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

Ricardo Delgado Santander; Katarina Gašić; Srđan Goran Aćimović

doi:10.1007/978-1-0716-2517-0_14

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

Methods Mol Biol. 2022:2536:231-249. doi: 10.1007/978-1-0716-2517-0_14.

Authors

Ricardo Delgado Santander¹, Katarina Gašić^{1

2}, Srđan Goran Aćimović³

Affiliations

¹ Cornell University, Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Sciences, Hudson Valley Research Laboratory, Highland, NY, USA.
² Institute for Plant Protection and Environment, Department of Plant Diseases, Laboratory for Phytopathology, Belgrade, Serbia.
³ CenterVirginia Polytechnic Institute and State University, School of Plant and Environmental Sciences, Alson H. Smith Jr. Agricultural Research and Extension Center, Winchester, VA, USA. acimovic@vt.edu.

PMID: 35819608
DOI: 10.1007/978-1-0716-2517-0_14

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.

Keywords: Apple; Asian pear; Bacterial survival; Bacterial viability; Cankers; Fire blight; Molecular detection methods; Pear; Propidium monoazide; dPCR.

Publication types

Research Support, U.S. Gov't, Non-P.H.S.
Research Support, Non-U.S. Gov't

MeSH terms

Erwinia amylovora* / genetics
Fruit / microbiology
Plant Diseases / microbiology
Polymerase Chain Reaction / methods
Trees