Flow cytometric analysis of immuno-stained cells (immuno-FCM) was compared to immunofluorescence microscopy (IF) and dilution plating on a semi-selective medium, for quantitative detection of Xanthomonas axonopodis pv. phaseoli (Xap) in bean seed extracts. Cell concentrations of Xap between 10³-10(7) CFU/mL were added to healthy bean seed extracts. A flow cytometry sorting procedure was developed to separate immuno-stained Xap cells from crude seed extracts and confirming by PCR. FCM was evaluated for direct viable counting (DVC) of Xap using combinations of propidium iodide (PI) and carboxy fluorescein diacetate (cFDA) or PI and SYTO 9 and also the combination of immuno-FCM and PI. Dilution plating and IF allowed detection of Xap in bean seed extracts in a range of 10³-10(6) CFU/mL and immuno-FCM from 10(4)-10(6) CFU/mL. Sorted cells could be detected in crude seed extracts by PCR without further extraction. FCM also allowed quantification of viable cells of Xap after DVC procedures; the red fluorescent dye propidium iodide was used to identify dead cells in combination with the green fluorescent dyes cFDA or SYTO 9, these identifying live cells. The combination of immuno-FCM and PI could be more promising and reliable to detect this pathogen in seeds.
seed pathology; flow sorting; PCR-amplification; viability probes; immunofluorescence; bacteria
