Assessment of viable conidia of Monilinia fructicola in flower and stone fruit combining propidium monoazide (PMA) and qPCR

摘要

Brown rot is the most economically important fungal disease of stone fruits and is primarily caused by Monilinia laxa and M.fructicola. Conventional methods used to identify M.fructicola are mainly based on phenotypic characteristics and pathogen quantification is not always accurate. In contrast, methodologies based on molecular tools improve pathogen characterization and identification but are not able to differentiate between live and dead conidia. In this study the PMA-qPCR methodology was optimized, validated and applied to quantify viable cells of M.fructicola in artificially and naturally infected samples. qPCR methodology showed good primer efficiency and sensitivity with quantification limits lower than obtained using a plate count method. The conditions of propidium monoazide (PMA) pretreatment were 60m PMA for 20min incubation and 30min of light-emitting diode (LED) exposure that, combined with qPCR, measured live cells accurately without overestimation of dead cells. Using this methodology in naturally infected samples, M.fructicola live cells were quantified specifically, in contrast to other traditional methodologies that cannot distinguish among Monilinia spp. The developed methodology based on combined PMA-qPCR will be a new tool to quantify viable M.fructicola in further epidemiological and ecological studies of this fungus.