Showing papers on "Trichoderma harzianum published in 1995"

A novel endo-beta -1,3-glucanase, bgn13.1, involved in the mycoparasitism of trichoderma harzianum

Abstract: The mycoparasitic fungus Trichoderma harzianum CECT 2413 produces at least three extracellular beta-1,3-glucanases. The most basic of these extracellular enzymes, named BGN13.1, was expressed when either fungal cell wall polymers or autoclaved mycelia from different fungi were used as the carbon source. BGN13.1 was purified to electrophoretic homogeneity and was biochemically characterized. The enzyme was specific for beta-1,3 linkages and has an endolytic mode of action. A synthetic oligonucleotide primer based on the sequence of an internal peptide was designed to clone the cDNA corresponding to BGN13.1. The deduced amino acid sequence predicted a molecular mass of 78 kDa for the mature protein. Analysis of the amino acid sequence indicates that the enzyme contains three regions, one N-terminal leader sequence; another, nondefined sequence; and one cysteine-rich C-terminal sequence. Sequence comparison shows that this beta-1,3-glucanase, first described for filamentous fungi, belongs to a family different from that of its previously described bacterial, yeast, and plant counterparts. Enzymatic-activity, protein, and mRNA data indicated that bgn13.1 is repressed by glucose and induced by either fungal cell wall polymers or autoclaved yeast cells and mycelia. Finally, experimental evidence showed that the enzyme hydrolyzes yeast and fungal cell walls.

Purification and characterization of an endo-beta-1,6-glucanase from Trichoderma harzianum that is related to its mycoparasitism.

Abstract: The enzymes from Trichoderma species that degrade fungal cell walls have been suggested to play an important role in mycoparasitic action against fungal plant pathogens. The mycoparasite Trichoderma harzianum produces at least two extracellular beta-1,6-glucanases, among other hydrolases, when it is grown on chitin as the sole carbon source. One of these extracellular enzymes was purified to homogeneity after adsorption to its substrate, pustulan, chromatofocusing, and, finally, gel filtration. The apparent molecular mass was 43,000, and the isoelectric point was 5.8. The first 15 amino acids from the N terminus of the purified protein have been sequenced. The enzyme was specific for beta-1,6 linkages and showed an endolytic mode of action on pustulan. Further characterization indicated that the enzyme by itself releases soluble sugars and produces hydrolytic halli on yeast cell walls. When combined with other T. harzianum cell wall-degrading enzymes such as beta-1,3-glucanases and chitinases, it hydrolyzes filamentous fungal cell walls. The enzyme acts cooperatively with the latter enzymes, inhibiting the growth of the fungi tested. Antibodies against the purified protein also indicated that the two identified beta-1,6-glucanases are not immunologically related and are probably encoded by two different genes.

Primary structure and expression pattern of the 33-kDa chitinase gene from the mycoparasitic fungus Trichoderma harzianum

Abstract: A gene (chit33) from the mycoparasitic fungus Trichoderma harzianum, coding for a chitinase of 33 kDa, has been isolated and characterized. Partial amino-acid sequences from the purified 33-kDa chitinase were obtained. The amino-terminal peptide sequence was employed to design an oligonucleotide probe and was used as a primer to isolate a 1.2-kb cDNA. The cDNA codes for a protein of 321 amino acids, which includes a putative signal peptide of 19 amino acids. All microsequenced peptides found in this sequence, indicate that this cDNA codes for the 33-kDa chitinase. A high homology (approximately 43% identity) was found with fungal and plant chitinases, including yeast chitinases. However enzyme characteristics suggest a nutritional (saprophytic or mycoparasitic), rather than a morphogenetic, role for this chitinase. The chit33 gene appears as a single copy in the T. harzianum genome, is strongly suppressed by glucose, and de-repressed under starvation conditions as well as in the presence of autoclaved mycelia and/or fungal cell walls. The 33-kDa chitinase seems to be very stable except under starvation conditions. The independent regulation of each of the chitinases in T. harzianum indicates different specific roles.

The role of recognition in the induction of specific chitinases during mycoparasitism by Trichoderma harzianum

Abstract: Summary: The induction of chitinolytic enzymes in the biocontrol agent Trichoderma harzianum during parasitism on Sclerotium rolfsii and the role of fungal-fungal recognition in this process were studied. A change in the chitinolytic enzyme profile was detected during the interaction between the fungi, grown in dual culture on synthetic medium. Before coming into contact with each other, both fungi contained a protein with constitutive 1,4-†bT-N-acetylglucosaminidase activity. As early as 12 h after contact, the chitinolytic activity in S. rolfsii disappeared, while that of T. harzianum (a protein with a molecular mass of 102 kDa, CHIT 102) greatly increased. After 24 h of interaction, the activity of CHIT 102 diminished concomitantly with the appearance of a 73 kDa 1,4-β-N-acetylglucosaminidase, which became clear and strong at 48 h. This phenomenon did not occur if the S. rolfsii mycelium was autoclaved prior to incubation with T. harzianum, suggesting its dependence on vital elements from the host. Cycloheximide inhibited this phenomenon, indicating that de novo synthesis of enzymes is taking place in Trichoderma during these stages of the parasitism. A biomimetic system based on the binding of a purified surface lectin from the host S. rolfsii to nylon fibres was used to dissect the effect of recognition. An increase in CHIT 102 activity was detected, suggesting that the induction of chitinolytic enzymes in Trichoderma is an early event which is elicited by the recognition signal (i.e. lectin-carbohydrate interactions). It is postulated that recognition is the first step in a cascade of antagonistic events which triggers the parasitic response in Trichoderma.

Chronological Events Associated with the Antagonistic Properties of Trichoderma harzianum against Botrytis cinerea: Indirect Evidence for Sequential Role of Antibiosis and Parasitism

Abstract: Chronological events associated with the degradation of Botrytis cinerea by a strain of Trichoderma harzianum selected for superior biocontrol ability were studied using ultrastructure and cytochemical investigations in an attempt to define the relative roles of antibiosis and parasitism in the antagonistic process. The first ultrastructural changes were observed 12 h before contact between the organisms, and were characterized by punctuated invaginations of the Botrytis plasmalemma. These reactions were followed by a gradual retraction of the plasmalemma, disorganization of the cytoplasm, loss of turgor pressure and cell death within 48 h of contact between hyphae of the interacting fungi. The first evidence of penetration of B. cinerea by T. harzianum was recorded 72 h after contact. This penetration was apparently mediated by either mechanical pressure or localized wall digestion at points of entry, as there was no clear evidence of chitinolytic degradation of the B. cinerea cell wall, as determined by...

Molecular characterization and heterologous expression of an endo-β-1,6-glucanase gene from the mycoparasitic fungus Trichoderma harzianum

Abstract: Hydrolytic enzymes from the filamentous fungus Trichoderma harzianum have been described as critical elements of the mycoparasitic action of Trichoderma against fungal plant pathogens. In this report we describe the first genomic and cE)NA clones encoding a β-1,6-endoglucanase gene. The deduced protein sequence has limited homology with other β-glucanases. Northern experiments show a marked repression of mRNA accumulation by glucose. The protein has been successfully produced in Saccharomyces cerevisiae upon construction of a transcriptional fusion of the cDNA with a yeast promoter. This S. cerevisiae recombinant strain shows a strong lytic action on agar plates containing β-1,6-glucan.

Comparison of aerial and submerged spore properties for Trichoderma harzianum

Abstract: Spores produced by aerial mycelium of Trichoderma harzianum PI, a potential biocontrol agent, showed both higher UV-resistance and longer viability after storage than those produced within liquid media (‘submerged’ spores). Aerial spores were produced in clusters, had a thick outer wall, and few organelles. Trehalose content was significantly lower than in submerged spores. Conversely, submerged spores were mostly collapsed, not clustered and larger than aerial spores. They had many cytoplasmic organelles and a thinner outer wall. These spores were hydrophilic, while aerial ones were highly hydrophobic. On analysis, the latter was related with the presence of a single major low molecular mass protein (< 14 kDa). This protein was nearly absent in extracts from walls of submerged spores but was found in the extracellular medium. An involvement of the outer wall layer in the resting state of T. harzianum spores is proposed.

Randomly amplified polymorphic DNA fingerprinting identifies subgroups of Trichoderma viride and other Trichoderma sp. capable of chestnut blight biocontrol

Abstract: Eleven strains of Trichoderma viride, 2 strains of the putative teleomorph Hypocrea rufa and 9 of several other Trichoderma sp. were characterized by random polymorphic DNA amplification (RAPD) fingerprinting and screened for their ability to antagonize growth of European strains of the chestnut blight causing fungus Cryphonectria parasitica, using a dual-culture assay. The best strains were found in the species T. harzianum, T. parceramosum, a distinguishable subgroup of T. viride and a not named Trichoderma sp. The successful application of these strains against chestnut blight in vivo is demonstrated.

A tool for monitoring Trichoderma harzianum. II. The use of a GUS transformant for ecological studies in the rhizosphere.

Abstract: Population development, activity, and stability of a β-glucuronidase (GUS)-transformed strain of Trichoderma harzianum were studied in the rhizosphere of cucumber plants grown in sphagnum peat. When compared to the wild-type isolate, there was no significant difference in terms of population development (CFU/g of sphagnum peat). In a treatment with a combination of the wild-type isolate and the transformant, the proportion of GUS-active colonies on plates was constant throughout the experiment. On that basis, we concluded that the transformant was genetically stable when grown in a natural potting mixture. GUS was extracted from infested rhizosphere peat and assaved spectrophotometrically. The activity curve was inversely related to the population curve. Quantification of activated conidia by fluorescein diacetate staining indicated that GUS activity correlated with the activity of the fungus. Hyphae of T. harzianum stained with 5-bromo-4-chloro-3-indoyl-β-D-glucuronide were easily detected on the roots, especially around small wounds, such as damaged epidermal cells. Colonization of wounds may be important for the antagonistic effect of the organism, because it may block a possible way for pathogens to infect root tissue. Our results demonstrate the suitability of using a GUS transformant for detecting and monitoring a specific strain of T. harzianum deliberately released into the environment.

Trichorzins HA and MA, antibiotic peptides from Trichoderma harzianum. I. Fermentation, isolation and biological properties.

Abstract: Trichorzins HA and MA, original 18-residue peptides, were isolated from two strains of the widespread soil fungus Trichoderma harzianum which have been shown to exhibit antibiotic activity against phytopathogenic fungi. These linear peptides belonging to the peptaibol class are biosynthesized as a complex of closely related analogues. Nine major pure peptides, six trichorzins HA and three trichorzins MA, were isolated by reversed-phase HPLC. The isolated peptides exhibited antibacterial activity against S. aureus and increased the membrane permeability of egg phosphatidylcholine/cholesterol (7/3) liposomes, as measured by monitoring leakage kinetics of a fluorescent probe. Structure-activity relationships were deduced from the antibiotic and membrane-modifying properties.

A Tool for Monitoring Trichoderma harzianum: I. Transformation with the GUS Gene by Protoplast Technology

Abstract: To obtain a genetically marked strain of Trichoderma harzianum that can be used as a tool for studies of population dynamics, T. harzianum was cotransformed with the Escherichia coli uidA β-glucuronidase (GUS) gene and the hygromycin B (hygB) gene as the selective marker. To improve the efficiency of conditions used for the transformation, the isolation, reversion, and germination of mycelial protoplasts were studied by scanning electron microscopy (SEM). Hexamethyldisilzane was useful for preparing protoplasts from suspensions for SEM. It was essential to obtain a transformant that phenotypically resembled the wild-type. After mitotic stabilization of the transformants by single-spore isolations, three transformants were compared to the wild-type by measurement of spore germination and mycelial growth rates, identification of secondary metabolite profiles, and studies of extracellular proteins. One transformant, T3c, was dissimilar to the wild-type in most tests, whereas transformant T3a was physiologically very similar to the wild-type. Furthermore, transformant T3a remained genetically stable during nonselective cultivation on plates and in sterile peat-bran.

Molecular cloning and expression in S. cerevisiae of two exochitinases from Trichoderma harzianum.

Abstract: The synthetic exochitinase substrate 4-methylumbelliferyl N-acetylglucosamine was used to identify seven full-length exochitinase-encoding cDNAs from a Trichoderma harzianum cDNA library by expression in yeast. The cDNA clones represented transcripts of two exochitinase genes, designated as exc1 and exc2, which cross-hybridized under moderate stringency conditions in genomic Southern blots. The exc1 cDNA encodes a 578 amino acid polypeptide showing 72% similarity to the exc2-encoded 602-residue polypeptide. The deduced exochitinase amino acid sequences were found to be homologous with mammalian and fungal hexosaminidases as well as a bacterial chitobiosidase. The substrate specificity of the recombinant enzymes expressed in S. cerevisiae indicates that the enzymes are N-acetylglucosaminidases releasing single N-acetylglucosamine residues from the non-reducing end of the chitin substrate.

Comparative antagonistic properties of Gliocladium virens and Trichoderma harzianum on Sclerotium rolfsii and Rhizoctonia solani: its relevance to understanding the mechanisms of biocontrol

Abstract: An isolate of Trichoderma harzianum which is less effective than G. virens in suppressing S. rolfsii and R. solani was compared with G. virens for various mechanisms of antagonism in vitro, viz., antagonism in dual culture/hyphal parasitism, parasitism of sclerotia, and antibiosis. G. virens and T. harzianum were equally effective in parasitizing the hyphae of R. solani. Only T. harzianum parasitized the hyphae of S. rolfsii, and the two antagonists were comparable with respect to antibiosis on the test pathogens. However, G. virens readily parasitized the sclerotia of the test pathogens and was found to be more effective than T. har±ianuni in destroying the sclerotia. Under SEM, G. virens was found to colonize, penetrate, and sporulate inside the sclerotia of the test pathogens. Parasitism of sclerotia is suggested as the principal mechanism of biological control of S. rolfsii and R. solani by G. virens.

Trichorzins HA and MA, antibiotic peptides from Trichoderma harzianum. II. Sequence determination.

Abstract: A series of 18-residue antibiotic-antifungal peptides, trichorzins HA and MA, were isolated from Trichoderma harzianum strains exhibiting antagonistic properties against phytopathogenic fungi. The sequences of the nine major pure peptides isolated by HPLC were determined by positive ion FAB-MS data and two-dimensional NMR measurements, including COSY, HOHAHA, ROESY and 1H-13C LRCOSY experiments.

Fungal metabolites. XVIII. New membrane-modifying peptides, trichorozins I-IV, from the fungus Trichoderma harzianum.

Abstract: New membrane-modifying peptides, trichorozing I-IV, have been isolated from conidia of the fungus Trichoderma harzianum. Their amino acid sequences were clearly determined by spectrometric methods and, furthermore, they were synthesized by the solution-phase method. Trichorozins are a family of the class of peptaibols and are composed of 11 residues including an amino alcohol. Trichorozins exhibited voltage-dependent ion channel-like activity in lipid bilayers.

Urban and agricultural wastes for use as mulches on avocado and citrus and for delivery of microbial biocontrol agents

Abstract: SummaryUrban and agricultural waste products generally available to avocado and citrus growers in southern California were analyzed for their suitability for use as bioenhanced mulches on citrus and avocado. Of the mulches tested only yard waste (consisting of wood chips, grass and leaves), rice hulls and rice hulls-and-paper were not harmful to any growth parameter of citrus or avocado and were also adequate substrates for the growth of three biocontrol agents: Trichoderma harzianum, Gliocladium virens, and Pseudomonas fluorescens. Several mulches such as milled peanut hulls, milled almond hulls, chicken manure, a horse/cow manure mixture, cow manure and alfalfa hay were poor substrates for growth of the biocontrol agents and were damaging to at least one growth parameter of avocado and citrus. These mulches released toxic amounts of ammonia upon degradation, some in excess of 1000 µg NH3−1 dry wt. The percentage of healthy citrus roots, percentage of healthy avocado roots and growth of T. harzianum and ...

Trichoderma harzianum genes induced during growth on Rhizoctonia solani cell walls

Abstract: Trichoderma harzianum is a biocontrol agent that attacks a range of economically important phytopathogenic fungi. In an attempt to identify genes specifically expressed by T. harzianum during growth on cell walls of Rhizoctonia solani, we carried out differential screening of an induced cDNA library. In this paper we report the analysis of the sequence and expression of two cDNA clones that encode putative mycoparasitism-related proteins of T. harzianum. One of these clones corresponds to a gene, inda 1, that encodes a protein of 570 amino acids with a predicted molecular mass of 62853 Da. The predicted amino acid sequence of inda1 showed a high degree of similarity with amino acid permeases from several other organisms. The other cDNA clone corresponds to a gene, indc11, that encodes a novel protein of 340 ami acids with a predicted molecular mass of 37010 Da. The use of this methodology should provide specific genetic markers to follow mycoparasitism by Trichoderma spp.

Vital fluorochromes as tracers for fungal growth studies.

Abstract: Eight fluorescent dyes were tested for staining the spores or mycelia of six fungi and for their translocation into new growth when the preloaded spores or mycelia were incubated on agar coated coverslips. The dyes studied were Cellufluor, Nile red, fluorescein diacetate (FDA), carboxyfluorescein diacetate (CFDA), chloromethylfluorescein diacetate (CMFDA), aminochloromethyl coumarin (CMAC), and the carbocyamines DiIC18(3) and DiOC18(3). The fungi on which the dyes were tested included Botrytis cinerea, Fusarium oxysporum f.sp. lycopersici, Idriella bolleyi, Pythium oligandrum, Sclerotium cepivorum and Trichoderma harzianum. Most of the fluorochromes gave good initial staining of mycelia or spores; however, FDA fluorescence faded rapidly during excitation, making it impractical for use. Also, the spores and mycelia of B. cinerea and T. harzianum sometimes gave weak fluorescence with Nile red, and the spores and mycelia of I. bolleyi gave unusually weak fluorescence with Cellufluor. There were other variations of staining among the different dye/fungus combinations, but each fungus showed strong fluorescence at least one dye. Cellufluor, CMFDA, CMAC and, to a lesser extent, CFDA and Nile red, were efficiently translocated into new growth from preloaded spores or mycelia, whereas FDA, DiIC18(3) and DiOC18(3) were not. The extent of translocation ranged from 0.1 to 1.2 mm in germ tubes arising from spores, and from 0.9 to 9.2 mm in mycelia extending from dye-loaded agar blocks. The findings suggest that fluorescent dyes could be used as markers or tracers in studies of fungal growth and differentiation.

MR304A, a new melanin synthesis inhibitor produced by Trichoderma harzianum.

Abstract: In the screening for new melanin synthesis inhibitors, we found a novel isonitrile inhibitor, MR304Afrom the culture broth of Trichoderma harzianum. There were someisonitrile antibiotics that were reported to have been isolated from Trichoderma sp. 1 ~ 4) and trichoviridin, one of these isonitriles, showed a melanin biosynthesis inhibitory activity5). In this paper we described the fermentation, isolation, physico-chemical properties, structure determination and biological activities of MR304A. The producing microorganism, strain MR304,was isolated from a soil collected in Taejon, Korea. The fungus was identified as Trichoderma harzianum on the basis of its cultural properties and has been deposited with Korean Collection for Type Cultures, KCTC 0123BP.

Enhanced Root and Shoot Growth of Chrysanthemum Cuttings Propagated with the Fungus Trichoderma harzianum

Abstract: Trichoderma harzianum Rifai, a fungus that controls soilborne pathogens, can enhance growth of several vegetable and floriculture crops. Zero,5,or 25 g ofT. harzianum (isolate T-12) peat-bran amendment was added per kilogram medium in an effort to enhance the rooting of four chrysanthemum [Dendranthema xgrandiflorum (Ramat.) Kitamura] cultivars, two considered easy to root ('Davis' and 'White Marble') and two considered hard to root ('Dark Bronze Charm' and 'Golden Bounty'). Adding the T. harzianum amendment at both rates tested increased root and shoot fresh weights during 21 days of rooting, relative to the control. Supplementary treated cuttings were transplanted into nontreated growing medium after 21 days. Midway between transplant to flowering, increases in height, shoot dry weight, and root fresh and dry weight were detected in 'Dark Bronze Charm' with T-12, relative to the control ; increases in height, shoot fresh and dry weight, and number of nodes were detected in 'Golden Bounty' with T-12. By this time, there were no detectable differences in 'Davis' or 'White Marble'.