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A Duplex-Droplet Digital PCR Assay for Simultaneous Quantitative Detection of Monilinia fructicola and Monilinia laxa on Stone Fruits

Brown rot, caused by different Monilinia species, is a most economically important disease of pome and stone fruits worldwide. In Europe and in Italy, the quarantine pathogen M. fructicola was recently introduced and rapidly spread and, by competing with the main indigenous species Monilinia fructig...

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Autores principales: Raguseo, Celeste, Gerin, Donato, Pollastro, Stefania, Rotolo, Caterina, Rotondo, Palma Rosa, Faretra, Francesco, De Miccolis Angelini, Rita Milvia
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8667764/
https://www.ncbi.nlm.nih.gov/pubmed/34912308
http://dx.doi.org/10.3389/fmicb.2021.747560
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author Raguseo, Celeste
Gerin, Donato
Pollastro, Stefania
Rotolo, Caterina
Rotondo, Palma Rosa
Faretra, Francesco
De Miccolis Angelini, Rita Milvia
author_facet Raguseo, Celeste
Gerin, Donato
Pollastro, Stefania
Rotolo, Caterina
Rotondo, Palma Rosa
Faretra, Francesco
De Miccolis Angelini, Rita Milvia
author_sort Raguseo, Celeste
collection PubMed
description Brown rot, caused by different Monilinia species, is a most economically important disease of pome and stone fruits worldwide. In Europe and in Italy, the quarantine pathogen M. fructicola was recently introduced and rapidly spread and, by competing with the main indigenous species Monilinia fructigena and Monilinia laxa, caused relevant changes in Monilinia populations. As a result, in most areas, the pathogen almost replaced M. fructigena and now coexists with M. laxa. The availability of specific and easy-of-use quantification methods is essential to study the population dynamics, and in this work, a new method for the simultaneous quantification of M. fructicola and M. laxa based on droplet digital PCR (ddPCR) technique was established. Under the optimized reaction conditions, consisting of 250/500 nM of primers/probe sets concentration, 58°C as annealing temperature and 50 PCR cycles, the duplex-ddPCR assay was 200-fold more sensitive than duplex-real-time quantitative PCR (qPCR) assay, quantifying < 1 copy μL(–1) of target DNA in the PCR mixture. The results obtained with the validation assay performed on apricot and peach fruits, artificially inoculated with conidial suspensions containing different ratios of M. fructicola and M. laxa, showed a high correlation (R(2) = 0.98) between the relative quantity of DNA of the two species quantified by ddPCR and qPCR and a more accurate quantification by ddPCR compared to qPCR at higher concentrations of M. fructicola. The herein described method represents a useful tool for the early detection of Monilinia spp. on stone fruits and for the improving knowledge on the epidemiology of brow rot and interactions between the two prevalent Monilinia species.
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spelling pubmed-86677642021-12-14 A Duplex-Droplet Digital PCR Assay for Simultaneous Quantitative Detection of Monilinia fructicola and Monilinia laxa on Stone Fruits Raguseo, Celeste Gerin, Donato Pollastro, Stefania Rotolo, Caterina Rotondo, Palma Rosa Faretra, Francesco De Miccolis Angelini, Rita Milvia Front Microbiol Microbiology Brown rot, caused by different Monilinia species, is a most economically important disease of pome and stone fruits worldwide. In Europe and in Italy, the quarantine pathogen M. fructicola was recently introduced and rapidly spread and, by competing with the main indigenous species Monilinia fructigena and Monilinia laxa, caused relevant changes in Monilinia populations. As a result, in most areas, the pathogen almost replaced M. fructigena and now coexists with M. laxa. The availability of specific and easy-of-use quantification methods is essential to study the population dynamics, and in this work, a new method for the simultaneous quantification of M. fructicola and M. laxa based on droplet digital PCR (ddPCR) technique was established. Under the optimized reaction conditions, consisting of 250/500 nM of primers/probe sets concentration, 58°C as annealing temperature and 50 PCR cycles, the duplex-ddPCR assay was 200-fold more sensitive than duplex-real-time quantitative PCR (qPCR) assay, quantifying < 1 copy μL(–1) of target DNA in the PCR mixture. The results obtained with the validation assay performed on apricot and peach fruits, artificially inoculated with conidial suspensions containing different ratios of M. fructicola and M. laxa, showed a high correlation (R(2) = 0.98) between the relative quantity of DNA of the two species quantified by ddPCR and qPCR and a more accurate quantification by ddPCR compared to qPCR at higher concentrations of M. fructicola. The herein described method represents a useful tool for the early detection of Monilinia spp. on stone fruits and for the improving knowledge on the epidemiology of brow rot and interactions between the two prevalent Monilinia species. Frontiers Media S.A. 2021-11-29 /pmc/articles/PMC8667764/ /pubmed/34912308 http://dx.doi.org/10.3389/fmicb.2021.747560 Text en Copyright © 2021 Raguseo, Gerin, Pollastro, Rotolo, Rotondo, Faretra and De Miccolis Angelini. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Microbiology
Raguseo, Celeste
Gerin, Donato
Pollastro, Stefania
Rotolo, Caterina
Rotondo, Palma Rosa
Faretra, Francesco
De Miccolis Angelini, Rita Milvia
A Duplex-Droplet Digital PCR Assay for Simultaneous Quantitative Detection of Monilinia fructicola and Monilinia laxa on Stone Fruits
title A Duplex-Droplet Digital PCR Assay for Simultaneous Quantitative Detection of Monilinia fructicola and Monilinia laxa on Stone Fruits
title_full A Duplex-Droplet Digital PCR Assay for Simultaneous Quantitative Detection of Monilinia fructicola and Monilinia laxa on Stone Fruits
title_fullStr A Duplex-Droplet Digital PCR Assay for Simultaneous Quantitative Detection of Monilinia fructicola and Monilinia laxa on Stone Fruits
title_full_unstemmed A Duplex-Droplet Digital PCR Assay for Simultaneous Quantitative Detection of Monilinia fructicola and Monilinia laxa on Stone Fruits
title_short A Duplex-Droplet Digital PCR Assay for Simultaneous Quantitative Detection of Monilinia fructicola and Monilinia laxa on Stone Fruits
title_sort duplex-droplet digital pcr assay for simultaneous quantitative detection of monilinia fructicola and monilinia laxa on stone fruits
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8667764/
https://www.ncbi.nlm.nih.gov/pubmed/34912308
http://dx.doi.org/10.3389/fmicb.2021.747560
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