The UNITE database for molecular identification and taxonomic communication of fungi and other eukaryotes: sequences, taxa and classifications reconsidered.

UNITE (https://unite.ut.ee) is a web-based database and sequence management environment for molecular identification of eukaryotes. It targets the nuclear ribosomal internal transcribed spacer (ITS) region and offers nearly 10 million such sequences for reference. These are clustered into ∼2.4M species hypotheses (SHs), each assigned a unique digital object identifier (DOI) to promote unambiguous referencing across studies. UNITE users have contributed over 600 000 third-party sequence annotations, which are shared with a range of databases and other community resources. Recent improvements facilitate the detection of cross-kingdom biological associations and the integration of undescribed groups of organisms into everyday biological pursuits. Serving as a digital twin for eukaryotic biodiversity and communities worldwide, the latest release of UNITE offers improved avenues for biodiversity discovery, precise taxonomic communication and integration of biological knowledge across platforms.

Non-destructive DNA extraction from herbarium specimens: a method particularly suitable for plants with small and fragile leaves.

Protocols for DNA extraction from plants generally involve physical and chemical destruction of tissues. Use of these conventional methods precludes preservation of morphological information from herbarium specimens, especially for small plants with few leaves, and reduces the voucher value of specimens. Here, we developed a new, non-destructive DNA extraction protocol (Protocol 1) that only needs a small piece of leaf (< 25 mm2) to obtain DNA suitable for DNA sequencing from fragile herbarium specimens. The protocol was very simple and rapid; an extraction buffer was placed on the leaf surface of an intact specimen for 30 min at room temperature (20 °C). The quality of extracted DNA was checked by PCR amplification of two standard plant DNA barcode regions, the maturase K gene (matK, ca. 850 bp) and the ribulose-1,5-bisphosphatecarboxylase/oxygenase gene (rbcL, ca. 550 bp), for 14 vascular plant species encompassing various taxonomic groups. The protocol retrieved sequences from 80.0% of specimens for matK and 46.2% of specimens for rbcL. Placing of the extraction buffer onto specimens did not cause any tears or deformation, but caused discoloration in some plants. To improve DNA yield for specimens incompatible with Protocol 1, we developed an alternative protocol for DNA extraction with minimally invasive destruction of specimens (Protocol 2). In this protocol, a cut leaf was immersed in the extraction buffer for 30 min and stored subsequently in a fragment pocket on the specimen sheet. This alternative method retrieved matK sequences from 80.0% of specimens and rbcL sequences from 92.8% of specimens. The combination of Protocols 1 and 2 enabled us to obtain matK sequences from 90.0% of specimens and rbcL sequences form 92.8% of specimens. The new protocols facilitate the use of museum specimens for use of DNA of museum specimens while still preserving morphological information.

Sydowianumols A, B, and C, Three New Compounds from Discomycete Poculum pseudosydowianum.

Three new compounds, sydowianumols A (1), B (2), and C (3), were isolated from culture broth and mycelial extracts of Poculum pseudosydowianum (TNS-F-57853), an endophytic fungus isolated from fresh leaves of Quercus crispula. The structures of new compounds 1-3 were elucidated from spectroscopic data. Sydowianumols A (1) and B (2) exhibited antimicrobial activity against methicillin-resistant Staphylococcus aureus (MRSA) with 90% minimum inhibitory concentration (MIC90) values of 12.5 µg/mL.

Early-diverging wood-decaying fungi detected using three complementary sampling methods.

Wood-decaying fungi are essential components of degradation systems in forest ecosystems. However, their species diversity and ecological features are largely unknown. Three methods are commonly used to investigate fungal diversity: fruiting body collection, culturing, and environmental DNA analysis. Because no single method fully characterises fungal diversity, complementary approaches using two or more methods are required. However, few studies have compared the different methods and determined the best way to characterise fungal diversity. To this end, we investigated wood-decomposing Dacrymycetes (Agaricomycotina, Basidiomycota) using a complementary approach combining fruiting body collection, culturing, and environmental DNA analysis, thereby offering an effective approach for investigating the diversity of saprotrophic mushrooms. Fruiting body collection, culturing, and environmental DNA analysis detected 11, 10, and 16 operational taxonomic units (OTUs; 25 OTUs in total) and identified three, seven, and seven novel lineages, respectively. The three methods were complementary to each other to detect greater Dacrymycetes diversity. The culturing and environmental DNA analysis identified three early-diverging lineages that were not identified in the fruiting body collection suggesting that diverse lineages lacking observable fruiting bodies remain undiscovered. Such lineages may be important to understand Dacrymycetes evolution. To detect early branches of Dacrymycetes more efficiently, we recommend a combined approach consisting of a primary environmental DNA survey to detect novel lineages and a secondary culture survey to isolate their living mycelia. This approach would be helpful for identifying otherwise-undetectable lineages, and could thus uncover missing links that are important for understanding the evolution of mushroom-forming fungi.

First report of Veronaea botryosa as a causal agent of chromomycosis in frogs.

A dematiaceous hyphomycete, isolated from frogs, was determined as the possible etiologic agent of a case of systemic chromomycosis this cold-blooded animal. The fungus was identified as Veronaea botryosa on the basis of morphological features observed in histopathological examination and molecular phylogenetic evidence. Although V. botryosa is known to be distributed widely in litter and as a human pathogen, this is the first confirmed report of its involvement in a lethal infection in a cold-blooded animal, including an anuran.

Population structure of the invasive forest pathogen Hymenoscyphus pseudoalbidus.

Understanding the genetic diversity and structure of invasive pathogens in source and in introduced areas is crucial to the revelation of hidden biological features of an organism, to the reconstruction of the course of invasions and to the establishment of effective control measures. Hymenoscyphus pseudoalbidus (anamorph: Chalara fraxinea) is an invasive and highly destructive fungal pathogen found on common ash Fraxinus excelsior in Europe and is native to East Asia. To gain insights into its dispersal mechanisms and history of invasion, we used microsatellite markers and characterized the genetic structure and diversity of H. pseudoalbidus populations at three spatial levels: (i) between Europe and Japan, (ii) in Europe and (iii) at the epidemic's front in Switzerland. Phylogenetic and network analysis demonstrated that individuals from both regions are conspecific. However, populations from Japan harboured a higher genetic diversity and were genetically differentiated from European ones. No evident population structure was found among the 1208 European strains using Bayesian and multivariate clustering analysis. Only the distribution of genetic diversity in space, pairwise population differentiation (GST) and the spatial analysis of principal components revealed a faint geographical pattern around Europe. A significant allele deficiency in most European populations pointed to a recent genetic bottleneck, whereas no pattern of isolation by distance was found. Our data suggest that H. pseudoalbidus was introduced just once by at least two individuals. The potential source region of H. pseudoalbidus is vast, and further investigations are required for a more accurate localization of the source population.

Seven new mycoviruses identified from isolated ascomycetous macrofungi.

Mycoviruses have been described in all major fungal taxonomic groups. There has been much focus on commercially cultivated basidiomycetous macrofungi, while attention to viruses from ascomycetous macrofungi is lacking. Therefore, in this study, we conducted viral screening against fungal mycelia that were regenerated from ascomycetous macrofungi using agarose gel electrophoresis (AGE) and fragmented and primer-ligated dsRNA sequencing (FLDS). Among the 57 isolates, four isolates were detected with virus-like bands through screening with AGE, and subsequent FLDS analyses determined the viral sequences. Other isolates without virus-like bands in AGE were pooled to check for viral sequences. Using FLDS analysis, a total of seven new mycoviruses were identified, including two double-stranded RNA (dsRNA) viruses belonging to Quadriviridae and Partitiviridae, five positive-sense single-stranded RNA (ssRNA) viruses (three belonging to Mitoviridae, one belonging to Endornaviridae and one belonging to Virgaviridae). All viruses characterized in this study are novel species, and all the hosts are firstly reported to be infected by mycoviruses. These findings expand our knowledge of the diversity of mycoviruses from macrofungi in natural environments.

Paranazzamides A and B, new cyclic dipeptides containing a C7-prenylated tryptophan, produced by pathogenic reptile fungi Paranannizziopsis sp. UH-21.

Two new cyclic dipeptides, paranazzamides A (1) and B (2) containing a C7-prenylated tryptophan, were isolated from a culture broth of snake fungal disease-isolate Paranannizziopsis sp. UH-21. This is the first report on the new secondary metabolites from Paranannizziopsis sp. The planar structures of 1 and 2 were elucidated using various spectroscopic techniques including MS and 1D/2D NMR. The absolute configuration of 1 was assigned by comparison with the synthesized compound. Compounds 1 and 2 exhibited no antifungal activity, no antibacterial activity, and no cytotoxic activity even at a concentration of 128 µg ml-1, whereas 1 and 2 exhibited amphotericin B potentiating activity against Candida auris in combination treatment.

A new genus Neobelonopsis and two new species of Trichobelonium (Helotiales, Ascomycota) discovered mainly from poaceous grasses native to Asia in Japan.

Mollisioid fungi, represented by Mollisia (Fr.) P. Karst., are characterized by soft, sessile apothecia with globose, dark-celled excipula, hyaline ascospores, and worldwide distribution in temperate regions. Their generic and species delimitation is difficult due to the lack of distinct features, and studies based on DNA sequences are urgently required. Two genera of mollisioid fungi, Belonopsis and Trichobelonium, comprise relatively few species and are recognized by (0-)1-3-septate ascospores, medullary excipulum composed of loosely interwoven hyphae, and calcium oxalate crystals in the excipulum. Specimens of undescribed species that are morphologically assignable to Belonopsis or Trichobelonium were collected from various sites in Japan and their assignment to the proper genera was attempted. According to a molecular phylogenetic analysis involving members of Mollisiaceae based on concatenated sequences of ITS, LSU, and RPB1, eight taxonomic entities were placed in a strongly supported single clade with Mollisiadiesbachiana, separated from the type species of Belonopsis, B.excelsior. A new genus Neobelonopsis was thus proposed to accommodate the undescribed species. In this study, eight new species of Neobelonopsis and two new species of Trichobelonium were described. A new combination was also proposed for M.diesbachiana. The generic distinction of Neobelonopsis and Trichobelonium was supported by molecular analysis. Some additional characteristics to delimit Trichobelonium were identified, such as the presence of anchoring hyphae between the base of the apothecium and subiculum, and the production of abundant crystals and soluble pigments on the colonies. Derivative species of Neobelonopsis were found to have multi-septa in ascospores.

Reassessment of type specimens of Cordyceps and its allies, described by Dr. Yosio Kobayasi and preserved in the mycological herbarium of the National Museum of Nature and Science. Part 4. Cordyceps s. l. on Lepidoptera.

Dr. Kobayasi and Mr. Shimizu described 31 species of Cordyceps infecting Lepidoptera. Holotype specimens of 14 species and two authentic specimens of one of the 31 species were rediscovered from a herbarium of the National Museum of Nature and Science (TNS). Registration numbers (TNS-F-number) were given to these 16 specimens, and one was lectotypified as follows. Holotypes: Metarhizium indigoticum TNS-F-230337; Yosiokobayasia kusanagiensis TNS-F-197994 (Clavicipitaceae); Beauveria hepialidicola (Kobayasi & Shimizu) Hirok. Sato, S. Ban & Hosoya, comb. nov. TNS-F-197986; Cordyceps ampullacea TNS-F-197981, Cordyceps militaris f. alba TNS-F-230340, Cordyceps ochraceostromata TNS-F-195471, and Cordyceps rosea TNS-F-197972 (Cordyceps sensu stricto, Cordycipitaceae); Ophiocordyceps aurantia TNS-F-195485, Ophiocordyceps cochlidiicola TNS-F-195470, and Ophiocordyceps hiugensis TNS-F-197978 (Ophiocordy-cipitaceae); and Cordyceps changpaishanensis TNS-F-195501, Cordyceps ootakiensis TNS-F-197976, Cordyceps shimizui TNS-F-197995, and Cordyceps sulfurea TNS-F-197974 (Cordyceps sensu lato). Lectotype: Cordyceps bulolensis TNS-F-230327 (Cordyceps sensu lato). A new combination Beauveria hepialidicola comb. nov., is proposed for Cordyceps hepialidicola based on morphological observations.

Host jumping onto close relatives and across kingdoms by Tyrannicordyceps (Clavicipitaceae) gen. nov. and Ustilaginoidea_(Clavicipitaceae).

PREMISE OF STUDY: This research seeks to advance understanding of conditions allowing movement of fungal pathogens among hosts. The family Clavicipitaceae contains fungal pathogens exploiting hosts across three kingdoms of life in a pattern that features multiple interkingdom host shifts among plants, animals, and fungi. The tribe Ustilaginoideae potentially represents a third origin of plant pathogenesis, although these species remain understudied. Fungal pathogens that cause ergot are linked morphologically with Clavicipitaceae, but are not yet included in phylogenetic studies. The placement of Ustilaginoideae and ergot pathogens will allow differentiation between the host habitat and host relatedness hypotheses as mechanisms of phylogenetic diversification of Clavicipitaceae. METHODS: A multigene data set was assembled for Clavicipitaceae to test phylogenetic placement and ancestral character-state reconstructions for Ustilaginoidea virens and U. dichromonae as well as the ergot mycoparasite Cordyceps fratricida. Microscopic morphological observations of sexual and asexual states were also performed. KEY RESULTS: Phylogenetic placement of U. virens and U. dichromonae represents a third acquisition of the plant pathogenic lifestyle in Clavicipitaceae. Cordyceps fratricida was also placed in Clavicipitaceae and recognized as a new genus Tyrannicordyceps. Ancestral character state reconstructions indicate initially infecting hemipteran insect hosts facilitates subsequent changes to a plant pathogenic lifestyle. The ancestor of T. fratricida is inferred to have jumped from grasses to pathogens of grasses. CONCLUSIONS: The host habitat hypothesis best explains the dynamic evolution of host affiliations seen in Clavicipitaceae and throughout Hypocreales. Co-occurrence in the same habitat has allowed for host shifts from animals to plants, and from plants to fungi.

Examination of the generic concept and species boundaries of the genus Erioscyphella (Lachnaceae, Helotiales, Ascomycota) with the proposal of new species and new combinations based on the Japanese materials.

The genus Erioscyphella Kirschst., which was morphologically confused with Lachnum, was herein examined. Based on molecular phylogenetic analyses using a combined dataset of ITS, LSU, mtSSU, and RPB2 and morphological examinations, Erioscyphella was distinguished from Lachnum and redefined by longer ascospores and the presence of apical amorphous materials and/or resinous materials equipped on hairs. Species boundaries recognized by morphology/ecology and phylogenetic analyses were cross-checked using species delimitation analyses based on DNA barcode sequences downloaded from UNITE, resulting in that species' taxonomic problems being uncovered. Six new species (E.boninensis, E.insulae, E.otanii, E.papillaris, E.paralushanensis, and E.sasibrevispora) and two new combinations (E.hainanensis and E.sinensis) were proposed.

Pyrenopeziza orientalipetiolaris sp. nov. in Japan and morphological and genetic comparison with its relevant species P. petiolaris in Europe.

Some Asian fungi are morphologically very similar to European species but belong to different species. A fungus that resembles Pyrenopeziza petiolaris, which commonly occurs on the petioles of Acer pseudoplatanus in Europe, was found on the petioles of Acer spp. and other tree leaves in Japan. The apothecia of this fungus were smaller than those of P. petiolaris, suggesting that it is a different species. To examine this possibility, specimens of this fungus were collected from various hosts in Japan. A detailed morphological examination elucidated that this fungus differed from P. petiolaris in smaller apothecia, marginal cells of the ectal excipulum, and conidia. The ITS sequence difference between this fungus and P. petiolaris was 3.3-4.3%, and they formed distinct clades in the phylogenetic analysis, supporting that they are different species. Consequently, a new species, P. orientalipetiolaris is described. Since an undescribed phialophora-state was observed in the cultures of P. petiolaris for the first time, the morphology under culture is also reported in detail.

Euantennaria pleioblasti sp. nov. (Euantennariaceae) and Metacapnodium cf. quinqueseptatum (Metacapnodiaceae), two mixed sooty moulds in subicula on Pleioblastus sp. in Taiwan.

Black subicula, comprising a mixture of two sooty moulds of Euantennariaceae and Metacapnodiaceae, on Pleioblastus were collected in Batongguan, alt. ca. 2800 m, Nantou County, Taiwan in 1984. The former sooty mould is described and illustrated as Euantennaria pleioblasti sp. nov., an asexually typified species of the genus, as currently circumscribed with the application of the single name nomenclature for pleomorphic fungi. It is characterized by cylindrical, finely to coarsely roughened hyphae and black synnemata bearing massive fusiform, straight, mostly 11-14-septate phragmoconidia in a subglobose to obovoid head; its reliable sexual morph is obscure. The latter was identified as Metacapnodium cf. quinqueseptatum. It features the capnobotrys- and capnophialophora-like asexual morphs, in addition to the sexual morph with 5-7-septate ascospores. These sooty mould taxa are newly added to the mycobiota of Taiwan.

De Novo Genome Assembly of Stinkhorn Mushroom Clathrus columnatus (Basidiomycota, Fungi) Using Illumina and Nanopore Sequencing Data.

We report the reference genome of Clathrus columnatus isolate MO-923, which was isolated from Chichijima Island, the Ogasawara (Bonin) Islands, Japan. Oxford Nanopore Technologies MinION and Illumina sequence reads were assembled using NECAT and polished using Pilon to yield a 36.51-Mb genome with 10,625 predicted protein-coding genes.

Palmaenones A and B, two new antimicrobial chlorinated cyclopentenones from discomycete Lachnum palmae.

Two new antimicrobial chlorinated cyclopentenones, palmaenones A (1) and B (2) were isolated from the culture broth of discomycete Lachnum palmae (NBRC-106495), and the structures of 1 and 2 were elucidated by spectroscopic data and the stereochemistry of 1 was directly determined by a single-crystal X-ray diffraction analysis. Palmaenones A (1) and B (2) are cyclopentenones containing three chlorines. Compound 1 exhibited potent antimicrobial activity against Micrococcus luteus, Mycobacterium smegmatis, Escherichia coli, Xanthomonas campestris, and Mucor racemosus, while the activities of compound 2 were weaker than 1.

Systematics, ecology, and application of Helotiales: Recent progress and future perspectives for research with special emphasis on activities within JapanHelotiales: Recent progress and future perspectives for research with special emphasis on activities within Japan.

Helotiales is one of the most diverse groups of apothecial ascomycetes, including 3000-4000 taxa. Recent progress in the systematics, ecology, and their applications through research is herein reviewed based on the experiences of the author with a special emphasis on activities in Japan. In the past 30 y, more than 50 helotialean taxa have been added to the mycobiota of Japan, including new taxa. With the advent of molecular phylogeny, some families have been revisited, such as members with stroma (Sclerotiniaceae and Rutstroemiaceae) or hairs (Hyaloscyphaceae and Lachnaceae). Although the monophyly of Helotiales has not yet been demonstrated, our understanding of its phylogeny has greatly advanced. The unexpected ecological nature represented by endophytism has been revealed through barcoding and other molecular techniques. The research history of ash dieback is also reviewed, and the endophytism/saprophytism of the pathogen on its original host is discussed. Drug discoveries within Helotiales are reviewed, and successful examples are presented. As future perspectives, both the cumulation of occurrence and sequence data of Helotiales is greatly encouraged to elucidate this important group of fungi.

Lifecycle of Pyrenopeziza protrusa (Helotiales, Dermateaceae sensu lato) in Magnolia obovata revealed by field observation and molecular quantification.

Fungi exhibit saprophytic, parasitic, and symbiotic lifestyles, and flexibly switching between them by the environmental changes and host conditions. However, only a few studies have elucidated the detailed changes in fungal DNA or morphology, including the formation of reproductive structures along with lifestyle switching. We hypothesized that Pyrenopeziza protrusa, which occurs abundantly and specifically on Magnolia obovata as a saprophyte, is also associated with living hosts and switches its lifestyles as part of its lifecycle. To elucidate this hypothesis, we periodically sampled the fresh/fallen leaves of M. obovata to observe the seasonal occurrence of reproductive structures for the isolation and detection/quantification of P. protrusa DNA with newly developed species-specific primers. The isolation frequency and amount of P. protrusa DNA drastically increased in the fresh leaves just before defoliation in autumn, but remained high in fallen leaves from autumn to spring. Abundant production of conidiomata and apothecia was also observed in the fallen leaves with increasing DNA content. These results clarified a large part of the lifecycle of P. protrusa, suggesting that the lifestyle is switched from symbiotic to saprophytic stage by significantly increasing the amount of DNA in response to host conditions according to the seasonal variations.

Microstoma longipilum sp. nov. (Sarcoscyphaceae, Pezizales) from Japan.

Microstoma longipilum sp. nov. collected from two localities in Japan is described. It is characterized by long apothecial hairs and salmon pink discs. Molecular phylogenetic analyses supported the novelty of the fungus. We additionally reported the overlooked morphology of hyphal mats, conidiogenous cells produced directly from ascospores, and conidia. With the addition of M. longipilum, now six species of Microstoma are documented in Japan.

Podogigants A and B, two new potentiators of amphotericin B activity, from Sordariomycete Podostroma giganteum.

Two new compounds, podogigants A (1) and B (2), were isolated from the culture broth of Podostroma giganteum. This is the first report on the identification of secondary metabolites in P. giganteum. The structures of 1 and 2 were elucidated through spectroscopic analysis, including 2D NMR spectroscopy assisted by chemical derivatization, which revealed the presence of farnesyl- and geranyl-hydroquinone structures, respectively. Compounds 1 and 2 exhibited no antifungal activity even at a concentration of 64 µg/mL, whereas they potentiated amphotericin B (AmB) activity against several species of fungi. In particular, 1 potentiated AmB activity against C. albicans and R. oryzae by up to 32-fold (MIC value of AmB decreased from 1.0 to 0.032 µg/mL), while 2 potentiated AmB activity against C. albicans by up to 16-fold.