Selective Quantification of Erwinia amylovora Live Cells in Pome Fruit Tree Cankers by Viability Digital PCR

Abstract

The accurate assessment of Erwinia amylovora live cell populations in fire blight cankers by classic microbiology methods has major limitations. Some of them are the presence of competitive microbiota in samples that inhibit E. amylovora’s growth and the release of toxic compounds by plant material during sample processing, which may hamper the pathogen’s ability to form colonies on solid media. Digital PCR (dPCR) combined with the photo-reactive DNA-binding dye propidium monoazide (PMA) allows selective detection and quantification of live E. amylovora cells in woody samples while overcoming the constraints of culture-dependent methods. This work describes a reliable viability dPCR procedure to determine E. amylovora live cell concentrations in fire blight cankers from pome fruit trees. This protocol can be adapted for the analysis of other types of plant material and enables investigation of ecological, epidemiological, and management significance of cankers as a relatively underexplored part of the fire blight disease cycle.