Flow cytometry in plant pathology: a case study on Pseudomonas cichorii

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

Host adaptation and speciation through hybridization and polyploidy in Phytophthora.

It is becoming increasingly evident that interspecific hybridization is a common event in phytophthora evolution. Yet, the fundamental processes underlying interspecific hybridization and the consequences for its ecological fitness and distribution are not well understood. We studied hybridization events in phytophthora clade 8b. This is a cold-tolerant group of plant pathogenic oomycetes in which six host-specific species have been described that mostly attack winter-grown vegetables. Hybrid characterization was done by sequencing and cloning of two nuclear (ITS and Ypt1) and two mitochondrial loci (Cox1 and Nadh1) combined with DNA content estimation using flow cytometry. Three different mtDNA haplotypes were recovered among the presumed hybrid isolates, dividing the hybrids into three types, with different parental species involved. In the nuclear genes, additivity, i.e. the presence of two alleles coming from different parents, was detected. Hybrid isolates showed large variations in DNA content, which was positively correlated with the additivity in nuclear loci, indicating allopolyploid hybridization followed by a process of diploidization. Moreover, indications of homeologous recombination were found in the hybrids by cloning ITS products. The hybrid isolates have been isolated from a range of hosts that have not been reported previously for clade 8b species, indicating that they have novel pathogenic potential. Next to this, DNA content measurements of the non-hybrid clade 8b species suggest that polyploidy is a common feature of this clade. We hypothesize that interspecific hybridization and polyploidy are two linked phenomena in phytophthora, and that these processes might play an important and ongoing role in the evolution of this genus

Aberrant genome size and instability of Phytophthora ramorum oospore progenies

The functionality of the sexual cycle in the heterothallic pathogen Phytophthora ramorum, causal agent of Sudden Oak Death, has recently been demonstrated. Sexual reproduction could create genotypic variation and increase the pathogen’s ability to adapt to other host plants or changing environments. Genetic characterization using co-dominant microsatellite markers and flow cytometry of single-oospore progeny of crosses between a European A1 isolate and North American or European A2 isolates revealed a considerable number of non-Mendelian inheritance events. This includes inheritance of more than two alleles at a locus and non-inheritance of alleles from one parent at another locus. The progenies were mitotically unstable: zoospore and hyphal tip derivatives of the progenies showed genotypic rearrangements and phenotypic variation. Flow cytometry confirmed variation and instability in DNA content of the single-oospore progenies. This indicates that single-oospore progenies not only display aberrant genomic and phenotypic variation due to meiotic irregularities, but also extra variation as a result of post-meiotic genomic rearrangements

De schaduw op het onderzoek

Fraude is absoluut laakbaar, ook in de wetenschap, maar het kan toch geen kwaad stil te staan bij de omstandigheden waarin onderzoek anno 2013 gebeurt, schrijven NEGENTIEN JONGE ACADEMICI. De druk op wetenschappers om spectaculaire resultaten te boeken, is immers niet meer gezond.status: publishe