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Viridepyronone, a new antifungal 6-substituted 2H-pyran-2-one produced by Trichoderma viride.

A new antifungal 6-substituted 2H-pyran-2-one, named viridepyronone, has been isolated from a cultural filtrate of a strain of Trichoderma viride showing antagonistic activity in vitro toward Sclerotium rolfsii, which is the causal agent of crown and stem rot of artichoke. Viridepyronone was characterized as 6-(4-oxopentyl)-2H-pyran-2-one 2 with spectroscopic methods. Bioassays showed that viridepyronone had a good antifungal activity against S. rolfsii, and its minimum inhibitory concentration (over 90% inhibition) was found to be 196 microg/mL. This is the first report of viridepyronone produced by any species of fungi.

Fungicides, Industrial↗

Trichothecene production by Trichoderma brevicompactum.

Trichoderma brevicompactum, T. viride, T. harzianum, T. atroviride, T. longibrachiatum, T. erinaceum, T. citrinoviride, and Hypocrea lutea were screened for production of trichothecenes after growth on one or several solid and liquid media. Trichothecenes were detected by liquid chromatography combined with online UV/vis spectroscopy and electrospray high-resolution mass spectrometry. T. brevicompactum produced trichodermin and/or harzianum A on all media investigated, with liquid media yielding the largest amounts. Detection of octa-2Z,4E,6E-trienedioic acid in the harzianum-A-producing strains indicated that harzianum A was synthesized directly by esterification of trichodermol with octa-2Z,4E,6E-trienedioic acid. Both the T. viride strain from which trichodermin was originally isolated and the T. harzianum strain from which harzianum A was originally isolated were shown to belong to T. brevicompactum based on four independent criteria: metabolite profiles, micromorphology, macromorphology on yeast extract sucrose agar and potato dextrose agar, and DNA sequences of the ITS1/ITS2 regions of the nuclear ribosomal DNA.

Chromatography, Liquid↗

Viridenepoxydiol, a new pentasubstituted oxiranyldecene produced by Trichoderma viride.

A new pentasubstituted oxiranyldecene, named viridenepoxydiol, has been isolated (0.9 mg/L) from the culture filtrate of a strain of Trichoderma viride showing in vitro and in vivo antagonistic activity against Sclerotium rolfsii, which is the causal agent of crown and stem rot of artichoke. Viridenepoxydiol was characterized as 3,5,9-trimethyl-2-oxiranyl-dec-8-ene-2,5-diol (3) using spectroscopic methods. It showed inhibitor effect on mycelial growth of S. rolfsii and its minimum inhibitory concentration (over 90% inhibition) was found to be 396 mug/mL. This is the first time that viridenepoxydiol was reported.

Alkenes↗

Heterologous expression in Aspergillus nidulans of a Trichoderma longibrachiatum endoglucanase of enological relevance.

An Aspergillus nidulans transformant expressing the Trichoderma longibrachiatum endoglucanase 1 gene (egl1) has been constructed. The extracellular production of EGL1 in different culture media has been studied, and a medium has been found in which EGL1 is the predominant extracellular protein produced. The enzymatic properties of the heterologously produced EGL1 are very similar to those of the native enzyme. Grape maceration in the presence of culture filtrate enriched in EGL1 resulted in increased release of aroma precursors, particularly in the case of aromatic grapes. Cryoscanning electron microscopy of the flesh of grapes treated with EGL1-enriched culture filtrate revealed degradation of the cell wall matrix.

Aspergillus nidulans↗

Bioactive carotanes from Trichoderma virens.

Four new metabolites with carotane skeletons, trichocaranes A (1), B (2), C (3), and D (4), were isolated from Trichoderma virens and their structures established by the interpretation of NMR and mass spectroscopic data. The trichocaranes significantly inhibited the growth of etiolated wheat coleoptiles: 40% at 10(-4) M with 1 and 2 and 86% at 10(-3) M with 3.

Magnetic Resonance Spectroscopy↗

Sequences and antimycoplasmic properties of longibrachins LGB II and LGB III, two novel 20-residue peptaibols from Trichoderma longibrachiatum.

Longibrachins are members of the class of natural Aib-containing peptides designated as peptaibols. Six longibrachins, LGA I-IV and LGB II and III, were purified from a Trichoderma longibrachiatum strain by a procedure employing several chromatography steps including reversed-phase HPLC. The amino acid sequence determination was based on a combination of liquid secondary ion mass spectrometry (LSIMS) and two-dimensional 1H and 13C NMR spectroscopy. Longibrachins are 20-residue peptaibols with a C-terminal phenylalaninol and either neutral (LGA; Gln18) or acidic (LGB; Glu18) character. Longibrachins LGB II and III have novel sequences. Both longibrachins LGA and LGB show significant bactericidal activity against mycoplasmas (Acholeplasma, Mycoplasma, and Spiroplasma), with minimal inhibitory concentrations in the range 1.56-12.5 microM (3-25 micrograms/mL), and also perturb the permeability of membrane bilayers. Longibrachin LGA IV is the most potent of the presently known 18-20-residue peptaibols. The antimicrobial and membrane-perturbing properties of longibrachins, which are described here for the first time, were shown to be correlated.

Amino Acid Sequence↗

Trichodermamides A and B, cytotoxic modified dipeptides from the marine-derived fungus Trichoderma virens.

Trichodermamides A (1) and B (2), two modified dipeptides, have been isolated from cultures of the marine-derived fungus Trichoderma virens. The trichodermamides possess a rare cyclic O-alkyl-oxime functionality incorporated into a six-membered ring. The structure of trichodermamide B was established by X-ray diffraction analysis, while the structure assignment of trichodermamide A, and determination of the absolute stereochemistry, was accomplished by spectral and chemical methods. Trichodermamide B displayed significant in vitro cytotoxicity against HCT-116 human colon carcinoma with an IC(50) of 0.32 microg/mL.

Antineoplastic Agents↗

Isolation and structure of harzianum A: a new trichothecene from Trichoderma harzianum.

A new trichothecene, harzianum A [1], was isolated from the soil-borne fungus Trichoderma harzianum. The structure of 1 was determined by extensive spectral analyses including the nmr techniques of PS-COSY, HMQC, HMBC, and NOESY. Harzianum A [1] contains a (Z,E,E)-2,4,6-octatriendioic acid esterified on the 4 beta hydroxyl group of trichodermol and is structurally related to the trichoverroids. Harzianum A [1] showed no cytotoxicity against baby hamster kidney cells, no activity against Gram-negative and Gram-positive bacteria, but modest antifungal activity at 100 micrograms/ml.

Animals↗

L-735,334, a novel sesquiterpenoid potassium channel-agonist from Trichoderma virens.

A novel oleic acid ester of the carotane sesquiterpene 14-hydroxy CAF-603 was isolated from Trichoderma virens grown in a solid brown rice-based medium, a solid millet-based medium, or a mannitol-based liquid medium. Its structure was determined on the basis of ms and nmr analysis. It retains distinct biological activity on the high conductance calcium-activated potassium channel, unlike its analogues 14-hydroxy CAF-603, CAF-603 3-oleate, or CAF-603 3-linoleate.

Animals↗

Hirsutellone F, a dimer of antitubercular alkaloids from the seed fungus Trichoderma species BCC 7579.

[structure: see text] Hirsutellone F (7), a novel alkaloid dimer, was isolated together with known monomers, hirsutellones A (1), B (2), and C (3), from the seed fungus Trichoderma sp. BCC 7579. The structure of 7 was elucidated by spectroscopic analysis. Studies on biomimetic chemistry, using the dimer 7, suggested that compound 8 (17,1'-dehydrohirsutellone B) should be the precursor for all hirsutellones.

Alkaloids↗

Structural analyses of new tri- and tetrasaccharides produced from disaccharides by transglycosylation of purified Trichoderma viride beta-glucosidase.

A new beta-glucosidase was partially purified from Trichoderma viride cellulase. This beta-glucosidase catalyzed a transglycosylation reaction of cellobiose to give beta-D-Glc-(1-->6)-beta-D-Glc-(1-->4)-D-Glc (1, yield: 18.8%) and beta-D-Glc-(1-->6)-beta-D-Glc-(1-->6)-beta-D-Glc-(1-->4)-D-Glc (2, 3.7%), regioselectively. Furthermore, the enzyme regioselectively converted laminaribiose and gentiobiose into beta-D-Glc-(1-->6)-beta-D-Glc-(1-->3)-D-Glc (3, 15.3%) and beta-D-Glc-(1-->6)-beta-D-Glc-(1-->6)-D-Glc (4, 20.2%), respectively. The structures (1-4) of the products were determined by 1H and 13C NMR spectroscopies. This high regio- and stereoselectively of the beta-glucosidase could be applied for oligosaccharide synthesis.

Carbohydrate Sequence↗

Fractionation of cellulase and beta-glucosidase in a Trichoderma reesei culture liquid by use of two-phase partitioning.

An aqueous two-phase system based on the two polymers poly(ethylene glycol) and dextran has been used for the fractionation of cellulase enzymes present in culture liquid obtained by fermentation with Trichoderma reesei. The activities of beta-glucosidase and glucanases were separated to high degree by using the two-phase systems for a counter-current distribution process in nine transfer steps. While the glucanases had high affinity to the poly(ethylene glycol) rich top phase the beta-glucosidase was enriched in the dextran-containing bottom phase. Multiple counter-current distribution performed indicates the heterogeneity of beta-glucosidase activities assuming at least four isoenzyme forms. One step concentration of beta-glucosidase by using system with 46:1 phase volume ratio resulted in 16 times higher enzyme activity.

Cellulase↗

In vitro evaluation of trichoderma and gliocladium antagonism against the symbiotic fungus of the leaf-cutting ant Atta cephalotes.

The antagonistic activity of Trichoderma and Gliocladium isolates against Attamyces sp., a symbiotic fungus of the leaf-cutting ant Atta cephalotes, was investigated. A. cephalotes cultures this fungus as the primary food source. Most of the Trichodema and Gliocladium isolates tested in vitro (82.6%) inhibited the Attamyces sp. mycelial growth. which was probably due to their colonization ability and competition for nutrients, both of them known mechanisms of some species of these genera. T. lignorum strain T-26 was the strongest inhibitor achieving a colonization of 23%. Microscopical observations indicate that the inhibitory effect was caused by an interaction that took place in close contact with the host hypha, causing wall deformation that led to the collapse of the turgor pressure.

Agaricales↗

Synergistic activity of endochitinase and exochitinase from Trichoderma atroviride (T. harzianum) against the pathogenic fungus (Venturia inaequalis) in transgenic apple plants.

Genes from the biocontrol fungus Trichoderma atroviride encoding the antifungal proteins endochitinase or exochitinase (N-acetyl-beta-D-hexosaminidase) were inserted into 'Marshall McIntosh' apple singly and in combination. The genes were driven by a modified CaMV35S promoter. The resulting plants were screened for resistance to Venturia inaequalis, the causal agent of apple scab, and for effects of enzyme expression on growth. Disease resistance was correlated with the level of expression of either enzyme when expressed alone but exochitinase was less effective than endochitinase. The level of expression of endochitinase was negatively correlated with plant growth while exochitinase had no consistent effect on this character. Plants expressing both enzymes simultaneously were more resistant than plants expressing either single enzyme at the same level; analyses indicated that the two enzymes acted synergistically to reduce disease. Selected lines, especially one expressing low levels of endochitinase activity and moderate levels of exochitinase activity, were highly resistant in growth chamber trials and had negligible reduction in vigor relative to control plants. We believe that this is the first report of resistance in plants induced by expression of an N-acetylhexosaminidase and is the first report of in planta synergy between an exochitinase and an endochitinase.

Blotting, Western↗

The carbohydrate moiety of alpha-galactosidase from Trichoderma reesei.

Alpha-galactosidase from Trichoderma reesei is a glycoprotein that contains O- and N-linked carbohydrate chains. There are 6 O-linked glycans per protein molecule that are linked to serine and threonine and can be released by beta-elimination. Among these are monomers: D-glucose, D-mannose, and D-galactose; dimers: alpha1-6 D-mannopyranosyl-alpha-D-glycopyranoside and alpha1-6 D-glucopyranosyl-alpha-D-galactopyranoside and one trimer: alpha-D-glucopyranosyl-alpha1-2 D-mannopyranosyl-alpha1-6 D-galactopyranoside. N-linked glycans are of the mannose-rich type and may be released by treating the protein with Endo-beta-N-acetyl glycosaminidase F or by hydrozinolysis. The enzyme was deglycosylated with Endo-beta-N-acetyl glycosaminidase F as well as with a number of exoglycosidases that partially remove the terminal residues of O-linked glycans. The effect of enzymatic deglycosylation on the properties of alpha-galactosidase has been considered. The effects of tunicamycin and 2-deoxyglucose on the secretion and glycosylation of the enzyme during culture growth have been analysed. The presence of two glycoforms of alpha-galactosidase differing in the number of N-linked carbohydrate chains and the microheterogeneity of the carbohydrate moiety of the enzyme are described.

Amino Acid Sequence↗

Intrinsic fluorescence in endoglucanase and cellobiohydrolase from Trichoderma pseudokiningii S-38: effects of pH, quenching agents, and ligand binding.

To gain further insight into the difference in substrate specificity between endoglucanase and cellobiohydrolase, the intrinsic fluorescence properties of cellobiohydrolase I (CBH I) and endoglucanase I (EG I) from Trichoderma pseudokiningii S-38 were investigated. The results for the spectral characteristics, ligand binding and fluorescence quenching suggest that the fluorescence of two enzymes comes from tryptophan residues, and that tryptophan residue(s) may be involved in the function of the two enzymes. The results also suggest that the binding tryptophan in EG I may be more exposed to solvent than that in CBH I. This interpretation is supported by the observations that the effects of pH upon the fluorescence of EG I are greater than that of CBH I; spectral shifts are different in EG I and CBH I under various conditions, and fluorescence lifetime changes caused by cellobiose binding are larger for EG I than for CBH I.

Acrylamide↗