Please use this identifier to cite or link to this item: http://repositorio.ufla.br/jspui/handle/1/41988
Title: Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting
Other Titles: Detecção de Xanthomonas axonopodis pv. phaseoli em sementes de feijão usando citometria de fluxo em combinação com anticorpo e sondas fluorescentes de viabilidade
Keywords: Seed pathology
Flow sorting
Sondas de viabilidade
PCR-amplification
Immunofluorescence
Patologia de sementes
Issue Date: 2010
Publisher: Sociedade Brasileira de Fitopatologia
Citation: TEBALDI, N. D. et al. Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting. Tropical Plant Pathology, Brasília, DF, v. 35, n. 4, p. 213-222, 2010.
Abstract: 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 103-107 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 103-106 CFU/mL and immuno-FCM from 104-106 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.
URI: http://repositorio.ufla.br/jspui/handle/1/41988
Appears in Collections:DFP - Artigos publicados em periódicos



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