Abstract
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.
Highlights
Almost complete saturation of positive droplets was always obtained at 25 ng of target DNA. For both the primers/probe sets, increased droplet digital polymerase chain reaction (PCR) (ddPCR) for Monilinia spp. quantification concentrations up to 500/250 nM compared to those used in quantitative PCR (qPCR) (240/160 nM) resulted in higher fluorescence amplitude and better separation between positive and negative droplets, which was clearly visible for M. fructicola but not M. laxa (Figure 1A)
The introduction of the quarantine species M. fructicola into new areas caused significant changes in Monilinia populations responsible for brown rot on stone fruit (Villarino et al, 2013; Abate et al, 2018), so intensive and careful monitoring programs are crucial for evaluating the effectiveness of integrated crop protection strategies and whether adaptation is need (Côté et al, 2004)
The recent availability of ddPCR for the quantification of fungal pathogens allows the absolute quantification of the target, without the use of a standard curve, and an improvement in accuracy and sensitivity as compared with qPCR assays (Palumbo et al, 2016; del Pilar Martínez-Diz et al, 2020)
Summary
Monilinia laxa is considered indigenous to Europe, while M. fructicola was first introduced in 2001 in France (OEPP/EPPO, 2002), reported in 2009 in Northern Italy (OEPP/EPPO, 2009; Pellegrino et al, 2009), and later in other regions of the country (Abate et al, 2018). It is currently included in the A2 list of pathogens recommended for regulation as quarantine pests in the EPPO areas.. The shift caused by M. fructicola introduction in new geographic areas could be explained by its faster growth rate, infection, and adaptation to hot–dry conditions, associated with more abundant sporulation compared with M. laxa and M. fructigena (Byrde and Willetts, 1977; Lichtemberg et al, 2014; Villarino et al, 2016; Abate et al, 2018)
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