Showing papers on "Trichoderma harzianum published in 2005"

Cloning of Beauveria bassiana chitinase gene Bbchit1 and its application to improve fungal strain virulence.

Abstract: Entomopathogenic fungi can produce a series of chitinases, some of which act synergistically with proteases to degrade insect cuticle. However, chitinase involvement in insect fungus pathogenesis has not been fully characterized. In this paper, an endochitinase, Bbchit1, was purified to homogeneity from liquid cultures of Beauveria bassiana grown in a medium containing colloidal chitin. Bbchit1 had a molecular mass of about 33 kDa and pI of 5.4. Based on the N-terminal amino acid sequence, the chitinase gene, Bbchit1, and its upstream regulatory sequence were cloned. Bbchit1 was intronless, and there was a single copy in B. bassiana. Its regulatory sequence contained putative CreA/Crel carbon catabolic repressor binding domains, which was consistent with glucose suppression of Bbchit1. At the amino acid level, Bbchit1 showed significant similarity to a Streptomyces avermitilis putative endochitinase, a Streptomyces coelicolor putative chitinase, and Trichoderma harzianum endochitinase Chit36Y. However, Bbchit1 had very low levels of identity to other chitinase genes previously isolated from entomopathogenic fungi, indicating that Bbchit1 was a novel chitinase gene from an insect-pathogenic fungus. A gpd-Bbchit1 construct, in which Bbchit1 was driven by the Aspergiullus nidulans constitutive promoter, was transformed into the genome of B. bassiana, and three transformants that overproduced Bbchit1 were obtained. Insect bioassays revealed that overproduction of Bbchit1 enhanced the virulence of B. bassiana for aphids, as indicated by significantly lower 50% lethal concentrations and 50% lethal times of the transformants compared to the values for the wild-type strain.

Enhancing biological control of basal stem rot disease (Ganoderma boninense) in oil palm plantations

Abstract: Basal Stem Rot (BSR) disease caused by Ganoderma boninense is the most destructive disease in oil palm, especially in Indonesia and Malaysia. The available control measures for BSR disease such as cultural practices and mechanical and chemical treatment have not proved satisfactory due to the fact that Ganoderma has various resting stages such as melanised mycelium, basidiospores and pseudosclerotia. Alternative control measures to overcome the Ganoderma problem are focused on the use of biological control agents and planting resistant material. Present studies conducted at Indonesian Oil Palm Research Institute (IOPRI) are focused on enhancing the use of biological control agents for Ganoderma. These activities include screening biological agents from the oil palm rhizosphere in order to evaluate their effectiveness as biological agents in glasshouse and field trials, testing their antagonistic activities in large scale experiments and eradicating potential disease inoculum with biological agents. Several promising biological agents have been isolated, mainly Trichoderma harzianum, T. viride, Gliocladium viride, Pseudomonas fluorescens, and Bacillus sp. A glasshouse and field trial for Ganoderma control indicated that treatment with T. harzianum and G. viride was superior to Bacillus sp. A large scale trial showed that the disease incidence was lower in a field treated with biological agents than in untreated fields. In a short term programme, research activities at IOPRI are currently focusing on selecting fungi that can completely degrade plant material in order to eradicate inoculum. Digging holes around the palm bole and adding empty fruit bunches have been investigated as ways to stimulate biological agents.

Variability of non-mutualistic filamentous fungi associated with Atta sexdens rubropilosa nests.

Abstract: A survey of the filamentous fungi other than the symbiotic one found in association withAtta sexdens rubropilosa colonies was carried out. Different fungal species (27 taxa) were isolated a few days after treating the workers with toxic baits (sulfluramid; Mirex-S®), from 40 laboratory and 20 field nests.Syncephalastrum racemosum (54 %) andEscovopsis weberi (21 %),Trichoderma harzianum (38 %) andFusarium oxysporum (23 %) were the prevalent species in laboratory and field nests, respectively.Acremonium kiliense, Acremonium strictum, E. weberi, F. oxysporum, Fusarium solani, Moniliella suaveolens andT. harzianum were found in both nests’ groups. We revealed that many filamentous fungi can co-exist in a dormant state inside the nests of these insects and some of them appear to be tightly associated with this environment.

Production of Cellulase from Oil Palm Biomass as Substrate by Solid State Bioconversion

Abstract: Solid state bioconversion (SSB) of lignocellulosic material oil palm biomass (OPB) generated from palm oil industries as waste was conducted by evaluating the enzyme production through filamentous fungus in lab-scale experiment. OPB in the form of empty fruit bunches (EFB) was used as the solid substrate and treated with the fungus Trichoderma harzianum to produce cellulase. The results presented in this study revealed that the higher cellulase activity of 0.0413 unit was achieved at the day 3 of fermentation. While the optimum study indicated the enzyme production of 0.0433 unit with moisture content of 50%, 0.0413 unit with 5% v/w of inoculum size and 0.0413 unit with co-substrate concentration of 2% (w/w) at days 9, 9 and 12 of fungal treatment, respectively. The parameters glucosamine and reducing sugar were observed to evaluate the growth and substrate utilization in the experiment.

Xylanase Production from Trichoderma harzianum 1073 D3 with Alternative Carbon and Nitrogen Sources

Abstract: Summary The effect of some natural wastes (orange pomace, orange peel, lemon pomace, lemon peel, apple pomace, pear peel, banana peel, melon peel and hazelnut shell) on the production of xylanase from Trichoderma harzianum 1073 D3 has been studied and maximum activity has been observed on melon peel (26.5 U/mg of protein) followed by apple pomace and hazelnut shell. Also, molasses could be used as an additional carbon source as it decreased the production time approximately by 50 %. Finally, potential alternatives of organic nitrogen source (cotton leaf and soybean residue wastes) were analyzed and it was concluded that peptone could be replaced with these residues especially when economics of the process is the major objective.

Protection of grapevine pruning wounds from infection by Eutypa lata using Trichoderma harzianum and Fusarium lateritium

Abstract: Trichoderma harzianum applied to grapevine pruning wounds in a spore suspension and in the commercial formulations of Trichoseal, Trichoseal spray and Vinevax pruning wound dressing reduced recovery of Eutypa lata in the glasshouse and in the field. Recovery of E. lata was significantly reduced (P < 0.001) when fresh wounds were treated with viable T. harzianum 2 or 7 days before inoculation with ascospores of the pathogen in the glasshouse. In field experiments, recovery of E. lata was significantly reduced (P < 0.001) when fresh pruning wounds were treated with spores of T. harzianum, Fusarium lateritium or Vinevax 1 or 14 days before ascospores were applied. In general, a delay of 14 days between wounding and inoculation with ascospores of E. lata reduced recovery of the pathogen compared with inoculation on the day after wounding.

Symposium-in-Print: Ultraviolet Radiation and Terrestrial Ecosystems The Use of Wavelength-selective Plastic Cladding Materials in Horticulture: Understanding of Crop and Fungal Responses Through the Assessment of Biological Spectral Weighting Functions

Abstract: Plant responses to light spectral quality can be exploited to deliver a range of agronomically desirable end points in protected crops. This can be achieved using plastics with specific spectral properties as crop covers. We have studied the responses of a range of crops to plastics that have either (a) increased transmission of UV compared with standard horticultural covers, (b) decreased transmission of UV or (c) increased the ratio of red (R) : far-red (FR) radiation. Both the UV-transparent and R : FR increasing films reduced leaf area and biomass, offering potential alternatives to chemical growth regulators. The UV-opaque film increased growth, but while this may be useful in some crops, there were tradeoffs with elements of quality, such as pigmentation and taste. UV manipulation may also influence disease control. Increasing UV inhibited not only the pathogenic fungus Botrytis cinerea but also the disease biocontrol agent Trichoderma harzianum .U nlikeB. cinerea, T. harzianum was highly sensitive to UV-A radiation. These fungal responses and those for plant growth in the growth room and the field under different plastics are analyzed in terms of alternative biological spectral weighting functions (BSWF). The role of BSWF in assessing general patterns of response to UV modification in horticulture is also discussed.

The Use of Wavelength-selective Plastic Cladding Materials in Horticulture: Understanding of Crop and Fungal Responses Through the Assessment of Biological Spectral Weighting Functions

Abstract: Plant responses to light spectral quality can be exploited to deliver a range of agronomically desirable end points in protected crops This can be achieved using plastics with specific spectral properties as crop covers We have studied the responses of a range of crops to plastics that have either (a) increased transmission of UV compared with standard horticultural covers, (b) decreased transmission of UV or (c) increased the ratio of red (R) : far-red (FR) radiation Both the UV-transparent and R : FR increasing films reduced leaf area and biomass, offering potential alternatives to chemical growth regulators The UV-opaque film increased growth, but while this may be useful in some crops, there were tradeoffs with elements of quality, such as pigmentation and taste UV manipulation may also influence disease control Increasing UV inhibited not only the pathogenic fungus Botrytis cinerea but also the disease biocontrol agent Trichoderma harzianum Unlike B cinerea, T harzianum was highly sensitive to UV-A radiation These fungal responses and those for plant growth in the growth room and the field under different plastics are analyzed in terms of alternative biological spectral weighting functions (BSWF) The role of BSWF in assessing general patterns of response to UV modification in horticulture is also discussed

Single and double inoculation with Azospirillum/Trichoderma: the effects on dry bean and wheat

Abstract: A plant growth-promoting rhizobacterium (Azospirillum brasilense Sp7) and a bio-control fungus, which can solubilize insoluble phosphorus (Trichoderma harzianum Rifai 1295-22), were evaluated for their single and combined effects on dry bean (Phaseolus vulgaris) and wheat (Triticum aestivum L.) grown in soil. A pot experiment with bean and a field experiment with both bean and wheat were established. In contrast to single inoculation of Trichoderma, the single inoculation of Azospirillum and the double inoculation did not significantly (P >0.05) increase nodule numbers and nodule mass at 45 days after planting in pot grown beans. However, the Azospirillum inoculation with supplementary phosphorus significantly (P 0.05) differences among the inoculation treatments for plant dry weight, total plant nitrogen, and total plant phosphorus at 45 days after planting in both pot and field experiments with bean. However, the combined inoculation and rock phosphate application at 1 Mg ha−1 significantly (P 0.05). The combined inoculation improves many plant and yield parameters and, therefore, has some advantages over single inoculation provided that rock phosphate was supplied at an amount not exceeding 1 Mg ha−1. Higher rock phosphate application rates decreased many plant and yield parameters in our study.

Specific PCR Assays for the Detection and Quantification of DNA from the Biocontrol Strain Trichoderma harzianum 2413 in Soil

Abstract: Strain identification in situ is an important factor in the monitoring of microorganisms used in the field. In this study, we demonstrated the use of sequence-characterized amplified region (SCAR) markers to detect genomic DNA from Trichoderma harzianum 2413 from soil. Two primers (SCAR A1/SCAR A1c) were tested against DNA of 27 isolates of Trichoderma spp. and amplified a 990-bp fragment from T. atroviride 11 and a 1.5-kb fragment from T. harzianum 2413, using an annealing temperature of 68°C. These fragments showed no significant homology to any sequence deposited in the databases. The primer pair, BR1 and BR2, was designed to the 1.5-kb fragment amplified from T. harzianum 2413, generating a SCAR marker. To test the specificity of these primers, experiments were conducted using the DNA from 27 Trichoderma spp. strains and 22 field soil samples obtained from four different countries. PCR results showed that BR1 and BR2 amplified an 837-bp fragment unique to T. harzianum 2413. Assays in which total DNA was extracted from sterile and nonsterile soil samples, inoculated with spore or mycelium combinations of Trichoderma spp. strains, indicated that the BR1 and BR2 primers could specifically detect T. harzianum 2413 in a pool of mixed DNA. No other soil-microorganisms containing these sequences were amplified using these primers. To test whether the 837-bp SCAR marker of T. harzianum 2413 could be used in real-time PCR experiments, new primers (Q2413f and Q2413r) conjugated with a TaqMan fluorogenic probe were designed. Real-time PCR assays were applied using DNA from sterile and nonsterile soil samples inoculated with a known quantity of spores of Trichoderma spp. strains.

Effet inhibiteur in vitro et in vivo du Trichoderma harzianum sur Fusarium oxysporum f. sp. radicis-lycopersici

Abstract: Les essais de confrontation directe, sur milieu de culture ou a distance, entre Fusarium oxysporum f. sp. radicis-lycopersici et Trichoderma harzianum ont revele que ce dernier a pu inhiber la croissance mycelienne du F. oxysporum f. sp. radicis-lycopersici de plus de 65 % par rapport au temoin et ce apres quatre jours d’incubation a 25 °C. De plus, au dela de cette periode et au terme de six jours, le T. harzianum envahit les colonies de F. oxysporum f. sp. radicis-lycopersici sur lesquelles il sporule meme, revelant ainsi son pouvoir hautement myco-parasitaire. Des resultats interessants ont egalement ete obtenus in vivo : en effet, le repiquage des plants de tomate dans un melange de perlite inoculee par une suspension sporale de F. oxysporum f. sp. radicis-lycopersici et de T. harzianum a reduit l’incidence de la fusariose des racines et du collet comparativement aux plants repiques uniquement dans de la perlite inoculee par le pathogene. Les plants repiques dans de la perlite contenant le pathogene et l’antagoniste etudie, ne montrent aucune difference comparativement a ceux du temoin sain (non inocule et non traite) ; mieux encore, les plants traites par le T. harzianum presentent une croissance vegetative meilleure et un systeme racinaire vigoureux.

Investigation of factors affecting xylanase activity from Trichoderma harzianum 1073 D3

Abstract: In this study, some physiological conditions affecting the activity of xylanase enzyme produced from Trichoderma harzianum 1073 D3 were determined. In addition, stabilization of pH and temperature in liquid and semi-solid state cultivation media were investigated. It was concluded that for maximum xylanase activity, incubation at 60°C in an enzyme incubation medium with pH 5 that contained 1 % xylan was appropriate. The stability studies showed that the enzyme was relatively stable in the pH range 3-7 and retained more than 50 % of its original activity after four months.

6‐pentyl‐α‐pyrone production by Trichoderma harzianum: The influence of energy dissipation rate and its implications on fungal physiology

Abstract: The influence of the agitation conditions on biomass growth, morphology, carbon metabolism, viability, and 6-pentyl-alpha-pyrone (6PP) production by Trichoderma harzianum were studied in an extractive fermentation system. Batch spore-inoculated cultures developed at dissolved oxygen concentrations above 35% of air saturation were carried out in a 14 L bioreactor. The effect of energy dissipation rate over culture performance was assessed using two sets of three Rushton turbines (having different diameters) operated at different agitation speeds. Higher mechanical stress enhanced cellular differentiation (i.e., sporulation), while yielding lower specific growth rates and increased specific CO(2) production rates (CPRs) at relatively constant specific glucose consumption rates. In addition, fungal viability and clump mean diameter decreased gradually at higher energy dissipation rates. 6PP biosynthesis was growth associated and its specific productivity showed a bell-shaped relationship with the energy dissipation rate. T. harzianum physiology was, therefore, strongly influenced by the prevailing hydrodynamic conditions as it triggered cellular metabolism and differentiation shifts.

Studies on the management of root-knot nematode, Meloidogyne incognita-wilt fungus, Fusarium oxysporum disease complex of green gram, Vigna radiata cv ML-1108.

Abstract: Studies were conducted under pot conditions to determine the comparative efficacy of carbofuran at 1 mg a.i./kg soil, bavistin at 1 mg a.i./kg soil, neem (Azadirachta indica) seed powder at 50 mg/kg soil, green mould (Trichoderma harzianum) at 50.0 ml/kg soil, rhizobacteria (Pseudomonas fluorescens) at 50.0 ml/kg soil against root-knot nematode, Meloidogyne incognita-wilt fungus, Fusarium oxysporum disease complex on green gram, Vigna radiata cv ML-1108. All the treatments significantly improved the growth of the plants as compared to untreated inoculated plants. Analysis of data showed that carbofuran and A. indica seed powder increased plant growth and yield significantly more in comparison to bavistin and P. fluorescens. Carbofuran was highly effective against nematode, bavistin against fungus, A. indica seed powder against both the pathogens and both the bioagents were moderately effective against both the pathogens.

BGN16.3, a novel acidic β-1,6-glucanase from mycoparasitic fungus Trichoderma harzianum CECT 2413

Abstract: A new component of the β-1,6-glucanase (EC 3.2.1.75) multienzymatic complex secreted by Trichoderma harzianum has been identified and fully characterized. The protein, namely BGN16.3, is the third isozyme displaying endo-β-1,6-glucanase activity described up to now in T. harzianum CECT 2413. BGN16.3 is an acidic β-1,6-glucanase that is specifically induced by the presence of fungal cell walls in T. harzianum growth media. The protein was purified to electrophoretical homogenity using its affinity to β-1,6-glucan as first purification step, followed by chomatofocusing and gel filtration. BGN16.3 has a molecular mass of 46 kDa in SDS/PAGE and a pI of 4.5. The enzyme only showed activity against substrates with β-1,6-glycosidic linkages, and it has an endohydrolytic mode of action as shown by HPLC analysis of the products of pustulan hydrolysis. The expression profile analysis of BGN16.3 showed a carbon source control of the accumulation of the enzyme, which is fast and strongly induced by fungal cell walls, a condition often regarded as mycoparasitic simulation. The likely involvement β-1,6-glucanases in this process is discussed.

Antagonistic potential of Trichoderma spp. against Rhizoctonia solani and use of M13 microsatellite-primed PCR to evaluate the antagonist genetic variation

Abstract: Rhizoctonia solani is the most important pathogen involved in cotton seedling disease in Egypt. Variations in the antagonistic activity of 20 isolates belonging to Trichoderma harzianum and T. longi-brachiatum were evaluated in vitro, against three isolates of Rhizoctonia solani in dual culture. There was highly significant interaction between isolates of Trichoderma and R. solani. All isolates of Trichoderrma spp. showed varying levels of antagonism against R. solani. Similarly, the interaction between Trichoderma isolates and R. solani was a highly significant source of variation in surviving seedlings under greenhouse conditions. In vitro and in vivo antagonism were not correlated, which indicated that, in vitro inhibition of the pathogen was not predictive of in vivo biocontrol by an individual antagonist. Microsatellite-primed PCR analysis was used to evaluate the genetic variation among Trichoderma isolates. The analysis divided the isolates into two main groups. Grouping the isolates based on cluster analysis of their DNA profiles matched that based on their morphological taxonomy. However, no congruency was found between cluster analyses obtained by PCR and cluster analysis by efficiency parameters of the biocontrol agents.

Seed-borne mycoflora of sunflower (helianthus annuus l.)

Abstract: Using standard blotter and deep-freezing techniques, seed-borne mycoflora of 35 samples of sunflower (Helianthus annuus L.) were studied. Acremonium fusidioides, Arthrobotrys oligospora, Aspergillus ochraceus, Bipolaris bisepta, Cephaliophora tropica, Chaetomium spinosum, Cladobotryum varium, Cladosporium cladosporioides, Emericella nidulans, Gonatobotrys simplex, Humicola grisea, Memnoniella echinata, Mucor mucedo, Myrothecium verrucaria, Phialophora verrucosa and Syncephalastrum racemosum were found to be new seed-borne fungal species on sunflower. Absidia corymbifera, Alternaria alternata, Aspergillus flavus, A. niger, A. terreus, Chaetomium bostrychodes, C. globosum, Emericella nidulans, Fusarium pallidoroseum, F. solani, Macrophomina phaseolina, Penicillium spp., Rhizoctonia solani and Rhizopus stolonifer were predominantly isolated by both techniques. During seed component plating, Aspergillus awamori, A. ustus and Exerohilum halodes were found to be new reported species. Macrophomina phaseolina, Rhizoctonia solani and Trichoderma harzianum were isolated from all component parts, whereas, Fusarium solani was isolated only from cotyledons and axis.

Synergistic effects of Trichoshield on enhancement of growth and resistance to downy mildew in pearl millet

Abstract: Trichoshield, a talc formulation consisting of spores of Trichoderma harzianum, Trichoderma lignorum, Gliocladium virens and Bacillus subtilis was tested, following different application methods, for its ability to promote growth of pearl millet plants and to induce resistance to downy mildew of pearl millet. Under laboratory conditions, trichoshield seed treatment enhanced seed germination and seedling vigor of pearl millet significantly over the control; under greenhouse conditions vegetative growth parameters like height, fresh and dry weight, leaf area and number of tillers were significantly enhanced over the control: Trichoshield formulation offered greater protection against downy mildew in comparison with individual strains of T. harzianum, T. lignorum, G. virensand B. subtilis. Among the methods of application, foliar spray was found to be a more efficient delivery method than seed treatment or slurry treatment. Combinations of foliar spray with seed treatment and slurry treatment produced the same effect as foliar spray alone. Under field conditions, trichoshield treatment enhanced reproductive parameters like number of earheads, length and girth of earheads, 1000 seed weight and yield significantly over the control. Days required for 50% flowering was reduced by 4 days compared to the control. Yield enhancement of 28% over the control was highly significant. Trichoshield treatment offered protection ranging from 52 to 71% under field conditions, depending on the application method. However, the chemical fungicide metalaxyl Apron provided the highest protection against downy mildew, both under greenhouse and field conditions.

The chemistry of the bio-control agent, Trichoderma harzianum.

Abstract: The role of 6-n-pentyl-2H-pyran-2-one and other metabolites of the fungus Trichoderma harzianum, in its application as a bio-control agent, is discussed.

Proteins Related to the Biocontrol of Pythium Damping‐off in Maize with Trichoderma harzianum Rifai

Abstract: Induced resistance has been evidenced as one of mechanisms of Trichoderma to control plant diseases, however, no study showed the change of host proteomics in Trichoderma-induced resistance of maize against damping-off caused by Pythium ultimum Trow. The mechanism of Trichoderma harzianum Rifai for controlling maize seedling disease caused by Pythium ultimum Trow was investigated firstly by proteome technique and the result suggested that T. harzianum strain T22 was not only able to promote seedling growth but also protein accumulation. One-dimensional electrophoresis assay showed that more bands appeared on the gel with T22 or T22 combined with P. ultimum (T22 +P. ultimum) treatment than with other treatments. Enzyme assay showed that two chitinases of the root sample were more activated in the treatments with T22 than in the other treatments without T22. Proteins in the seedling roots from the various treatments were separated through protein extraction and 2-D electrophoresis technique. In the seedlings produced from the T22-treated seeds, there were 104 up-regulated proteins and 164 down-regulated proteins relative to the control, and 97 and 150, respectively, after treatment with T22 +P. ultimum; however, with P. ultimum alone the values were much lower than with the other two treatments. The correlation coefficient values were 0.72, 0.51 and 0.49 for the comparison of protein spot distribution on gel among control with T22, P. ultimum and T22 +P. ultimum, respectively. So it seemed that P. ultimum infection was more effective than T22 in interfering with the host proteome profile. Furthermore, analysis with MALDI-TOF-MAS showed that some important proteins associated with defensive reactions were identified in T22 or T22 +P. ultimum treatments, including endochitinase, pathogenesis-related protein PRMS (pathogenesis-related maize seed), GTP-binding protein, isoflavone reductase and other proteins related to respiration. All those proteins are probably part of the network of resistance or development-related proteins. Interestingly, P. ultimum treatment resulted in elimination of pathogenesis-related protein PRMS on gel, and therefore damping-off could be in part attributed to inhibition of the expression of this protein by P. ultimum infection. Some unknown proteins are also related to the defensive reaction of the host. (Managing editor: Li-Hui ZHAO)