Cabbage defense response provoked by Trichoderma Th-LAAO.

To investigate the molecular mechanism of Trichoderma L-amino acid oxidase (Th-LAAO) in protecting and in promoting growth of cabbage infected with Botrytis cinerea, a three-way interaction system was established. Cabbage leaves treated with purified Th-LAAO significantly constrained damaged leaf area caused by B. cinerea infection. In response to Th-LAAO treatment, the expression levels of genes involved in photosynthesis, such as ribulose-1,5-bisphosphate carboxylase oxygenase, Rubisco activase, and ATP synthase increased 2.54, 2.18, and 1.41 folds, respectively. The transcription levels of sucrose transport protein 1 increased 7.6 fold. As to the expression of defense-related genes, the transcription level of ascorbate peroxidase increased 1.46 fold. On the contrary, pathogenesis-related protein 1, chitinase, ß-1,3 glucanase, and glutathione S-transferase decreased significantly. Overall, the results indicated that Th-LAAO may stimulate CO2 fixation and sucrose transport and elicit host defense responses in cabbage against B. cinerea, and this elicitation of defense response is likely to contribute to induced systemic resistance of host plant.

Gene-to-Gene Network Analysis of the Mediation of Plant Innate Immunity by the Eliciting Plant Response-Like 1 (Epl1) Elicitor of Trichoderma formosa.

A new clade, Trichoderma formosa, secretes eliciting plant response-like 1 (Epl1), a small peptide elicitor that stimulates plant immunity. Nicotiana benthamiana pretreated with Epl1 for 3 days developed immunity against Tomato mosaic virus (ToMV) infection. The transcriptome profiles of T. formosa and N. benthamiana were obtained by deep sequencing; the transcript of Epl1 is 736 nt in length and encodes a 12-kDa peptide. Identifying critical genes in Epl1-mediated immunity was challenging due to high similarity between the transcriptome expression profiles of Epl1-treated and ToMV-infected N. benthamiana samples. Therefore, an efficient bioinformatics data mining approach was used for high-throughput transcriptomic assays in this study. We integrated gene-to-gene network analysis into the ContigViews transcriptome database, and genes related to jasmonic acid and ethylene signaling, salicylic acid signaling, leucine-rich repeats, transcription factors, and histone variants were hubs in the gene-to-gene networks. In this study, the Epl1 of T. formosa triggers plant immunity against various pathogen infections. Moreover, we demonstrated that high-throughput data mining and gene-to-gene network analysis can be used to identify critical candidate genes for further studies on the mechanisms of plant immunity.

Concentrations and analysis of health risks of ambient air metallic elements at Longjing site in central Taiwan.

The concentrations of particulates and metallic elements that were bound to total suspended particulates in ambient air at Long Cyuan Elementary School (LCYES), Lung Ching Elementary School (LCHES) and Long Shan Primary School (LSPS) sampling sites in the Longjing area were measured. Significant difference tests were conducted at LSPS, LCYES and LCHES sites. Finally, carcinogenic and non-carcinogenic risk values for LSPS, LCYES and LCHES sites in the Longjing district were evaluated. The results show that the most average particulate and metallic element concentrations were highest in October, November, January, February, March, April, August, and September The average particulate and metallic element concentrations at LCHES were higher than at the other sampling sites. The Concentration Scatter Diagrams reveal the absence of significant variation among the LSPS, LCYES and LCHES sampling sites in the Longjing district. Therefore, these sampling sites are inferred to have similar emission sources. The children and adults inhalation carcinogenic risks which referenced US EPA method were all within acceptable ranges. Non-carcinogenic risks revealed that all metallic elements considered herein were harmless to human health.

Annual ambient atmospheric mercury speciation measurement from Longjing, a rural site in Taiwan.

The main purpose of this study was to monitor ambient air particulates and mercury species [RGM, Hg(p), GEM and total mercury] concentrations and dry depositions over rural area at Longjing in central Taiwan during October 2014 to September 2015. In addition, passive air sampler and knife-edge surrogate surface samplers were used to collect the ambient air mercury species concentrations and dry depositions, respectively, in this study. Moreover, direct mercury analyzer was directly used to detect the mercury Hg(p) and RGM concentrations. The result indicated that: (1) The average highest RGM, Hg(p), GEM and total mercury concentrations, and dry depositions were observed in January, prevailing dust storm occurred in winter season was the possible major reason responsible for the above findings. (2) The highest average RGM, Hg(p), GEM and total mercury concentrations, dry depositions and velocities were occurred in winter. This is because that China is the largest atmospheric mercury (Hg) emitter in the world. Its Hg emissions and environmental impacts need to be evaluated. (3) The results indicated that the total mercury ratios of Kaohsiung to that of this study were 5.61. This is because that Kaohsiung has the largest industry density (~60 %) in Taiwan. (4) the USA showed average lower mercury species concentrations when compared to those of the other world countries. The average ratios of China/USA values were 89, 76 and 160 for total mercury, RGM and Hg(p), respectively, during the years of 2000-2012.

Seasonal variations and sources study by way of back trajectories and ANOVA for ambient air pollutants (particulates and metallic elements) within a mixed area at Longjing, central Taiwan: 1-year observation.

This study measured the concentrations of particulates and metallic elements in ambient air by using PS-1 sampler (TSP) at Longjing area. And this study focuses on the collection of ambient air particulates, metallic elements, particulate-bound mercury Hg(p), concentrations. In addition, the sources of ambient pollutants by way of back trajectory analysis are found. Moreover, test mean concentration variance differences for metallic elements (PM, Hg(p), Mn, Fe, Zn, Cr, Cu, and Pb) among the four seasons (spring, summer, autumn and winter) through ANOVA are calculated. The result indicates that the average highest particulate concentration occurred in winter season, and the order was winter > spring > autumn > summer, and the mostly highest average metallic element (Mn, Fe, Zn, Cr, Cu, Pb) concentrations occurred in autumn. Moreover, the mostly average lowest metallic element concentrations occurred in summer. In addition, the above results of backward trajectories that the major particulate pollutants parcel mainly come from northeastern Taiwan. Moreover, when comparing the results of the first half year to that of the second half year, the they indicated that all metallic elements displayed significant differences in concentrations except those of Hg(p), Mn, Fe, Zn. Finally, metallic element Hg(p) is the only one which showed no significant concentration difference from either seasonal variations or half-year observations.

L-amino acid oxidase-induced apoptosis in filamentous Botrytis cinerea.

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.

Purification, identification and characterization of Nag2 N-acetylglucosaminidase from Trichoderma virens strain mango.

BACKGROUND: N-acetylglucosaminidase (NAGase) could liberate N-acetylglucosamine (GlcNAc) from GlcNAc-containing oligosaccharides. Trichoderma spp. is an important source of chitinase, particularly NAGase for industrial use. nag1 and nag2 genes encoding NAGase, are found in the genome in Trichoderma spp. The deduced Nag1 and Nag2 shares ~ 55% homology in Trichoderma virens. Most studies were focus on Nag1 and nag1 previously. RESULTS: The native NAGase (TvmNAG2) was purified to homogeneity with molecular mass of ~ 68 kDa on SDS-PAGE analysis, and identified as Nag2 by MALDI/MS analysis from an isolate T. virens strain mango. RT-PCR analyses revealed that only nag2 gene was expressed in liquid culture of T. virens, while both of nag1 and nag2 were expressed in T. virens cultured on the plates. TvmNAG2 was thermally stable up to 60 °C for 2 h, and the optimal pH and temperature were 5.0 and 60-65 °C, respectively, using p-nitrophenyl-N-acetyl-ß-D-glucosaminide (pNP-NAG) as substrate. The hydrolytic product of colloidal chitin by TvmNAG2 was suggested to be GlcNAc based on TLC analyses. Moreover, TvmNAG2 possesses antifungal activity, inhibiting the mycelium growth of Sclerotium rolfsii. And it was resistant to the proteolysis by papain and trypsin. CONCLUSIONS: The native Nag2, TvmNAG2 was purified and identified from T. virens strain mango, as well as enzymatic properties. To our knowledge, it is the first report with the properties of native Trichoderma Nag2.

Atmospheric-particulates-bound mercury Hg(p) study at five characteristic sampling sites in Taiwan.

The main purpose for this study is to observe the seasonal and month variations for particulates-bound mercury Hg(p) in total suspended particulates (TSP) concentration, dry deposition at five characteristic sampling sites during years of 2009-2010 in central Taiwan. The results show that the highest and lowest monthly average particulates-bound mercury Hg(p) concentrations in TSP were occurred in Dec. and Oct. at Gao-mei (wetland), Chang-hua (downtown) and He-mei (residential) sampling site. In addition, the results show that the highest and lowest monthly average particulates-bound mercury Hg(p) dry deposition was occurred in Feb. and Oct. at Quan-xing (industrial) sampling site. This study reflected that the mean highest particulates-bound mercury Hg(p) concentrations in TSP and mean highest particulates-bound mercury Hg(p) dry deposition were occurred at Gao-mei (wetland) and Quan-xing (industrial). However, the mean lowest particulates-bound mercury Hg(p) concentrations in TSP and mean lowest particulates-bound mercury Hg(p) dry deposition were also occurred at Gao-mei (wetland). Regarding seasonal variation, the order of mean-particulates-bound mercury Hg(p) concentrations in TSP in winter and spring were Gao-mei (wetland) > Quan-xing (industrial) > Bei-shi (suburban/coastal) > Chang-hua (downtown) > He-mei (residential). Finally, the order of mean-particulates-bound mercury Hg(p) dry deposition in fall, spring and summer were Quan-xing (industrial) > Bei-shi (suburban/coastal) > Chang-hua (downtown) > He-mei (residential) > Gao-mei (wetland).

Expression of L-amino acid oxidase of Trichoderma harzianum in tobacco confers resistance to Sclerotinia sclerotiorum and Botrytis cinerea.

L-amino acid oxidase (ThLAAO) secreted by Trichoderma harzianum ETS323 is a flavoenzyme with antimicrobial characteristics. In this study, we transformed the ThLAAO gene into tobacco to elucidate whether ThLAAO can activate defense mechanisms and confer resistance against phytopathogens. Transgenic tobacco overexpressing ThLAAO showed enhanced resistance against Sclerotinia sclerotiorum and Botrytis cinerea and activated the expression of defense-related genes and the genes involved in salicylic acid, jasmonic acid, and ethylene biosynthesis accompanied by substantial accumulation of H2O2 in chloroplasts, cytosol around chloroplasts, and cell membranes of transgenic tobacco. Scavenge of H2O2 with ascorbic acid abolished disease resistance against B. cinerea infection and decreased the expression of defense-related genes. ThLAAO-FITC application on tobacco protoplast or overexpression of ThLAAO-GFP in tobacco revealed the localization of ThLAAO in chloroplasts. Chlorophyll a/b binding protein (CAB) was isolated through ThLAAO-ConA affinity chromatography. The pull down assay results confirmed ThLAAO-CAB binding. Application of ThLAAO-Cy5.5 on cabbage roots promptly translocated to the leaves. Treatment of ThLAAO on cabbage roots induces systemic resistance against B. cinerea. Overall, these results demonstrate that ThLAAO may target chloroplast and activate defense mechanisms via H2O2 signaling to confer resistance against S. sclerotiorum and B. cinerea.

Plate assay for fungal enzymes using cellophane membranes.

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.

Induced proteome of Trichoderma harzianum by Botrytis cinerea.

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.

Efficient isolation of anthraquinone-derivatives from Trichoderma harzianum ETS 323.

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.

Medium optimization for polysaccharide production of Cordyceps sinensis.

As a potential anticarcinogenic agent, polysaccharides from Cordyceps sinensis have been demonstrated to possess strong antioxidation activity. The aim of the present research was to study the optimal medium to produce polysaccharides of C. sinensis by using response surface methodology (RSM). The composition of optimized medium for polysaccharide production calculated from the regression model of RSM was 6.17% sucrose, 0.53% corn steep powder, 0.5% (NH4)2HPO4, and 0.15% KH2PO4 at pH 4.44, with a predicted maximum polysaccharide production of 3.17 g/L. When applying this optimal medium, the maximum polysaccharide production was 3.05 and 3.21 g/L in a shake flask and a 5-L jar fermentor, respectively. When the pH was controlled at a higher level such as pH 5.0, both cell growth and polysaccharide production were inhibited. A low pH of 2.85 was required for maximum production of polysaccharides.

Characterization of a novel resistance-related deoxycytidine deaminase from Brassica oleracea var. capitata.

Brassica oleracea deoxycytidine deaminase (BoDCD), a deoxycytidine deaminase (DCD, EC 3.5.4.14) enzyme, is known to play an important role in the Trichoderma harzianum ETS 323 mediated resistance mechanism in young leaves of B. oleracea var. capitata during Rhizoctonia solani infection. BoDCD potentially neutralizes cytotoxic products of host lipoxygenase activity, and thereby BoDCD restricts the hypersensitivity-related programmed cell death induced in plants during the initial stages of infection. To determine the biochemical characteristics and to partially elucidate the designated functional properties of BoDCD, the enzyme was cloned into an Escherichia coli expression system, and its potential to neutralize the toxic analogues of 2'-deoxycytidine (dC) was examined. BoDCD transformants of E. coli cells were found to be resistant to 2'-deoxycytidine analogues at all of the concentrations tested. The BoDCD enzyme was also overexpressed as a histidine-tagged protein and purified using nickel chelating affinity chromatography. The molecular weight of BoDCD was determined to be 20.8 kDa as visualized by SDS-PAGE. The substrate specificity and other kinetic properties show that BoDCD is more active in neutralizing cytotoxic cytosine ß-d-arabinofuranoside than in deaminating 2'-deoxycytinde to 2'-deoxyuridine in nucleic acids or in metabolizing cytidine to uridine. The optimal temperature and pH of the enzyme were 27 °C and 7.5. The Km and Vmax values of BoDCD were, respectively, 91.3 µM and 1.475 mM for its natural substrate 2'-deoxycytidine and 63 µM and 2.072 mM for cytosine ß-d-arabinofuranoside. The phenomenon of neutralization of cytotoxic dC analogues by BoDCD is discussed in detail on the basis of enzyme biochemical properties.

Involvement of pachybasin and emodin in self-regulation of Trichoderma harzianum mycoparasitic coiling.

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.

Monomeric L-amino acid oxidase-induced mitochondrial dysfunction in Rhizoctonia solani Reveals a novel antagonistic mechanism of Trichoderma harzianum ETS 323.

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.

Identification of antibacterial mechanism of L-amino acid oxidase derived from Trichoderma harzianum ETS 323.

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.

A novel L-amino acid oxidase from Trichoderma harzianum ETS 323 associated with antagonism of Rhizoctonia solani.

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.

Cloning of a novel L-amino acid oxidase from Trichoderma harzianum ETS 323 and bioactivity analysis of overexpressed L-amino acid oxidase.

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% ß-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.

Purification and characterization of novel glucanases from Trichoderma harzianum ETS 323.

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.