The production and characterisation of trichotoxin peptaibols, by Trichoderma asperellum

Trichoderma spp. are regularly found as a constituent of the mycoflora of many soils and are noted for their antagonistic activity against bacteria and other fungi. This latter property is the basis for the widespread interest in their use in the biological control of soil-borne fungal plant pathogens. This antagonism is partly based on their ability to produce an impressive inventory of secondary metabolites. An important group of bioactive metabolites produced by Trichoderma spp. are the non-ribosomal peptides (NRPs), especially the peptaibols. A virulent antagonistic strain, T. asperellum, which had been used in biological control strategies in Malaysia and previously examined for mycolytic enzyme production, has been studied for its potential for peptaibol production. The present research demonstrated the ability of T. asperellum to produce at least two metabolites which were identified as acid trichotoxin 1704E (Ac-Aib-Gly-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Ala-Ala-Aib-Pro-Leu-Aib-Iva-Glu-Vol) and neutral trichotoxin 1717A (Ac-Aib-Gly-Aib-Leu-Aib-Gln-Aib-Aib-Aib-Ala-Aib-Aib-Pro-Leu-Aib-Iva-Gln-Vol). Addition of free Aib to the culture medium enhanced the production of trichotoxins. Biological activity of these substances was investigated against Bacillus stearothermophilus. The general characteristics of peptaibols, also found in the trichotoxins, include the presence of high proportions of the uncommon amino acid Aib, the protection of the N- and C-termini by an acetyl group and reduction of the C-terminus to 2-amino alcohols, respectively, amphipathy and microheterogeneity.     

Novel elongase of Pythium sp. with high specificity on Δ-18C desaturated fatty acids

We identified a novel elongase gene from a selected strain of the Oomycete, Pythium sp. BCC53698. Using a PCR approach, the cloned gene (PyElo) possessed an open reading frame (ORF) of 834 bp encoding 277 amino acid residues. A similarity search showed that it had homology with the PUFA elongases of several organisms. In addition, the signature characteristics, including four conserved motifs, a histidine-rich catalytic motif and membrane-associated feature were present in the Pythium gene. Heterologous expression in Saccharomyces cerevisiae showed that it was specific for fatty acid substrates, having a double bond at Δ⁶-position, which included γ-linolenic acid (GLA) and stearidonic acid (STA), and preferentially elongated the n3-18C PUFA. This is an elongase in Oomycete fungi, which displays very high specificity on Δ⁶-18C desaturated fatty acids. This will be a powerful tool to engineer PUFA biosynthesis in organisms of interest through the n-6 series pathway for producing value-added fatty acids.     

Ascherxanthone B from Aschersonia luteola, a new antifungal compound active against rice blast pathogen Magnaporthe grisea

Aims:  Fungicide resistance now exists in the rice blast fungus, Magnaporthe grisea , necessitating the need for new active agents. Fungi isolated from habitats in Thailand were screened with reference to this problem.
Methods and Results:  A new, reliable in vitro screening system based on a microdilution plate format was set up using a virulent strain of M. grisea THL 16. Culture broth extracts from approximately 800 fungal strains were investigated, one of these, Aschersonia luteola BCC 8774, was found to produce an active fungicidal compound, ascherxanthone B, with an IC₉₀ value of 0·58 μg ml⁻¹ (0·95 μmol l⁻¹). An in vivo study of anti-blast efficacy of ascherxanthone B showed a positive effect in disease reduction.
Conclusions:  Previous report has shown that a species of Aschersonia produces ascherxanthone A. Research on the species, A. luteola BCC 8774, led to the discovery of related novel metabolite, ascherxanthone B with fungicidal properties.
Significance and Impact of the Study:  Current methods of rice blast control seem to fail leading to increase in crop losses. Our discovery of the anti-blast activity shown by ascherxanthone B is the first step in the development of a potentially novel fungicide.     

Isolation and characterisation of a partial peptide synthetase gene from Trichoderma asperellum

Many species of Trichoderma have attracted interest as agents for the biological control of soil borne fungal pathogens of a range of crop plants. Research on the biochemical mechanisms associated with this application has focused on the ability of these fungi to produce enzymes which lyse fungal cell walls, and antifungal antibiotics. An important group of the latter are the non-ribosomal peptides called peptaibols. In this study Trichoderma asperellum, a strain used in biological control in Malaysia, was found to produce the peptaibol, trichotoxin. This type of peptide molecule is synthesised by a peptide synthetase (PES) enzyme template encoded by a peptide synthetase (pes) gene. Using nucleotide sequences amplified from adenylation (A-) domains as probes, to hybridise against a lambda FIXII genomic library from T. asperellum, 25 clones were recovered. These were subsequently identified as representative of four groups based on their encoding properties for specific amino acid incorporation modules in a PES. This was based on analysis of their amino acid sequences which showed up to 86% identity to other PESs including TEX 1.