Cargando…

Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR

Clavibacter michiganensis subsp. michiganensis (Cmm) is a seed-borne pathogen that causes bacterial canker disease of tomato. Cmm is typically detected in tomato seeds using quantitative real-time polymerase chain reaction (qPCR) combined with culture-based isolation. The viable but nonculturable (V...

Descripción completa

Detalles Bibliográficos
Autores principales: Han, Sining, Jiang, Na, Lv, Qingyang, Kan, Yumin, Hao, Jianjun, Li, Jianqiang, Luo, Laixin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933903/
https://www.ncbi.nlm.nih.gov/pubmed/29723290
http://dx.doi.org/10.1371/journal.pone.0196525
_version_ 1783320028548956160
author Han, Sining
Jiang, Na
Lv, Qingyang
Kan, Yumin
Hao, Jianjun
Li, Jianqiang
Luo, Laixin
author_facet Han, Sining
Jiang, Na
Lv, Qingyang
Kan, Yumin
Hao, Jianjun
Li, Jianqiang
Luo, Laixin
author_sort Han, Sining
collection PubMed
description Clavibacter michiganensis subsp. michiganensis (Cmm) is a seed-borne pathogen that causes bacterial canker disease of tomato. Cmm is typically detected in tomato seeds using quantitative real-time polymerase chain reaction (qPCR) combined with culture-based isolation. The viable but nonculturable (VBNC) state of Cmm may result in the underestimation or false negative detection of the pathogen. In the present study, propidium monoazide (PMA) and its improved structure PMAxx were used to pretreat Cmm prior to DNA extraction, followed by qPCR. Both PMA and PMAxx could bind to the chromosomal DNA of dead bacterial cells and therefore block DNA amplification by PCR. This effect, however, does not occur in living bacterial cells, as the chemicals cannot penetrate through the undamaged cell membrane. Both viable and dead Cmm cells were treated with PMA and PMAxx at various concentrations. With this treatment, the range of the cell population was determined for effective detection. PMAxx showed a better discrimination effect than PMA on the viable and dead cells of Cmm and was therefore used throughout the present study. VBNC cells of Cmm (10(8) CFU mL(-1)) was induced by 50 μM copper sulfate, which was detected at different sampling times up to a month by using both PMAxx-qPCR and flow cytometry assays. The optimal PMAxx concentration was 20 μM for detecting membrane-intact Cmm cells. High specificity and sensitivity were obtained at Cmm concentrations ranging from 10(3) to 10(7) CFU mL(-1). The accurate and robust results of PMAxx-qPCR were confirmed by flow cytometry method to detect viable Cmm cells. Furthermore, the PMAxx-qPCR assay was successfully used in detecting VBNC Cmm cells in tomato seeds with as few as 10 seeds per set.
format Online
Article
Text
id pubmed-5933903
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-59339032018-05-18 Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR Han, Sining Jiang, Na Lv, Qingyang Kan, Yumin Hao, Jianjun Li, Jianqiang Luo, Laixin PLoS One Research Article Clavibacter michiganensis subsp. michiganensis (Cmm) is a seed-borne pathogen that causes bacterial canker disease of tomato. Cmm is typically detected in tomato seeds using quantitative real-time polymerase chain reaction (qPCR) combined with culture-based isolation. The viable but nonculturable (VBNC) state of Cmm may result in the underestimation or false negative detection of the pathogen. In the present study, propidium monoazide (PMA) and its improved structure PMAxx were used to pretreat Cmm prior to DNA extraction, followed by qPCR. Both PMA and PMAxx could bind to the chromosomal DNA of dead bacterial cells and therefore block DNA amplification by PCR. This effect, however, does not occur in living bacterial cells, as the chemicals cannot penetrate through the undamaged cell membrane. Both viable and dead Cmm cells were treated with PMA and PMAxx at various concentrations. With this treatment, the range of the cell population was determined for effective detection. PMAxx showed a better discrimination effect than PMA on the viable and dead cells of Cmm and was therefore used throughout the present study. VBNC cells of Cmm (10(8) CFU mL(-1)) was induced by 50 μM copper sulfate, which was detected at different sampling times up to a month by using both PMAxx-qPCR and flow cytometry assays. The optimal PMAxx concentration was 20 μM for detecting membrane-intact Cmm cells. High specificity and sensitivity were obtained at Cmm concentrations ranging from 10(3) to 10(7) CFU mL(-1). The accurate and robust results of PMAxx-qPCR were confirmed by flow cytometry method to detect viable Cmm cells. Furthermore, the PMAxx-qPCR assay was successfully used in detecting VBNC Cmm cells in tomato seeds with as few as 10 seeds per set. Public Library of Science 2018-05-03 /pmc/articles/PMC5933903/ /pubmed/29723290 http://dx.doi.org/10.1371/journal.pone.0196525 Text en © 2018 Han et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Han, Sining
Jiang, Na
Lv, Qingyang
Kan, Yumin
Hao, Jianjun
Li, Jianqiang
Luo, Laixin
Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR
title Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR
title_full Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR
title_fullStr Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR
title_full_unstemmed Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR
title_short Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR
title_sort detection of clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qpcr
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933903/
https://www.ncbi.nlm.nih.gov/pubmed/29723290
http://dx.doi.org/10.1371/journal.pone.0196525
work_keys_str_mv AT hansining detectionofclavibactermichiganensissubspmichiganensisinviablebutnonculturablestatefromtomatoseedusingimprovedqpcr
AT jiangna detectionofclavibactermichiganensissubspmichiganensisinviablebutnonculturablestatefromtomatoseedusingimprovedqpcr
AT lvqingyang detectionofclavibactermichiganensissubspmichiganensisinviablebutnonculturablestatefromtomatoseedusingimprovedqpcr
AT kanyumin detectionofclavibactermichiganensissubspmichiganensisinviablebutnonculturablestatefromtomatoseedusingimprovedqpcr
AT haojianjun detectionofclavibactermichiganensissubspmichiganensisinviablebutnonculturablestatefromtomatoseedusingimprovedqpcr
AT lijianqiang detectionofclavibactermichiganensissubspmichiganensisinviablebutnonculturablestatefromtomatoseedusingimprovedqpcr
AT luolaixin detectionofclavibactermichiganensissubspmichiganensisinviablebutnonculturablestatefromtomatoseedusingimprovedqpcr