Mycoparasitic Fungus Research Articles

Expression of two major chitinase genes of Trichoderma atroviride (T. harzianum P1) is triggered by different regulatory signals.

Regulation of the expression of the two major chitinase genes, ech42 (encoding the CHIT42 endochitinase) and nag1 (encoding the CHIT73 N-acetyl-beta-D-glucosaminidase), of the chitinolytic system of the mycoparasitic biocontrol fungus Trichoderma atroviride (= Trichoderma harzianum P1) was investigated by using a reporter system based on the Aspergillus niger glucose oxidase. Strains harboring fusions of the ech42 or nag1 5' upstream noncoding sequences with the A. niger goxA gene displayed a glucose oxidase activity pattern that was consistent under various conditions with expression of the native ech42 and nag1 genes, as assayed by Northern analysis. The expression product of goxA in the mutants was completely secreted into the medium, detectable on Western blots, and quantifiable by enzyme-linked immunosorbent assay. nag1 gene expression was triggered during growth on fungal (Botrytis cinerea) cell walls and on the chitin degradation product N-acetylglucosamine. N-Acetylglucosamine, di-N-acetylchitobiose, or tri-N-acetylchitotriose also induced nag1 gene expression when added to mycelia pregrown on different carbon sources. ech42 expression was also observed during growth on fungal cell walls but, in contrast, was not triggered by addition of chitooligomers to pregrown mycelia. Significant ech42 expression was observed after prolonged carbon starvation, independent of the use of glucose or glycerol as a carbon source, suggesting that relief of carbon catabolite repression was not involved in induction during starvation. In addition, ech42 gene transcription was triggered by physiological stress, such as low temperature, high osmotic pressure, or the addition of ethanol. Four copies of a putative stress response element (CCCCT) were found in the ech42 promoter.

EXG1p, a novel exo-β1,3-glucanase from the fungus Cochliobolus carbonum, contains a repeated motif present in other proteins that interact with polysaccharides

Genomic and cDNA copies of EXG1, a gene encoding an exo-β1,3-glucanase from the plant pathogenic fungus Cochliobolus carbonum, were isolated. The gene contains two introns of 50 and 53 bp, and the mRNA has a 5′-untranslated region of 90 nt and a 3′-untranslated region of 159 nt. The deduced protein product, EXG1p, has a predicted signal peptide of 17 amino acids, but based on the known N-terminus of the mature protein is further processed to remove an additional 25 amino acids. The sequence of EXG1p is not closely related to any other known protein, but has a low similarity (29% overall amino acid identity) to BGN13.1, an endo-β1,3-glucanase from the mycoparasitic fungus Trichoderma harzianum. EXG1p contains two imperfect copies of a 23-amino acid motif that is found in several other proteins that interact with polysaccharides, including plant and bacterial polygalacturonases, phage neck appendage protein, phage endoneuramidase, and bacterial mannuronan epimerase.

Genes from mycoparasitic fungi as a source for improving plant resistance to fungal pathogens.

Disease resistance in transgenic plants has been improved, for the first time, by the insertion of a gene from a biocontrol fungus. The gene encoding a strongly antifungal endochitinase from the mycoparasitic fungus Trichoderma harzianum was transferred to tobacco and potato. High expression levels of the fungal gene were obtained in different plant tissues, which had no visible effect on plant growth and development. Substantial differences in endochitinase activity were detected among transformants. Selected transgenic lines were highly tolerant or completely resistant to the foliar pathogens Alternaria alternata, A. solani, Botrytis cinerea, and the soilborne pathogen Rhizoctonia solani. The high level and the broad spectrum of resistance obtained with a single chitinase gene from Trichoderma overcome the limited efficacy of transgenic expression in plants of chitinase genes from plants and bacteria. These results demonstrate a rich source of genes from biocontrol fungi that can be used to control diseases in plants.

Unexpected homology between inducible cell wall protein QID74 of filamentous fungi and BR3 salivary protein of the insect Chironomus.

A gene, qid74, of mycoparasitic filamentous fungus Trichoderma harzianum and its allies encodes a cell wall protein that is induced by replacing glucose in the culture medium with chitin (simulated mycoparasitism conditions). Because no trace of this gene can be detected in related species such as Gibberella fujikuroi and Saccharomyces cerevisiae, the qid74 gene appears to have arisen de novo within the genus Trichoderma. Qid74 protein, 687 residues long, is now seen as highly conserved tandem repeats of the 59-residue-long unit. This unit itself, however, may have arisen as tandem repeats of the shorter 13-residue-long basic unit. Within the genus Trichoderma, the amino acid sequence of Qid74 proteins has been conserved in toto. The most striking is the fact that Qid74 shares 25.3% sequence identity with the carboxyl-terminal half of the 1,572-residue-long BR3 protein of the dipteran insect Chironomus tentans. BR3 protein is secreted by the salivary gland of each aquatic larva of Chironomus to form a tube to house itself. Furthermore, the consensus sequence derived from these 59-residue-long repeating units resembles those of epidermal growth factor-like domains found in divergent invertebrate and vertebrate proteins as to the positions of critical cysteine residues and homology of residues surrounding these cysteines.

Cladobotryal: a fungal metabolite with a novel ring system.

In screening for antifungal metabolites a novel compound, cladobotryal, was isolated from the mycoparasitic fungus Cladobotryum varium. Its structure was established as (+)-(2R*,3R*)-2-[(Z)-2-buten-2-yl]-3, 7-dihydro-3-formyl-3-methyl-5-phenylfuro[2,3-b]pyridin-4(2H)-one on the basis of spectroscopic evidence and single crystal X-ray analysis of its methyl hemiacetal. The fused furo[2,3-b]pyridinone skeleton of cladobotryal seems unprecedented within the chemical literature.

Degradation of sclerotia of Sclerotinia sclerotiorum by fungus gnats ( Bradysia coprophila) and the biocontrol fungi Trichoderma spp.

Our purpose was to evaluate the ability of fungus gnats ( Bradysia coprophila) and mycoparasitic fungi, alone or in combination, to degrade sclerotia of the plant pathogen Sclerotinia sclerotiorum. A collection of potential mycoparasites was evaluated for the ability to degrade sclerotia of S. sclerotiorum in soil. The most effective isolate, Trichoderma hamatum TMCS-3, was combined with fungus gnats in laboratory pot tests. Fewer sclerotia survived in the combination treatments, compared to treatments with either organism alone, and the viability of sclerotia was reduced.

Improved biocontrol activity of Trichoderma harzianum by over-expression of the proteinase-encoding gene prb1.

Transformation systems developed for Trichoderma spp. were utilized to improve the biocontrol efficiency of the mycoparasitic fungus Trichoderma harzianum by increasing the copy number of the basic proteinase gene prb1. The transformants were stable and carried from two to ten copies of prb1. High levels of expression of prb1 during fungus-fungus interaction were detected when T. harzianum and Rhizoctonia solani were confronted in vitro. In liquid cultures the proteinase was induced by cell walls of R. solani. Under greenhouse conditions, incorporation of T. harzianum transformants into pathogen-infested soil significantly reduced the disease caused by R. solani in cotton plants.

Transformation of a mycoparasitic Trichoderma harzianum strain with the argB gene of Aspergillus nidulans

A transformation system was developed for the mycoparasitic filamentous fungus Trichoderma harzianum, based upon complementation of auxotrophic mutants. Prototrophic transformants were obtained using plasmids pSal23 and pAN5-41B, carrying the Aspergillus nidulans argB gene, and both the Aspergillus nidulans argB and Escherichia coli lacZ genes, respectively.

Molecular cloning and expression of the nag1 gene (N-acetyl-beta-D-glucosaminidase-encoding gene) from Trichoderma harzianum P1.

A 72-kDa N-acetyl-beta-D-glucosaminidase was purified from the mycoparasitic fungus Trichoderma harzianum P1; antibodies were raised against it, and aa-sequences were obtained. The antibody reacted with a single 72-kDa protein band in culture filtrates of T. harzianum grown on chitin, and was subsequently used to clone the corresponding nag1 gene from a lambdagt11 cDNA expression library. It was interrupted by two short introns and encoded a protein of 580 amino acids. The deduced protein sequence contained aa-sequence areas of high similarity to N-acetyl-glucosaminidases from other eukaryotes such as Candida albicans, and invertebrate and vertebrate animal tissues. The highest similarity was observed with the corresponding gene from the silkworm. The aa-sequence of a tryptic fragment of purified N-acetyl-beta-D-glucosaminidase from T. harzianum corresponded to a deduced aa sequence from a portion of the cloned gene, thus verifying that the protein is encoded by nag 1. Southern analysis showed that nag 1 is present as a single-copy gene in T. harzianum. Expression of nag1-mRNA was strongly induced upon growth on chitin, N-acetyl-glucosamine and the cell walls of Botrytis cinerea used as a carbon source. The appearance of the corresponding N-acetyl-beta-D-glucosaminidase protein, as determined by Western analysis, paralleled the pattern of nag 1 expression, thereby suggesting that its formation is regulated at the level of transcription.

The role of lectins in recognition and adhesion of the mycoparasitic fungus Trichoderma spp. to its host.

The destructive parasitic mode in Trichoderma, a natural antagonist to other fungi and well-known biocontrol agent of plant pathogenic fungi, appears to be a complex process, made up from several successive steps. Upon contact with the host Trichoderma coils around or grows along the host hyphae and forms hook-like structures that aid in penetrating the host’s cell wall (Chet, 1987). In Trichoderma, this reaction has been found to be rather specific (Dennis and Webster, 1971), and lectin-carbohydrate interactions were assumed to mediate the attachment and recognition between Trichoderma and soil-borne plant pathogenic fungi (Barak et al., 1985).KeywordsTrichoderma HarzianumInfection StructureAdhesive MaterialNylon FiberChitinolytic EnzymeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

A novel endo-beta-1,3-glucanase, BGN13.1, involved in the mycoparasitism of Trichoderma harzianum

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.

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

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.

A rind-associated factor from sclerotia of Sclerotinia minor stimulates germination of a mycoparasite

Compounds that specifically stimulate germination of Sporidesmium sclerotivorum macroconidia have been isolated from the melanized outer layer of Sclerotinia minor sclerotia. The compound is a small hydrophobic molecule that is able to pass through a 3000 Da filter and is heat-labile in water. Three fractions were resolved using reversed-phase hplc. The component with presumed biological activity in soil is sparingly soluble in water. It has been given the trivial name ‘sporigermin’. This is the first report describing the isolation of a compound from a host fungus that acts as a specific signal to stimulate germination of a mycoparasitic fungus.

Sequence analysis and expression studies of a gene encoding a novel serine + alanine-rich protein in Trichoderma harzianum.

The promoters of constitutive genes have proven themselves to be useful parts of expression vectors for genetic engineering in different organisms. Based on a differential screening of an induced cDNA library, we have used a new approach to isolate constitutively expressed genes which can be used for this purpose. Following this strategy, we have isolated constitutively expressed cDNA clones of the mycoparasitic fungus Trichoderma harzianum. One of these cDNA clones corresponds to a gene (cob4) that encodes a novel serine + alanine-rich protein. The predicted amino acid (aa) sequence of this protein (171 aa, deduced M(r) 15,564, pI 12.49) displays a motif that is also found in structural proteins of different species and in a yeast thermoinducible protein. Northern (RNA) blot analysis has demonstrated that cob4 is expressed during growth when glucose or cell walls of a phytopathogenic fungus are provided as carbon source. Hybridizations of T. harzianum genomic DNA indicate that this gene most probably belongs to a multigene family.

Cellulose-triggered sporulation in the galactose oxidase-producing fungus Cladobotryum (Dactylium) dendroides NRRL 2903 and its re-identification as a species of Fusarium

The production of extracellular galactose oxidase is limited to a few fungal species, including the important plant pathogens Fusarium graminearum and F. moniliforme . The best-studied enzyme is the one produced by the mycoparasitic fungus Cladobotryum (Dactylium) dendroides NRRL 2903. The NRRL 2903 strain was first mis-identified as Polyporus circinatus and later re-determined as Dactylium dendroides , although sporulation was never observed and the fungus was regarded as sterile. Upon growth at 25 °C, 50 rpm, in liquid medium containing 2% cellulose as the sole carbon source, and in the presence of 0·5–0·75% yeast extract, conidial production was induced in NRRL 2903, which was re-identified as Fusarium sp. The only other known commercial strain of Cladobotryum (Dactylium) dendroides able to produce galactose oxidase, ATCC 46032, also produced fusiform conidia upon growth in cellulose-containing medium, and was shown to be genetically identical to the NRRL 2903 strain. Genetic comparison with six different representative strains of Cladobotryum dendroides (teleomorph: Hypomyces rosellus ), and four strains of the closely related Hypomyces aurantius , based on the analysis of the presence or absence of a homologous galactose oxidase gene ( gaoA ), RAPD-PCR and RFLP analysis, confirm the distinct nature of the NRRL 2903 strain and Cladobotryum dendroides . Despite the resemblance of NRRL 2903 conidia and conidiophores to those of Fusarium chlamydosporum genetic comparison, with three different strains, suggests NRRL 2903 cannot be re-identified as F. chlamydosporum . Two of the strains, however, contain a region in their genome that is highly homologous to the galactose oxidase gene ( gaoA ), and one strain exhibits extracellular galactose oxidase activity but only partial homology to the gaoA gene of NRRL 2903.

A putative catabolite-repressed cell wall protein from the mycoparasitic fungus Trichoderma harzianum

A cDNA clone encoding a putative cell wall protein (Qid3) was isolated from a library prepared from chitin-induced mRNA in cultures of the mycoparasitic fungus Trichoderma harzianum. The predicted 14 kDa protein shows a potential signal peptide, several hydrophobic domains and certain motifs that are structurally similar to proline-rich and glycine-rich plant cell wall proteins. Expression of the qid3 gene is derepressed in the absence of glucose. When introduced in yeast, qid3 expression causes cell division arrest into cytokinesis and cell separation, probably due to its cell wall localization.

Studies on the Metabolites of Mycoparasitic Fungi. I. Metabolites of Cladobotryum varium.

Six new α-pyrone derivatives, named cladobotrins I-VI (1-3, 5, 10, 11), and a know one, rosellisin aldehyde (6), were isolated from the culture broth of a mycoparasitic fungus, Cladobotryum varium NEES et DUBY (=Didymocladium ternatum BON.). Their structures were established by spectroscopic methods including two-dimensional NMR techniques. Among the compounds obtained, three α-pyrone derivatives, having a formyl group at the C-5 position, showed inhibitory activity against the mycelial growth of a fungus, Ganoderma lucidum (FR.) KARST., the fruit bodies of which are used as the oriental crude drug Lin-Chi.

Efficient production of phialoconidia of Verticillium lecanii for biocontrol of cucumber powdery mildew, Sphaerotheca fuliginea

A method is described that yields over 3 • 10 9 phialoconidia/ml of VerticiIlium lecanii. Additional keyword: mycoparasites. Spore production of mycoparasitic fungi may vary considerably and mass production of fungal conidia for biological control often meets with problems. Some mycoparasites do not produce sufficient inoculum for small-scale experiments (Hijwegen, 1988). Usually, special studies on cultivation methods are required as demonstrated for Ampelomyces quisqualis by Schmitz-Elsherif (1990). Verticillium lecanii (Zimm.) Vi6gas strain Fyto 88.1, isolated from Sphaerotheca fuliginea (Schlecht.: Fr.) Poll. (Hijwegen, 1988), can easily be grown and sporulates well on most solid media such as agars containing potato-dextrose broth, malt extract plus mycological peptone, oat meal, chitin or skimmed milk. In this way productions of 1-4 • 107 conidia/cm 2 (0.5-2 • 109 conidia/petridish) can be obtained. When, however, 8 • 101~ conidia were required every week for a greenhouse experiment, (Verhaar and Hijwegen, unpublished) surface grown cultures were inadequate and other methods of cultivation were investigated. V. lecanii was grown at 20 ~ and 135 rpm in liquid media containing milled, autoclaved oat meal, skimmed milk or malt extract plus mycological peptone. Yields of conidia were rather low. Conidium production was enhanced by raising the incubation temperature to 25 ~ Yeast cell walls and malt extract plus mycological peptone gave a good yield of conidia. These media were, however, surpassed by far by growing V. lecanii in autoclaved oat meal suspension. V. lecanii has been reported to produce only blastoconidia in liquid culture (Latg6 et al., 1986). After consulting Dr W. Gums, CBS, Baam this was investigated using our strain Fyto 88.1. In our experiments submerged mycelium, producing micro-verticils with micro-phialides carrying normal phialoconidia, was formed in liquid culture. Budding was not observed. Conidia produced in liquid culture could not be distinguished microscopically in size or shape from conidia produced on solid oat meal medium. In 1% oat meal more mycelium was formed than in 3% oat meal. This raises the question whether oat meal contains substances that influence the mycelium--conidia ratio. To investigate conidium production quantitatively, 0.8 cm 2 pieces of agar with mycelium from the margin of a growing culture of V. lecanii were added to 300-ml Erlenmever flasks containing 100 ml of 1, 2, or 3% milled and autoclaved oat meal (Quaker I~IO

Growth of the mycoparasitic fungus Verticillium biguttatum from different geographical origins at near-minimum temperatures

Sclerotia ofRhizoctonia solani collected from potato tubers from different countries were assayed for the presence of mycoparasites. Among the mycoparasites observedVerticillium biguttatum predominated. Its geographical distribution was not restricted to certain latitudes or soil types;V. biguttatum occurred worldwide in potato fields.

Colonization of roots and stolons of potato by the mycoparasitic fungus Ver ticillium biguttatum

Potential host plants of the mycoparasitic fungus yerticillium biguttatum were screened. The fungus was found on many different plant genotypes, but the largest populations were found on potato, the natural substrate of the phytopathogenic Rhizoctonia solani. The ability of V. bigutlatum to colonize potato stolons and roots was investigated. After inoculation of seed tubers, V. biguttatum occurred in higher frequences on the stolons than without inoculation. There was no indication of epiphytic growth under natural conditions. Mycoparasitic growth and passive transport of inoculum by advancing roots, stolons and sprouts through the soil may explain the observed colonization pattern of V. biguttatum. Further evidence on the obligate parasitic nature of V. biguttatum is presented.