Non-invasive determination of plant-associated bacteria in the phyllosphere of plants

Epiphytic living Pseudomonas strains isolated from different Malus domestica cultivars were transformed with two reporter genes [green fluorescent protein (gfp) and luciferase (luxAB)]. The establishment and distribution of these bacteria on sterile, in vitro-propagated, and thus genetically identical, Malus domestica plants were continuously analysed with a cooled, back-illuminated, charge-coupled-device (CCD) camera system. The combination of the assessment of bioluminescence and the use of a CCD camera offer an intriguing method to study, non-invasively and in real time, plant-microbe interactions as well as the colonization of the phyllosphere by microorganisms. Here we report on the applicability and sensitivity of the method with the goal to investigate quantitatively the interaction of symbiotic and pathogenic microorganisms with the corresponding host plant. It will be shown that the three bacterial isolates of the genus Pseudomonas studied, differ considerably with respect to their establishment on the host plants. It will also be shown that the chosen host apple variety has an impact on the activity of the bacterial cultivars. Analysis by a laser scanning fluorescence microscope provides the first evidence for the mode by which the epiphytic microorganisms interact with the plant.