Publications (14)39.84 Total impact
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Article: Involvement of pachybasin and emodin in self-regulation of Trichoderma harzianum mycoparasitic coiling.
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ABSTRACT: Our aim was to determine the effects of two secondary metabolites secreted by Trichoderma harzianum, pachybasin and emodin, on the mycoparasitic coiling behavior and cAMP content of T. harzianum. The number of T. harzianum coils around Nylon 66 fiber was increased in the presence of R. solani. The number of T. harzianum coils around R. solani hyphae and Nylon 66 fiber were significantly increased in the presence of pachybasin and emodin. The cAMP level in T. harzianum was significantly increased by close contact with R. solani and much higer cAMP level in the presence of exogenous pachybasin and emodin. A cAMP inhibitor diminished the effect of pachybasin and emodin on T. harzianum coiling around Nylon 66 fiber. The results suggest that pachybasin and emodin mediate the increase in the number of Trichoderma mycoparasitic coils via cAMP signaling. This is the first report to suggest that pachybasin and emodin play roles in the biocontrol mechanism of Trichoderma.Journal of Agricultural and Food Chemistry 03/2012; 60(9):2123-8. · 2.82 Impact Factor -
Article: Monomeric L-amino acid oxidase-induced mitochondrial dysfunction in Rhizoctonia solani Reveals a novel antagonistic mechanism of Trichoderma harzianum ETS 323.
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ABSTRACT: The monomeric L-amino acid oxidase (mTh-LAAO) of Trichoderma harzianum ETS 323 has been suggested to antagonize Rhizoctonia solani by an unknown mechanism. Here, the mTh-LAAO-treated R. solani exhibited hyphal lysis and apoptotic characteristics such as DNA fragmentation, reactive oxygen species (ROS) accumulation, lipid peroxidation, and mitochondrial membrane potential depolarization. This hyphal lysis was suppressed by the mitochondria-dependent apoptosis inhibitor oligomycin while accompanied by reduction of ROS accumulation. This result suggested that mitochondria-mediated apoptosis in R. solani was involved in mTh-LAAO-induced growth inhibition, which was supported by the evidence of cytocheome c release and activation of caspases 9 and 3. Furthermore, the data indicated that the mTh-LAAO-induced fungal cell death was also closely interrelated with the interaction of mTh-LAAO with R. solani hyphal cell wall proteins. These results illuminate the biological function and mechanism underlying the antagonistic action of T. harzianum mTh-LAAO against fungal pathogens.Journal of Agricultural and Food Chemistry 03/2012; 60(10):2464-71. · 2.82 Impact Factor -
Article: L-amino acid oxidase-induced apoptosis in filamentous Botrytis cinerea.
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ABSTRACT: As opposed to single-cell yeast and mammalian cell lines, apoptosis has not been greatly investigated in filamentous fungi because antibodies to the relevant fungal apoptosis-related proteins are not available commercially and because multicellular organisms cannot be studied using flow cytometry. Here we demonstrate how antibodies from a nonfungal source could be used to investigate this pathway. We show that apoptosis in the filamentous fungus Botrytis cinerea is triggered by the mitochondria-mediated caspase pathway, with release of the apoptotic factors cytochrome c, caspase 3, and caspase 9, on treatment with Trichoderma harzianum-derived L-amino acid oxidase.Analytical Biochemistry 09/2011; 420(1):93-5. · 3.00 Impact Factor -
Article: Cloning of a novel L-amino acid oxidase from Trichoderma harzianum ETS 323 and bioactivity analysis of overexpressed L-amino acid oxidase.
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ABSTRACT: L-amino acid oxidases (L-AAOs) have been isolated from many organisms, such as snake, and are known to have antibacterial activity. To the best of the authors' knowledge, this is the first report of the cloning of cDNA encoding a novel Trichoderma harzianum ETS 323 L-amino acid oxidase (Th-L-AAO). The protein was overexpressed in Escherichia coli and purified to homogeneity. Comparisons of its deduced amino acid sequence with the sequence of other L-AAOs revealed the similarity to be between 9 and 24%. The molecular mass of the purified protein was 52 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme substrate specificity was highest for L-phenylalanine, and its optimal pH and temperature for activity were 7 and 40 °C, respectively; exogenous metal ions had no significant effect on activity. Circular dichroism spectroscopy indicated that the secondary structure of Th-L-AAO is composed of 17% α-helices, 28% β-sheets, and 55% random coils. The bacterially expressed Th-L-AAO also mediated antibacterial activity against both gram-positive and gram-negative food spoilage microorganisms. Furthermore, a three-dimensional protein structure was created to provide more information about the structural composition of Th-L-AAO, suggesting that the N-terminal sequence of Th-L-AAO may have contributed to the antibacterial activity of this protein.Journal of Agricultural and Food Chemistry 08/2011; 59(17):9142-9. · 2.82 Impact Factor -
Article: Identification of antibacterial mechanism of L-amino acid oxidase derived from Trichoderma harzianum ETS 323.
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ABSTRACT: Although L-amino oxidase (LAAO; EC 1.4.3.2) has been reported to be a potent antibacterial agent, the mechanism responsible for its antibacterial activity has not been identified. The present study aimed to identify the mechanism responsible for the antibacterial activity of Th-LAAO, an LAAO recently isolated from the extracellular proteins of Trichoderma harzianum ETS 323, at the same time as elucidating the nature of this enzyme. The results obtained indicate that the enzyme activity and structure of Th-LAAO are stable at pH 6-8 and less stable at both pH 4-5.5 and pH 9. At pH 7.0, the optimum temperature for Th-LAAO was found to be 40 °C, comprising the temperature at which enzymatic activity is greatest, with enzymatic activity deceasing with further increases in temperature as a result of thermal denaturation of the enzyme, leading to partial denaturation at 50 °C. The results obtained by confocal microscopy and flow cytometry indicate that Th-LAAO interacts with bacteria to cause membrane permeabilization, and this interaction may be promoted by the amphipathic sequence in Th-LAAO and other cytotoxic LAAOs located at the N-terminus. The findings of increased exogenous H(2) O(2) production and reactive oxidative species accumulation in Th-LAAO-treated bacteria indicate that reactive oxidative species accumulation may trigger forms of cell damage, including lipid peroxidation and DNA strand breakage that results in bacterial growth inhibition. Taken together, the results indicate that the processes of bacterial interaction, membrane permeabilization and H(2)O(2) production are involved in the mechanism responsible for the antibacterial activity of Th-LAAO.FEBS Journal 07/2011; 278(18):3381-94. · 3.79 Impact Factor -
Article: A novel L-amino acid oxidase from Trichoderma harzianum ETS 323 associated with antagonism of Rhizoctonia solani.
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ABSTRACT: Trichoderma spp. are used as biocontrol agents against phytopathogens such as Rhizoctonia solani, but their biocontrol mechanisms are poorly understood. A novel L-amino oxidase (Th-LAAO) was identified from the extracellular proteins of Trichoderma harzianum ETS 323. Here, we show a FAD-binding glycoprotein with the best substrate specificity constant for L-phenylalanine. Although the amino acid sequence of Th-LAAO revealed limited homology (16-24%) to other LAAO members, a highly conserved FAD-binding motif was identified in the N-terminus. Th-LAAO was shown to be a homodimeric protein, but the monomeric form was predominant when grown in the presence of deactivated Rhizoctonia solani. Furthermore, in vitro assays demonstrated that Th-LAAO had an antagonistic effect against Rhizoctonia solani and a stimulatory one on hyphal density and sporulation in T. harzianum ETS 323. These findings further our understanding of T. harzianum as a biocontrol agent and provide insight into the biological function of l-amino acid oxidase.Journal of Agricultural and Food Chemistry 04/2011; 59(9):4519-26. · 2.82 Impact Factor -
Article: Purification and characterization of novel glucanases from Trichoderma harzianum ETS 323.
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ABSTRACT: Trichoderma harzianum ETS 323 secretes two glucanases, a 23.5 kDa endoglucanase (EG Th1) and a 61 kDa exoglucanase (ExG Th1). They were identified by their hydrolysis products and were purified to homogeneity. The optimal temperature and pH for both EG Th1 (7.3-fold purification, 5.0% yield) and ExG Th1 (33.7-fold purification, 0.15% yield) were 50 °C and pH 4.5, respectively. The kinetic parameters of EG Th1 (K(m) = 23 mg mL(-1), V(max) = 294 μM min(-1), specific activity = 7.4 U mg(-1)) and ExG Th1 (K(m) = 85 mg mL(-1), V(max) = 385 μM min(-1), specific activity = 24.6 U mg(-1)) toward carboxymethyl cellulose were determined. Both enzymes favored CMC and maintained 100% activity for 10 days at 38 °C. KCl, MgCl(2), HgCl(2), and FeCl(3) showed approximately 30% inhibition against EG Th1 but not ExG Th1. They catalyzed transglycosylation of glucose in the presence of cellobiose, but ExG Th1 exhibited better activity and higher product diversity.Journal of Agricultural and Food Chemistry 10/2010; 58(19):10309-14. · 2.82 Impact Factor -
Article: Study on the anthraquinones separated from the cultivation of Trichoderma harzianum strain Th-R16 and their biological activity.
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ABSTRACT: The biocontrol fungal species of Trichoderma, which colonizes plant roots, are well-known for their potential to control plant pathogens. Six anthraquinones, of which four have been identified for the first time from Trichoderma and two have already been reported in other strains, were purified from Trichoderma harzianum strain Th-R16 to evaluate their biological activities. The structures of the compounds were determined by one- and two-dimensional NMR. The compounds were shown to exhibit stronger antifungal activity than antibacterial activity. Low yield compounds, like 1,5-dihydroxy-3-hydroxymethyl-9,10-anthraquinone, were found to be more active against fungal pathogens than pachybasin and crysophanol, which were found to be the major extracellular metabolites. Test anthraquinones with higher oxidation numbers had better antifungal activity, and their activities were concentration-dependent.Journal of Agricultural and Food Chemistry 09/2009; 57(16):7288-92. · 2.82 Impact Factor -
Article: Plate assay for fungal enzymes using cellophane membranes.
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ABSTRACT: Fungal mycelia mass and pigments are major obstacles to investigating the secretion of bioactive substances such as enzyme activities using a plate assay. In this study, we applied a cellophane membrane and demonstrated that it can block mycelia mass and conidia (especially pigmented spores that would likely interfere with any subsequent color development-based activity detection) while allowing secreting enzymes to pass through. Visual observation after lifting the cellophane membrane and the collected mycelia and conidia indicated that the bioactivities on specific plates were improved significantly, although some fungal growth hurdle was noted. This proved to be true whether the assays were color development based or not.Analytical Biochemistry 09/2009; 397(1):121-3. · 3.00 Impact Factor -
Article: Screen and genetic assessment of tea germplasms with elevated methylated catechin, (-)-epigallocatechin-3-O-(3-O-methyl)gallate.
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ABSTRACT: The aim of this research was to identify tea varieties containing high levels of catechin methyl ester that could be used as important sources for genetic breeding and in the production of high quality tea. We examined 113 species that have been preserved in the Taitung Branch of the Tea Research and Extension Station of Taiwan (TTES). The average level of (-)-epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3''Me) was 0.45% for all varieties screened. Among them, 16 varieties with higher EGCG3''Me content (>0.8%) were considered good candidates for manufacturing partially fermented tea. Analysis of these tea varieties revealed that the EGCG3''Me content in leaves did not correlate with the caffeine content. Genetic assessments revealed that the lengths of their internal transcribed spacers (ITS) were in the range of 638-670 bp and that the sequence identities were in the range of 0.690-1. Two major groups were constructed by phylogenetic analysis, I and II, with a genetic distance of 0.08 based on the ITS1-5.8S-ITS2 sequences between the ribosomal genes. Our results provide genetic information about tea varieties with elevated EGCG3''Me content and indicate the need for a comprehensive genetic assessment of tea germplasms preserved in the TTES to better serve the future of tea breeding.Journal of Agricultural and Food Chemistry 09/2009; 57(19):8906-12. · 2.82 Impact Factor -
Article: Induced proteome of Trichoderma harzianum by Botrytis cinerea.
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ABSTRACT: As a notable biocontrol agent, Trichoderma harzianum can antagonize a diverse array of phytopathogenic fungi, including Botrytis cinerea, Rhizoctonia solani and Fusarium oxysporum. Elucidating the biocontrol mechanism of T. harzianum in response to the pathogens enables it to be exploited in the control of plant diseases. Two-dimensional gel electrophoresis (2-DE) was performed to obtain secreted protein patterns of T. harzianum ETS 323, grown in media that contained glucose, a mixture of glucose and deactivated B. cinerea mycelia, deactivated B. cinerea mycelia or deactivated T. harzianum mycelia. Selected protein spots were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ninety one out of 100 excised protein spots were analyzed and some proteins were sequence identified. Of these, one l-amino acid oxidase (LAAO) and two endochitinases were uniquely induced in the media that contained deactivated B. cinerea mycelia as the sole carbon source. Activities of the cell wall-degrading enzymes (CWDEs), including beta-1,3-glucanases, beta-1,6-glucanases, chitinases, proteases and xylanases, were significantly higher in media with deactivated B. cinerea mycelia than in other media. This finding suggests that the cell wall of B. cinerea is indeed the primary target of T. harzianum ETS 323 in the biocontrol mechanism. The possible roles of LAAO and xylanase were also discussed.Mycological Research 06/2009; 113(Pt 9):924-32. · 2.81 Impact Factor -
Article: Proteomic study of biocontrol mechanisms of Trichoderma harzianum ETS 323 in response to Rhizoctonia solani.
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ABSTRACT: To elucidate the entire range of proteins that are secreted by Trichoderma harzianum ETS 323 in its antagonism with Rhizoctonia solani, an in vivo interaction between them was mimicked and not only the secreted cell wall-degrading enzymes (CWDEs) but also all of the proteome were investigated. Seven CWDEs, chitinase, cellulase, xylanase, beta-1,3-glucanase, beta-1,6-glucanase, mannanase, and protease,were revealed by activity assay, in-gel activity stain, 2-DE, and LC-MS/MS analysis. Extracellular protein extracts from media that contained R. solani exhibited much higher CWDE activities than media that did not contain R. solani. Cellulase and mannanase activity, however, were insignificant. Activity stain also revealed that beta-1,3-glucanase, beta-1,6-glucanase, and xylanase activity occurred exclusively in media that contained R. solani. Furthermore, 35 of the 43 excised spots on the 2-DE gel were successfully analyzed by LC-MS/MS, and eight proteins were identified. They were two glycoside hydrolases, two proteases, two beta-glucosidases, one endochitinase and, interestingly, one amino acid oxidase. Additionally, a possible mechanism was proposed to elucidate how the cell walls of R. solani are systematically enveloped and disintegrated.Journal of Agricultural and Food Chemistry 08/2008; 56(16):6914-22. · 2.82 Impact Factor -
Article: Efficient isolation of anthraquinone-derivatives from Trichoderma harzianum ETS 323.
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ABSTRACT: Anthraquinone-derivatives, chrysophanol and pachybasin, were purified by a silica column chromatography with two different solvent systems from Trichoderma harzianum ETS 323. The fungus was incubated in sugarcane bagasse solid medium at room temperature without rotation. Structure of chrysophanol was solved by X-ray diffraction and pachybasin by NMR spectra. About 233+/-13 mg of pure chrysophanol and 773+/-40 mg of pure pachybasin were recovered per kg of solid cultural medium, with yields 1.7+/-0.2% and 5.6+/-0.5%, respectively.Journal of Biochemical and Biophysical Methods 05/2007; 70(3):391-5. · 2.33 Impact Factor -
Article: Circular dichroism analysis of destruxins from Metarhizium anisopliae.
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ABSTRACT: Destruxins are secondary metabolites secreted by Metarhizium anisopliae [Y. Kodaira, Toxic substances to insects, produced by Aspergillus ochraceus and Oopsra destructor, Agric. Biol. Chem., 25 (1961) 261-262. D.W. Roberts, Toxins from the entomogenous fungus Metarhizium anisoplaie: Isolation from submerged cultures, J. Invertebr. Pathol., 14 (1969) 82-88. D.W. Roberts, Toxins from the entomogenic fungi in microbial control of pest and plant disease, Academic press, New York, 1981, pp441-464.]. In recent research, other than being used as insecticides, destruxins exhibited great potential in therapeutical applications such as antitumor, antivirus, and animal cell immunization effectiveness, etc. In this study, the conformations purified destruxins were determined by circular dichroism (CD). The results indicated that these cyclic peptides have the type I beta-turn conformation. In addition, different types of destruxins exhibited different CD spectra in acetonitrile. Therefore, these characters can be used as fingerprints to identify each type of destruxin. To further investigate the interactions among destruxins, various combinations of destruxins in 10 mM phosphate-buffered saline (PBS) were also studied by CD. The results strongly suggested that destruxins might work independently in vivo. To our knowledge, this is the first report presenting the CD analysis of purified destruxins.Journal of Biochemical and Biophysical Methods 02/2005; 62(1):41-50. · 2.33 Impact Factor
Top co-authors
Top Journals
Institutions
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2011–2012
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Tzu Chi University
- Institute of Medical Sciences
Keelung, Taiwan, Taiwan
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2005–2012
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National Dong Hwa University
- Institute of Biotechnology
Hualian, Taiwan, Taiwan
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2007–2010
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Da-Yeh University
Taiwan
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2009
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Buddhist Tzu Chi General Hospital
Taipei, Taipei, Taiwan
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