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.

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.

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.

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.

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).

Diversity and Distribution of Aspergillus fumigatus and Its Related Species in Izu and Ogasawara Islands, Japan.

The taxon Aspergillus section Fumigati comprises several causative agents of aspergillosis. Here, the distribution of Aspergillus sect. Fumigati in outdoor environments of Izu and Ogasawara Islands was investigated. Different strains were isolated from soil samples collected from 68 sites on 9 islands (Izu-oshima, Toshima, Shikinejima, Kozushima, Miyakejima, Hachijojima, Mukojima, Chichijima, and Hahajima), including different landscapes, and identified using morphological characteristics and calmodulin (CaM) sequences. Seven Aspergillus sect. Fumigati species were identified. The occurrence frequency of Aspergillus fumigatus was higher in forest sites on the islands, except for Ogasawara Islands, whereas that of species other than A. fumigatus was higher in bare land and grassland sites on all islands. The occurrence frequency of A. fumigatus was more than 50% on islands between Izu-oshima and Toshima, decreased on islands between Shikinejima and Hachijojima, and was zero on Ogasawara Islands. Considering other Aspergillus species, Aspergillus felis showed high occurrence frequency on islands between Izu-oshima and Shikinejima, Aspergillus pseudoviridinutans on islands between Kozushima and Hachijojima, and Aspergillus udagawae on Ogasawara Islands. At two study sites (grassland and forest sites), the soil was sampled throughout the year to evaluate whether the occurrence frequency of each fungal species was affected by sampling season. At the grassland site, A. pseudoviridinutans was isolated at more than 90% frequency, regardless of the sampling season. A. fumigatus occurrence frequency at the forest site ranged from 0% to 60% and greatly varied among sampling seasons. Thus, differences in island location and landscape affected the distribution of Aspergillus sect. Fumigati.

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.

Species Diversity based on Revised Systematics of Xerophilic Aspergillus section Restricti Isolated from Storage Rooms and Houses in Japan.

Eighty-seven strains of Aspergillus section Restricti were isolated from five storage rooms (50 strains) and 21 houses (37 strains) between 2014 and 2020. Eleven species were identified based on their morphological characteristics and molecular phylogeny using the rRNA internal transcribed spacer (ITS) region, calmodulin (CaM), ß-tubulin (benA), and RNA polymerase II second largest subunit (RPB2) sequences. A. penicillioides, which was known to cause the deterioration of cultural assets, was isolated at high frequency (73%) from the surfaces of 11 cultural assets in the storage rooms; A. clavatophorus and A. magnivesiculatus, which are closely related to A. penicillioides, were also isolated frequently (45 and 64%, respectively). Five species [A. clavatophorus (42.8%), A. penicillioides (42.8%), A. magnivesiculatus (14.3%), A. reticulatus (28.6%), and A. vitricola (28.6%)] were isolated from dust on the carpets in seven houses. Five species [A. clavatophorus (33.3%), A. penicillioides (55.5%), A. magnivesiculatus (44.4%), A. restrictus (44.4%), and A. gracilis (11.1%)] were isolated from dust on the bedding in nine houses. Using the taxonomic system described by Sklenár et al. (2017), five species (A. clavatophorus, A. magnivesiculatus, A. hordei, A. reticulatus, and A. glabripes) previously identified as A. penicillioides were confirmed as new to Japan.

Ubiquitous Distribution of Azole-Resistant Aspergillus fumigatus- Related Species in Outdoor Environments in Japan.

Aspergillus fumigatus-related species are responsible for causing aspergillosis, which is a fatal infectious disease. Recently, there has been a series of reports of A. fumigatus-related species that are resistant to azole drugs used in clinical practice for the treatment of fungal infections. Some of these species have been isolated from outdoor environments. Testing the drug susceptibility of the strains from outdoor environments, therefore, is important. In this study, we isolated and cultured 72 strains of A. fumigatus-related species from the outdoor environment in Japan. The isolates identified via morphological observation and molecular phylogenetic analysis were Aspergillus felis, Aspergillus lentulus, Aspergillus pseudoviridinutans, Aspergillus udagawae, and Aspergillus wyomingensis. The results of the drug susceptibility testing revealed that A. felis (6 of 14 strains) and A. pseudoviridinutans (13 of 17 strains) were resistant to itraconazole (ITCZ), with 4 mg/L or higher minimum inhibitory concentrations (MICs). The voriconazole (VRCZ)-resistant strains with 4 mg/L or higher MICs were A. felis (14 of 14), A. lentulus (4 of 4), A. pseudoviridinutans (15 of 17), A. udagawae (23 of 34), A. wyomingensis (1 of 3), and A. pseudoviridinutans (1 of 3). Among them, A. felis (1 of 14) and A. pseudoviridinutans (7 of 17) demonstrated 8 mg/L or higher MICs for ITCZ and VRCZ. These results indicate that A. fumigatus-related species resistant to ITCZ and VRCZ are widely distributed in outdoor environments in Japan.

Slow-growing fungi belonging to the unnamed lineage in Chaetothyriomycetidae form hyphal coils in vital ericaceous rhizodermal cells in vitro.

The diversity and functionality of ericoid mycorrhizal (ErM) fungi are still being understudied. Members of Chaetothyriomycetidae evolved a specific lifestyle of inhabiting extreme, poor, or toxic environments. Some taxa in this subclass, especially in Chaetothyriales, are also putative ErM taxa, but their mycorrhizal ability is mostly unknown because the members are generally hard to isolate from roots. This study herein focused on eight root isolates and provided their phylogeny and morphology of root colonization. Phylogenetic analysis based on rRNA sequences clarified that the isolated strains were not classified into Chaetothyriales, but in an unnamed lineage in Chaetothyriomycetidae. This lineage also contains rock isolates, bryosymbionts, and a resinicolous species as well as various environmental sequences obtained from soil/root samples. All strains grew extremely slow by mycelia on cornmeal or malt extract agar (2.9-8.5 mm/month) and formed hyphal coils in vital rhizodermal cells of sterile blueberry seedlings in vitro. This study illustrated the presence of a novel putative ErM lineage in Chaetothyriomycetidae.

Genetic variations and in vitro root-colonizing ability for an ericaceous host in Sarcoleotia globosa (Geoglossomycetes).

We discovered that Sarcoleotia globosa (Geoglossomycetes) fruited on the soil of ornamental Erica pot cultures, and its ascospores can germinate on plain agar. These findings prompted us to collect isolates from horticultural and natural environments in Japan and analyze their phylogeny and root colonizing ability. Pure cultures were successfully obtained from ascospores and surface-sterilized ericaceous roots. Phylogenetic analysis based on rRNA internal transcribed spacer sequences revealed that Japanese samples were separated into three strongly supported clades. Individual clade consisted of samples derived from (1) Erica pot cultures, (2) Rhododendron planted in a garden or Vaccinium pot culture, and (3) natural habitats in Hokkaido. Colony characteristics and in vitro root-colonizing morphology observed may correspond to these phylogenetic variations. Irrespective of the clades, all tested strains formed hyphal coils in vital rhizodermal cells of V. virgatum seedlings, which resembled those of ericoid mycorrhizae. Our results represent novel findings that can be the first step in unraveling the currently unknown ecology of geoglossoid fungi.

Variability of decomposing ability among fungi associated with the bleaching of subtropical leaf litter.

A diverse array of fungi take part in decomposition, but the variability of their decomposing ability is not fully understood. A total of 49 isolates of Ascomycota and Basidiomycota were obtained from bleached areas of leaf litter in a subtropical forest in southern Japan, and their ability to decompose leaves and recalcitrant compounds was quantified in pure culture. Mass loss of leaves of Castanopsis sieboldii (Fagaceae) and Schima wallichii (Theaceae) was evaluated at 20 C for 12 wk in vitro. Fungi caused a loss of -0.4% to 34.3% of the original leaf mass. The greatest mass loss was caused by four isolates of Mycena (Tricholomataceae). Isolates of Lachnocladiaceae caused greater mass loss of recalcitrant compounds, registered as acid-unhydrolyzable residue (AUR), which includes lignin, relative to Xylariaceae. Isolates of Rhytismataceae, Lachnocladiaceae, Marasmiaceae, and Omphalotaceae exhibited selective decomposition of AUR, Mycena exhibited simultaneous decomposition of AUR and components other than AUR, and Xylariaceae exhibited selective decomposition of components other than AUR. Mass losses of leaves and AUR caused by each fungal isolate were similar for C. sieboldii and S. wallichii. The Mycena isolates obtained from C. sieboldii caused greater mass losses of leaves and AUR than those obtained from the other tree species, suggesting a "home-field advantage" of decomposing potentialities, with implications for natural interactions beyond the in vitro system evaluated here.

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.

Diversity and Geographic Distribution of Ligninolytic Fungi Associated With Castanopsis sieboldii Leaf Litter in Japan.

The diversity and geographic pattern of ligninolytic fungi were investigated within the distribution range of an evergreen tree, Castanopsis sieboldii (Fagaceae), in Japan. Fungal isolates obtained from 18 sites in subtropical and temperate regions in Japan were classified into 50 operational taxonomic units in Ascomycota and Basidiomycota according to the base sequence of the rDNA internal transcribed spacer region. Ordination by nonmetric multidimensional scaling showed the separation of fungal compositions between the study sites which was significantly related to the latitude, longitude, and mean annual temperature (MAT) of the study sites. We applied variation partitioning to separate the magnitude of the climatic, spatial, and leaf property factors and found the roles of MAT and spatial factors in structuring fungal assemblages, suggesting the importance of both niche processes and such non-niche processes as priority effect and dispersal limitation. The bleached area on leaf litter was greater at sites with higher MAT and precipitation located at lower latitudes and at sites where some major ligninolytic fungi occurred at greater relative frequencies, indicating that not only the climatic conditions but also the biogeographic patterns of distribution of ligninolytic fungi influence the decomposition of lignin in leaf litter.

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.

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.

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.

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.

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.

Mycorrhizal Formation and Diversity of Endophytic Fungi in Hair Roots of Vaccinium oldhamii Miq. in Japan.

The root diameters as well as colonization and diversity of the root-associating fungi of Vaccinium oldhamii Miq. were investigated in order to obtain information on their mycorrhizal properties. The distal regions of roots had typical hair roots with diameters of less than 100 µm. Ericoid mycorrhizal fungi (ErMF) and dark septate endophytes (DSE) were frequently observed in the roots. Ascomycetes, particularly helotialean fungi, appeared to be dominant among the endophytic fungi of V. oldhamii roots. Furthermore, Rhizoscyphus ericae (Read) Zhuang & Korf and Oidiodendron maius Barron known as ErMF were detected more frequently than other fungal species.