[USE OF TRICHODERMA 5320 (TRICHODERMA KOMINGI OUD.) AGAINST PSEUDOMONAS LACHRYMANS (E. SMITH, A. BRYAN) CARSNER, A CAUSATIVE AGENT OF ANGULAR LEAF SPOT OF CUCUMBER].
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Cucumis↗
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[Cytostatic effect of L-lysine-alpha-oxidase from Trichoderma harzianum Rifai and Trichoderma viride].
L-lysine-alpha-oxidase, a new fungal enzyme catalyzing oxidative L-lysine deamination, was shown to have an inhibitory effect on the in vitro synthesis of DNA, RNA and proteins in human carcinoma ovarian (CaOv) cells.
Amino Acid Oxidoreductases↗
[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.
Amino Acid Oxidoreductases↗
[Isolation and comparative properties of serine proteinases of the microscopic fungi Trichoderma lignorum and Trichoderma koningii].
Using affinity chromatography on bacitracin-Sepharose combined with ion-exchange chromatography on aminosilochrome and isoelectrofocusing, individual serine proteases have been isolated for the first time from surface cultures of T. lignorum and T. koningii. The proteinases have pI values at 6.8 and 6.7 and pH optima of 10.5. Both proteinases are stable within the pH range of 4-11 and have molecular weight of 21 000. The amino acid composition of T. lignorum enzyme is Lys3His4Arg9Asx23Thr17Ser23Glx10Pro8Gly27Ala25Cys3Val13Met2Ile11Leu11Tyr7Phe6Tr p3, that of the T. koningii enzyme is Lys3His4 Arg9Asx23Thr16Ser26Glx10Pro9Gly29Ala26Cys3Val14Met2Ile9Leu11Tyr6Phe5Trp3. The enzymes are completely inhibited by phenylmethylsulfonylfluoride, diphenylcarbamoylchloride and trypsin inhibitors from beans, Actinomyces janthinus and potato tubers. The enzymatic and molecular properties of the enzymes are similar to those of subtilisins and previously described fungal serine proteinases, especially to those of proteinase K of the fungus Tritirachium album Limber.
Amino Acid Sequence↗
Genetic relatedness of Trichoderma sect. Pachybasium species based on molecular approaches.
Molecular approaches including internal transcribed spacer (ITS) sequences of ribosomal DNA, universal primer polymerase chain reaction (UP-PCR) fingerprinting, and DNA-DNA hybridization were used to study the genetic relatedness of species within Trichoderma sect. Pachybasium. In the analysis of ITS and 5.8S sequences of ribosomal DNA, parsimony analysis demonstrated that forty-one strains were distributed into five main groups supported by high bootstrap values. The species of Trichoderma sect. Pachybasium were clustered into groups I, II, and IV, with the strains of Trichoderma fasculatum and Trichoderma strictipile forming a separate branch, an independent group V. Some species within each group showed nearly identical sequence differences (fewer than 1-3 bp). UP-PCR and DNA-DNA hybridization were further used to clarify the genetic relatedness of these species with highly similar ITS sequences. Highly similar or identical UP-PCR profiles and high values of DNA complementarity (>70%) were observed among some species, Trichoderma hamatum and Trichoderma pubescens; Trichoderma croceum, Trichoderma polysporum and Trichoderma album, Trichoderma crassum and Trichoderma flavofuscum; and Trichoderma strictipile and Trichoderma fasciculatum. Although every species can be differentiated morphologically, the species showed highly similar molecular characteristics in the above cases, indicating that they could be conspecific. However, in some cases (Trithoderma longipile, T. crassum and T. flavofuscum; Trichoderma fertile and Trichoderma minutisporum; Trichoderma tomentosum, Trichoderma inhamatum and Trichoderma harzianum) there were discriminative patterns of UP-PCR and (or) low levels (<50%) of DNA-DNA hybridization; even their ITS sequences were similar, suggesting a closely phylogenetic relationship.
DNA Fingerprinting↗
Genetic and metabolic diversity of Trichoderma: a case study on South-East Asian isolates.
We have used isolates of Trichoderma spp. collected in South-East Asia, including Taiwan and Western Indonesia, to assess the genetic and metabolic diversity of endemic species of Trichoderma. Ninety-six strains were isolated in total, and identified at the species level by analysis of morphological and biochemical characters (Biolog system), and by sequence analysis of their internal transcribed spacer regions 1 and 2 (ITS1 and 2) of the rDNA cluster, using ex-type strains and taxonomically established isolates of Trichoderma as reference. Seventy-eight isolates were positively identified as Trichoderma harzianum/Trichoderma inhamatum (37 strains) Trichoderma virens (16 strains), Trichoderma spirale (8 strains), Trichoderma koningii (3 strains), Trichoderma atroviride (3 strains), Trichoderma asperellum (4 strains), Hypocrea jecorina (anamorph: Trichoderma reesei; 2 strains), Trichoderma viride (2 strains), Trichoderma hamatum (1 strain), and Trichoderma ghanense (1 strain). Analysis of biochemical characters revealed that T. virens, T. spirale, T. asperellum, T. koningii, H. jecorina, and T. ghanense formed clearly defined clusters, thus exhibiting species-specific metabolic properties. In biochemical character analysis T. atroviride and T. viride formed partially overlapping clusters, indicating that these two species may share overlapping metabolic characteristics. This behavior was even more striking with T. harzianum/T. inhamatum where genotypes defined on the basis of ITS1 and 2 sequences overlapped significantly with adjacent genotypes in the biochemical character analysis, and four strains from the same location (Bali, Indonesia) even clustered with species from section Longibrachiatum. The data indicate that the T. harzianum/T. inhamatum group represents species with high metabolic diversity and partially unique metabolic characteristics. Nineteen strains yielded three different ITS1/2 sequence types which were not alignable with any known species. They were also uniquely characterized by morphological and biochemical characters and therefore represent three new taxa of Trichoderma.
Asia, Southeastern↗
Tricalcium phosphate solubilizing abilities of Trichoderma spp. in relation to P uptake and growth and yield parameters of chickpea (Cicer arietinum L.).
Nine isolates of Trichoderma spp. were investigated for their ability to solubilize insoluble phosphate in Pikovskaya's broth and were compared with an efficient phosphate-solubilizing bacterium Bacillus megaterium subsp. phospaticum PB that was used as the reference strain. All 9 Trichoderma isolates were found to solubilize insoluble tricalcium phosphate to various extents. Trichoderma viride (TV 97) (9.03 microg x mL(-1)), Trichoderma virens (PDBCTVs 12) (9.0 microg x mL(-1)), and Trichoderma virens (PDBCTVs 13) (8.83 microg x mL(-1)) solubilized 70% of that solubilized by the reference strain Bacillus megaterium (12.43 microg x mL(-1)). Pot culture and field evaluations with Trichoderma harzianum (PDBCTH 10), Trichoderma viride (TV 97), and Trichoderma virens (PDBCTVs 12) using chickpea (Cicer arietinum L.) 'Annegeri-1' as the test plant and rock phosphate as the phosphorus source showed significantly increased P uptake in plants treated with Trichoderma harzianum (PDBCTH 10) followed by Trichoderma virens (PDBCTVs 12) and Trichoderma viride (TV 97). Inoculation of Trichoderma spp. also showed increased growth and yield parameters of chickpea compared with the uninoculated controls under both glasshouse and field conditions.
Agriculture↗
An immunological approach to quantifying the saprotrophic growth dynamics of Trichoderma species during antagonistic interactions with Rhizoctonia solani in a soil-less mix.
Studies of the saprotrophic growth dynamics of Trichoderma species and their fungal hosts during antagonistic interactions are severely hampered by the absence of methods that allow the unambiguous identification and quantification of individual genera in complex environments such as soil or compost containing mixed populations of fungi. Furthermore, methods are required that allow discrimination between active hyphal growth and other components of fungal biomass such as quiescent spores that are produced in large numbers by Trichoderma species. This study details the use of monoclonal antibodies to quantify the saprotrophic growth dynamics of the soil-borne plant pathogen Rhizoctonia solani and biological control strains of Trichoderma asperellum and Trichoderma harzianum during antagonistic interactions in peat-based microcosms. Quantification was based on the immunological detection of constitutive, extracellular antigens that are secreted from the growing tip of Rhizoctonia and Trichoderma mycelium and, in the case of Trichoderma harzianum, from quiescent phialoconidia also. The Trichoderma-specific monoclonal antibody (MF2) binds to a protein epitope of the enzyme glucoamylase, which was shown by immunofluorescence and immunogold electron gold microscopy studies of Trichoderma virens in vitro to be produced at the origin of germ tube emergence in phialoconidia and from the growing tip of germ tubes. In addition, a non-destructive immunoblotting technique showed that the enzyme was secreted during active growth of Trichoderma asperellum mycelium in peat. The Rhizoctonia solani-specific monoclonal antibody (EH2) similarly binds to a protein epitope of a glycoprotein that is secreted during active mycelial growth. Extracts derived from lyophilized mycelium were used as a quantifiable and repeatable source of antigens for construction of calibration curves. These curves were used to convert the absorbance values obtained in ELISA tests of peat extracts to biomass equivalents, which allowed comparisons of the saprotrophic growth dynamics of the pathogen and antagonists to be made in single or mixed species microcosms. Trichoderma species were able to compete successfully with R. solani for nutrients and to prevent saprotrophic growth of the pathogen. Specificity of the Trichoderma quantitative assay was tested in non-sterile soil-based microcosms artificially inoculated with T. asperellum. The assay was highly specific and only detected T. asperellum population dynamics. No cross-reactivity was found with extracts from soil samples containing contaminant fungi.
Animals↗
Changes in activity of extracellular enzymes in dual cultures of Lentinula edodes and mycoparasitic Trichoderma strains.
AIMS: The main problem that arises during the cultivation of Lentinula edodes, the Asian Shiitake mushroom, is that the logs on which the cultivation is performed are contaminated by competing micro-organisms, especially Trichoderma spp. The aim of this study was to examine the changes in activity of extracellular enzymes in dual cultures of Trichoderma spp. and L. edodes. METHODS AND RESULTS: Extracellular enzyme activities were determined spectrophotometrically. Trichoderma enzymes important for the degradation of fungal cell walls (N-acetyl-beta-glucosaminidase and laminarinase) were shown to be induced by inactive L. edodes mycelia in liquid culture. The changes that occurred in the extracellular enzyme activities of L. edodes and mycoparasitic Trichoderma spp. (T. aureoviride, T. harzianum and T. viride) were examined during antagonistic interactions on solid medium. The extracellular enzyme patterns of both partners proved to be altered. Trichoderma spp. were induced to produce N-acetyl-beta-glucosaminidase and laminarinase in the presence of active L. edodes mycelia, similarly as observed in liquid culture. The activities of both laccase and manganese peroxidase of L. edodes decreased after physical contact with active Trichoderma mycelia, possibly in consequence of the beginning of degradation of L. edodes by the Trichoderma enzymes. However, besides a decrease in manganese peroxidase activity, an enhancement of L. edodes laccase activity was observed on solid media containing crude culture fluids from Trichoderma liquid cultures. The metabolites responsible for these effects proved to be heat stable. CONCLUSIONS: Induction and inhibition of several extracellular enzymes of both partners were shown in dual cultures of L. edodes and Trichoderma strains, indicating the important role of these enzymes in the antagonistic interaction between the two species. SIGNIFICANCE AND IMPACT OF THE STUDY: As the main problem during the large-scale cultivation of L. edodes is the contamination of the growth substrate by Trichoderma mycelia, the particular knowledge of the mechanism of this competition might be relevant.
Antibiosis↗
Suppression of Pythium spp. by Trichoderma spp. during germination of tomato seeds in soilless growing media.
In the Flemish horticulture Pythium spp. is an important pathogen of tomato plants (Lycopersicon esculenthum) in soilless growing media. Therefore some experiments were conducted to evaluate the possibility of decreasing the damage caused by Pythium spp. by Trichoderma spp. In a tray with several growing media, a suspension of Trichoderma conidia (10(6)/ml growing medium) was applied two weeks before sowing. On some objects, a compost extract (Biostimulus) was added. The growing media used in the experiment were rockwool, recycled rockwool and recycled coconut fibre. After sowing, the trays were covered with perlite. Three isolates of Trichoderma spp.: T. asperellum (Biofungus), T. harzianum (Tri 003) and Trichoderma sp. (KHK) and two isolates of Pythium spp.: P. ultimum (MUCL) en P. aphanidermatum (HRI, UK) were used. Propamocarb was used as a chemical standard. The use of coconut fibre growing medium resulted in a higher percentage (36%) of germination than the rockwool media when only Pythium spp. was used. The presence of the spontaneous developing microflora in the coconut fibre medium gave probably also a suppression of Pythium spp. For that reason the results of the suppression by Trichoderma spp. are not easy to explain and very variable on the different objects. Pythium ultimum was more suppressed than P. aphanidermatum on all the growing media and the application of all the Trichoderma isolates increased the germination percentage of tomato seeds. T. asperellum (Biofungus) gave on rockwool also a good result for the suppression of P. aphanidermatum (increasing of germination with 48%). This effect was comparable with the propamocarb treatment (48%). T. harzianum (Tri 003) gave a small suppression (22%) and Trichoderma sp. (KHK) gave almost no suppression of P. aphanidermatum (7%). When less Trichoderma conidia were applied the germination percentage decreased. The adding of a compost extract (Biostimulus) had no influence on the results. This experiment shows that application of Trichoderma conidia in the presence of Pythium spp. increases the germination percentage of tomato seeds, sowed in the used soilless growing media.
Belgium↗
Fungal and plant gene expression during the colonization of cacao seedlings by endophytic isolates of four Trichoderma species.
Endophytic isolates of Trichoderma species are being considered as biocontrol agents for diseases of Theobroma cacao (cacao). Gene expression was studied during the interaction between cacao seedlings and four endophytic Trichoderma isolates, T. ovalisporum-DIS 70a, T. hamatum-DIS 219b, T. harzianum-DIS 219f, and Trichoderma sp.-DIS 172ai. Isolates DIS 70a, DIS 219b, and DIS 219f were mycoparasitic on the pathogen Moniliophthora roreri, and DIS 172ai produced metabolites that inhibited growth of M. roreri in culture. ESTs (116) responsive to endophytic colonization of cacao were identified using differential display and their expression analyzed using macroarrays. Nineteen cacao ESTs and 17 Trichoderma ESTs were chosen for real-time quantitative PCR analysis. Seven cacao ESTs were induced during colonization by the Trichoderma isolates. These included putative genes for ornithine decarboxylase (P1), GST-like proteins (P4), zinc finger protein (P13), wound-induced protein (P26), EF-calcium-binding protein (P29), carbohydrate oxidase (P59), and an unknown protein (U4). Two plant ESTs, extensin-like protein (P12) and major intrinsic protein (P31), were repressed due to colonization. The plant gene expression profile was dependent on the Trichoderma isolate colonizing the cacao seedling. The fungal ESTs induced in colonized cacao seedlings also varied with the Trichoderma isolate used. The most highly induced fungal ESTs were putative glucosyl hydrolase family 2 (F3), glucosyl hydrolase family 7 (F7), serine protease (F11), and alcohol oxidase (F19). The pattern of altered gene expression suggests a complex system of genetic cross talk occurs between the cacao tree and Trichoderma isolates during the establishment of the endophytic association.
Cacao↗
Effect of biocontrol strains of Trichoderma on plant growth, Pythium ultimum polulations, soil microbial communities and soil enzyme activities.
Five strains of Trichoderma with known biocontrol activities were assessed for their effect upon pea growth and their antagonistic activity against large Pythium ultimum inocula. The effect of Trichoderma inocula upon the indigenous soil microflora and soil enzyme activities in the presence and absence of Pythium is assessed. In the absence of Pythium, Trichoderma strain N47 significantly increased the wet shoot weight by 15% but did not significantly affect the dry weight, whilst strains T4 and N47 significantly increased the root weights by 22% and 80%) respectively. Strains TH1 and N47 resulted in significantly greater root lengths. Pythium inoculation significantly reduced the root length and the number of lateral roots and nodules, and significantly increased the root and rhizosphere soil fungal populations. Pythium inoculation significantly reduced the plant wet and dry shoot weights and significantly increased the wet and the dry shoot/root ratio. All the Trichoderma strains reduced the number of lesions caused by Pythium and increased the number of lateral roots. The effect of the Pythium on emergence and shoot growth was significantly reduced by all the Trichoderma strains except strain To10. Inoculation with Trichoderma strains TH1 and T4 resulted in significantly greater wet root weights (62% and 57%, respectively) in the presence of Pythium compared to the Pythium control. Strain N47 significantly increased the shoot/root ratio compared to the Pythium control. Inoculation with Trichoderma strains T4, T12 and N47 significantly reduced Pythium populations. Pythium increased the activity of C, N and P cycle enzymes, whilst four Trichoderma strains reduced this effect, indicating reduced plant damage and C leakage. Overall, strains T4 and N47 had the greatest beneficial characteristics, as both these strains improved plant growth in the absence of Pythium and reduced plant damage in the presence of Pythium. The dual properties of these strains improve the commercial application, giving them an advantage over single action inocula, especially in the absence of plant pathogens.
Acetylglucosaminidase↗
A mini-bag technique for evaluation of fungicide effects on Trichoderma spp in mushroom compost.
An in vivo technique was developed to observe colonisation of mushroom compost by Trichoderma spp. Isolates of T. harzianum (Th2), T. harzianum (Th1), T. koningii (Tk) and T. viride (Tv) were artificially introduced into compost using a mini-bag system. Wheat grains, colonised by Trichoderma spp, were placed centrally on a layer of compost at the bottom of 1-litre polythene bags which were then filled with 350 g of spawned or un-spawned compost, and partially sealed. After 14 and 21 days incubation at 27 degrees C, the bags were assessed for recovery of Trichoderma from middle and top zones using a needle stab re-isolation technique and a visual colonisation scoring system. Visible green mould contamination, similar to that observed in practice, developed within 21 days. The visual colonisation scoring was reliably related to the re-isolation success. In this evaluation, Trichoderma spp showed considerable differences in their relative abilities to colonise spawned and un-spawned compost, with Th2 isolates being consistently superior to the other isolates of Th1, Tk and Tv in colonising spawned compost. This technique was employed to evaluate the effects of fungicides on the colonisation of mushroom compost by three Trichoderma spp: Th2, Th1 and Tk, using 1-litre and 5-litre mini-bag systems. Aqueous suspensions of benomyl, carbendazim, thiabendazole, prochloraz and prochloraz+carbendazim incorporated into the compost at 50 mg litre(-1), or applied to spawn at 50 mg kg(-1), reduced the colonisation by Trichoderma spp. Prochloraz and prochloraz+carbendazim were superior to benomyl, carbendazim or thiabendazole in reducing compost colonisation by Th2, Th1 and Tk, with Th2 being the most persistent type, capable of colonising treated compost in the presence of all five fungicides. The prochloraz+carbendazim mixture, not normally used in mushroom production, was equal to or better than prochloraz alone. The incidence of green mould colonisation by Th2 was as extensive in the 5-litre compost bags as in the 1-litre bags, but colonisation by Th1 and Tk was more apparent in the 5-litre bags. The in vivo mini-bag evaluations using wheat grain Trichoderma inoculum and needle stab re-isolation procedures proved an efficient method for studying colonisation and screening for effectiveness of fungicides applied to mushroom compost or spawn.
Agaricales↗
Quantification and characterisation of Trichoderma spp. from different ecosystems.
Basal stem rot of oil palm caused by Ganoderma boninense is of major economic importance. Observations of the low incidence of disease due to Ganoderma species in natural stands, suggest that the disease is kept under control by some biological means. Trichoderma spp. are saprophytic fungi with high antagonistic activities against soil-borne pathogens. However, their abundance and distribution are soil and crop specific. Trichoderma species have been found to be concentrated in the A1 (0-30 cm) and Be soil horizons (30-60 cm), although the abundance of Trichoderma was not significantly different between the oil palm and non-oil palm ecosystems. Characterisation of Trichoderma isolates based on cultural, morphological and DNA polymorphism showed that T. harzianum, T. virens, T. koningii and T. longibrachiatum made up 72, 14, 10 and 4% of the total Trichoderma isolates isolated. As Trichoderma species are present in the oil palm ecosystem, but at lower numbers and in locations different from those desired, soil augmentation with antagonistic Trichoderma spp. can be developed as a strategy towards integrated management of basal stem rot of oil palm.
Arecaceae↗
In vitro activity and synergism of amphotericin B, azoles and cationic antimicrobials against the emerging pathogen Trichoderma spp.
OBJECTIVES: The uncommon fungal pathogen Trichoderma shows increasing medical importance particularly in immunocompromised patients. Despite systemic antifungal therapy, prognosis of Trichoderma infection is poor regardless of the type of infection and the therapy used. The aim of the present study was to evaluate the in vitro activity and synergism of double antifungal combinations including amphotericin B, voriconazole, fluconazole, chlorhexidine digluconate and Akacid plus against 15 isolates of Trichoderma longibrachiatum and 1 isolate of Trichoderma harzianum. METHODS: Individual MICs were determined by using broth microdilution method following the NCCLS M38-A guidelines with standard RPMI 1640 broth. Synergy tests were performed using the chequerboard method. RESULTS: All clinical Trichoderma strains showed reduced susceptibility to fluconazole (MICs>or=64 mg/L) and amphotericin B (MICs=2 mg/L), whereas lower MICs of 0.5-1 mg/L were detected for voriconazole. Akacid plus reached the lowest MIC values in a range of 0.06-0.5 mg/L, 4- to 32-fold higher MICs were found for chlorhexidine. No antagonism was observed for any of the antifungal combinations tested. Interaction of amphotericin B and azoles was indifferent (fractional inhibitory concentration index, FICI 2-4). The combination of one azole and one cationic biocide showed different degree of synergism (FICI 0.07-2.03). Interaction of Akacid plus and chlorhexidine resulted in synergism for each Trichoderma isolate (FICI-range 0.05-0.5). CONCLUSIONS: These results demonstrate no interaction between antifungals and some degree of synergism between azoles and cationic antimicrobials against Trichoderma spp.
Amphotericin B↗
Identification of Trichoderma strains by image analysis of HPLC chromatograms.
Forty-four Trichoderma strains from water-damaged building materials or indoor dust were classified with chromatographic image analysis on full chromatographic matrices obtained by high performance liquid chromatography with UV detection of culture extracts. The classes were compared with morphological identification and rDNA sequence data, and for each class all strains were of the same identity. With all three techniques each strain--except one--was identified as the same species. These strains belonged to Trichoderma atroviride (nine strains), Trichoderma viride (three strains), Trichoderma harzianum (10 strains), Trichoderma citrinoviride (12 strains), and Trichoderma longibrachiatum (nine strains). The odd strain was identified as Trichoderma hamatum by morphology and rDNA sequencing, but not by image analysis as no reference strains of this species were included. It is concluded that the secondary metabolite profile contains sufficient information for classification and species identification.
Air Pollution, Indoor↗
Interactions of Phytophthora cinnamomi and Trichoderma spp. in relation to propagule production in soil cultures at 26 degrees C1.
Effects of Trichoderma harzianum and T. polysporum on chlamydospore production by two isolates of Phytophthora cinamomi were determined over a 21-day period in nonsterile, sterilized, and sterilized amended soil. Trichoderma was either coinoculated with P. cinnamomi or added to the cultures on day 3 of the incubation period. In nonsterile soil, conversion of mycelial fragments in the P. cinnamomi inoculum to chlamydospores resulted in an initial slight increase in chlamydospore numbers. In cultures where either of the Trichoderma isolates was added on day 3, a significiant reduction in chlamydospore numbers was observed on day 4; however, neither of the Trichoderma isolates at either inoculation time significantly affected clamydospore numbers by day 21. Results from studies with sterilized amended soil indicated that a reduction in P. cinnamomi chlamydospore numbers by either of the Trichoderma isolates was dependent upon availability of simple carbohydrates. In sterilized nonamended soil, the number of chlamydospores was increased in cultures containing either Trichoderma isolate; in sterilized amended soil chlamydospore numbers were initially reduced, followed by a general increase as the nutrients were depleted. Neither of the Trichoderma isolates exhibited significant antagonistic qualities toward P. cinnamomi.
Fungi↗
Characterization of a protease produced by a Trichoderma harzianum isolate which controls cocoa plant witches' broom disease.
BACKGROUND: Several Trichoderma strains have been reported to be effective in controlling plant diseases, and the action of fungal hydrolytic enzymes has been considered as the main mechanism involved in the antagonistic process. However, although Trichoderma strains were found to impair development of Crinipellis perniciosa, the causal agent of cocoa plant witches' broom disease, no fungal strain is available for effective control of this disease. We have then undertaken a program of construction of hydrolytic enzyme-overproducing Trichoderma strains aiming improvement of the fungal antagonistic capacity. The protease of an indian Trichoderma isolate showing antagonistic activity against C. perniciosa was purified to homogeneity and characterized for its kinetic properties and action on the phytopathogen cell wall. RESULTS: A protease produced by the Trichoderma harzianum isolate 1051 was purified to homogeneity by precipitation with ammonium sulfate followed by hydrophobic chromatography. The molecular mass of this protease as determined by SDS-polyacrylamide gel electrophoresis was about 18.8 kDa. Its N-terminal amino acid sequence shares no homology with any other protease. The purified enzyme substantially affected the cell wall of the phytopathogen C. perniciosa. Western-blotting analysis showed that the enzyme was present in the culture supernatant 24 h after the Trichoderma started to grow in casein-containing liquid medium. CONCLUSIONS: The capacity of the Trichoderma harzianum protease to hydrolyze the cell wall of C. perniciosa indicates that this enzyme may be actually involved in the antagonistic process between the two fungi. This fact strongly suggest that hydrolytic enzyme over-producing transgenic fungi may show superior biocontrol capacity.
Agaricales↗