Trichoderma in its natural environment competes for nutrient uptake and is required to protect itself from adverse natural toxic compounds, such as those produced by plants and other microbes in the soil community, or...Trichoderma in its natural environment competes for nutrient uptake and is required to protect itself from adverse natural toxic compounds, such as those produced by plants and other microbes in the soil community, or synthetic toxic compounds released human activity. One of the most important metabolic pathways for drug resistance and substrate uptake, both in prokaryotes and eukaryotes, is ATP dependent. The role of ABC transporter proteins in the biology of Trichoderma is still not known. We present the cloning of the first four ABC transporter genes (TABC1, TABC2, TABC3, TABC4 ) in Trichoderma, and in particular T. atroviride P1, and the characterization of TABC2 The complete sequence of this gene is 6535 bp, which includes a promoter of 1624 bp, a terminator of 642 bp and a coding region of 4264 bp. The promoter contains many of the potential transcription factor binding sites found in the 5’ upstream region of the ech42 gene of T. atroviride P1. These included: heat shock factors (HSF), a nitrogen-regulating factor (Nit-2), a stress-response element (STRE), a GCR1 elements, and a Cre BP1 motif. Northern analysis and RT-PCR demonstrated that TABC2 is highly expressed when Trichoderma is subjected to nitrogen starvation, grown in the presence of culture filtrates of Botrytis cinerea, Rhizoctonia solani, and Pythium ultimum, or when N-acetylglucosamine is added to the substrate. TABC2 appears to be co-regulated with some CWDE-encoding genes, suggesting that this is the first ABC transporter encoding gene involved in mycoparasitic events. It’s role in the interaction of Trichoderma with fungal hosts or plants is being investigated by targeted gene disruption and overexpression.展开更多
In the late 1980’s the deve lop ment of a severe epidemic of green mold caused by Trichoderma spp. was not ed in the commercial production of Agaricus bisporus (champignon) in the U nited Kingdom, North America, Spai...In the late 1980’s the deve lop ment of a severe epidemic of green mold caused by Trichoderma spp. was not ed in the commercial production of Agaricus bisporus (champignon) in the U nited Kingdom, North America, Spain and Holland, which caused extensive economic losses. The parasitic fungi isolated from the edible mushroom belonged to four biotypes, Th1, Th2, Th3 and Th4 of T. harzianum. However, among these biotypes, only Th2 (since c lassified as T. aggressivum f. europaeum) and Th4 (T. aggressivum f. aggressivum) were identified as the fungi causing problems in Agaricus production. In general, mushroom compost hosts both aggressive and innocuous is olates of Trichoderma, which are not morphologically distinguishable. Abo ut four years ago, a problem with green mold became apparent in the production o f Pleurotus ostreatus in Northern Italy, which eventually developed to a c risis situation in the South two years later and threatened to seriously comprom ise the Pleurotus market. This study was initiated to: isolate and identif y the aggressive fungi, then morphologically, physiologically and genetically characterize the isolates, dete rmine the source and phases of infection, and study methods of control. Samples were obtained from different phases of compost preparation at the locality of a major producer and supplier of compost to the mushroom industry in Southern Ital y, and microbial counts were conducted. Although the presence of Trichoderma was detected in the initial stages of composting, this value was reduced to zero from the phase of pasteurization to seeding with Pleurotus. Trichoderma infestations were noted in the packaged Pleurotus bales at various time s during the incubation phase (7-15 days after seeding) and after shipping to th e mushroom greenhouses, where the pathogen infestations greatly reduced the qual ity and quantity of the mushroom yield, as well as the number of potential harvest cycles. Preliminary r esults from the morphological and genetic characterization of Trichoderma isolates parasitic to Pleurotus indicated that they are different from bot h T. aggressivum forms parasitic to Agaricus, and the majority of the isolates probably belong to the species T. harzianum. In vitr o confrontation plates were performed with 26 isolates of aggressive Trich oderma obtained from compost, three Trichoderma isolates used in biolog ical control and 12 varieties of Pleurotus. No inhibitory effect was obse rved between any of the Trichoderma isolates with Pleurotus, althou gh some growth inhibition was caused by the biocontrol isolates of Trichoderm a on some of the aggressive isolates. The temperature optimum for Pleurotus growth was at 28 ℃, whereas Trichoderma grew well at a wider range (20- 28 ℃), and exceeded the growth rate of Pleurotus by three times at 25 ℃. T he pH optimum for the growth of Pleurotus was alkaline (pH 8-9) whereas Trichoderma preferred acidic-neutral pH (5-7) . Various commercial fungicides used in agriculture (procloraz, thiabendazole, dichloran, benomyl, p r opiconazole, thiofanatomethyl) were tested against the aggressive and biocontro l isolates of Trichoderma, as well as the different varieties of Pleuro tus to determine dose response curves and combinations that would inhibit spo re germination, mycelial growth and subsequent sporulation. Both procloraz and thiabendazole, which are pesticides allowed in e dible mushroom production, were found to control the growth of the aggressive Trichoderma isolates and did not have a negative effect on Pleurotus.展开更多
The beneficial applications of Trichoderma spp. in agriculture include not only the control of plant pathogens, but also the improvement of plant growth, micronutrient availability, and plant tolerance to abiotic stre...The beneficial applications of Trichoderma spp. in agriculture include not only the control of plant pathogens, but also the improvement of plant growth, micronutrient availability, and plant tolerance to abiotic stress. In addition, it has been suggested that these fungi are able to increase plant disease resistance by activating induced systemic resistance (ISR) . The mode of action of these beneficial fungi in the Trichoderma -plant-pathogen interaction are many, complex and not completely understood. Numerous lytic enzymes have been characterized, the encoding genes (ech42 gluc78, nag1 from T. atroviride strain P1) cloned, and their role in biocontrol demonstrated. The corresponding biocontrol-related inducible promoters have been used in a reporter system based on the Aspergillus niger glucose oxidase gene (goxA) to monitor biocontrol activity. Glucose oxidase catalyzes the oxygen-dependent oxidation of D-glucose to D-glucono-1,5-lactone and hydrogen peroxide; this latter compound is known to have an antifungal effect and activate the plant defence cascade, thus increasing resistance to pathogen attack. T. atroviride P1 transformants with various promoters gox were tested as seed coating treatments on bean seeds planted in soil infested with a soilborne fungal pathogen. Successively, the emergent leaves were inoculated with a foliar pathogen to determine the effect of the GOX transformants on biocontrol and resistance to pathogen attack. Inoculations with the P1-GOX transformants not only reduced disease symptoms caused by a soil pathogen, but also the lesions of various foliar pathogens applied far from the Trichoderma colonization, thus activating ISR. A similar approach is being use to genetically improve T. harzianum T22, a rhizosphere competent and commercially marketed strain not transformed yet, by using four different gox gene constructs under the control of constitutive and inducible promoters. Plasmids have been introduced in Trichoderma by protoplasts co-transformation. hygromicin resistant progeny selected, and mitotically stable transformants analysed to confirm the presence of the novel enzyme activity. Progenies are being tested for biocontrol ISR inducing activity.展开更多
Numerous Trichoderma spp. are mycoparasites and commercially applied as biological control agents against a large number of plant pathogenic fungi. The mycoparasitic interaction is host-specific and several research s...Numerous Trichoderma spp. are mycoparasites and commercially applied as biological control agents against a large number of plant pathogenic fungi. The mycoparasitic interaction is host-specific and several research strategies have been applied to identify the main genes and compounds involved in the antagonist-plant-pathogen three-way interaction. During mycoparasitism, signals from the host fungus are recognised by Trichoderma, stimulating antifungal activities that are accompanied by morphological changes and the secretion of hydrolytic enzymes and antibiotics. Interestingly some morphological changes appeared highly conserved in the strategy of pathogenicity within the fungal world, i.e. the formation of appressoria as well as the secretion of hydrolytic enzymes seem to be general mechanisms of attack both for plant pathogens and mycoparasitic antagonists. This knowledge is being used to identify receptors and key components of signalling pathways involved in fungus-fungus interaction. For this purpose we have cloned the first genes (tmk1, tga1, tga3) from T. atroviride showing a high similarity to MAP kinase and G protein subunits (see abstract by Zeilinger et al.), which have been found to have an important role in pathogenicity by Magnaporthe grisea. To identify the function and involvement of these factors in mycoparasitism by T. atroviride, tmk1, tga1, tga3 disruptant strains were produced. The knock-out mutants were tested by in vivo biocontrol assays for their ability to inhibit soil and foliar plant pathogens such as Rhizoctonia solani, Pythium ultimum and Botrytis cinerea . Disruption of these genes corresponded to a complete loss of biocontrol ability, suggesting a significant role in mycoparasitism. In particular, it has been suggested that tga3 regulates the expression of chitinase-encoding genes, the secretion of the corresponding enzymes and the process of conidiation. Comparative proteome analysis of wild type and disruptants supported this hypothesis, and indicated many changes in the protein profiles of T. atroviride in different interaction conditions with plants and pathogenic hosts.展开更多
The molecular basis of Trichoderma -plant interaction is very complex and still not completely understood. The colonization of the root system by rhizosphere competent strains of Trichoderma results in increased devel...The molecular basis of Trichoderma -plant interaction is very complex and still not completely understood. The colonization of the root system by rhizosphere competent strains of Trichoderma results in increased development of root/aerial systems, in improved yields and in plant disease control. Other beneficial effects, such as the induction of plant systemic resistance, have also been described. To understand the mechanisms involved we are using different approaches, including the making of transformants expressing genes that encode for compounds able to affect plant response to pathogens. Trichoderma transformants carrying the avirulence gene Avr4 from Cladosporium fulvum under the control of constitutive and inducible promoters were obtained and tested on tomato plants having the Cf4 resistance gene. Necrosis and suberification zones, similar to the symptoms appearing during Cladosporium-tomato interaction, were found when the roots of the Cf4 plants were treated with Avr4-Trichoderma. This demonstrates that selected Trichoderma strains are able to transfer to the plant molecules that may deeply affect metabolism, disease resistance etc. Therefore, these beneficial fungi can be regarded as biotechnological tools to provide a variety of crops with useful compounds. Moreover, in in vitro competition assays the transformants were found to be more effective as antagonists against Alternaria alternata than the wild type. Trichoderma sends a variety of biochemical signals to the plants including avirulence molecules; therefore the presence of avr-like proteins in the fungus proteome was investigated. Proteome analysis has permitted us to isolate and sequence many proteins potentially having this function. From the extracellular protein extracts, we have purified and sequenced a protein with structural characteristics similar to Avr4 of C. fulvum. The protein, Hytra1, was found to be a hydrophobin with chitin binding activity, the typical 8 cysteine residues, and 4 disulfide bridges. Infiltrations of the extracellular protein fractions containing Hytra1 resulted in a strong HR reaction on tobacco and tomato leaves, and induction of a novel phytoalexin.展开更多
Fusarium spp. are pathogens of many important agricultural crops, and are often strong mycotoxin producers. Fusarium proliferatum, in particular, causes disease in cereals and secretes the toxin Beauvaricin that conta...Fusarium spp. are pathogens of many important agricultural crops, and are often strong mycotoxin producers. Fusarium proliferatum, in particular, causes disease in cereals and secretes the toxin Beauvaricin that contaminates livestock feed and cereals, producing a variety of toxicity symptoms ranging from poor weight gain to mortality. Beauvaricin is a cyclodepsipeptide and acts as a potent mycotoxin known to have insecticidal properties. This compound is highly toxic to human cell lines, where it induces apoptosis and specifically inhibits cholesterol acetyltransferase. Nothing is known about the role of this mycotoxin during the interaction of F. proliferatum with other microorganisms, including the fungal antagonists Trichoderma spp. In vitro tests have demonstrated that the antagonistic and mycoparasitic activity of Trichoderma is not inhibited by the presence of Beauvaricin at concentrations up to 10 mg/kg in the substrate. In vivo biocontrol assays on barley and wheat with Trichoderma against F. proliferatum isolates, producing and non-producing Beauvaricin, confirmed the ability of the antagonist to control this pathogen in all cases. Also Trichoderma culture filtrates obtained in conditions that promote Cell Wall Degrading Enzyme (CWDE) secretion, were able to inhibit. spore. germination. of. different. F.. proliferatum. isolates.. These. results. suggest. the. possibility. of. using. Trichoderma. and/or. its. metabolites. to. control. contaminants. of. livestock. feed. by. mycotoxin-producing. Fusarium..展开更多
The molecular factors involved in the three-way interaction between plant, pathogenic fungi and antagonistic/biocontrol fungi, such as Trichoderma, are still poorly understood, even if they represent a matter of inter...The molecular factors involved in the three-way interaction between plant, pathogenic fungi and antagonistic/biocontrol fungi, such as Trichoderma, are still poorly understood, even if they represent a matter of interest for improving crop management and developing new strategies for plant diseases control. The aim of this work is to investigate the components involved in this interaction and, for this purpose, a proteomic approach was used. 2-D maps of the protein extracts from the single components in various interactions between plants (potato, bean, tobacco or tomato), pathogens (Botrytis cinerea, Rhizoctonia solani or Pythium ultimum) and biocontrol fungi (Trichoderma atroviride strain P1 or Trichoderma harzianum strain T22) were obtained. The proteome of each partner was collected separately and extracted by acetone precipitation in presence of trichloroacetic acid and a reducing agent (DTT). The extracted proteins were separated by isoelectrofocusing (IEF), using IPG (Immobilized pH gradient) strips, followed by SDS-PAGE. In order to improve resolution the separations were performed both on wide than narrow pH range and on different gel lengths. Differential spots were noted in the proteome of the three-way interaction when compared to each single component. These were further characterized by mass spectrometry and in silico analysis with the aim of identifying and cloning the relative genes. During the in vitro interaction of T. harzianum strain T22 with tomato and the culture filtrate or cell walls of pathogens, the spot number was higher than in the presence of pathogen biomass. In terms of Trichoderma differential proteins displayed on 2D gels, the most important changes were obtained in the presence of P. ultimum . During the in vivo interaction with tomato, the antagonist proteome changed much more in presence of soilborne fungi R. solani and P. ultimum than with the foliar fungus B. cinerea, both in terms of total and increased or novel spots. In silico analysis of some of those spots revealed homology with intracellular enzymes (GTPases, hydrolases) and with stress-related proteins (heat shock proteins HSP70, bacteriocin cloacin). Specific proteins in the plant proteome, i.e. pathogenesis-related proteins, have been identified during the in vivo interaction of bean with R. solani and T. atroviride strain P1. This is in agreement with the demonstrated ability of these beneficial fungi to induce plant systemic disease resistance by activating expression of defence-related genes. Proteins extracted from T. atrovride strain P1 which were analysed by mass spectrometry, revealed some interesting homologies with a fungal hydrophobin of Pleurotus ostreatus and an ABC transporter of Ralstonia metallidurans. These could represent molecular factors involved in the antagonistic mechanisms of Trichoderma and play a role in the three-way interaction with the plant and other microbes.展开更多
Trichoderma atroviride strain P1 has been used extensively to study the mycoparasitic mechanisms in the interaction between plant pathogenic host and beneficial antagonistic fungi. Mutants of P1 containing the green f...Trichoderma atroviride strain P1 has been used extensively to study the mycoparasitic mechanisms in the interaction between plant pathogenic host and beneficial antagonistic fungi. Mutants of P1 containing the green fluorescent protein (gfp) or glucose oxidase (gox) reporter systems and different inducible promoters (from the exochitinase nag1 gene, or the endochitinase ech42 gene of P1) were used to determine the factors that activate the biocontrol gene expression cascade in the antagonist. The following compounds were tested singly and in various combinations: purified Trichoderma P1 enzymes (endochitinase, exochitinase, chitobiosidase, glucanase); antagonist culture filtrates (T. atroviride P1 wild-type and relative knock-out mutants, T. harzianum, T. reesei); pathogen culture filtrates (Botrytis, Pythium, Rhizoctonia); purified fungal cell walls (CWs) from Trichoderma, Botrytis, Pythium, Rhizoctonia; colloidal crab shell chitin; and plant extracts from cucumber leaves, stems or roots. Strong induction of mycoparasitism was found with the various digestion products produced by treating fungal CWs and colloidal chitin with purified enzymes or fungal culture filtrates. Filtrates from chitinase knock-out mutants, as well as CWs from Oomycetes fungi, were less active in producing the stimulus for mycoparasitism. The host CW digestion products were separated by molecular weight (MW) to determine which compounds were able to activate Trichoderma. Micromolecules of MW less than 3 kDa were found to trigger mycoparasitism gene expression before physical contact with the host pathogen. These compounds stimulated mycelial growth and spore germination of the antagonist. Purification of these host-derived compounds was conducted by HPLC and in vivo assay. The obtained inducers were able to stimulate both the production of endochitinase and exochitinase enzymes, even under repressing conditions in the presence of glucose. Inducers stimulated the biocontrol effect of P1 in the presence of host fungi. The disease symptom development on bean leaves inoculated with Botrytis and Trichoderma spores was clearly reduced by the addition of the inducers, unless these molecules were not specifically inactivated. Finally, purified inducers added to liquid cultures of T. atroviride P1 stimulated the production of low MW antibiotics and metabolites which inhibited Botrytis spore germination. Mass spectrometry analysis (ESI-MS) of the inducers indicated the presence of hexose oligomers, like cellobiose, while MS/MS analysis by selective fragmentation of peaks in the spectrum demonstrated the presence of at least three distinct compounds that were biologically active.展开更多
文摘Trichoderma in its natural environment competes for nutrient uptake and is required to protect itself from adverse natural toxic compounds, such as those produced by plants and other microbes in the soil community, or synthetic toxic compounds released human activity. One of the most important metabolic pathways for drug resistance and substrate uptake, both in prokaryotes and eukaryotes, is ATP dependent. The role of ABC transporter proteins in the biology of Trichoderma is still not known. We present the cloning of the first four ABC transporter genes (TABC1, TABC2, TABC3, TABC4 ) in Trichoderma, and in particular T. atroviride P1, and the characterization of TABC2 The complete sequence of this gene is 6535 bp, which includes a promoter of 1624 bp, a terminator of 642 bp and a coding region of 4264 bp. The promoter contains many of the potential transcription factor binding sites found in the 5’ upstream region of the ech42 gene of T. atroviride P1. These included: heat shock factors (HSF), a nitrogen-regulating factor (Nit-2), a stress-response element (STRE), a GCR1 elements, and a Cre BP1 motif. Northern analysis and RT-PCR demonstrated that TABC2 is highly expressed when Trichoderma is subjected to nitrogen starvation, grown in the presence of culture filtrates of Botrytis cinerea, Rhizoctonia solani, and Pythium ultimum, or when N-acetylglucosamine is added to the substrate. TABC2 appears to be co-regulated with some CWDE-encoding genes, suggesting that this is the first ABC transporter encoding gene involved in mycoparasitic events. It’s role in the interaction of Trichoderma with fungal hosts or plants is being investigated by targeted gene disruption and overexpression.
文摘In the late 1980’s the deve lop ment of a severe epidemic of green mold caused by Trichoderma spp. was not ed in the commercial production of Agaricus bisporus (champignon) in the U nited Kingdom, North America, Spain and Holland, which caused extensive economic losses. The parasitic fungi isolated from the edible mushroom belonged to four biotypes, Th1, Th2, Th3 and Th4 of T. harzianum. However, among these biotypes, only Th2 (since c lassified as T. aggressivum f. europaeum) and Th4 (T. aggressivum f. aggressivum) were identified as the fungi causing problems in Agaricus production. In general, mushroom compost hosts both aggressive and innocuous is olates of Trichoderma, which are not morphologically distinguishable. Abo ut four years ago, a problem with green mold became apparent in the production o f Pleurotus ostreatus in Northern Italy, which eventually developed to a c risis situation in the South two years later and threatened to seriously comprom ise the Pleurotus market. This study was initiated to: isolate and identif y the aggressive fungi, then morphologically, physiologically and genetically characterize the isolates, dete rmine the source and phases of infection, and study methods of control. Samples were obtained from different phases of compost preparation at the locality of a major producer and supplier of compost to the mushroom industry in Southern Ital y, and microbial counts were conducted. Although the presence of Trichoderma was detected in the initial stages of composting, this value was reduced to zero from the phase of pasteurization to seeding with Pleurotus. Trichoderma infestations were noted in the packaged Pleurotus bales at various time s during the incubation phase (7-15 days after seeding) and after shipping to th e mushroom greenhouses, where the pathogen infestations greatly reduced the qual ity and quantity of the mushroom yield, as well as the number of potential harvest cycles. Preliminary r esults from the morphological and genetic characterization of Trichoderma isolates parasitic to Pleurotus indicated that they are different from bot h T. aggressivum forms parasitic to Agaricus, and the majority of the isolates probably belong to the species T. harzianum. In vitr o confrontation plates were performed with 26 isolates of aggressive Trich oderma obtained from compost, three Trichoderma isolates used in biolog ical control and 12 varieties of Pleurotus. No inhibitory effect was obse rved between any of the Trichoderma isolates with Pleurotus, althou gh some growth inhibition was caused by the biocontrol isolates of Trichoderm a on some of the aggressive isolates. The temperature optimum for Pleurotus growth was at 28 ℃, whereas Trichoderma grew well at a wider range (20- 28 ℃), and exceeded the growth rate of Pleurotus by three times at 25 ℃. T he pH optimum for the growth of Pleurotus was alkaline (pH 8-9) whereas Trichoderma preferred acidic-neutral pH (5-7) . Various commercial fungicides used in agriculture (procloraz, thiabendazole, dichloran, benomyl, p r opiconazole, thiofanatomethyl) were tested against the aggressive and biocontro l isolates of Trichoderma, as well as the different varieties of Pleuro tus to determine dose response curves and combinations that would inhibit spo re germination, mycelial growth and subsequent sporulation. Both procloraz and thiabendazole, which are pesticides allowed in e dible mushroom production, were found to control the growth of the aggressive Trichoderma isolates and did not have a negative effect on Pleurotus.
文摘The beneficial applications of Trichoderma spp. in agriculture include not only the control of plant pathogens, but also the improvement of plant growth, micronutrient availability, and plant tolerance to abiotic stress. In addition, it has been suggested that these fungi are able to increase plant disease resistance by activating induced systemic resistance (ISR) . The mode of action of these beneficial fungi in the Trichoderma -plant-pathogen interaction are many, complex and not completely understood. Numerous lytic enzymes have been characterized, the encoding genes (ech42 gluc78, nag1 from T. atroviride strain P1) cloned, and their role in biocontrol demonstrated. The corresponding biocontrol-related inducible promoters have been used in a reporter system based on the Aspergillus niger glucose oxidase gene (goxA) to monitor biocontrol activity. Glucose oxidase catalyzes the oxygen-dependent oxidation of D-glucose to D-glucono-1,5-lactone and hydrogen peroxide; this latter compound is known to have an antifungal effect and activate the plant defence cascade, thus increasing resistance to pathogen attack. T. atroviride P1 transformants with various promoters gox were tested as seed coating treatments on bean seeds planted in soil infested with a soilborne fungal pathogen. Successively, the emergent leaves were inoculated with a foliar pathogen to determine the effect of the GOX transformants on biocontrol and resistance to pathogen attack. Inoculations with the P1-GOX transformants not only reduced disease symptoms caused by a soil pathogen, but also the lesions of various foliar pathogens applied far from the Trichoderma colonization, thus activating ISR. A similar approach is being use to genetically improve T. harzianum T22, a rhizosphere competent and commercially marketed strain not transformed yet, by using four different gox gene constructs under the control of constitutive and inducible promoters. Plasmids have been introduced in Trichoderma by protoplasts co-transformation. hygromicin resistant progeny selected, and mitotically stable transformants analysed to confirm the presence of the novel enzyme activity. Progenies are being tested for biocontrol ISR inducing activity.
文摘Numerous Trichoderma spp. are mycoparasites and commercially applied as biological control agents against a large number of plant pathogenic fungi. The mycoparasitic interaction is host-specific and several research strategies have been applied to identify the main genes and compounds involved in the antagonist-plant-pathogen three-way interaction. During mycoparasitism, signals from the host fungus are recognised by Trichoderma, stimulating antifungal activities that are accompanied by morphological changes and the secretion of hydrolytic enzymes and antibiotics. Interestingly some morphological changes appeared highly conserved in the strategy of pathogenicity within the fungal world, i.e. the formation of appressoria as well as the secretion of hydrolytic enzymes seem to be general mechanisms of attack both for plant pathogens and mycoparasitic antagonists. This knowledge is being used to identify receptors and key components of signalling pathways involved in fungus-fungus interaction. For this purpose we have cloned the first genes (tmk1, tga1, tga3) from T. atroviride showing a high similarity to MAP kinase and G protein subunits (see abstract by Zeilinger et al.), which have been found to have an important role in pathogenicity by Magnaporthe grisea. To identify the function and involvement of these factors in mycoparasitism by T. atroviride, tmk1, tga1, tga3 disruptant strains were produced. The knock-out mutants were tested by in vivo biocontrol assays for their ability to inhibit soil and foliar plant pathogens such as Rhizoctonia solani, Pythium ultimum and Botrytis cinerea . Disruption of these genes corresponded to a complete loss of biocontrol ability, suggesting a significant role in mycoparasitism. In particular, it has been suggested that tga3 regulates the expression of chitinase-encoding genes, the secretion of the corresponding enzymes and the process of conidiation. Comparative proteome analysis of wild type and disruptants supported this hypothesis, and indicated many changes in the protein profiles of T. atroviride in different interaction conditions with plants and pathogenic hosts.
文摘The molecular basis of Trichoderma -plant interaction is very complex and still not completely understood. The colonization of the root system by rhizosphere competent strains of Trichoderma results in increased development of root/aerial systems, in improved yields and in plant disease control. Other beneficial effects, such as the induction of plant systemic resistance, have also been described. To understand the mechanisms involved we are using different approaches, including the making of transformants expressing genes that encode for compounds able to affect plant response to pathogens. Trichoderma transformants carrying the avirulence gene Avr4 from Cladosporium fulvum under the control of constitutive and inducible promoters were obtained and tested on tomato plants having the Cf4 resistance gene. Necrosis and suberification zones, similar to the symptoms appearing during Cladosporium-tomato interaction, were found when the roots of the Cf4 plants were treated with Avr4-Trichoderma. This demonstrates that selected Trichoderma strains are able to transfer to the plant molecules that may deeply affect metabolism, disease resistance etc. Therefore, these beneficial fungi can be regarded as biotechnological tools to provide a variety of crops with useful compounds. Moreover, in in vitro competition assays the transformants were found to be more effective as antagonists against Alternaria alternata than the wild type. Trichoderma sends a variety of biochemical signals to the plants including avirulence molecules; therefore the presence of avr-like proteins in the fungus proteome was investigated. Proteome analysis has permitted us to isolate and sequence many proteins potentially having this function. From the extracellular protein extracts, we have purified and sequenced a protein with structural characteristics similar to Avr4 of C. fulvum. The protein, Hytra1, was found to be a hydrophobin with chitin binding activity, the typical 8 cysteine residues, and 4 disulfide bridges. Infiltrations of the extracellular protein fractions containing Hytra1 resulted in a strong HR reaction on tobacco and tomato leaves, and induction of a novel phytoalexin.
文摘Fusarium spp. are pathogens of many important agricultural crops, and are often strong mycotoxin producers. Fusarium proliferatum, in particular, causes disease in cereals and secretes the toxin Beauvaricin that contaminates livestock feed and cereals, producing a variety of toxicity symptoms ranging from poor weight gain to mortality. Beauvaricin is a cyclodepsipeptide and acts as a potent mycotoxin known to have insecticidal properties. This compound is highly toxic to human cell lines, where it induces apoptosis and specifically inhibits cholesterol acetyltransferase. Nothing is known about the role of this mycotoxin during the interaction of F. proliferatum with other microorganisms, including the fungal antagonists Trichoderma spp. In vitro tests have demonstrated that the antagonistic and mycoparasitic activity of Trichoderma is not inhibited by the presence of Beauvaricin at concentrations up to 10 mg/kg in the substrate. In vivo biocontrol assays on barley and wheat with Trichoderma against F. proliferatum isolates, producing and non-producing Beauvaricin, confirmed the ability of the antagonist to control this pathogen in all cases. Also Trichoderma culture filtrates obtained in conditions that promote Cell Wall Degrading Enzyme (CWDE) secretion, were able to inhibit. spore. germination. of. different. F.. proliferatum. isolates.. These. results. suggest. the. possibility. of. using. Trichoderma. and/or. its. metabolites. to. control. contaminants. of. livestock. feed. by. mycotoxin-producing. Fusarium..
文摘The molecular factors involved in the three-way interaction between plant, pathogenic fungi and antagonistic/biocontrol fungi, such as Trichoderma, are still poorly understood, even if they represent a matter of interest for improving crop management and developing new strategies for plant diseases control. The aim of this work is to investigate the components involved in this interaction and, for this purpose, a proteomic approach was used. 2-D maps of the protein extracts from the single components in various interactions between plants (potato, bean, tobacco or tomato), pathogens (Botrytis cinerea, Rhizoctonia solani or Pythium ultimum) and biocontrol fungi (Trichoderma atroviride strain P1 or Trichoderma harzianum strain T22) were obtained. The proteome of each partner was collected separately and extracted by acetone precipitation in presence of trichloroacetic acid and a reducing agent (DTT). The extracted proteins were separated by isoelectrofocusing (IEF), using IPG (Immobilized pH gradient) strips, followed by SDS-PAGE. In order to improve resolution the separations were performed both on wide than narrow pH range and on different gel lengths. Differential spots were noted in the proteome of the three-way interaction when compared to each single component. These were further characterized by mass spectrometry and in silico analysis with the aim of identifying and cloning the relative genes. During the in vitro interaction of T. harzianum strain T22 with tomato and the culture filtrate or cell walls of pathogens, the spot number was higher than in the presence of pathogen biomass. In terms of Trichoderma differential proteins displayed on 2D gels, the most important changes were obtained in the presence of P. ultimum . During the in vivo interaction with tomato, the antagonist proteome changed much more in presence of soilborne fungi R. solani and P. ultimum than with the foliar fungus B. cinerea, both in terms of total and increased or novel spots. In silico analysis of some of those spots revealed homology with intracellular enzymes (GTPases, hydrolases) and with stress-related proteins (heat shock proteins HSP70, bacteriocin cloacin). Specific proteins in the plant proteome, i.e. pathogenesis-related proteins, have been identified during the in vivo interaction of bean with R. solani and T. atroviride strain P1. This is in agreement with the demonstrated ability of these beneficial fungi to induce plant systemic disease resistance by activating expression of defence-related genes. Proteins extracted from T. atrovride strain P1 which were analysed by mass spectrometry, revealed some interesting homologies with a fungal hydrophobin of Pleurotus ostreatus and an ABC transporter of Ralstonia metallidurans. These could represent molecular factors involved in the antagonistic mechanisms of Trichoderma and play a role in the three-way interaction with the plant and other microbes.
文摘Trichoderma atroviride strain P1 has been used extensively to study the mycoparasitic mechanisms in the interaction between plant pathogenic host and beneficial antagonistic fungi. Mutants of P1 containing the green fluorescent protein (gfp) or glucose oxidase (gox) reporter systems and different inducible promoters (from the exochitinase nag1 gene, or the endochitinase ech42 gene of P1) were used to determine the factors that activate the biocontrol gene expression cascade in the antagonist. The following compounds were tested singly and in various combinations: purified Trichoderma P1 enzymes (endochitinase, exochitinase, chitobiosidase, glucanase); antagonist culture filtrates (T. atroviride P1 wild-type and relative knock-out mutants, T. harzianum, T. reesei); pathogen culture filtrates (Botrytis, Pythium, Rhizoctonia); purified fungal cell walls (CWs) from Trichoderma, Botrytis, Pythium, Rhizoctonia; colloidal crab shell chitin; and plant extracts from cucumber leaves, stems or roots. Strong induction of mycoparasitism was found with the various digestion products produced by treating fungal CWs and colloidal chitin with purified enzymes or fungal culture filtrates. Filtrates from chitinase knock-out mutants, as well as CWs from Oomycetes fungi, were less active in producing the stimulus for mycoparasitism. The host CW digestion products were separated by molecular weight (MW) to determine which compounds were able to activate Trichoderma. Micromolecules of MW less than 3 kDa were found to trigger mycoparasitism gene expression before physical contact with the host pathogen. These compounds stimulated mycelial growth and spore germination of the antagonist. Purification of these host-derived compounds was conducted by HPLC and in vivo assay. The obtained inducers were able to stimulate both the production of endochitinase and exochitinase enzymes, even under repressing conditions in the presence of glucose. Inducers stimulated the biocontrol effect of P1 in the presence of host fungi. The disease symptom development on bean leaves inoculated with Botrytis and Trichoderma spores was clearly reduced by the addition of the inducers, unless these molecules were not specifically inactivated. Finally, purified inducers added to liquid cultures of T. atroviride P1 stimulated the production of low MW antibiotics and metabolites which inhibited Botrytis spore germination. Mass spectrometry analysis (ESI-MS) of the inducers indicated the presence of hexose oligomers, like cellobiose, while MS/MS analysis by selective fragmentation of peaks in the spectrum demonstrated the presence of at least three distinct compounds that were biologically active.
