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
During an investigation of mycoparasitic fungi on sclerotia of Sclerotinia sclerotiorum in China, Gliocladium species was consistently encountered and isolated from natural soils collected over the country. Colonizati...During an investigation of mycoparasitic fungi on sclerotia of Sclerotinia sclerotiorum in China, Gliocladium species was consistently encountered and isolated from natural soils collected over the country. Colonization frequencies on sclerotia of S. sclerotiorum by the fungi ranged from 40% to 100% when the sclerotia were introduced into soils and coincubated at 22-24 ℃ for 4 weeks. Identification showed that G. roseum, G. virens and G. catenulatum were the dominant species among the 300 isolates gained. Molecular taxonomy of some Gliocladium isolates analysed by rDNA ITS sequence used ITS1 and ITS4 as primers was demonstrated to be identical with morphological classification. Reinoculation tests by placing surface-sterilized sclerotia onto colony of Gliocladium isolates for 7 days and then surface-sterilized again resulted in 100% sclerotia colonized. Dipping sclerotia with spores suspension of isolates for 5 min and incubated the sclerotia in Petri dishes at 25 ℃ for 24 h resulted in infection of the sclerotia. Microscopic observations indicated that Gliocladium isolates grew along the host hyphae, coiled around, formed appressorium-like structures, penetrated and degraded hyphae of S. sclerotiorum when dual-cultured in slides. Paraffin-section showed that sclerotial tissue collapsed when infected by Gliocladium isolates. Host range test demonstrated that the isolates suppressed hyphal growth of several plant pathogenic fungi, including S. sclerotiorum, Botrytis cinerea, Fusarium oxysporum, Alternaria tenuis, and Rhizoctonia solani. Activities of fungal cell wall degrading enzymes in cultural filtrates and parasitized sclerotia were detected. Results indicated that chitinase and glucanase were important factors involved in the mycoparasitism. A 51 kDa chitinase was isolated and purified from G. catenulatum HL-1-1 culture filtrate. A part of amino acid sequences of the enzyme has been analysed. It was found to have a high homogeneity to chitinase A from Stenotrophomonas maltophilia, while no homogeneity to chitinases from Trichoderma and other fungi. It was considered to be a new chitinase quite different from those produced by other mycoparasitic fungi. Optimum temperature and pH for the enzyme activity were 60 ℃ and 6.0. respectively. Ca 2+. promoted the activity, while Fe 3+., Cu 2+. and Ag+ inhibited it. Km of the chitinase was 2.832. The chitanase significantly inhibited the growth of hyphae, germination of conidia and sclerotia of some plant pathogenic fungi. Gene encoding this chitinase is being cloned from the total RNA of HL-1-1 isolates by RT-PCR. Biological characteristics of some Gliocladium isolates were studied. Results showed that temperature ranged from 22 ℃ to 30 ℃ was optimum for isolates hyphae growth and spore germination, while they could endure high temperature. Spores could not germinate below 5 ℃ or over 40 ℃ and lethal temperature of spores was 52 ℃. Optimum pH was 5.0 for hyphae growth and 6.0 for sporulation. Dark condition enhanced hyphae growth and light promoted good for sporulation. Glucose, sucrose and mannose were optimum C source for hyphal growth while glucose, xylose, soluble starch and chitin optimum for sporulation. Soybean cake powder, yeast extract and casein hydrolysate were good for hyphal growth while soybean cake powder, beef extract, urea and NaNO 3 were good N sources for sporulation. Extract of sclerotia promoted spore germination of the isolate,but D-glucose inhibited. Greenhouse tests confirmed that Gliocladium isolates GJ-1-1, SS-1-1, HL-1-1, SH-1-1, GW-1-1 promoted the seedling growth of soybean, cucumber, wheat and rice. Our primary studies showed that some Gliocladium isolates are of great potential in biocontrol of plant fungal diseases, such as soybean stem rot caused by S. sclerotiorum and cucumber root disease caused by R. solani.展开更多
Trichoderma species are currently used as biocontrol agents for crop diseases caused by a number of fungal plant pathogens. However, their biocontrol performance in the field can be unreliable and it is likely that mo...Trichoderma species are currently used as biocontrol agents for crop diseases caused by a number of fungal plant pathogens. However, their biocontrol performance in the field can be unreliable and it is likely that more consistent performance could be achieved through knowledge and manipulation of the genes involved. For example, induction of the genes could be optimised for variable environmental and physiological conditions, superior strains could be selected more effectively and novel strains could be created. One method by which Trichoderma species accomplish biocontrol is mycoparasitism. Several genes involved in the mycoparasitic interaction have previously been characterised, however these consist predominantly of those that encode enzymes that degrade fungal cell walls. In the current study subtractive hybridisation was used to target genes expressed when Trichoderma hamatum and the plant pathogen Sclerotinia sclerotiorum were cultured together, subtracting genes expressed when each are grown individually. This experimental design has the potential to yield T. hamatum genes involved in mycoparasitism of S. sclerotiorum, and S. sclerotiorum genes up-regulated in host defence. The cDNA fragments yielded by the subtraction were characterised with respect to expression, sequence and species of origin. A number of novel T. hamatum genes which were up-regulated during mycoparasitism were identified.展开更多
The relationship between taxonomic status of Trichoderma spp., chitinase production in solid substrate fermentation (SSF) on four media and mycoparasitism in dual culture (confrontation assay) against four plant patho...The relationship between taxonomic status of Trichoderma spp., chitinase production in solid substrate fermentation (SSF) on four media and mycoparasitism in dual culture (confrontation assay) against four plant pathogenic fungi was studied. Seventy five Trichoderma isolates belonging to 35 species have been screened. The plant pathogenic fungi used in confrontation assay were Botrytis cinerea, Fusarium oxysporum f.sp. dianthi, Rhizoctonia solani and Sclerotinia sclerotiorum . The SSF media contained wheat bran, crude chitin (from crab shells, SIGMA) and salt solutions. The best performing isolates in mycoparasitism tests were Trichoderma flavofuscum, T.harzianum, T.inhamatum, T.koningii and T.strigosum. Some isolates exhibiting good mycoparasitism produced chitinase in SSF only at low or medium level. In contrary there were isolates with excellent extracellular chitinase production but their biocontrol potential did not belong to the leading group. Statistical methods have been used to evaluate the data.展开更多
Mycoparasitic species of Trichoderma are commercially applied as biological control agents against various fungal pathogens. The mycoparasitic interaction is host specific and includes recognition, attack and subseque...Mycoparasitic species of Trichoderma are commercially applied as biological control agents against various fungal pathogens. The mycoparasitic interaction is host specific and includes recognition, attack and subsequent penetration and killing of the host. Investigations on the underlying events revealed that Trichoderma responds to multiple signals from the host (e.g. lectins or other ligands such as low molecular weight components released from the host’s cell wall) and host attack is accompanied by morphological changes and the secretion of hydrolytic enzymes and antibiotics. Degradation of the cell wall of the host fungus is-besides glucanases and proteases-mainly achieved by chitinases. In vivo studies showed that the ech42 gene (encoding endochitinase 42) is expressed before physical contact of Trichoderma with its host, probably representing one of the earliest events in mycoparasitism, whereas Nag1 (N-acetylglucosaminidase) plays a key role in the general induction of the chitinolytic enzyme system of T. atroviride . Investigations on the responsible signal transduction pathways of T. atroviride led to the isolation of several genes encoding key components of the cAMP and MAP kinase signaling pathways, as alpha and β subunits of heterotrimeric G proteins, the regulatory subunit of cAMP-dependent protein kinase, adenylate cyclase, and three MAP kinases. Analysis of knockout mutants, generated by Agrobacterium-mediated transformation, revealed that at least two alpha-subunits of heterotrimeric G proteins are participating in mycoparasitism-related signal transduction. The Tga1 G alpha subunit was shown to be involved in mycoparasitism-related processes such as chitinase expression and overproduction of toxic secondary metabolites, whereas Tga3 was found to be completely avirulent showing defects in chitinase formation and host recognition.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During an investigation of mycoparasitic fungi on sclerotia of Sclerotinia sclerotiorum in China, Gliocladium species was consistently encountered and isolated from natural soils collected over the country. Colonization frequencies on sclerotia of S. sclerotiorum by the fungi ranged from 40% to 100% when the sclerotia were introduced into soils and coincubated at 22-24 ℃ for 4 weeks. Identification showed that G. roseum, G. virens and G. catenulatum were the dominant species among the 300 isolates gained. Molecular taxonomy of some Gliocladium isolates analysed by rDNA ITS sequence used ITS1 and ITS4 as primers was demonstrated to be identical with morphological classification. Reinoculation tests by placing surface-sterilized sclerotia onto colony of Gliocladium isolates for 7 days and then surface-sterilized again resulted in 100% sclerotia colonized. Dipping sclerotia with spores suspension of isolates for 5 min and incubated the sclerotia in Petri dishes at 25 ℃ for 24 h resulted in infection of the sclerotia. Microscopic observations indicated that Gliocladium isolates grew along the host hyphae, coiled around, formed appressorium-like structures, penetrated and degraded hyphae of S. sclerotiorum when dual-cultured in slides. Paraffin-section showed that sclerotial tissue collapsed when infected by Gliocladium isolates. Host range test demonstrated that the isolates suppressed hyphal growth of several plant pathogenic fungi, including S. sclerotiorum, Botrytis cinerea, Fusarium oxysporum, Alternaria tenuis, and Rhizoctonia solani. Activities of fungal cell wall degrading enzymes in cultural filtrates and parasitized sclerotia were detected. Results indicated that chitinase and glucanase were important factors involved in the mycoparasitism. A 51 kDa chitinase was isolated and purified from G. catenulatum HL-1-1 culture filtrate. A part of amino acid sequences of the enzyme has been analysed. It was found to have a high homogeneity to chitinase A from Stenotrophomonas maltophilia, while no homogeneity to chitinases from Trichoderma and other fungi. It was considered to be a new chitinase quite different from those produced by other mycoparasitic fungi. Optimum temperature and pH for the enzyme activity were 60 ℃ and 6.0. respectively. Ca 2+. promoted the activity, while Fe 3+., Cu 2+. and Ag+ inhibited it. Km of the chitinase was 2.832. The chitanase significantly inhibited the growth of hyphae, germination of conidia and sclerotia of some plant pathogenic fungi. Gene encoding this chitinase is being cloned from the total RNA of HL-1-1 isolates by RT-PCR. Biological characteristics of some Gliocladium isolates were studied. Results showed that temperature ranged from 22 ℃ to 30 ℃ was optimum for isolates hyphae growth and spore germination, while they could endure high temperature. Spores could not germinate below 5 ℃ or over 40 ℃ and lethal temperature of spores was 52 ℃. Optimum pH was 5.0 for hyphae growth and 6.0 for sporulation. Dark condition enhanced hyphae growth and light promoted good for sporulation. Glucose, sucrose and mannose were optimum C source for hyphal growth while glucose, xylose, soluble starch and chitin optimum for sporulation. Soybean cake powder, yeast extract and casein hydrolysate were good for hyphal growth while soybean cake powder, beef extract, urea and NaNO 3 were good N sources for sporulation. Extract of sclerotia promoted spore germination of the isolate,but D-glucose inhibited. Greenhouse tests confirmed that Gliocladium isolates GJ-1-1, SS-1-1, HL-1-1, SH-1-1, GW-1-1 promoted the seedling growth of soybean, cucumber, wheat and rice. Our primary studies showed that some Gliocladium isolates are of great potential in biocontrol of plant fungal diseases, such as soybean stem rot caused by S. sclerotiorum and cucumber root disease caused by R. solani.
文摘Trichoderma species are currently used as biocontrol agents for crop diseases caused by a number of fungal plant pathogens. However, their biocontrol performance in the field can be unreliable and it is likely that more consistent performance could be achieved through knowledge and manipulation of the genes involved. For example, induction of the genes could be optimised for variable environmental and physiological conditions, superior strains could be selected more effectively and novel strains could be created. One method by which Trichoderma species accomplish biocontrol is mycoparasitism. Several genes involved in the mycoparasitic interaction have previously been characterised, however these consist predominantly of those that encode enzymes that degrade fungal cell walls. In the current study subtractive hybridisation was used to target genes expressed when Trichoderma hamatum and the plant pathogen Sclerotinia sclerotiorum were cultured together, subtracting genes expressed when each are grown individually. This experimental design has the potential to yield T. hamatum genes involved in mycoparasitism of S. sclerotiorum, and S. sclerotiorum genes up-regulated in host defence. The cDNA fragments yielded by the subtraction were characterised with respect to expression, sequence and species of origin. A number of novel T. hamatum genes which were up-regulated during mycoparasitism were identified.
文摘The relationship between taxonomic status of Trichoderma spp., chitinase production in solid substrate fermentation (SSF) on four media and mycoparasitism in dual culture (confrontation assay) against four plant pathogenic fungi was studied. Seventy five Trichoderma isolates belonging to 35 species have been screened. The plant pathogenic fungi used in confrontation assay were Botrytis cinerea, Fusarium oxysporum f.sp. dianthi, Rhizoctonia solani and Sclerotinia sclerotiorum . The SSF media contained wheat bran, crude chitin (from crab shells, SIGMA) and salt solutions. The best performing isolates in mycoparasitism tests were Trichoderma flavofuscum, T.harzianum, T.inhamatum, T.koningii and T.strigosum. Some isolates exhibiting good mycoparasitism produced chitinase in SSF only at low or medium level. In contrary there were isolates with excellent extracellular chitinase production but their biocontrol potential did not belong to the leading group. Statistical methods have been used to evaluate the data.
文摘Mycoparasitic species of Trichoderma are commercially applied as biological control agents against various fungal pathogens. The mycoparasitic interaction is host specific and includes recognition, attack and subsequent penetration and killing of the host. Investigations on the underlying events revealed that Trichoderma responds to multiple signals from the host (e.g. lectins or other ligands such as low molecular weight components released from the host’s cell wall) and host attack is accompanied by morphological changes and the secretion of hydrolytic enzymes and antibiotics. Degradation of the cell wall of the host fungus is-besides glucanases and proteases-mainly achieved by chitinases. In vivo studies showed that the ech42 gene (encoding endochitinase 42) is expressed before physical contact of Trichoderma with its host, probably representing one of the earliest events in mycoparasitism, whereas Nag1 (N-acetylglucosaminidase) plays a key role in the general induction of the chitinolytic enzyme system of T. atroviride . Investigations on the responsible signal transduction pathways of T. atroviride led to the isolation of several genes encoding key components of the cAMP and MAP kinase signaling pathways, as alpha and β subunits of heterotrimeric G proteins, the regulatory subunit of cAMP-dependent protein kinase, adenylate cyclase, and three MAP kinases. Analysis of knockout mutants, generated by Agrobacterium-mediated transformation, revealed that at least two alpha-subunits of heterotrimeric G proteins are participating in mycoparasitism-related signal transduction. The Tga1 G alpha subunit was shown to be involved in mycoparasitism-related processes such as chitinase expression and overproduction of toxic secondary metabolites, whereas Tga3 was found to be completely avirulent showing defects in chitinase formation and host recognition.
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.
