Plant diseases heavily affct plant growth and crop yield even in modern agriculture. Control its difficult because pathogens mutate frequently, and this leads in frequent breaking of disease resistance in commercial c...Plant diseases heavily affct plant growth and crop yield even in modern agriculture. Control its difficult because pathogens mutate frequently, and this leads in frequent breaking of disease resistance in commercial cultivars. The excessive application of chemical pesticides is not only producing pesticide-resistant pathogens, but it is harming the environment threatening the health of human beings. Therefore, the use of biological control agents (BCA) may provide an environmental friendly alternative to chemicals for plant disease control. Hypersensitive response (HR) and systemic acquired resistance (SAR) are the typical expressions of plant defense reactions. Once SAR is established,, the plants exhibits a broad-spectrum of disease resistance against pathogen attack. Researchers have identified elicitor proteins, such as elicitins and harpins, which activate plant defense reactions. It would be useful to explore the possibility of using biological control agents to induce a status of SAR in crop plants. Trichoderma viride is an ubiquitous soil saprophyte and a biological control agent acting by competition for nutrients, antibiosis, and mycoparasitism. If T. viride could be used as a producer and carrier of an elicitor protein, it may be used as a novel BCA specifically active on some plants. To test this possibility, we used cryptogein, a proteinaceous elicitor secreted by Phytophthora cryptogea, to bio-engineering T. viride . The plasmid containing the Crypt gene or its mutated form, was introduced into T. viride genome by using the restriction enzyme mediated integration (REMI) method. The transformed T. viride was able to produce the Crypt protein and to improve disease resistance when the mutants were applied on tobacco plants. In summary our study included: 1. Construction of pCSNTCC and pCSNTCCm plasmids: Crypt gene was mutated by changing the K at position 13 of Crypt into a V (the mutated form was named CryK13V) as described elsewhere. In order allow secretion of the transgenic protein in T. viride cells, a signal sequence of a chitinase gene from Trichoderma (ThChi) was fused to the 5’ end of Crypt and CryK13V. The chimeric genes were placed under the control of trpC promoter in the vector pCSN43. A hygromycin resistant gene was introduced into the vectors, thus obtaining the plasmids pCSNTCC (for Crypt gene) and pCSNTCCm (CrypK13V) . 2. Establishment of a T. viride transformation system:The optimum conditions for T. viride protoplasts isolation and regeneration from were determined. For protoplast isolation, 24 hours-old hyphae of T. viride were digested with 4 mg/mL of Glucanex in phosphate buffer (pH 6.98) for 4 hours at 30 ℃, with a protoplast yield of 4.7×107 colony forming unit/mL. The maximum regeneration rate (14.5%) was obtained in the CM medium containing 0.3 mol/L KCl and 0.3 mol/L inositol. Plasmids pCSNTCC and pCSNTCCm were transformed into the protoplasts of T. viride by a Xho I restriction enzyme-mediated integration, with an efficiency of 1-2 transformants per microgram of DNA. Thirty transformants were obtained, TV-1 to TV-20 for Crypt gene and TV-21 to TV-30 for CrypK13V gene. The presence of the hygromycin resistance gene in the transformants was determined by polymerase chain reactions. The elicitor protein was detected in the culture media by western blot analysis but not inside the cells. The result indicated that the exogenous gene was expressed in T. viride , but the transgenic protein was entirely secreted into the culture media. 3. Expression of Crypt gene in T. viride enhanced plant disease resistance:Tobacco plants (4-6 week-old) were treated with spores of the transgenic or the wild-type T. viride applied to the soil. After ten days the plants or detached leaves were inoculated with Phytophthora parasitica var nicotianae, Alternaria alternata, Pseudomonas syringae pv. tabaci (Pst), or Tobacco mosaic virus (TMV). The lesions caused by TMV were suppressed by the treatment with the transgenic T. viride as compared with the wild-type展开更多
Fusarium moniliforme Sheld.is a rice pathogenic fungus and causes the disease called Bakanae,which has increasingly damaged rice production in the recent years. Trichoderma spp. has been one of the most widely used bi...Fusarium moniliforme Sheld.is a rice pathogenic fungus and causes the disease called Bakanae,which has increasingly damaged rice production in the recent years. Trichoderma spp. has been one of the most widely used biological control agent of plant disease. By geneticaly labelling F. moniliforme with the GFP reporter gene, we have studied the antagonistic action of Trichoderma viride against this pathogenic fungus. The binary GFP reporter vector pCHF3-35S∷GFP was constructed, which carries the gfp gene driven by the CaMv35S promoter. The vector was transformed into F. moniliforme via Agrobacterium.The mycoparasitism of T.viride against F.moniliforme was tested by dual culture and examined with fluorescence microscope. The result of the dual culture showed that the T.viride maintained a strong competitive ability against F. moniliforme , by growing on the top of the pathogen colony. Fluorescence microscope observation indicated that attacked hyphae of F. moniliform were distorted, swollen or broken. This indicate an enzymatic by T.viride to degrade the host cell walls and used the cell contents as a source of nutrients (Fig 1) .展开更多
The pectin is a backbone of the plant cell wall, its network structure will systemicly resolve when the plant cell splits up and forms. The pectinase produced by Rhizoctonia mainly acts on the pectin of cell wall, and...The pectin is a backbone of the plant cell wall, its network structure will systemicly resolve when the plant cell splits up and forms. The pectinase produced by Rhizoctonia mainly acts on the pectin of cell wall, and causes the maceration of tissue and the death of protoplast. Polygalacturonase (PG) can decompose the galacturonic acid of disease tissue. The research defined the PG activities of extracellular metabolite of the different virulence Rhizoctonia isolates, and testifid the effect of Trichoderma viride to PG activities, and clarified the mechanisms of biocontrol by Trichoderma. The test methods as following: Firstly, to select the isolates of different virulence: WK-47, WK-141 and WK-160 strain of Rhizoctonia AG-D and YW-2 strain of Rhizoctonia AG1-IA and TCS-1 strain of Trichoderma viride. Secondly, to culture TCS-1 on PD, and draw a group of fermented liquid in every 24 hours, and draw 7 times. Thirdly, to culture quietly Rhizoctonia isolates with Czapek-Dox at 25℃ for 15 days, filter and centrifuge (2350 g×30 min), fetch the clear liquid, put it into the ammonium sulfate according to 60% saturation degree, put it quietly for 30 min at 4℃, centrifuge (21000 g×30 min) at 4℃, remove the clear liquid, dissolve the deposit with sodium acetate buffer (25 mmol/L, pH5.5), dialysis for 48 h in the same buffer,and change the buffer every 12 h, Fourthly, to put TCS-1 fermented broth of different times in the tubes, one mL per a tube, add 0.5 mL PG to every tube, react for 4 h in 30 ℃ water, the same time fetch the test tube filled with the same treated liquid that was not dealed in 30℃ water.Finally,to testify PG activities with DNS’s test. In all, PG of Rhizoctonia had high activities and virulence. The conrtrol efficacy of T.viride to PG activities of WK-47, WK-141, WK-160 and YW-2 were 95%,94%,95%,92% separately, fermented time had a great influence to control efficacy, the third fermented broth did the best. Through effect to PG activities T. viride can reduce the virulence of Rhizoctonia, and protect the hosts. The specific mechanism, qualitative and quantitative research of antagonistic substance in the fermented broth will be further carried out.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 aureoviride mutant T1010 was cultured in media containing different nutrients. Statistics showed significant differences and the results described below: The C/N tested were 3∶1, 6∶1, 12∶1, 24∶1,48∶1....Trichoderma aureoviride mutant T1010 was cultured in media containing different nutrients. Statistics showed significant differences and the results described below: The C/N tested were 3∶1, 6∶1, 12∶1, 24∶1,48∶1. The best C/N was 24∶1. The amount of spores was 4.0×109 cfu/plate. The best carbon source among glucose, sucrose, maltose, lactose, cellulose and starch was the last one. With a starch-containing medium, T1010 produced 5.3×109 cfu/plate. The asparagine was the best nitrogen source. T1010 absorbed it easily and produced 9.6×109 cfu/plate. K, Mg, P, S, Fe were important to support T1010 growth and sporulation. In the absence of K, P, and Fe, Trichoderma grew poorly, while Mg didn’t seem to help Trichoderma colony development. A little amount of vitamins allwed T1010 to grow better. This data allowed us to improve the cultivation of Trichoderma at industrial level.展开更多
A transgenic strain of Trichoderma atroviride that expresses the Aspergillus niger glucose oxidase gene goxA under a homologous pathogen-inducible promoter (nag1) has been constructed, with the aim of increasing the a...A transgenic strain of Trichoderma atroviride that expresses the Aspergillus niger glucose oxidase gene goxA under a homologous pathogen-inducible promoter (nag1) has been constructed, with the aim of increasing the ability of this biocontrol agent (BCA) to attack phytopathogenic fungi and enhance plant systemic disease resistance. The sporulation and growth rate of the transgenic progenies were similar to the wild-type strain P1. goxA expression occurred immediately after contact with the plant pathogen, and the glucose oxidase formed was secreted extracellularly. The transformed strain SJ3 4, containing 12-14 copies of the transgene, produced significantly less N-acetyl-glucosaminidase and endochitinase then wild type. However, the ability of its culture filtrate to inhibit the germination of Botrytis cinerea spores was increased by about 3-fold. In comparison to P1, the transgenic strain more quickly overgrew and lysed in vitro the pathogens Rhizoctonia solani and Pythium ultimum. In assays in vivo SJ3 4 showed a highly improved biocontrol ability in soil heavily infested with those pathogens, where the wild type was unable to protect the plant and allow seeds to germinate. The Trichoderma-gox was able to induce a much higher level of systemic resistance against the foliar pathogen B. cinerea, as compared to the parent strain. This work demonstrate that i) heterologous genes driven by pathogen-inducible promoters can improve the biocontrol and Induced Systemic Resistance properties of fungal BCAs such as Trichoderma spp., and ii) these microbes can be used as vectors to provide the plant with useful molecules able, for instance, to increase pathogen展开更多
The quantitative expression and the regulation of chitinase-encoding genes ech30, ech42 and nag1 in Trichoderma atroviride P1 under varying growth conditions were investigated using real-time RT-PCR, principle compone...The quantitative expression and the regulation of chitinase-encoding genes ech30, ech42 and nag1 in Trichoderma atroviride P1 under varying growth conditions were investigated using real-time RT-PCR, principle component and multivariate analyses. Twelve media combinations including 0.1% and 3% glucose as carbon source and no (0 mmol/L), low (10 mmol/L) and high (100 mmol/L) ammonium acetate as nitrogen source combined with or without colloidal chitin at 3 time intervals and 2 replications were applied to current study. The real-time RT-PCR analysis showed that the expression of ech30, ech42 and nag1 was regulated by the interaction of nitrogen, glucose and chitin under different growth conditions. The highest and earliest expressions of ech30 were induced by glucose and nitrogen starvation i.e. 0.1% glucose and 10 mmol/L ammonium acetate in the growth media. This was also the case for ech42 and nag1 but at a relatively low level. In contrast, high (3%) glucose and high (100 mmol/L) ammonium acetate concentrations repressed the expression of all the genes studied. These results were confirmed by principle component and multivariate analyses. The effect of chitin on ech30, ech42 and nag1 expression varied depending on the concentrations of glucose and ammonium acetate.展开更多
Three genes encoding for fungal cell wall degrading enzymes (CWDE), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possib...Three genes encoding for fungal cell wall degrading enzymes (CWDE), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations. The coding sequences were placed downstream of the rice actin promoter and all vectors were used to transform rice plants. A total of more than 1,800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. Expression in plant was obtained for all the fungal genes used singly or in combinations. The ech42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani, while the exochitinase-encoding gene, nag70, had a lesser effect. The expression level of endochitinase but not of the exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the positive effect of endochitinase on disease resistance when two genes were co-expressed in transgenic rice. Improved resistance to Magnaporthe grisea was found in all types of regenerated plants, including those with the gluc78 gene alone, while a few lines expressing either ech42 or nag70 appeared to be immune to this pathogen. Transgenic plants expressing the gluc78 gene alone were stunted and only few of them survived, even though they showed resistance to M. grisea. However, combination with either one of the two other genes (ech42, nag70) as included in the same T-DNA region, reduced the negative effect of gluc78 on plant growth. This is the first report of single or multiple of expression of transgens encoding CWDEs that results in resistance to blast and sheath blight in rice.展开更多
文摘Plant diseases heavily affct plant growth and crop yield even in modern agriculture. Control its difficult because pathogens mutate frequently, and this leads in frequent breaking of disease resistance in commercial cultivars. The excessive application of chemical pesticides is not only producing pesticide-resistant pathogens, but it is harming the environment threatening the health of human beings. Therefore, the use of biological control agents (BCA) may provide an environmental friendly alternative to chemicals for plant disease control. Hypersensitive response (HR) and systemic acquired resistance (SAR) are the typical expressions of plant defense reactions. Once SAR is established,, the plants exhibits a broad-spectrum of disease resistance against pathogen attack. Researchers have identified elicitor proteins, such as elicitins and harpins, which activate plant defense reactions. It would be useful to explore the possibility of using biological control agents to induce a status of SAR in crop plants. Trichoderma viride is an ubiquitous soil saprophyte and a biological control agent acting by competition for nutrients, antibiosis, and mycoparasitism. If T. viride could be used as a producer and carrier of an elicitor protein, it may be used as a novel BCA specifically active on some plants. To test this possibility, we used cryptogein, a proteinaceous elicitor secreted by Phytophthora cryptogea, to bio-engineering T. viride . The plasmid containing the Crypt gene or its mutated form, was introduced into T. viride genome by using the restriction enzyme mediated integration (REMI) method. The transformed T. viride was able to produce the Crypt protein and to improve disease resistance when the mutants were applied on tobacco plants. In summary our study included: 1. Construction of pCSNTCC and pCSNTCCm plasmids: Crypt gene was mutated by changing the K at position 13 of Crypt into a V (the mutated form was named CryK13V) as described elsewhere. In order allow secretion of the transgenic protein in T. viride cells, a signal sequence of a chitinase gene from Trichoderma (ThChi) was fused to the 5’ end of Crypt and CryK13V. The chimeric genes were placed under the control of trpC promoter in the vector pCSN43. A hygromycin resistant gene was introduced into the vectors, thus obtaining the plasmids pCSNTCC (for Crypt gene) and pCSNTCCm (CrypK13V) . 2. Establishment of a T. viride transformation system:The optimum conditions for T. viride protoplasts isolation and regeneration from were determined. For protoplast isolation, 24 hours-old hyphae of T. viride were digested with 4 mg/mL of Glucanex in phosphate buffer (pH 6.98) for 4 hours at 30 ℃, with a protoplast yield of 4.7×107 colony forming unit/mL. The maximum regeneration rate (14.5%) was obtained in the CM medium containing 0.3 mol/L KCl and 0.3 mol/L inositol. Plasmids pCSNTCC and pCSNTCCm were transformed into the protoplasts of T. viride by a Xho I restriction enzyme-mediated integration, with an efficiency of 1-2 transformants per microgram of DNA. Thirty transformants were obtained, TV-1 to TV-20 for Crypt gene and TV-21 to TV-30 for CrypK13V gene. The presence of the hygromycin resistance gene in the transformants was determined by polymerase chain reactions. The elicitor protein was detected in the culture media by western blot analysis but not inside the cells. The result indicated that the exogenous gene was expressed in T. viride , but the transgenic protein was entirely secreted into the culture media. 3. Expression of Crypt gene in T. viride enhanced plant disease resistance:Tobacco plants (4-6 week-old) were treated with spores of the transgenic or the wild-type T. viride applied to the soil. After ten days the plants or detached leaves were inoculated with Phytophthora parasitica var nicotianae, Alternaria alternata, Pseudomonas syringae pv. tabaci (Pst), or Tobacco mosaic virus (TMV). The lesions caused by TMV were suppressed by the treatment with the transgenic T. viride as compared with the wild-type
文摘Fusarium moniliforme Sheld.is a rice pathogenic fungus and causes the disease called Bakanae,which has increasingly damaged rice production in the recent years. Trichoderma spp. has been one of the most widely used biological control agent of plant disease. By geneticaly labelling F. moniliforme with the GFP reporter gene, we have studied the antagonistic action of Trichoderma viride against this pathogenic fungus. The binary GFP reporter vector pCHF3-35S∷GFP was constructed, which carries the gfp gene driven by the CaMv35S promoter. The vector was transformed into F. moniliforme via Agrobacterium.The mycoparasitism of T.viride against F.moniliforme was tested by dual culture and examined with fluorescence microscope. The result of the dual culture showed that the T.viride maintained a strong competitive ability against F. moniliforme , by growing on the top of the pathogen colony. Fluorescence microscope observation indicated that attacked hyphae of F. moniliform were distorted, swollen or broken. This indicate an enzymatic by T.viride to degrade the host cell walls and used the cell contents as a source of nutrients (Fig 1) .
文摘The pectin is a backbone of the plant cell wall, its network structure will systemicly resolve when the plant cell splits up and forms. The pectinase produced by Rhizoctonia mainly acts on the pectin of cell wall, and causes the maceration of tissue and the death of protoplast. Polygalacturonase (PG) can decompose the galacturonic acid of disease tissue. The research defined the PG activities of extracellular metabolite of the different virulence Rhizoctonia isolates, and testifid the effect of Trichoderma viride to PG activities, and clarified the mechanisms of biocontrol by Trichoderma. The test methods as following: Firstly, to select the isolates of different virulence: WK-47, WK-141 and WK-160 strain of Rhizoctonia AG-D and YW-2 strain of Rhizoctonia AG1-IA and TCS-1 strain of Trichoderma viride. Secondly, to culture TCS-1 on PD, and draw a group of fermented liquid in every 24 hours, and draw 7 times. Thirdly, to culture quietly Rhizoctonia isolates with Czapek-Dox at 25℃ for 15 days, filter and centrifuge (2350 g×30 min), fetch the clear liquid, put it into the ammonium sulfate according to 60% saturation degree, put it quietly for 30 min at 4℃, centrifuge (21000 g×30 min) at 4℃, remove the clear liquid, dissolve the deposit with sodium acetate buffer (25 mmol/L, pH5.5), dialysis for 48 h in the same buffer,and change the buffer every 12 h, Fourthly, to put TCS-1 fermented broth of different times in the tubes, one mL per a tube, add 0.5 mL PG to every tube, react for 4 h in 30 ℃ water, the same time fetch the test tube filled with the same treated liquid that was not dealed in 30℃ water.Finally,to testify PG activities with DNS’s test. In all, PG of Rhizoctonia had high activities and virulence. The conrtrol efficacy of T.viride to PG activities of WK-47, WK-141, WK-160 and YW-2 were 95%,94%,95%,92% separately, fermented time had a great influence to control efficacy, the third fermented broth did the best. Through effect to PG activities T. viride can reduce the virulence of Rhizoctonia, and protect the hosts. The specific mechanism, qualitative and quantitative research of antagonistic substance in the fermented broth will be further carried out.
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 aureoviride mutant T1010 was cultured in media containing different nutrients. Statistics showed significant differences and the results described below: The C/N tested were 3∶1, 6∶1, 12∶1, 24∶1,48∶1. The best C/N was 24∶1. The amount of spores was 4.0×109 cfu/plate. The best carbon source among glucose, sucrose, maltose, lactose, cellulose and starch was the last one. With a starch-containing medium, T1010 produced 5.3×109 cfu/plate. The asparagine was the best nitrogen source. T1010 absorbed it easily and produced 9.6×109 cfu/plate. K, Mg, P, S, Fe were important to support T1010 growth and sporulation. In the absence of K, P, and Fe, Trichoderma grew poorly, while Mg didn’t seem to help Trichoderma colony development. A little amount of vitamins allwed T1010 to grow better. This data allowed us to improve the cultivation of Trichoderma at industrial level.
文摘A transgenic strain of Trichoderma atroviride that expresses the Aspergillus niger glucose oxidase gene goxA under a homologous pathogen-inducible promoter (nag1) has been constructed, with the aim of increasing the ability of this biocontrol agent (BCA) to attack phytopathogenic fungi and enhance plant systemic disease resistance. The sporulation and growth rate of the transgenic progenies were similar to the wild-type strain P1. goxA expression occurred immediately after contact with the plant pathogen, and the glucose oxidase formed was secreted extracellularly. The transformed strain SJ3 4, containing 12-14 copies of the transgene, produced significantly less N-acetyl-glucosaminidase and endochitinase then wild type. However, the ability of its culture filtrate to inhibit the germination of Botrytis cinerea spores was increased by about 3-fold. In comparison to P1, the transgenic strain more quickly overgrew and lysed in vitro the pathogens Rhizoctonia solani and Pythium ultimum. In assays in vivo SJ3 4 showed a highly improved biocontrol ability in soil heavily infested with those pathogens, where the wild type was unable to protect the plant and allow seeds to germinate. The Trichoderma-gox was able to induce a much higher level of systemic resistance against the foliar pathogen B. cinerea, as compared to the parent strain. This work demonstrate that i) heterologous genes driven by pathogen-inducible promoters can improve the biocontrol and Induced Systemic Resistance properties of fungal BCAs such as Trichoderma spp., and ii) these microbes can be used as vectors to provide the plant with useful molecules able, for instance, to increase pathogen
文摘The quantitative expression and the regulation of chitinase-encoding genes ech30, ech42 and nag1 in Trichoderma atroviride P1 under varying growth conditions were investigated using real-time RT-PCR, principle component and multivariate analyses. Twelve media combinations including 0.1% and 3% glucose as carbon source and no (0 mmol/L), low (10 mmol/L) and high (100 mmol/L) ammonium acetate as nitrogen source combined with or without colloidal chitin at 3 time intervals and 2 replications were applied to current study. The real-time RT-PCR analysis showed that the expression of ech30, ech42 and nag1 was regulated by the interaction of nitrogen, glucose and chitin under different growth conditions. The highest and earliest expressions of ech30 were induced by glucose and nitrogen starvation i.e. 0.1% glucose and 10 mmol/L ammonium acetate in the growth media. This was also the case for ech42 and nag1 but at a relatively low level. In contrast, high (3%) glucose and high (100 mmol/L) ammonium acetate concentrations repressed the expression of all the genes studied. These results were confirmed by principle component and multivariate analyses. The effect of chitin on ech30, ech42 and nag1 expression varied depending on the concentrations of glucose and ammonium acetate.
文摘Three genes encoding for fungal cell wall degrading enzymes (CWDE), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations. The coding sequences were placed downstream of the rice actin promoter and all vectors were used to transform rice plants. A total of more than 1,800 independently regenerated plantlets in seven different populations (for each of the three genes and each of the four gene combinations) were obtained. Expression in plant was obtained for all the fungal genes used singly or in combinations. The ech42 gene encoding for an endochitinase increased resistance to sheath blight caused by Rhizoctonia solani, while the exochitinase-encoding gene, nag70, had a lesser effect. The expression level of endochitinase but not of the exochitinase was correlated with disease resistance. Nevertheless, exochitinase enhanced the positive effect of endochitinase on disease resistance when two genes were co-expressed in transgenic rice. Improved resistance to Magnaporthe grisea was found in all types of regenerated plants, including those with the gluc78 gene alone, while a few lines expressing either ech42 or nag70 appeared to be immune to this pathogen. Transgenic plants expressing the gluc78 gene alone were stunted and only few of them survived, even though they showed resistance to M. grisea. However, combination with either one of the two other genes (ech42, nag70) as included in the same T-DNA region, reduced the negative effect of gluc78 on plant growth. This is the first report of single or multiple of expression of transgens encoding CWDEs that results in resistance to blast and sheath blight in rice.
