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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, β-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.

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-β-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.

The study was to ascertain the feasibility of using rice straw fermented by Trichoderma as ruminant roughage. Three species or three strains of Trichoderma (NTLg-Te S5-1, NTLg-Te S9-1, and Tien Chung Ca6-1) were used to inoculate hot water-sterilized rice straw. Result showed that rice straw fermented with Trichoderma NTLg-Te S5-1 had the highest cellulase and xylanase activity. Antioxidant ferulic acid content of water extract was improved after fermentation. Eighteen Barbados sheep were randomly distributed into one of three dietary groups. Ration of Bermuda hay group (BER) is 90% Bermuda hay with 10% concentrate. Unfermented rice straw (RS) group and fermented rice straw (FRS) group substituted 25% of Bermuda hay with rice straw or fermented rice straw. Dry matter intake, daily weight gain, blood analysis, and feed conversion ratio were not significantly different among groups after 4 weeks of trial. Dry matter digestibility of BER was the highest, and of FRS was significantly higher than RS. Malondialdehyde (MDA) content in serum of FRS and BER was lower than RS. These results indicated that 25% substitution of Bermuda hay with Trichoderma-fermented rice straw in the diet of Barbados sheep could increase digestibility and inhibit lipid oxidation when compared to unfermented rice straw.

This investigation analyzes the particulate-bound mercury (PBM) compositions in soil and vegetation at a traffic sampling site in Taichung, Taiwan, during a sampling period from October to December, 2015. A direct mercury analyzer (DMA-80) was used to measure the particulate-bound mercury (PBM). A T-test was conducted to determine the mean differences between the PBM composition in soil and that in vegetation at the site. The results indicate that 1) the mean particulate-bound mercury compositions in soil and vegetation were the lowest in November, when the (mean OR average) wind speed was the highest (4.1 m/s); 2) Particulate-bound mercury compositions (PBM) in both soil and vegetation correlated weakly with temperature, humidity and wind speed; 3) T-test statistical results denoted that the PBM compositions did not significantly differ between soil and vegetation in the three-month sampling period.

This study was conducted to evaluate the dietary supplementation of Trichoderma pseudokoningii enzyme powder (EP) and its effect on the growth performance, intestinal morphology and antioxidative status of broilers. In vitro experiments showed that xylanase and cellulase activities of EP were 487.96 ± 14.14 U/g and 18.55 ± 0.54 U/g, respectively. Total phenolic and ferulic acid content was 4.79 ± 0.35 mg GAE/g DW and 4.38 ± 0.32 μM/g DW, respectively. A total of 240 d-old Ross 308 male broiler chicks were randomly subjected to one of four treatments, namely diets supplemented with dry EP at 0% (control), 0.1%, 0.2% or 0.4% for 35d. Results showed that in the starter phase (1–21 d), the EP supplemented groups had improved body weight, weight gain and FCR compared to the control group. The villus:crypt ratio of both jejunum and ileum in all EP-treated groups was significantly higher than that of the control group. When testing serum antioxidant activity, results showed that 0.2% and 0.4% EP supplementation significantly increased superoxide dismutase activity in both the finisher phase and the overall experimental period. In addition, catalase activity was elevated in all EP supplemented groups in both the finisher phase and throughout the experimental period. 0.1% EP seemed to have no effect during the starter period. Serum malondialdehyde concentration was not affected by EP supplementation. These results suggest that EP is potential feed additive in terms of improving broiler performance and enhancing antioxidative status, while also providing an optimal intestinal environment.

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.

The effect of chrysophanol, a secondary metabolite of Trichoderma harzianum , on the growth and disease resistance of cabbage against Botrytis cinerea was investigated. Chrysophanol assisted cabbage growth and promoted resistance against B . cinerea . Proteins related to photosynthesis were expressed in T . harzianum - and chrysophanol-treated cabbages. The transcription levels of photosynthesis- and sucrose transport-related genes increased in chrysophanol-treated cabbages. The transcription levels of chitinase increased in T . harzianum -treated cabbages and those of ascorbate peroxidase in T . harzianum - and chrysophanol-treated cabbages. Chrysophanol is involved in stimulating plant growth and photosynthesis and in eliciting host defense responses during the colonization of T . harzianum .

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.

This experiment investigated the effects of wheat bran fermented with Trichoderma pseudokoningii (FWB) on growth performance, intestinal morphology and the incidence of non-specific pathological lesions in broilers. In vitro experimental results showed that cellulase and xylananse activity of fermented wheat bran reached its peak at day 4 and solid-state fermentation (SSF) enhanced the reducing sugar content. In addition, the possibility of Trichoderma adhering to the broiler’s crop epitheliums was also discovered. A total of 180 day-old Ross 308 male broilers were randomly distributed into one of the three dietary groups until 35 d of age: basal diet (control), 10% of basal diet replaced with wheat bran (10% WB) or 10% of diet replaced with fermented wheat bran (10% FWB). Results showed that 10% FWB group had lower feed consumption than the others, but improved feed conversion ratio (FCR) for starter phase (1 to 21 d) when compared to the control group. Furthermore, 10% FWB group had significantly increased villus height and villus height/crypt depth ratio in the ileum compared to the control group. Coliform bacteria count in the ileum was lower in the 10% WB group than the control, however, there were no differences between the 10% WB and 10% FWB treatment groups in the C. perfringens count. There were no morphological changes or incidences of non-specific pathological lesions in the 10% FWB group. These results suggested that replacing 10% of a basal diet with fermented wheat bran could not only improve growth performance but also provide optimal intestinal morphology in broilers.

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.

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.

This study analyzes the particulate-bound mercury (PBM) contents of two parts (leaf and stem) of different crops (white cabbage, Peking cabbage, and chili) at a sampling site in the coastal zone in the Taichung area from October 7 to 15, 2014. A direct mercury analyzer (DMA-80) was used to measure mercury content. The Mann-Whitney rank sum test was utilized to determine the mean differences between the PBM in two parts of various crops (white cabbage leaf, white cabbage stem, Peking cabbage leaf, Peking cabbage stem, and chili) at the Long-Jing sampling site. The results indicated the following: 1) The average mercury content was lowest in the Peking cabbage stem. The ratio of the mercury content in the stem to that in the leaf of the white cabbage was 5 and that of Peking cabbage was 3.5.2) No significant difference was observed between median mercury content in white cabbage leaf and Peking cabbage leaf, white cabbage stem and Peking cabbage stem, and Peking cabbage leaf and chili. 3) The averag...

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.

Endophytic bacterial diversity in four rice cultivars grown in two soil-types, their plant-probiotic features and rhizospheric deployment under P-stress were investigated. Oryza sativa cvs. TCN1, TCS10, TK8, and TN71 were cultivated in greenhouse using non-sterile acidic and near-neutral paddy soils for 60 days. Root, stem and leaf tissues were screened for culturable bacterial endophytes using nutrient agar. Endophytes were identified and profiled for plant-probiotic features. The richness, Shannon-Weiner diversity, evenness and Venn’s distribution in terms of endophytic strains were evaluated. Seed-borne endophytes were characterized through DGGE. The deployment of endophytes into the rhizosphere in TCN1 and TK8 under gnotobiotic P-stress was assessed. A total of 52 distinct endophytic bacterial strains affiliated to 5 classes and 20 discrete genera exhibiting differential plant-probiotic features were isolated from various tissues of four different rice cultivars. The diversity and distribution of endophytes fluctuated with soil-type, tissue-type and rice genotype. Gnotobiotic insoluble P treatment revealed significantly enhanced deployment of P-solubilizing rhizobacteria in TCN1 as compared to soluble P and P-lacking control. Rice endophytic bacteria are diverse, and their distribution within the plant and deployment as rhizobacteria were found to be influenced by host genotype, edaphic factors and nutrient stress.

The main purpose of this study was to monitor ambient air particulates and particulate-bound Hg (Hg[p]) compositions in different crops in the coastal zone in the Taichung, Taiwan, area at a sampling site during 2013 October to 2013 December. In addition, a direct mercury analyzer (DMA-80) was used to directly detect the concentration with measured results by the regression equation of Hg in the ambient air. The results indicated that: 1) the mean values of ambient air Hg(p) compositions in rice displayed no significant differences for weeks one, two, and three versus those of the other crops (white cabbage, Arden lettuce, and Gynura procumbens); 2) for white cabbage, the higher the humidity, the lower the average ambient air Hg(p) compositions that were obtained, a phenomenon that was also reflected on the crops of white cabbage and G. procumbens; 3) Arden lettuce displayed no significant differences in the leaf Hg(p) compositions during weeks one, two, and three; however, the stem portions displayed sig...

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 β-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 β-d-arabinofuranoside. The phenomenon of neutralization of cytotoxic dC analogues by BoDCD is discussed in detail on the basis of enzyme biochemical properties.

This study concerns the concentrations of particulates in ambient air, dry deposited particulates, particulate-bound mercury (Hg(p)), and Hg(p) that is dry deposited in both total suspended particulates and on a dry deposition plate at Long Cyuan Elementary School (LCYES), Lung Ching Elementary School (LCHES), and Long Shan Primary School (LSPS) in Longjing. The LCHES site had the highest particulate Hg(p) concentrations in October and November, and the least dry deposition of particulates in October. The LSPS site had the lowest Hg(p) dry depositions in October. The LCYES site had the lowest particulate and Hg(p) concentrations in November and October, and had the highest dry deposition of particulates and Hg(p) in November and December. These particulates originated from the northwest of Taiwan and were transported by way of mainland China in October. A back trajectory analysis supported this finding. Therefore, particulate concentrations in ambient air were highest in October and the major sources of particulate-bound mercury Hg(p) in ambient air were in mainland China, especially in October and December. Mainland China and Taichung Thermal Power Plant were the main sources of particles and Hg(p) in ambient air.

This study focuses on particulate-bound Hg (Hg[p]) composition (ng/g) (defined as ng/g), concentrations (ng/m3) and dry deposition (ng/m2*min) variations for PM18, PM10, PM2.5, and PM1.0. Three characteristic sampling sites collected 1-day ambient suspended particles during November and December 2010 using MOUDI-100S4 samplers. Additionally, Hg(p) size distributions are also characterized. Composition variations of Hg(p) in PM18 were in the order of 0.158 ± 0.055 ng/g (Quan-xing sampling site) > 0.086 ± 0.097 ng/g (Hung-kuang sampling site) > 0.083 ± 0.065 ng/g (Gao-mei sampling site). Further, composition variations of Hg(p) in PM10 were in the order of 0.115 ± 0.038 ng/g (Quan-xing site) > 0.107 ± 0.080 ng/g (Gao-mei site) > 0.103 ± 0.060 ng/g (Hung-kuang site). Composition variations of Hg(p) in PM2.5 were in the order of 0.885 ± 0.423 ng/g (Quan-xing site) > 0.458 ± 0.105 ng/g (Gao-mei site) > 0.372 ± 0.157 ng/g (Hung-kuang site). Finally, composition variations of Hg(p) in PM1.0 were in the order of ...

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.

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.

The focus of this study is to observe the seasonal variations of particulate bound mercury Hg(p) concentration and amount of Hg(p) (ng/g) in total suspended particulates (TSP) and dry deposition at two (industrial and wetland) characteristic sampling sites in Taiwan, during the years 2009–2010. Concentrations of Hg(p) from each sample (quartz filter, overhead projection film) were analyzed by a direct Hg(p) analyzer DMA-80. Approximately 30 mg of the filter sample was loaded directly into the DMA-80 and analyzed. The results indicated that the mean highest Hg(p) in TSP (expressed in ng/m3) and dry deposition (expressed in ng/m2/min) both occurred at the industrial site (Quan-xing) while the corresponding mean lowest Hg(p) concentrations occurred at the wetland site (Gao-mei). Also, the mean highest amount of Hg(p) (expressed in ng/g) in TSP and dry deposition occurred at the industrial site (Quan-xing), while the mean lowest amount of Hg(p) occurred at the wetland site (Gao-mei). Metal products manufactur...

The main purpose of this study was to monitor ambient air particles and particulate-bound mercury Hg(p) in total suspended particulates (TSP) concentration, dry deposition at five characteristic-sampling sites. In addition, the calculated/measured dry deposition flux ratios of ambient air particles and particulate-bound mercury Hg(p) using three dry deposition models during the years of 2009–2010 were also studied. The results showed that the order of seasonal variations for mean particulate-bound mercury Hg(p) concentrations in TSP at Bei-shi (suburban/coastal), Chang-hua (downtown) and He-mei (residential) were all shown as spring > fall, winter > summer, respectively. However, the order of seasonal variations mean particulate-bound mercury Hg(p) concentrations in TSP that occurred at Gao-mei (wetland) were shown as spring > fall > winter > summer, respectively. Finally, the results of this study also indicated that using the Noll and Fang model exhibited batten prediction results for ambient air particles and particulate-bound mercury Hg(p) dry deposition when compared with the other depositions models at any of the characteristic-sampling sites for this study.

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).

From September 3, 2009, to March 5, 2010, the dry deposition, total suspended particulate (TSP) matter, coarse and fine particulates, and bound mercury (Hg[p]) in the atmosphere were investigated at Bei-Shi sampling site in central Taiwan. When using projection film as collection medium, the average ambient air particulates for day dry deposition fluxes and TSPs were 162.09 ± 97.69 (μg/(min/cm2)) and 78.71 ± 34.42 (μg/m3), respectively. The average fine particulate matter (PM2.5) and coarse particulate matter (PM2.5–10) were 20.5 ± 6.30 μgm−3 and 11.2 ± 4.44 μg m−3, respectively. Average atmospheric particulates Hg(p) in dry depositions, TSPs, PM2.5−10, and PM2.5were 0.156 ± 0.082 ng/m2*min, 0.07 ± 0.036 ng/m3, 0.011 ± 0.006 ng/m3,and 0.007 ± 0.005, respectively. The average compositions of atmospheric bound Hg Hg(p) in dry depositions, TSPs, PM2.5−10 and PM2.5 were 1.02, 36.8, 4.16 and 2.821, respectively. Also, the highest composition for particulate Hg(p) in TSP was observed in spring (50.74 ng/g), and...

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.

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.

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.

In this research, the factors of oxygen concentration (21%, 30%, and 40%) and olive oil addition were studied on the production of mycelial biomass and polysaccharides of Grifola frondosa in a 5-L jar fermentor. The 40% oxygen was found to inhibit both cell growth and polysaccharide production with a higher rate of glucose consumption. The highest cell concentration was found at 9.29 g/L at day 9 with 1 vvm at 21% O2. With 30% O2 the inter-cellular polysaccharide (IPS) level increased earlier than 21% O2 and reached the maximum concentration at day 8 with 21.9 mg/g. On the other hand, 21% O2 reached the maximum concentration at day 11 with 19.5 mg/g. With 1% olive oil addition in 21% O2 and 40% O2, the production of mycelial biomass was enhanced and increased to 10.1 and 14.9 g/L, respectively, after 9 days’ cultivation. And the extra-cellular polysaccharide (EPS) production increased from 0.7–0.9 g/L to 2.24 g/L and 3.00 g/L at day 13 with 21% O2 and 40% O2 aeration, respectively. In addition, the IPS increased rapidly and reached the maximum level of 28.2 mg/g at day 7 and this level remained till day 13 through the whole fermentation. The scanning electron microscopy (SEM) of the mycelia and cell activity were studied to investigate the role of olive oil under high concentration of oxygen aeration. The vegetative cell was observed from both the 7-day-old culture and the 9-day-old culture from both 21% O2 and 40% O2 with olive oil addition. With 40% O2 aeration, low growth activity and significant wrinkles appeared on the surface of mycelia on both 7-day-old culture and 9-day-old culture. The olive oil addition in media was observed to reduce the wrinkles appeared on the surface of mycelia under high concentration oxygen and made the cell be aging more slowly.

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.

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.

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.

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.

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.

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.

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.

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.