Simultaneous Imaging of Pseudomonas fluorescens WCS365 Populations Expressing Three Different Autofluorescent Proteins in the Rhizosphere: New Perspectives for Studying Microbial Communities

Abstract

To visualize simultaneously different populations of pseudomonads in the rhizosphere at the single cell level in a noninvasive way, a set of four rhizosphere-stable plasmids was constructed expressing three different derivatives of the green fluorescent protein (GFP), namely enhanced cyan (ECFP), enhanced green (EGFP), enhanced yellow (EYFP), and the recently published red fluorescent protein (RFP; DsRed). Upon tomato seedling inoculation with Pseudomonas fluorescens WCS365 populations, each expressing a different autofluorescent protein followed by plant growth for 5 days, the rhizosphere was inspected using confocal laser scanning microscopy. We were able to visualize simultaneously and clearly distinguish from each other up to three different bacterial populations. Microcolonies consisting of mixed populations were frequently observed at the base of the root system, whereas microcolonies further toward the root tip predominantly consisted of a single population, suggesting a dynamic behavior of microcolonies over time. Since the cloning vector pME6010 has a broad host range for gram-negative bacteria, the constructed plasmids can be used for many purposes. In particular, they will be of great value for the analysis of microbial communities, for example in processes such as biocontrol, biofertilization, biostimulation, competition for niches, colonization, and biofilm formation.

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.…”

Isolation of Fluorescent Pseudomonas spp. from Diverse Agro-Ecosystems of India and Characterization of their PGPR Traits

“…Isolate P17 produced only 85.5 μg mLG 1 EPS (Table 3). The plant-growthpromoting pseudomonads have been reported to discontinuously colonize the root surface, developing as small biofilms along epidermal fissures (Bloemberg et al, 2000). Plant growth promoting effect of IAA and EPS producing Pseudomonas spp.…”

Safety Assessment of Transgenic Organisms, Volume 4

“…Detection of marked cells can be achieved by epifluorescence microscopy, laser confocal microscopy, flow cytometry and spectrofluorometry (Tombolini and Jansson, 1998). It is possible to study the location of the cells marked by gfp on plant roots by studying samples with confocal laser scanning microscopy (Normander et al, 1999;Bloemberg et al, 2000). Because of these advantages gfp is chosen as a marker instead of lacZ and XylE.…”

“…Using different derivatives of the green fluorescent protein, namely enhanced cyan (ecfp), enhanced green (egfp), enhanced yellow (eyfp) and the red fluorescent protein (rfp), it is also now possible to simultaneously visualize different populations of micro-organisms. Bloemberg et al (2000) could visualize and distinguish ecfp, egfp and rfp marked P. fluorescens cells in the rhizosphere of tomato plants by triple imaging using a confocal laser scanning microscope with negligible cross talk.…”

“…Although microcopy is not particularly suited for cell enumeration the method has been successfully applied for this purpose (Putland and Rivkin, 1999;Unge and Jansson, 2001) The strength of the method is in its use to study the in situ localization of cells (Bloemberg et al, 2000;Unge and Jansson, 2001). …”