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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...

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Bacteria belonging to the genus Methylobacterium are characterized by being able to rely on methanol as a sole carbon and energy source and by presenting a more or less intense pink reddish pigmentation. These bacteria, also referred to as pink-pigmented methylotrophic bacteria (PPFMs), are common inhabitants of the phyllosphere and are found in many other environmental samples. Since they grow slowly they are often overlooked and their impact on phyllosphere microbial communities and on the plants harboring them is not well studied nor has their ecology been elucidated. In a survey of PPFM colonization in three different agricultural sites, PPFM populations were identified on both red clover and winter wheat, but red clover was more consistently colonized. Isolations from collected leaves showed PPFM populations to decrease from spring towards summer, but they increased again towards the end of the cropping season. Isolates from red clover readily colonized winter wheat leaves and vice versa in greenhouse experiments, but population sizes were dependent on the application procedure. Tested isolates had also good potential to colonize the rhizosphere, especially after seed inoculations. Confocal scanning laser microscopy showed gfp-tagged isolates to colonize the surface of clover leaves by forming large aggregates.