Showing papers on "Trichoderma harzianum published in 2009"

Harzianic acid, an antifungal and plant growth promoting metabolite from Trichoderma harzianum

Abstract: A Trichoderma harzianum strain, isolated from composted hardwood bark in Western Australia, was found to produce a metabolite with antifungal and plant growth promoting activity The structure and absolute configuration of the fungal compound, harzianic acid (1), were determined by X-ray diffraction studies Harzianic acid showed antibiotic activity against Pythium irregulare, Sclerotinia sclerotiorum, and Rhizoctonia solani A plant growth promotion effect was observed at low concentrations of 1

The ThPG1 Endopolygalacturonase Is Required for the Trichoderma harzianum-Plant Beneficial Interaction

Abstract: Considering the complexity of the in vivo interactions established by a mycoparasitic biocontrol agent at the plant rhizosphere, proteomic, genomic, and transcriptomic approaches were used to study a novel Trichoderma gene coding for a plant cell wall (PCW)-degrading enzyme. A proteome analysis, using a three-component (Trichoderma spp.–tomato plantlets–pathogen) system, allowed us to identify a differentially expressed Trichoderma harzianum endopolygalacturonase (endoPG). Spot 0303 remarkably increased only in the presence of the soilborne pathogens Rhizoctonia solani and Pythium ultimum, and corresponded to an expressed sequence tag from a T. harzianum T34 cDNA library that was constructed in the presence of PCW polymers and used to isolate the Thpg1 gene. Compared with the wild-type strain, Thpg1-silenced transformants showed lower PG activity, less growth on pectin medium, and reduced capability to colonize tomato roots. These results were combined with microarray comparative data from the transcriptome of Arabidopsis plants inoculated with the wild type or a Thpg1-silenced transformant (ePG5). The endoPG-encoding gene was found to be required for active root colonization and plant defense induction by T. harzianum T34. In vivo assays showed that Botrytis cinerea leaf necrotic lesions were slightly smaller in plants colonized by ePG5, although no statistically significant differences were observed.

Factors affecting the production of Trichoderma harzianum secondary metabolites during the interaction with different plant pathogens

Abstract: Aims: Strains of Trichoderma spp. produce numerous bioactive secondary metabolites. The in vitro production and antibiotic activities of the major compounds synthesized by Trichoderma harzianum strains T22 and T39 against Leptosphaeria maculans, Phytophthora cinnamomi and Botrytis cinerea were evaluated. Moreover, the eliciting effect of viable or nonviable biomasses of Rhizoctonia solani, Pythium ultimum or B. cinerea on the in vitro production of these metabolites was also investigated. Methods and Results: T22azaphilone, 1-hydroxy-3-methyl-anthraquinone, 1,8-dihydroxy-3-methyl-anthraquinone, T39butenolide, harzianolide, harzianopyridone were purified, characterized and used as standards. In antifungal assays, T22azaphilone and harzianopyridone inhibited the growth of the pathogens tested even at low doses (1–10 μg per plug), while high concentrations of T39butenolide and harzianolide were needed (>100 μg per plug) for inhibition. The in vitro accumulation of these metabolites was quantified by LC/MS. T22azaphilone production was not enhanced by the presence of the tested pathogens, despite its antibiotic activity. On the other hand, the anthraquinones, which showed no pathogen inhibition, were stimulated by the presence of P. ultimum. The production of T39butenolide was significantly enhanced by co-cultivation with R. solani or B. cinerea. Similarly, viable and nonviable biomasses of R. solani or B. cinerea increased the accumulation of harzianopyridone. Finally, harzianolide was not detected in any of the interactions examined. Conclusions: The secondary metabolites analysed in this study showed different levels of antibiotic activity. Their production in vitro varied in relation to: (i) the specific compound; (ii) the phytopathogen used for the elicitation; (iii) the viability of the elicitor; and (iv) the balance between elicited biosynthesis and biotransformation rates. Significance and Impact of the Study: The use of cultures of phytopathogens to enhance yields of Trichoderma metabolites could improve the production and application of novel biopesticides and biofertilizers based on the active compounds instead of the living microbe. This could have a significant beneficial impact on the management of diseases in crop plants.

Production and purification of cellulose- degrading enzymes from a filamentous fungus trichoderma harzianum

Abstract: The major components of plant cell walls are cellulose, hemicellulose and lignin, with cellulose being the most abundant component. Cellulose degrading-enzymes provide a key opportunity for achieving tremendous benefits of biomass utilization. Trichoderma harzianum, a filamentous fungus, produces cellulose degrading enzymes and has been exploited by the industry. In this study, three cellulases, exoglucanase (EXG), endoglucanase (EG) and β-glucosidase (BGL) were partially purified from T. harzianum. The optimal pH, temperature and incubation time for cellulases production was found to be 5.5, 28 o C and 120 h respectively. To promote cellulases production, we evaluated the effects of glucose, carboxymethylcellulose (CMC), corn cobs, birchwood xylan and wheat bran as carbon sources for cellulase production. The results showed that CMC induced cellulases production whereas glucose repressed the synthesis of cellulases. The fungus was cultivated with 1% CMC for 120 h at 28oC, pH 5.5 and the resulting culture filtrate was used for cellulase purification. EXG, EG and BGL were partially purified from culture filtrate of fungus by Ammonium sulfate precipitation followed by gel filtration chromatography on Sephadex G200 and on Sephadex G50. After final purification step specific activities (IU/mg -1 ) of the enzymes were; EXG: 49.22, EG: 0.63 and BGL: 0.35 with 21.87-, 7.15- and 1.74- fold purification, respectively.

Suppression of Fusarium wilt of watermelon by a bio-organic fertilizer containing combinations of antagonistic microorganisms

Abstract: Fusarium wilt of watermelon commonly occurs in locations where the crop has been grown for many seasons. Its occurrence results in a severely decreased watermelon crop. The goal of this study was to assess the capability of a new product (bio-organic fertilizer) to control the wilt in Fusarium-infested soil. Pot experiments were conducted under growth chamber and greenhouse conditions. The results showed that the fertilizer controlled the wilt disease. Compared with control pots, the incidence rates of Fusarium wilt at 27 and 63 days following treatment of the plants with the bio-organic fertilizer at a rate of 0.5% (organic fertilizer + antagonistic microorganisms, including 3 × 109 CFU g−1 Paenibacillus polymyxa and 5 × 107 CFU g−1 Trichoderma harzianum) were reduced by 84.9 and 75.0%, respectively, in both the growth chamber and greenhouse settings. The activities of antioxidases (catalase, superoxide dismutase and peroxidase) in watermelon leaves increased by 38.9, 150 and 250%, respectively. In the roots, stems and leaves, the activity of β-1,3-glucanase (pathogenesis-related proteins) increased by 80, 1140 and 100% and that of chitinase increased by 240, 80, and 20%, respectively, while the contents of malondialdehyde fell by 56.8, 42.1 and 45.9%, respectively. These results indicate that this new fertilizer formula is capable of protecting watermelon from Fusarium oxysporum f.sp. niveum. The elevated levels of defense-related enzymes are consistent with the induction and enhancement of systemic acquired resistance of plant.

Effect of some rare earth elements on the growth and lanthanide accumulation in different Trichoderma strains

Abstract: Accumulation of rare earth elements (REE) in the soil may be due to the use of REE enriched fertilizers and to contamination by REE containing wastes. Although widely used in China for soil and foliar dressing of crops, little is known about the effect of REE applications on the soil microbial community. The effect of REE on the growth of biological control strains of Trichoderma atroviride and Trichoderma harzianum was investigated in vitro using either a mix of different REE containing different amounts of lanthanum, cerium, praseodymium, neodymium, gadolinium nitrate and lanthanum nitrate alone in comparison to treatments with potassium nitrate and water. In plate tests applied concentrations ranged from 0.1 mM to 300 mM for lanthanum and REE mix and from 0.1 mM to 900 mM for the potassium solution. In liquid culture tests applied concentrations ranged from 0.001 mM to 100 mM for lanthanum and REE mix and from 0.003 mM to 900 mM for the potassium solution. ICP-MS, TEM and TEM X-ray microanalysis were used to study the accumulation of REE in fungal biomass. All the Trichoderma strains showed a good tolerance to the presence of REE in the culture media. Some growth enhancing effects were observed in liquid cultures of T. harzianum strains but not in T. atroviride. Accumulation of REE in fungal biomass, both at intracellular level and in the extracellular matrix, was observed.

Interactions between arbuscular mycorrhizal fungi and Trichoderma harzianum and their effects on Fusarium wilt in melon plants grown in seedling nurseries

Abstract: BACKGROUND: Biological control through the use ofTrichoderma spp. and arbuscular mycorrhizal fungi (AMF) could contribute to a reduction of the inputs of environmentally damaging agrochemical products. The objective of this study was to evaluate the interactions between four AMF (Glomus intraradices, Glomus mosseae, Glomus claroideum and Glomus constrictum )a nd Trichodermaharzianum for their effects on melon plant growth and biocontrol of Fusarium wilt in seedling nurseries. RESULTS: AMF colonisation decreased fresh plant weight, which was unaffected by the presence of T. harzianum. Dual inoculation resulted in a decrease in fresh weight compared with AMF-inoculated plants, except for G. intraradices .A MF colonisation level varied with the AM endophyte and was increased by T. harzianum ,e xcept inG. mosseae-inoculated plants. Negative effects of AMF on T. harzianum colony-forming units were found, except with G. intraradices. AMF alone were less effective than T.harzianum in suppressing disease development. Combined inoculation resulted in a general synergistic effect on disease control. CONCLUSION: Selection of the appropriate AMF species and its combination with T. harzianum were significant both in the formation and effectiveness of AM symbiosis and the reduction of Fusarium wilt incidence in melon plants. The combination of G.intraradices andT.harzianum provided better results than any other tested. c � 2009 Society of Chemical Industry

Morphological, biochemical and molecular characterization of Trichoderma harzianum isolates for their efficacy as biocontrol agents.

Abstract: The random amplified polymorphic DNA (RAPD) procedure was used to examine the genetic variability among 8 isolates of Trichoderma harzianum, and their ability to antagonize Sclerotium rolfsii using a dual culture assay was correlated with RAPD profiles. Eight oligodeoxynucleotide primers were selected for the RAPD assays, which resulted in 86 bands for 8 isolates of T. harzianum. The data were entered into a binary matrix and a similarity matrix was constructed using the DICE similarity (SD) index. An unweighted pair grouping mathematical averaging (UPGMA) cluster based on SD values was generated using the NTSYS computer program. A mean coefficient of similarity obtained for pairwise comparisons was c. 30% and it showed that the variability among the isolates of T. harzianum was very high. Using the dual culture method in antagonism experiments, the T. harzianum isolates were classified in to antagonism classes. Further, T. harzianum isolates were screened for chitinase and β-1,3-glucanase activity. RAPD was efficient in demonstrating the high intraspecific genetic variation among isolates. The dendrogram did not show the grouping of isolates by their level of antagonism. Relationship among polymorphism existent, the aggressiveness and the origin of isolates were not found.

Trichoderma harzianum elicits defence response genes in roots of potato plantlets challenged by Rhizoctonia solani

Abstract: Biological control of Rhizoctonia solani with Trichoderma harzianum has been demonstrated in several studies. However, none have reported the dynamics of expression of defence response genes. Here we investigated the expression of these genes in potato roots challenged by R. solani in the presence/absence of T. harzianum Rifai MUCL 29707. Analysis of gene expression revealed an induction of PR1 at 168 h post-inoculation (hpi) and PAL at 96 hpi in the plants inoculated with T. harzianum Rifai MUCL 29707, an induction of PR1, PR2 and PAL at 48 hpi in the plants inoculated with R. solani and an induction of Lox at 24 hpi and PR1, PR2, PAL and GST1 at 72 hpi in the plants inoculated with both organisms. These results suggest that in the presence of T. harzianum Rifai MUCL 29707, the expression of Lox and GST1 genes are primed in potato plantlets infected with R. solani at an early stage of infection.

Trichoderma harzianum L1 as a potential source for lytic enzymes and elicitor of defense responses in chickpea (Cicer arietinum L.) against wilt disease caused by Fusarium oxysporum f. sp. ciceri.

Abstract: The effect of some natural lignocellulosic substances on the production of �-glucanase, chitinase, protease and xylanase from Trichoderma harzianum L1 has been studied under solid state fermentation conditions. Maximum activities of all these enzymes were observed in the fermentation medium containing the mixture of 1% rice bran, neem cake and 0.1% crab shell powder. The induction of plant defense response was investigated by inoculating the roots of chickpea cv JG62 with the biocontrol agent, T. harzianum L1. A root extract of chickpea inoculated with T. harzianum L1 showed increased activities of phenylalanine ammonia lyase and polyphenol oxidase, as well as induction of new trypsin and chymotrypsin inhibitors. The Fusarium oxysporum protease-2 was inhibited completely by root extract of chickpea inoculated with T. harzianum L1 and showed maximum resistance to rotting of roots caused by wilt disease

Gene expression analysis of the biocontrol fungus Trichoderma harzianum in the presence of tomato plants, chitin, or glucose using a high-density oligonucleotide microarray.

Abstract: It has recently been shown that the Trichoderma fungal species used for biocontrol of plant diseases are capable of interacting with plant roots directly, behaving as symbiotic microorganisms. With a view to providing further information at transcriptomic level about the early response of Trichoderma to a host plant, we developed a high-density oligonucleotide (HDO) microarray encompassing 14,081 Expressed Sequence Tag (EST)-based transcripts from eight Trichoderma spp. and 9,121 genome-derived transcripts of T. reesei, and we have used this microarray to examine the gene expression of T. harzianum either alone or in the presence of tomato plants, chitin, or glucose. Global microarray analysis revealed 1,617 probe sets showing differential expression in T. harzianum mycelia under at least one of the culture conditions tested as compared with one another. Hierarchical clustering and heat map representation showed that the expression patterns obtained in glucose medium clustered separately from the expression patterns observed in the presence of tomato plants and chitin. Annotations using the Blast2GO suite identified 85 of the 257 transcripts whose probe sets afforded up-regulated expression in response to tomato plants. Some of these transcripts were predicted to encode proteins related to Trichoderma-host (fungus or plant) associations, such as Sm1/Elp1 protein, proteases P6281 and PRA1, enchochitinase CHIT42, or QID74 protein, although previously uncharacterized genes were also identified, including those responsible for the possible biosynthesis of nitric oxide, xenobiotic detoxification, mycelium development, or those related to the formation of infection structures in plant tissues. The effectiveness of the Trichoderma HDO microarray to detect different gene responses under different growth conditions in the fungus T. harzianum strongly indicates that this tool should be useful for further assays that include different stages of plant colonization, as well as for expression studies in other Trichoderma spp. represented on it. Using this microarray, we have been able to define a number of genes probably involved in the transcriptional response of T. harzianum within the first hours of contact with tomato plant roots, which may provide new insights into the mechanisms and roles of this fungus in the Trichoderma-plant interaction.

Detection of viridiofungin A and other antifungal metabolites excreted by Trichoderma harzianum active against different plant pathogens

Abstract: Antibiosis is assumed to be an essential mechanism exerted by potential biocontrol agents (BCAs) of Trichoderma spp. Therefore, in the present study, we report for the first time on the elucidation and production of viridiofungin A (VFA) from T. harzianum isolate T23 cultures and investigate the antifungal potential of VFA and some other secondary metabolites purified from T. harzianum cultures against Fusarium moniliforme. The bioautography assay revealed that T. harzianum isolates T16 and T23 excreted several secondary metabolites with antifungal activity. Following isolation and purification of the antifungal zones, three fractions (F223, F323 and F423) from extracts of isolate T23 and two fractions (F416 and F516) from extracts of isolate T16 exhibited pronounced fungitoxic activity in the bioautography and antibiotic disk assays against Cladosporium spp. and F. moniliforme, respectively. The structure of the antifungal metabolite in fraction F323 was identified as viridiofungin A (VFA), the first report of production of VFA by isolate T23 of T. harzianum. Following cultivation of isolate T23 in PDB medium for 9 days, 94.6 mg l−1 of VFA were determined. VFA and fraction F516 retarded the mycelial growth of F. moniliforme in the non-volatile phase assay by >90% for each 250 μg ml−1 7 days post-inoculation (dpi). While VFA and fraction F416 showed both volatile and non-volatile effects, fraction F516 seemed to exhibit mainly non-volatile activity. Microscopic examination revealed that hyphae of F. moniliforme grown on VFA-amended medium were less branched and appeared thicker than untreated hyphae. Furthermore, in the presence of VFA, formation of chlamydospores by F. moniliforme was increased. Finally, the antifungal spectrum of VFA towards various important plant pathogens was evaluated. Germination of propagules of a variety of fungal pathogens in vitro was differentially inhibited by VFA. While in the presence of 100 μg ml−1 VFA conidial germination of V. dahliae was completely inhibited, a slightly higher concentration (150 μg ml−1) of the inhibitor was required to suppress germination of Phytophthora infestans sporangia or sclerotia of Sclerotinia sclerotiorum. Contrary to several reports in the literature, VFA proved to be fungistatic rather than fungicidal. However, neither VFA nor the other Trichoderma metabolites, such as 6PAP, F416 and F516, exhibited any antibacterial activity against Gram-positive and Gram-negative bacteria.

Biological control of root-knot nematode; Meloidogyne incognita in the medicinal plant; Withania somnifera and the effect of biocontrol agents on plant growth.

Abstract: The medicinally important plant; Withania somnifera, is highly susceptible to root knot nematode; Meloidogyne incognita. Various nematode antagonistic fungi have been studied for their use as biocontrol agents. In the experiment, the potential of fungi Trichoderma harzianum, Paecilomyces lilacinus and Arthrobotrys oligospora along with natural organic compound (Neem compound mix) to control the nematode; M. incognita was evaluated. Also, their potential to control nematodes was compared with that achieved by using the chemical control agent; carbofuran. The fungal agents evaluated significantly controlled nematode population and enhanced plant growth.

In vitro studies on the potential Trichoderma harzianum for antagonistic properties against Ganoderma boninense

Abstract: Ganoderma boninense is a bracket fungus that is pathogenic on oil palms causing basal stem rot, while some members of the genus Trichoderma are good biological control agents against numerous plant fungal diseases. We isolated and tested T. harzianum for in-vivo nursery to suppress diseases caused by G. boninense. A total of 48 local isolates of T. harzianum were selected for screening of their antagonistic properties against G. boninense (strain PER 71) by dual culture techniques. The results showed that all of the isolates of T. harzianum inhibited the growth of PER 71 with percentage of inhibition radial growth (PIRG) values ranging from 47.86 to 72.06%. The best eight samples from the dual cultures were then tested for their production of volatile antifungal compounds against PER 71, which gave PIRG values between 24.53 and 58.70% over 6 days. The values ranged from 18.35 to 40.16% over 6 days for the antifungal activities of their non-volatile compounds. Isolate FA 30 was demonstrated to be the best isolate not only to the dual culture inhibition tests but also the best for the production of inhibitory properties from both volatile and non-volatile antifungal compounds.

Molecular identification of Trichoderma species associated with Pleurotus ostreatus and natural substrates of the oyster mushroom.

Abstract: Green mold of Pleurotus ostreatus, caused by Trichoderma species, has recently resulted in crop losses worldwide. Therefore, there is an emerging need for rapid means of diagnosing the causal agents. A PCR assay was developed for rapid detection of Trichoderma pleurotum and Trichoderma pleuroticola, the two pathogens causing green mold of P. ostreatus. Three oligonucleotide primers were designed for identifying these species in a multiplex PCR assay based on DNA sequences within the fourth and fifth introns in the translation elongation factor 1α gene. The primers detected the presence of T. pleurotum and/or T. pleuroticola directly in the growing substrates of oyster mushrooms, without the need for isolating the pathogens. The assay was used to assess the presence of the two species in natural environments in which P. ostreatus can be found in Hungary, and demonstrated that T. pleuroticola was present in the growing substrates and on the surface of the basidiomes of wild oyster mushrooms. Other Trichoderma species detected in these substrates and habitats were Trichoderma harzianum, Trichoderma longibrachiatum and Trichoderma atroviride. Trichoderma pleurotum was not found in any of the samples from the forested areas tested in this study.

Study on the anthraquinones separated from the cultivation of Trichoderma harzianum strain Th-R16 and their biological activity.

Abstract: The biocontrol fungal species of Trichoderma, which colonizes plant roots, are well-known for their potential to control plant pathogens. Six anthraquinones, of which four have been identified for the first time from Trichoderma and two have already been reported in other strains, were purified from Trichoderma harzianum strain Th-R16 to evaluate their biological activities. The structures of the compounds were determined by one- and two-dimensional NMR. The compounds were shown to exhibit stronger antifungal activity than antibacterial activity. Low yield compounds, like 1,5-dihydroxy-3-hydroxymethyl-9,10-anthraquinone, were found to be more active against fungal pathogens than pachybasin and crysophanol, which were found to be the major extracellular metabolites. Test anthraquinones with higher oxidation numbers had better antifungal activity, and their activities were concentration-dependent.

Vermicompost suppresses Rhizoctonia solani Kühn in cucumber seedlings

Abstract: Disease suppressiveness of vermicompost produced from agricultural wastes consisting of cattle manure, tree bark (Salix spp.), potato culls, and apples was assayed on damping-off of two days-old cucumber (Cucumis sativus cv. Cevher) seedlings infected by Rhizoctonia solani Kuhn (AG-4). Suppression effect was assessed at the rates of 0, 10, 20 and 30% (v/v) vermicompost, either blended with Trichoderma harzianum Rifai (KRL-AG2), amended with potting mixtures consisting of sand and garden soil (1:1, v/v). Effect of water extracts of vermicompost on growth of R. solani mycelium in Petri dishes was also analyzed. Disease suppression effect increased in proportion to the pot amendment rate of vermicompost. Vermicomposts not blended with T. h arz ian u m effectively controlled damping-off of cucumber by R. solani (AG-4) at the rate of 20% and 30%. Vermicompost not blended with T. harzianum improved plant growth as well as that blended with T. harzianum. Analysis of the effect of water extracts of vermicompost on growth of R. solani mycelium in Petri dishes revealed antagonistic activity of a putative bacterium. Heat sterilization eliminated the suppressive and antagonistic effect by vermicompost and its water extracts, respectively. Activity of an antagonistic bacterium, which expressed a strong inhibition of growth of the pathogen mycelium, indicated that the type of suppressiveness against Rhizoctonia disease by the vermicompost is specific.

Antifungal and allelopathic effects of Asafoetida against Trichoderma harzianum and Pleurotus spp.

Abstract: Methanol extract (MeOH) of Asafoetida oleogum-resin was assayed for its in-vitro ability to control Trichoderma harzianum. The thirty-two components of MeOH-extracted resin were identified by GC-MS analysis. The antifungal and allelopathic effects of the MeOH extracts concentrations against T. harzianum and Pleurotus spp., were investigated in dual culture experiments on an agar-based medium. MeOH extract showed fungistatic and fungicidal properties against T. harzianum strains and Pleurotus spp. at higher concentrations. In dual culture, all strains of T. harzianum were antagonistic to Pleurotus spp. than in control. When MeOH extracts concentrations was added to the substrate culture, the antagonistic activity of T. harzianum against the Pleurotus spp. was moderate (0.625 μg/ml of MeOH extract) or weak (1.25 μg/ml of MeOH extract) against the Pleurotus spp. that either completely or partially replaced T. harzianum. TEM observations revealed that fungal growth inhibition from the MeOH extract was accompanied by marked morphological and cytological changes.