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.

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.

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.

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.

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.

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.

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.

First report of ophidiomycosis in Asia caused by Ophidiomyces ophiodiicola in captive snakes in Japan.

Ophidiomycosis is an emerging infectious disease caused by the fungus Ophidiomyces ophiodiicola, which has been affecting wild and captive snakes in North America, Europe, and Australia. We report 12 cases of suspected ophidiomycosis in captive colubrid snakes in Japan. Pathological and microbiological examinations were performed, and the results confirmed the diagnosis of ophidiomycosis in two snakes, which indicated that the remaining sympatrically raised snakes also had ophidiomycosis since they exhibited similar lesions. This is the first report of ophidiomycosis in Asia caused by O. ophiodiicola. To prevent the expansion of ophidiomycosis in the natural environment in Japan, there is a need to evaluate the ophidiomycosis carrier status of imported snakes, the pathogenicity of the infection in native snakes, and the prevalence and distribution of O. ophiodiicola in wild and captive snakes. Measures also must be taken to prevent endemicity globally.

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.

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.

Phylogeography of post-Pleistocene population expansion in Dasyscyphella longistipitata (Leotiomycetes, Helotiales), an endemic fungal symbiont of Fagus crenata in Japan.

During the Last Glacial Maximum (LGM), drastic environmental changes modified the topology of the Japanese Archipelago, impacting species distributions. An example is Fagus crenata, which has a present continuous distribution throughout Japan. However, by the end of the LGM it was restricted to southern refugia. Similarly, Dasyscyphella longistipitata (Leotiomycetes, Helotiales, Lachnaceae) occurs strictly on cupules of F. crenata, sharing currently an identical distribution. As the effects of the LGM remain poorly understood for saprobiotic microfungal species, herein we identified past structuring forces that shaped the current genetic diversity within D. longistipitata in relation to its host using a phylogeographic approach. We inferred present and past potential distributions through species distribution modeling, identifying environmental suitability areas in mid-southern Japan from which subsequent colonizations occurred. Our findings suggest that current high genetic diversity and lack of genetic structure within D. longistipitata are the result of recent multiple re-colonization events after the LGM.

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.

A multigene phylogeny toward a new phylogenetic classification of Leotiomycetes.

Fungi in the class Leotiomycetes are ecologically diverse, including mycorrhizas, endophytes of roots and leaves, plant pathogens, aquatic and aero-aquatic hyphomycetes, mammalian pathogens, and saprobes. These fungi are commonly detected in cultures from diseased tissue and from environmental DNA extracts. The identification of specimens from such character-poor samples increasingly relies on DNA sequencing. However, the current classification of Leotiomycetes is still largely based on morphologically defined taxa, especially at higher taxonomic levels. Consequently, the formal Leotiomycetes classification is frequently poorly congruent with the relationships suggested by DNA sequencing studies. Previous class-wide phylogenies of Leotiomycetes have been based on ribosomal DNA markers, with most of the published multi-gene studies being focussed on particular genera or families. In this paper we collate data available from specimens representing both sexual and asexual morphs from across the genetic breadth of the class, with a focus on generic type species, to present a phylogeny based on up to 15 concatenated genes across 279 specimens. Included in the dataset are genes that were extracted from 72 of the genomes available for the class, including 10 new genomes released with this study. To test the statistical support for the deepest branches in the phylogeny, an additional phylogeny based on 3156 genes from 51 selected genomes is also presented. To fill some of the taxonomic gaps in the 15-gene phylogeny, we further present an ITS gene tree, particularly targeting ex-type specimens of generic type species. A small number of novel taxa are proposed: Marthamycetales ord. nov., and Drepanopezizaceae and Mniaeciaceae fams. nov. The formal taxonomic changes are limited in part because of the ad hoc nature of taxon and specimen selection, based purely on the availability of data. The phylogeny constitutes a framework for enabling future taxonomically targeted studies using deliberate specimen selection. Such studies will ideally include designation of epitypes for the type species of those genera for which DNA is not able to be extracted from the original type specimen, and consideration of morphological characters whenever genetically defined clades are recognized as formal taxa within a classification.

A check list of non-lichenised fungi occurring on Fagus crenata, a tree endemic to Japan.

Non-lichenised fungi from Fagus crenata, an endemic and major temperate tree species, were enumerated based on three approaches: fungarium specimens at the National Museum of Nature and Science; isolates obtained mainly from leaves and roots, and their molecular identification by barcoding region; and literature. In total, 209, 49, and 232 taxa were recognised from the fungarium specimens, isolates, and literature, respectively. Only three taxa were commonly observed using all three approaches. Moreover, the results demonstrate the diversity of fungi occurring on a single host plant species, and provide the basis for comparisons between fungi from Fagus spp. in other regions of the world.

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.

The enigmatic Mixia osmundae revisited: a systematic review including new distributional data and recent advances in its phylogeny and phylogenomics.

The enigmatic basidiomycete genus Mixia includes intracellular parasites of Osmunda and Osmundastrum ferns. Here, the authors review the systematic and phylogenetic history of M. osmundae, originally known as Taphrina osmundae, and provide new data from investigations of specimens of Osmunda japonica collected in Yunnan Province, China, which we determine to be conspecific with M. osmundae. In addition, Taphrina higginsii, a parasite on fronds of Osmundastrum cinnamomeum described from Georgia, USA, was confirmed to be phenotypically identical with M. osmundae. The name T. higginsii is lectotypified with a Mix specimen. Collections examined to date document four localities for M. osmundae: Japan (Honshu and Kyushu), Taiwan (Taichung), USA (Georgia), and China (Yunnan), and host-parasite relationships with the old extant ferns Osmunda japonica and its relatives and with Osmundastrum cinnamomeum. The phylogenetic placement of M. osmundae on the fungal tree of life, its evolutionary implications, and recent advances in the phylogenomics of this fungus are briefly reviewed and discussed.