Brown rot is the most serious fungus affecting stone fruit at an economic level and is caused mainly by Monilinia laxa e M. fructicola. Conventional methods used to identify M. fructicola are based mainly on phenotypic characteristics and pathogen quantification is not always accurate. In contrast, methods based on molecular tools improve pathogen characterisation and identification but do not differentiate between live and dead conidia.
Researchers from the IRTA Research Centre in Lleida (Spain), optimised the PMA-qPCR method and applied it to quantify viable cells of M. fructicola in artificially and naturally infected samples. qPCR methodology showed good efficiency and sensitivity with quantification limits lower than those obtained with the plate count method.
To develop this method, researchers used propidium monoazide (PMA) as a pretreatment for 20 minutes incubation followed by 30 minutes exposure to light-emitting diodes (LED). Combined with qPCR, this treatment enabled the measuring of live cells accurately without overestimating dead cells.
"By using this methodology in naturally infected samples, live M. fructicola cells were quantified with precision, unlike with other traditional methods. This method will be new tool to quantify live M. fructicola cells in additional studies."
Source: Vilanova L., Usall J., Teixidó N., Torres R., 'Assessment of viable conidia of Monilinia fructicola in flower and stone fruit combining propidium monoazide (PMA) and qPCR', 2017, Plant Pathology. doi:10.1111/ppa.12676