AUTHOR=Raguseo Celeste , Gerin Donato , Pollastro Stefania , Rotolo Caterina , Rotondo Palma Rosa , Faretra Francesco , De Miccolis Angelini Rita Milvia 

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

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.747560

DOI=10.3389/fmicb.2021.747560

ISSN=1664-302X

ABSTRACT=<p>Brown rot, caused by different <italic>Monilinia</italic> species, is a most economically important disease of pome and stone fruits worldwide. In Europe and in Italy, the quarantine pathogen <italic>M. fructicola</italic> was recently introduced and rapidly spread and, by competing with the main indigenous species <italic>Monilinia fructigena</italic> and <italic>Monilinia laxa</italic>, caused relevant changes in <italic>Monilinia</italic> populations. As a result, in most areas, the pathogen almost replaced <italic>M. fructigena</italic> and now coexists with <italic>M. laxa</italic>. 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 <italic>M. fructicola</italic> and <italic>M. laxa</italic> 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 &lt; 1 copy μL<sup>–1</sup> 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 <italic>M. fructicola</italic> and <italic>M. laxa</italic>, showed a high correlation (<italic>R</italic><sup>2</sup> = 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 <italic>M. fructicola</italic>. The herein described method represents a useful tool for the early detection of <italic>Monilinia</italic> spp. on stone fruits and for the improving knowledge on the epidemiology of brow rot and interactions between the two prevalent <italic>Monilinia</italic> species.</p>