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.

Influence of arbuscular mycorrhizal fungi (AMF) on zinc biogeochemistry in the rhizosphere of Lindenbergia philippensis growing in zinc-contaminated sediment.

The association of arbuscular mycorrhizal fungi (AMF) with the roots of Lindenbergia philippensis (Cham.) Benth., sampled from a Zn-contaminated settling pond at a zinc smelter, significantly enhanced Zn accumulation (72,540 ± 5,092 mg kg⁻¹ dry weight) in rhizosphere sediment amended with 1,000 mg L⁻¹ of Zn sulfate solution compared to fungicide-treatments that suppressed AMF colonization. This can be explained by a significant proportion of Zn being found in rectangular crystals that were associated with the root mucilaginous sheath. Despite this, all treatments maintained the same Zn coordination geometry in both Zn oxidation state and the coordinated neighbouring atoms. X-ray absorption spectroscopy (XAS) showed a Zn(II) oxidation state as a core atom and associated with six oxygen atoms symmetrically arranged in an octahedral coordination and coordinated with sulfur. The results may indicate a role for AMF in enhancing Zn immobilization in the rhizosphere of indigenous plants that successfully colonize Zn mining and smelting disposal sites.

Fungal biotransformation of zinc silicate and sulfide mineral ores.

In this work, several fungi with geoactive properties, including Aspergillus niger, Beauveria caledonica and Serpula himantioides, were used to investigate their potential bioweathering effects on zinc silicate and zinc sulfide ores used in zinc extraction and smelting, to gain understanding of the roles that fungi may play in transformations of such minerals in the soil, and effects on metal mobility. Despite the recalcitrance of these minerals, new biominerals resulted from fungal interactions with both the silicate and the sulfide, largely resulting from organic acid excretion. Zinc oxalate dihydrate was formed through oxalate excretion by the test fungi and the mineral surfaces showed varying patterns of bioweathering and biomineral formation. In addition, calcium oxalate was formed from the calcium present in the mineral ore fractions, as well as calcite. Such metal immobilization may indicate that the significance of fungi in effecting metal mobilization from mineral ores such as zinc silicate and zinc sulfide is rather limited, especially if compared with bacterial sulfide leaching. Nevertheless, important bioweathering activities of fungi are confirmed which could be of local significance in soils polluted by such materials, as well as in the mycorrhizosphere.

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.

Biotransformation of ent-kaur-16-en-19-oic acid by Psilocybe cubensis.

Biotranformation of ent-kaur-16-en-19-oic acid (1) using Psilocybe cubensis resulted in hydroxylated products. After two days of incubation, ent-16beta,17-dihydroxy-kauran-19-oic acid (2) was isolated. After further incubation for nine days, two novel metabolites, ent-12alpha,16beta,17-trihydroxy-kauran-19-oic acid (3) and ent-11alpha,16beta,17-trihydroxy-kauran-19-oic acid (4), were obtained. The metabolites were identified by spectroscopic methods and X-ray crystallography. Compounds 1-4 were evaluated for their cytotoxic properties against the human leukaemia K562 cell line; only compound 1 showed moderate activity.

Isolation and identification of mucinolytic actinomycetes.

Biochemical and physiological tests, and 16S rRNA gene sequences, were used to classify nine Actinomycete strains isolated from soil and sand samples in Thailand. These strains were isolated based on their ability to readily degrade mucin glycoproteins. A turbidometric based mucinolytic assay was developed to confirm this. In addition all strains showed significant production of proteases. Phylogenetic analysis of the strains revealed that from the nine isolated Actinomycete strains eight were closely related to Streptomyces species and one was identified as belonging to the genus Kitasatospora. The biochemical and physiological tests performed identified two strain pairs that were similar (with only 3.9% difference observed) and this was in accordance with the phylogenetic results obtained. The remaining strains were distinct from each other, with the soil-isolated strains forming a separate clade to the sand-isolated strains in the inferred phylogenetic trees. The isolated mucinolytic Actinomycete strains will be the subject of further investigations into their proteolytic and glycosidic activity. Mucin degrading enzymes such as these are studied for their potential to be used for the development of a drug delivery system.

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.

Biodegradation of benzo(a)pyrene by a newly isolated Fusarium sp.

Benzo(a)pyrene (BaP) is a five-ring polycyclic aromatic hydrocarbon produced by the incomplete combustion of organic materials. It is one of the priority pollutants listed by the US Environmental Protection Agency. This study describes a fungal isolate that is able to biodegrade benzo(a)pyrene. The filamentous fungus, isolated from leaves of Pterocarpus macrocarpus Kurz., was identified as a Fusarium sp. (strain E033). Fusarium sp. E033 was able to survive in the presence of benzo(a)pyrene concentrations up to 1.2 mM (300 mg L(-1)). Biodegradation experiments using 0.4 mM (100 mg L(-1)) benzo(a)pyrene demonstrated that Fusarium sp. E033 was able to degrade 65-70% of the initial benzo(a)pyrene provided, and two transformation products, a dihydroxy dihydro-benzo(a)pyrene and a benzo(a)pyrene-quinone, were detected within 30 days of incubation at 32 degrees C. The factors affecting biodegradation efficiency were also investigated. While increasing aeration promoted better fungal growth and benzo(a)pyrene biodegradation, increasing the glucose concentration from 5 to 50 mM had an adverse effect on biodegradation. Ethanol and methanol, provided at 5 mM to increase benzo(a)pyrene water solubility, increased the fungal biomass yield but did not promote degradation. The Fusarium sp. E033 isolated in this study can tolerate and degrade relatively high concentrations of benzo(a)pyrene, suggesting its potential application in benzo(a)pyrene bioremediation.

Cytotoxic activity of four xanthones from Emericella variecolor, an endophytic fungus isolated from Croton oblongifolius.

Four xanthones were isolated from mycelia of Emericella variecolor, an endophytic fungus isolated from the leaves of Croton oblongifolius. Their structures were elucidated by spectroscopic analysis to be shamixanthone, 14-methoxytajixanthone-25-acetate, tajixanthone methanoate, and tajixanthone hydrate. All compounds were tested for cytotoxic activity against various human tumor cell lines including gastric carcinoma, colon carcinoma, breast carcinoma, human hepatocarcinoma, and lung carcinoma. The antitumor activities of these xanthones were compared with that of doxorubicin hydrochloride, a chemotherapeutic substance. All of them showed moderate activities and were selective against gastric carcinoma, colon carcinoma, and breast carcinoma. Only tajixanthone hydrate exhibited moderate activity against all cancer cell lines. Furthermore, under the test conditions it was found that 14-methoxytajixanthone-25-acetate and tajixanthone hydrate are almost as active as doxorubicin hydrochloride against gastric carcinoma (KATO3) and breast carcinoma (BT474).