Trichoderma atroviride strain P1 has been used extensively to study the mycoparasitic mechanisms in the interaction between plant pathogenic host and beneficial antagonistic fungi. Mutants of P1 containing the green f...Trichoderma atroviride strain P1 has been used extensively to study the mycoparasitic mechanisms in the interaction between plant pathogenic host and beneficial antagonistic fungi. Mutants of P1 containing the green fluorescent protein (gfp) or glucose oxidase (gox) reporter systems and different inducible promoters (from the exochitinase nag1 gene, or the endochitinase ech42 gene of P1) were used to determine the factors that activate the biocontrol gene expression cascade in the antagonist. The following compounds were tested singly and in various combinations: purified Trichoderma P1 enzymes (endochitinase, exochitinase, chitobiosidase, glucanase); antagonist culture filtrates (T. atroviride P1 wild-type and relative knock-out mutants, T. harzianum, T. reesei); pathogen culture filtrates (Botrytis, Pythium, Rhizoctonia); purified fungal cell walls (CWs) from Trichoderma, Botrytis, Pythium, Rhizoctonia; colloidal crab shell chitin; and plant extracts from cucumber leaves, stems or roots. Strong induction of mycoparasitism was found with the various digestion products produced by treating fungal CWs and colloidal chitin with purified enzymes or fungal culture filtrates. Filtrates from chitinase knock-out mutants, as well as CWs from Oomycetes fungi, were less active in producing the stimulus for mycoparasitism. The host CW digestion products were separated by molecular weight (MW) to determine which compounds were able to activate Trichoderma. Micromolecules of MW less than 3 kDa were found to trigger mycoparasitism gene expression before physical contact with the host pathogen. These compounds stimulated mycelial growth and spore germination of the antagonist. Purification of these host-derived compounds was conducted by HPLC and in vivo assay. The obtained inducers were able to stimulate both the production of endochitinase and exochitinase enzymes, even under repressing conditions in the presence of glucose. Inducers stimulated the biocontrol effect of P1 in the presence of host fungi. The disease symptom development on bean leaves inoculated with Botrytis and Trichoderma spores was clearly reduced by the addition of the inducers, unless these molecules were not specifically inactivated. Finally, purified inducers added to liquid cultures of T. atroviride P1 stimulated the production of low MW antibiotics and metabolites which inhibited Botrytis spore germination. Mass spectrometry analysis (ESI-MS) of the inducers indicated the presence of hexose oligomers, like cellobiose, while MS/MS analysis by selective fragmentation of peaks in the spectrum demonstrated the presence of at least three distinct compounds that were biologically active.展开更多
文摘Trichoderma 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.
