Combining flow cytometry andgfpreporter gene for quantitative evaluation ofPectpbacterium carotovorumssp.carotovoruminOrnithogalum dubiumplantlets

Abstract

Aims: Ornithogalum dubium is a natural host of the soft rot pathogen Pectobacterium carotovorum ssp. carotovorum (Pcc). The present study was aimed to develop a quantification system for Pcc expressing a gfp reporter gene, using fluorescent activated cell sorter (FACS) in planta. Methods and Results: Several calibration steps were required to distinctly gate the GFP‐labelled bacteria at FL1 mode and count the bacteria. To validate the bacterial counts obtained by FACS analysis, an internal standard of polystyrene green fluorescent microsphere beads was employed, resulting in high correlation with serial dilutions and plate counting. This allowed quantification of the bacteria, with no further need to culture, dilute or plate the cells. Micropropagation tools were developed to produce uniform plantlets of O. dubium, which were either inoculated with increasing concentrations of Pcc or elicited for resistance towards Pcc using methyl jasmonate. The rapid counting procedure allowed recovering, gating and counting the bacterial population in planta, separately from the plant cells background and from the microsphere beads. Conclusions: The FACS based quantification approach of Pcc was found accurate, reproducible and time saving, thus useful for counting bacteria in planta. Significance and Impact of the Study: The combination of time‐ and cost‐saving approach for Pcc quantification with efficient screening tools during early stages of micropropagation may facilitate the preliminary process of selection for resistant cultivars.

Salicylic acid can reduce infection symptoms caused by Dickeya solani in tissue culture grown potato (Solanum tuberosum L.) plants

“…Kondor, highly susceptible to infections with Dickeya spp., resulted in a rapid colonization of plants. The use of the GFPtagged D. solani strain IPO2254 allowed microscopical observations of bacteria on root and stem base surface as it was described earlier by Golan et al (2010), who used a GFP-tagged P. carotovorum subsp. carotovorum to follow the fate and population dynamics of bacterial cells upon infection of in vitro grown Ornithogalum dubium plants.…”

Applications of flow cytometry in plant pathology for genome size determination, detection and physiological status

“…(2004) used FCM and plate counts to quantify and study the evolution of culturable and nonculturable PI‐stained Pseudomonas fluorescens cells in different root zones. Golan et al . (2010) counted GFP‐tagged Pectobacterium carotovorum ssp.…”

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

“…Plasmids from the pPROBE series were already successfully applied for tagging bacterial cells for flow cytometry and microscopy ( i.e. , [8,27,28]).…”

“…This can be accomplished after modification of the strain to obtain additional antibiotic resistance for selective growth and/or introduction of a reporter gene, such as a fluorescent protein-coding gene. Quantitative techniques for colonization study, providing cell count in colony forming units (CFU) per gram, cm or sq cm of root, include serial dilution plating on selective media and flow cytometry [8]. To visually demonstrate colonization and obtain qualitative data, such as the location of bacteria on plant roots, classical fluorescence and confocal laser scanning microscopy (CLSM) can be applied [9].…”