Showing papers on "Trichoderma harzianum published in 2006"

Major secondary metabolites produced by two commercial Trichoderma strains active against different phytopathogens.

Abstract: Aims: Trichoderma harzianum strains T22 and T39 are two micro-organisms used as active agents in a variety of commercial biopesticides and biofertilizers and widely applied amongst field and greenhouse crops. The production, isolation, biological and chemical characterization of the main secondary metabolites produced by these strains are investigated. Methods and Results: Of the three major compounds produced by strain T22, one is a new azaphilone that shows marked in vitro inhibition of Rhizoctonia solani, Pythium ultimum and Gaeumannomyces graminis var. tritici. In turn, filtrates from strain T39 were demonstrated to contain two compounds previously isolated from other T. harzianum strains and a new butenolide. The production of the isolated metabolites was also monitored by liquid chromatography/mass spectrometry during in vitro interaction with R. solani. Conclusions: This paper reports the isolation and characterization of the main secondary metabolites obtained from culture filtrates of two T. harzianum strains and their production during antagonistic interaction with the pathogen R. solani. Significance and Impact of the Study: This is the first work on secondary metabolites produced by the commercially applied strains T22 and T39. Our results provide a better understanding of the metabolism of these fungi, which are both widely used as biopesticides and/or biofertilizers in biocontrol.

Transgenic Tobacco Plants Overexpressing Chitinases of Fungal Origin Show Enhanced Resistance to Biotic and Abiotic Stress Agents

Abstract: Genes encoding defense-related proteins have been used to alter the resistance of plants to pathogens and other environmental challenges, but no single fungal gene overexpression has produced broad-spectrum stress resistance in transgenic lines. We have generated transgenic tobacco (Nicotiana tabacum) lines that overexpress the endochitinases CHIT33 and CHIT42 from the mycoparasitic fungus Trichoderma harzianum and have evaluated their tolerance to biotic and abiotic stress. Both CHIT33 and CHIT42, individually, conferred broad resistance to fungal and bacterial pathogens, salinity, and heavy metals. Such broad-range protective effects came off with no obvious detrimental effect on the growth of tobacco plants.

Bacteria in oral secretions of an endophytic insect inhibit antagonistic fungi

Abstract: Colonisation of host trees by an endophytic herbivore, the spruce beetle, Dendroctonus rufipennis , is accompanied by invasion of its galleries by a number of fungal species. Four of these associated species were identified as Leptographium abietinum , Aspergillus fumigatus , Aspergillus nomius , and Trichoderma harzianum . 2. Trichoderma and Aspergillus significantly reduced spruce beetle survival and reproduction in controlled assays. 3. A previously undescribed behaviour was observed, in which spruce beetle adults exuded oral secretions, especially within fungus-pervaded galleries. 4. These oral secretions inhibited the growth of fungi except A. nomius , and disrupted the morphology of the latter. Administration of these secretions indicated a dose- dependent inhibitory effect. 5. Oral secretions cultured on microbiological media yielded substantial bacterial growth. 6. Filter-sterilised secretions failed to inhibit fungal growth, evidence that the bacteria are responsible for the antifungal activity. 7. Nine bacterial isolates belonging to the Actinobacteria, Firmicutes, Gammapro- teobacteria, and Betaproteobacteria taxa were obtained from the secretions. 8. Bacterial isolates showed species-specific inhibitory activity against the four fungi antagonistic to spruce beetle. The bacterium with the strongest fungal inhibition activity was the actinomycete Micrococcus luteus . 9. The production of bark beetle secretions containing bacteria that inhibit fungal growth is a novel finding. This suggests an additional level of complexity to ecological associations among bark beetles, conifers, and microorganisms, and an important adaptation for colonising subcortical tissue.

Leaf hairs influence phytopathogenic fungus infection and confer an increased resistance when expressing a Trichoderma α-1,3-glucanase

Abstract: The leaf surface of a very large number of plant species are covered by trichomes. Non-glandular trichomes are specialized unicellular or multicellular structures that occur in many different plant species and function in xenobiotic detoxification and protecting the plant against pest attack. By analysing the susceptibility of trichome mutants, evidence is provided that indicates the influence of leaf trichomes on foliar fungal infections in Arabidopsis thaliana, probably by facilitating the adhesion of the fungal spores/hyphae to the leaf surface. A decreased trichome number in the hairless Arabidopsis mutant gl1 enhances tolerance against the necrotrophic fungus Botrytis cinerea. By contrast, the try mutant shows an increased susceptibility to both fungal infection and accumulation. Trichome density does not influence infection by the soil-borne pathogen Rhizoctonia solani. In addition, the influence of trichomes on foliar infection is supported by targeting the high-level expression of the Trichoderma harzianum alpha-1,3-glucanase protein to the specialized cell structures. Trichome expression of this anti-fungal hydrolase shows a significant resistance to infection by the foliar pathogen Botrytis cinerea. Resistance to this fungus is not dependent on the constitutive induction of the salicylic or jasmonic defence signalling pathways, but the presence of the alpha-1,3-glucanase protein in trichomes.

ThHog1 controls the hyperosmotic stress response in Trichoderma harzianum

Abstract: Trichoderma harzianum is a widespread mycoparasitic fungus, able to successfully colonize a wide range of substrates under different environmental conditions. Transcript profiling revealed a subset of genes induced in T. harzianum under hyperosmotic shock. The hog1 gene, a homologue of the MAPK HOG1 gene that controls the hyperosmotic stress response in Saccharomyces cerevisiae, was characterized. T. harzianum hog1 complemented the hog1Δ mutation in S. cerevisiae, but showed different features to yeast alleles: improved osmoresistance by expression of the hog1 allele and a lack of lethality when the hog1 F315S allele was overexpressed. ThHog1 protein was phosphorylated in T. harzianum under different stress conditions such as hyperosmotic or oxidative stress, among others. By using a ThHog1-GFP fusion, the protein was shown to be localized in nuclei under these stress conditions. Two mutant strains of T. harzianum were constructed: one carrying the hog1 F315S allele, and a knockdown hog1-silenced strain. The silenced strain was highly sensitive to osmotic stress, and showed intermediate levels of resistance against oxidative stress, indicating that the main role of ThHog1 protein is in the hyperosmotic stress response. Stress cross-resistance experiments showed evidences of a secondary role of ThHog1 in oxidative stress. The strain carrying the hog1 F315S allele was highly resistant to the calcineurin inhibitor cyclosporin A, which suggests the existence of links between the two pathways. The two mutant strains showed a strongly reduced antagonistic activity against the plant pathogens Phoma betae and Colletotrichum acutatum, which points to a role of ThHog1 protein in fungus–fungus interactions.

Inhibition of aflatoxin B1 production of Aspergillus flavus, isolated from soybean seeds by certain natural plant products

Abstract: Aims: The inhibitory effect of cowdung fumes, Captan, leaf powder of Withania somnifera, Hyptis suaveolens, Eucalyptus citriodora, peel powder of Citrus sinensis, Citrus medica and Punica granatum, neem cake and pongamia cake and spore suspension of Trichoderma harzianum and Aspergillus niger on aflatoxin B1 production by toxigenic strain of Aspergillus flavus isolated from soybean seeds was investigated. Methods and Results: Soybean seed was treated with different natural products and fungicide captan and was inoculated with toxigenic strain of A. flavus and incubated for different periods. The results showed that all the treatments were effective in controlling aflatoxin B1 production. Captan, neem cake, spore suspension of T. harzianum, A. niger and combination of both reduced the level of aflatoxin B1 to a great extent. Leaf powder of W. somnifera, H. suaveolens, peel powder of C. sinensis, C. medica and pongamia cake also controlled the aflatoxin B1 production. Conclusions: All the natural product treatments applied were significantly effective in inhibiting aflatoxin B1 production on soybean seeds by A. flavus. Significance and Impact of the Study: These natural plant products may successfully replace chemical fungicides and provide an alternative method to protect soybean and other agricultural commodities from aflatoxin B1 production by A. flavus.

Use of Trichoderma harzianum and Trichoderma viride for the biological control of Meloidogyne incognita on tomato.

Abstract: Trichoderma harzianum and Trichoderma viride were tested for their capacity to reduce the incidence of the root-knot nematode Meloidogyne incognita on tomato. In vitro studies demonstrated that all tested isolates were effective in causing nematode mortality compared with the control. Trichoderma slightly reduced nematode damage to tomato in vivo. Treatment of the soil with the biocontrol agents before transplanting, improved control over treatment directly at transplanting. The Trichoderma isolates could not be re-isolated from the endorhiza, but were successfully re-isolated from the rhizosphere 45 days after fungal inoculation. Only slight increases in plant growth could be measured. The mutualistic endophyte F. oxysporum 162, used as positive control, was more effective in root-knot nematode biocontrol than the Trichoderma isolates.

Generation, annotation and analysis of ESTs from Trichoderma harzianum CECT 2413

Abstract: The filamentous fungus Trichoderma harzianum is used as biological control agent of several plant-pathogenic fungi. In order to study the genome of this fungus, a functional genomics project called "TrichoEST" was developed to give insights into genes involved in biological control activities using an approach based on the generation of expressed sequence tags (ESTs). Eight different cDNA libraries from T. harzianum strain CECT 2413 were constructed. Different growth conditions involving mainly different nutrient conditions and/or stresses were used. We here present the analysis of the 8,710 ESTs generated. A total of 3,478 unique sequences were identified of which 81.4% had sequence similarity with GenBank entries, using the BLASTX algorithm. Using the Gene Ontology hierarchy, we performed the annotation of 51.1% of the unique sequences and compared its distribution among the gene libraries. Additionally, the InterProScan algorithm was used in order to further characterize the sequences. The identification of the putatively secreted proteins was also carried out. Later, based on the EST abundance, we examined the highly expressed genes and a hydrophobin was identified as the gene expressed at the highest level. We compared our collection of ESTs with the previous collections obtained from Trichoderma species and we also compared our sequence set with different complete eukaryotic genomes from several animals, plants and fungi. Accordingly, the presence of similar sequences in different kingdoms was also studied. This EST collection and its annotation provide a significant resource for basic and applied research on T. harzianum, a fungus with a high biotechnological interest.

Development of formulations of Trichoderma harzianum strain M1 for control of damping-off of tomato caused by Pythium aphanidermatum

Abstract: A carbendazim resistant Trichoderma harzianum strain M1, inhibitory to the growth of the damping-off pathogen Pythium aphanidermatum was used for the development of new carrier formulations. Seven different formulations (talc, lignite, lignite + fly ash-based powder formulation, wettable powder, bentonite paste, polyethylene glycol-paste and gelatin-glycerin-gel) were developed for seed treatment. Shelf life of the formulations was evaluated under storage at 24°C up to nine months. The population of propagules was optimum in all the formulations up to three months of storage. Seed treatment with Trichoderma formulations reduced the incidence of damping-off disease of tomato up to 74% and also enhanced the plant biomass under greenhouse and field conditions. Active colonization of T. harzianum in the rhizosphere of tomato plants was observed following seed treatment with the formulations.

Honey bee dispersal of the biocontrol agent Trichoderma harzianum T39: effectiveness in suppressing Botrytis cinerea on strawberry under field conditions

Abstract: Botrytis cinerea, which causes grey mould, is a major pathogen of many crops. On strawberry, isolates of Trichoderma spp. can effectively control B. cinerea, but frequent application is necessary. Bees can be used to disseminate biological control agents to the target crop. We tested the ability of honey bees to disseminate Trichoderma harzianum T39 to control B. cinerea in strawberry in the field during the winter in Israel over two consecutive seasons. We used the recently developed ‘Triwaks’ dispenser for loading the bees with the T. harzianum inoculum. During both years, grey mould developed in late January in untreated control plots; at low to medium disease levels it was partially controlled by fungicide treatment, and was best controlled in bee-visited plots. At high disease levels neither chemical nor biological control was effective. To assess the spatial distribution of inoculum by bees, we sampled flowers up to 200 m from the hives and found effective levels of T. harzianum even at 200 m. The approach used in this study provides an effective control of grey mould in strawberry in conditions of low to medium grey mould incidence.

Antimicrobial activities of various essential oils against foodborne pathogenic or spoilage moulds

Abstract: The use of essential oils in the food industry, as natural sanitizing agents, requires the definition of optimal conditions. The aim of the present work was to evaluate some antimicrobial activity parameters as mycelial growth inhibition, minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) of six essential oils against Aspergillus niger, Aspergillus terreus,Chaetomium globosum, Penicillium chrysogenum, Penicillium pinophilum, Trichoderma harzianum and Trichoderma viride. The antimicrobial activity of essential oils was monitored by the macrodiluition technique. The mycelial growth inhibition, fungistatic and fungicidal concentrations were recorded for each strain that showed sensitivity to the essential oils. The essential oils of catnip, cinnamon, tea tree and thyme essential oils exhibited a large spectrum antimicrobial activities; those of clary sage and laurel inhibited the mycelial growth in a few fungal strains. The essential oils of cinnamon and thyme had the lowest MIC and MFC values against all the fungi assayed, followed by catnip, tea tree, clary sage and laurel. The use of these natural products rather than, the currently used antifungal chemicals, may be of interest given that: i) essential oils are of natural origin which means they are safer for human health and the environment and ii) there is less chance that the pathogenic microorganisms will develop resistance.

Colonization of the rhizosphere, rhizoplane and endorhiza of garlic (Allium sativum L.) by strains of Trichoderma harzianum and their capacity to control allium white-rot under field conditions

Abstract: Five strains (C4, C13, C17, C30, C44) of Trichoderma harzianum that efficiently antagonise Sclerotium cepivorum in vitro, were used in central Mexico to inoculate field grown garlic (Allium sativum L.). Endorhiza, rhizosphere and soil colonization were evaluated at different times and at various root and soil depths. Experiments were conducted in both the 2000 and 2001 crop cycles to evaluate the capacity of five strains as biological control agents (BCA) against S. cepivorum. Significant differences were observed among the strains in terms of their ecological behavior. Strain C4 showed the best endorhizal and rhizosphere colonization. Strain C44, besides being a good rhizosphere colonizer, dispersed widely in the soil. In terms of all three variables, strain C30 had poor colonization ability, whereas the strains C4, C17 and C44 were best able to control white rot in the field. Of these, strain C4 showed the best performance over 2 years when it was applied as a pre-colonized substrate at planting time. High early endorhizal colonization was positively correlated with the BCA capacity of the strains. These data suggest that the ecological behavior of Trichoderma strains should be included as a criterion for strain selection for biological control purposes.

Biocontrol agents in combination with Moringa oleifera extract for integrated control of Sclerotium- caused cowpea damping-off and stem rot

Abstract: Damping-off and stem rot disease-causing Sclerotium rolfsii has been reported as a destructive soil-borne pathogen of numerous crops, especially in the tropics and subtropics. Trials were conducted to test the efficacy of biocontrol agents alone or combined with Moringa oleifera leaf extracts for the control of the disease. In the laboratory, PDA was amended with Moringa leaf extract, and mycelial growth of S. rolfsii was measured. In the greenhouse and field, Trichoderma Kd 63, Trichoderma IITA 508 and Bacillus subtilis were evaluated as seed treatments, soil drench or sprinkle, separately or combined with Moringa leaf extracts. Percentage disease incidence, severity and control were recorded. In the laboratory, the higher the extract concentration the less the mycelial growth and no mycelial growth occurred on extract at 15 or 20 g leaves 10 ml )1 water. In the greenhouse, the highest disease control was observed at a Moringa extract concentration of 15 kg leaves 10 l )1 water (w/v). Seed treatments using Trichoderma Kd 63, and soil sprinkle using Trichoderma IITA 508 had a significantly (P = 0.05) higher effect on a disease incidence than Bacillus. Disease severity followed the same pattern. Moringa seed treatment combined with Trichoderma soil sprinkle resulted in significantly more than 94% and 70% disease control in the greenhouse and field, respectively, with significant yield increase in the field. This is the first report of Moringa leaf extract combined with Trichoderma as an integrated control for Sclerotium damping-off and stem rot of cowpea in the field.

Synthesis of enzymes connected with mycoparasitism by ectomycorrhizal fungi.

Abstract: The production of enzymes involved in mycoparasitism by several strains of ectomycorrhizal fungi: Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), S. bovinus (15-3), S. luteus (14-7) on different substrates such as colloidal chitin, mycelia of Trichoderma harzianum, T. virens and Mucor hiemalis was examined. Chitinases and β-1,3-glucanases were assayed spectrophotometrically by measuring the amount of reducing sugars releasing from suitable substrate by means of Miller's method. β-glucosidases were determined by measuring the amount of p-nitrophenol released from p-nitrophenyl-β-D-glucopyranoside. It was observed that A. muscaria (16-3) and L. laccata (9-12) biosynthesized the highest activity of enzymes in contrast to the strains of S. bovinus and S. luteus. The mycelium of T. harzianum turned out to be the best substrate for the induction of β-1,3-glucanases and β-glucosidases for both strains of L. laccata, although the difference in the induction of chitinases in the presence of mycelia of different species of Trichoderma was not indicated.

In vitro activity and synergism of amphotericin B, azoles and cationic antimicrobials against the emerging pathogen Trichoderma spp.

Abstract: 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 64 mg/L) and amphotericin B (MICs = 2 mg/L), whereas lower MICs of 0.5–1 mg/L were detected for voriconazole.

Sensitivity of Clonostachys rosea and Trichoderma spp. as Potential Biocontrol Agents to Pesticides

Abstract: Clonostachys rosea 47 (CR47), Trichoderma atroviride 59 (TA59), T. atroviride 312 (TA312), Trichoderma harzianum 24 (TH24), Trichoderma longibrachiatum 9 (TL9), T. longibrachiatum 144 (TL144) and Trichoderma viride 15 (TV15) were tested to evaluate their in vitro sensitivity towards five fungicides (carboxin, guazatine, prochloraz, thiram and triticonazole) and four herbicides (chlorsulfuron, chlorotoluron, flufenacet and pendimethalin). All antagonists showed low sensitivity to carboxin and thiram and high sensitivity to prochloraz. For mycelial radial growth, TV15 was highly sensitive to guazatine, prochloraz and triticonazole and TH24 moderately insensitive to carboxin, guazatine and thiram. For conidial germination TL144 was the most sensitive to the fungicides, for mycelial radial growth and conidial germination CR47 was the least sensitive. None of the antagonists showed any mycelial radial growth inhibition in presence of the herbicides at field dose, except for TL144. Most antagonists did not show any conidial germination inhibition by the herbicides. The in vitro toxicity of prochloraz, guazatine and triticonazole towards the antagonists was confirmed by light and scanning electron microscope showing hyphal disruptions and extrusion of cytoplasmic content. A mixture of CR47 and/or TA312 with carboxin, thiram and triticonazole, applied to wheat seeds, was able to control Fusarium culmorum artificially inoculated to wheat seedlings in growth chambers. In the field, the antagonists applied along with triticonazole or thiram, at 1/10 of the field dose to seeds naturally infected by F. culmorum, gave a disease control comparable to that induced by triticonazole at full field dose. Our results demonstrate how an integration of microorganisms with pesticides makes the control of wheat foot rot possible.

Evaluation of Trichoderma Isolates for Biological Control of Charcoal Stem Rot in Melon Caused by Macrophomina phaseolina

Abstract: Trichoderma harzianum (T39), T.virens (DAR74290), T.viride (MO), T. harzianum (M) and TrichderminB a commercial formulation of T. harzianum(Bi) were evaluated as potential biological agents for the control of charcoal stem rot in melon caused by Macrophomina phaseolina. Cell-free metabolites of T. harzianum(M), T. harzianum (T39) and T. virens (DAR 74290) inhibited growth of M. phaseolina completely in vitro and appeared to be fungicidal. T. viride (MO) inhibited fungal growth from 34.9% to 71%. T. harzianum (T39), T. harzianum (M), T. virens (DAR 74290), T. viride(MO) and T. harzianum (Bi) were tested for their ability to protect melon plants from disease caused by M. phaseolina in a glasshouse experiment. The percentage of stand plants with the antagonist alone or in combination with the pathogen was significantly (p<0.05) greater than in plants inoculated with the pathogen alone. The percentages for the stand plants in treatments were as follows: T. harzianum (T39), T. harzianum (M), T. virens (DAR 74290), M. phaseolina, M. phaseolina + T. virens (DAR 74290), M. phaseolina + T. harzianum (T39) and M. phaseolina + T. harzianum (M),at 95, 100, 97.5, 15, 64.25, 75.25 and 47.55 percent respectively. The percentage for the stand plant in the commercial Trichodermin B + M. phaseolina treatment (96.7%) was greater than for M. phaseolina alone (46.7%) and demonstrated the best result in the control of charcoal stem rot in melon.