Fluorescence techniques to detect and to assess viability of plant pathogenic bacteria

Plant pathogenic bacteria cause major economic losses in commercial crop production worldwide every year. The current methods used to detect and to assess the viability of bacterial pathogens and to test seed lots or plants for contamination are usually based on plate assays or on serological techniques. Plating methods provide information about cell viability, but are generally laborious and time-consuming. Serological techniques, such as immunofluorescence microscopy (IF) and enzyme-linked immunosorbent assay (ELISA), are much faster than most of the plating assays. However, they provide the user only with semi-quantitative information, which for various tests is not satisfactory, and they do not distinguish between viable and non-viable cell. Flow cytometry (FCM) is a rapid, reliable, and sensitive technique that has been successfully applied to detect and to assess the viability of several microorganisms in the field of veterinary science, medicine, and microbiology, and it could be worth exploring in the field of plant pathology. The research described in this thesis focused on the development of a rapid, reliable, and accurate method for the detection and assessment of viability of the seed-borne organisms Xanthomonascampestris pv. campestris (Xcc), the causal agent of black rot on cabbage, and Clavibacter michiganensis subsp. michiganensis (Cmm), the cause of bacterial canker of tomato, by applying fluorescent probes in combination with flow cytometry or spectrofluorometry.The viability of Cmm cells was first determined by measuring the intracellular pH (pH in ), as a parameter for viability, applying the fluorescent probe 5(and 6-)-carboxyfluorescein succinimidyl ester (cFSE) in combination with fluorescence spectrofluorometry or flow cytometry. The growth of Cmm cells in Glucose-Nutrient-Broth medium supplemented with potassium chloride in the absence and presence of the ionophore nigericin was evaluated to determine the minimum pH in value at which cells are able to grow. In the presence of nigericin (0.1 µmol -1 ), which equilibrates the intracellular and the extracellular pH out (pH in = pH out ), Cmm was not able to grow at pH 5.5 and below. Therefore, viable cells should maintain their intracellular pH above this pH value. The pH in of Cmm cells exposed to acid treatments, 0.1, 0.2 or 0.6 mol l -1 of HCl for 1 hour, was determined using fluorescence spectrofluorometry. In HCl treated cells no pH gradient could be detected (pH in = pH out ). Fluorescence microscopy revealed that these cells were poorly labeled with cFSE, either due to a low esterase activity in the cytoplasm or due to an increased efflux of cFSE resulting from the damage caused by the acid treatment. The spectrofluorometry analysis for pH in measurements was not able to detect the signal of these weakly stained cells and only a small percentage of HCl treated cells (0.001%) could be recovered on plate. For cells exposed to elevated temperatures, 40, 45 or 50 °C for 1 hour, the pH in was determined using cFSE in combination with flow cytometry and fluorescence spectrofluorometry. A good correlation (r 2 ≥0.80) was found between the number of colony-forming units per ml (CFU ml -1 ) determined by plate counting and the magnitude of the pH gradient (pH out - pH in ) determined with spectrofluorometry for the heat-treated populations. However, with the spectrofluorometry technique the analysis is based on the whole cell population and the sensitivity of this technique was found to be rather low. In our experiments, cell numbers of at least 10 7 CFU ml -1 were needed for the analysis. Using flow cytometry, which measures fluorescence intensity of individual cells, heat-treated and non-treated Cmm cells could be distinguished based on differences in the fluorescence ratios (pH gradients) after labeling with cFSE. From the FL1/FL2 dot plots the ratio of the green and the orange signals (FL1/FL2) could be calculated (after back transformation from log to linear mode). From this ratio the intracellular pH was calculated. The heat-treated cells had a low fluorescence ratio (no pH gradient) and could not be recovered on plates, whereas the ratio of live cells was significantly higher (pH gradient present). The major advantages of flow cytometry when compared with spectrofluorometry were its sensitivity and speed, because the analysis could be performed in two hours.The fluorescent enzyme activity probes Calcein acetoxy methyl ester (Calcein AM) and carboxyfluorescein diacetate (cFDA), and the nucleic acid probe propidium iodide (PI), were evaluated for assessing the viability of Cmm cells when applied in combination with flow cytometry. Heat-treated (80 °C for 30 minutes) and viable (non-treated) Cmm cells were mixed in different ratios, 100/0, 50/50, 20/80, and 0/100% respectively, to create populations varying in viability. Non-treated and heat-treated Cmm cells labeled with Calcein AM, cFDA, PI, or combinations of Calcein AM and cFDA with PI, could be distinguished based on their fluorescence intensity in flow cytometry analyses. Non-treated cells showed relatively high green fluorescence intensity levels, as the result of staining with Calcein AM or cFDA. Once inside the cell, Calcein AM and cFDA are cleaved (hydrolysed) by non-specific esterases to release fluorescein, a polar fluorescent compound which is retained inside the cells. Thus, the ability of the cell to accumulate fluorescein due to esterase activity is used as a parameter for viability. Damaged cells (heat-treated) showed high red fluorescence intensity levels, as the result of PI entering the cells with damaged cell membranes, intercalating into RNA and DNA. Flow cytometry allowed a rapid quantification and separation of viable Cmm cells labeled with Calcein AM or cFDA from heat-treated cells labeled with PI. The results showed a good correlation (r 2 ≥0.95) between the percentage of non-treated cells present in the samples and the flow cytometry counts for Cmm cells labeled with Calcein AM or cFDA. A linear relation (r 2 ≥0.80) was also found between the percentage of heat-treated cells in the samples and the flow cytometry counts for Cmm cells labeled with PI. However, when PI was applied as a single stain, it was able to stain 18 to 56% of non-treated Cmm cells. These results suggest that PI cannot be considered a good viability indicator for viable Cmm cells when applied alone. However, itt was shown to be a good indicator for dead or damaged cells. Therefore, the application of flow cytometry in combination with fluorescent probes appears to be a promising technique for assessing viability of Cmm cells in suspensions when cells are labeled with Calcein AM or the combination of Calcein AM with PI.Flow cytometry was also evaluated for the rapid detection of Xcc cells labeled in pure suspensions and in suspensions containing mixtures of Xcc and the common saprophyte Pseudomonas fluorescens (Psf) with a specific FITC-labeled monoclonal antibody (Mab). The concentration of Mab affected the sensitivity of the flow cytometry measurements. This is based on the concept that the optimal concentration of Mab is the one that gives the greatest discrimination between the fluorescently stained target cells and cells stained as the result of non-specific binding. However, the Mab concentrations tested do not seem to be a limiting factor for the detection of Xcc by flow cytometry. A limitation, however, is the concentration of cells present in the samples. Xcc cells labeled with FITC-conjugated monoclonal antibodies could rapidly be detected at low numbers, i.e 10 3 colony-forming units per ml in pure suspensions and in suspensions containing both Xcc and saprophytic Psf cells. The detection limit for Xcc applying other serological techniques, such as immunoflorescence microscopy (IF) and enzyme-linked immunosorbent assay (ELISA), is approximately 10 3 and 10 5 CFU ml -1 , respectively. A good correlation (r 2 ≥0.95) was observed between the flow cytometry counts and plate counts, although flow counts were always higher than plate counts due to the fact that antibodies do not discriminate between viable and non-viable cells. The number of Psf cells, relative to the number of Xcc cells, did not interfere, neither in the flow cytometry measurements nor in plate counts. Thus, flow cytometry in combination with Xcc specific FITC-labeled monoclonal antibodies may provide a novel tool for rapid detection and quantification of this plant pathogenic bacterium.The flow cytometry method applied to bacterial suspensions was evaluated as a tool for a rapid detection of Xcc cells, labeled with a mixture of three specific FITC-monoclonal antibodies (18G12, 2F4, and 20H6), in crude seed extracts. Flow cytometry allowed a rapid detection and quantification of Xcc cells labeled with FITC-monoclonal antibodies in both artificially and naturally Xcc-contaminated samples tested. Flow cytometry was able to detect the labeled Xcc cells in the seed extracts based on their high green fluorescence levels. No cross-reactions were observed with related Xanthomonads or other microorganisms present in artificially contaminated samples. In conclusion, the application of the flow cytometry technique in combination with specific, FITC-labeled monoclonal antibodies was shown to be a rapid and reliable alternative for the detection and quantification of Xcc cells in seed extracts.The work described in this thesis showed that flow cytometry in combination with fluorescent probes can be a promising technique to detect and to assess viability of plant pathogenic bacteria. Nonetheless, the application of flow cytometry as a routine method to test seed lots or plants for contamination with bacteria has to be further explored, especially combining detection with viability assessment in the same assay.</p

The application of flow cytometry and fluorescent probe technology for detection and assessment of viability of plant pathogenic bacteria

Conventional methods to detect and assess the viability of plant pathogenic bacteria are usually based on plating assays or serological techniques. Plating assays provide information about the number of viable cells, expressed as colony-forming units, but are time-consuming and laborious. Serological methods, such as the enzyme-linked immunosorbent assay (ELISA) and immunofluorescence microscopy (IF), can be performed in a shorter timespan than most plating assays, but they do not discriminate between live and dead cells. Flow cytometry (FCM) in combination with fluorescent probe technology is a rapid, sensitive, and quantitative technique to detect microorganisms and assess their viability. Quantitative information on the presence and viability of plant pathogenic microorganisms is valuable for risk assessment regarding disease transmission and disease development. FCM has been applied successfully in the fields of food microbiology, veterinary science, and medical research to detect and distinguish between viable and non-viable bacteria. The aim of this review is to show the potential of FCM and fluorescent probe technology for the field of plant pathology

The use of fluorescent probes to assess viability of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis by flow cytometry

Determination of the viability of bacteria by the conventional plating technique is a time-consuming process. Methods based on enzyme activity or membrane integrity are much faster and may be good alternatives. Assessment of the viability of suspensions of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) using the fluorescent probes Calcein acetoxy methyl ester (Calcein AM), carboxyfluorescein diacetate (cFDA), and propidium iodide (PI) in combination with flow cytometry was evaluated. Heat-treated and viable (non-treated) Cmm cells labeled with Calcein AM, cFDA, PI, or combinations of Calcein AM and cFDA with PI, could be distinguished based on their fluorescence intensity in flow cytometry analysis. Non-treated cells showed relatively high green fluorescence levels due to staining with either Calcein AM or cFDA, whereas damaged cells (heat-treated) showed high red fluorescence levels due to staining with PI. Flow cytometry also allowed a rapid quantification of viable Cmm cells labeled with Calcein AM or cFDA and heat-treated cells labeled with PI. Therefore, the application of flow cytometry in combination with fluorescent probes appears to be a promising technique for assessing viability of Cmm cells when cells are labeled with Calcein AM or the combination of Calcein AM with P