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

Chitarra, L. G.; Breeuwer, P.; Abee, Tjakko; Bulk, R.W. van den

doi:10.1590/s0100-41582006000400004

Cited by 12 publications

(13 citation statements)

References 33 publications

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“…(2002) used FCM on plant pathogenic fungi to determine stress and viability on conidia during and after heat treatment. Several authors have used FCM on plant pathogenic bacteria to compare different fluorochromes for viability assessment (Chitarra et al ., 2006; Porter et al ., 1997b), whereas others have tested survival under stress (Ordax et al ., 2006; van Overbeek et al ., 2004). Most of the authors that have compared FCM with plate counts detected more bacteria using FCM (Chitarra et al ., 2006; Ordax et al ., 2006; Porter et al ., 1997b; van Overbeek et al ., 2004); this discrepancy could be as high as 10 8 /mL –1 for bacteria in the VBNC state (Ordax et al ., 2006).…”

Section: Applications Of Fcm In Plant Pathologymentioning

confidence: 99%

Applications of flow cytometry in plant pathology for genome size determination, detection and physiological status

D'hondt¹,

Höfte

Bockstaele

et al. 2011

Molecular Plant Pathology

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Flow cytometers are probably the most multipurpose laboratory devices available. They can analyse a vast and very diverse range of cell parameters. This technique has left its mark on cancer, human immunodeficiency virus and immunology research, and is indispensable in routine clinical diagnostics. Flow cytometry (FCM) is also a well-known tool for the detection and physiological status assessment of microorganisms in drinking water, marine environments, food and fermentation processes. However, flow cytometers are seldom used in plant pathology, despite FCM's major advantages as both a detection method and a research tool. Potential uses of FCM include the characterization of genome sizes of fungal and oomycete populations, multiplexed pathogen detection and the monitoring of the viability, culturability and gene expression of plant pathogens, and many others. This review provides an overview of the history, advantages and disadvantages of FCM, and focuses on the current applications and future possibilities of FCM in plant pathology.

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Section: Applications Of Fcm In Plant Pathologymentioning

confidence: 99%

Applications of flow cytometry in plant pathology for genome size determination, detection and physiological status

D'hondt¹,

Höfte

Bockstaele

et al. 2011

Molecular Plant Pathology

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“…On the other hand, propidium iodide (PI) can pass through damaged cell membranes only and form complexes with ds-DNA and RNA. PI-stained cells are assumed to be nonviable (Chitaara et al 2006).…”

Section: Flow Cytometrymentioning

confidence: 99%

“…However, both plate count and FCM results were positively correlated with the percentage of viable Cmm population. FCM analyses of Calcein AM stained cells may be used as a viability indicator, as this procedure was found to be superior to the plate count method, in addition to the shorter assay time required for FCM analyses (Chitaara et al 2006; Appendix 7).…”

Section: Flow Cytometrymentioning

confidence: 99%

Detection of Bacterial and Phytoplasmal Pathogens

Narayanasamy¹

2010

Microbial Plant Pathogens-Detection and Disease Diagnosis:

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Bacterial pathogens classified as prokaryotes are much simpler in structure and smaller in size and they have less complex structural features compared to fungal plant pathogens. As the morphological characteristics of bacterial cells are less variable, biological, biochemical, physiological, immunological and genomic characteristics have to be determined for reliable identification and meaningful classification of bacterial pathogens. Detection and identification of bacterial plant pathogens present in whole plants and in propagative plant materials have been possible by employing isolation on cultural media and metabolic fingerprinting methods. But they are labor-intensive and require long time. Results often are inconclusive. Isozyme analysis, direct colony thin layer chromatography and gel electrophoresis techniques have been successfully applied for the detection of some bacterial pathogens. Immunoassays and nucleic acid-based assays have become widely accepted techniques, providing more sensitive and specific detection and quantification of bacterial pathogens affecting a wide range of host plant species. However, the major limitation of the molecular techniques is their inability to discriminate living cells from the dead ones. Attempts have been made to overcome this limiting factor by using DNA binding dyes and estimating pathogen RNAs as an indicator of cell viability. The diagnostic methods have both merits and demerits and hence, appropriate method has to be selected based on cost-effectiveness and possibility of obtaining reliable results rapidly to suit the requirements of the investigation concerned.Phytoplasmas are cell wall-less, nonculturable bacteria belonging to Mollicutes. Lack of cultural characteristics has made obligatory to study the morphological characteristics of phytoplasmas present in the phloem cells of infected plant hosts by observing under electron microscopes. As most of the phytoplasmas look alike in the ultrathin sections, the morphological characters have no diagnostic value. Histochemical methods using DNA binding dyes have been employed to localize the phytoplasmas in the host cells. Application of immunoassays and nucleic acidbased techniques has been effective in providing information for the identification and differentiation of phytoplasams. Polyclonal and monoclonal antibodies have been generated for detection of phytoplasmas infecting several plant species. Universal and species-specific primers and probes have been designed based on

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“…michiganensis (Smith) Davis, Gillaspie, Vidaver & Harris in tomato seed extracts, and Xanthomonas campestris pv. campestris (Pammel) Dowson in cabbage seed extracts (Chitarra et al, 2006). In theory, FCM can replace visual observation and quantification of bacteria in IF cell-staining (Diaper & Edwards, 1994;Bunthof et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

“…michiganenis and X. campestris pv. campestris (Chitarra et al, 2006). The aim of this work was to evaluate FCM methods for the detection and quantification of Xap in crude bean seed extracts and to compare with IF and dilution plating; to evaluate different fluorescent probes for direct viable count staining of Xap and to develop a FCM sorting method for PCR amplification directly on sorted sample fluid, without the need of DNA purification.…”

Section: Introductionmentioning

confidence: 99%

Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting

Tebaldi¹,

Peters²,

Souza³

et al. 2010

Trop. plant pathol.

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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 10 3 -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 10 3 -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. Key words: seed pathology, flow sorting, PCR-amplification, viability probes, immunofluorescence, bacteria. RESUMO Detecção de Xanthomonas axonopodis pv. phaseoli em sementes de feijão usando citometria de fluxo em combinação com anticorpo e sondas fluorescentes de viabilidadeA combinação do uso do citômetro de fluxo (FCM) e de anticorpo policlonal (imuno-FCM) foi comparada à microscopia de imunofluorescência (IF) e ao plaqueamento em meio de cultura semi-seletivo, para a detecção de Xanthomonas axonopodis pv. phaseoli (Xap) em sementes de feijão. Concentrações de Xap variando de 10 3 a 10 7 CFU/mL foram adicionados aos extratos de sementes. Um método de separação pelo citômetro de fluxo foi desenvolvido para a detecção de Xap em extratos de semente e posterior confirmação por PCR. Para avaliação da viabilidade das células foram usadas sondas fluorescentes, iodeto de propídio (PI)/carboxi diacetato de fluoresceína (cFDA) e PI/SYTO 9 e também, a combinação de imuno-FCM e PI. Em meio semi-seletivo e IF foram detectadas 10 3 -10 6 UFC/mL e no FCM 10 4 -10 6 UFC/mL, em extratos de sementes artificialmente infestados. Xap somente foi detectada em extratos de sementes por PCR, após o processo de separação pelo FCM. Foi possível pelo FCM a quantificação e identificação de células viáveis (verde fluorescente) e células mortas (vermelho fluorescente) de Xap, pelas sondas cFDA/SYTO 9 e PI, respectivamente. A combinação de immuno-FCM e PI poderá ser uma técnica promissora e segura para a detecção deste patógeno em sementes. Palavras-chave: patologia de sementes, PCR, sondas de viabilidade, imuno-fluorescência, bactéria. INTRODUCTIONXanthomonas axonopodis pv. phaseoli (Smith) Vauterin, Hoste, Kersters & Swinges (Xap) is a seedtransmitted plant-pathogenic bacterium w...

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

Cited by 12 publications

References 33 publications

Applications of flow cytometry in plant pathology for genome size determination, detection and physiological status

Applications of flow cytometry in plant pathology for genome size determination, detection and physiological status

Detection of Bacterial and Phytoplasmal Pathogens

Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting

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