Monitoring population levels of Pseudomonas fluorescens labeled with green fluorescent protein, using fluorescence microscopy and direct fluorescence scanning techniques

Abstract

Pseudomonas fluorescens strain 1100-6 is an effective biocontrol agent for apple blue mold, but more information is needed on its interaction with pathogens. The objective of this study was to develop a technique to evaluate the population levels of strain 1100-6 on wounded apples, in the presence or absence of Penicillium expansum or Penicillium solitum, the apple blue mold pathogens, using the green fluorescent protein (GFP) as a marker. Cells of strain 1100-6 labeled with GFP (strain 1100-6 GFP) were monitored by fluorescence microscopy or direct fluorescence scanning (Storm® scanning system) with a digital imaging software for quantifying fluorescence produced by either technique. There was a strong linear correlation between cell number and fluorescence intensity of strain 1100-6 GFP-labeled cells, for standard cell suspensions, with both fluorescence microscopy and fluorescence scanning techniques. This relationship was maintained in ‘Gala’ apples inoculated with 1100-6 GFP, in the presence or absence of challenge inoculation with pathogenic Penicillium spp. Fluorescence intensity in apple wounds inoculated with 1100-6 GFP was determined after 4 and 9 days at 20 °C, and after 25, 35, and 40 days at 5 °C. The concentration of strain 1100-6 GFP increased over time at both temperatures, in the presence or absence of pathogens. Fluorescence due to GFP was not detected by fluorescence microscopy or scanning in apple tissue taken 1 cm away from the site of inoculation. Uses of fluorescence microscopy or direct fluorescence scanning for determining cell numbers of a GFP-labeled bacterium are discussed.