Ghent University. Faculty of Bioscience Engineering
Abstract
Flow cytometry (FCM) is a powerful and very versatile technique to measure cells in suspension. It is an indispensible method for routine diagnostics in the medical sector, but also for research purposes in very diverse fields of study. Despite its multiple and still increasing use in other sectors, FCM is scarcely used in plant pathology.
In this thesis, we explored the possibilities of flow cytometry in plant pathology, focussing on viability and specific detection of the waterborne lettuce pathogen Pseudomonas cichorii.
In a first phase we tested different fluorescent dyes and optimal instrument settings to stain, detect and count bacteria with FCM and determine their viability. In a next step, we wanted to develop a specific detection method for P. cichorii in irrigation water. As this pathogen can cause midrib rot on greenhouse-grown lettuce after a single overhead irrigation with water containing only 100 CFU ml-1, very sensitive detection was necessary. Moreover, P. cichorii is most found in rainwater, and this water often contains high bacterial backgrounds, as well as other organic and inorganic pollutants. Therefore we chose to develop a detection system based on immunomagnetic beads, which would allow specific capture and concentration of the target cells out of the water. We wanted to combine specific detection with viability assessment, in order to have a method that is also useful to research in vivo survival of P. cichorii and gain more insight into the epidemiology of the bacteria.
The combination of immunomagnetic separation (IMS) and live/dead staining is not easy and has seldom been tried. We tested different bead systems and defined the most important factors influencing IMS and nonspecific staining. We obtained best results with the relatively large (2.6 µm) non-fluorescent Compel beads. After optimizing this bead system, we came to a method in which beads were identified based on scatter properties and bacteria based on fluorescence properties. Bead-bacteria complexes had both the large scatter of the beads and the high fluorescence of the live/dead stained bacteria. Combining those two conditions in a logical combination of gates allowed the exclusion of most noise and resulted in the sensitive enumeration of bead-bacteria complexes. This method was further evaluated on mixed cultures and larger volumes and finally tested on different irrigation waters from commercial lettuce greenhouses.
Irrigation water proved to be a difficult and very variable matrix. Despite the extra sample pretreatment steps, we could not reliably detect P. cichorii cells below the infection threshold of 100 cells ml-1, except in one water type. The major problems we encountered were a too low recovery of P. cichorii, combined with a too high background remaining in the final samples. Besides the IMS method itself, of which the binding percentage and binding strength should be improved, both the bacteria and their matrix complicated detection. Their was a significant difference between some of the tested sampling dates and the analysis date had a significant effect on P. cichorii recovery: higher recovery was obtained in the same waters sampled in March, compared to the February samplings. Furthermore, recovery improved when a water sample was spiked and analysed after storage for at least a week. Also PCR recovery may be influenced by sampling date, but here recovery tended to be lower in spring samplings. The combination of low recovery and an unknown influence of water constitution on recovery, made that our IMS method is not (yet) suited as an alternative for the existing PCR detection of P. cichorii. However, when comparing the conventional real-time PCR detection of P. cichorii with our IMS-FCM method, or with IMS pretreatment followed by PCR analysis, conventional RT-PCR is by far the most expensive method. Not the PCR analysis itself, but the sample pretreatment and DNA extraction before PCR is laborious and has, besides very high labour cost, also high material costs. Although PCR will remain the most specific method, IMS and/or FCM could be brought to a comparable sensitivity and have the potential to become a more cost-effective alternative for sample pretreatment and/or PCR analysis.
The fact that P. cichorii is a difficult bacterium to detect is not only due to the IMS/FCM methodology, its low infection threshold, or to the complexity of its natural environment. Also the extremely high sensitivity of these bacteria to mechanical stress complicated detection. Mechanical stress seems to induce rapid apoptosis and autolysis, making P. cichorii cells disappear for both FCM or PCR detection. Medium constitution, especially salt concentration and the presence of nutrients, has a big influence on survival. In the absence of H2O2 and presence of 1% LB, recovery percentages of more than 90% could be obtained, while in saline solution, less than 10% was recovered after centrifugation. Although the enigmatic behaviour of P. cichorii complicated our research, such a far-reaching effect of common lab practices on bacterial viability has never been reported before and may be of considerable importance for microbiological practices
