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PubMed1991

Isolation of uridine auxotrophs from Trichoderma reesei and efficient transformation with the cloned ura3 and ura5 genes.

Uridine auxotrophs of the filamentous fungus Trichoderma reesei have been selected using a positive screening procedure with 5-fluoro orotate. Mutants deficient for the orotidine-5'-phosphate decarboxylase gene (ura3 mutants) and for the orotate phosphoribosyl transferase gene (ura5 mutants) have been characterized. The homologous ura3 and ura5 genes have been isolated and used to transform the auxotrophic mutants. Transformation efficiency with these homologous systems is very high (greater than 10(4) transformants per micrograms DNA). Transformation occurred by integration of vector DNA at homologous and ectopic loci. Mitotic instability was observed among some of the transformants. Sequence analysis at the protein level, of the T. reesei ura3 and ura5 genes showed extensive blocks of homology, with the corresponding genes from other organisms. The ura3 gene from T. reesei contains an insertion of 103 aa. A similar sequence is also found inserted in OMPdecase from the pyrenomycetes Neurospora crassa and Cephalosporium acremonium.

Amino Acid Sequence↗
PubMed1990

The development of a heterologous transformation system for the cellulolytic fungus Trichoderma reesei based on a pyrG-negative mutant strain.

Six uridine auxotroph mutants of Trichoderma reesei QM 9414 were isolated by resistance to 5-fluoroorotic acid and one strain was identified as OMP-decarboxylase negative (pyr-) by a radiometric enzyme assay. Transformation to uridine prototrophy was achieved with the pyr4 gene of Neurospora crassa (up to 1500 transformants/micrograms) and with pyrA of Aspergillus niger (700-800 transformants/micrograms). In many transformants the PYR+ function seems to be present as extrachromosomal DNA. There is evidence for a correlation between the stability of transformants and integration of the vector in the genome whereas unstable transformants are obtained when autonomous replication of the plasmid occurs.

Blotting, Southern↗
PubMed1992

Electrophoretic karyotyping of wild-type and mutant Trichoderma longibrachiatum (reesei) strains.

An electrophoretic karyotype of Trichoderma longibrachiatum (reesei) was obtained using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Seven chromosomal DNA bands were separated in the wild-type T. longibrachiatum strain QM6a. The sizes of the chromosomal DNA bands ranged from 2.8 to 6.9 Mb, giving an estimated total genome size of about 33 Mb. The electrophoretic karyotype of the strain QM6a was compared to three hyper-celluloytic mutant strains, QM9414, RutC30 and VTT-D-79125. The chromosome pattern of the mutant QM9414 was quite similar to that of the wild-type QM6a except that the smallest chromosome differed somewhat in size. The VTT-D-79125 and RutC30 strains, which have undergone several mutagenesis steps, showed striking differences in their karyotype compared to the initial parent. The chromosomal DNA bands were identified using the previously characterized T. longibrachiatum genes (egl1, egl2, cbh1, cbh2, pgk1, rDNA) and random clones isolated from a genomic library. In all strains the cellulase genes cbh1, cbh2 and egl2 were located in the same linkage group (chromosome II in the wild-type), while the main endoglucanase, egl1, hybridized to another chromosomal DNA band (chromosome VI in the wild-type).

Blotting, Southern↗
PubMed1991

Sequence of the cloned pyr4 gene of Trichoderma reesei and its use as a homologous selectable marker for transformation.

We have cloned and sequenced the Trichoderma reesei pyr4 gene encoding orotidine-5'-monophosphate decarboxylase. Comparison of this sequence with that of the equivalent gene from other filamentous fungi suggests that T. reesei is closely related to Cephalosporium acremonium and Neurospora crassa. The cloned pyr4 gene has been used as a homologous selectable marker for transformation of T. reesei. The majority of transformants obtained with circular plasmid were mitotically unstable and contained non-integrated plasmid molecules, sometimes in addition to plasmid integrated in the genome, Linearization of plasmid prior to transformation decreased the transformation frequency but increased the proportion of stable transformation obtained.

Amino Acid Sequence↗
PubMed1976

Starch degradation by the mould Trichoderma viride. I. The mechanism of starch degradation.

The mechanism of starch degradation by the fungus Trichoderma viride was studied in strain CBS 354.44, which utilizes glucose, starch and dextrins but is unable to assimilate maltose. It was shown that the amylolytic enzyme system is completely extracellular, equally well induced by starch, amylose or amylopectin and that it consists mainly of enzymes of the glucoamylase type which yield glucose as the main product of starch hydrolysis. Small amounts of alpha-amylase are produced also. The enzymes produced in starch cultures degrade starch, amylose and amylopectin equally well. Enzyme synthesis in starch media takes place to a considerable extent after exhaustion of the carbon source when maximum growth has been attained. Low-molecular dextrins are degraded by extracellular enzymes of the glucoamylase type. These enzymes are produced in media containing starch or dextrins. Maltotriose is consumed for only one third leaving maltose in the culture filtrate. Maltose is hardly attacked and hardly induces any amylolytic enzyme activity. No stable alpha-glucosidase appears to be produced.

Amylases↗
PubMed1976

Starch degradation by the mould Trichoderma viride. II. Regulation of enzyme synthesis.

The synthesis of amylolytic enzymes by the maltose not-utilizing Trichoderma viride strain CBS 354.44 requires the presence of starch or dextrins. Several readily utilizable carbon sources such as glucose and glutamic acid were shown to exert a strong catabolite repression which completely inhibited enzyme induction by starch or dextrins. Enzyme synthesis occurs in the exponential and in the stationary growth phase. In the latter, the ratio between saccharifying and dextrinizing enzyme activity is invariably high. In the exponential growth phase this ratio depends on the nature of the inducing substrate. Growth on starch results in an initially high production of dextrinizing activity, the saccharifying one becoming predominant in the course of exponential growth. The latter activity in dextrin DE 30 cultures is predominant from the very beginning. Thus, the amylolytic enzyme system of T. viride consists of at least two different enzymes, the synthesis of each being controlled specifically. The careful regulation of the synthesis of the dextrinizing enzyme is discussed with special reference to the production of non-utilizable maltose by the latter.

Amylases↗
PubMed1977

Sophorose metabolism and cellulase induction in Trichoderma.

The cellulase inducer sophorose was rapidly catabolized to CO2 and H2O by Trichoderma: only small amounts were used to induce the synthesis of cellulase. 3H-sophorose uptake began after a lag of 1 h and its half-life in the medium was less than 5 h. Cellulase activity in the medium did not increase till 6 h after the addition of sophorose and reached a half maximum value at 14 h. The presence of free sophorose in the medium was required for continuous cellulase production. Several small sophorose addition induced much more cellulase than an equivalent single dose. These results are attributed to two pathways of sophorose utilization, a catabolic pathway that has a high capacity but low affinity for sophorose and an inductive pathway having a lower capacity but higher affinity for sophorose.

Cellulase↗
PubMed1976

Effects of polyoxin D on germination, morphological development and biosynthesis of the cell wall of Trichoderma viride.

When polyoxin D is added to a spore suspension of Trichoderma viride at a concentration from 50-100 mug/ml, it inhibits from 40-60% of germination. This percentage increases if dimethylsulfoxide (DMSO) is added. Mycelium growing in the presence of polyoxin D becomes irregular and loses its rigidity, showing several bulges along the hypha. Under the electron microscope the features of the cell wall and cytoplasmic content are apparently normal. Nevertheless, after incubation with different lytic systems or with (14C) glucose, it can be seen that polyoxin D partially inhibits the biosynthesis of beta-(1-3) glucan and the biosynthesis of chitin to a greater extent attaining inhibition of 83% at 100 mug/ml of the antibiotic concentration. Rgenerating protoplasts are less affected by polyoxin D. They do regenerate slower but the percentage of regeneration is more than 80%. Aberrant tubes synthesized by these protoplasts are not affected, they manifest their usual morphology and lack of chitin is confirmed in their composition.

Antifungal Agents↗
PubMed1975

Antagonistic effects of Trichoderma harzianum on Pythium aphanidermatum causing the damping-off disease of tobacco in Nigeria.

Culture filtrates of Trichoderma harzianum Rifai have been found to inhibit zoospore germination, germ tube elongation and mycelial growth of Pythium aphanidermatum causing the damping-off disease of tobacco in Nigeria. Further, the invasion of the hyphae of P. aphanidermatum by hyphae of T. harzianum has been demonstrated in mixed cultures of the two fungi. The results of investigations on the role of T. harzianum as an agent of biological control of the damping-off disease in sterilized and unsterilzed soils are reported and discussed.

Antibiosis↗
PubMed1975

Protoplasts of Trichoderma viride: formation and regeneration.

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.

Bacteriolysis↗
PubMed1991

In vitro antagonism of bioluminescent fungi by Trichoderma harzianum.

Two species of bioluminescent fungi, Panellus stypticus and Omphalotus olearius were placed in contact with three different strains of interfungal pathogenic Trichoderma harzianum. Subsequent light emission by the luminous fungi and advance of the interfungal pathogens were compared. Relative differences among the pathogens were reflected in their rate of mycelial advance, the total area over which they produced spores upon the host fungi, and decreases in host bioluminescence. After ten days differences in the total surface areas of spore production varied from 1 to 53 per cent. Differences in the reduction of bioluminescence of the same material ranged over 2 orders of magnitude. Final reduction in luminescence ranged over 6 orders of magnitude. A marked reduction in bioluminescence was observed to precede the advance of spore production. The greatest reduction in luminescence was correlated with the presence of T. harzianum hyphae. Two strains of T. harzianum, NRRL 1698 and ATCC 58674, were effective against both bioluminescent fungi within the study period while a third strain, NRRL 13019, was only effective against Omphalotus olearius.

Agaricales↗
PubMed1990

Antagonistic potential of Gliocladium virens and Trichoderma longibrachiatum to phytopathogenic fungi.

Three isolates of Gliocladium virens (G1, G2 and G3) and two of Trichoderma longibrachiatum (T1 and T2) were screened against isolates of three soilborne plant pathogens namely Rhizoctonia solani, Sclerotium rolfsii and Pythium aphanidermatum. G. virens exhibited stronger hyperparasitism and wider biological spectrum than T. longibrachiatum. Further, similarities as well as variation was observed in the ability of the various isolates to invade the test pathogens in dual culture. For the hyperparasites, acidic pH range (5.0 to 5.5) favoured both growth and spore germination. The hyperparasites made direct contact with the pathogens followed by varied modes of attack invariably leading to cell disruption. Antagonists, G1 and G3 revealed strong antibiosis while T2 showed moderate effect. All the isolates produced enhanced levels of lytic enzymes adaptively and there were marked differences among them. However, no correlation was observed between these attributes and the hyperparasitic potential of the various isolates in dual culture. The relevance and the role of enzymes and toxic metabolite(s) in the antagonism of G. virens and T. longibrachiatum to these pathogens are discussed.

Chytridiomycota↗
PubMed1975

Chemical and structural differences in mycelial and regeneration walls of trichoderma viride.

When incubated in Winge medium, protoplasts from Trichoderma viride obtained by treatment with Micromonospora chalcea or Streptomyces venezuelae RA lytic systems first synthesized an aberrant wall, different from the normal one; it was aseptate, larger and irregular in size and length. They then regenerated a new wall, similar to the original one from which they were liberated. Analysis showed that the wall polymers were mainly beta-(1-3) glucan, beta-(1-6) glucan and chitin in the normal walls, whereas chitin was absent in aberrant tubes. These results are discussed below together with electron micrographs of aberrant and normal walls.

Carbohydrates↗
PubMed1975

Inhibition of growth of Aspergillus flavus and Trichoderma viride by peanut embryos.

Growth of Aspergillus flavus and Trichoderma viride on agar media was inhibited around embryos of green peanut seeds but not around embryos of cured seeds, intact peanut seeds, or testae. Both fungi were able to colonize intact seeds and testae. Substances inhibitory to A. flavus and T. viride were extracted with acetone from cotyledons of freshly harvested peanut seeds. Four compounds inhibitory to A. flavus were detected in crude acetone extracts. Three of the compounds demonstrated properties of phenolics. Results of this study suggest that inhibitory compounds in peanut cotyledons may play a role in protecting the peanut embryo from fungal infection.

Antifungal Agents↗
PubMed1992

Molecular cloning of the imidazoleglycerolphosphate dehydratase gene of Trichoderma harzianum by genetic complementation in Saccharomyces cerevisiae using a direct expression vector.

The Trichoderma harzianum imidazoleglycerolphosphate dehydratase gene (igh) has been isolated by complementation of a Saccharomyces cerevisiae his3 mutant using a direct expression vector. This Escherichia coli-yeast shuttle vector was developed to allow efficient cloning and expression of cDNA libraries. The cDNA is 627 nucleotides long and codes for a protein of 209 amino acids with an apparent molecular mass of 22,466 daltons. The predicted protein sequence showed 63.6%, 58.7%, and 38.4% identity respectively to the corresponding enzymes from S. cerevisiae, Pichia pastoris and E. coli. Northern analysis showed that the expression of the igh gene in T. harzianum is not inhibited by external histidine and the level of igh mRNA was about threefold higher in cells starved of histidine.

Amino Acid Sequence↗
PubMed1975

The isolation of protoplasts of the fission yeast Schizosaccharomyces by Trichoderma viride and snail enzymes.

The formation of protoplasts of the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces versatilis after the combined application of snail enzymes and Trichoderma viride enzymes in an osmotic stabilizer (0.4M KCl, pH 5.5) was studied by light and electron microscopy. The effect of the enzymes used leads during 30 min to the formation of 100% protoplast population. Using electron microscopy no original walls or wall remnants were detected in the suspension of protoplasts. Protoplasts are viable and in liquid nutrient medium they regenerate cell walls and revert into normal cells. Such a protoplast population may be useful for biochemical study of protoplast metabolism by quantitative methods as well as for the chemical study of regenerating cell walls.

Animals↗
PubMed1978

Regulation of periodicity and intensity of photo-induced conidiation of Trichoderma viride.

After a short irradiation at 366 nm with 200 lx, the intensity of conidiation of Trichoderma viride colonies grown in the dark increased for the first 10 s proportionally with time. The increase slowed down after 10 s-5 min of exposure and after 10-60 min of irradiation the conidiation intensity began to decrease. When photo-induced by daylight, the conidiation started at a high rate after 25 h and persisted even after 48 h. The conidiation had no circadian character and its periodicity depended on the periodicity of photo-induction. Its intensity was also influenced by the carbon sources used, a maximum being reached with glucose (1-2%). Higher glucose concentrations inhibited conidiation but had no influence on growth of colonies.

Culture Media↗