In Vivo Study of Trichoderma -Pathogen-Plant Interactions, Using Constitutive and Inducible Green Fluorescent Protein Reporter Systems

Abstract

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.Trichoderma spp. are active ingredients in a variety of commercial biofungicides used to control a range of economically important aerial and soilborne fungal plant pathogens (17,19). The antagonistic activity of biocontrol Trichoderma strains is attributable to one or more complex mechanisms, including nutrient competition, antibiosis, the activity of cell wall-lytic enzymes, induction of systemic resistance, and increased plant nutrient availability (16,18,19,24,30,31,42). Many studies, primarily in vitro studies, have shed light on the molecular basis of the three-way relationship among the pathogen, the plant, and the biocontrol agent (14, 35). However, the complexities of these interactions have been poorly studied in situ. For example, many of the factors involved in biocontrol are known (32,34,51,55), but the antifungal mechanisms, including mycoparasitism, and the fate of Trichoderma in the soil and on the plant are not well understood. Effective monitoring of biocontrol-related processes in vivo based on the use of vital markers (1, 20, 40) provides a basis for development of new selection methods and improved applications.The green fluorescent protein (GFP)-encoding gene (gfp) (8) is a powerful tool for monitoring the fate and behavior of bacterial and fungal inoculants in situ (1,5,29,43,(48)(49)(50).GFP, unlike other biomarkers (22), does not require any substrate or additional cofactors in order to fluoresc...

The rhizosphere: a playground and battlefield for soilborne pathogens and beneficial microorganisms

“…Advances in molecular techniques, fluorescence labeling, and imaging such as confocal laser microscopy, may be useful in the future. These techniques have been used to study colonization of roots by biocontrol bacteria and fungi (Gamalero et al 2005;Bloemberg et al 2000;Lu et al 2004) and infection of roots by Fusarium (Lagopodi et al 2002;Bolwerk et al 2005;Olivain et al 2006), but these have been descriptive and have not employed spatial statistics.…”

Biocontrol of root and crown rot in tomatoes under greenhouse conditions using Trichoderma harzianum and Paenibacillus lentimorbus. Additional effect of solarization

“…It was cultured on potato-dextrose-agar, PDA (DIFCO), for the obtention of conidia (Lu et al 2004). These were used as inoculum for liquid cultures of the fungus for: a) its characterization in terms of the secretion of biocontrol enzymes; b) for obtention of the inoculum to be applied to tomato seeds and c) for formulations.…”

ThHog1 controls the hyperosmotic stress response in Trichoderma harzianum

“…This versatile genus antagonizes other fungal cells (mycoparasitism), which is the basis for its use in agriculture to control fungal plant pathogens. Furthermore, increasing evidences point to a symbiotic mycorrhiza-like relationship of Trichoderma with plants (Harman et al, 2004;Lu et al, 2004). Finally, a variety of hydrolytic enzymes are used by Trichoderma to obtain nutrients from soil and plant material (Kubicek & Penttila, 1998), which makes this genus a unique source to identify polymer-degrading enzymes with industrial value (Penttila, 1998).…”