distinct strategies to deconstruct wood that can significantly vary carbon release rates and fates. White and brown rot-type fungi attack lignin as a prerequisite to access carbohydrates (white rot) or selectively remove carbohydrates (brown rot). Soft rot fungi use less well-studied mechanisms to deconstruct wood (e.g., cavitation and erosion). These fungi often co-exist in nature, creating a balance in carbon turnover that could presumably "tip" in a changing climate. There is no simple genetic marker, however, to distinguish fungi by rot types, and traditional black and white distinctions (brown and white, in this case) cannot explain a spectrum of "gray" carbon loss possibilities. In this study, we tested 39 wood-degrading fungal strains along this spectrum of rot types. We tracked wood mass loss and chemical changes in aspen blocks in early- to mid-decay stages, including three signatures of fungal nutritional mode measured from wood rather than from fungus: dilute alkali solubility, water-soluble monosaccharides, and lignin loss (%) relative to density loss (%) (L/D). Results were then plotted relative to rot types and correlated with gene counts, combining new data with past results in some cases. Results yielded a novel distinction in soluble monosaccharide patterns for brown rot fungi, and reliable distinctions between white and brown rot fungi, although soft rot fungi were not as clearly distinguished as suggested in past studies. Gene contents (carbohydrate-active enzymes and peroxidases) also clearly distinguished brown and white rot fungi, but did not offer reliable correlation with lignin vs. carbohydrate selectivity. These results support the use of wood residue chemistry to link fungal genes (with known or unknown function) with emergent patterns of decomposition. Wood signatures, particularly L/D, not only confirm the rot type of dominant fungi, but they offer a more nuanced, continuous variable to which we can correlate genomic, transcriptomic, and secretomic evidence rather than limit it to functional categories as distinct "bins."

Diverse subterranean fungi of an underground iron ore mine.

Mines and caves are unusual ecosystems containing unique fungi and are greatly understudied compared to other environments. The Soudan Mine in Tower, MN, an iron ore mine that closed in 1963 after operating for 80 years, was sampled to explore fungal diversity and to investigate taxa that tolerate heavy metals for potential bioprocessing technologies or as sources of bioactive molecules for drug discovery and possible biocontrol for white-nose syndrome (WNS) of bats. The mine is 714 m deep, has 18 levels and contains large quantities of wooden timbers, in contrast to many other oligotrophic subterranean environments. Fungi were cultured from samples and the ITS region was sequenced for identification and phylogenetic analysis. Results show Ascomycota are the dominant fungi followed by Basidiomycota and Mucoromycota. Out of 164 identified taxa, 108 belong to the Ascomycota and 26 and 31 to Basidiomycota and Mucoromycota, respectively. There are also 46 taxa that do not match (<97% BLAST GenBank identity) sequenced fungal species. Examples of the most commonly isolated Ascomycota include Scytalidium sp., Mariannaea comptospora, Hypocrea pachybasidioides, Oidiodendron griseum and Pochonia bulbillosa; Basidiomycota include Postia sp., Sistotrema brinkmannii, Calocera sp., Amylocorticiellum sp.; Mucoromycota include Mortierella parvispora, M. gamsii, M. hyaline, M. basiparvispora and Mortierella sp. Unusual growth forms were also found including large quantities of black rhizomorphs of Armillaria sinapina and white mycelial cords of Postia sp. mycelium, as well as Pseudogymnoascus species growing over large areas of mine walls and ceiling. The mine environment is a relatively extreme environment for fungi, with the presence of high levels of heavy metals, complete darkness and poor nutrient availability. Several genera are similar to those isolated in other extreme environments but phylogenetic analyses show differences in species between these environments. Results indicate this subterranean environment hosts a wide diversity of fungi, many of them not found in above ground environments.

Antifungal Norditerpene Oidiolactones from the Fungus Oidiodendron truncatum, a Potential Biocontrol Agent for White-Nose Syndrome in Bats.

White-nose syndrome (WNS) is a devastating disease of hibernating bats caused by the fungus Pseudogymnoascus destructans. We obtained 383 fungal and bacterial isolates from the Soudan Iron Mine, an important bat hibernaculum in Minnesota, then screened this library for antifungal activity to develop biological control treatments for WNS. An extract from the fungus Oidiodendron truncatum was subjected to bioassay-guided fractionation, which led to the isolation of 14 norditerpene and three anthraquinone metabolites. Ten of these compounds were previously described in the literature, and here we present the structures of seven new norditerpene analogues. Additionally, this is the first report of 4-chlorophyscion from a natural source, previously identified as a semisynthetic product. The compounds PR 1388 and LL-Z1271α were the only inhibitors of P. destructans (MIC = 7.5 and 15 µg/mL, respectively). Compounds were tested for cytotoxicity against fibroblast cell cultures obtained from Myotis septentrionalis (northern long eared bat) and M. grisescens (gray bat) using a standard MTT viability assay. The most active antifungal compound, PR 1388, was nontoxic toward cells from both bat species (IC50 > 100 µM). We discuss the implications of these results in the context of the challenges and logistics of developing a substrate treatment or prophylactic for WNS.

Assessment of biodegradation in ancient archaeological wood from the Middle Cemetery at Abydos, Egypt.

Abydos is a large, complex archaeological site located approximately 500 km south of Cairo in Upper Egypt. The site has served as a cemetery for thousands of years and is where most of the Early Dynastic royal tombs are located. North Abydos includes the Middle Cemetery and the North Cemetery, which are separated from each other by a wadi. The Middle Cemetery was the burial ground for important Sixth Dynasty (2407-2260 BC) officials and over time for thousands of elite and non-elite individuals as well. Excavations at the core area of the Old Kingdom mortuary landscape have revealed many culturally important wooden objects but these are often found with extensive deterioration that can compromise their preservation. The objectives of this study were to characterize the biodegradation that has taken place in excavated wooden objects, elucidate the type of wood degradation present, obtain information on soil properties at the site and identify fungi currently associated with the wood and soils. Light and scanning electron microscopy studies were used to observe the micromorphological characteristics of the wood, and culturing on different media was done to isolate fungi. Identification of the fungi was done by examining morphological characteristics and extracting rDNA from pure cultures and sequencing the ITS region. Wooden objects, made from Cedrus, Juniperus and Acacia as well as several unidentified hardwoods, were found with extensive degradation and were exceedingly fragile. Termite damage was evident and frass from the subterranean termites along with sand particles were present in most woods. Evidence of soft rot attack was found in sections of wood that remained. Fungi isolated from wood and soils were identified as species of Aspergillus, Chaetomium, Cladosporium, Fusarium, Penicillium, Stemphylium Talaromyces and Trichoderma. Results provide important information on the current condition of the wood and gives insights to the identity of the fungi in wood and soils at the site. These results provide needed information to help develop conservation plans to preserve these degraded and fragile wooden objects.