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PubMed2005

Bioaccumulation of copper by Trichoderma viride.

Studies were carried out on interaction of Trichoderma viride with copper and reports bioaccumulation as a mechanism of copper tolerance during growth. There was a marked increase in the lag phase of the growth, which was concentration dependent. At a concentration of 100 mg/L of CuCl2.2H2O, 81% of Cu(II) were removed by 3.4 g/L of the biomass in 72 h. The process was temperature and pH dependent. The maximum copper bioaccumulation occurred at 30 degrees C, pH 5.0. Metabolic inhibitors such as sodium azide (NaN3) and 2,4-dinitrophenol (2,4-DNP) drastically reduced the extent of Cu(II) bioaccumulation. Electron microscopy and cell fractionation studies revealed that 70-80% of copper was present as a layer on the cell wall surface.

2,4-Dinitrophenol↗

PubMed2006

Effect of substrates on growth and shelf life of Trichoderma harzianum and its use in biocontrol of diseases.

A study on the effect of different agrowastes on the population density and shelf life of Trichoderma harzianum indicated that all the substrates showed effective propagation of T. harzianum; however, the population count after 30 days was maximum in used tea leaves (8 x 10(8) cfu/g) and shelf life was found to be maximum (2.9 x 10(5) cfu/g after 210 days) in wheat bran-sawdust. The application of these formulations on chickpea and groundnut plants significantly reduced the percent mortality due to chickpea wilt complex and groundnut collar rot disease, respectively.

Arachis↗

PubMed2006

Factors affecting foaming behavior in cellulase fermentation by Trichoderma reesei Rut C-30.

Coupling fermentation with in situ foam fractionation may be beneficial to cellulase production in optimizing oligomer inducer generation, minimizing catabolite repression and reducing cellulase degradation by proteases. In this study, the potential factors that may affect the foaming behavior of broth from Trichoderma reesei Rut C-30 fermentation were examined. These factors included solid (both cell and cellulose) concentrations, cellulase activity and extracellular protein concentration. The loss of cellulase activity caused by the foaming process was minimal. The foamate generation was lower in the presence of higher solids (cell and/or cellulose) concentrations. Cellulase appeared to promote the broth foaming ability but its enrichment ratio was not high (lower than 1.2). The enrichment ratios for the individual component enzymes (beta-glucosidase, endo- and exo-glucanases) were found to be similarly low. None of the cellulase components were likely the primary foaming factors. The foam also carried out cells and cellulose solids. The hydrophobicity of cell surface, studied at various fermentation stages and in both media with and without cellulose, increased as the fermentation approached the stationary phase and then decreased gradually after entering the stationary phase.

Cellulase↗

PubMed2006

Starch industry wastewater as a substrate for antagonist, Trichoderma viride production.

Starch industry wastewater was investigated to assess and improve its potential as a raw material for the conidia production of biocontrol fungi, Trichoderma viride. The wastewater was tested with and without supplements of glucose, soluble starch, meat peptone and probable conidiation inducer chemicals in shake flask culture. Addition of complex carbon source (soluble starch, 1% and 2% w/v) produced maximum conidia ( approximately 3.02 and 4.2 x 10(10)CFU/mL, respectively). On the other hand, glucose addition as a simpler carbon source was either ineffective or, reduced conidia production (from 1.6 x 10(8) in control to 3.0 x 10(7)CFU/mL in 5% w/v glucose supplement). Supplement of nitrogen source showed a small increase of conidia concentration. Propionic, maleic and humic acids, EDTA, pyridine, glycerol and CaCO(3) were examined as probable conidiation inducers and showed effect only on initial rate of conidiation with no increase in final conidia concentration. Intra and extracellular ATP correlation with spore production showed dependence on growth media used and conidia concentration at the end of fermentation. Addition of carbon and nitrogen sources showed an increase in protease activity (from 0.4985 to 2.43 IU/mL) and entomotoxicity (from 10448 to 12335 spruce budworm unit (SBU)/microL). Entomotoxicity was improved by 11% in fermenter over shake flask when starch industry wastewater was supplemented with meat peptone.

Animals↗

PubMed2006

Degradation of cyanide by Trichoderma mutants constructed by restriction enzyme mediated integration (REMI).

REMI technique was used to construct mutants with improved cyanide-degradation ability from biocontrol fungus Trichoderma koningii strain T30. The plasmid pV2 transformation was confirmed by PCR and Southern blot analysis. Out of 21 transformants, 15 single-copied transformants (71.4%) were found. To compare enzyme activities of rhodanese and cyanide hydratase, T. atroviride T23, T. harzianum T21 and their transformants constructed by REMI previously were also included. Transformants TkB6 (0.173 micromols thiocyanate formed min(-1)mg protein(-1)) from T30 and TaK1 (0.174 micromols thiocyanate formed min(-1)mg protein(-1)) from T23 showed higher rhodanese activity than other transformants and their wild strains. TkA9 (5.53 micromols formamide formed h(-1)mg protein(-1)) from T30 and Th64 (5.35 micromols formamide formed h(-1)mg protein(-1)) from T21 had higher cyanide hydratase activity than other transformants and their wild strains.

Base Sequence↗

PubMed2007

Evaluation of culture conditions for cellulase production by two Trichoderma reesei mutants under solid-state fermentation conditions.

Response surface methodology (RSM) was used to evaluate the effects of fermentation parameters for cellulase production by Trichoderma reesei QM9414 and T. reesei MCG77 in solid-state fermentation using rice bran as substrate. Initial pH, moisture content and temperature were optimized using filter paper activity (FPA) as response. Statistical analysis of the results for T. reesei QM9414 showed that only moisture content had significant effect on cellulase activity and had a linear effect on enzyme activity (maximum enzyme activities were obtained at 70% moisture content). The results for T. reesei MCG77 showed that temperature and moisture content were the most significant parameters for cellulase activity. The optimum cellulase production was in the temperature range of 25-30 degrees C and moisture content between 55% and 70%. After the optimization, the FPA in T. reesei MCG77 was increased by 2.5 folds compared to that of T. reesei QM9414.

Bioreactors↗

PubMed2005

Microbial transformation of silybin by Trichoderma koningii.

Microbial transformation of silybin A (1) and silybin B (2), the major hepatoprotective flavonolignan diastereomers from the fruits of Silybum marianum, with the culture broth of Trichoderma koningii gave two pairs of glucosylated derivatives. Their structures were identified as silybin A 3-O-beta-D-glucopyranoside (3), silybin A 7-O-beta-D-glucopyranoside (4), silybin B 3-O-beta-D-glucopyranoside (5) and silybin B 7-O-beta-D-glucopyranoside (6) by spectroscopic methods.

Fruit↗

PubMed2006

Mechanism of action of the endo-(1-->3)-alpha-glucanase MutAp from the mycoparasitic fungus Trichoderma harzianum.

(1-->3)-alpha-glucanases catalyze the hydrolysis of fungal cell wall (1-->3)-alpha-glucan, and function during cell division of yeasts containing this cell wall component or act in mycoparasitic processes. Here, we characterize the mechanism of action of the (1-->3)-alpha-glucanase MutAp from the mycoparasitic fungus Trichoderma harzianum. We observed that MutAp releases predominantly beta-glucose upon hydrolysis of crystalline (1-->3)-alpha-glucan, indicating inversion of the anomeric configuration. After having identified (1-->3)-alpha-glucan tetrasaccharide as the minimal substrate for MutAp, we showed that reduced (1-->3)-alpha-glucan pentasaccharide is cleaved into a trisaccharide and a reduced disaccharide, demonstrating that MutAp displays endo-hydrolytic activity. We propose a model for the catalytic mechanism of MutAp, whereby the enzyme breaks an intrachain glycosidic linkage of (1-->3)-alpha-glucan, and then continues its hydrolysis towards the non-reducing end by releasing beta-glucose residues in a processive manner.

Carbohydrate Conformation↗

PubMed2005

The Trichoderma reesei hydrophobin genes hfb1 and hfb2 have diverse functions in fungal development.

Hydrophobins are fungal self-assembling proteins. Here, the hydrophobin genes hfb1 and hfb2 were deleted in Trichoderma reesei and their biological roles studied. Our results suggest that HFBI has a role in hyphal development and HFBII in sporulation. Sporulating colonies of the Deltahfb2 strain were wettable and sporulation was only 50% of the parent strain. Colonies of Deltahfb1 showed wettable and fluffy phenotype. In shaken liquid cultures, the hyphae of Deltahfb1 were thinner and biomass formation was slower compared to the parent strain while in static liquid cultures no aerial hyphae were formed. Expressing the Schizophyllum commune hydrophobin SC3 in the Deltahfb1 strain restored the formation of aerial hyphae.

Fungal Proteins↗

PubMed2004

Functional analysis of tvsp1, a serine protease-encoding gene in the biocontrol agent Trichoderma virens.

Serine proteases are highly conserved among fungi and considered to play a key role in different aspects of fungal biology. These proteases can be involved in development and have been related to pathogenesis or biocontrol processes. A gene (tvsp1) encoding an extracellular serine protease was cloned from Trichoderma virens, a biocontrol agent effective against soilborne fungal pathogens. The gene was expressed in Escherichia coli and a polyclonal antibody was raised against the recombinant protein. The expression pattern of tvsp1 was determined and its physiological role was addressed by mutational analysis. Strains of T. virens in which tvsp1 was deleted (PKO) or constitutively overexpressed (POE) were not affected in growth rate, conidiation, extracellular protein accumulation, antibiotic profiles nor in their ability to induce phytoalexins in cotton seedlings. Tvsp1 overexpression, however, significantly increased the ability of some strains to protect cotton seedlings against Rhizoctonia solani. Our data show that Tvsp1 is not necessary for the normal growth or development of T. virens, but plays a role in the biocontrol process.

Base Sequence↗

PubMed2004

Insight into Trichoderma reesei's genome content, organization and evolution revealed through BAC library characterization.

Trichoderma reesei is an important industrial fungus known for its ability to efficiently secrete large quantities of protein as well as its wide variety of biomass degrading enzymes. Past research on this fungus has primarily focused on extending its protein production capabilities, leaving the structure of its 33 Mb genome essentially a mystery. To begin to address these deficiencies and further our knowledge of T. reesei's secretion and cellulolytic potential, we have created a genomic framework for this fungus. We constructed a BAC library containing 9216 clones with an average insert size of 125 kb which provides a coverage of 28 genome equivalents. BAC ends were sequenced and annotated using publicly available software which identified a number of genes not seen in previously sequenced EST datasets. Little evidence was found for repetitive sequence in T. reesei with the exception of several copies of an element with similarity to the Podospora anserina transposon, PAT. Hybridization of 34 genes involved in biomass degradation revealed five groups of co-located genes in the genome. BAC clones were fingerprinted and analyzed using fingerprinted contigs (FPC) software resulting in 334 contigs covering 28 megabases of the genome. The assembly of these FPC contigs was verified by congruence with hybridization results.

Chromosome Mapping↗

PubMed2004

The fungal STRE-element-binding protein Seb1 is involved but not essential for glycerol dehydrogenase (gld1) gene expression and glycerol accumulation in Trichoderma atroviride during osmotic stress.

Fungi counteract extracellular osmotic pressure by producing intracellular polyols to prevent loss of water. In yeast osmotic signaling involves a MAP-kinase pathway culminating at the STRE-binding transcription factors Msn2/4. We investigated the role of a putative STRE-binding orthologue of Trichoderma atroviride, Seb1, in osmotic stress signaling. T. atroviride, subjected to osmotic stress (10% glucose or glycerol, 1M KCl or NaCl), responds by raising its intracellular glycerol level. In contrast to Aspergillus nidulans, no erythritol is accumulated. Accumulation of glycerol levels under osmotic stress is strongly reduced in a seb1 deletion strain. To investigate glycerol biosynthesis in T. atroviride, the genes encoding glycerol dehydrogenase (gld1) and glycerol-3-phosphate dehydrogenase (gfd1) were cloned and characterized. Although both genes contain STRE-elements in their 5'-non-coding regions, only gld1 mRNA accumulates in response to osmotic stress, whereas expression of gfd1 remains at a constitutive level. In comparison to A. nidulans gld1 transcript levels in T. atroviride rise very slowly under conditions of salt stress. Deletion of seb1 results in a delayed accumulation of the gld1 transcript, but final levels match those in the wild-type whereas gfd1 transcript accumulation remains unaffected. Assays for glycerol dehydrogenase and glycerol-3-phosphate dehydrogenase enzymatic activities reveal an increase of the former--whereas the latter remains mainly unaffected--in the wild-type and the Deltaseb1 strain under different kinds of osmotic stress. The data suggest that Seb1 is only involved in, but not essential for osmotic stress response which is in contrast to the yeast orthologues Msn2/4.

5' Untranslated Regions↗

PubMed2005

Proteomic analysis of secreted proteins from Trichoderma harzianum. Identification of a fungal cell wall-induced aspartic protease.

Trichoderma mycoparasitic activity depends on the secretion of complex mixtures of hydrolytic enzymes able to degrade the host cell wall. We have analysed the extracellular proteome secreted by T. harzianum CECT 2413 in the presence of different fungal cell walls. Significant differences were detected in 2DE maps, depending on the use of specific cell walls or chitin. A combination of MALDI-TOF and liquid chromatography mass spectrometry allowed the identification of a novel aspartic protease (P6281: MW 33 and pI 4.3) highly induced by fungal cell walls. A broad EST library from T. harzianum CECT 2413 was used to obtain the full-length sequence. The protein showed 44% identity with the polyporopepsin (EC 3.4.23.29) from the basidiomycete Irpex lacteus. Lower identity percentages were found with other pepsin-like proteases from filamentous fungi (<31%) and animals (<29%). Northern blot and promoter sequence analyses support the implication of the protease P6281 in mycoparasitism.

Amino Acid Sequence↗

PubMed2006

Cloning and characterization of the erg1 gene of Trichoderma harzianum: effect of the erg1 silencing on ergosterol biosynthesis and resistance to terbinafine.

Trichoderma species are commonly used as biocontrol agents of different plant-pathogenic fungi. Terpene compounds are involved in the biocontrol process due to their antifungal properties (e.g., ergokonins and viridins) but additionally their structural function in the cell membranes (ergosterol) is essential. We report here the characterization of the T. harzianum erg1 gene, encoding a squalene epoxidase, a key enzyme in the biosynthesis of triterpene derivatives such as ergosterol. In T. harzianum the partial silencing of the erg1 gene gave rise to transformants with a higher level of sensitivity to terbinafine, an antifungal compound that acts specifically over the squalene epoxidase activity. In addition, these silenced transformants produced lower levels of ergosterol than the wild type strain. Finally, the silencing of the erg1 gene resulted in an increase in the expression level of the erg7 gene that encodes the oxidosqualene lanosterol-cyclase, another enzyme of the terpene biosynthesis pathway.

Antifungal Agents↗

PubMed2006

Overexpression of the Saccharomyces cerevisiae RER2 gene in Trichoderma reesei affects dolichol dependent enzymes and protein glycosylation.

Protein secretion in Trichoderma reesei could be stimulated by overexpression of the yeast Saccharomyces cerevisiae DPM1 gene encoding dolichyl phosphate mannose synthase (DPMS) a key enzyme in the O-glycosylation pathway. The secreted proteins were glycosylated to the wild type level. On the other hand, the elevated concentration of GDP-mannose, a direct substrate for DPMS, resulting from overexpression in T. reesei of the mpg1 gene coding for guanyltransferase, did not affect secretion of proteins but did affect the degree of their O- and N-glycosylation. In this paper, we examined the effects of dolichol, an indispensable carrier of sugar residues in protein glycosylation, on the synthesis of glycosylated proteins. An increase in dolichol synthesis was obtained by overexpression of the yeast gene encoding cis-prenyltransferase, the first enzyme of the mevalonate pathway committed to dolichol biosynthesis. We observed that, an increased concentration of dolichol resulted in an increased expression of the dpm1 gene and DPMS activity and in overglycosylation of secreted proteins.

Chitin↗

PubMed2007

Partial silencing of a hydroxy-methylglutaryl-CoA reductase-encoding gene in Trichoderma harzianum CECT 2413 results in a lower level of resistance to lovastatin and lower antifungal activity.

In the present article, we describe the cloning and characterization of the Trichoderma harzianum hmgR gene encoding a hydroxymethylglutaryl CoA reductase (HMGR), a key enzyme in the biosynthesis of terpene compounds. In T. harzianum, partial silencing of the hmgR gene gave rise to transformants with a higher level of sensitivity to lovastatin, a competitive inhibitor of the HMGR enzyme. In addition, these hmgR-silenced transformants produced lower levels of ergosterol than the wild-type strain in a minimal medium containing lovastatin. The silenced transformants showed a decrease in hmgR gene expression (up to a 8.4-fold, after 72h of incubation), together with an increase in the expression of erg7 (up to a 15.8-fold, after 72h of incubation), a gene involved in the biosynthesis of triterpenes. Finally, hmgR-silenced transformants showed a reduction in their antifungal activity against the plant-pathogen fungi Rhizoctonia solani and Fusarium oxysporum.

Antibiosis↗

PubMed2004

Isolation, characterization and expression of the hex1 gene from Trichoderma reesei.

Polymers of the HEX1 protein produce Woronin bodies in filamentous fungi. We have isolated and sequenced the hex1 gene and flanking regions from the industrially exploited fungus Trichoderma reesei. Multiple transcription start sites (TSS) and the 5' untranslated region (UTR) were identified by 5'RACE PCR. There are three hex1 transcript types, two of which originate from two TSSs at approximately -320 and -1335 from the start codon, which are separated by a 500-bp intron within the 5'UTR. The third transcript type results from alternative splicing of the intron within the coding sequence at the 3' end, which results in the inclusion or exclusion of an unconserved histidine-rich coding region. The three transcripts code for two forms of HEX1 protein. N-terminal sequencing of HEX1 separated by 2D gel electrophoresis confirms that there are two forms of HEX1 protein which are modified further by alternative cleavage of the N-terminus. The dominant form of HEX1 is coded by a cDNA with TSS at position -1335. Expression of hex1 on cellulase-inducing medium peaks strongly within 24 h of growth but the protein is expressed at a lower and more consistent level in medium containing glucose. This is the first investigation of expression of the hex1 gene encoding a protein unique to filamentous fungi.

Amino Acid Sequence↗

PubMed2004

Effect of treatment with Trichoderma harzianum Rifai formulated in invert emulsion on postharvest decay of apple blue mold.

Conidia of Trichoderma harzianum were formulated in invert emulsion (water-in-oil type) at a concentration of 6.0 x 10(7) conidia/ml of the formulation. Treatments with conidia in the formulated emulsion and conidia in sterile distilled water were conducted to assess the effect on Penicillium expansum Link inoculated on apple fruits. Comparisons were made with control treatments of an uninoculated emulsion and uninoculated distilled water. The T. harzianum-containing emulsion significantly (P <or= 0.05) reduced the decay-lesion diameter compared to the T. harzianum-containing water or the uninoculated emulsion or distilled water controls (17.5, 25.7, 34.0, and 34.2 mm, respectively). Significant differences (P <or= 0.05) were also obtained between means of percent reduction in decay-lesion diameter relative to sterile distilled water control in the treatments with formulated and nonformulated conidia and control with uninoculated invert emulsion (48.8%, 24.8%, and 0.6%, respectively). Mean time period needed for sporulation of P. expansum was significantly (P <or= 0.05) increased when the treatments with formulated T. harzianum conidia were applied in comparison with the other types of treatments (14.2 versus 12.2, 9.0, and 8.8 days, respectively, for the other treatments). However, no significant differences (P <or= 0.05) were obtained in means of decay-lesion diameter or in means of time period needed for sporulation of P. expansum in the control treatments (uninoculated invert emulsion versus sterile distilled water). This indicates that the effect of the formulation ingredients on decay-lesion development of P. expansum or its sporulation was not significant. A significant (P <or= 0.05) long period of protection from P. expansum infection (up to 2 months) was also obtained when unwounded apple fruits were dipped for 30-s period in formulated T. harzianum conidia before being inoculated by P. expansum compared to the wounded fruits. This indicates the importance of the latter type of treatment in protecting apple fruits from blue mold infection for long time at postharvest stage without refrigeration.

Antibiosis↗