In vivo study of Trichoderma-pathogen-plant interactions, using constitutive and inducible green fluorescent protein reporter systems

• Published in: Applied and Environmental Microbiology 2004-05, Vol.70 (5), p.3073-3081
• Main Authors: Lu, Z, Tombolini, R, Woo, S, Zeilinger, S, Lorito, M, Jansson, J.K
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; biological control; biological control agents; cell adhesion; chitin; Chitin - metabolism; Chitinases - genetics; Chitinases - metabolism; Cucumbers; Cucumis sativus; Cucumis sativus - microbiology; disease control; Fundamental and applied biological sciences. Psychology; fungal antagonists; fungal diseases of plants; fungal spores; Fungi; Gene expression; Gene Expression Regulation, Fungal; Genes, Reporter; genetically engineered microorganisms; green fluorescent protein; Green Fluorescent Proteins; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Microbiology; Microscopy, Confocal; Microscopy, Fluorescence; mycelium; Mycology; mycoparasites; mycoparasitism; Pathogens; Pest Control, Biological; Plant Diseases - microbiology; plant pathogenic fungi; Plant Roots - microbiology; Proteins; Pythium - growth & development; Pythium ultimum; reporter genes; Rhizoctonia - growth & development; Rhizoctonia solani; seed inoculation; seeds; Seeds - microbiology; Soil Microbiology; Spores, Fungal; Thanatephorus cucumeris; Trichoderma; Trichoderma - genetics; Trichoderma - growth & development; Trichoderma - metabolism; Trichoderma atroviride
• ISSN: 0099-2240; 1098-5336; 1098-5336
• DOI: 10.1128/AEM.70.5.3073-3081.2004

Plant tissue colonization by Trichoderma atroviride plays a critical role in the reduction of diseases caused by phytopathogenic fungi, but this process has not been thoroughly studied in situ. We monitored in situ interactions between gfp-tagged biocontrol strains of T. atroviride and soilborne plant pathogens that were grown in cocultures and on cucumber seeds by confocal scanning laser microscopy and fluorescence stereomicroscopy. Spores of T. atroviride adhered to Pythium ultimum mycelia in coculture experiments. In mycoparasitic interactions of T. atroviride with P. ultimum or Rhizoctonia solani, the mycoparasitic hyphae grew alongside the pathogen mycelia, and this was followed by coiling and formation of specialized structures similar to hooks, appressoria, and papillae. The morphological changes observed depended on the pathogen tested. Branching of T. atroviride mycelium appeared to be an active response to the presence of the pathogenic host. Mycoparasitism of P. ultimum by T. atroviride occurred on cucumber seed surfaces while the seeds were germinating. The interaction of these fungi on the cucumber seeds was similar to the interaction observed in coculture experiments. Green fluorescent protein expression under the control of host-inducible promoters was also studied. The induction of specific Trichoderma genes was monitored visually in cocultures, on plant surfaces, and in soil in the presence of colloidal chitin or Rhizoctonia by confocal microscopy and fluorescence stereomicroscopy. These tools allowed initiation of the mycoparasitic gene expression cascade to be monitored in vivo.