Two new Mycena section Calodontes species: One newly discovered and the other new to Japan.

In 2017, two candidate species of Mycena were reported from Japan, with the Japanese names "Togari-sakura-take" and "Mitsuhida-sakura-take". However, to date, no taxonomic study or formal description has been undertaken for these two species. In the present study, we conducted comprehensive morphological and molecular phylogenetic examinations of "Togari-sakura-take" and "Mitsuhida-sakura-take", and compared them to known species within the genus Mycena. We performed phylogenetic analyses on a concatenated dataset, including the internal transcribed spacer region of ribosomal RNA, RNA polymerase II largest subunit, and translation elongation factor-1 alpha genes. "Togari-sakura-take" formed a clade with Mycena subulata, which was recently described from China, whereas "Mitsuhida-sakura-take" formed a distinct independent clade. We identified the former as M. subulata based on molecular phylogenetic analyses and morphological observations. However, the Japanese specimens displayed dextrinoid cheilocystidia and caulocystidia as well as the inamyloidity of basidiospores, which differed from the original description of M. subulata based on the materials from China. "Mitsuhida-sakura-take" was characterized by its remarkably dense lamellae and could be distinguished from known Mycena species by the combination of absent pleurocystidia and presence of bowling pin-shaped cheilocystidia. Here, we describe "Mitsuhida-sakura-take" as a new species, named Mycena densilamellata, in the section Calodontes.

Taxonomic and ecological significance of synnema-like structures/acanthophyses produced by Physisporinus (Meripilaceae, Polyporales) species.

Physisporinus, a genus in Polyporales, Basidiomycota, is a versatile fungus that lives as a wood decomposer, a potential pathogen of standing trees, and an orchid mycobiont. We previously reported that some Physisporinus species inhabiting wet wood in aquatic environments such as streams and waterfalls form synnema-like structures (SSs) bearing acanthophyses at their apices, and that they produce acanthophyses on vegetative hyphae when cultured on agar media. In this study, we investigated the acanthophysis-forming ability in Physisporinus and allied genera, and experimentally demonstrated the function of SSs. Phylogenetic analyses and observations of Meripilus, Physisporinus and Rigidoporus cultures showed that all of the strains forming acanthophyses belonged to Physisporinus, whereas strains of Meripilus and Rigidoporus did not produce acanthophyses. These findings suggest that SS/acanthophysis formation is a useful taxonomic character for members of Physisporinus. When Physisporinus strains were cultured under oxygen (O2) concentrations of 5, 10, 20 and 40%, most of those cultured under 20% O2 formed the most acanthophyses. According to these experimental data, the SSs/acanthophyses in Physisporinus were considered to have a respiratory function. Physisporinus probably acquired the SS/acanthophysis-forming ability to adapt to moist and/or aquatic habitats and to decay wet wood in which the O2 concentration is often low.

A new species of the genus Aleurodiscus sensu lato (Russulales, Basidiomycota) from Hachijo Island, Japan.

Aleurodiscus sagittisporus sp. nov. is described and illustrated. This species is characterized by producing basidiomata with a monomitic hyphal system, clampless-septate hyphae, arrowhead-shaped, amyloid, finely verrucose basidiospores, gloeocystidia, dendrohyphidium-like branched paraphysoid hyphae, and variously shaped swelling cells in the hymenium. Phylogenetic analyses based on nuclear rDNA LSU and ITS sequences revealed that the species is distinct from the lineage of Aleurodiscus s. str. and related genera in the Aleurodiscus s. lat. clade. Basidiomata of A. sagittisporus have been collected only from dead petioles attached to living trees of Livistona chinensis var. subglobosa on Hachijo Island, Japan.

Mycorrhizal synthesis, morpho-anatomical characterization of mycorrhizae, and evaluation of mycorrhiza-forming ability of Hydnum albidum-like species using monokaryotic and dikaryotic cultures.

Despite the economic and ecological importance of Hydnum species, in vitro synthesis of ectomycorrhizae of this genus has not been reported due to difficulties in establishing pure cultures. We inoculated pure cultures of 12 monokaryotic and 3 dikaryotic mycelial strains of an undescribed Hydnum albidum-like species on roots of axenic Pinus densiflora seedlings to synthesize ectomycorrhizae and to evaluate their mycorrhiza-forming ability. Six months after inoculation, both monokaryotic and dikaryotic strains formed ectomycorrhizae with Hartig net hyphae at the root cortex. Monokaryotic and dikaryotic strains exhibited similar morpho-anatomical characteristics of ectomycorrhizae, with the exception for clamped septa of emanating and outer mantle hyphae in the latter. Between monokaryotic and descendant dikaryotic strains, there were no significant differences in number of mycorrhizae in pine seedlings, whereas monokaryotic strains showed a greater total number of root tips and lower colonization rates than the descendant dikaryotic strains. These results indicate that both monokaryotic and dikaryotic mycelia of the H. albidum-like species can form mycorrhizae under axenic condition, and that can be applied toward the cultivation of hedgehog mushrooms.

Four mycelial strains of Entoloma clypeatum species complex form ectomycorrhiza-like roots with Pyrus betulifolia seedlings in vitro, and one develops fruiting bodies 2 months after inoculation.

Entoloma clypeatum species complex (ECSC) forms ectomycorrhiza-like roots (EMLR) with host plant species of Rosaceae or Ulmaceae. The EMLR colonized with ECSC are characterized by a thick fungal mantle, absence of a Hartig net structure, and collapse of the apical meristem caused by hyphal invasion. Some researchers have suggested parasitism of ECSC because of this unique mode of colonization; however, the nature of the interaction between ECSC and host plants has not been investigated in co-culture because of the difficulty of culturing this group of fungi. We established a procedure to synthesize EMLR of ECSC on pear seedlings using fungal cultures. Three conspecific strains of ECSC isolated from basidiospores and one strain isolated from EMLR were tested. Cultured mycelia were inoculated onto a modified Norkrans' C (MNC) or Hyponex-yeast-glucose (HYG) medium slant on the bottom of a polycarbonate jar and covered with autoclaved andosol or a vermiculite/sphagnum moss mixture (VSM); an axenically cultivated Pyrus betulifolia seedling was then planted in the jar. Five months after inoculation, the formation of EMLR with Hartig net-like hyphae was confirmed in all of the experimental plots. However, the rate of root colonization was significantly higher in experimental plots using andosol than in those using VSM. The growth of pear seedlings was similar irrespective of the level of root colonization, suggesting commensalism rather than parasitism of ECSC. One experimental plot using strain A3, an MNC slant, and andosol as a substrate produced ECSC fruiting bodies with mature basidia and basidiospores. The results suggested that our procedure enables the synthesis of EMLR of ECSC and cultivation of their fruiting bodies.

Novel tyrosinase inhibitors from liquid culture of Neolentinus lepideus.

Tyrosinase is the key enzyme that controls melanin formation in the human skin. We performed a screening of 96 extracts of mushroom cultures and fruiting bodies for examining their inhibitory activity against mushroom tyrosinase. The ethyl acetate extracts of culture filtrate of Neolentinus lepideus exhibited the strongest inhibitory activity. The active compounds 1 and 2 were purified by repeated chromatographic separations from the extract. On the basis of spectroscopic analyses, 1 and 2 were identified to be 1,3-dihydroisobenzofuran-4,5,7-triol and 5-methoxy-1,3-dihydroisobenzofuran-4,7-diol, respectively. Lineweaver-Burk plot of the enzyme reaction in the presence of 1 indicated that 1 was a potent competitive inhibitor. The respective IC50 values of 1 and 2 were 173 and 263 µg/mL. Compound 1 at 15 µg/mL suppressed melanin accumulation stimulated by α-MSH in the murine melanoma B16 cells, as well as the induced accumulation of both tyrosinase transcript and protein without inhibiting cell proliferation.

Taxonomy and phylogenetic position of Fomitiporia torreyae, a causal agent of trunk rot on Sanbu-sugi, a cultivar of Japanese cedar (Cryptomeria japonica) in Japan.

Trunk rot poses a substantial threat to Sanbu-sugi, one of the most economically important cultivars of Japanese cedar (Cryptomeria japonica). The etiology of this disease, including its main agents, is incompletely known. This trunk rot was attributed to Fomitiporia (Phellinus) hartigii or F. (Phellinus) punctata. Here we phylogenetically analyzed DNA sequences of four markers from a set of strains isolated from trunk-rot symptoms and recovered a single, monophyletic clade, indicating that a single taxon is involved. This clade was identified as Fomitiporia torreyae, a species described from eastern China. This analysis also proved that trunk rot and/or dieback of other conifers and broadleaf trees, including Sawara cypress (Chamaecyparis pisifera), Japanese umbrella pine (Sciadopitys verticillata) and Japanese pear (Pyrus pyrifolia var. culta), were caused by the same species. The phylogenetic approach to Fomitiporia revealed that the F. torreyae clade was closely related to F. bannaensis but clearly distinct from F. punctata, which originally was thought to be the cause of trunk rot in Sanbu-sugi. Fomitiporia torreyae is redescribed on the basis of more than 40 specimens from multiple hosts from Japan and China. Fomitiporia juniperina comb. nov. is proposed.

Hypochnicium sensu lato (Polyporales, Basidiomycota) from Japan, with descriptions of a new genus and three new species.

Species of Hypochnicium (Polyporales, Basidiomycota) collected from Japan were studied on their taxonomy by morphological and phylogenetic approaches. Phylogenetic analyses based on a nrDNA LSU and ITS dataset including the Japanese specimens and other publicly available ones show that Hypochnicium is polyphyletic. Since the clade containing the type species H. bombycinum was well-supported, we defined this clade as Hypochnicium s. str., and emended Hypochnicium to include restricted taxa with only smooth basidiospores. The new genus Neohypochnicium is proposed to accommodate the remaining taxa excluded from the genus Hypochnicium s. str., which includes both species with smooth basidiospores and ornamented ones. Three new species, Gyrophanopsis japonica, N. asiaticum and N. perlongicystidiosum are described and illustrated based on morphological and phylogenetic analyses using an ITS region dataset. In addition, the following 15 new combinations are proposed: N. albostramineum, N. aotearoae, N. capitulateum, N. cremicolor, N. cystidiatum, N. geogenium, N. guineense, N. huinayense, N. michelii, N. microsporum, N. patagonicum, N. pini, N. punctulatum, N. subrigescens and N. wakefieldiae. An identification key to Japanese species of Bulbillomyces, Gyrophanopsis, Hypochnicium and Neohypochnicium is provided.

Trichoderma mienum sp. nov., isolated from mushroom farms in Japan.

During an investigation of Hypocrea/Trichoderma species inhabiting mushroom bedlogs, we found five strains of an undescribed species from a culture collection. These were analyzed using a combined approach, including morphology of holomorph, cultural studies, and phylogenetic analyses of the rRNA gene cluster of the internal transcribed spacer region, translation elongation factor 1-α, and RNA polymerase subunit II gene sequences. Distinctive morphological characters include stromata with green ascospores produced on potato dextrose agar medium, and Gliocladium-like to irregularly Verticillium-like conidiophores. In phylogenetic analyses, this species belongs to the Semiorbis clade, but its morphological characteristics do not match the other members of this clade. Based on morphological observations and phylogenetic analyses, we describe this as a new species, Trichoderma mienum, representing its Hypocrea teleomorph and Trichoderma anamorph.

Two new species of Fulvifomes (Basidiomycota, Hymenochaetaceae) on threatened or near threatened tree species in Japan.

We describe two new species of Fulvifomes based on morphological observations and phylogenetic investigations. Both species were identified as Phellinus rimosus by former mycologists, but both are morphologically distinct from authenticated specimen of P. rimosus. Fulvifomes boninensis is characterized by perennial basidiomata, a sulcate pileus surface becoming rimose, lack of a distinct crust on the pileus surface, subdimitic hyphal system in the context, and ellipsoid basidiospores. This species is endemic to the Bonin Islands, Japan, and is specific to the host Morus boninensis, a red-listed tree species. Fulvifomes imazekii is characterized by perennial basidiomata, sulcate and velutinous pileus surface, lack of a crust on the pileus surface, dimitic hyphal system in the context, and broadly ellipsoid basidiospores. This species is specific to Berchemiella berchemiaefolia, and is known only from Mt. Yokogura-yama, in Kochi Prefecture, Japan. Fulvifomes boninensis and F. imazekii are considered threatened, because of their high host specificity each with a threatened tree species as well as the limited distribution of the former and the extremely small number of "mature individuals" of the latter. The following new combinations were also proposed: Fulvifomes aulaxinus, F. pappianus, and F. tepperi.

Systematic revision of Hydnum species in Japan.

Hydnum (Hydnaceae, Basidiomycota) exhibits endemic species diversity in East Asia; however, few comprehensive systematic studies have been conducted to date. Here, we performed morphological, ecological, phylogenetic, and biological evaluations of the taxonomy of Hydnum species in Japan. In total, 186 Japanese Hydnum specimens were used for morphological observations. Phylogenetic trees were constructed using sequence data of nuc rDNA internal transcribed spacer ITS1-5.8S-ITS2 (ITS) region and a portion of translation elongation factor 1-α (tef1). Intra- and interspecific mating tests using 78 monokaryotic strains of 13 species did not conflict with species delimitation inferred from their ITS and tef1 phylogenetic relationships. This study provides detailed morphological descriptions of 15 rigorously identified species from Japan, nine of which are described as new: H. alboluteum, H. albopallidum, H. pinicola, H. itachiharitake, H. minospororufescens, H. orientalbidum, H. subberkeleyanum, H. tomaense, and H. tottoriense. Three species documented in this work are new to Japan: H. boreorepandum, H. mulsicolor, and H. umbilicatum. The remaining three species (H. cremeoalbum, H. minus, and H. repando-orientale), previously reported from Japan, are redescribed using data from newly collected materials. We also transferred two old species (Hericium fimbrillatum and Sarcodon nauseofoetidus) from East Asian Hydnum into other genera.

Description of a new species of Gerhardtia (Lyophyllaceae, Agaricales) from Japan based on morphological and molecular phylogenetic analyses and live culture characteristics.

We describe a new species of Gerhardtia from Japan based on basidiomata morphology, live culture characteristics, and molecular phylogenetic analyses. Gerhardtia venosolamellata is found on broad-leaf litter, and is characterized by tricholomatoid to marasmioid basidiomata, an off-white to pale salmon-pink pileus surface with faint marginal striae, subdistant lamellae with lateral veins, a tomentose to strigose stipe base with hyphal strands generating arthroconidia measuring 4-7 × 2-3 µm, cyanophilic, elongate-ellipsoid to cylindrical, slightly verrucose or undulate basidiospores measuring 4.5-6 × 2.5-3 µm, and cyanophilic basidia measuring 25-35 × 5-6 µm and containing siderophilous granules. Phylogenetic analyses based on the internal transcribed spacer and large subunit regions of the fungal nrDNA indicates that G. venosolamellata is related to G. sinensis and G. highlandensis, but differs from the former with respect to basidiomata color, basidiospore shape, and habitat. An isotype specimen of G. highlandensis exhibited relatively close lamellae without veins, and slightly larger basidiospores (4.5-6.5 × 2.5-3 µm). Cultured mycelia of G. venosolamellata produced arthroconidia measuring 4.5-8.5 × 2.5-3 µm with both schizolytic and rhexolytic secession on MA and PDA media, and chlamydospores occasionally covered with crystals on MA and MYG media.

Taxonomy and ecology of Physisporinus forming synnema-like structures in freshwater environments.

Since 2011, we have collected fungi that form synnema-like structures (SSs) bearing many acanthophyses at the apex on water-splashed wood in streams in various regions of Japan. A provisional phylogenetic analysis of strains isolated from SSs based on their nrDNA sequences implied affinity with Physisporinus (Polyporales, Basidiomycota). However, it has not been reported that this genus forms SSs in freshwater habitats. We found a fungus forming not only SSs on the water-boundary part of wood but also resupinate basidiocarps with poroid hymenophores on nonsubmerged parts, and the morphological characteristics of the basidiocarps matched those of Physisporinus. Therefore, we investigated the relationship between SS-forming fungi and their sexual states by taxonomic approaches. Phylogenetic analyses based on nrDNA internal transcribed spacer (ITS) and large subunit (LSU) sequences indicated that SS-forming fungi diverged into five clades in Physisporinus. Each clade was discriminated by the color of SSs, morphology of acanthophyses, and cultural characteristics. Of the five clades, Clade 1, which consisted only of sequences of strains isolated from SSs and basidiocarps produced on rhizomorphs, was closely related to P. eminens and P. undatus, but the morphology of basidiocarps and the manner of basidiocarp development differed. Clade 5 was closely related to P. castanopsidis, P. crocatus, P. pouzarii, P. sanguinolentus, P. subcrocatus, P. tibeticus, and P. vitreus, but the basidiocarp morphology differed. Therefore, Clades 1 and 5 were described as two new species. Regarding Clades 2, 3, and 4, further taxonomic studies with additional specimens are required. SS and acanthophysis formation in wet habitats in streams and in culture could be recognized as new taxonomic and ecological characters of Physisporinus.

Two new species of Sistotrema s.l. (Cantharellales) from Japan with descriptions of their ectomycorrhizae.

We describe two new species of resupinate Sistotrema sensu lato (Cantharellales) collected in Japan: S. flavorhizomorphae and S. chloroporum. Both species have urniform basidia with more than four sterigmata and monomitic hyphal system, oil-rich hyphae in subiculum, which is typical for this genus. Sistotrema chloroporum is characterized by poroid hymenophore partly yellowish-green, basidia 4-6-spored, medium-sized basidiospores (4.5-6.5 × 3.5-6 µm), and broadleaf forest habitat. Sistotrema flavorhizomorphae is characterized by hydnoid-irpicoid hymenophore, bright yellowish rhizomorphs, basidia 6-8-spored, small basidiospores (3-3.5 × 2.5-3 µm), and pine forest habitat. Phylogenetic trees inferred from the fungal nrDNA ITS and LSU and the rpb2 sequences supported that both species were distinct and grouped with other ectomycorrhizal Sistotrema and Hydnum species, but their generic boundary was unclear. Mycorrhizae underneath basidiomes of both species were identified and described via molecular techniques. Mycorrhizae of S. chloroporum have similar characteristics to those of other Sistotrema s.l. and Hydnum species, i.e., S. confluens and H. repandum, whereas S. flavorhizomorphae has a distinct morpho-anatomy, for example, a distinct pseudoparenchymatous mantle. Comprehensive characterizations of basidiomes and mycorrhizae improve the taxonomic analysis of mycorrhizal species of Sistotrema s.l.

The genus Laetiporus (Basidiomycota, Polyporales) in East Asia.

Relationships among East Asian, North American and European Laetiporus sulphureus s. lat., a cosmopolitan brown rot species complex, were assessed with phylogenetic analyses and incompatibility tests. Three East Asian taxa, Laetiporus cremeiporus sp. nov., Laetiporus montanus and Laetiporus versisporus, are described and illustrated as well as compared with related taxa from Southeast Asia, North America and Europe. Phylogenetic analyses showed that L. cremeiporus and L. versisporus are clearly distinct species among Laetiporus taxa. The three conifer inhabiting species, Laetiporus conifericola, Laetiporus huroniensis and L. montanus, are closely related to each other. The European population of L. montanus exhibits two sequence variants of the EF1alpha: one is the same as observed in L. sulphureus in Europe and the other is that observed in East Asian population of L. montanus. A key to the known species of Laetiporus in the northern hemisphere is provided.

Isolation of isolactarane sesquiterpenes from a Phlebia tremellosa culture filtrate and their growth promotion effects on lettuce roots.

The ethyl acetate extract of the culture filtrate of Phlebia tremellosa promoted elongation of the lateral roots of lettuce seedlings at 250 µg/mL. We purified two compounds that promote root elongation by using activity-guided chromatographic fractionation. On the basis of spectroscopic analyses, these compounds were identified to be isolactarane sesquiterpenes derived from the dehydrogenation of merulactone, which was previously isolated from the same species. We named the purified compounds phlelactones A and B. Phlelactones A and B promoted primary root elongation at 100-300 and 10-30 µg/mL and the elongation and formation of lateral roots at 300-1000 and 30-100 µg/mL, respectively.

Phylogenetic relationships of Polyporus and morphologically allied genera.

Polyporus accommodates morphologically heterogeneous species and is divided into six infrageneric groups based on macromorphological characters. On the other hand allied genera have macro- and microscopic characters similar to those of Polyporus. The phylogenetic relationships of Polyporus and allied genera were established from sequences of RNA polymerase II second largest subunit (RPB2), nuclear ribosomal large subunit (nucLSU) and mitochondrial ATPase subunit 6 (ATP6). The molecular phylogenetic trees confirmed that Polyporus is a polyphyletic genus and recognized six major clades (1-6) containing species of Polyporus and several allied genera. Among the clades one contained three infrageneric groups of Polyporus and two allied genera, Datronia and Pseudofavolus while one other contained group Polyporellus and Lentinus. Five of the six major clades contained species belonging to a single infrageneric group, Favolus, Melanopus, Polyporellus or Polyporus. This suggests that morphological characters used to define these groups have phylogenetic significance and reveals the need for a taxonomic revision of Polyporus and its allied genera.