Exploring the Xylariaceae and its relatives.

The Xylariaceae and its relatives rank as one of the best-known members of the Ascomycota. They are now well recognized for their diversity, global distribution, ecological activities and their outstanding novel metabolites with wide ranging bioactivity.

Antioxidant Activity and Cytotoxicity against Cancer Cell Lines of the Extracts from Novel Xylaria Species Associated with Termite Nests and LC-MS Analysis.

Xylaria species associated with termite nests or soil have been considered rare species in nature and the few which have been reported upon have been found to act as a rich source of bioactive metabolites. This study evaluated 10 ethyl acetate extracts of five new Xylaria species associated with termite nests or soil for their antioxidant activity, and cytotoxicity against different cancer and normal cell lines. DPPH and ABTS radical scavenging activities of the extracts demonstrated strong capacity with low IC50 values. The highest observed activities belonged to X. vinacea SWUF18-2.3 having IC50 values of 0.194 ± 0.031 mg/mL for DPPH assay and 0.020 ± 0.004 mg/mL for ABTS assay. Total phenolic content ranged from 0.826 ± 0.123 to 3.629 ± 0.381 g GAE/g crude extract which correlated with antioxidant activities. The high total phenolic content could contribute to the high antioxidant activities. Cytotoxicity was recorded against A549, HepG2, HeLa and PNT2 and resulted in broad spectrum to specific activity depending on the cell lines. The highest activities were observed with X. subintraflava SWUF16-11.1 which resulted in 11.15 ± 0.32 to 13.17 ± 2.37% cell viability at a concentration of 100 µg/mL. Moreover, LC-MS fingerprints indicated over 61 peaks from all isolates. There were 18 identified and 43 unidentified compounds compared to mass databases. The identified compounds were from various groups of diterpenoids, diterpenes, cytochalasin, flavones, flavonoids, polyphenols, steroids and derivatives, triterpenoids and tropones. These results indicate that Xylaria spp. has abundant secondary metabolites that could be further explored for their therapeutic properties.

Twelve New Taxa of Xylaria Associated with Termite Nests and Soil from Northeast Thailand.

The diversity of Xylaria species associated with termite nests in northeast Thailand was investigated. Among the 14 taxa included in this study, 11 species and one variety were described as new, and another two species resemble the existing taxa, X. escharoidea and X. nigripes. The newly described taxa are X. chaiyaphumensis, X. conica, X. fulvescens, X. ischnostroma, X. margaretae, X. minima, X. reinkingii var. microspora, X. siamensis, X. sihanonthii, X. subintraflava, X. thienhirunae, and X. vinacea. Their morphological and cultural characteristics are described and illustrated, and their ITS, α-actin and ß-tubulin sequences were analysed. A dichotomous key to the 17 species of Xylaria occurring in Thailand is provided.

Utilization of oil palm decanter cake for valuable laccase and manganese peroxidase enzyme production from a novel white-rot fungus, Pseudolagarobasidium sp. PP17-33.

Oil palm decanter cake (OPDC) in the current study was converted to valuable products as laccase and manganese peroxidase (MnP) by an undescribed strain of the white-rot fungus, Pseudolagarobasidium sp. PP17-33. The optimization to enhance the production of enzymes through solid-state fermentation was performed using Plackett-Burman design and response surface methodology. The highest observed laccase was 5.841 U/gds and observed MnP was 5.156 U/gds, which enhanced yield by 2.59-fold and 1.94-fold from the non-optimization. The optimized medium (mg/g of OPDC) consisted of 0.852 mg CuSO4·5H2O, 13.512 mg glucose, 2 mg yeast extract, 0.2 mg KH2PO4, 1.5 mg MgSO4·7H2O, 0.01 mg FeSO4·7H2O, 0.15 mg MnSO4·H2O, 0.01 mg ZnSO4·7H2O and 0.3 mg Tween 80 (pH 5.0) when incubated at 30 °C for 7 days. The most significant variables of laccase and MnP productions were CuSO4·5H2O and glucose concentrations. This study is the first to report on the production of ligninolytic enzymes from OPDC waste using white-rot fungi. In addition, five different white-rot fungi, Coriolopsis aspera, C. retropicta, Dentipellis parmastoi, Nigroporus vinosus and Tyromyces xuchilensis, are newly observed producers of ligninolytic enzymes in Thailand. The results obtained from this study are significant not only for agro-industrial waste management but also for value-added enzyme production.

Gelatinomyces siamensis gen. sp. nov. (Ascomycota, Leotiomycetes, incertae sedis) on bamboo in Thailand.

Gelatinomyces siamensis gen. sp. nov., incertae sedis within Leotiomycetes, the Siamese jelly-ball, is described. The fungus was collected from bamboo culms and branches in Nam Nao National Park, Phetchabun, Thailand. It presents as a ping-pong ball-sized and golf ball-like gelatinous ascostroma. The asci have numerous ascospores, are thick-walled, and arise on discoid apothecia which are aggregated and clustered to form the spherical gelatinous structures. An hyphomycete asexual morph is morphologically somewhat phialophora-like, and produces red pigments. On the basis of phylogenetic analysis based on rRNA, SSU, and LSU gene sequences, the lineage is closest to Collophora rubra. However, ITS sequences place the fungus on a well-separated branch from that fungus, and the morphological and ecological differences exclude it from Collophora.

Fungi in Thailand: a case study of the efficacy of an ITS barcode for automatically identifying species within the Annulohypoxylon and Hypoxylon genera.

Thailand, a part of the Indo-Burma biodiversity hotspot, has many endemic animals and plants. Some of its fungal species are difficult to recognize and separate, complicating assessments of biodiversity. We assessed species diversity within the fungal genera Annulohypoxylon and Hypoxylon, which produce biologically active and potentially therapeutic compounds, by applying classical taxonomic methods to 552 teleomorphs collected from across Thailand. Using probability of correct identification (PCI), we also assessed the efficacy of automated species identification with a fungal barcode marker, ITS, in the model system of Annulohypoxylon and Hypoxylon. The 552 teleomorphs yielded 137 ITS sequences; in addition, we examined 128 GenBank ITS sequences, to assess biases in evaluating a DNA barcode with GenBank data. The use of multiple sequence alignment in a barcode database like BOLD raises some concerns about non-protein barcode markers like ITS, so we also compared species identification using different alignment methods. Our results suggest the following. (1) Multiple sequence alignment of ITS sequences is competitive with pairwise alignment when identifying species, so BOLD should be able to preserve its present bioinformatics workflow for species identification for ITS, and possibly therefore with at least some other non-protein barcode markers. (2) Automated species identification is insensitive to a specific choice of evolutionary distance, contributing to resolution of a current debate in DNA barcoding. (3) Statistical methods are available to address, at least partially, the possibility of expert misidentification of species. Phylogenetic trees discovered a cryptic species and strongly supported monophyletic clades for many Annulohypoxylon and Hypoxylon species, suggesting that ITS can contribute usefully to a barcode for these fungi. The PCIs here, derived solely from ITS, suggest that a fungal barcode will require secondary markers in Annulohypoxylon and Hypoxylon, however. The URL http://tinyurl.com/spouge-barcode contains computer programs and other supplementary material relevant to this article.

Xylariaceae on the fringe.

The Xylariaceae is one of the best-known pyrenomycete families (Ascomycota) and is distributed throughout the world. The majority are wood inhabitants and are prevalent in tropical and subtropical regions. Halorosellinia oceanicum is the most widely distributed in mangroves and can be regarded as truly manglicolous being frequently recorded as the dominant member of the family in such environments in S.E. Asia. In Malaysian mangroves, members of the Xylariaceae have been found to be numerically important with up to 9% present in one mangrove ecosystem. A further twelve xylariaceous genera are reported as occurring as their teleomorphs in mangrove forest and their immediate surroundings including Anthostomella, Astrocystis, Biscogniauxia, Camillea, Daldinia, Fasciatispora, Hypoxylon, Kretzschmaria, Nemania, Nipicola, Rosellinia and Xylaria. Furthermore, the presence of species from a number of these taxa, especially species of Anthostomella and Xylaria, are regularly isolated as endophytes from a variety of mangrove plant species. Mangrove Xylariaceae are also well known for their ability to produce novel and often bioactive metabolites.

Ascus apical apparatus and ascospore characters in Xylariaceae.

Members of Xylariaceae (Ascomycota) are recognized and classified mainly on the morphological features of their sexual state. In a number of genera high morphological variation of stromatal characters has made confident recognition of generic and specific boundaries difficult. There are, however, a range of microscopical characteristics which can in most cases make distinctions, especially at generic level, even in the absence of molecular data. These include details of the apical apparatus in the ascus (e.g. disc-shaped, inverted hat-shaped, rhomboid, composed of rings, amyloid, non-amyloid); position and length of the germ slit; and presence and type of ascospore wall ornamentation as seen by scanning electron microscopy (SEM). Unfortunately many of the classical studies on xylariaceous genera omitted these features and were undertaken long before the development of scanning electron microscopy. More recent studies have, however, demonstrated their value as diagnostic characters in the family. Camillea is for example, instantly recognizable by its rhomboid or diamond shaped apical apparatus, and the distinctive inverted hat or urniform type is usually prominent in Xylaria, Rosellinia, Kretzschmaria, and Nemania. At least six categories of apical apparatus based on shape and size can be recognized. Ascospore ornamentation as seen by SEM has been exceptionally useful and provided the basis for separating Camillea from Biscogniauxia and other xylariaceous genera.

Ascospore discharge, germination and culture of fungal partners of tropical lichens, including the use of a novel culture technique.

A total of 292 lichen samples, representing over 200 species and at least 65 genera and 26 families, were collected, mainly in Thailand; 170 of the specimens discharged ascospores in the laboratory. Generally, crustose lichens exhibited the highest discharge rates and percentage germination. In contrast, foliose lichen samples, although having a high discharge rate, had a lower percentage germination than crustose species tested. A correlation with season was indicated for a number of species. Continued development of germinated ascospores into recognizable colonies in pure culture was followed for a selection of species. The most successful medium tried was 2 % Malt-Yeast extract agar (MYA), and under static conditions using a liquid culture medium, a sponge proved to be the best of several physical carriers tested; this novel method has considerable potential for experimental work with lichen mycobionts.

Purification and biochemical characterization of an extracellular beta-glucosidase from the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm.

An extracellular beta-glucosidase was purified from culture filtrates of the wood-decaying fungus Daldinia eschscholzii (Ehrenb.:Fr.) Rehm grown on 1.0% (w/v) carboxymethyl-cellulose using ammonium sulfate precipitation, ion-exchange, hydrophobic interaction and gel filtration chromatography. The enzyme is monomeric with a molecular weight of 64.2 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and has a pI of 8.55. The enzyme catalyzes the hydrolysis of p-nitrophenyl-beta-D-glucopyranoside (PNPG) as the substrate, with a K(m) of 1.52 mM, and V(max) of 3.21 U min mg(-1) protein. Glucose competitively inhibited beta-glucosidase with a K(i) value of 0.79 mM. Optimal activity with PNPG as the substrate was at pH 5.0 and 50 degrees C. The enzyme was stable at pH 5.0 at temperatures up to 50 degrees C. The purified beta-glucosidase was active against PNPG, cellobiose, sophorose, laminaribiose and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel or o-nitrophenyl-beta-d-galactopyranoside. The activity of beta-glucosidase was stimulated by Ca(2+), Co(2+), Mg(2+), Mn(2+), glycerol, dimethyl sulfoxide (DMSO), dithiothreitol and EDTA, and strongly inhibited by Hg(2+). The internal amino acid sequences of D. eschscholziibeta-glucosidase have similarity to the sequences of the family 3 beta-glucosyl hydrolase.

Molecular study of the genus Astraeus.

The aim of the present work was to determine phylogenetic relationships among Astraeus species and to support macroscopic and microscopic characters of Astraeus with analysis of the ITS rDNA region. Collections of Astraeus basidiomes were made from different geographical areas in Thailand and compared with existing collections made worldwide. The marriage of observations on morphological features, including basidiospore ornamentation and molecular data demonstrated the presence of several Astraeus species. Sequences for 41 Astraeus collections were compared and the phylogenetic analyses grouped Thai Astraeus collections into two distinct groups. One contained A. odoratus and an Asian species described herein as A. asiaticus. There are at least two additional species: A. pteridis, and one so far un-named from North America. Our results show that molecular data can be used in combination with traditional morphological characteristics to resolve taxonomic uncertainties in the genus Astraeus.