Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting
Autor(a) principal: | |
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Data de Publicação: | 2010 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UFLA |
Texto Completo: | http://repositorio.ufla.br/jspui/handle/1/41988 |
Resumo: | 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. |
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Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable countingDetecção de Xanthomonas axonopodis pv. phaseoli em sementes de feijão usando citometria de fluxo em combinação com anticorpo e sondas fluorescentes de viabilidadeSeed pathologyFlow sortingSondas de viabilidadePCR-amplificationImmunofluorescencePatologia de sementesFlow 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.A 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 103 a 107 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 103-106 UFC/mL e no FCM 104-106 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.Sociedade Brasileira de Fitopatologia2020-07-15T17:57:40Z2020-07-15T17:57:40Z2010info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfTEBALDI, 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.http://repositorio.ufla.br/jspui/handle/1/41988Tropical Plant Pathologyreponame:Repositório Institucional da UFLAinstname:Universidade Federal de Lavras (UFLA)instacron:UFLAAttribution-NonCommercial 4.0 Internationalhttp://creativecommons.org/licenses/by-nc/4.0/info:eu-repo/semantics/openAccessTebaldi, Nilvanira D.Peters, JeroenSouza, Ricardo M.Chitarra, Luiz G.Van der Zouwen, PatriciaBergervoet, JanVan der Wolf, Janeng2023-05-26T19:53:09Zoai:localhost:1/41988Repositório InstitucionalPUBhttp://repositorio.ufla.br/oai/requestnivaldo@ufla.br || repositorio.biblioteca@ufla.bropendoar:2023-05-26T19:53:09Repositório Institucional da UFLA - Universidade Federal de Lavras (UFLA)false |
dc.title.none.fl_str_mv |
Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting Detecção de Xanthomonas axonopodis pv. phaseoli em sementes de feijão usando citometria de fluxo em combinação com anticorpo e sondas fluorescentes de viabilidade |
title |
Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting |
spellingShingle |
Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting Tebaldi, Nilvanira D. Seed pathology Flow sorting Sondas de viabilidade PCR-amplification Immunofluorescence Patologia de sementes |
title_short |
Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting |
title_full |
Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting |
title_fullStr |
Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting |
title_full_unstemmed |
Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting |
title_sort |
Detection of Xanthomonas axonopodis pv. phaseoli in bean seeds by flow cytometry, immunostaining and direct viable counting |
author |
Tebaldi, Nilvanira D. |
author_facet |
Tebaldi, Nilvanira D. Peters, Jeroen Souza, Ricardo M. Chitarra, Luiz G. Van der Zouwen, Patricia Bergervoet, Jan Van der Wolf, Jan |
author_role |
author |
author2 |
Peters, Jeroen Souza, Ricardo M. Chitarra, Luiz G. Van der Zouwen, Patricia Bergervoet, Jan Van der Wolf, Jan |
author2_role |
author author author author author author |
dc.contributor.author.fl_str_mv |
Tebaldi, Nilvanira D. Peters, Jeroen Souza, Ricardo M. Chitarra, Luiz G. Van der Zouwen, Patricia Bergervoet, Jan Van der Wolf, Jan |
dc.subject.por.fl_str_mv |
Seed pathology Flow sorting Sondas de viabilidade PCR-amplification Immunofluorescence Patologia de sementes |
topic |
Seed pathology Flow sorting Sondas de viabilidade PCR-amplification Immunofluorescence Patologia de sementes |
description |
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. |
publishDate |
2010 |
dc.date.none.fl_str_mv |
2010 2020-07-15T17:57:40Z 2020-07-15T17:57:40Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
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. http://repositorio.ufla.br/jspui/handle/1/41988 |
identifier_str_mv |
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. |
url |
http://repositorio.ufla.br/jspui/handle/1/41988 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.rights.driver.fl_str_mv |
Attribution-NonCommercial 4.0 International http://creativecommons.org/licenses/by-nc/4.0/ info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Attribution-NonCommercial 4.0 International http://creativecommons.org/licenses/by-nc/4.0/ |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Sociedade Brasileira de Fitopatologia |
publisher.none.fl_str_mv |
Sociedade Brasileira de Fitopatologia |
dc.source.none.fl_str_mv |
Tropical Plant Pathology reponame:Repositório Institucional da UFLA instname:Universidade Federal de Lavras (UFLA) instacron:UFLA |
instname_str |
Universidade Federal de Lavras (UFLA) |
instacron_str |
UFLA |
institution |
UFLA |
reponame_str |
Repositório Institucional da UFLA |
collection |
Repositório Institucional da UFLA |
repository.name.fl_str_mv |
Repositório Institucional da UFLA - Universidade Federal de Lavras (UFLA) |
repository.mail.fl_str_mv |
nivaldo@ufla.br || repositorio.biblioteca@ufla.br |
_version_ |
1807835165953622016 |