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Monitoring Viable Cells of the Biological Control Agent Lactobacillus plantarum PM411 in Aerial Plant Surfaces by Means of a Strain-Specific Viability Quantitative PCR Method

A viability quantitative PCR (v-qPCR) assay was developed for the unambiguous detection and quantification of Lactobacillus plantarum PM411 viable cells in aerial plant surfaces. A 972-bp region of a PM411 predicted prophage with mosaic architecture enabled the identification of a PM411 strain-speci...

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Autores principales: Daranas, Núria, Bonaterra, Anna, Francés, Jesús, Cabrefiga, Jordi, Montesinos, Emilio, Badosa, Esther
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society for Microbiology 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930365/
https://www.ncbi.nlm.nih.gov/pubmed/29523544
http://dx.doi.org/10.1128/AEM.00107-18
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author Daranas, Núria
Bonaterra, Anna
Francés, Jesús
Cabrefiga, Jordi
Montesinos, Emilio
Badosa, Esther
author_facet Daranas, Núria
Bonaterra, Anna
Francés, Jesús
Cabrefiga, Jordi
Montesinos, Emilio
Badosa, Esther
author_sort Daranas, Núria
collection PubMed
description A viability quantitative PCR (v-qPCR) assay was developed for the unambiguous detection and quantification of Lactobacillus plantarum PM411 viable cells in aerial plant surfaces. A 972-bp region of a PM411 predicted prophage with mosaic architecture enabled the identification of a PM411 strain-specific molecular marker. Three primer sets with different amplicon lengths (92, 188, and 317 bp) and one TaqMan probe were designed. All the qPCR assays showed good linearity over a 4-log range and good efficiencies but differed in sensitivity. The nucleic acid-binding dye PEMAX was used to selectively detect and enumerate viable bacteria by v-qPCR. The primer set amplifying a 188-bp DNA fragment was selected as the most suitable for v-qPCR. The performance of the method was assessed on apple blossoms, pear, strawberry, and kiwifruit leaves in potted plants under controlled environmental conditions, as well as pear and apple blossoms under field conditions, by comparing v-qPCR population estimations to those obtained by qPCR and specific plate counting on de Man-Rogosa-Sharpe (MRS)-rifampin. The population estimation did not differ significantly between methods when conditions were conducive to bacterial survival. However, under stressful conditions, differences between methods were observed due to cell death or viable-but-nonculturable state induction. While qPCR overestimated the population level, plate counting underestimated this value in comparison to v-qPCR. PM411 attained stable population levels of viable cells on the flower environment under high relative humidity. However, the unfavorable conditions on the leaf surface and the relatively dryness in the field caused an important decrease in the viable population. IMPORTANCE The v-qPCR method in combination with plate counting and qPCR is a powerful tool for studies of colonization and survival under field conditions, to improve formulations and delivery strategies of PM411, and to optimize the dose and timing of spray schedules. It is expected that PEMAX v-qPCR could also be developed for monitoring other strains on plant surfaces not only as biological control agents but also beneficial bacteria useful in the sustainable management of crop production.
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spelling pubmed-59303652018-05-11 Monitoring Viable Cells of the Biological Control Agent Lactobacillus plantarum PM411 in Aerial Plant Surfaces by Means of a Strain-Specific Viability Quantitative PCR Method Daranas, Núria Bonaterra, Anna Francés, Jesús Cabrefiga, Jordi Montesinos, Emilio Badosa, Esther Appl Environ Microbiol Plant Microbiology A viability quantitative PCR (v-qPCR) assay was developed for the unambiguous detection and quantification of Lactobacillus plantarum PM411 viable cells in aerial plant surfaces. A 972-bp region of a PM411 predicted prophage with mosaic architecture enabled the identification of a PM411 strain-specific molecular marker. Three primer sets with different amplicon lengths (92, 188, and 317 bp) and one TaqMan probe were designed. All the qPCR assays showed good linearity over a 4-log range and good efficiencies but differed in sensitivity. The nucleic acid-binding dye PEMAX was used to selectively detect and enumerate viable bacteria by v-qPCR. The primer set amplifying a 188-bp DNA fragment was selected as the most suitable for v-qPCR. The performance of the method was assessed on apple blossoms, pear, strawberry, and kiwifruit leaves in potted plants under controlled environmental conditions, as well as pear and apple blossoms under field conditions, by comparing v-qPCR population estimations to those obtained by qPCR and specific plate counting on de Man-Rogosa-Sharpe (MRS)-rifampin. The population estimation did not differ significantly between methods when conditions were conducive to bacterial survival. However, under stressful conditions, differences between methods were observed due to cell death or viable-but-nonculturable state induction. While qPCR overestimated the population level, plate counting underestimated this value in comparison to v-qPCR. PM411 attained stable population levels of viable cells on the flower environment under high relative humidity. However, the unfavorable conditions on the leaf surface and the relatively dryness in the field caused an important decrease in the viable population. IMPORTANCE The v-qPCR method in combination with plate counting and qPCR is a powerful tool for studies of colonization and survival under field conditions, to improve formulations and delivery strategies of PM411, and to optimize the dose and timing of spray schedules. It is expected that PEMAX v-qPCR could also be developed for monitoring other strains on plant surfaces not only as biological control agents but also beneficial bacteria useful in the sustainable management of crop production. American Society for Microbiology 2018-05-01 /pmc/articles/PMC5930365/ /pubmed/29523544 http://dx.doi.org/10.1128/AEM.00107-18 Text en Copyright © 2018 Daranas et al. https://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Plant Microbiology
Daranas, Núria
Bonaterra, Anna
Francés, Jesús
Cabrefiga, Jordi
Montesinos, Emilio
Badosa, Esther
Monitoring Viable Cells of the Biological Control Agent Lactobacillus plantarum PM411 in Aerial Plant Surfaces by Means of a Strain-Specific Viability Quantitative PCR Method
title Monitoring Viable Cells of the Biological Control Agent Lactobacillus plantarum PM411 in Aerial Plant Surfaces by Means of a Strain-Specific Viability Quantitative PCR Method
title_full Monitoring Viable Cells of the Biological Control Agent Lactobacillus plantarum PM411 in Aerial Plant Surfaces by Means of a Strain-Specific Viability Quantitative PCR Method
title_fullStr Monitoring Viable Cells of the Biological Control Agent Lactobacillus plantarum PM411 in Aerial Plant Surfaces by Means of a Strain-Specific Viability Quantitative PCR Method
title_full_unstemmed Monitoring Viable Cells of the Biological Control Agent Lactobacillus plantarum PM411 in Aerial Plant Surfaces by Means of a Strain-Specific Viability Quantitative PCR Method
title_short Monitoring Viable Cells of the Biological Control Agent Lactobacillus plantarum PM411 in Aerial Plant Surfaces by Means of a Strain-Specific Viability Quantitative PCR Method
title_sort monitoring viable cells of the biological control agent lactobacillus plantarum pm411 in aerial plant surfaces by means of a strain-specific viability quantitative pcr method
topic Plant Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930365/
https://www.ncbi.nlm.nih.gov/pubmed/29523544
http://dx.doi.org/10.1128/AEM.00107-18
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