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

November 2000 , Volume 13 , Number  11
Pages  1,170 - 1,176

Guido V. Bloemberg , André H. M. Wijfjes , Gerda E. M. Lamers , Nico Stuurman , and Ben J. J. Lugtenberg

Leiden University, Institute of Molecular Plant Sciences, Wassenaarseweg 64, Leiden 2333 AL, The Netherlands


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Accepted 20 July 2000.

To visualize simultaneously different populations of pseu-domonads 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. Micro-colonies consisting of mixed populations were frequently observed at the base of the root system, whereas micro-colonies 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.


Additional keywords: fluorescence, triple color imaging.

© 2000 The American Phytopathological Society