One-Step Detection of Monilinia fructicola, M. fructigena, and M. laxa on Prunus and Malus by a Multiplex Real-Time PCR Assay

Brown rot is an economically important fungal disease affecting stone and pome fruit orchards, as well as harvested fruit during storage and on the market. Monilinia fructicola, M. laxa, and M. fructigena are the main causal agents of this disease and each have a different regulatory status depending on regional regulations. In this study, a new multiplex tool based on real-time polymerase chain reaction was developed to detect the three pathogenic fungi in a single reaction on fruit, twigs, and flowers of Prunus and Malus spp. Species-specific primer-hydrolysis probe combinations were designed to amplify a region located in a previously described MO368 sequenced characterized amplified region marker, and used in a quadruplex format coupled with the 18S Uni universal primer-probe test in order to check the quality of the DNA template. The assay was designed and optimized with the objective to provide high performance values. Experimental data supported its sensitivity, specificity, reproducibility, and robustness. In addition, a set of quality controls was implemented to minimize the risk of false-positive and false-negative results, thus making this new test fit for use in serial analyses and reliable in the framework of official controls.