[Objective] This study aimed to investigate the feasibility of agricultural irri- gation with reclaimed water in Urumqi City. [Method] Chinese cabbages were used as experimental materials and irrigated with control wa...[Objective] This study aimed to investigate the feasibility of agricultural irri- gation with reclaimed water in Urumqi City. [Method] Chinese cabbages were used as experimental materials and irrigated with control water, 50% reclaimed water and 100% reclaimed water, and then the number of endophytic bacteria in Chinese cab- bages was measured. [Result] Using 50% reclaimed water, the fresh weight of Chi- nese cabbage was improved by 68.94%; however, with the deepening internalization of exogenous microorganism internalization, the total number of endophytic bacteria and coliform flock in Chinese cabbages treated with 50% reclaimed water was sig- nificantly higher than the other two treatment groups (P〈0.05). [Conclusion] Re- claimed water enhances both the weight of Chinese cabbage and the number of endophytic bacteria, which is unsuitable for irrigation of edible crops such as Chi- nese cabbage. Strengthening disinfection during the process of sewerage treatment could solve this problem.展开更多
For exploring the influences of application and residue of chemical fungi- cides on chlamydospore preparations of Trichoderma spp., the effects of seven chemical fungicides on chlamydospore germination and mycelia gro...For exploring the influences of application and residue of chemical fungi- cides on chlamydospore preparations of Trichoderma spp., the effects of seven chemical fungicides on chlamydospore germination and mycelia growth of two bio- control fungi T. harzianum 610 and T. Iongibrachiatum 758 were studied. Carben- dazim, tebuconazole and difenoconazole showed strong toxicities, thiram and car- bexin showed moderate toxicities, and metalaxyl showed Mycelia growth of the two strains was more sensitive to most tested fungicides than those of chlamydospore germination. Chlamydospore germination of 610 was more sensitive to tested fungicides than those of 758, and mycelia growth of 758 was more sensitive to most tested fungi- cides than those of 610. Among the seven fungicides, 98% carbendazim had the strongest effects (ECru values were 1.64 and 0.05μpg/ml), and 70% pentachloroni- trobenzene had the weakest effects (EC50 values were 1.64 and 0,05 μg/ml) for chlamydospore germination and mycelia growth of 610. As for 758, 98% carbendaz- im had the strongest inhibitory effects and 95% metalaxyl had the weakest inhibitory for chlamydospore germination of 756 (EC50 values were 0.62 and 1 108.61 μg/ml respectively), whereas 96.2% tebuconazole showed the strongest inhibitory effects for mycelia growth of 758 (EC= value was 0.32μg/ml), and 95% metalaxyl was the weakest (EC= value was 1 206.29 μg/ml). According to the applied concentration of different fungicides in practice, we concluded that chlamydospore preparations of 610 and 758 could not be combined with carbendazim, tebuconazole, thiram and carboxin for controlling plant diseases, and the pesticide residues to the biocontrol effects should be kept in mind. Chlamydospore preparations of 610 and 758 can be and difenoconazole for controlling plant dis- eases, 758 chlamydospore preparations and germinated chlamydospore of 610 can be combined with metalaxyl for controlling plant diseases, and pesticide residue risk was not serious.展开更多
In order to explore whether the endophytic Trichoderma strain P3.9 of loquat has an adverse effect on indigenous fungi in loquat rhizosphere soil, the quantitative change of aboriginal fungi is determined by dish dilu...In order to explore whether the endophytic Trichoderma strain P3.9 of loquat has an adverse effect on indigenous fungi in loquat rhizosphere soil, the quantitative change of aboriginal fungi is determined by dish dilution and plate colony-counting method with time changing in one season. The results showed that after the inoculation of endophytic Trichoderma strain P3.9, the total number of indigenous fungi in loquat rhizosphere soil had an obviously downward trend in contrast with the control which was without inoculation. For treatment groups, 5~60 d after inoculation, the number of indigenous fungi showed a cyclic upward-downward trend at a 10-d interval except for the insignificant changes from 35 to 40 d;60~90 d after inoculation, the upward-downward trend repeated at a 30-d interval. For the control group, the number of indigenous fungi first presented a downward-upward trend every 15 d during the period of 5~50 d, and then an upward-downward trend from the period of 50~70 d and the 75~85 d, and lastly continuous growth from 85 to 90 d;particularly, it did not vary greatly from 70 to 75 d. In general, the quantity of indigenous fungi is unstable in the control group which fluctuates more significantly than in the treatment group. The number of indigenous fungi in the treatment group was significantly lower than that in the control group. The Trichoderma strain P3.9 can inhibit indigenous fungi in loquat rhizosphere soil effectively.展开更多
The choice of active Trichoderma strains is important in designing effective and safe biocontrol applications. Many species of Trichoderma have multiple strategies for fungal antagonism and indirect effects on plant h...The choice of active Trichoderma strains is important in designing effective and safe biocontrol applications. Many species of Trichoderma have multiple strategies for fungal antagonism and indirect effects on plant health, such as growth promotion, systemic resistance induction and fertility improvements. Some strains are powerful antibiotic producers, and their suitability for use in biocontrol systems must be carefully assessed. However, many other active strains have no antibiotic capacity, and these are likely to be more useful in food production systems since they have not adverse effects on important groups of beneficial soil organisms. We have assessed the performance of selected naturally occurring Trichoderma strains (singly and in combination) and developed TUSAL, a mixture of Trichoderma harzianum and T. viride that has demonstrated to be effective against major pathogens in sugar beet and horticulture. TUSAL, has been bulked up and tested under field conditions, showing positive effects on precocity and root development, and increasing the crop production in field trials carried out in different pathosystems. The environmental impact of TUSAL strains on beneficial organisms in the environment were assessed before release, and molecular detection methods were developed to monitor the presence and performance of strains in the field. In addition, Trichoderma protein extracts with high glucanase and chitinase activities, have also been obtained from wild type strains and their effectiveness as biofungicides was tested in laboratory and field conditions, defining the concentration of protein necessary to produce fungicide effects. The genes coding for protein production were introduced into suitable organisms for large-scale production in the laboratory, never released to the environment. The effect of these novel biofungicide proteins was studied separately and synergistically with Trichoderma conidia, and with minimal doses of chemical fungicides. Suitable active Trichoderma strains are being registered in the EU by the company NBT. Both Trichoderma strains and proteins are included in formulations patented as biocontrol agents.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
Study of plant roots and the diversity of soil micro biota, such as bacteria, fungi and microfauna associated with them, is important for understanding the ecological complexities between diverse plants, microbes, soi...Study of plant roots and the diversity of soil micro biota, such as bacteria, fungi and microfauna associated with them, is important for understanding the ecological complexities between diverse plants, microbes, soil and climates and their role in phytoremediation of contaminated soils. The arbuscular mycorrhizal fungi (AMF) are universal and ubiquitous rhizosphere mi-croflora forming symbiosis with plant roots and acting as biofertilizers, bioprotactants, and biodegraders. In addition to AMF, soils also contain various antagonistic and beneficial bacteria such as root pathogens, plant growth promoting rhizobacteria including free-living and symbiotic N-fixers, and mycorrhiza helping bacteria. Their potential role in phytoremediation of heavy metal (HM) contaminated soils and water is becoming evident although there is need to completely understand the ecological complexities of the plant-microbe-soil interactions and their better exploitation as consortia in remediation strategies employed for contaminated soils. These multitrophic root microbial associations deserve multi-disciplinary investigations using molecular, biochemical, and physiological techniques. Ecosystem restoration of heavy metal contaminated soils practices need to incorporate microbial bio-technology research and development. This review highlights the ecological complexity and diversity of plant-microbe-soil combinations, particularly AM and provides an overview on the recent developments in this area. It also discusses the role AMF play in phytorestoration of HM contaminated soils, i.e. mycorrhizoremediation.展开更多
Trichoderma is a fungal genus of great and demonstrable biotechnological value, but its genome is poorly surveyed compared with other model microorganisms. Due to their ubiquity and rapid substrate colonization, Trich...Trichoderma is a fungal genus of great and demonstrable biotechnological value, but its genome is poorly surveyed compared with other model microorganisms. Due to their ubiquity and rapid substrate colonization, Trichoderma species have been widely used as biocontrol organisms for agriculture, and their enzyme systems are widely used in industry. Therefore, there is a clear interest to explore beyond the phenotype to exploit the underlying genetic systems using functional genomics tools. The great diversity of species within the Trichoderma genus, the absence of optimized systems for its exploration, and the great variety of genes expressed under a wide range of ambient conditions are the main challenges to consider when starting a comprehensive functional genomics study. An initial project started by three Spanish groups has been extended into the project TRICHOEST, funded by the EU (FP5, QLRT-2001-02032) to target the transcriptome analysis of selected Trichoderma strains with biocontrol potential, in conditions related to antagonism, nutrient stress and plant interactions. Once specific conditions were defined, cDNA libraries were produced and used for EST sequencing. Nine strains from seven Trichoderma species have been considered in this study and an important amount of gene sequence data has been generated, analyzed and used to compare the gene expression in different strains. In parallel to sequencing, genomic expression studies were carried out by means of macro-arrays to identify genes expressed in specific conditions. In silico analysis of DNA sequencing data together with macro-array expression results have lead to a selection based on the potential use of the gene sequences. The selected clone sequences were completed and cloned in appropriate vectors to initiate functional analysis by means of expression studies in homologous and heterologous systems.展开更多
As it was rear to find out detailed description, a study on biology and control measures ofP. marginatus was carried out at National Plant Quarantine Service, Katunayake Sri Lanka. Average length and width of differen...As it was rear to find out detailed description, a study on biology and control measures ofP. marginatus was carried out at National Plant Quarantine Service, Katunayake Sri Lanka. Average length and width of different stages, number of eggs in an egg sac, hatchability rate and duration of life cycle were studied. Control measures were tested using herbal oils and it was arranged in Completely Randomized Design with six replicates. Experiments were carried out in laboratory conditions under 28 ±2℃ and 70% RH. Range of length and width of different life stages revealed that, egg 0.3-0.1 mm ×0.15-0.10 mm, 1st instar 0.4-0.2 mm × 0.20-0.10 mm, 2nd instar 0.6-0.5 mm × 0.29-0.20 mm, 3rd instar male 0.8-0.5 mm × 0.30-0.20 mm, 3rd instar female 0.7-0.5 mm × 0.29-0.20 mm, adult male 0.9-0.7 mm× 0.20-0.10 mm and adult female 2.8-1.9 mm × 1.40-0.80 mm. A range of 100-200 eggs were in an ovisac and hatchability rate was 76-80%. Twenty to twenty-four days were taken to complete their life cycle. Cinnamon and Neem oil in cooperated with Surfactant and Kerosene oil could be effectively used as potential chemical agents for control of P. marginatus.展开更多
Different from ribosomal genes, which contain highly conserved sequences that are detected in all organisms, the intergenic spacer of rDNA (IGS) appears to be the most rapidly-evolving spacer region. For this reason w...Different from ribosomal genes, which contain highly conserved sequences that are detected in all organisms, the intergenic spacer of rDNA (IGS) appears to be the most rapidly-evolving spacer region. For this reason we tested this region for phylogenetic studies. This report focuses on the study of IGS sequences of isolates belonging to Trichoderma section (T. viride, T. koningii, T. hamatum, T. erinaceus, T. asperellum) and Pachybasium section (T. harzianum, T. crassum, T. fasciculatum, T. oblongisporum, T. virens). Using the primer pair 28STD and CNS1, the Fast Start Taq DNA Polymerase (Roche), and a three temperature PCR protocol, products ranging from ca 1900 to 2400 bp were obtained from all tested isolates. The PCR product of 16 Trichoderma spp. isolates was cloned into a pGEM-TEeasy Vector (Promega) and sequenced. Based on a BLAST search we can conclude that the PCR product represents the whole IGS region. Multiple alignments of IGS sequences revealed two portions with different homology level. Portion A (ca 1660 bp) is the portion that contains 3’ end of 28S gene and is the more variable, while portion B (ca 830 bp), that contains the 3’ end of IGS region and the 5’ end of 18S gene, is the less variable. Comparing all sequences in region A 705 identical pairs occur out of 1704 total nucleotides (41.4%), while in region B identical pairs were 723 out of 832 total nucleotides (86.9%) . Sequence comparison of the two regions at intraspecific level (where it was possible) showed higher variability in region A (0.17%-6.8%) than in region B (0.0%-1.0%) . At interspecific level, performing all possible comparisons, the variability of region A (19.5%-52.7%) and B (0.8%- 16.9%). were significantly higher. Comparing sequences of species belonging to Trichoderma section variability of the two regions appears reduced if compared with that obtained from comparisons of species belonging to Pachybasium section. On the basis of sequence alignment, phylogenetic trees were obtained either with entire IGS, with region A, and with region B. Results of this analysis revealed that all isolates belonging to Trichoderma section grouped separately from isolates belonging to Pachybasium section. IGS region allowed us to group species according to their taxonomic position. The topology of the tree did not change substantially, varying in genetic distance only. Performing a GenBank search sequences representing the final portion of the IGS region of other fungal species were found, and we carried out a multiple alignment using also our sequences of Trichoderma spp. and Diaporthe helianthi. The phylogeny inferred from sequence alignment matched the generally accepted morphology-based classification and was identical to other molecular schemes at high taxonomic level. Data analysis was useful in establishing a broad-scale phylogeny of Ascomycota and was also useful in sorting them into statistically-supported clades. The tree showed that Trichoderma occurred in a well-supported terminal subclade of a larger clade that also contained other genera belonging to Hypocreales order. Sequence analysis of the Trichoderma spp. IGS region allowed us to design a specific PCR primer that was successfully used to amplify region A. The new reverse primer LCR2, that recognize all Trichoderma isolates, was identified in region B and confirmed for its specificity on the DNA of fungi belonging to other Ascomycota genera. Results obtained showed that IGS region seems to be an interesting and versatile tool for phylogenetic analysis, for resolving some taxonomic problems and for constructing specific primer useful for different purposes.展开更多
A 60-day pot experiment was carried out using di-(2-ethylhexyl) phthalate (DEHP) as a typical organic pollutant phthalic ester and cowpea (Vigna sinensis) as the host plant to determine the effect of arbuscular mycorr...A 60-day pot experiment was carried out using di-(2-ethylhexyl) phthalate (DEHP) as a typical organic pollutant phthalic ester and cowpea (Vigna sinensis) as the host plant to determine the effect of arbuscular mycorrhizal inoculation on plant growth and degradation of DEHP in two contaminated soils, a yellow-brown soil and a red soil. The air-dried soils were uniformly sprayed with different concentrations of DEHP, inoculated or left uninoculated with an arbuscular mycorrhizal (AM) fungus, and planted with…展开更多
Phytophthora species are particularly aggressive plant pathogens and are often associated with the decline of many tree species, including oak and beech. Several fungi and bacteria species are known as potential antag...Phytophthora species are particularly aggressive plant pathogens and are often associated with the decline of many tree species, including oak and beech. Several fungi and bacteria species are known as potential antagonists usable as biological control agents. Phosphonate (H3PO3), commonly branded as phosphite, has also been used in the past years to protect trees against invasive Phytophthora spp.. This study aimed at comparing the effects of selected antagonist microorganisms and phosphonate, when applied by microinjection or leaf treatment. Antagonistic species were first selected for their high inhibitory activity against problematic Phytophthora species, such as Phytophthora cactorum, P. quercina and P. plurivora attacking Quercus robur and Fagus sylvatica in Polish forests. Three endophytic species Trichoderma atroviride (two strains), T. harzianum and Bacillus amyloliquefaciens showed a high control activity, and their efficacy was then assessed in comparison with a phosphonate treatment. Two application methods were experimented in this study: injection of a solution of spores or phosphonate into the sap vessels of beech or a foliar treatment on oak. Phosphonate and two strains of Trichoderma significantly reduced the necrotic area on oak leaves inoculated with P. plurivora and one strain of T. atroviride significantly reduced necrotic areas on beech branches. These results are therefore promising of a novel way to control Phytophthora spp. in forest stands and nurseries.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
基金Supported by National Natural Science Foundation of China(Grant No.30960178)Natural Science Foundation of Xinjiang Province(Grant No.200821126)~~
文摘[Objective] This study aimed to investigate the feasibility of agricultural irri- gation with reclaimed water in Urumqi City. [Method] Chinese cabbages were used as experimental materials and irrigated with control water, 50% reclaimed water and 100% reclaimed water, and then the number of endophytic bacteria in Chinese cab- bages was measured. [Result] Using 50% reclaimed water, the fresh weight of Chi- nese cabbage was improved by 68.94%; however, with the deepening internalization of exogenous microorganism internalization, the total number of endophytic bacteria and coliform flock in Chinese cabbages treated with 50% reclaimed water was sig- nificantly higher than the other two treatment groups (P〈0.05). [Conclusion] Re- claimed water enhances both the weight of Chinese cabbage and the number of endophytic bacteria, which is unsuitable for irrigation of edible crops such as Chi- nese cabbage. Strengthening disinfection during the process of sewerage treatment could solve this problem.
基金Supported by Special Fund for Agro-scientific Research in the Public Interest(201303057)948 Program(2011-G4)~~
文摘For exploring the influences of application and residue of chemical fungi- cides on chlamydospore preparations of Trichoderma spp., the effects of seven chemical fungicides on chlamydospore germination and mycelia growth of two bio- control fungi T. harzianum 610 and T. Iongibrachiatum 758 were studied. Carben- dazim, tebuconazole and difenoconazole showed strong toxicities, thiram and car- bexin showed moderate toxicities, and metalaxyl showed Mycelia growth of the two strains was more sensitive to most tested fungicides than those of chlamydospore germination. Chlamydospore germination of 610 was more sensitive to tested fungicides than those of 758, and mycelia growth of 758 was more sensitive to most tested fungi- cides than those of 610. Among the seven fungicides, 98% carbendazim had the strongest effects (ECru values were 1.64 and 0.05μpg/ml), and 70% pentachloroni- trobenzene had the weakest effects (EC50 values were 1.64 and 0,05 μg/ml) for chlamydospore germination and mycelia growth of 610. As for 758, 98% carbendaz- im had the strongest inhibitory effects and 95% metalaxyl had the weakest inhibitory for chlamydospore germination of 756 (EC50 values were 0.62 and 1 108.61 μg/ml respectively), whereas 96.2% tebuconazole showed the strongest inhibitory effects for mycelia growth of 758 (EC= value was 0.32μg/ml), and 95% metalaxyl was the weakest (EC= value was 1 206.29 μg/ml). According to the applied concentration of different fungicides in practice, we concluded that chlamydospore preparations of 610 and 758 could not be combined with carbendazim, tebuconazole, thiram and carboxin for controlling plant diseases, and the pesticide residues to the biocontrol effects should be kept in mind. Chlamydospore preparations of 610 and 758 can be and difenoconazole for controlling plant dis- eases, 758 chlamydospore preparations and germinated chlamydospore of 610 can be combined with metalaxyl for controlling plant diseases, and pesticide residue risk was not serious.
文摘In order to explore whether the endophytic Trichoderma strain P3.9 of loquat has an adverse effect on indigenous fungi in loquat rhizosphere soil, the quantitative change of aboriginal fungi is determined by dish dilution and plate colony-counting method with time changing in one season. The results showed that after the inoculation of endophytic Trichoderma strain P3.9, the total number of indigenous fungi in loquat rhizosphere soil had an obviously downward trend in contrast with the control which was without inoculation. For treatment groups, 5~60 d after inoculation, the number of indigenous fungi showed a cyclic upward-downward trend at a 10-d interval except for the insignificant changes from 35 to 40 d;60~90 d after inoculation, the upward-downward trend repeated at a 30-d interval. For the control group, the number of indigenous fungi first presented a downward-upward trend every 15 d during the period of 5~50 d, and then an upward-downward trend from the period of 50~70 d and the 75~85 d, and lastly continuous growth from 85 to 90 d;particularly, it did not vary greatly from 70 to 75 d. In general, the quantity of indigenous fungi is unstable in the control group which fluctuates more significantly than in the treatment group. The number of indigenous fungi in the treatment group was significantly lower than that in the control group. The Trichoderma strain P3.9 can inhibit indigenous fungi in loquat rhizosphere soil effectively.
文摘The choice of active Trichoderma strains is important in designing effective and safe biocontrol applications. Many species of Trichoderma have multiple strategies for fungal antagonism and indirect effects on plant health, such as growth promotion, systemic resistance induction and fertility improvements. Some strains are powerful antibiotic producers, and their suitability for use in biocontrol systems must be carefully assessed. However, many other active strains have no antibiotic capacity, and these are likely to be more useful in food production systems since they have not adverse effects on important groups of beneficial soil organisms. We have assessed the performance of selected naturally occurring Trichoderma strains (singly and in combination) and developed TUSAL, a mixture of Trichoderma harzianum and T. viride that has demonstrated to be effective against major pathogens in sugar beet and horticulture. TUSAL, has been bulked up and tested under field conditions, showing positive effects on precocity and root development, and increasing the crop production in field trials carried out in different pathosystems. The environmental impact of TUSAL strains on beneficial organisms in the environment were assessed before release, and molecular detection methods were developed to monitor the presence and performance of strains in the field. In addition, Trichoderma protein extracts with high glucanase and chitinase activities, have also been obtained from wild type strains and their effectiveness as biofungicides was tested in laboratory and field conditions, defining the concentration of protein necessary to produce fungicide effects. The genes coding for protein production were introduced into suitable organisms for large-scale production in the laboratory, never released to the environment. The effect of these novel biofungicide proteins was studied separately and synergistically with Trichoderma conidia, and with minimal doses of chemical fungicides. Suitable active Trichoderma strains are being registered in the EU by the company NBT. Both Trichoderma strains and proteins are included in formulations patented as biocontrol agents.
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Study of plant roots and the diversity of soil micro biota, such as bacteria, fungi and microfauna associated with them, is important for understanding the ecological complexities between diverse plants, microbes, soil and climates and their role in phytoremediation of contaminated soils. The arbuscular mycorrhizal fungi (AMF) are universal and ubiquitous rhizosphere mi-croflora forming symbiosis with plant roots and acting as biofertilizers, bioprotactants, and biodegraders. In addition to AMF, soils also contain various antagonistic and beneficial bacteria such as root pathogens, plant growth promoting rhizobacteria including free-living and symbiotic N-fixers, and mycorrhiza helping bacteria. Their potential role in phytoremediation of heavy metal (HM) contaminated soils and water is becoming evident although there is need to completely understand the ecological complexities of the plant-microbe-soil interactions and their better exploitation as consortia in remediation strategies employed for contaminated soils. These multitrophic root microbial associations deserve multi-disciplinary investigations using molecular, biochemical, and physiological techniques. Ecosystem restoration of heavy metal contaminated soils practices need to incorporate microbial bio-technology research and development. This review highlights the ecological complexity and diversity of plant-microbe-soil combinations, particularly AM and provides an overview on the recent developments in this area. It also discusses the role AMF play in phytorestoration of HM contaminated soils, i.e. mycorrhizoremediation.
文摘Trichoderma is a fungal genus of great and demonstrable biotechnological value, but its genome is poorly surveyed compared with other model microorganisms. Due to their ubiquity and rapid substrate colonization, Trichoderma species have been widely used as biocontrol organisms for agriculture, and their enzyme systems are widely used in industry. Therefore, there is a clear interest to explore beyond the phenotype to exploit the underlying genetic systems using functional genomics tools. The great diversity of species within the Trichoderma genus, the absence of optimized systems for its exploration, and the great variety of genes expressed under a wide range of ambient conditions are the main challenges to consider when starting a comprehensive functional genomics study. An initial project started by three Spanish groups has been extended into the project TRICHOEST, funded by the EU (FP5, QLRT-2001-02032) to target the transcriptome analysis of selected Trichoderma strains with biocontrol potential, in conditions related to antagonism, nutrient stress and plant interactions. Once specific conditions were defined, cDNA libraries were produced and used for EST sequencing. Nine strains from seven Trichoderma species have been considered in this study and an important amount of gene sequence data has been generated, analyzed and used to compare the gene expression in different strains. In parallel to sequencing, genomic expression studies were carried out by means of macro-arrays to identify genes expressed in specific conditions. In silico analysis of DNA sequencing data together with macro-array expression results have lead to a selection based on the potential use of the gene sequences. The selected clone sequences were completed and cloned in appropriate vectors to initiate functional analysis by means of expression studies in homologous and heterologous systems.
文摘As it was rear to find out detailed description, a study on biology and control measures ofP. marginatus was carried out at National Plant Quarantine Service, Katunayake Sri Lanka. Average length and width of different stages, number of eggs in an egg sac, hatchability rate and duration of life cycle were studied. Control measures were tested using herbal oils and it was arranged in Completely Randomized Design with six replicates. Experiments were carried out in laboratory conditions under 28 ±2℃ and 70% RH. Range of length and width of different life stages revealed that, egg 0.3-0.1 mm ×0.15-0.10 mm, 1st instar 0.4-0.2 mm × 0.20-0.10 mm, 2nd instar 0.6-0.5 mm × 0.29-0.20 mm, 3rd instar male 0.8-0.5 mm × 0.30-0.20 mm, 3rd instar female 0.7-0.5 mm × 0.29-0.20 mm, adult male 0.9-0.7 mm× 0.20-0.10 mm and adult female 2.8-1.9 mm × 1.40-0.80 mm. A range of 100-200 eggs were in an ovisac and hatchability rate was 76-80%. Twenty to twenty-four days were taken to complete their life cycle. Cinnamon and Neem oil in cooperated with Surfactant and Kerosene oil could be effectively used as potential chemical agents for control of P. marginatus.
文摘Different from ribosomal genes, which contain highly conserved sequences that are detected in all organisms, the intergenic spacer of rDNA (IGS) appears to be the most rapidly-evolving spacer region. For this reason we tested this region for phylogenetic studies. This report focuses on the study of IGS sequences of isolates belonging to Trichoderma section (T. viride, T. koningii, T. hamatum, T. erinaceus, T. asperellum) and Pachybasium section (T. harzianum, T. crassum, T. fasciculatum, T. oblongisporum, T. virens). Using the primer pair 28STD and CNS1, the Fast Start Taq DNA Polymerase (Roche), and a three temperature PCR protocol, products ranging from ca 1900 to 2400 bp were obtained from all tested isolates. The PCR product of 16 Trichoderma spp. isolates was cloned into a pGEM-TEeasy Vector (Promega) and sequenced. Based on a BLAST search we can conclude that the PCR product represents the whole IGS region. Multiple alignments of IGS sequences revealed two portions with different homology level. Portion A (ca 1660 bp) is the portion that contains 3’ end of 28S gene and is the more variable, while portion B (ca 830 bp), that contains the 3’ end of IGS region and the 5’ end of 18S gene, is the less variable. Comparing all sequences in region A 705 identical pairs occur out of 1704 total nucleotides (41.4%), while in region B identical pairs were 723 out of 832 total nucleotides (86.9%) . Sequence comparison of the two regions at intraspecific level (where it was possible) showed higher variability in region A (0.17%-6.8%) than in region B (0.0%-1.0%) . At interspecific level, performing all possible comparisons, the variability of region A (19.5%-52.7%) and B (0.8%- 16.9%). were significantly higher. Comparing sequences of species belonging to Trichoderma section variability of the two regions appears reduced if compared with that obtained from comparisons of species belonging to Pachybasium section. On the basis of sequence alignment, phylogenetic trees were obtained either with entire IGS, with region A, and with region B. Results of this analysis revealed that all isolates belonging to Trichoderma section grouped separately from isolates belonging to Pachybasium section. IGS region allowed us to group species according to their taxonomic position. The topology of the tree did not change substantially, varying in genetic distance only. Performing a GenBank search sequences representing the final portion of the IGS region of other fungal species were found, and we carried out a multiple alignment using also our sequences of Trichoderma spp. and Diaporthe helianthi. The phylogeny inferred from sequence alignment matched the generally accepted morphology-based classification and was identical to other molecular schemes at high taxonomic level. Data analysis was useful in establishing a broad-scale phylogeny of Ascomycota and was also useful in sorting them into statistically-supported clades. The tree showed that Trichoderma occurred in a well-supported terminal subclade of a larger clade that also contained other genera belonging to Hypocreales order. Sequence analysis of the Trichoderma spp. IGS region allowed us to design a specific PCR primer that was successfully used to amplify region A. The new reverse primer LCR2, that recognize all Trichoderma isolates, was identified in region B and confirmed for its specificity on the DNA of fungi belonging to other Ascomycota genera. Results obtained showed that IGS region seems to be an interesting and versatile tool for phylogenetic analysis, for resolving some taxonomic problems and for constructing specific primer useful for different purposes.
基金Project supported by the National Natural Science Foundation of China (No. 40101015) the National Key Basic Research Support foundation of China (No. G1999011806)
文摘A 60-day pot experiment was carried out using di-(2-ethylhexyl) phthalate (DEHP) as a typical organic pollutant phthalic ester and cowpea (Vigna sinensis) as the host plant to determine the effect of arbuscular mycorrhizal inoculation on plant growth and degradation of DEHP in two contaminated soils, a yellow-brown soil and a red soil. The air-dried soils were uniformly sprayed with different concentrations of DEHP, inoculated or left uninoculated with an arbuscular mycorrhizal (AM) fungus, and planted with…
文摘Phytophthora species are particularly aggressive plant pathogens and are often associated with the decline of many tree species, including oak and beech. Several fungi and bacteria species are known as potential antagonists usable as biological control agents. Phosphonate (H3PO3), commonly branded as phosphite, has also been used in the past years to protect trees against invasive Phytophthora spp.. This study aimed at comparing the effects of selected antagonist microorganisms and phosphonate, when applied by microinjection or leaf treatment. Antagonistic species were first selected for their high inhibitory activity against problematic Phytophthora species, such as Phytophthora cactorum, P. quercina and P. plurivora attacking Quercus robur and Fagus sylvatica in Polish forests. Three endophytic species Trichoderma atroviride (two strains), T. harzianum and Bacillus amyloliquefaciens showed a high control activity, and their efficacy was then assessed in comparison with a phosphonate treatment. Two application methods were experimented in this study: injection of a solution of spores or phosphonate into the sap vessels of beech or a foliar treatment on oak. Phosphonate and two strains of Trichoderma significantly reduced the necrotic area on oak leaves inoculated with P. plurivora and one strain of T. atroviride significantly reduced necrotic areas on beech branches. These results are therefore promising of a novel way to control Phytophthora spp. in forest stands and nurseries.
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.