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Colonization of Potato Rhizosphere by GFP-Tagged Bacillus subtilis MB73/2, Pseudomonas sp. P482 and Ochrobactrum sp. A44 Shown on Large Sections of Roots Using Enrichment Sample Preparation and Confocal Laser Scanning Microscopy

1
Laboratory of Biological Plant Protection, Intercollegiate Faculty of Biotechnology UG&MUG, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland
2
Laboratory of Molecular Bacteriology, Intercollegiate Faculty of Biotechnology UG&MUG, Medical University of Gdansk, 80-822 Gdansk, Poland
3
Laboratory of Virus Molecular Biology, Intercollegiate Faculty of Biotechnology UG&MUG, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland
*
Author to whom correspondence should be addressed.
Sensors 2012, 12(12), 17608-17619; https://doi.org/10.3390/s121217608
Received: 30 September 2012 / Revised: 19 November 2012 / Accepted: 21 November 2012 / Published: 18 December 2012
(This article belongs to the Special Issue Sensor-Based Technologies and Processes in Agriculture and Forestry)
The ability to colonize the host plants’ rhizospheres is a crucial feature to study in the case of Plant Growth Promoting Rhizobacteria (PGPRs) with potential agricultural applications. In this work, we have created GFP-tagged derivatives of three candidate PGPRs: Bacillus subtilis MB73/2, Pseudomonas sp. P482 and Ochrobactrum sp. A44. The presence of these strains in the rhizosphere of soil-grown potato (Solanum tuberosum L.) was detected with a classical fluorescence microscope and a confocal laser scanning microscope (CLSM). In this work, we have used a broad-field-of-view CLMS device, dedicated to in vivo analysis of macroscopic objects, equipped with an automated optical zoom system and tunable excitation and detection spectra. We show that features of this type of CLSM microscopes make them particularly well suited to study root colonization by microorganisms. To facilitate the detection of small and scattered bacterial populations, we have developed a fast and user-friendly enrichment method for root sample preparation. The described method, thanks to the in situ formation of mini-colonies, enables visualization of bacterial colonization sites on large root fragments. This approach can be easily modified to study colonization patterns of other fluorescently tagged strains. Additionally, dilution plating of the root extracts was performed to estimate the cell number of MB73/2, P482 and A44 in the rhizosphere of the inoculated plants. View Full-Text
Keywords: bacteria visualization; fluorescent protein; PGPR; plant-associated bacteria; soil environment; transformation bacteria visualization; fluorescent protein; PGPR; plant-associated bacteria; soil environment; transformation
MDPI and ACS Style

Krzyzanowska, D.; Obuchowski, M.; Bikowski, M.; Rychlowski, M.; Jafra, S. Colonization of Potato Rhizosphere by GFP-Tagged Bacillus subtilis MB73/2, Pseudomonas sp. P482 and Ochrobactrum sp. A44 Shown on Large Sections of Roots Using Enrichment Sample Preparation and Confocal Laser Scanning Microscopy. Sensors 2012, 12, 17608-17619.

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