Detection of plant pathogenic bacteria by flow cytometry (FLOWBACT)

Can applications of flow cytometry known from medical and food sciences be used for fytopathology? What are the possibilities of live/dead staining of plant pathogens? Can we detect plant pathogens by flow cytometry? What are the possibilities of specific detection of low concentrations of pathogens? What can be learned from the comparison qPCR – flow cytometry concerning technical possibilities and economics? The detection of Pseudomonas cichorii was used as a case study for this topic. P. cichorii is a waterborne lettuce pathogen prevalent in low concentrations in irrigation water. Although it causes  serious crop damage, the pathogen is still difficult to detect.    

The improvement of live/dead staining is the first step in the development of detection of bacteria. Next we evaluate bead systems for detection of specific bacteria. Immunomagnetic separation (IMS) is used to this means. Antibody coupling makes specific detection possible, while the magnetic properties of the beads allow for concentration of bacteria by magnetic separation. The combination of IMS and live/dead staining makes it possible to analyze the bacteria with the flow cytometer. The combination of these techniques is experimental and rarely tested. First lab cultures of bacteria are tested with this model. Later on the technique is validated with greenhouse samples of irrigation water. Irrigation water is a very difficult and complex matrix for analysis.

Flow cytometry offers opportunities for fytopathological applications. Unexpected results surfaced with the test organism Pseudomonas cichorii. This bacterium is hard to find in greenhouse water samples. It appeared that mechanical stress has a negative influence on the survival of the bacteria. The combination of qPCR and flow cytometry was needed to come to this conclusion. The occurrence of this type of stress in P. cichorii was unknown before the start of the project. Furthermore, in general apoptose and autolysis are not well studied in bacteria. The results obtained by flow cytometry will probably lead to more applications because of its wide scope of possibilities. Based on our results, live/dead staining turned out to be easy to apply and evaluate microbial life in soil- and water samples. Beyond the scope of the project there were very positive results for the evaluation of diversity in fungi by genome size analysis. Other tests suggest the flow cytometric detection of plant viruses by IMS is more easily compared to bacterial detection.