Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan

Abstract This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.

Isolation, sequence, and conformation of seven trichorzianines B from Trichoderma harzianum.

From the antagonistic fungus Trichoderma harzianum, a group of acidic new peptides, trichorzianines B (TB), was isolated in addition to neutral trichorzianines A (TA) previously studied. TA and TB exhibit various biological activities related to their membrane properties and a different behaviour of the two groups was noticed. As observed for other peptaibols, TB consist in a microheterogeneous mixture which was resolved into pure peptides by reversed-phase C18 HPLC. The sequence of the seven main isolated TB, namely TB IIa, TB IIIc, TB IVb, TB Vb, TB VIa, TB VIb, TB VII, was determined by the combined use of positive ion FAB mass spectrometry and 2D 1H n.m.r. spectroscopy, including COSY and NOESY experiments. TB differ from the corresponding TA only by the replacement of Gln 18 in the TA sequence by a glutamic acid. The 1H n.m.r. data suggested that the TB are mainly organized in an alpha helix.

Isolation and sequence determination of trichorzianines A antifungal peptides from Trichoderma harzianum.

Trichorzianines A, membrane active peptides of the peptaibol class, were isolated from cultures of the mould Trichoderma harzianum. Trichorzianines A were separated into pure components by HPLC on octadecyl bonded and SiO2 phases successively. Nine trichorzianines A (IIa, IIIa, IIIb, IIIc, IVb, Vb, VIa, VIb and VII) were isolated from the complex microheterogeneous mixture. Their N-terminal amino acid is acetylated, the C-terminal amino alcohol is either tryptophanol or phenylalaninol, 7 to 8 of the 19 residues are alpha-aminoisobutyric acid. Gas chromatography on a chiral phase showed isovaline to have the D-configuration and all the other optically active amino acids and amino alcohols to have the L-configuration. The amino acid sequences were determined from their positive ion FAB mass spectra which exhibited the preferential cleavage of the Aib 12-Pro 13 amide bond as a main fragmentation. The resulting fragments subsequently underwent amide bond ruptures that generated two series of abundant acylium ions which enabled direct determination of the 1-19 sequence. The relative position of the isomeric amino acids in the sequence of trichorzianine AVII was assigned from analysis of the N- and C-terminal oligopeptides yielded by its selective acidic hydrolysis. The microheterogeneity of trichorzianines A results mainly from single or multiple substitution of amino acids at the specific positions 5, 14, 16 and 19.

Crystal structure of L-Pro-L-Leu-Aib-Aib-L-Glu-L-Valol, the C-terminal hexapeptide fragment of trichotoxin.

The crystal structure of L-prolyl-L-leucyl-alpha-aminoisobutyryl-alpha- aminoiso-butyryl-alpha-L-glutamyl-L-valinol (L-Pro-L-Leu-Aib-Aib-L-Glu-L-Valol), the C-terminal hexapeptide fragment of trichotoxin, has been determined by X-ray crystallography. The hexapeptide forms a right-handed 3(10)-helix consisting of two 10-atom hydrogen-bonded beta-turns of type III and one beta-turn of type I. Two of the intramolecular hydrogen-bonds are particularly weak, thus suggesting that the title compound may adopt non-helical conformations in solution, as observed by circular dichroism. In the crystal the molecules are hydrogen-bonded head-to-tail, forming infinitely long helical columns.

Evidence that the mechanisms of fungitoxicity of 8-quinolinol and its bischelate with copper(II) are different.

The copper(II) contents of the growth media, Sabouraud dextrose and Czapek-Dox broths, and of the spore inocula of Aspergillus niger (ATCC 1004), Aspergillus oryzae (ATCC 1011), Trichoderma viride (ATCC 8678), and Myrothecium verrucaria (ATCC 9095) were determined by atomic absorption spectrophotometry in a graphite furnace. The test systems composed of Sabouraud dextrose broth and spore inocula of the four fungi contained only a little over 3% of the copper(II) required to form a minimal inhibitory concentration of bis(8-quinolinolato)copper(II). The test system of Czapek-Dox broth and A. oryzae contained slightly less than 65% of the copper(II) required to form a minimal inhibitory concentration of the bischelate of 8-quinolinol with copper(II). When the minimal inhibitory concentrations of 8-quinolinol and bis(8-quinolinolato)copper(II) were added simultaneously to the test system of Czapek-Dox broth and A. oryzae, 10% of the combined mixture of toxicants caused complete inhibition of growth indicating synergism between the toxicants. These results together with the observation that alpha-lipoic acid as well as small aliphatic thiol-containing compounds (cysteine, glutathione, dithioerythritol, and dithiothreitol) reversed the toxicity of 8-quinolinol but not the toxicity of bis(8-quinolinolato)copper(II) led to the conclusion that the mechanisms of fungitoxicity of both toxicants are different.

Trichotoxin A40. Purification by counter-current distribution and sequencing of isolated fragments.

The isolation of the membrane-modifying polypeptide antibiotics from the mycelium of Trichoderma viride 5242 was optimized via extraction with dichloromethane and chromatography on Sephadex LH-20. The components trichotoxin A40 and A50 were separated from each other and purified by multiplicative counter-current distribution. The sequence of proteinase-resistant trichotoxin A40 was determined by combined gas chromatography and mass spectrometry of three isolated N-acetylated dodecapeptides and two N-prolylhexapeptides obtained after selective trifluoroacetolysis. Including amino acid exchanges due to natural microheterogeneity, the sequence is Ac-Aib-Gly(LAla)-Aib-LLeu-Aib-LGln-Aib-Aib-Aib(LAla )-LAla-Aib-Aib-LPro-LLeu -Aib-DIva(Aib)-LGlu-LValol. In contrast to the eicosapeptide alamethicin, trichotoxin A40 contains only 18 residues, with a higher proportion of alpha-aminoisobutyric acid (Aib), C-terminal L-valinol (Vol), one D-isovaline (Iva) and no proline at the N-terminal part.

A method for the detection and differentiation of cellulase components in polyacrylamide gels.

Endoglucanase and exoglucanase components of cellulase can be detected and differentiated after polyacrylamide gel electrophoresis by performing activity stains. Endoglucanase activity was visualized in carboxymethyl cellulose agar replicas of gels by staining with Congo red. General beta-1,4-glucanase activity was located by soaking the gel in a solution of NaBH4-reduced cellulooligosaccharides, and detecting the formation of reducing sugars by reaction with triphenyl tetrazolium chloride. Endoglucanases are active in both assays, while exoglucanases can be distinguished by their activity in the cellulo-oligosaccharide assay only. This methodology has facilitated the purification and characterization of cellulase components from Trichoderma reesei and Microbispora bispora.

Crystallization and preliminary X-ray diffraction results of trichorzianine A 1, a peptide with nineteen residues from Trichoderma harzianum.

Trichorzianine A 1 is one of the main components of a mixture of related antibiotic peptides (trichorzianines) produced by the fungus Trichoderma harzianum. Good crystals were obtained and allowed X-ray diffraction up to 0.8 A resolution. The space group is orthorhombic, C222(1), Z = 8, a = 64.8 (1) A, b = 9.33 (3) A, c = 39.9 (1) A. The solvent content is only 12%, preventing a heavy ion diffusion. So, we are trying to obtain the structure by direct methods.

Possible role of lectins in mycoparasitism.

Lectin activity in a host-mycoparasite relationship was demonstrated with Rhizoctonia solani and Trichoderma harzianum. Attachment of O but not A and B erythrocytes to hyphae occurred on R. solani but not on its mycoparasite. This phenomenon, which was Ca2+ and Mn2+ dependent, was prevented by galactose, present in T. harzianum cell walls, and by fucose.

Paracelsin, a peptide antibiotic containing alpha-aminoisobutyric acid, isolated from Trichoderma reesei Simmons. Part A.

A peptide antibiotic has been isolated from Trichoderma reesei QM 9414. Although crystalline and uniform in TLC, this antibiotic could be resolved by HPLC into 3 sequence analogues. The close relationship to alamethicin was proved by chemical and spectroscopic methods, and the formation of ion-conducting pores in lipid bilayers.

Effects of cereals and culture filtrate of Trichoderma viride on lipid metabolism of swine.

Swine were fed corn- or barley-based diets with, or without, culture filtrate (CF) of Trichoderma viride for 21 days. Weight gains were nonsignificantly but slightly increased by CF. The activities of beta-hydroxy-beta-methylglutaryl coenzyme A (HMG-CoA) reductase, cholesterol 7 alpha-hydroxylase, acetyl-CoA carboxylase (ACX), fatty acid synthetase (FAS) and other lipogenic enzymes in several tissues were determined. Significant decreases in the activities of HMG-CoA reductase and cholesterol 7 alpha-hydroxylase in all tissues of swine fed the CF-diets were observed. The major site for the regulation of cholesterol biosynthesis was adipose tissue followed by the intestine, liver, lung and muscle in order of activity. The concentrations of cholesterol in serum and muscle were decreased 27% and 23%, respectively, by CF. ACX and FAS activities increased ca. 2-fold when CF was fed with either of the cereal-based diets. The major sites for fatty acid synthesis was the adipose tissue and, to a lesser extent, the liver. Very low rates of synthesis were detected in intestine, lung and muscle. Similar distributions of activities were found for related lipogenic enzymes.

Poly(A)-associated RNA from the mitochondrial fraction of the fungus Trichoderma.

Total RNA was extracted from purified mitochondrial and cytoplasmic fractions of germinating conidia of Trichoderma viride and bound to oligo(dT)-cellulose at 22 and 4 degrees C. Under chromatographic conditions which retained very short poly(A) segments (i.e., 4 degrees C), up to 10% of short-term 32PO4-lebeled RNA from the mitochondrial fraction were selectively bound. The poly(A)-associated RNAs from the mitochondrial and cytoplasmic fractions showed the following characteristics. (a) On polyacrylamide gels mitochondrial fraction RNA had a distinctive pattern with a major peak at about 22 S and a smaller one at about 29 S; in contrast, cytoplasmic fraction RNA was heterogenously distributed along the gel. (b) The poly(A) segment released by RNAase digestion of mitochondrial fraction poly(A)-associated RNA migrated on polyacrylamide gels as molecules 20-25-nucleotides long, while that of the cytoplasmic fraction showed an apparent size of 50-60 nucleotides. (c) Mitochondrial fraction RNA bound to oligo(dT)-cellulose in the cold had a guanine + cytosine content of 21% versus 34% for bulk mitochondrial RNA and 48% for cytoplasmic poly(A)-associated RNA; the oligo(dT)-bound RNAs were further identified by their high percentages of adenine residues (46% for the mitochondria and 30% for the cytoplasm). (d) The poly(A)-associated RNA fraction was translated, in vitro, in a cell-free protein-synthesizing system from wheat germ. The products induced by cytoplasmic RNA showed a complex pattern on polyacrylamide gels of many polypeptides ranging in molecular weights from 10000 to 40000. The pattern induced by mitochondrial fraction RNA however, was much simpler, revealing two discrete, main products: a major one at Mr approximately 13000 and a minor one at Mr approximately 20000.

Some chemical and structural features of the conidial wall of Trichoderma viride.

Cell wall of spores of Trichoderma viride contains polymers similar to those of mycelial cell wall, such as beta-(1 leads to 3), beta-(1 leads to 6) glucans and protein, but chitin, always present in the mycelium, cannot be found in spores. Melanin, which in other fungi appears associated with chitin, replaces this polymer in the spore wall of T. viride and is located in the outermost layer. Attempts to characterize the pigment of the spore wall indicate that it is a non-indolic melanin-like polyphenol.

Characterization of polyadenylate from the fungus Trichoderma viride.

Polyribonucleotide segments, about 60 nucleotides long and consisting of about 95% adenylic acid residues, were isolated from whole cell ribonucleic acid of the deuteromyceteous fungus Trichoderma viride. Similar findings in two other groups of the true fungi raise the possibility that short polyadenylate sequences may be a feature of these relatively simple organisms.