A real‐time (TaqMan) PCR assay to differentiate Monilinia fructicola from other brown rot fungi of fruit crops

Abstract

To prevent the entry and spread of the brown rot fungus Monilinia fructicola in Europe, a fast and reliable method for detection of this organism is essential. In this study, an automated DNA extraction method combined with a multiplex real-time PCR based on TaqMan chemistry was developed for fast, convenient and reliable detection of both the EU quarantine organism Monilinia fructicola and the three other brown rot fungi M. fructigena, M. laxa and Monilia polystroma. Using the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene repeat, a Monilinia genus-specific primer pair and two differently labelled fluorogenic probes specific for M. fructicola and the group M. fructigena ⁄ M. laxa ⁄ Monilia polystroma were developed. The analytical specificity of the assay was assessed by testing 33 isolates of the four brown rot fungi and 13 isolates of related fungal species or other fungal species that can be present on stone and pome fruit. No crossreactions were observed. The assay was found to have a detection limit of 0AE6 pg of DNA, corresponding to 27 haploid genomes or four conidia. Comparison of a manual DNA isolation followed by a conventional PCR with an automated DNA isolation combined with the presently developed real-time PCR showed that the latter method gave improved results when tested with 72 naturally infected stone fruit samples. The detection rate increased from 65 to 97%.

Peach Brown Rot: Still in Search of an Ideal Management Option

“…Since the first decade of this century, several methods of molecular characterization to distinguish Monilinia species [4,40,85,87,88] exist. Most of the molecular methods for characterization or disease detection include the Polymerase Chain Reaction (PCR), which is an in vitro, primer-directed, enzymatic reaction capable of exponential amplification of DNA.…”

“…in stone fruit trees combining a set of universal primers with the inclusion of an internal control for the diagnosis of brown rot caused by the three more important species. More recently, the RT-PCR method developed by Brouwershaven et al [88] was validated against all four brown rot-causing Monilinia species (M. laxa, M. fructicola, M. fructigena, and M. polystroma). In 2016, Guinet et al [93] used a multiplex real-time PCR (RT-PCR) to detect and discriminate the three common species of Monilinia (M. laxa, M. fructicola, and M. fructigena) on Prunus and Malus.…”

“…Recently, Garcia-Benitez [95] compared the overnight freezing-incubation technique (ONFIT) and the modified RT-PCR quantitative polymerase chain reaction (qPCR)-based methods of van Brouwershaven et al [88] to detect latent brown rot infections and, subsequently, distinguish between the Monilinia spp. in flowers and peach fruits.…”

Epidemiología y control de la podredumbre parda del melocotón en postcosecha

“…We detected bigger amounts of M. fructicola than M. laxa DNA and M. fructigena DNA in latently infected flowers using qPCR. We found that latent M. laxa infections had bigger DNA amounts Although conventional PCR and qPCR-based methods have already been developed for identifying and discriminating Monilinia species, these methods rely on sampling plant material with visible disease (Côté et al, 2004;Gell et al, 2007;Guinet et al, 2016;Hughes et al, 2000;Ioos and Frey, 2000;van Brouwershaven et al, 2010). We found that the qPCR-based method can detect the pathogen in artificial and natural latent brown rot infections.…”

“…Three different groups have developed qPCR methods for detection of Monilinia spp. from fungal cells collected from symptomatic plant material (Guinet et al, 2016;Luo et al, 2007;van Brouwershaven et al, 2010). But they have not been used to detect latent infection.…”