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PubMed2002

Comparative degradation of oomycete, ascomycete, and basidiomycete cell walls by mycoparasitic and biocontrol fungi.

Fourteen fungi (primarily representing mycoparasitic and biocontrol fungi) were tested for their ability to grow on and degrade cell walls (CWs) of an oomycete (Pythium ultimum), ascomycete (Fusarium equisetii), and basidiomycete (Rhizoctonia solani), and their hydrolytic enzymes were characterized. Protein was detected in the cultural medium of eleven of the test isolates, and these fungi significantly degraded CWs over the 14-day duration of the experiment. In general, a greater level of CW degradation occurred for F. equisetii and P. ultimum than for R. solani. Fungi that degraded F. equisetii CWs were Coniothyrium minitans, Gliocladium roseum, Myrothecium verrucaria, Talaromyces flavus, and Trichoderma harzianum. Taxa degrading P ultimum CWs included Chaetomium globosum, Coniothyrium minitans, M. verrucaria, Seimatosporium sp., Talaromyces flavus, Trichoderma hamatum, Trichoderma harzianum, and Trichoderma viride. Production of extracellular protein was highly correlated with CW degradation. Considerable variation in the molecular weights of CW-degrading enzymes were detected among the test fungi and the CW substrates in zymogram electrophoresis. Multivariate analysis between CW degradation and hydrolysis of barley beta-glucan (beta1,3- and beta1,4-glucanases), laminarin (beta1,3- and beta1,6-glucanases), carboxymethyl cellulose (endo-beta1,4-glucanases), colloidal chitin (chitinases), and chitosan (chitosanases) was conducted. For F. equisetii CWs, the regression model accounted for 80% of the variability, and carboxymethyl cellulases acting together with beta-glucanases contributed an R2 of 0.52, whereas chitinases and beta-glucanases alone contributed an R2 of 0.11 and 0.12, respectively. Only 61% of the variability observed in the degradation of P. ultimum CWs was explained by the enzyme classes tested, and primarily beta-glucanases (R2 of 0.53) and carboxymethyl cellulases (R2 of 0.08) alone contributed to CW break down. Too few of the test fungi degraded R. solani CWs to perform multivariate analysis effectively. This study identified several fungi that degraded ascomyceteous and oomyceteous, and to a lesser extent, basidiomycetous CWs. An array of enzymes were implicated in CW degradation.

Carbohydrate Metabolism↗

PubMed2002

Bioconversion of poultry wastes I-factors influencing the assay and productivity of crude uricase by three uricolytic filamentous fungi.

The optimum temperature for biomass yield and uricase production by uricolytic fungi, Aspergillus terreus. A. flavus and Trichoderma sp. was at 30 degrees C. The time required for maximum production of uricase and biomass yield was 4 days for two Aspergillus species and 6 days for Trichoderma sp. The optimum pH was at 6.4 for A. terreus and pH 6.6 for both A. flavus and Trichoderma sp. The maximum fungal biomass yield was achieved in medium supplemented with 4% poultry waste. The best carbon sources for the production of uricase and mycelia yield were glycerol, sucrose and maltose by A. terreus, A. flavus and Trichoderma sp., respectively. Uric acid was found to be the best nitrogen source for production and activity of uricase by the three tested fungi. The addition of some vitamins to the culture media increased the maximum biomass yield of all the isolates, but did not significantly increase uricase production.

Animals↗

PubMed1996

Fungitoxic activity of saponins: practical use and fundamental principles.

Saponins, as aescin or alfalfa saponins have fungistatic activity on Trichoderma viride G fungal strain. (Biacs and Gruiz, 1982) The principle of fungistatic activity is an interaction between saponin and membrane constituents, such as sterols, proteins and phospholipids. The interaction results in the destruction of the cell membrane and an increase of the ion permeability (Gruiz and Biacs, 1989). Some Trichoderma strains are sensitive, whereas others are resistant to saponins. Their lipid composition after treatment differs considerably (Biacs and Gruiz, 1984), although ergosterol content of the membrane increased in both sensitive and nonsensitive strains. The possible metabolic changes in fatty acid composition of membrane phospholipids influence sensitivity of fungi to saponins. An important role is attributed to linoleic acid (Gruiz and Biacs, 1989). Since the fungal membrane composition can be changed by exogenously added sterols and fatty acids or triglycerides (Gruiz and Biacs, 1990), experiments were made to decrease saponin sensitivity of Trichoderma viride by giving linoleic-acid-containing oil and ergosterol. In such a way the elimination of the inhibitory effect of saponins was successful. The ergosterol content after saponin treatment is almost equal value not depending on the sensitivity and the ergosterol content of control or modified cells. This equalizing behavior of saponins is remarkable; its regulatory effect affects not only the ergosterol content, but also the fatty acid composition of the fungal membrane.

Antifungal Agents↗

PubMed2006

Detection of peptaibols and partial cloning of a putative peptaibol synthetase gene from T. harzianum CECT 2413.

The characterization of 11- and 18-residue peptaibols (peptides synthesized by peptide synthetases) at Trichoderma harzianum CECT 2413 (a filamentous fungus) was performed. Using a heterologous probe from tex1, the only peptaibol synthetase cloned and characterized so far in Trichoderma species, was cloned; a region that comprised 11676 bp of a second peptide synthetase gene detected in these strain (called salps2) and sequenced. The deduced sequence of Salps2 (3891 amino acids) contained three complete and a fourth incomplete module of a peptide synthetase, in which the typical adenylation, thiolation and condensation domains were found, but also an additional dehydrogenase/reductase domain in the C-terminus of the last module. Based on sequence similarity and analysis of its modular structure, it is proposed that Salps2 is a peptaibol synthetase. Additionally, analysis of =4.4-kb sequence downstream of salps2 was done and the signature sequences of Salps2 were identified and compared with those of available sequences of the other Trichoderma peptaibol synthetases.

Cloning, Molecular↗

PubMed1981

Affinity chromatography of proteolytic enzymes on silica-based biospecific sorbents.

New biospecific sorbents for affinity chromatography of proteolytic enzymes were prepared by the attachment of the cyclopeptide antibiotics bacitracin, bacilliquin or gramicidin S to aminosilochrom via a reaction with p-benzoquinone. The content of the cyclopeptide ligands within the sorbents varied from 2 to 46 mumol/g. The sorbents prepared by this reaction were successfully applied in the purification of the carboxylic proteinases produced by fungi, Russula decolorans (a basidiomycete) and Trichoderma lignorum, as well as crude pepsin. Serine proteinases from Thermoactinomyces vulgaris, Trichoderma koningii, Trichoderma lignorum and bacilli (subtilisins) were also submitted to chromatography on these materials. The yields of purified enzymes approached quantitative levels, sometimes being higher as a result of elimination of inhibitors. An important advantage of these sorbents is their stability against the enzymes degrading the carbohydrate matrixes of affinity sorbents synthesized on the basis of agarose, dextran or cellulose derivatives.

Animals↗

PubMed2005

Fungal solid state culture of palm kernel cake.

Palm kernel cake (PKC), an agro-industrial by-product used extensively in the animal feed industry, has limited use in fish feeds due to its high fiber and low protein contents. In this study, PKC was processed under solid state culture conditions with five fungal strains and the effect of this fungal culturing on the amino acid, fatty acid, cellulose and hemicellulose fractions was evaluated. Fungal strains used were Sclerotium rolfsii, Trichoderma harzianum, Trichoderma longiobrachiatum, Trichoderma koninggi and Aspergillus niger. Fungal growth was carried out at 50% moisture level and 1% inoculum level for 7 days. A significant increase in protein content from 18.76% to 32.79% was obtained by growing T. longibrachiatum on PKC. Cellulose level decreased significantly from 28.31% to 12.11% for PKC cultured with T. longibrachiatum, and hemicellulose from 37.03% to 19.01% for PKC cultured with A. niger. Fungal culturing of PKC brought about an increase in the level of unsaturated- and a decrease in the level of the saturated-fatty acids.

Amino Acids↗

PubMed2000

Aroma compounds recovery from mycelial cultures in aqueous two-phase processes.

This paper presents the evaluation of the potential use of aqueous two-phase systems (ATPS) for the recovery of 6-pentyl-alpha-pyrone (6PP) produced by Trichoderma harzianum. The partition behaviour of 6PP and Trichoderma harzianum mycelium (biomass) in polyethylene glycol (PEG)-salt (phosphate and sulphate) and PEG-dextran ATPS was investigated. The influence of defined system parameters (e.g. molecular mass of PEG and dextran, volume ratio, etc.) on the partition behaviour of 6PP and Trichoderma harzianum mycelium was evaluated to select under which conditions 6PP and mycelium partition to opposite phases. In PEG-dextran systems either large extraction phases were required or mycelium and 6PP partitioned to the same phase. ATPS comprising V(r)=0.23, PEG 8000 6.6% w/w and sulphate 14.0% w/w provided the best conditions to satisfy the process requirement of biomass accumulation into the bottom phase and 6PP concentration in the top phase.

Dextrans↗

PubMed2003

Efficacy of microorganisms antagonistic to Rhizoctonia cerealis and their cell wall degrading enzymatic activities.

The effect of Trichoderma atroviride, T. harzianum, T. longibrachiatum, Clonostachys rosea and Bacillus subtilis isolates applied to wheat seeds against Rhizoctonia cerealis disease of seedlings was investigated under controlled greenhouse conditions. Most Trichoderma isolates significantly reduced the incidence of disease compared with the infected control. Bacillus subtilis was also effective against sharp eyespot, although less active than Trichoderma spp. Interactions between the antagonistic microorganisms and the cereal pathogenic fungus in dual culture experiments on agar growth medium were also studied. Almost all tested antagonists showed competitive activity against R. cerealis: inhibition of its mycelial growth and hyphal interaction. The production of extracellular beta-N-acetylhexosaminidase, chitin 1,4-beta-chitobiosidase, glucan 1,3-beta-glucosidase and protease activity by the tested microorganisms in the presence of cell walls of R. cerealis was then determined. All isolates showed glucosaminidase and chitobiosidase activity. They also produced glucosidase activity, except B. subtilis, whereas only C. rosea, B. subtilis and one isolate of T. harzianum showed detectable levels of protease activity.

Antibiosis↗

PubMed2006

New approaches to the analysis of enzymatically hydrolyzed methyl cellulose. Part 2. Comparison of various enzyme preparations.

In this part of our studies, dealing with new approaches to the analysis of enzymatically hydrolyzed methyl cellulose, five different enzymes or enzyme preparations containing endoglucanases (from Bacillus agaradhaerens Cel 5A, Trichoderma reesei, Trichoderma viride, and two obtained from Trichoderma longibrachiatum) were used to hydrolyze six different methyl celluloses (MCs). The main goal was to investigate whether enzymes could be used for determination of the heterogeneity of the substituent distribution along the cellulose chain. To obtain information about the heterogeneity, it was necessary to gather information on how the enzymes affect hydrolysis. Size exclusion chromatography with multi-angle light scattering and refractive index detection (SEC-MALS/RI) was used to estimate the molar mass distribution of the MCs before and after hydrolysis. A novel internal standard addition method in combination with electrospray ionization ion trap mass spectrometry (ESI-ITMS) was used to determine the amount of formed oligomers. Two MCs, one with a degree of substitution (DS) of 1.8 and one with DS 1.3, were hydrolyzed with all of the five enzymes. The yield of summarized di- and trisaccharides was approximately 2% of the hydrolysis products for the MC with DS 1.8, whereas the product mixture, obtained from a MC with a DS of 1.3, contained 7-16% di- and trisaccharides. By a novel sample preparation method in combination with ESI-IT tandem MS, outlined in part 1 of this work, it was shown that the enzymes produced oligomers with the reducing end bearing no or only one substituent. Comparison of the methyl pattern at the nonreducing ends of the dimers and trimers indicated that the -2 subsite of the active complex is less tolerant than subsites -3 and +1. All enzymes had similar general selectivity toward the methyl substituents but also showed some differences. From both SEC-MALS/RI and ESI-ITMS, differences with respect to substituent distribution of MCs could be recognized but not for each enzyme used. Basic considerations for enzymatic hydrolysis and analysis of methyl cellulose were listed as a consequence of the results from the work.

Bacillus↗

PubMed2004

Recombinant enzymes from thermophilic micro-organisms expressed in fungal hosts.

Cost-effective production of enzymes for industrial processes makes the appropriate selection of the host/vector expression system critical. We have tested two fungal systems for the bulk production of enzymes from thermophiles. The yeast Kluyveromyces lactis has been developed as a secretion host employing expression vectors based on the 2u-like plasmid pKD1 of Kluyveromyces drosophilarium. Our second system involves the filamentous fungus Trichoderma reesei. Signal and protein fusion vectors have been constructed using the strong cellobiohydrolase 1 ( cbh1 ) promoter and recombinant plasmid DNAs introduced into various high-secreting T. reesei strains using biolistic particle delivery. In some cases (e.g. the xynB gene of Dictyoglomus thermophilum) we have reconstructed the genes according to Trichoderma codon preferences and demonstrated a dramatic increase in the production of the enzymes. The heterologous XynB enzyme is glycosylated differently in different Trichoderma strains. A proteomics approach has been taken to identify strongly expressed proteins produced by T. reesei under various cultivation conditions in order to identify condition-specific promoters driving the production of these proteins. Analyses indicated that HEX1, the major protein of the fungal Woronin body, is a dominant protein under both cellulase-inducing and -repressing conditions. The hex1 gene together with its promoter and terminator sequences has been isolated and the promoter function studied relative to cultivation time and medium.

Biochemistry↗

PubMed1998

Trichoderma/pathogen interactions: measurement of antagonistic chemicals produced at the antagonist/pathogen interface using a tubular bioassay.

A tubular bioassay was used to measure analytically the local production and concentration of the antifungal Trichoderma secondary metabolite 6-n-pentyl-2H-pyran-2-one (6PAP) at the Trichoderma antagonist/pathogen interface. 6PAP levels significantly increased in the presence of the pathogen Botrytis cinerea, typically 300-700%, and were highest near the pathogen source. The level of response for a particular Trichoderma isolate was found to vary with the test organism used. Two products produced by biotransformation of 6PAP by B. cinerea in response to the interaction were also detected.

Antibiosis↗

PubMed2002

The effects of volatile microbial secondary metabolites on protein synthesis in Serpula lacrymans.

The effects of volatile secondary metabolites produced by Trichoderma pseudokoningii, Trichoderma viride and Trichoderma aureoviride on growth rate and protein synthesis in two Serpula lacrymans isolates were investigated. Mycelial growth was affected to differing degrees, depending on the specific interactive microbial couplet involved. Protein synthesis by both S. lacrymans (Forfar) and S. lacrymans (H28) was affected by the volatile secondary metabolites of T. aureoviride and T. viride, but not by those of T. pseudokoningii. Mycelial growth and the original pattern of protein synthesis resumed when the antagonists were removed. It is probable that volatile secondary metabolites have played an important role during the evolution of microorganisms in the context of community, population and functional dynamics.

Basidiomycota↗

PubMed2002

Cork taint of wines: role of the filamentous fungi isolated from cork in the formation of 2,4,6-trichloroanisole by o methylation of 2,4,6-trichlorophenol.

Cork taint is a musty or moldy off-odor in wine mainly caused by 2,4,6-trichloroanisole (2,4,6-TCA). We examined the role of 14 fungal strains isolated from cork samples in the production of 2,4,6-TCA by O methylation of 2,4,6-trichlorophenol (2,4,6-TCP). The fungal strains isolated belong to the genera Penicillium (four isolates); Trichoderma (two isolates); and Acremonium, Chrysonilia, Cladosporium, Fusarium, Mortierella, Mucor, Paecilomyces, and Verticillium (one isolate each). Eleven of these strains could produce 2,4,6-TCA when they were grown directly on cork in the presence of 2,4,6-TCP. The highest levels of bioconversion were carried out by the Trichoderma and Fusarium strains. One strain of Trichoderma longibrachiatum could also efficiently produce 2,4,6-TCA in liquid medium. However, no detectable levels of 2,4,6-TCA production by this strain could be detected on cork when putative precursors other than 2,4,6-TCP, including several anisoles, dichlorophenols, trichlorophenols, or other highly chlorinated compounds, were tested. Time course expression studies with liquid cultures showed that the formation of 2,4,6-TCA was not affected by a high concentration of glucose (2% or 111 mM) or by ammonium salts at concentrations up to 60 mM. In T. longibrachiatum the O methylation of 2,4,6-TCP was catalyzed by a mycelium-associated S-adenosyl-L-methionine (SAM)-dependent methyltransferase that was strongly induced by 2,4,6-TCP. The reaction was inhibited by S-adenosyl-L-homocysteine, an inhibitor of SAM-dependent methylation, suggesting that SAM is the natural methyl donor. These findings increase our understanding of the mechanism underlying the origin of 2,4,6-TCA on cork, which is poorly understood despite its great economic importance for the wine industry, and they could also help us improve our knowledge about the biodegradation and detoxification processes associated with chlorinated phenols.

Anisoles↗

PubMed2005

Biotic relation between Fusarium oxysporum schlecht. and fungi isolated from the substrate of Stewartia pseudocamellia (max.).

The effect of the fungi community colonizing the substrate of Stewartia plants on the growth of Fusarium oxysporum Schlecht. was investigated. The soil samples from 2 years old Stewartia cuttings were taken for analysis in the second decade of October 2002 and 2003, when the symptoms of disease appeared on Stewartia plant. Fungi were isolated from the substrate using the sand method (Mańka K. 1974). Fusarium oxysporum were isolated from root system of Stewartia pseudocamellia plant. The isolates selected for the investigation proved pathogenic influence on Stewartia in an infection experiment. For estimation of biotic effect of saprobiotic fungi community on Stewartia pathogen F. oxysporum the biotic series method (Mańka K. 1974) was applied. The results showed that species of genera: Penicillium and Trichoderma were the most frequent in the community of fungi in the substrate. Both fungi communities of these fungi could not limit the growth of investigated pathogen F. oxysporum. It showed negative ABSTRACT biotic effect. The strongest antagonistic effect displayed Trichoderma viride and Trichoderma harzianum.

Fusarium↗

PubMed2001

Viability and stability of biological control agents on cotton and snap bean seeds.

Cotton and snap bean were selected for a multi-year, multi-state regional (south-eastern USA) research project to evaluate the efficacy of both commercial and experimental bacterial and fungal biological control agents for the management of damping-off diseases. The goal for this portion of the project was to determine the viability and stability of biological agents after application to seed. The biological seed treatments used included: (1) Bacillaceae bacteria, (2) non-Bacillaceae bacteria, (3) the fungus Trichoderma and (4) the fungus Beauveria bassiana. Seed assays were conducted to evaluate the following application factors: short-term (< or = 3 months) stability after seed treatment; quality (i.e. isolate purity); compatibility with chemical pesticides and other biocontrol agents; application uniformity between years and plant species. For the bacterial treatments, the Bacillaceae genera (Bacillus and Paenibacillus) maintained the greatest population of bacteria per seed, the best viability over time and the best application uniformity across years and seed type. The non-Bacillaceae genera Burkholderia and Pseudomonas had the least viability and uniformity. Although Beauveria bassiana was only evaluated one year, the seed fungal populations were high and uniform. The seed fungal populations and uniformity for the Trichoderma isolates were more variable, except for the commercial product T-22. However, this product was contaminated with a Streptomyces isolate in both the years that it was evaluated. The study demonstrated that Bacillaceae can be mixed with Trichoderma isolates or with numerous pesticides to provide an integrated pest control/growth enhancement package.

Bacillaceae↗

PubMed2004

Application of Toxkit microbiotests for toxicity assessment in soil and compost.

The potential of Toxkit microbiotests to detect and analyze pollution in agricultural soil and the quality of compost was studied. The toxicity tests used included seed germination biotests using cress salad (Lepidum sativum L.), tomato (Lycopersicum esculentum L.), and cucumber (Cucumis sativus L.), and the Toxkit microbiotests included those with microalgae (Selenastrum capricornutum), protozoa (Tetrahymena thermophila), crustaceans (Daphnia magna, Thamnocephalus platyurus, and Heterocypris incongruens), and rotifers (Brachionus calyciflorus). Experiments on compost were undertaken in a modified solid-state fermentation system (SSF) and under field conditions (in a windrow). To promote the composting process, two strains of Trichoderma (Trichoderma lignorum and Trichoderma viride), as well as a nitrification association that regulated the nitrogen-ammonification and nitrification processes were applied.

Animals↗

PubMed1975

The effect of volatile and gaseous metabolites of swelling seeds on germination of fungal spores.

Effects of volatile and gaseous metabolites of swelling seeds of pea, bean, wheat, corn cucumber, tomato, lentil, carrot, red papper and lettuce on germination of spores of five genera of fungi were found to depend rather on the fungal than on the plant genus. Germination of spores of Botrytis cinerea, Mucor racemosus and Trichoderma viride was most severely inhibited. Spores of Verticillium dahliae were less sensitive and germination of spores of Fusarium oxysporum was inhibited only in two cases. On the other hand, exudates of pea and bean stimulated germination of spores of Fusarium oxysporum. Also spores of Trichoderma viride germinated better in an atmosphere enriched with exuded metabolites of swelling lettuce seeds. When carbon dioxide produced by the swelling seeds was absorbed in potassium hydroxide, spores of Trichoderma viride and Verticillium dahliae did not germinate at all, the inhibitory effects of volatile and gaseous exudates on germination of spores of Mucor racemosus were accentuated, and also the percentage of germinated spores of Fusarium oxysporum decreased. Germination of spores of Botrytis cinerea was not influenced. Absorption of volatile and gaseous metabolites in a solution of potassium permanganate decreased in most cases their inhibitory effects, particularly in Botrytis cinerea.

Carbon Dioxide↗

PubMed1986

Isolation and partial characterization of two antifungal proteins from barley.

We have developed a simple assay for detecting antifungal compounds utilizing impregnated paper discs on agar to inhibit mycelial spread of an indicator organism, Trichoderma reesei. Using this assay we have isolated and purified to apparent homogeneity two antifungal proteins from dehusked barley grain. Both proteins are present at high concentrations: over 10 mg of each protein can be isolated per 100 g of grain. The first protein has a molecular weight of 30 000 and is identical to the 30 kDa ribosome-inactivating protein previously isolated from barley. This protein very effectively inactivates fungal ribosomes and this may explain its antifungal activity and biological role. The second antifungal protein has a molecular weight of 28 000 and is 20-fold more potent than the 30 kDa protein in inhibiting growth of Trichoderma. In addition to Trichoderma, the 28 kDa protein also efficiently inhibits growth of Phycomyces blakesleeanus, Alternaria alternaria and a protoplast-forming mutant of Neurospora crassa. The 28 kDa protein does not inactivate fungal ribosomes and we are currently investigating other possible enzymatic activities of this protein.

Antifungal Agents↗