Nuttika Suwannasai

Diversity and community composition of ectomycorrhizal fungi in a dry deciduous dipterocarp forest in Thailand

Large forest areas of South-East Asia, are dominated by the Dipterocarpaceae tree family, which contains many important timber species. Unlike many other tropical trees, Dipterocarpaceae rely on ectomycorrhizal (ECM) root symbiosis for their mineral nutrition. This study aims to document the richness and community composition of ECM fungi in a dry deciduous forest in Thailand. Combining morphological and molecular identification methods revealed 69 species of ECM fungi that belong to 17 phylogenetic lineages. The /russula-lactarius, /tomentella-thelephora, /sordariales, /sebacina and /cantharellus lineages were the most species-rich. The fungal richness is comparable to other tropical rain forest sites, but the phylogenetic community structure has elements of both tropical and temperate ecosystems. Unlike tropical rain forests, the Cenococcum geophilum complex was one of the most frequent fungal taxa that had a relatively high ITS genetic diversity over the small sampling area. This study provides the first snapshot insight into the fungal community of dry dipterocarp forests. However, it is necessary to broaden the spatial and temporal scales of sampling to improve our understanding of the below-ground relations of dry and humid tropical forests.

Astraodoric acids A-D: new lanostane triterpenes from edible mushroom Astraeus odoratus and their anti-Mycobacterium tuberculosis H37Ra and cytotoxic activity.

Tuberculosis (TB) is one of the chronic infectious diseases caused by Mycobacterium tuberculosis that causes about 2-3 million deaths per year. Isoniazid and rifampicin are examples of first line drugs used for TB treatment; however, they are potentially hepatotoxic. More effective and safer drugs are urgently needed, especially from natural products. Basidiomycete mushrooms are known as important sources of pharmaceutically active metabolites including an anti-TB agent. In this work, the chemical constituents of the edible mushroom Astraeus odoratus were isolated and investigated for antibacterial activity against M. tuberculosis H(37)Ra. The cytotoxic activity against cancerous cell lines was also evaluated. Four new lanostane triterpenes, astraodoric acids A-D, and new 5-hydroxyhypaphorine have been isolated together with four known compounds. The structures were elucidated by NMR spectroscopic methods, HR-ESI-MS results, and X-ray crystallographic analysis. Astraodoric acids A and B exhibited moderate antibacterial (MICs of 50 and 25 μg/mL) and cytotoxic activities (IC(50) values of 34.69 and 18.57 μg/mL against KB and 19.99 and 48.35 μg/mL against NCI-H187), respectively. The results of this study show that A. odoratus could be a significant natural source for safer antitubercular and anticancer agents.

Microbial metabolism of α-mangostin isolated from Garcinia mangostana L.

α-Mangostin (1), a prenylated xanthone isolated from the fruit hull of Garcinia mangostana L., was individually metabolized by two fungi, Colletotrichum gloeosporioides (EYL131) and Neosartorya spathulata (EYR042), repectively. Incubation of 1 with C. gloeosporioides (EYL131) gave four metabolites which were identified as mangostin 3-sulfate (2), mangostanin 6-sulfate (3), 17,18-dihydroxymangostanin 6-sulfate (4)and isomangostanin 3-sulfate (5). Compound 2 was also formed by incubation with N. spathulata (EYR042). The structures of the isolated compounds were elucidated by spectroscopic data analysis. Of the isolated metabolites, 2 exhibited significant anti-mycobacterial activity against Mycobacterium tuberculosis.

Isolation and characterisation of a novel Podoviridae-phage infecting Weissella cibaria N 22 from Nham, a Thai fermented pork sausage

A novel Podoviridae lactic acid bacteria (LAB) phage from Nham, a Thai fermented pork sausage, is reported. From a total of 36 samples, 41 isolates of LAB were obtained and employed as hosts for the isolation of phages. From these LAB, only one phage, designated Φ 22, was isolated. The lactic acid bacterial isolate named N 22, sensitive to phage Φ 22 infection was identified by an API 50 CHL kit and N 22s complete sequence of the 16S rDNA sequence. BLASTN analysis of the 16S rDNA sequence revealed a 99% similarity to the 16S rDNA sequence of Weissella cibaria in the GenBank database. Electron micrographs indicated that the phage head was icosahedral with head size and tail length of 92 × 50 nm and 27 nm, respectively. On the basis of the morphology, this phage belongs to the family Podoviridae. Host-range determination revealed that the phage Φ 22 was not capable of infecting the other 40 isolates of LAB and referenced Weissella strains used. A one-step growth experiment showed that the latent period and burst size were estimated at 110 min and 55 phage particles/infected cell, respectively. Furthermore, the phage was infective over a wide range of pH (pH 5.0-8.0) and the D time of Φ 22 was calculated as 88 s at 70 °C and 15s at 80 °C. Phage titers decreased below the detection limit (20 PFU/ml) after heating for more than 60s at 80 °C, or 20s at 90 °C or less than 10s at 100 °C. The results from the study of Nham revealed that Φ 22 was active against the potential starter culture (W. cibaria N 22) for Nham fermentation. Phage infection could adversely affect the fermentation process of Nham by delaying acidification when using W. cibaria N 22 as a starter. However, the results from a sensory test revealed that the panelists did not detect any defects in the final products. This is the first report on the isolation of W. cibaria phage.

Antimalarial and cytotoxic constituents of Xylaria cf. cubensis PK108

Xylaria cf. cubensis PK108 was identified by its distinctive morphological characteristics and its internal transcribed spacers sequence analysis. The chromatographic separation and structural elucidation based on spectroscopic analysis of fungal crude extracts led to 10 compounds; tryptoquivaline L (1), fiscalin C (2), epi-fiscalin C (3), cytochalasin D (4), ergosterol (5), ergosterol peroxide (6), chevalone C (7), xylaranol B (8), helvolic acid (9) and cyclo-(l-Pro-l-Leu) (10). The bioassay screening showed that 4 displayed cytotoxicity against KB and NCI-H187 cancer cell lines with IC50 values of 3.25 and 5.95 μg mL− 1. 6 exhibited cytotoxicity against NCI-H187 with an IC50 value of 5.81 μg mL− 1. 7 and 9 showed antimalarial activity with IC50 values of 25.00 and 6.25 μg mL− 1, respectively. This result establishes Xylaria as broad spectrum bioactive compound producers.

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.

A new cerebroside and the cytotoxic constituents isolated from Xylaria allantoidea SWUF76.

Abstract A new cerebroside, namely allantoside (1), and 10 known compounds (2–11) were isolated from Xylaria allantoidea SWUF76. The structure of compound 1 was determined by comprehensive spectroscopic analysis including 1D and 2D nuclear magnetic resonance (NMR) as well as high-resolution electron ionisation mass spectrometry (HREIMS) and electrospray ionisation mass spectrometry (ESIMS). Compounds 1, 4, 5, 6, 7, 8 and 11 were evaluated for cytotoxic activities against cancer cell lines (Hela, HT29, HCT116 and MCF-7) and normal Vero cell lines by MTT assay. Compounds 6 and 7 exhibited anticancer activity after 24 h of treatment. Compound 7 showed significant cytotoxicity against Hela (IC50 = 2.24 μg/mL), HT29 (IC50 = 2.51 μg/mL), HCT116 (IC50 = 3.50 μg/mL) and MCF-7 (IC50 = 3.77 μg/mL) and Vero (IC50:3.65 μg/mL) cells. Compound 6 showed slight cytotoxicity against all tested cancer cell lines.

A New Representative of Star-Shaped Fungi: Astraeus sirindhorniae sp. nov. from Thailand

Phu Khieo Wildlife Sanctuary (PKWS) is a major hotspot of biological diversity in Thailand but its fungal diversity has not been thouroughly explored. A two-year macrofungal study of this remote locality has resulted in the recognition of a new species of a star-shaped gasteroid fungus in the genus Astraeus. This fungus has been identified based on a morphological approach and the molecular study of five loci (LSU nrDNA, 5.8S nrDNA, RPB1, RPB2 and EF1-a). Multigene phylogenetic analysis of this new species places it basal relative to other Astraeus, providing additional evidence for the SE Asian orgin of the genus. The fungus is named in honour of Her Majesty Princess Sirindhorn on the occasion the 84th birthday of her father, who have both been supportive of natural heritage studies in Thailand.

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.