Qualitative metabolomics-based characterization of a phenolic UDP-xylosyltransferase with a broad substrate spectrum from Lentinus brumalis.

Wood-decaying fungi are the major decomposers of plant litter. Heavy sequencing efforts on genomes of wood-decaying fungi have recently been made due to the interest in their lignocellulolytic enzymes; however, most parts of their proteomes remain uncharted. We hypothesized that wood-decaying fungi would possess promiscuous enzymes for detoxifying antifungal phytochemicals remaining in the dead plant bodies, which can be useful biocatalysts. We designed a computational mass spectrometry-based untargeted metabolomics pipeline for the phenotyping of biotransformation and applied it to 264 fungal cultures supplemented with antifungal plant phenolics. The analysis identified the occurrence of diverse reactivities by the tested fungal species. Among those, we focused on O-xylosylation of multiple phenolics by one of the species tested, Lentinus brumalis. By integrating the metabolic phenotyping results with publicly available genome sequences and transcriptome analysis, a UDP-glycosyltransferase designated UGT66A1 was identified and validated as an enzyme catalyzing O-xylosylation with broad substrate specificity. We anticipate that our analytical workflow will accelerate the further characterization of fungal enzymes as promising biocatalysts.

Cyclohumulanoid Sesquiterpenes Induced by the Noncompetitive Coculture of Phellinus orientoasiaticus and Xylodon flaviporus.

Microbial cocultivation has been applied as a strategy to induce the biosynthesis of specialized metabolites. However, most previous studies have focused on competitive interactions between test strains. During our LC-MS-based chemical screening of randomized cocultures of Basidiomycetous fungi, we discovered that the coculture of Phellinus orientoasiaticus (Hymenochaetaceae) and Xylodon flaviporus (Schizoporaceae) induces multiple metabolites, although they did not show any competitive morphology. Targeted isolation yielded three new sesquiterpenes (1-3) along with five known analogues (4-8). The structures of the isolates were determined by MS and NMR experiments as well as electronic circular dichroism analysis. LC-MS analysis suggested that cyclohumulanoids of illudane-, sterpurane-, and tremulane-type scaffolds (1-7) were produced by P. orientoasiaticus, whereas a drimane-type sesquiterpene (8) was produced by X. flaviporus. None of the isolates exhibited antifungal activity or cytotoxicity, and compounds 1-7 exhibited NO production of LPS-treated RAW276.4 cells in a range of 15.9% to 38.0% at 100 µM.

Plastic-inhabiting fungi in marine environments and PCL degradation activity.

Plastic waste has a negative impact on marine ecosystems and the quantity of this source of anthropogenic pollution continues to increase. Several studies have investigated plastic biodegradation using various microorganisms. In this study, we isolated fungi from polyethylene terephthalate (PET) waste on Korean seacoasts and evaluated their ability to degrade plastic by comparing the diameters of the clear zones they formed on polycaprolactone (PCL) agar. We isolated 262 strains from 47 plastic waste sources and identified 108 fungal species via molecular methods. The PCL agar assay revealed that 87 species presented with varying degrees of PCL degradation capacity. Among them, certain fungal species were strong PCL degraders. The present study demonstrated the possibility that some fungi inhabiting plastic could potentially degrade it in the marine environment. We believe that the discoveries made herein lay theoretical and practical foundations for the development of novel bioremediation systems for marine plastispheres and help mitigate the environmental pollution issues related to plastic wastes.

Taxonomy, comparative genomics and evolutionary insights of Penicillium ucsense: a novel species in series Oxalica.

The genomes of two Penicillium strains were sequenced and studied in this study: strain 2HH was isolated from the digestive tract of Anobium punctatum beetle larva in 1979 and the cellulase hypersecretory strain S1M29, derived from strain 2HH by a long-term mutagenesis process. With these data, the strains were reclassified and insight is obtained on molecular features related to cellulase hyperproduction and the albino phenotype of the mutant. Both strains were previously identified as Penicillium echinulatum and this investigation indicated that these should be reclassified. Phylogenetic and phenotype data showed that these strains represent a new Penicillium species in series Oxalica, for which the name Penicillium ucsense is proposed here. Six additional strains (SFC101850, SFCP10873, SFCP10886, SFCP10931, SFCP10932 and SFCP10933) collected from the marine environment in the Republic of Korea were also classified as this species, indicating a worldwide distribution of this new taxon. Compared to the closely related strain Penicillium oxalicum 114-2, the composition of cell wall-associated proteins of P. ucsense 2HH shows five fewer chitinases, considerable differences in the number of proteins related to ß-D-glucan metabolism. The genomic comparison of 2HH and S1M29 highlighted single amino-acid substitutions in two major proteins (BGL2 and FlbA) that can be associated with the hyperproduction of cellulases. The study of melanin pathways shows that the S1M29 albino phenotype resulted from a single amino-acid substitution in the enzyme ALB1, a precursor of the 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis. Our study provides important knowledge towards understanding species distribution, molecular mechanisms, melanin production and cell wall biosynthesis of this new Penicillium species.

Exploring fine-scale assembly of ectomycorrhizal fungal communities through phylogenetic and spatial distribution analyses.

Ectomycorrhizal fungi (EMF) form symbiotic relationship with the roots of host plants. EMF communities are composed of highly diverse species; however, how they are assembled has been a long-standing question. In this study, we investigated from a phylogenetic perspective how EMF communities assemble on Pinus densiflora seedlings at different spatial scales (i.e., seedling scale and root tip scale). P. densiflora seedlings were collected from different habitats (i.e., disturbed areas and mature forests), and their EMF communities were investigated by morphotype sequencing and next-generation sequencing (NGS). To infer assembly mechanisms, phylogenetic relatedness within the community (i.e., phylogenetic structure) was estimated and spatial distribution of EMF root tips was analyzed. The EMF communities on pine seedlings were largely different between the two habitats. Phylogenetically restricted lineages (Amphinema, /suillus-rhizopogon) were abundant in the disturbed areas, whereas species from diverse lineages were abundant in the mature forests (Russula, Sebacina, /tomentella-thelephora, etc.). In the disturbed areas, phylogenetically similar EMF species were aggregated at the seedling scale, suggesting that disturbance acts as a powerful abiotic filter. However, phylogenetically similar species were spatially segregated from each other at the root tip scale, indicating limiting similarity. In the mature forest seedlings, no distinct phylogenetic signals were detected at both seedling and root tip scale. Collectively, our results suggest that limiting similarity may be an important assembly mechanism at the root tip scale and that assembly mechanisms can vary across habitats and spatial scales.

Species Prioritization Based on Spectral Dissimilarity: A Case Study of Polyporoid Fungal Species.

Biological species collections are critical for natural product drug discovery programs. However, prioritization of target species in massive collections remains difficult. Here, we introduce an untargeted metabolomics-based prioritization workflow that uses MS/MS molecular networking to estimate scaffold-level distribution. As a demonstration, we applied the workflow to 40 polyporoid fungal species. Nine species were prioritized as candidates based on the chemical structural and compositional similarity (CSCS) metric. Most of the selected species showed relatively higher richness and uniqueness of metabolites than those of the others. Cryptoporus volvatus, one of the prioritized species, was investigated further. The chemical profiles of the extracts of C. volvatus culture and fruiting bodies were compared, and it was shown that derivative-level diversity was higher in the fruiting bodies; meanwhile, scaffold-level diversity was similar. This showed that the compounds found from a cultured fungus can also be isolated in wild mushrooms. Targeted isolation of the fruiting body extract yielded three unknown (1-3) and six known (4-9) cryptoporic acid derivatives, which are drimane-type sesquiterpenes with isocitric acid moieties that have been reported in this species. Cryptoporic acid T (1) is a trimeric cryptoporic acid reported for the first time. Compounds 2 and 5 exhibited cytotoxicity against HCT-116 cell lines with IC50 values of 4.3 and 3.6 µM, respectively.

Reviewing the world's edible mushroom species: A new evidence-based classification system.

Wild mushrooms are a vital source of income and nutrition for many poor communities and of value to recreational foragers. Literature relating to the edibility of mushroom species continues to expand, driven by an increasing demand for wild mushrooms, a wider interest in foraging, and the study of traditional foods. Although numerous case reports have been published on edible mushrooms, doubt and confusion persist regarding which species are safe and suitable to consume. Case reports often differ, and the evidence supporting the stated properties of mushrooms can be incomplete or ambiguous. The need for greater clarity on edible species is further underlined by increases in mushroom-related poisonings. We propose a system for categorizing mushroom species and assigning a final edibility status. Using this system, we reviewed 2,786 mushroom species from 99 countries, accessing 9,783 case reports, from over 1,100 sources. We identified 2,189 edible species, of which 2,006 can be consumed safely, and a further 183 species which required some form of pretreatment prior to safe consumption or were associated with allergic reactions by some. We identified 471 species of uncertain edibility because of missing or incomplete evidence of consumption, and 76 unconfirmed species because of unresolved, differing opinions on edibility and toxicity. This is the most comprehensive list of edible mushrooms available to date, demonstrating the huge number of mushrooms species consumed. Our review highlights the need for further information on uncertain and clash species, and the need to present evidence in a clear, unambiguous, and consistent manner.

A proposed stepwise screening framework for the selection of polycyclic aromatic hydrocarbon (PAH)-degrading white rot fungi.

This study suggests a simple three-step screening protocol for the selection of white rot fungi (WRF) capable of degrading polycyclic aromatic hydrocarbons (PAHs), which combines easily applicable bioassay techniques, and verifies that protocol by evaluating the PAH degradation activity, ligninolytic enzyme secretion, and relevant gene expressions of the selected PAH-degraders. Using 120 fungal strains, a sequence of bioassay techniques was applied: Bavendamm's reaction (Step 1), remazol brilliant blue R (RBBR) decolorization (Step 2); assays for tolerance to four mixed PAHs-phenanthrene, anthracene, fluoranthene, and pyrene (Step 3). This stepwise protocol selected 14 PAH-degrading WRF, including Microporus vernicipes, Peniophora incarnata, Perenniporia subacida, Phanerochaete sordida, Phlebia acerina, and Phlebia radiata. Of these, P. incarnata exhibited the highest PAH degradative activity, ranging from 40 to > 90%, which was related to the time-variable secretions of three extracellular ligninolytic enzymes: laccase, manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP). Laccase and MnP production by P. incarnata tended to be greater in the early stages of PAH degradation, whereas its LiP production became intensified with decreasing laccase and MnP production. Pilc1 and pimp1 genes encoding laccase and MnP were expressed, indicating the occurrence of extracellular enzyme-driven biodegradation of PAH by the fungal strains.

Diversity and Ecology of Marine Algicolous Arthrinium Species as a Source of Bioactive Natural Products.

In our previous study, all Arthrinium isolates from Sargassum sp. showed high bioactivities, but studies on marine Arthrinium spp. are insufficient. In this study, a phylogenetic analysis of 28 Arthrinium isolates from seaweeds and egg masses of Arctoscopus japonicus was conducted using internal transcribed spacers, nuclear large subunit rDNA, ß-tubulin, and translation elongation factor region sequences, and their bioactivities were investigated. They were analyzed as 15 species, and 11 of them were found to be new species. Most of the extracts exhibited radical-scavenging activity, and some showed antifungal activities, tyrosinase inhibition, and quorum sensing inhibition. It was implied that marine algicolous Arthrinium spp. support the regulation of reactive oxygen species in symbiotic algae and protect against pathogens and bacterial biofilm formation. The antioxidant from Arthrinium sp. 10 KUC21332 was separated by bioassay-guided isolation and identified to be gentisyl alcohol, and the antioxidant of Arthrinium saccharicola KUC21221 was identical. These results demonstrate that many unexploited Arthrinium species still exist in marine environments and that they are a great source of bioactive compounds.

Effect of fairy ring bacteria on the growth of Tricholoma matsutake in vitro culture.

Tricholoma matsutake (pine mushroom) (Basidiomycota, Agaricales) is a valuable edible fungal species that cannot be cultivated artificially. As an ectomycorrhizal fungus, T. matsutake interacts with trees belonging to the Pinaceae and Fagaceae, and forms fairy rings around host trees that are arc-shaped areas with dense hyphae of T. matsutake in the soil. Because the fairy rings maintain their dense hyphae for several years and form fruiting bodies, the characteristics of the fairy ring may be important in understanding the ecology of T. matsutake. Recent studies have shown that diverse bacteria co-exist in the fairy ring, and suggest that the fairy ring bacteria may influence on the growth of T. matsutake. However, the effect of the fairy ring bacteria on the growth of T. matsutake is largely unknown. In this study, we isolated fairy ring bacteria and investigated their effect on the growth of T. matsutake in co-culture experiments. In addition, the relationship between bacterial effects and nutrient conditions was tested using different media with varying glucose concentrations. A total of 237 bacteria (28 species) were isolated from fairy rings of four different T. matsutake producing areas: Proteobacteria (17 species), Firmicutes (7 species), and Actinobacteria (4 species). Burkholderiaceae (Burkholderia and Paraburkholderia) was most abundant in the fairy ring bacteria communities. Most bacteria showed a negative effect on the growth of T. matsutake when it grew on glucose rich medium (20 g/L). In glucose deficient medium (2 g/L), however, some bacteria promoted the growth of T. matsutake. In addition, the mode of interaction between bacteria and T. matsutake is different, depending on the glucose concentration.

Diversity and abundance of human-pathogenic fungi associated with pigeon faeces in urban environments.

Pathogenic fungi are a growing health concern worldwide, particularly in large, densely populated cities. The dramatic upsurge of pigeon populations in cities has been implicated in the increased incidence of invasive fungal infections. In this study, we used a culture-independent, high-throughput sequencing approach to describe the diversity of clinically relevant fungi (CRF) associated with pigeon faeces and map the relative abundance of CRF across Seoul, Korea. In addition, we tested whether certain geographical, sociological and meteorological factors were significantly associated with the diversity and relative abundance of CRF. Finally, we compared the CRF diversity of fresh and old pigeon faeces to identify the source of the fungi and the role of pigeons in dispersal. Our results demonstrated that both the composition and relative abundance of CRF are unevenly distributed across Seoul. The green area ratio and the number of multiplex houses were positively correlated with species diversity, whereas wind speed and number of households were negatively correlated. The number of workers and green area ratio were positively correlated with the relative abundance of CRF, whereas wind speed was negatively correlated. Because many CRF were absent in fresh faeces, we inferred that most species cannot survive the gastrointestinal tract of pigeons and instead are likely transmitted through soil or air and use pigeon faeces as a substrate for proliferation.

Metschnikowia cf. typographi and other pathogens from the bark beetle Ips sexdentatus - Prevalence, histological and ultrastructural evidence, and molecular characterization.

Ips sexdentatus (six-spined engraver beetle) from Austria and Poland were dissected and examined for the presence of pathogens. Specimens collected in Austria were found to contain the ascomycetous fungus Metschnikowia cf. typographi. Infection rates ranged from 3.6% to 26.8% at different collection sites. M. cf. typographi infected midguts were investigated by histological, ultrastructural and molecular techniques. Extraordinary ultrastructural details are shown, such as ascospores with bilateral flattened flanks resembling alar rims at both sides of their attenuating tube-like ends. These have not yet been described in other yeast species. Molecular investigations showed a close phylogenetic relationship to the fungi Metschnikowia agaves and Candida wancherniae. Presence of the entomopathogenic fungus Beauveria bassiana found in Austria was confirmed both morphologically and molecularly. The eugregarine Gregarina typographi was diagnosed most frequently. Infection rates of all I. sexdentatus specimens ranged from 21.4% to 71.9% in Austria and 54.1% to 68.8% in Poland. Other entomopathogenic protists, bacteria, or viruses were not detected.

Comparison of the Diversity of Basidiomycetes from Dead Wood of the Manchurian fir (Abies holophylla) as Evaluated by Fruiting Body Collection, Mycelial Isolation, and 454 Sequencing.

In this study, three different methods (fruiting body collection, mycelial isolation, and 454 sequencing) were implemented to determine the diversity of wood-inhabiting basidiomycetes from dead Manchurian fir (Abies holophylla). The three methods recovered similar species richness (26 species from fruiting bodies, 32 species from mycelia, and 32 species from 454 sequencing), but Fisher's alpha, Shannon-Wiener, Simpson's diversity indices of fungal communities indicated fruiting body collection and mycelial isolation displayed higher diversity compared with 454 sequencing. In total, 75 wood-inhabiting basidiomycetes were detected. The most frequently observed species were Heterobasidion orientale (fruiting body collection), Bjerkandera adusta (mycelial isolation), and Trichaptum fusco-violaceum (454 sequencing). Only two species, Hymenochaete yasudae and Hypochnicium karstenii, were detected by all three methods. This result indicated that Manchurian fir harbors a diverse basidiomycetous fungal community and for complete estimation of fungal diversity, multiple methods should be used. Further studies are required to understand their ecology in the context of forest ecosystems.

Penicillium jejuense sp. nov., isolated from the marine environments of Jeju Island, Korea.

Three strains of an unidentified Penicillium species were isolated during a fungal diversity survey of marine environments in Korea. These strains are described here as a new species following a multigene phylogenetic analyses of nuc rDNA internal transcribed spacer barcodes (ITS1-5.8S-ITS2), genes for ß-tubulin, calmodulin and RNA polymerase II second largest subunit, and observation of macro-and micromorphological characters. Phylogenetic analyses revealed that the three strains formed a strongly supported monophyletic group distinct from previously reported species of section Aspergilloides. Morphologically this species can be distinguished from its sister species, P. crocicola, by the reverse color on Czapek yeast autolysate agar, abundant production of sclerotia on malt extract agar and colony characters on yeast extract sucrose agar. We name this new species P. jejuense, after the locality where it was discovered. At 25 C for 7 d, P. jejuense colonies grew to 55-60 mm on CYA, 45-48 mm on MEA, 48-52 mm on YES and 23-26 mm on CREA. Conidia (2.2-3.4 × 2.0-2.6 µm) and sclerotia (160-340 × 125-210 µm) were globose to ellipsoidal.

Trichoderma songyi sp. nov., a new species associated with the pine mushroom (Tricholoma matsutake).

A new species, Trichoderma songyi, was found to be associated with the pine mushroom (Tricholoma matsutake) in Korea. This species was isolated from three different substrates: Tricholoma matsutake basidiomata, as well as roots of Pinus densiflora and soil in the fairy ring. Based on its molecular and phenotypic characteristics, we demonstrate that Trichoderma songyi is unique and distinguishable from closely related species. We performed phylogenetic analyses based on two molecular markers, the genes for both translation elongation factor 1-alpha and the second largest subunit of RNA polymerase II. Phylogenetic analyses showed that Trichoderma songyi is closely related to Trichoderma koningii aggregate and Trichoderma caerulescens. Morphologically, Trichoderma songyi can be distinguished from these closely related taxa by its growth rates, colony morphology on PDA in darkness, and coconut-like odour. Due to the economic importance of the pine mushroom, the relationship between Trichoderma songyi and Tricholoma matsutake should be studied further.

Marine-derived Penicillium in Korea: diversity, enzyme activity, and antifungal properties.

The diversity of marine-derived Penicillium from Korea was investigated using morphological and multigene phylogenetic approaches, analyzing sequences of the internal transcribed spacer region, ß-tubulin gene, and RNA polymerase subunit II gene. In addition, the biological activity of all isolated strains was evaluated. We tested for the extracellular enzyme activity of alginase, endoglucanase, and ß-glucosidase, and antifungal activity against two plant pathogens (Colletotrichum acutatum and Fusarium oxysporum). A total of 184 strains of 36 Penicillium species were isolated, with 27 species being identified. The most common species were Penicillium polonicum (19.6 %), P. rubens (11.4 %), P. chrysogenum (11.4 %), and P. crustosum (10.9 %). The diversity of Penicillium strains isolated from soil (foreshore soil and sand) and marine macroorganisms was higher than the diversity of strains isolated from seawater. While many of the isolated strains showed alginase and ß-glucosidase activity, no endoglucanase activity was found. More than half the strains (50.5 %) showed antifungal activity against at least one of the plant pathogens tested. Compared with other strains in this study, P. citrinum (strain SFC20140101-M662) showed high antifungal activity against both plant pathogens. The results reported here expand our knowledge of marine-derived Penicillium diversity. The relatively high proportion of strains that showed antifungal and enzyme activity demonstrates that marine-derived Penicillium have great potential to be used in the production of natural bioactive products for pharmaceutical and/or industrial use.

Phylogenetic Assessment of Understudied Families in Hymenochaetales (Basidiomycota, Fungi)-Reporting Uncovered Species and Reflecting the Recent Taxonomic Updates in the Republic of Korea.

Hymenochaetales Oberw. is an order classified in Basidiomycota of Fungi, and species in this order display notable diversity. They exhibit various fruiting body shapes, including clavarioid, effused-reflexed, and resupinate basidiomes. Few mycorrhizal species have been reported in Hymenochaetales, but wood-decaying species dominate the order. Hymenochaetaceae Imazeki & Toki and Schizoporaceae Jülich are the most species-rich families within Hymenochaetales, and most species in the Republic of Korea belong to these two families. As such, current taxonomic classification and nomenclature are not reflected upon species in the remaining Hymenochaetales families. For this study, a multifaceted morphological and multigenetic marker-based phylogenetic investigation was conducted to, firstly, comprehensively identify understudied Hymenochaetales specimens in Korea and, secondly, reflect the updates on the species classification. Five genetic markers were assessed for the phylogenetic analysis: nuclear small subunit ribosomal DNA (nSSU), internal transcribed spacer (ITS), nuclear large subunit ribosomal DNA (nLSU), RNA polymerase II subunit 2 gene (RPB2), and translation elongation factor 1 gene (TEF1). The results from phylogenetic analysis supported 18 species classified under eight families (excluding Hymenochaetaceae and Schizoporaceae) in Korea. Species formerly placed in Rickenellaceae and Trichaptum sensu lato have been systematically revised based on recent taxonomic reconstructions. In addition, our findings revealed one new species, Rickenella umbelliformis, and identified five formerly nationally unreported species classified under five understudied families. Our findings contribute to a better understanding of Hymenochaetales diversity and highlight the need for continued research.

Global phylogeographical distribution of Gloeoporus dichrous.

Phylogeographic analyses are efficient in ecological and evolutionary studies to discover the origin of a lineage, its dispersal routes, and the divergence of ancestral traits. Studies on widespread wood-decay fungi have revealed the phylogenetic division of several polypores based on geographical distribution. In this study, specimens of Gloeoporus dichrous, a cosmopolitan polypore species, were collected globally and analyzed for their geographic distribution. Multi-marker Bayesian molecular clock and haplotype analyses revealed a clear division of G. dichrous populations by continent. The species diverged from its neighboring clades 10.3 (16.0-5.6) million years ago, with Asian and North American populations at the center of divergence. Possible dispersal mechanisms and pathways are predicted and discussed based on the evaluated transfer routes. The biogeography of G. dichrous analyzed in this study represents a fraction of the polypore evolution and may advance the understanding of the overall evolution of wood-decay fungi.

Long-Term Investigation of Marine-Derived Aspergillus Diversity in the Republic of Korea.

Aspergillus species play a crucial role in terrestrial environments as degraders and are well known for producing various secondary metabolites. Recently, Aspergillus species have been discovered in marine environments, exhibiting adaptability to high salinity and producing diverse secondary metabolites with valuable properties. However, limited research has focused on their marine diversity, leading to inaccurate species identification. The current study addresses this gap by investigating diverse marine habitats in the Republic of Korea, including sediment, seawater, seaweed, and marine animals. From three coasts of the Korean Peninsula, 472 Aspergillus strains were isolated from the various marine habitats. A total of 41 species were accurately identified using multigenetic markers: internal transcribed spacer, calmodulin, and ß-tubulin. The findings underscore the importance of accurate identification and provide a basis for elucidating the functional role of marine-derived Aspergillus species in marine ecosystems.