Comparative study of the effect of L-lysine-L-oxidase from Trichoderma harzianum Rifai and Trichoderma viride on nucleic acid synthesis in human tumor cells in vitro

L-lysine-alpha-oxidase, a new fungal enzyme catalyzing oxidative deamination of L-lysine, exerts an inhibitory effect on DNA, RNA and protein synthesis in human cells of carcinoma ovarius (CaOv) in vitro.     

Gypsum Biomineralization in Sulphide-rich Hardpans by a Native Trichoderma harzianum Rifai Strain

Natural acid mine drainage affects abandoned sulfide mines worldwide, causing hardpan formation and heavy metal leaching. Hardpans are characterized by very low permeability, thus representing a significant limiting factor for oxygen and water circulation in the rhizosphere. Our work shows the results concerning the gypsum biomineralization capability of a native Trichoderma harzianum Rifai strain in sulfide -rich hardpans. Two months after the fungal inoculum, hardpan clasts resulted, completely covered by T. harzianum mycelium, where newly formed gypsum crystals occur. Our study provided the first evidence about interaction between T. harzianum and sulfide -mineralized hardpans as well as about its influence on gypsum genesis.     

Antifungal activity of a novel endochitinase gene (chit36) from Trichoderma harzianum Rifai TM

A novel 36-kDa endochitinase named chit36 has been isolated and characterized from Trichoderma harzianum Rifai TM. Partial amino acid sequences from the purified protein were used to clone the fungal cDNA, based on polymerase chain reaction with degenerate primers. The complete open reading frame encodes a 344-amino acid protein which shows 84% similarity to a putative chitinase from Streptomyces coelicolor. Chit36 was overexpressed under the pki1 constitutive promoter from Trichoderma reesei via biolistic transformation of T. harzianum TM. Stable transformants showed expression and endochitinase activity of chit36 in glucose-rich medium. Culture filtrates containing secreted CHIT36 as the sole chitinolytic enzyme completely inhibited the germination of Botrytis cinerea conidia. Growth of Fusarium oxysporum f. sp. melonis and Sclerotium rolfsii were significantly inhibited on agar plates on which the Trichoderma transformants had previously been grown.     

Proteins Related to the Biocontrol of Pythium Damping-off in Maize with Trichoderma harzianum Rifai

Induced resistance has been evidenced as one of mechanisms of Trichoderma to control plant diseases, however, no study showed the change of host proteomics in Trichoderma-induced resistance of maize against damping-off caused by Pythium ultimum Trow. The mechanism of Trichoderma harzianum Rifai for controlling maize seedling disease caused by Pythium ultimum Trow was investigated firstly by proteome technique and the result suggested that T. harzianum strain T22 was not only able to promote seedling growth but also protein accumulation. One-dimensional electrophoresis assay showed that more bands appeared on the gel with T22 or T22 combined with P. ultimum （T22 ＋ P. ultimum） treatment than with other treatments. Enzyme assay showed that two chitinases of the root sample were more activated in the treatments with T22 than in the other treatments without T22. Proteins in the seedling roots from the various treatments were separated through protein extraction and 2-D electrophoresis technique. In the seedlings produced from the T22-treated seeds, there were 104 up-regulated proteins and 164 down-regulated proteins relative to the control, and 97 and 150, respectively, aftel treatment with T22 ＋ P. ultimum; however, with P. ultimum alone the values were much lower than with the other two treatments. The correlation coefficient values were 0.72, 0.51 and 0.49 for the comparison of protein spot distribution on gel among control with T22, P. ultimum and T22 ＋ P. ultimum, respectively. So it seemed that P. ultimum infection was more effective than T22 in interfering with the host proteome profile. Furthermore, analysis with MALDITOF-MAS showed that some important proteins associated with defensive reactions were identified in T22 or T22 ＋ P. ultimum treatments, including endochitinase, pathogenesis-related protein PRMS （pathogenesis-related maize seed）, GTP-binding protein, isoflavone reductase and other proteins related to respiration. All those proteins are probably part of the network of resistance or development-related proteins. Interestingly, P. ultimum treatment resulted in elimination of pathogenesis-related protein PRMS on gel, and therefore damping-off could be in part attributed to inhibition of the expression of this protein by P. ultimum infection. Some unknown proteins are also related to the defensive reaction of the host.     

Effects of different inoculum densities of Trichoderma harzianum and Trichoderma viride against Meloidogyne javanica on tomato

A greenhouse experiment was conducted to evaluate the effects of different inoculum densities of two Saudi isolates of Trichoderma harzianum and Trichoderma viride against Meloidogyne javanica on tomato. Four densities (10⁴, 10⁶, 10⁸ and 10¹⁰ spores/g of soil) of each fungus were used. The results indicate that all four inoculum densities of the two Trichoderma species suppressed the nematode reproduction and root galling; and increased the growth of tomato plants, compared to controls. Efficacy of both fungi increased as their inoculum densities increased. Generally, efficacy of T. harzianum was better than that of T. viride, especially at the highest used density (10¹⁰ spore/g soil) which resulted in the best control.     

L-lysine-alpha-oxidase activity of some Trichoderma species

Trichoderma cultures were tested for their ability to produce L-lysine-alpha-oxidase. The highest enzyme activity was manifested by T. harzianum (MGU), T. longibrachiatum Rifai VKM F-2025 and T. aureoviride Rifai VKM F-2026. The biosynthesis of the enzyme did not depend on the growth of the cultures and did not vary among the species.     

Solubilization of Phosphates and Micronutrients by the Plant-Growth-Promoting and Biocontrol Fungus Trichoderma harzianum Rifai 1295-22

We investigated the capability of the plant-growth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22 (T-22) to solubilize in vitro some insoluble or sparingly soluble minerals via three possible mechanisms: acidification of the medium, production of chelating metabolites, and redox activity. T-22 was able to solubilize MnO₂, metallic zinc, and rock phosphate (mostly calcium phosphate) in a liquid sucrose-yeast extract medium, as determined by inductively coupled plasma emission spectroscopy. Acidification was not the major mechanism of solubilization since the pH of cultures never fell below 5.0 and in cultures containing MnO₂ the pH rose from 6.8 to 7.4. Organic acids were not detected by high-performance thin-layer chromatography in the culture filtrates. Fe₂O₃, MnO₂, Zn, and rock phosphate were also solubilized by cell-free culture filtrates. The chelating activity of T-22 culture filtrates was determined by a method based on measurement of the equilibrium concentration of the chrome azurol S complex in the presence of other chelating substances. A size exclusion chromatographic separation of the components of the culture filtrates indicated the presence of a complexed form of Fe but no chelation of Mn. In liquid culture, T. harzianum T-22 also produced diffusible metabolites capable of reducing Fe(III) and Cu(II), as determined by the formation of Fe(II)-Na₂-bathophenanthrolinedisulfonic acid and Cu(I)-Na₂-2,9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulfonic acid complexes. This is the first report of the ability of a Trichoderma strain to solubilize insoluble or sparingly soluble minerals. This activity may explain, at least partially, the ability of T-22 to increase plant growth. Solubilization of metal oxides by Trichoderma involves both chelation and reduction. Both of these mechanisms also play a role in biocontrol of plant pathogens, and they may be part of a multiple-component action exerted by T-22 to achieve effective biocontrol under a variety of environmental conditions.     

Low-molecular-weight xylanase from Trichoderma viride

An endo-1,4-beta-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) has been isolated from a commercial preparation of Trichoderma viride. The molecular weight was 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the pI value was 9.3. The xylanase was a true xylanase without cellulase activity. When the N-terminal amino acid sequence of the first 50 residues was compared with that of a xylanase from Schizophyllum commune, strong evidence for homology was found, with more than 50% amino acid identity. T. viride xylanase also possessed extensive identity with a proposed amino-terminal consensus sequence of xylanases from bacteria.     

Acetophenone derivatives from Chilean isolate of Trichoderma pseudokoningii Rifai

The novel acetophenone derivative 2′,4′-dihydroxy-3′-methoxymethyl-5′-methylacetophenone and the known 2′,4′-dihydroxy-3′,5′-dimethylacetophenone (clavatol) were isolated from the culture filtrate of a Chilean strain of Trichoderma pseudokoningii. This revised version was published online in July 2006 with corrections to the Cover Date.     

Low-molecular-weight xylanase from Trichoderma viride.

An endo-1,4-beta-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) has been isolated from a commercial preparation of Trichoderma viride. The molecular weight was 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the pI value was 9.3. The xylanase was a true xylanase without cellulase activity. When the N-terminal amino acid sequence of the first 50 residues was compared with that of a xylanase from Schizophyllum commune, strong evidence for homology was found, with more than 50% amino acid identity. T. viride xylanase also possessed extensive identity with a proposed amino-terminal consensus sequence of xylanases from bacteria.     

Raffinose-induced mutanase production from Trichoderma harzianum

Abstract The enzyme α (1 → 3),3-glucanohydrolase (referred to as mutanase) from the filamentous fungus Trichoderma harzianum OMZ 779 is capable of degrading the water-insoluble glucan in dental plaque. Previously, it was necessary to produce the glucan (referred to as mutan) in vitro for use as the sole carbon source and inducer of mutanase synthesis in fungal cultures. We report here that raffinose also induces the production of mutanase. The metabolism of raffinose differed from that of other sugars in metabolic end products and secreted protein profile. In addition to mutanase, we observed an approximately 15 000 M _r protein that was also regulated by carbon source and by illumination conditions.     

Specific and nonspecific glucanases from Trichoderma viride

Six endoglucanases (Endo I, II, III, IV, V, and VI), three exoglucanases (Exo I, II, and III), and a beta-glucosidase (beta-gluc I) isolated from a commercial cellulase preparation of Trichoderma viride origin were examined as to their activities on xylan ex oat spelts. Endo I, II, and III as well as Exo II and III showed no activity toward xylan and were classified as specific glucanases. Less specificity was found for the endoglucanases Endo IV, V, and VI, Exo I, and beta-gluc I, whose enzymes were able to hydrolyze xylan. With respect to product formation these xylanolytic cellulases fit the classification of xylanases generally accepted in the literature. Kinetic experiment with xylan, CM-cellulose, and p-nitrophenyl-beta-D-glucoside revealed that Endo IV, V, an VI and Exo I prefer to hydrolyze beta-1, 4-D-glucosidic linkages. beta-Gluc I showed no clear substrate preference.     

Light-activated adenyl cyclase from Trichoderma viride

The effect of light on adenyl cyclase (E.C. 4.6.1.1) and 3':5'-cyclic-AMP-phosphodiesterase (E.C. 3.1.4.17) activity of Trichoderma viride was investigated. Adenyl cyclase proved to be a membrane-associated enzyme, requiring Mn2+ and was activated by light. In contrast, 3':5'-cyclic-AMP-phosphodiesterase showed no light-stimulated activity. The activity of 3':5'-cyclic-AMP-phosphodiesterase was present mainly in the cytosol and was stimulated by Mg2+.     

Biological spectrum of Trichoderma harzianum Rifai isolates to control fungal diseases of tomato (Solanum lycopersicon L.)

Three Trichoderma harzianum isolates viz., Th-Sks, Th-Ke and Th-Ar collected from respective states of India viz., Rajasthan, Kerala and Andhra Pradesh were evaluated for the management of six fungal diseases namely damping off, Fusarium wilt, Rhizoctonia wilt, early leaf spot, late blight and Septoria leaf spot in tomato. During in vitro analysis, T. harzianum isolates inhibited the pathogens’ growth. Isolate Th-Sks was the most virulent antagonist against all the test pathogens and exhibited maximum of 79.47% growth inhibition of Phytophthora infestans. Isolate Th-Sks proved most effective at suppression efficacy in the range of 95–100% and 91–100% against all diseases under glasshouse and in the field conditions, respectively. Tomato seeds treatment with isolate Th-Sks also promoted plant height (78.23 cm) and fruits yield (290 g/plant) during field trial and data were found to be not-significantly different from other isolates. Thus, it is concluded that isolate Th-Sks can be utilised as a biocontrol agent for management of fungal diseases in tomato.     

Characterization of a chitinase and an endo-beta-1,3-glucanase from Trichoderma harzianum Rifai T24 involved in control of the phytopathogen Sclerotium rolfsii

Of 24 Trichoderma isolates, T harzianum Rifai (T24) showed a potential for control of the phytopathogenic basidiomycete Sclerotium rolfsii. When T24 was grown on different carbon sources, growth inhibition of S. rolfsii by the T24 culture filtrate correlated with the activity of extracellular chitinase and beta-1,3-glucanase. The 43-kilodalton (kDa) chitinase and the 74-kDa beta-1,3-glucanase were purified from the T24 culture filtrate in two and three steps, respectively, using ammonium sulphate precipitation followed by hydrophobic interaction chromatography (phenyl-Sepharose) and gel filtration (beta-1,3-glucanase). Km and Kcat were 3.8 g l(-1) and 0.71 s(-1) for the chitinase (chitin) and 1.1 g(-1) and 52 s(-1) for the beta-1,3-glucanase (laminarin). The chitinase showed higher activity on chitin than on less-acetylated substrate analogues (chitosan), while the beta-1,3-glucanase was specific for beta-1,3-linkages in polysaccharides. Both enzymes were stable at 30 degrees C, while at 60 degrees C the chitinase and the beta-1,3-glucanase were rapidly inactivated, showing half-lives of 15 and 20 min, respectively. The enzymes inhibited growth of S. rolfsii in an additive manner showing a promising ED50 (50% effective dose) value of 2.7 microg/ml.     

Protoplasts of Trichoderma viride

High yields of protoplasts from the 18-hr old mycelium of Trichoderma viride were obtained by using the lytic system, produced by Streptomyces venezuelae RA and Micromonospora chalcea grown on a synthetic medium containing laminarin and chitin, when 0.7 M MgSO₄ or (NH₄)₂SO₄ were used as osmotic stabilizers. Regeneration of these protoplasts occurred through the production of an abortive tube and direct germination of the protoplasts. Regeneration could also take place in the medium used to produce protoplasts, but the process was different in many details.     

Biodegradation of wastepaper by cellulase from Trichoderma viride

Environmental issues such as the depletion of non-renewable energy resources and pollution are topical. The extent of solid waste production is of global concern and development of its bioenergy potential can combine issues such as pollution control and bioproduct development, simultaneously. Various wastepaper materials, a major component of solid waste, were treated with the cellulase enzyme from Trichoderma viride, thus bioconverting their cellulose component into fermentable sugars. All wastepaper materials exhibited different susceptibilities towards the cellulase as well as the production of non-similar sugar releasing patterns when increasing amounts of paper were treated with a fixed enzyme concentration. The hydrolysis of wastepaper with changing enzyme concentrations and incubation periods also resulted in dissimilar sugar-producing tendencies. A general decline in hydrolytic efficiency was observed when increasing sugar concentrations were produced during biodegradation of all wastepaper materials.     

Purification and characterization of laminaran hydrolases from Trichoderma viride

At least three extracellular laminaran hydrolases which hydrolyzed laminaran (beta-1,3:1,6-glucan) from Eisenia bicyclis were secreted in wheat bran solid medium by Trichoderma viride U-1. These three enzymes, lam AI, AII, and B, were purified to electrophoretic homogeneity. Their molecular masses were estimated to be 70.1, 70.4, and 45.0 kDa for lam AI, AII, and B, respectively, by SDS-PAGE. Whereas both lam AI and AII could hydrolyze laminarin from Laminaria digitata, lam AII showed higher activity against Laminaria laminarin rather than Eisenia laminaran. On the other hand, lam B preferentially hydrolyzed pustulan, a beta-1,6-glucan. Laminarioligosaccharide was hydrolyzed by lam AI and AII but not B, whereas gentiooligosaccharide was hydrolyzed by only lam B. It showed that lam AI and AII were specific for beta-1,3-linkages, but lam B was specific for beta-1,6-linkages. These results indicated that T. viride U-1 has a multiple glucanolytic enzyme system.