Talaromyces mellisjaponici sp. nov., a xerophilic species isolated from honey in Japan.

Five isolates of a xerophilic Talaromyces species were obtained from honey in Japan. Molecular phylogenetic analysis based on a combined dataset for four regions (rRNA internal transcribed spacer, ß-tubulin, calmodulin and RNA polymerase II second largest subunit) revealed that the strains formed an independent clade in section Trachyspermi, which is sister to Talaromyces affinitatimellis, Talaromyces basipetosporus and Talaromyces speluncarum. The strains and their relatives have different growth on creatine agar, yeast extract sucrose agar and dichloran 18 % glycerol agar, different branching patterns (mostly monoverticillate or biverticillate, less frequently divaricate or terverticillate), and different sizes and surface structures of conidia. Xerotolerance tests were also conducted using media adjusted to five different sucrose concentrations (0, 20, 40, 60 and 80 %). The colony diameters of the strains were larger than those of T. affinitatimellis, T. basipetosporus and T. speluncarum at each sucrose concentration. Altogether, the obtained morphological, molecular and physiological data allowed the proposal of Talaromyces mellisjaponici sp. nov. for this novel species, with NBRC 116048T as the type strain.

Aspergillus verrucosus sp. nov., a xerophilic species isolated from house dust and honey in Japan.

Three strains of a xerophilic Aspergillus species were isolated from house dust and honey in Japan. A molecular phylogenetic analysis based on the combined dataset for four regions (internal transcribed spacer rDNA, calmodulin, ß-tubulin, and RNA polymerase II second largest subunit) revealed that the strains formed an independent lineage, sister to Aspergillus halophilicus classified in section Restricti. Morphological comparisons show that the strains differ from A. halophilicus in three aspects: (i) the size of cleistothecia, as well as the surface structure and size of ascospores, (ii) the ability to grow on Harrold's agar and dichloran 18 % glycerol agar, and (iii) the lack of conidiophore formation on potato dextrose agar +20 % NaCl. These strains could be clearly distinguished from all known Aspergillus section Restricti species. Therefore, we consider it to be a novel species and propose the name Aspergillus verrucosus sp. nov. (NBRC 115547T).

A cryptic root isolate belonging to Geoglossales from potted Rhododendron: its molecular phylogeny and ability to colonize an ericoid mycorrhizal host in vitro.

Although the lifestyle of Geoglossales remains largely unknown, recent advancements have established a hypothesis regarding the ericoid mycorrhizal lifestyle of geoglossoid fungi. In this study, we focused on one isolate of Geoglossales sp. obtained from surface-sterilized roots of potted Rhododendron transiens. We aimed to reveal the phylogenetic position and in vitro colonizing ability of this species in the hair roots of ericoid mycorrhizal plants. Based on our multigene phylogenetic tree, this species is a sister of the genus Sarcoleotia which has not been reported from either other studies or field environment. Its ascocarps could not be obtained, and conspecific sequences were not found in the databases and repositories examined. The Geoglossales sp. colonized the vital rhizodermal cells of blueberries in vitro with hyphal coils. There were relatively large morphological variations of coils consistent with extraradical hyphae; however, overall, the colonization morphologically resembled those by Sarcoleotia globosa and representative ericoid mycorrhizal fungi. The taxonomy and ecological significance of the species remain to be resolved; nevertheless, our results suggest that the ericoid mycorrhizal lifestyle may be widespread within Geoglossales.

A case of allergic fungal rhinosinusitis caused by Schizophyllum commune identified in both patient's nasal sputum and veranda's soil samples.

This is a case report of allergic fungal rhinosinusitis caused by Schizophyllum commune (S. commune) identified in a patient's nasal mucus and environmental soil sample using (r)DNA sequencing. Although filamentous basidiomycetes, including S. commune, are known as environmental pathogens causing allergic respiratory diseases worldwide, many patients with infections caused by S. commune have not been correctly diagnosed. Repeated exposures to environmental floating fungi supposedly make an easy sensitization and colonization of fungi in the nasal passages, resulting in the onset of allergic fungal rhinosinusitis due to S. commune in our living environments. This report indicates the importance of reconsidering allergic respiratory diseases associated with our living environments.

Mariannamides A and B, new cyclic octapeptides isolated from Mariannaea elegans NBRC102301.

Two new cyclic octapeptides, mariannamides A (1) and B (2), have been isolated from Mariannaea elegans NBRC102301, a Pinus densiflora-derived filamentous fungus. Their structures were elucidated to be cyclo-(l-Leu1-l-Pro1-l-Pro2-l-Leu2-l-Ile1-l-Pro3-l-Val1-l-Ile2) and cyclo-(l-Leu1-l-Pro1-l-Pro2-l-Leu2-l-Ile1-l-Pro3-l-Val1-l-Val2) based on spectroscopic data and Marfey's method. Mariannamide A (1) promoted mRNA expression of sirtuin 1 (SIRT1) in C2C12 cells, a mouse skeletal muscle myoblast cell line, and showed the antimicrobial activity against Escherichia coli and Cryptococcus neoformans.

Decaturenol A and known oxalicine related meroterpenoids isolated from Penicillium decaturense RO050, and their new biological activities.

Decaturenol A (1), a new oxalicine related meroterpenoid, has been isolated from Penicillium decaturense RO050 along with seven known compounds (2-8). The structure of 1 was elucidated by spectroscopic data. The effects of isolated compounds (1-8) on endoplasmic reticulum (ER) stress-induced cell death in HT22 hippocampal nerve cells and on the interleukin 10 (IL-10)-induced expression of CD163, a M2 phenotype marker, in human monocyte-derived macrophages were evaluated. While decaturenol A (1) exhibited a protective effect on ER stress-induced cell death in HT22 cells at 10 µM, on the other hand oxalicine A (7) showed cytotoxic activity (IC50 = 5.9 µM). Additionally, decaturenol A (1), decaturins D (2), E (3), and B (4) inhibited the IL-10-induced expression of CD163 each at a concentration of 20 µg/mL.

Identification of Lycopodium Alkaloids Produced by an Ultraviolet-Irradiated Strain of Paraboeremia, an Endophytic Fungus from Lycopodium serratum var. longipetiolatum.

12- epi-Lycopodine (1), a Lycopodium alkaloid, along with lycopodine (2) and huperzine A (3), were discovered in the mycelium of Paraboeremia sp. Lsl3KI076, a UV-irradiated strain of Paraboeremia sp. Lsl3, an endophytic fungus from Lycopodium serratum Thunb. var. longipetiolatum Spring. Additionally, a trace of 1 was isolated from Phlegmariurus nummulariifolius (Blume) Ching, and the structure was elucidated on the basis of spectroscopic data. This is the first report proving that a new naturally occurring Lycopodium alkaloid can be obtained from an endophytic fungus.

Identification of Two Phenanthrene Derivatives from Australasian Allied Species in Genus Dendrobium.

Genus Dendrobium (Orchidaceae) contains numerous species. Phylogenetic analyses based on morphological characteristics and DNA sequences indicated that this genus is divided into two major groups: Asian and Australasian clades. On the other hand, little is known about the phytochemical differences and similarities among the species in each clade. In this study, we selected 18 Dendrobium species (11 from the Asian clade and 7 from the Australasian clade) and constructed HPLC profiles, arrays composed of relative intensity of the chromatographic peaks. Next, orthogonal partial least square discriminant analysis (OPLS-DA) was applied to the profile matrix to classify Dendrobium species into the Asian and Australasian clades in order to identify the peaks that significantly contribute to the class separation. In the end, two phenanthrenes, 4,9-dimethoxyphenanthrene-2,5-diol 1 and 1,5-dimethoxyphenanthrene-2,7-diol 2, which contributed to the class separation, were isolated from the HPLC peaks. The existence of 2 was limited to the genetically related Australasian species.

Increased Expression of 15-Hydroxyprostaglandin Dehydrogenase in Spinal Astrocytes During Disease Progression in a Model of Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive, and fatal neurodegenerative disease caused by selective loss of motor neurons. Both ALS model mice and patients with sporadic ALS have increased levels of prostaglandin E2 (PGE2). Furthermore, the protein levels of microsomal PGE synthase-1 and cyclooxygenase-2, which catalyze PGE2 biosynthesis, are significantly increased in the spinal cord of ALS model mice. However, it is unclear whether PGE2 metabolism in the spinal cord is altered. In the present study, we investigated the protein level of 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a key enzyme in prostaglandin metabolism, in ALS model mice at three different disease stages. Western blotting revealed that the 15-PGDH level was significantly increased in the lumbar spinal cord at the symptomatic stage and end stage. Immunohistochemical staining demonstrated that 15-PGDH immunoreactivity was localized in glial fibrillary acidic protein (GFAP)-positive astrocytes at the end stage. In contrast, 15-PGDH immunoreactivity was not identified in NeuN-positive large cells showing the typical morphology of motor neurons in the anterior horn. Unlike 15-PGDH, the level of PGE2 in the spinal cord was increased only at the end stage. These results suggest that the significant increase of PGE2 at the end stage of ALS in this mouse model is attributable to an imbalance of the synthetic pathway and 15-PGDH-dependent scavenging system for PGE2, and that this drives the pathogenetic mechanism responsible for transition from the symptomatic stage.

In vitro resistance of Aspergillus fumigatus to azole farm fungicide.

Azole resistance in Aspergillus fumigatus is mainly due to a point mutation in the 14α-sterol demethylase (CYP51A) gene, which encodes the target of azole fungicides. Moreover, overexpression of CYP51B or multidrug resistance (MDR) gene is supposedly related to the mechanism of azole resistance in A. fumigatus. In this study, we tried to induce resistance to tetraconazole, an azole fungicide, in strains of A. fumigatus from a farm and then investigated mutation and expression of their CYP51A, CYP51B, and multidrug resistance (MDR) genes. Three tetraconazole resistant strains were induced and their minimum inhibitory concentration (MIC) for tetraconazole was 145 mg/L. However, the MICs of itraconazole (ITZ), posaconazole (POS), and voriconazole (VRZ) obtained by an E-test of the three tetraconazole resistant strains were 0.064-0.19 mg/L for ITZ, 0.023-0.32 mg/L for POS, and 0.047-0.064 mg/L for VRZ. No gene mutations were detected in the CYP 51A sequence amplified in these strains. RT-PCR of cyp51A and cyp51B indicated that the tetraconazole resistant strains more highly expressed these genes than the susceptible strain in tetraconazole containing medium.

Distinct phylogeographic structure recognized within Desmazierella acicola.

Desmazierella acicola (anamorph Verticicladium trifidum, Chorioactidaceae) represents a frequent colonizer of pine needles in litter. Considering the global diversity and distribution of pine species, we expected different phylogenetic lineages to exist in different geographical and climatic areas inhabited by these hosts. We compared DNA sequence data with phenotypic characteristics (morphology of the anamorph and growth at three different temperatures) of 43 strains isolated mostly from pine and also spruce needle litter sampled in various geographical areas. Analyses of ITS rDNA recovered eight geographically structured lineages. Fragments of genes for the translation elongation factor 1-α, and the second largest subunit of RNA polymerase II reproduced similar lineages, although not all of them were monophyletic. The similarity in ITS sequences among the clade with samples from Continental-Atlantic Europe and four other clades was lower than 95%. Several lineages exhibit also a tendency toward host specificity to a particular pine species. Growth tests at different temperatures indicated a different tolerance to specific climatic conditions in different geographic areas. However, the surveyed phenotypic characteristics also showed high variation within lineages, most evident in the morphology of the anamorph. Until a morphological study of the teleomorph is carried out, all of these lineages should be treated as distinct populations within a single species.

Does farm fungicide use induce azole resistance in Aspergillus fumigatus?

Azole resistance of Aspergillus fumigatus isolates has been reported worldwide and it would appear to be mainly due to a point mutation in the 14α-sterol demethylase (CYP51A) gene, which is the target enzyme for azoles. The mutation has been confirmed in isolates from patients who received long-term itraconazole (ITZ) therapy and from agricultural fields where high levels of azole fungicides were employed. However, the relationship between farm environments and azole-resistant A. fumigatus has not been fully studied. In this investigation, 50 isolates of A. fumigatus were obtained from a farm where tetraconazole has been sprayed twice a year for more than 15 years. The mean minimum inhibitory concentration (MIC) of isolates was 0.74 (0.19-1.5) mg/L against ITZ, which was below the medical resistance level of ITZ. The sequence of CYP51A from isolates indicated no gene mutations in isolates from the farm. Antifungal susceptibility of isolates to tetraconazole showed that spraying with tetraconazole did not induce resistance to tetraconazole or ITZ in A. fumigatus.

Bleaching of leaf litter and associated microfungi in subboreal and subalpine forests.

Fungal decomposition of lignin leads to the whitening, or bleaching, of leaf litter, especially in temperate and tropical forests, but less is known about such bleaching in forests of cooler regions, such as boreal and subalpine forests. The purposes of the present study were to examine the extent of bleached area on the surface of leaf litter and its variation with environmental conditions in subboreal and subalpine forests in Japan and to examine the microfungi associated with the bleaching of leaf litter by isolating fungi from the bleached portions of the litter. Bleached area accounted for 21.7%-32.7% and 2.0%-10.0% of total leaf area of Quercus crispula and Betula ermanii, respectively, in subboreal forests, and for 6.3% and 18.6% of total leaf area of B. ermanii and Picea jezoensis var. hondoensis, respectively, in a subalpine forest. In subboreal forests, elevation, C/N ratio and pH of the FH layer, and slope aspect were selected as predictor variables for the bleached leaf area. Leaf mass per area and lignin content were consistently lower in the bleached area than in the nonbleached area of the same leaves, indicating that the selective decomposition of acid unhydrolyzable residue (recalcitrant compounds such as lignin, tannins, and cutins) enhanced the mass loss of leaf tissues in the bleached portions. Isolates of a total of 11 fungal species (6 species of Ascomycota and 5 of Basidiomycota) exhibited leaf-litter-bleaching activity under pure culture conditions. Two fungal species (Coccomyces sp. and Mycena sp.) occurred in both subboreal and subalpine forests, which were separated from each other by approximately 1100 km.

Two novel Archaeorhizomyces species isolated from ericoid mycorrhizal roots and their association with ericaceous plants in vitro.

Archaeorhizomyces is a diverse and ubiquitous genus of the subphylum Taphrinomycotina, which contains soil-inhabiting/root-associated fungi. Although ecological importance and root-associating lifestyles of Archaeorhizomyces can be postulated, morphological aspects of fungal body and root colonization are largely unknown due to the scarcity of cultures. We obtained three unidentified Archaeorhizomyces isolates from ericoid mycorrhizal (ErM) roots of Rhododendron scabrum and Rhododendron × obtusum collected in Japan. To advance our understanding of lifestyle of the genus, we investigated their general morphology, phylogeny, and in vitro root-colonizing ability in ericoid mycorrhizal hosts, Vaccinium virgatum and Rhododendron kaempferi. Some morphological characteristics, such as slow glowing white-to-creamy-colored colonies and formation of yeast-like or chlamydospore-like cells, were shared between our strains and two described species, Archaeorhizomycesfinlayi and Archaeorhizomyces borealis, but they were phylogenetically distant. Our strains were clearly distinguished as two undescribed species based on morphology and phylogenetic relationship. As seen in typical ErM fungi, both species frequently formed hyphal coils within vital rhizodermal cells of ErM plants in vitro. The morphology of hyphal coils was also different between species. Consequently, two novel species, Archaeorhizomyces notokirishimae sp. nov. and Archaeorhizomyces ryukyuensis sp. nov., were described.

Species diversity of xerophilic Aspergillus and Penicillium in marine surface waters revealed by isolation using osmophilic medium.

The species diversity of xerophilic and halophilic fungi distributed in marine surface water was studied at four local sites located in two geographically distant regions in Japan. At each site, 5-10 samples were collected and isolated using an osmophilic medium. Species identification was conducted based on nucleotide sequence of calmodulin or ß -tubulin and morphological characteristics for Aspergillus, Penicillium, and Talaromyces, and on the sequences of rRNA internal transcribed spacer for the other taxa. Overall, 231 strains were isolated from all sites and classified into 85 species belonged to 12 orders and 33 genera. The isolates that showed better mycelial growth than the control（no NaCl added) in the halotolerance test were defined as halophilic fungi, and only 22 species（10 Aspergillus species and 12 Penicillium species) were halophilic. Comparison of the halophilic fungal flora of the two regions revealed that four species common to both regions were isolated for Aspergillus, but no such species were isolated for Penicillium. Given that 15 halophilic species（10 Aspergillus and 5 Penicillium species) are known to be xerophilic species distributed in indoor environments, it can be inferred that indoor xerophilic species are likely to be widely distributed in marine surface water.

2(3H)-Dihydrofranolactone metabolites from Pleosporales sp. NUH322 as anti-amyotrophic lateral sclerosis drugs.

Amyotrophic lateral sclerosis (ALS) is a devastating motor disease with limited treatment options. A domestic fungal extract library was screened using three assays related to the pathophysiology of ALS with the aim of developing a novel ALS drug. 2(3H)-dihydrofuranolactones 1 and 2, and five known compounds 3-7 were isolated from Pleosporales sp. NUH322 culture media, and their protective activity against the excitotoxicity of ß-N-oxalyl-L-α,ß-diaminopropionic acid (ODAP), an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamatergic agonist, was evaluated under low mitochondrial glutathione levels induced by ethacrynic acid (EA) and low sulfur amino acids using our developed ODAP-EA assay. Additional assays evaluated the recovery from cytotoxicity caused by transfected SOD1-G93A, an ALS-causal gene, and the inhibitory effect against reactive oxygen species (ROS) elevation. The structures of 1 and 2 were elucidated using various spectroscopic methods. We synthesized 1 from D-ribose, and confirmed the absolute structure. Isolated and synthesized 1 displayed higher ODAP-EA activities than the extract and represented its activity. Furthermore, 1 exhibited protective activity against SOD1-G93A-induced toxicity. An ALS mouse model, SOD1-G93A, of both sexes, was treated orally with 1 at pre- and post-symptomatic stages. The latter treatment significantly extended their lifespan (p = 0.03) and delayed motor deterioration (p = 0.001-0.01). Our result suggests that 1 is a promising lead compound for the development of ALS drugs with a new spectrum of action targeting both SOD1-G93A proteopathy and excitotoxicity through its action on the AMPA-type glutamatergic receptor.

Assessment of the fungal diversity and succession of ligninolytic endophytes in Camellia japonica leaves using clone library analysis.

Fungal assemblages in live, newly shed and partly decomposed leaves of Camellia japonica were investigated with a clone library analysis to assess the fungal diversity and succession in a subtropical forest in southern Japan. Partly decomposed leaves were divided into bleached and adjacent nonbleached portions to estimate the fungi functionally associated with lignin decomposition in the bleached portions, with an emphasis on Coccomyces sinensis (Rhytismataceae, Ascomycota). From 144 cloned 28S ribosomal DNA (rDNA) sequences, 48 operational taxonomic units (OTUs) were defined based on a sequence similarity threshold of 98%. Forty-one (85%) of the 48 OTUs belonged to the Ascomycota and seven OTUs (15%) to the Basidiomycota. Twenty-six OTUs (54%) were detected only once (singletons). The number of OTUs and the diversity indices of the fungal assemblages in the different leaves were in this order: live leaves > newly shed leaves > bleached portions > nonbleached portions of partly decomposed leaves. The fungal assemblages were similar in newly shed leaves and the bleached portions of partly decomposed leaves. Ligninolytic fungi of the genera Coccomyces, Lophodermium and Xylaria were frequently detected in the bleached portions. OTU3, identified as Coccomyces sinensis, was detected in live and newly shed leaves and the bleached portions of partly decomposed leaves, suggesting that this fungus latently infects live leaves, persists after leaf fall and takes part in lignin decomposition.

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.

Acremomannolipin A, the potential calcium signal modulator with a characteristic glycolipid structure from the filamentous fungus Acremonium strictum.

By the newly developed assay method, the glycolipid Acremomannolipin A (1) was isolated from a filamentous fungus Acremonium strictum as a potential calcium signal modulator. The structure of 1 elucidated on the basis of intensive spectroscopic analyses as well as its degradation studies is quite unique: the d-mannopyranose is connected to d-mannitol through a ß-glycoside linkage; all the hydroxyls in the mannose are highly masked as peresters with aliphatic acids, and this moiety is made hydrophobic, whereas the mannitol part exhibits a highly hydrophilic property. The compound (1) showed the characteristic bioactivity property, enabling calcineurin deletion cells to grow in the presence of Cl(-), which would be caused by calcium signal modulating. The activity was so potent as to exert the effect at a concentration of 200 nM.

Metabolic Diversity of Xylariaceous Fungi Associated with Leaf Litter Decomposition.

Fungi in the family Xylariaceae are primary agents of leaf litter decomposition. However, the diversity of carbon source utilization by xylariaceous fungi and the relative effects on this from environmental and phylogenetic factors are largely unknown. This study assessed the metabolic diversity and redundancy of xylariaceous fungi, associated with leaf litter decomposition, by measuring their in vitro capacity to utilize multiple carbon sources. The work identified the relative influences of geographic and climatic sources, as well as the taxonomic and phylogenetic relatedness, of the fungi. Using Biolog EcoPlateTM, 43 isolates belonging to Nemania, Xylaria, Nodulisporium, Astrocystis, and Hypoxylon, isolated from Castanopsis sieboldii leaf litter at eight sites in Japan, were found to have the capacity to utilize a variety of carbohydrates, amino acids/amines, carboxylic acids, and polymers. The genera of xylariaceous fungi and their origins significantly affected their metabolic diversity and utilization of carbon sources. Variation partitioning demonstrated that dissimilarities in carbon utilization among fungal isolates were mostly attributable to site differences, especially climatic factors: mean annual temperature and precipitation, and maximum snow depth. Moreover, xylariaceous isolates that originated from adjacent sites tended to have similar patterns of carbon source utilization, suggesting metabolic acclimation to local environmental conditions.