Prakitsin Sihanonth

Endophytic fungi from mangrove plant species of Thailand: their antimicrobial and anticancer potentials

Abstract We examined the distribution of endophytic fungi in the leaves of mangrove forest trees growing at three different locations (Chanthaburi Province, Prachuap Khiri Khan Province and Ranong Province) in Thailand. Three thousand and nine-hundred leaf segments from 10 different hosts belonging to seven families, Rhizophoraceae (Rhizophora apiculata, R. mucronata, Ceriops decandra), Sonneratiaceae (Sonneratia alba), Combretaceae (Lumnitzera littorea), Avicenniaceae (Avicennia alba), Acanthaceae (Acanthus ilicifolius), Meliaceae (Xylocarpus granatum and Xylocarpus moluccensis) and Malvaceae (Thespesia populneoides), were screened for the presence of fungal endophytes. The dominant endophytes varied by host type. Phyllosticta was the most frequently isolated fungus from plants at all sites. The common fungal endophyte genera were Cladosporium, Colletotrichum, Phomopsis and Xylaria. Most endophytic isolates in mangrove leaves were recovered from Ranong Province. The antimicrobial potential of 71 endophytic fungi isolated from mangrove plants towards selected bacteria (Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus) was tested using ethyl acetate extracts of fungi cultivated under static conditions. All test bacteria were inhibited by a Cladosporium sp. isolated from the leaves of T. populneoides and an endophytic Xylaria sp. 1 isolated from A. ilicifolius leaves caused considerable inhibition to Gram-positive and Gram-negative bacteria. Additionally, the crude extracts of 84 endophytic fungi were tested for anticancer activities by the MTT assay against A375 (human malignant melanoma), SW620 (human colorectal adenocarcinoma), Kato III (human gastric carcinoma), HepG2 (human liver hepatoblastoma) and Jurkat (human acute T cell leukemia). Most extracts had cytotoxicity against some cancer cell lines.

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.

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

Four xanthones were isolated from mycelia ofEmericella variecolor, an endophytic fungus isolated from the leaves ofCroton 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).

Purification and characterization of a xylanase from the endophytic fungus Alternaria alternata isolated from the Thai medicinal plant, Croton oblongifolius Roxb.

1 Biotechnology Program, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand. 2 Department of Microbiology, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand. 3 Department of Botany, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand. 4 Department of Chemistry, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand. 5 Institute of Biotechnology and Genetic Engineering, Chulalongkorn University, 254 Phayathai Road, Pathumwan, Bangkok 10330, Thailand.

Elucidation of distribution patterns and possible infection routes of the neurotropic black yeast Exophiala dermatitidis using AFLP.

Distribution of populations of the opportunistic black yeast Exophiala dermatitidis was studied using AFLP. This fungus has been hypothesized to have a natural habitat in association with frugivorous birds and bats in the tropical rain forest, and to emerge in the human-dominated environment, where it occasionally causes human pulmonary or fatal disseminated and neurotropic disease. The hypothesis of its natural niche was investigated by comparing a set of 178 strains from natural and human-dominated environments in Thailand with a worldwide selection of 107 strains from the reference collection of the CBS Fungal Biodiversity Centre, comprising 75.7% clinical isolates. Many isolates had unique AFLP patterns and were too remote for confident comparison. Eight populations containing multiple isolates could be distinguished, enabling determination of geographic distributions of these populations. Some of the populations were confined to Thailand, while others occurred worldwide. The local populations from Thailand contained strains from natural and urban environments, suggesting an environmental jump of the fungus. Strains from human brain belonged to widely dispersed populations. In some cases cerebral isolates were identical to isolates from the human intestinal tract. The possibility of cerebral infection through intestinal translocation was thus not excluded.

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 approach to isolating siderophore‐producing actinobacteria

Aims: This study was conducted to investigate the application of 2,2′‐dipyridyl as a new approach to isolating siderophore‐producing actinobacteria.

Molecular phylogeny of ectomycorrhizal Pisolithus fungi associated with pine, dipterocarp, and eucalyptus trees in Thailand

The phylogenetic relationships among 135 Pisolithus basidiomes and two isolates collected from three pine forests, a pine-dipterocarp forest, two dipterocarp forests, and 29 eucalyptus plantations in Thailand were investigated. Internal transcribed spacer (ITS) polymorphism analyses, including terminal RFLP, divided them into 26 groups. The ITS in a representative basidiome of each group was sequenced, and a phylogenetic analysis was performed. The dendrogram suggested that at least three Pisolithus species are present in Thailand. Pisolithus basidiomes collected in the pine forests and under some Shorea roxburghii trees in a pine-dipterocarp forest corresponded to species 5 as previously described by Martin et al. in 2002. Those collected under S. roxburghii and Dipterocarp alatus trees in the dipterocarp forests did not match any previously reported species. Basidiomes collected from the eucalyptus plantations were all identified as Pisolithus albus.

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-16β,17-dihydroxy-kauran-19-oic acid (2) was isolated. After further incubation for nine days, two novel metabolites, ent-12α,16β,17-trihydroxy-kauran-19-oic acid (3) and ent-11α,16β,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.

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.