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Genetic improvement of Trichoderma ability to induce systemic resistance

浙江大学学报(农业与生命科学版) 2004年第4期30卷 423-423页

作者： ciliento r Woo S L Di Benedetto P ruocco M Scala F Soriente I Ferraioli S Brunner K Zeilinger S Mach r L Lorito M Dip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 Italy and Microbial Biochemistry-Gene Technology Department Institute for Chemical Engineering Technical University of Vienna Getreidemarkt 9/166-5 1060 Wien Austria

The beneficial applications of Trichoderma spp. in agriculture include not only the control of plant pathogens, but also the improvement of plant growth, micronutrient availability, and plant tolerance to abiotic stress. In addition, it has been suggested that these fungi are able to increase plant disease resistance by activating induced systemic resistance (ISr) . The mode of action of these beneficial fungi in the Trichoderma -plant-pathogen interaction are many, complex and not completely understood. Numerous lytic enzymes have been characterized, the encoding genes (ech42 gluc78, nag1 from T. atroviride strain P1) cloned, and their role in biocontrol demonstrated. The corresponding biocontrol-related inducible promoters have been used in a reporter system based on the Aspergillus niger glucose oxidase gene (goxA) to monitor biocontrol activity. Glucose oxidase catalyzes the oxygen-dependent oxidation of D-glucose to D-glucono-1,5-lactone and hydrogen peroxide; this latter compound is known to have an antifungal effect and activate the plant defence cascade, thus increasing resistance to pathogen attack. T. atroviride P1 transformants with various promoters gox were tested as seed coating treatments on bean seeds planted in soil infested with a soilborne fungal pathogen. Successively, the emergent leaves were inoculated with a foliar pathogen to determine the effect of the GOX transformants on biocontrol and resistance to pathogen attack. Inoculations with the P1-GOX transformants not only reduced disease symptoms caused by a soil pathogen, but also the lesions of various foliar pathogens applied far from the Trichoderma colonization, thus activating ISr. A similar approach is being use to genetically improve T. harzianum T22, a rhizosphere competent and commercially marketed strain not transformed yet, by using four different gox gene constructs under the control of constitutive and inducible promoters. Plasmids have been introduced in Trichoderma by protoplast

关键词： glucose oxidase induced systemic resistance lytic enzymes

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Use of Trichoderma spp. for biological control of the livestock feed contaminant fungus Fusarium proliferatum

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浙江大学学报（农业与生命科学版） 2004年第4期30卷 414-414页

作者： ruocco M Pane F ritieni A Lanzuise S Ambrosino P Marra r Woo S L ciliento r Soriente I Ferraioli S Scala F Lorito M Dip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 ItalyDip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 ItalyDip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 Italy Dip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 Italy Dip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 ItalyDip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 ItalyDip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 Italy Dip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 ItalyDip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 Italy Dip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 Italy Dip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Uni

Fusarium spp. are pathogens of many important agricultural crops, and are often strong mycotoxin producers. Fusarium proliferatum, in particular, causes disease in cereals and secretes the toxin Beauvaricin that contaminates livestock feed and cereals, producing a variety of toxicity symptoms ranging from poor weight gain to mortality. Beauvaricin is a cyclodepsipeptide and acts as a potent mycotoxin known to have insecticidal properties. This compound is highly toxic to human cell lines, where it induces apoptosis and specifically inhibits cholesterol acetyltransferase. Nothing is known about the role of this mycotoxin during the interaction of F. proliferatum with other microorganisms, including the fungal antagonists Trichoderma spp. In vitro tests have demonstrated that the antagonistic and mycoparasitic activity of Trichoderma is not inhibited by the presence of Beauvaricin at concentrations up to 10 mg/kg in the substrate. In vivo biocontrol assays on barley and wheat with Trichoderma against F. proliferatum isolates, producing and non-producing Beauvaricin, confirmed the ability of the antagonist to control this pathogen in all cases. Also Trichoderma culture filtrates obtained in conditions that promote Cell Wall Degrading Enzyme (CWDE) secretion, were able to inhibit. spore. germination. of. different. F.. proliferatum. isolates.. These. results. suggest. the. possibility. of. using. Trichoderma. and/or. its. metabolites. to. control. contaminants. of. livestock. feed. by. mycotoxin-producing. Fusarium..

关键词：家畜霉菌饲料污染物镰刀霉木霉属生物防治

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Cloning of first abc transporter encoding gene from Trichoderma spp. and its expression during stress and mycoparasitism

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浙江大学学报（农业与生命科学版） 2004年第4期30卷 450-450页

作者： Lanzuise S ruocco M Scala V Catapano L Woo S ciliento r Ferraioli S Soriente I Vinale F Scala F Del Sorbo G Lorito M Dip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 ItalyDip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 ItalyDip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 Italy Dip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 Italy Dip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 ItalyDip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 Italy Dip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 Italy Dip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 ItalyDip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 Italy Dip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Università di Napoli "Federico II" and CNR-IPP Via Università 100 Portici (NA) 80055 Italy Dip. Arboricoltura Botanica e Patologia Vegetale-Sez. Patologia Vegetale Lab. di Lotta Biologica Un

Trichoderma in its natural environment competes for nutrient uptake and is required to protect itself from adverse natural toxic compounds, such as those produced by plants and other microbes in the soil community, or synthetic toxic compounds released human activity. One of the most important metabolic pathways for drug resistance and substrate uptake, both in prokaryotes and eukaryotes, is ATP dependent. The role of ABC transporter proteins in the biology of Trichoderma is still not known. We present the cloning of the first four ABC transporter genes (TABC1, TABC2, TABC3, TABC4 ) in Trichoderma, and in particular T. atroviride P1, and the characterization of TABC2 The complete sequence of this gene is 6535 bp, which includes a promoter of 1624 bp, a terminator of 642 bp and a coding region of 4264 bp. The promoter contains many of the potential transcription factor binding sites found in the 5’ upstream region of the ech42 gene of T. atroviride P1. These included: heat shock factors (HSF), a nitrogen-regulating factor (Nit-2), a stress-response element (STrE), a GCr1 elements, and a Cre BP1 motif. Northern analysis and rT-PCr demonstrated that TABC2 is highly expressed when Trichoderma is subjected to nitrogen starvation, grown in the presence of culture filtrates of Botrytis cinerea, rhizoctonia solani, and Pythium ultimum, or when N-acetylglucosamine is added to the substrate. TABC2 appears to be co-regulated with some CWDE-encoding genes, suggesting that this is the first ABC transporter encoding gene involved in mycoparasitic events. It’s role in the interaction of Trichoderma with fungal hosts or plants is being investigated by targeted gene disruption and overexpression.

关键词：真菌寄生现象应激反应木霉属真菌克隆病原体

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Heterologous expression of the glucose oxidase gene in Trichoderma atroviride leads enhanced ability to attack phytopathogenic fungi and induction of plant systemic disease resistance

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浙江大学学报（农业与生命科学版） 2004年第4期30卷 418-418页

作者： robert L Mach Brunner Kurt Matteo Lorito Susanne Zeilinger rosalia ciliento Sheridan Woo Gene Technology Group Institute of Chemical Engineering TU-Vienna Getreidemarkt 9/166/5 A-1060 Wien Austria Gene Technology Group Institute of Chemical Engineering TU-Vienna Getreidemarkt 9/166/5 A-1060 Wien AustriaDipartimento di Arboricoltura Botanica e Patologia Vegetale Sezione di. Patologia Vegetale Università degli Studi di Napoli "Federico II" Via Università 100 80050. Portici (NA) Italy Gene Technology Group Institute of Chemical Engineering TU-Vienna Getreidemarkt 9/166/5 A-1060 Wien AustriaDipartimento di Arboricoltura Botanica e Patologia Vegetale Sezione di. Patologia Vegetale Università degli Studi di Napoli "Federico II" Via Università 100 80050. Portici (NA) Italy Dipartimento di Arboricoltura Botanica e Patologia Vegetale Sezione di. Patologia Vegetale Università degli Studi di Napoli "Federico II" Via Università 100 80050. Portici (NA) Italy

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

关键词：植物致病真菌抗性病害植物异种表达葡萄糖氧化酶基因

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