A transcriptomic database was constructed to study the biocontrol mechanisms of Trichoderma harzianum ACCC30371 using high quality UniGenes following growth in eight culture media[(1/2PD,minimal medium MM(containing d...A transcriptomic database was constructed to study the biocontrol mechanisms of Trichoderma harzianum ACCC30371 using high quality UniGenes following growth in eight culture media[(1/2PD,minimal medium MM(containing dextrose 10 g L^-1),C starvation medium(derived from MM without dextrose),N starvation medium(derived from MM without ammonium sulphate),and four kinds of phytopathogenic fungi cell wall media].A 4 Gbp transcriptome was generated and 96.7%of the database had a sequencing error rate less than 1%.A total of 25,013 UniGene sequences were obtained with a mean length of 1135 nt.There were 2571 sequences longer than 3000 nt.The National Center for Biotechnology Information Accession number of this transcriptome is SRR8382572.There were 16,360 Unigenes annotated to the Nr protein database,9875 to the SwissProt database,10,266 to the KEGG database,7164 to the COG database,and 1508 to the GO database along with their protein functional annotations.There were 16,723 functional genes identified.We identified 402 bio-control genes,including 14 related to competition,311 to mycoparasitism,76 to antibiosis,and one related to eliciting a plant response.This shows that T.harzianum ACCC30371 has integrated biocontrol mechanisms,and of these mechanisms,mycoparasitism is the most prevalent.Antibiosis and induced systemic resistance also play important roles.These results provide a foundation for further research into the biocontrol mechanisms of Trichoderma,as well as the development and utilization of biological fungicides.展开更多
The palm, Elaeis guineensis, has its origins in Africa but is planted on a commercial basis in several countries. Statistics for 2002 showed that in the lead for land mass under oil palm cultivation is Indonesia, at 3...The palm, Elaeis guineensis, has its origins in Africa but is planted on a commercial basis in several countries. Statistics for 2002 showed that in the lead for land mass under oil palm cultivation is Indonesia, at 3,769,000. ha, followed by Malaysia at 3,376,000. ha; however, the world’s leading producer of palm oil is still Malaysia, since the 1970’s. Both countries are predicted to produce 82.4% of the world’s palm oil production by the year 2005. However, the palm is susceptible to basal stem rot, a devastating disease which results in direct loss of field stands and to which no effective chemical control is yet available. Caused by Ganoderma boninense, infected palms appear symptomless, at the first sign of disease, at least 50% of the internal trunk tissue stem would have actually rotted. This study investigated the efficacy of Trichoderma harzianum (isolate FA 1132) as a biological control agent, using 6-month old oil palm seedlings as models and the experiment performed in a greenhouse at 29- 30 ℃. ambient conditions. The plants were artificially infected with G. boninense and a conidial suspension of 1×109-9×109 spores/mL was applied as a soil drench at 1L/plant every 2 weeks for 20 weeks. The parameters examined were efficacy of the biocontrol agent and the effect of Trichoderma- incorporated mulch in addition to the soil drench. Efficacy was assessed in terms disease severity index (DSI) where a higher percentage indicates a higher severity. Results showed that infection first sets in on untreated plants at week 12 and got worse progressively. The completely untreated plants were all infected and the DSI at 20 weeks after infection (wa.i.) was 92.5%. Plants given only a Trichoderma -infused food base supplement without conidial suspension gave a DSI of 70% whereas those given a conidial soil drench without supplemental food base gave a DSI of 85% at 20 w.a.i. Infected plants given a conidial treatment together with a food base supplement gave a DSI of 5% at 20 w.a.i. This investigation showed that there is potential in the use of T. harzianum (isolate FA 1132) as a biological control agent of basal stem rot and warrant further studies in its mass propagation for field trials.展开更多
In commercial greenhouses, senescent flower petals or flowers of vegetables such as tomato, strawberry, hot pepper and zucchini squash were blighted to be removed from fruits within five days after spraying of Trichod...In commercial greenhouses, senescent flower petals or flowers of vegetables such as tomato, strawberry, hot pepper and zucchini squash were blighted to be removed from fruits within five days after spraying of Trichoderma harzianum YC459 (TORY, JGreen Inc.), a biocontrol agent with good and consistent efficacy as chemical fungicides for the control of gray mold rot caused by B. cinerea. The mechanism for selective colonization of senescent floral tissues by T. harzianum YC459 was elucidated using fresh and senescent (4 days and 14 days after pollination, respectively) floral tissues of zucchini squash (Cucurbita moschata Duchesne) . The spores of T. harzianum YC459 were produced much more on water agar and liquid culture media supplemented with 5% dry powder of senescent floral tissues than with fresh tissues during 15 days incubation. Mycelial growth was also much better in the media with senescent tissues than with fresh tissues. Enzyme activities of carboxymethyl cellulase, amylase and polygalacturonase in the liquid media, which might be involved in the colonization and degradation of tissues by T. harzianum YC459 were compared. The activities of three enzymes were significantly higher in the media with senescent floral tissues than with fresh floral tissues reaching to the maximum during 9 to 12 days of incubation. Especially, the activities of carboxymethyl cellulase and polygalacturonase of T. harzianum YC459 were much higher than those of other Trichoderma species, T. asperellum, T. viride and T. koningii in the liquid media with senescent floral tissues. Based on the results, the selective colonization and degradation of senescent floral tissues, an important habitat for B. cinerea, may be another mechanism for the biocontrol of gray mold rot of vegetables by T. harzianum YC459.展开更多
Over forty Trichoderma harzianum isolates have been screened in solid substrate fermentation (SSF) for chitinase production. Strains were isolated from Asian soil and tree bark samples. Identification was performed in...Over forty Trichoderma harzianum isolates have been screened in solid substrate fermentation (SSF) for chitinase production. Strains were isolated from Asian soil and tree bark samples. Identification was performed in Canada and Austria by classical and molecular taxonomical methods. Four SSF media were used for the screening. They contained wheat bran, crude chitin from crab shells (SIGMA) and different salt solutions for wetting of the substrate. In a five day fermentation at 30°C the best chitinase producers were T. harzianum TUB F-691, TUB F-693, TUB F-699, TUB F-700, TUB F-927, TUB F-947 and TUB F-972 isolates (TUB = Technical University of Budapest culture collection) . The best producers yielded between 3.5-5.5 International Unit/g dry matter (DM) chitinase. T. harzianum TUB F-947 was selected for further optimization. This strain was isolated from a soil of Thailand. The optimum moisture content (67%), optimal alternative substrate-carrier for SSF (wheat straw) and chitin content of the substrate (30%) were determined. Temperature and pH optima of chitinase produced by T. harzianum TUB F-947 were found to be at 50°C and 5.0, respectively. The enzyme complex was thermally not stable at 50 ℃. Medium optimization experiments to enhance the chitinase production using statistical methods also have been performed.展开更多
After comparison of Trichoderma population density and test of colonization ability in rhizospheres were conducted. Auxotrophic mutants of T. harzianum tolerant to carbendazim and UV-light were obtained by UV-light mu...After comparison of Trichoderma population density and test of colonization ability in rhizospheres were conducted. Auxotrophic mutants of T. harzianum tolerant to carbendazim and UV-light were obtained by UV-light mutagenesis and carbendazim stress on PDA medium and a basis medium with hot pepper root exudation by adding the fungicide. The results showed: all four different isolates of Trichoderma had certain colonization ability in rhizosphere with the characteristic of growing as roots and distributing many more around root tips. The ability, however, was different for the 4 isolates, showing the wild isolates colonized weakly in rhizosphere. Around 5% spores alive and mutants could be obtained after the isolates were irradiated under a 20W UV-light at 25 cm distance for 5 min. The mutants tolerant to carbendazim were screened out by adding the fungicide into PDA medium, which increased resistance to the fungicide 100 times higher than their original isolates and showed auxotrophic. Three mutants,G7n,G20n and G5n, grew very well on PDA and a basic medium with hot pepper root exudation. Therefore, these mutants could be used as the isolates with good colonization ability for further research.展开更多
Trichoderma harzianum strain T22 controls various diseases of maize and other crops, including seedling and root rots caused by Pythium ultimum. Seedlings of inbred line Mo17 were grown from T22-treated or untreated s...Trichoderma harzianum strain T22 controls various diseases of maize and other crops, including seedling and root rots caused by Pythium ultimum. Seedlings of inbred line Mo17 were grown from T22-treated or untreated seeds in field soil or in field soil intested with the pathogen. Five days after planting, seedlings of Mo17 (5-days-old) were smaller in the presence of P. ultimum and larger in the presence of T22 relative to the control. The combination of T22 with P. ultimum (T22+ P. ultimum ) resulted in plants as large as T22 alone. Methods for protein extraction and 2-D gel electrophoresis were developed. Proteins in seedlings roots from the various treatments were separated on 2-D gels and analyzed using PDQuest TM. 2-D software. With seedlings produced from T22-treated seeds, there were 104 unmatched proteins and 164 matched proteins relative to the control, and 97 and 150 from the treatment with T22+ P. ultimum, respectively, however, with P. ultimum alone the numbers were much lower than above two treatments. Comparatively, there was very lower similarity of proteome patterns of seedling roots with T22 or P. ultimum or both to control seedlings, the correlative coefficient values were 0.72, 0.51 and 0.49 for the comparisons among control with T22, P. ultimum and T22+ P. ultimum, respectively. Moreover, correlative coefficient of proteome patterns between T22 with P. ultimum was only 0.65, and T22 fungal proteome were also not same as any one of seedling roots with various treatments. Taken together, the components in seedling root proteome seemed to be mostly coming from Mo17 plants themselves and affected strongly by either microbes, but the effects appeared to be stronger by P. ultimum than by T22. 41 spots were selected for protein mass fingerprinting identification, and most detected-spots were intensified in abundance by T22 or T22+ P.ultimum treatments such as pathogenesis-related protein and endochitnase etc. SOD (Mn) was found to be involved in the defensive reaction of host against P. ultimum because the protein only appeared in the treatment with T22 or T22+ P.ultimum. Besides, some proteins associated with host respiration, nutrition synthesis and transport appeared to be in coordination with defensive-related proteins against the damping off.展开更多
The effect of preliminary nutrient activation on the ability of conidia of the antagonist Trichoderma harzianum P1 (ThP1) to suppress Botrytis cinerea was investigated in laboratory, greenhouse and field trials. Preli...The effect of preliminary nutrient activation on the ability of conidia of the antagonist Trichoderma harzianum P1 (ThP1) to suppress Botrytis cinerea was investigated in laboratory, greenhouse and field trials. Preliminary nutrient activation at 21 ℃ accelerated subsequent germination of the antagonist at temperatures from 9 ℃ to 21 ℃; at ≥ 18 ℃ the germination time of preactivated ThP1 conidia did not differ significantly from that of B. cinerea. When coinoculated with B. cinerea, concentrated inocula of preactivated but ungerminated ThP1 conidia reduced in vitro germination of the pathogen by ≥ 87% at 12 ℃ to 25 ℃; initially-quiescent conidia achieved this level of suppression only at 25 ℃. Application of quiescent ThP1 conidia to detached strawberry flowers in moist chambers reduced infection by B. cinerea by ≥85% at 24 ℃, but only by 35% at 12 ℃. Preactivated conidia reduced infection by ≥60% at 12 ℃. Both quiescent and preactivated conidia significantly reduced latent infection in greenhouse-grown strawberries at a mean temperture of 19 ℃, while only preactivated conidia were effective in the field at a mean temperature of 14 ℃ on the day of treatment application. Trichoderma biocontrol isolates are most effective as highly concentrated inocula. Their antagonism to other fungi may be a result of pregermination respiration. In a nutrient-rich medium, almost all Trichoderma. atroviride P1 (P1) conidia initiated germination processes and increased respiration, even in dense suspensions. When 1×107 P1 conidia per mL were coinoculated with 1×105 B. cinerea conidia per mL, dissolved oxygen fell to <1% within 2 h and the pathogen failed to germinate. More dilute P1 suspensions consumed oxygen slowly enough to allow coinoculated B. cinerea to germinate. On nutrient-poor media, fewer P1 conidia initiated germination. Oxygen consumption by the inoculum and inhibition of B. cinerea were enhanced when P1 conidia were nutrient-activated before inoculation. Pregermination respiration also affected the competitive capacity of the antagonist on solid substrates, where respiratory CO 2 stimulated germination rate and initial colony growth. These parameters were directly correlated with inoculum concentration (R2≥ 0.97, P<0.01). After initiating germination, Trichoderma conidia became more sensitive to desiccation and were killed by drying after only two hours incubation on a nutrient-rich substrate at 23 ℃. Consideration of these effects of germination initiation on the competitive potential of Trichoderma in biological control will bee discussed.展开更多
基金The work was supported by the Fundamental Research Funds of the Central University,China(Grant Number 2572017AA03 and Grant Number 2572014BA15).
文摘A transcriptomic database was constructed to study the biocontrol mechanisms of Trichoderma harzianum ACCC30371 using high quality UniGenes following growth in eight culture media[(1/2PD,minimal medium MM(containing dextrose 10 g L^-1),C starvation medium(derived from MM without dextrose),N starvation medium(derived from MM without ammonium sulphate),and four kinds of phytopathogenic fungi cell wall media].A 4 Gbp transcriptome was generated and 96.7%of the database had a sequencing error rate less than 1%.A total of 25,013 UniGene sequences were obtained with a mean length of 1135 nt.There were 2571 sequences longer than 3000 nt.The National Center for Biotechnology Information Accession number of this transcriptome is SRR8382572.There were 16,360 Unigenes annotated to the Nr protein database,9875 to the SwissProt database,10,266 to the KEGG database,7164 to the COG database,and 1508 to the GO database along with their protein functional annotations.There were 16,723 functional genes identified.We identified 402 bio-control genes,including 14 related to competition,311 to mycoparasitism,76 to antibiosis,and one related to eliciting a plant response.This shows that T.harzianum ACCC30371 has integrated biocontrol mechanisms,and of these mechanisms,mycoparasitism is the most prevalent.Antibiosis and induced systemic resistance also play important roles.These results provide a foundation for further research into the biocontrol mechanisms of Trichoderma,as well as the development and utilization of biological fungicides.
文摘The palm, Elaeis guineensis, has its origins in Africa but is planted on a commercial basis in several countries. Statistics for 2002 showed that in the lead for land mass under oil palm cultivation is Indonesia, at 3,769,000. ha, followed by Malaysia at 3,376,000. ha; however, the world’s leading producer of palm oil is still Malaysia, since the 1970’s. Both countries are predicted to produce 82.4% of the world’s palm oil production by the year 2005. However, the palm is susceptible to basal stem rot, a devastating disease which results in direct loss of field stands and to which no effective chemical control is yet available. Caused by Ganoderma boninense, infected palms appear symptomless, at the first sign of disease, at least 50% of the internal trunk tissue stem would have actually rotted. This study investigated the efficacy of Trichoderma harzianum (isolate FA 1132) as a biological control agent, using 6-month old oil palm seedlings as models and the experiment performed in a greenhouse at 29- 30 ℃. ambient conditions. The plants were artificially infected with G. boninense and a conidial suspension of 1×109-9×109 spores/mL was applied as a soil drench at 1L/plant every 2 weeks for 20 weeks. The parameters examined were efficacy of the biocontrol agent and the effect of Trichoderma- incorporated mulch in addition to the soil drench. Efficacy was assessed in terms disease severity index (DSI) where a higher percentage indicates a higher severity. Results showed that infection first sets in on untreated plants at week 12 and got worse progressively. The completely untreated plants were all infected and the DSI at 20 weeks after infection (wa.i.) was 92.5%. Plants given only a Trichoderma -infused food base supplement without conidial suspension gave a DSI of 70% whereas those given a conidial soil drench without supplemental food base gave a DSI of 85% at 20 w.a.i. Infected plants given a conidial treatment together with a food base supplement gave a DSI of 5% at 20 w.a.i. This investigation showed that there is potential in the use of T. harzianum (isolate FA 1132) as a biological control agent of basal stem rot and warrant further studies in its mass propagation for field trials.
文摘In commercial greenhouses, senescent flower petals or flowers of vegetables such as tomato, strawberry, hot pepper and zucchini squash were blighted to be removed from fruits within five days after spraying of Trichoderma harzianum YC459 (TORY, JGreen Inc.), a biocontrol agent with good and consistent efficacy as chemical fungicides for the control of gray mold rot caused by B. cinerea. The mechanism for selective colonization of senescent floral tissues by T. harzianum YC459 was elucidated using fresh and senescent (4 days and 14 days after pollination, respectively) floral tissues of zucchini squash (Cucurbita moschata Duchesne) . The spores of T. harzianum YC459 were produced much more on water agar and liquid culture media supplemented with 5% dry powder of senescent floral tissues than with fresh tissues during 15 days incubation. Mycelial growth was also much better in the media with senescent tissues than with fresh tissues. Enzyme activities of carboxymethyl cellulase, amylase and polygalacturonase in the liquid media, which might be involved in the colonization and degradation of tissues by T. harzianum YC459 were compared. The activities of three enzymes were significantly higher in the media with senescent floral tissues than with fresh floral tissues reaching to the maximum during 9 to 12 days of incubation. Especially, the activities of carboxymethyl cellulase and polygalacturonase of T. harzianum YC459 were much higher than those of other Trichoderma species, T. asperellum, T. viride and T. koningii in the liquid media with senescent floral tissues. Based on the results, the selective colonization and degradation of senescent floral tissues, an important habitat for B. cinerea, may be another mechanism for the biocontrol of gray mold rot of vegetables by T. harzianum YC459.
文摘Over forty Trichoderma harzianum isolates have been screened in solid substrate fermentation (SSF) for chitinase production. Strains were isolated from Asian soil and tree bark samples. Identification was performed in Canada and Austria by classical and molecular taxonomical methods. Four SSF media were used for the screening. They contained wheat bran, crude chitin from crab shells (SIGMA) and different salt solutions for wetting of the substrate. In a five day fermentation at 30°C the best chitinase producers were T. harzianum TUB F-691, TUB F-693, TUB F-699, TUB F-700, TUB F-927, TUB F-947 and TUB F-972 isolates (TUB = Technical University of Budapest culture collection) . The best producers yielded between 3.5-5.5 International Unit/g dry matter (DM) chitinase. T. harzianum TUB F-947 was selected for further optimization. This strain was isolated from a soil of Thailand. The optimum moisture content (67%), optimal alternative substrate-carrier for SSF (wheat straw) and chitin content of the substrate (30%) were determined. Temperature and pH optima of chitinase produced by T. harzianum TUB F-947 were found to be at 50°C and 5.0, respectively. The enzyme complex was thermally not stable at 50 ℃. Medium optimization experiments to enhance the chitinase production using statistical methods also have been performed.
文摘After comparison of Trichoderma population density and test of colonization ability in rhizospheres were conducted. Auxotrophic mutants of T. harzianum tolerant to carbendazim and UV-light were obtained by UV-light mutagenesis and carbendazim stress on PDA medium and a basis medium with hot pepper root exudation by adding the fungicide. The results showed: all four different isolates of Trichoderma had certain colonization ability in rhizosphere with the characteristic of growing as roots and distributing many more around root tips. The ability, however, was different for the 4 isolates, showing the wild isolates colonized weakly in rhizosphere. Around 5% spores alive and mutants could be obtained after the isolates were irradiated under a 20W UV-light at 25 cm distance for 5 min. The mutants tolerant to carbendazim were screened out by adding the fungicide into PDA medium, which increased resistance to the fungicide 100 times higher than their original isolates and showed auxotrophic. Three mutants,G7n,G20n and G5n, grew very well on PDA and a basic medium with hot pepper root exudation. Therefore, these mutants could be used as the isolates with good colonization ability for further research.
文摘Trichoderma harzianum strain T22 controls various diseases of maize and other crops, including seedling and root rots caused by Pythium ultimum. Seedlings of inbred line Mo17 were grown from T22-treated or untreated seeds in field soil or in field soil intested with the pathogen. Five days after planting, seedlings of Mo17 (5-days-old) were smaller in the presence of P. ultimum and larger in the presence of T22 relative to the control. The combination of T22 with P. ultimum (T22+ P. ultimum ) resulted in plants as large as T22 alone. Methods for protein extraction and 2-D gel electrophoresis were developed. Proteins in seedlings roots from the various treatments were separated on 2-D gels and analyzed using PDQuest TM. 2-D software. With seedlings produced from T22-treated seeds, there were 104 unmatched proteins and 164 matched proteins relative to the control, and 97 and 150 from the treatment with T22+ P. ultimum, respectively, however, with P. ultimum alone the numbers were much lower than above two treatments. Comparatively, there was very lower similarity of proteome patterns of seedling roots with T22 or P. ultimum or both to control seedlings, the correlative coefficient values were 0.72, 0.51 and 0.49 for the comparisons among control with T22, P. ultimum and T22+ P. ultimum, respectively. Moreover, correlative coefficient of proteome patterns between T22 with P. ultimum was only 0.65, and T22 fungal proteome were also not same as any one of seedling roots with various treatments. Taken together, the components in seedling root proteome seemed to be mostly coming from Mo17 plants themselves and affected strongly by either microbes, but the effects appeared to be stronger by P. ultimum than by T22. 41 spots were selected for protein mass fingerprinting identification, and most detected-spots were intensified in abundance by T22 or T22+ P.ultimum treatments such as pathogenesis-related protein and endochitnase etc. SOD (Mn) was found to be involved in the defensive reaction of host against P. ultimum because the protein only appeared in the treatment with T22 or T22+ P.ultimum. Besides, some proteins associated with host respiration, nutrition synthesis and transport appeared to be in coordination with defensive-related proteins against the damping off.
文摘The effect of preliminary nutrient activation on the ability of conidia of the antagonist Trichoderma harzianum P1 (ThP1) to suppress Botrytis cinerea was investigated in laboratory, greenhouse and field trials. Preliminary nutrient activation at 21 ℃ accelerated subsequent germination of the antagonist at temperatures from 9 ℃ to 21 ℃; at ≥ 18 ℃ the germination time of preactivated ThP1 conidia did not differ significantly from that of B. cinerea. When coinoculated with B. cinerea, concentrated inocula of preactivated but ungerminated ThP1 conidia reduced in vitro germination of the pathogen by ≥ 87% at 12 ℃ to 25 ℃; initially-quiescent conidia achieved this level of suppression only at 25 ℃. Application of quiescent ThP1 conidia to detached strawberry flowers in moist chambers reduced infection by B. cinerea by ≥85% at 24 ℃, but only by 35% at 12 ℃. Preactivated conidia reduced infection by ≥60% at 12 ℃. Both quiescent and preactivated conidia significantly reduced latent infection in greenhouse-grown strawberries at a mean temperture of 19 ℃, while only preactivated conidia were effective in the field at a mean temperature of 14 ℃ on the day of treatment application. Trichoderma biocontrol isolates are most effective as highly concentrated inocula. Their antagonism to other fungi may be a result of pregermination respiration. In a nutrient-rich medium, almost all Trichoderma. atroviride P1 (P1) conidia initiated germination processes and increased respiration, even in dense suspensions. When 1×107 P1 conidia per mL were coinoculated with 1×105 B. cinerea conidia per mL, dissolved oxygen fell to <1% within 2 h and the pathogen failed to germinate. More dilute P1 suspensions consumed oxygen slowly enough to allow coinoculated B. cinerea to germinate. On nutrient-poor media, fewer P1 conidia initiated germination. Oxygen consumption by the inoculum and inhibition of B. cinerea were enhanced when P1 conidia were nutrient-activated before inoculation. Pregermination respiration also affected the competitive capacity of the antagonist on solid substrates, where respiratory CO 2 stimulated germination rate and initial colony growth. These parameters were directly correlated with inoculum concentration (R2≥ 0.97, P<0.01). After initiating germination, Trichoderma conidia became more sensitive to desiccation and were killed by drying after only two hours incubation on a nutrient-rich substrate at 23 ℃. Consideration of these effects of germination initiation on the competitive potential of Trichoderma in biological control will bee discussed.
