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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Frontiers Media S.A.
2021
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oai:doaj.org-article:00fa14a78849482b96f95207f50195862021-12-01T13:58:59ZA Duplex-Droplet Digital PCR Assay for Simultaneous Quantitative Detection of Monilinia fructicola and Monilinia laxa on Stone Fruits1664-302X10.3389/fmicb.2021.747560https://doaj.org/article/00fa14a78849482b96f95207f50195862021-11-01T00:00:00Zhttps://www.frontiersin.org/articles/10.3389/fmicb.2021.747560/fullhttps://doaj.org/toc/1664-302XBrown 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 (R2 = 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.Celeste RaguseoDonato GerinStefania PollastroCaterina RotoloPalma Rosa RotondoFrancesco FaretraRita Milvia De Miccolis AngeliniFrontiers Media S.A.articleddPCRquantitative PCR (qPCR)brown rotdetectionplant pathogensfungiMicrobiologyQR1-502ENFrontiers in Microbiology, Vol 12 (2021) |
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| collection |
DOAJ |
| language |
EN |
| topic |
ddPCR quantitative PCR (qPCR) brown rot detection plant pathogens fungi Microbiology QR1-502 |
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ddPCR quantitative PCR (qPCR) brown rot detection plant pathogens fungi Microbiology QR1-502 Celeste Raguseo Donato Gerin Stefania Pollastro Caterina Rotolo Palma Rosa Rotondo Francesco Faretra Rita Milvia De Miccolis Angelini A Duplex-Droplet Digital PCR Assay for Simultaneous Quantitative Detection of Monilinia fructicola and Monilinia laxa on Stone Fruits |
| 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 (R2 = 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. |
| format |
article |
| author |
Celeste Raguseo Donato Gerin Stefania Pollastro Caterina Rotolo Palma Rosa Rotondo Francesco Faretra Rita Milvia De Miccolis Angelini |
| author_facet |
Celeste Raguseo Donato Gerin Stefania Pollastro Caterina Rotolo Palma Rosa Rotondo Francesco Faretra Rita Milvia De Miccolis Angelini |
| author_sort |
Celeste Raguseo |
| title |
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_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_sort |
duplex-droplet digital pcr assay for simultaneous quantitative detection of monilinia fructicola and monilinia laxa on stone fruits |
| publisher |
Frontiers Media S.A. |
| publishDate |
2021 |
| url |
https://doaj.org/article/00fa14a78849482b96f95207f5019586 |
| work_keys_str_mv |
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| _version_ |
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