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.

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.

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.

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.

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.

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.

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.

Taxonomy of corticioid fungi in Japan : Present status and future prospects.

Corticioid fungi form effused basidiomata with various hymenophore configurations, mostly on woody substrates, and they are presently classified into 15 orders in Agaricomycotina in the phylum Basidiomycota. In this review, the corticioid fungi of Japan are treated on the basis of the current classification system, and all currently known species are listed and classified by order. Japanese corticioid fungi number 442 species in 160 genera belonging to 14 orders. Analysis of the distribution of Japanese corticioid species reveals that 375 species (85% of the total) have wide distributions, and that the Japanese corticioid fungal flora is composed of species with diverse patterns of distribution. In the subtropical regions, 146 corticioid species belonging to 77 genera are listed, including 100 species from the Nansei Islands and 76 from the Ogasawara Islands. Although these two island groups are located at similar latitudes, they share only 30 corticioid species-21% of the total reported from the subtropical regions. Along with this summary of the current status, this study presents future directions for the taxonomic research of the Japanese corticioid fungi.

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.

Sulzbacheromyces sinensis, an Unexpected Basidiolichen, was Newly Discovered from Korean Peninsula and Philippines, with a Phylogenetic Reconstruction of Genus Sulzbacheromyces.

Most of lichens are formed by Ascomycota, less than 1% are lichenized Basidiomycota. The flora investigation of lichenized Ascomycota of South Korea has been well studied in the past three decades; however, prior to this study, none of basidiolichens was discovered. During the recent excursion, an unexpected clavarioid basidiolichen, Sulzbacheromyces sinensis was collected. Morphology and ecology has been recorded in detail. DNA was extracted, and ITS, 18S, 28S nuclear rDNA were generated. In order to further confirm the systematic position of the Korean specimens, maximum likelihood and Bayesian inference analysis including all the species of the order Lepidostromatales were conducted based on the ITS. As a result, the phylogenetic tree of the order Lepidostromatales was reconstructed, which differed from the previous studies. The inferred phylogenetic tree showed that species of Sulzbacheromyces in three different continents (Asia, South Africa and South America) were separated into three clades with support. In this study, the species worldwide distribution map of Lepidostromatales was illustrated, and S. sinensis had a widest distribution range (paleotropical extend to the Sino-Japanese) than other species (paleotropical or neotropical). Prior to this study, the range of distribution, southernmost and northernmost points and the fruiting time of S. sinensis were recorded, and the genus Sulzbacheromyces was firstly reported from Korean peninsula and Philippines.

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.

The giant mycoheterotrophic orchid Erythrorchis altissima is associated mainly with a divergent set of wood-decaying fungi.

The climbing orchid Erythrorchis altissima is the largest mycoheterotroph in the world. Although previous in vitro work suggests that E. altissima has a unique symbiosis with wood-decaying fungi, little is known about how this giant orchid meets its carbon and nutrient demands exclusively via mycorrhizal fungi. In this study, the mycorrhizal fungi of E. altissima were molecularly identified using root samples from 26 individuals. Furthermore, in vitro symbiotic germination with five fungi and stable isotope compositions in five E. altissima at one site were examined. In total, 37 fungal operational taxonomic units (OTUs) belonging to nine orders in Basidiomycota were identified from the orchid roots. Most of the fungal OTUs were wood-decaying fungi, but underground roots had ectomycorrhizal Russula. Two fungal isolates from mycorrhizal roots induced seed germination and subsequent seedling development in vitro. Measurement of carbon and nitrogen stable isotope abundances revealed that E. altissima is a full mycoheterotroph whose carbon originates mainly from wood-decaying fungi. All of the results show that E. altissima is associated with a wide range of wood- and soil-inhabiting fungi, the majority of which are wood-decaying taxa. This generalist association enables E. altissima to access a large carbon pool in woody debris and has been key to the evolution of such a large mycoheterotroph.

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.

Discovery or Extinction of New Scleroderma Species in Amazonia?

The Amazon Forest is a hotspot of biodiversity harboring an unknown number of undescribed taxa. Inventory studies are urgent, mainly in the areas most endangered by human activities such as extensive dam construction, where species could be in risk of extinction before being described and named. In 2015, intensive studies performed in a few locations in the Brazilian Amazon rainforest revealed three new species of the genus Scleroderma: S. anomalosporum, S. camassuense and S. duckei. The two first species were located in one of the many areas flooded by construction of hydroelectric dams throughout the Amazon; and the third in the Reserva Florestal Adolpho Ducke, a protected reverse by the INPA. The species were identified through morphology and molecular analyses of barcoding sequences (Internal Transcribed Spacer nrDNA). Scleroderma anomalosporum is characterized mainly by the smooth spores under LM in mature basidiomata (under SEM with small, unevenly distributed granules, a characteristic not observed in other species of the genus), the large size of the basidiomata, up to 120 mm diameter, and the stelliform dehiscence; S. camassuense mainly by the irregular to stellate dehiscence, the subreticulated spores and the bright sulfur-yellow colour, and Scleroderma duckei mainly by the verrucose exoperidium, stelliform dehiscence, and verrucose spores. Description, illustration and affinities with other species of the genus are provided.

Combined molecular and morphological data for improving phylogenetic hypothesis in Dacrymycetes.

We analyzed the DNA sequences of four gene regions, 28S and 18S rDNA, the ITS region and rpb2, to obtain a high resolution phylogenetic tree of Dacrymycetes. In addition, we comparatively studied micro- and macromorphological characteristics of representative species. The traditional generic classification based on morphological characteristics was not reflected by our molecular phylogenies. Ancestral state reconstructions indicated that the morphology of basidia and clamp connections are evolutionarily stable. In contrast, basidiocarps and basidiospore septation patterns appear variable. Dacrymyces unisporus shares the dolipores with non-perforate parenthesomes typical of other dacrymycetous taxa but is a unique species having predominantly non-bifurcate basidia and subglobose to ovoid basidiospores with transverse and longitudinal septa. In molecular phylogenies this species is a member of Dacrymycetes but always occupies a sister position in relation to the rest of the Dacrymycetes. Based on our results we propose a new genus, Unilacryma, for D. unisporus. For proper accommodation of this taxon, we introduce the family Unilacrymaceae and the order Unilacrymales.

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.

Karyological characterization of meiosis, post-meiotic mitosis and nuclear migration in the ectomycorrhizal fungus Rhizopogon roseolus (= R. rubescens).

Karyological characteristics during basidiosporogenesis of Rhizopogon roseolus, a member of the hypogeous Agaricomycetes, were studied by light and transmission electron microscopy. More than 1000 tissue fragments of young basidiomata were stained with HCl-Giemsa and observed by a light microscopy to evaluate nuclear behavior. Basidium morphology in the hymenium was observed by transmission electron microscopy. Meiosis and post-meiotic mitosis took place in the center of the basidium. Sterigmata appeared when the first meiotic division occurred, and the center of the basidium became constricted when the second meiotic division occurred. Asynchronous nuclear migration from the basidium into the basidiospores occurred after post-meiotic mitosis, producing eight uninucleate basidiospores. The nucleus migrated patchily into basidiospores. The pattern of post-meiotic mitosis of R. roseolus, in which post-meiotic mitosis took place in the center of the basidium, is reported for the first time.

Direct ethanol production from starch, wheat bran and rice straw by the white rot fungus Trametes hirsuta.

The white rot fungus Trametes hirsuta produced ethanol from a variety of hexoses: glucose, mannose, cellobiose and maltose, with yields of 0.49, 0.48, 0.47 and 0.47 g/g of ethanol per sugar utilized, respectively. In addition, this fungus showed relatively favorable xylose consumption and ethanol production with a yield of 0.44 g/g. T. hirsuta was capable of directly fermenting starch, wheat bran and rice straw to ethanol without acid or enzymatic hydrolysis. Maximum ethanol concentrations of 9.1, 4.3 and 3.0 g/l, corresponding to 89.2%, 78.8% and 57.4% of the theoretical yield, were obtained when the fungus was grown in a medium containing 20 g/l starch, wheat bran or rice straw, respectively. The fermentation of rice straw pretreated with ball milling led to a small improvement in the ethanol yield: 3.4 g ethanol/20 g ball-milled rice straw. As T. hirsuta is an efficient microorganism capable of hydrolyzing biomass to fermentable sugars and directly converting them to ethanol, it may represent a suitable microorganism in consolidated bioprocessing applications.

Degradation of chlorinated pesticide DDT by litter-decomposing basidiomycetes.

One hundred and two basidiomycete strains (93 species in 41 genera) that prefer a soil environment were examined for screening of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) biodegradation. Three strains within two litter-decomposing genera, Agrocybe and Marasmiellus, were selected for their DDT biotransformation capacity. Eight metabolites; 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), two monohydroxy-DDTs, monohydroxy-DDD, 2,2-dichloro-1,1-bis(4-chlorophenyl)ethanol, putative 2,2-bis(4-chlorophenyl)ethanol and two unidentified compounds were detected from the culture with Marasmiellus sp. TUFC10101. A P450 inhibitor, 1-ABT, inhibited the formation of monohydroxy-DDTs and monohydroxy-DDD from DDT and DDD, respectively. These results indicated that oxidative pathway which was catalyzed by P450 monooxygenase exist beside reductive dechlorination of DDT. Monohydroxylation of the aromatic rings of DDT (and DDD) by fungal P450 is reported here for the first time.

Production of ethanol by the white-rot basidiomycetes Peniophora cinerea and Trametes suaveolens.

The white-rot basidiomycetes, Peniophora cinerea and Trametes suaveolens, can produce ethanol from hexoses. Although T. suaveolens produced negligible amounts of ethanol under aerobic conditions, P. cinerea produced ethanol under both aerobic and semi-aerobic conditions and assimilated glucose, mannose, fructose, galactose, sucrose, maltose and cellobiose with yields of ethanol of 0.41, 0.45, 0.44, 0.19, 0.41, 0.44 and 0.45 g per g hexose, respectively. The corresponding results for T. suaveolens were 0.39, 0.3, 0.13, 0.2, 0.37, 0.35 and 0.31 g ethanol/g hexose. Furthermore, P. cinerea exhibited simultaneous saccharification and fermentation of amorphous cellulose.