Kinetic Control of Microtubule Morphology Obtained by Assembling Gold Nanoparticles on Living Fungal Biotemplates.

Self-assembly of nanoparticles on living biotemplate surfaces is a promising route to fabricate nano- or microstructured materials with high efficiency and efficacy. We used filamentous fungi to fabricate microtubules of gold nanoparticles through a novel approach that consists of isolating the hyphal growth from the nanoparticle media. This improved methodology resulted in better morphological control and faster adsorption kinetics, which reduced the time needed to form homogeneous microtubules and allowed for control of microtubule thickness through successive additions of nanoparticles. Differences in the adsorption rates due to modifications in the chemical identity of colloidal gold nanoparticles indicated the influence of secondary metabolites and growth media in the fungi metabolism, which demonstrated the need to choose not only the fungus biotemplate but also the correct medium to obtain microtubules with superior properties.

Strawberry Fruit Rot Caused by Neopestalotiopsis iranensis sp. nov., and N. mesopotamica.

A new species of Neopestalotiopsis based on both morphological and molecular characteristics is described. Neopestalotiopsis iranensis sp. nov. isolated from rotted strawberry (Fragaria ananassa) fruits as well as from stolon and leaf lesions in Kurdistan province, Iran. Initially, light tan and sunken spots developed on fruits and resulted in a soft decay of the fruit flesh. The new species is morphologically distinguished from similar species with different conidium size and by possessing longer apical appendages, as well as some knobbed basal appendages. Phylogenetic analyses (Bayesian inference, maximum likelihood and maximum parsimony analyses) based on internal transcribed spacer, ß-tubulin, and partial translation elongation factor 1-alpha combined gene sequences also indicated that this species is phylogenetically distinct from others. Moreover, strawberry crop is introduced here as a new host for N. mesopotamica.

Genome analysis of Daldinia eschscholtzii strains UM 1400 and UM 1020, wood-decaying fungi isolated from human hosts.

BACKGROUND: Daldinia eschscholtzii is a wood-inhabiting fungus that causes wood decay under certain conditions. It has a broad host range and produces a large repertoire of potentially bioactive compounds. However, there is no extensive genome analysis on this fungal species. RESULTS: Two fungal isolates (UM 1400 and UM 1020) from human specimens were identified as Daldinia eschscholtzii by morphological features and ITS-based phylogenetic analysis. Both genomes were similar in size with 10,822 predicted genes in UM 1400 (35.8 Mb) and 11,120 predicted genes in UM 1020 (35.5 Mb). A total of 751 gene families were shared among both UM isolates, including gene families associated with fungus-host interactions. In the CAZyme comparative analysis, both genomes were found to contain arrays of CAZyme related to plant cell wall degradation. Genes encoding secreted peptidases were found in the genomes, which encode for the peptidases involved in the degradation of structural proteins in plant cell wall. In addition, arrays of secondary metabolite backbone genes were identified in both genomes, indicating of their potential to produce bioactive secondary metabolites. Both genomes also contained an abundance of gene encoding signaling components, with three proposed MAPK cascades involved in cell wall integrity, osmoregulation, and mating/filamentation. Besides genomic evidence for degrading capability, both isolates also harbored an array of genes encoding stress response proteins that are potentially significant for adaptation to living in the hostile environments. CONCLUSIONS: Our genomic studies provide further information for the biological understanding of the D. eschscholtzii and suggest that these wood-decaying fungi are also equipped for adaptation to adverse environments in the human host.

New and interesting penzigioid Xylaria species with small, soft stromata.

Six penzigioid Xylaria species that are characterized by small, discoid to pulvinate, soft stromata are included in this study. Xylaria albocinctoides, X. bicampaniformis and X. lechatii are described as new; Nummularia albocincta, Hypoxylon carabayense and H. discolor are moved to the genus Xylaria to form new combinations X. albocincta, X. carabayensis and X. discolor respectively. An identification key is provided for the major aggregates of Xylaria that harbor penzigioid species as well as the species of the X. frustulosa aggregate, to which the six studied penzigioid Xylaria species belong.

Three new Xylaria species from Russian Far East.

Three new species of Xylaria, X. lepidota, X. primorskensis and X. sibirica were collected from Primorsky Territory in Russian Far East and neighboring areas. Xylaria primorskensis is a species with erect, stipitate stromata, whereas X. lepidota and X. sibirica produce stunted, sessile stromata. Cultures were obtained from X. primorskensis and X. sibirica. Xylaria sibirica formed a Xylocoremium anamorph in culture, while X. primorskensis remained sterile.

A new leaf blight disease of turfgrasses caused by Microdochium poae, sp. nov.

A novel species of Microdochium was identified as the causal agent of a leaf blight of Poa pratensis (Kentucky blue grass) and Agrostis stolonifera (Creeping bentgrasses), two cold-season turfgrasses widely grown on golf courses in northern China. This disease first appears as small, water-soaked, and scattered leaf spots. Under conditions of high temperatures and successive days of rain, the infected leaves rapidly lose their integrity and large diseased patches appear. Fungal strains were isolated from blighted leaf spots. A phylogenetic analysis based on the nuc rDNA internal transcribed spacer regions and 5.8S rRNA gene (ITS1-5.8S-ITS2 = ITS) and parts of the ß-tubulin (TUB2) and RNA polymerase II second largest subunit (RPB2) genes strongly supported that these isolates are a distinct evolutionary lineage in Microdochium (Microdochiaceae, Xylariales) that represents a new taxonomic species, herein named as M. poae. Microscopic characters confirmed that these strains were morphologically distinct from known Microdochium species. The pathogenicity of M. poae was confirmed by inoculating spore suspension on both grasses and reisolation of the pathogen from symptomatic tissues. The optimal growth temperature suggests that the occurrence of the new leaf blight disease caused by M. poae was significantly different from the microdochium patch disease caused by M. nivale.

A MYST Histone Acetyltransferase Modulates Conidia Development and Secondary Metabolism in Pestalotiopsis microspora, a Taxol Producer.

Reverse genetics is a promising strategy for elucidating the regulatory mechanisms involved in secondary metabolism and development in fungi. Previous studies have demonstrated the key role of histone acetyltransferases in transcriptional regulation. Here, we identified a MYST family histone acetyltransferase encoding gene, mst2, in the filamentous fungus Pestalotiopsis microspora NK17 and revealed its role in development and secondary metabolism. The gene mst2 showed temporal expression that corresponded to the conidiation process in the wild-type strain. Deletion of mst2 resulted in serious growth retardation and impaired conidial development, e.g., a delay and reduced capacity of conidiation and aberrant conidia. Overexpression of mst2 triggered earlier conidiation and higher conidial production. Additionally, deletion of mst2 led to abnormal germination of the conidia and caused cell wall defects. Most significantly, by HPLC profiling, we found that loss of mst2 diminished the production of secondary metabolites in the fungus. Our data suggest that mst2 may function as a general mediator in growth, secondary metabolism and morphological development.

Xylaria karyophthora: a new seed-inhabiting fungus of Greenheart from Guyana.

In 2010-2011, a Xylaria sp. was documented growing from seeds of both Chlorocardium rodiei and Chlorocardium venenosum, a commercially valuable timber in Guyana. We conducted extensive surveys in 2015-2016, where this Xylaria sp. was observed fruiting from ca. 80% of dispersed seeds in both natural and logged forests in the Upper Demerara-Berbice, Potaro-Siparuni, and the Cuyuni-Mazaruni districts of central Guyana. Species of Xylaria are ascomycetous fungi generally characterized by black, carbonaceous, multiperitheciate ascoma commonly found growing on dead wood. Combined teleomorphic and molecular data indicate that the fungus represents a novel species, described here as Xylaria karyophthora.

Studies on Morinia: recognition of Morinia longiappendiculata sp. nov. as a new endophytic fungus, and a new circumscription of Morinia pestalozzioides.

A new coelomycete, Morinia longiappendiculata sp. nov., isolated from living stems of four plant species in central Spain, is described. The distinctive morphological characteristics of this fungus are the production of conidia with long basal and apical appendages on filiform conidiogenous cells that contrasts with the short-appendaged conidia and cylindrical conidiogenic cells of the type species, M. pestalozzioides. Comparative sequence analysis of the ITS rDNA region and fragments of the translation elongation factor 1alpha, actin and chitin synthase 1 genes and the study of the HPLC profiles of the M. longiappendiculata and M. pestalozzioides isolates supported the recognition of the new species. Comparison of the ITS rDNA sequences of the Morinia isolates with GenBank sequences indicated that the genus belongs to the Amphisphaeriaceae with the highest similarity to Bartalinia and Truncatella. Bresadola's original definition of M. pestalozzioides is updated by adding information on conidiogenesis and molecular data. A lectotype and epitype are designated for the species. A study of bioactive metabolites revealed that M. pestalozzioides cultures produced moriniafungin, a novel sordarin analog with potent antifungal activity.

Morphological and Molecular Analysis Identifies the Associated Fungus ("Xianghui") of the Medicinal White Jelly Mushroom, Tremella fuciformis, as Annulohypoxylon stygium.

White jelly mushroom, Tremella fuciformis, is a popular edible mushroom with interesting medicinal properties (e.g., immunostimulating, antidiabetic). The formation of T. fuciformis basidiomes is highly dependent on the presence of a specific host fungus, both in nature and for industrial production. This host has traditionally been indicated as "Xianghui" in China, yet which or how many fungal species Xianghui comprises is unclear, with various authorities claiming different species. At present, Annulohypoxilon archeri is generally assumed to be the main Xianghui species, but this has not yet been confirmed experimentally. The implementation of older, premolecular-based research data (i.e., morphological) with present, sequence-based data to solve the identity remains confusing and studies addressing both identification methods in combination are lacking. The unclear identity of Xianghui is a major obstacle for further understanding of the important relationship between the host(s) and T. fuciformis. In this study, we collected a wild specimen of T. fuciformis together with several nearby stroma of Xianghui, cocultivated T. fuciformis with the Xianghui isolates, and observed basidiome formation. Internal transcribed spacer (ITS) sequence analysis showed that all Xianghui spore isolates belonged to the same species and both morphological analysis of sexual stages and ITS ß-tubulin and actin gene sequences of the Xianghui specified it as Annulohypoxylon stygium. The ITS sequences of the newly identified Xianghui further closely matched those of the Xianghui strains used in the mushroom industry, showing that wild and culture spawn Xianghui in China consist of A. stygium. In contrast with previous conclusions, A. stygium, and not A. archeris, seems to be the preferred host of T. fuciformis.

New Hypoxylon and Nemania species from Costa Rica and Taiwan.

Six xylariaceous fungi, including two Hypoxylon taxa and four Nemania taxa, are described as new. They were collected from either Costa Rica or Taiwan. Two of the Nemania species--N. flavitextura and N. primolutea--were cultured and typical Geniculosporium anamorphs were produced.

The genus Pyrenomyxa and its affinities to other cleistocarpous Hypoxyloideae as inferred from morphological and chemical traits.

Types and authentic specimens of Hypoxylon piceum, Pulveria porrecta, and Pyrenomyxa invocans were studied for morphological traits and extrolite (= secondary metabolite) profiles generated by analytical HPLC with UV-visual and mass spectrometric detection. The orange stromatal pigments of P. invocans are rubiginosin A and mitorubrinol. It lacks three different types of extrolites (BNT, macrocarpone and hypomiltin) that are known from Hypoxylon taxa and occur in H. piceum and P. porrecta. In agreement with morphological traits, the latter two names are regarded as synonymous and transferred to Pyrenomyxa. Another species from Eastern Russia, Pyrenomyxa morganii sp. nov., is recognized. It contains yet unidentified azaphilones besides BNT and orsellinic acid, and its culture produces 5-methylmellein and a virgariella-like anamorph. These findings suggest a close relationship of Pyrenomyxa to Hypoxylon and emphasize the utility of chemotaxonomic traits for fungal taxonomy in general. Pyrenomyxa is accepted ad interim until the phylogenetic relationships among Hypoxylon have been further evaluated by means of chemotaxonomic, morphological and molecular methods.

Phylogeny of Rosellinia capetribulensis sp. nov. and its allies (Xylariaceae).

A new Rosellinia species, R. capetribulensis isolated from Calamus sp. in Australia is described. R. capetribulensis is characterized by perithecia immersed within a carbonaceous stroma surrounded by subiculum-like hyphae, asci with large, barrel-shaped amyloid apical apparatus and large dark brown spores. Morphologically, R. capetribulensis appears to be similar to R. bunodes, R. markhamiae and R. megalospora. To gain further insights into the phylogeny of this new taxon we analyzed the ITS-5.8S rDNA using maximum parsimony and likelihood methods. In addition, a morphological dataset also was analyzed phylogenetically to investigate possible affinities. ITS rDNA based phylogenies reveal that R. capetribulensis is closely related to other Rosellinia species showing closest affinity to R. arcuata, RL necatrix and R. pepo. However, analysis of R. capetribulensis forms an unsupported branch sister to these taxa. Results from the morphological matrix indicate a close morphological affinity to members of Rosellinia subgenus Rosellinia. Despite that ITS rDNA and morphological analyses present difficulties in constructing a proper phylogenetic framework among Rosellinia and allied genera, there is sufficient evidence to support the establishment of the new taxon in the genus Rosellinia. The morphological similarities and differences between R. capetribulensis and allied genera such as Astrocystis and Entoleuca are also briefly discussed.

Functional analysis of a melanin biosynthetic gene using RNAi-mediated gene silencing in Rosellinia necatrix.

Rosellinia necatrix causes white root rot in a wide range of fruit trees and persists for extended periods as pseudosclerotia on root debris. However, the pathogenesis of this disease has yet to be clarified. The functions of endogeneous target genes have not been determined because of the inefficiency in genetic transformation. In this study, the function of a melanin biosynthetic gene was determined to examine its role in morphology and virulence. A polyketide synthase gene (termed as RnPKS1) in the R. necatrix genome is homologous to the 1,8-dihydroxynaphthalene (DHN) melanin biosynthetic gene of Colletotrichum lagenarium. Melanin-deficient strains of R. necatrix were obtained by RNA interference-mediated knockdown of RnPKS1. The virulence of these strains was not significantly reduced compared with the parental melanin-producing strain. However, knockdown strains failed to develop pseudosclerotia and were degraded sooner in soil than the parental strain. Microscopic observations of albino conidiomata produced by knockdown strains revealed that melanization is involved in synnema integrity. These results suggest that melanin is not necessary for R. necatrix pathogenesis but is involved in survival through morphogenesis. This is the first report on the functional analysis of an endogenous target gene in R. necatrix.

An endophytic Nodulisporium sp. from Central America producing volatile organic compounds with both biological and fuel potential.

A Nodulisporium sp. (Hypoxylon sp.) has been isolated as an endophyte of Thelypteris angustifolia (Broadleaf Leaf Maiden Fern) in a rainforest region of Central America. It has been identified both on the basis of its morphological characteristics and by scanning electron microscopy as well as ITS sequence analysis. The endophyte produces volatile organic compounds (VOCs) that have both fuel (mycodiesel) and use for biological control of plant disease. When grown on potato dextrose agar, the organism uniquely produces a series of ketones, including acetone; 2-pentanone; 3-hexanone, 4-methyl; 3-hexanone, 2,4- dimethyl; 2-hexanone, 4-methyl, and 5-hepten, 2-one and these account for about 25% of the total VOCs. The most abundant identified VOC was 1,8 cineole, which is commonly detected in this group of organisms. Other prominent VOCs produced by this endophyte include 1-butanol, 2- methyl, and phenylethanol alcohol. Moreover, of interest was the presence of cyclohexane, propyl, which is a common ingredient of diesel fuel. Furthermore, the VOCs of this isolate of Nodulisporium sp. were selectively active against a number of plant pathogens, and upon a 24 h exposure caused death to Phytophthora palmivora, Rhizoctonia solani, and Sclerotinia sclerotiorum and 100% inhibition to Phytophthora cinnamomi with only slight to no inhibition of the other pathogens that were tested. From this work, it is becoming increasingly apparent that each isolate of this endophytic Nodulisporium spp., including the Daldina sp. and Hypoxylon spp. teleomorphs, seems to produce its own unique set of VOCs.

Potential antitumor agent from the endophytic fungus Pestalotiopsis photiniae induces apoptosis via the mitochondrial pathway in HeLa cells.

4-(3',3'-Dimethylallyloxy)-5-methyl-6-methoxy-phthalide (DMMP) has previously been isolated from the endophytic fungus Pestalotiopsis photiniae. Although the cytotoxic activities of DMMP have been reported, little is known concerning the molecular mechanism of its cytotoxic effect. In the present study, we investigated the effect of DMMP on the growth of several types of cancer cell lines and investigated the mechanism of its antiproliferative effect. DMMP caused the growth inhibition of human cancer lines HeLa, MCF7 and MDA-MB-231, but had little antiproliferative effect on MRC5 normal lung cells. DMMP also significantly caused cell cycle arrest in the G1 phase and upregulated the cyclin-dependent kinase inhibitor p27KIPI protein in the HeLa cells. Moreover DMMP was able to induce marked nuclear apoptotic morphology in HeLa cells. DMMP induced apoptosis and loss of mitochondrial membrane potential (ΔΨm) in the HeLa cells. Although the activated forms of caspase-9 and -3 in HeLa cells were detected, pretreatment with caspase inhibitors (Ac-DEVD-CHO and Z-VAD-FMK) failed to attenuate DMMP-induced cell death. In addition, protein levels of the p53 family members, p53 and p73, were upregulated, and DMMP significantly increased the mRNA expression of pro-apoptotic Bcl-2 family genes (PUMA, NOXA, Bax, Bad and Bim). HPV E6-E7 mRNA levels were reduced. In conclusion, DMMP demonstrates potential for use in the treatment of cervical cancer.

Cytological analysis of mycelial incompatibility in Rosellinia necatrix.

When the mycelia of Rosellinia necatrix encounter mycelia with a different genetic background, distinct barrage lines form. In this study, we observed hyphal interactions between compatible and incompatible R. necatrix pairs by means of light and electron microscopy. Although we observed perfect hyphal anastomosis in compatible pairs of isolates, the hyphae never anastomosed in incompatible pairs (i.e., the hyphae remained parallel or crossed over without merging). These behaviours appeared to result from the detection of or failure to detect one or more diffusible factors. The attraction to other hyphae in pairs of incompatible isolates was increased by supplementation of the growing medium with activated charcoal, although no anastomosis was observed and ultrastructural observation confirmed a lack of hyphal anastomosis. Programmed cell death (PCD) started with one of the two approaching hyphae. Heterochromatin condensation and genomic DNA fragmentation were not observed. Moreover, cell damage began with the tonoplast and continued with the plasma and nuclear membranes, suggesting that the PCD observed in heterogenic incompatibility of R. necatrix was a vacuole-mediated process.

Phylogenetic relationships of Nemania plumbea sp. nov. and related taxa based on ribosomal ITS and RPB2 sequences.

During a survey fungal diversity of xylariaceous fungi in Thailand, a new Nemania species, N. plumbea, was identified. Nemania plumbea is characterized by soft-textured grey stromata on a persistent mat of white hyphae, pale brown ascospores with a short germ-slit on the more convex side. It also produces a Geniculosporium-like anamorph in culture. In order to evaluate its phylogenetic relationships among related species and genera, ITS-5.8S rDNA and RPB2 were analysed separately and simultaneously. Results from the phylogenetic analyses indicate that there is close phylogenetic association between N. plumbea and N. aenea. A preliminary account into the natural grouping of Xylariaceae based on ITS-5.8S rDNA and RPB2 sequences is also discussed.

Changes in secondary metabolism during stromatal ontogeny of Hypoxylon fragiforme.

Stromata of Hypoxylon fragiforme were studied during the vegetation period by hplc profiling, revealing changes in the composition during stromatal development. Cytochalasin H and two new cytochalasins named fragiformins A-B were identified as major constituents of the young, maturing stromata, whereas mature, ascogenous material yielded large amounts of mitorubrin-type azaphilones. The above compounds, further cytochalasins from Xylariaceae and other fungi, and additional azaphilones of the mitorubrin type were assayed for their nematicidal effects against Caenorhabditis elegans and their antimicrobial activities against Bacillus subtilis, Yarrowia lipolytica, and various filamentous fungi. The results confirmed data in the literature on broad-spectrum non-selective activities of azaphilones and cytochalasins in biological systems. Most interestingly, laboratory cultures of the above Hypoxylon spp. mainly produced dihydroisocoumarin derivatives and were found devoid of mitorubrins and cytochalasins. These rather drastic changes in the secondary metabolism of H. fragiforme and the above biological activities are discussed in relation to the possible biological functions of secondary metabolites (extrolites) in the Hypoxyloideae.

Morphological studies in Rosellinia (Xylariaceae): the first step towards a polyphasic taxonomy.

More than 90 species are accepted in the xylariaceous genus Rosellinia. Thorough morphological characterisation of all known species will provide a taxonomic hypothesis that can be later tested by DNA sequence analysis. Exploratory analyses of ascospore morphology, shape of ascus apical structure, and anamorph genus suggest six morphological groups in the genus.