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.
Key words
- Fire blight
- Cankers
- Bacterial survival
- Bacterial viability
- Propidium monoazide
- dPCR
- Molecular detection methods
- Apple
- Pear
- Asian pear
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Acknowledgments
This material is based upon work supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, Hatch/Multistate Research Program projects NYG-625835 and NYG-625857 under 1009897 and 1014444, the NY State Farm Viability Institute grant number 81927/A001-FVI 17 006, and the NY State Specialty Crop Block Grant Program grant number SCG 82535/A001-SCG 17 005 to SGA, and by the Ministry of Education, Science and Technological Development of the Republic of Serbia, Contract No. 451-03-9/2021-14/200010 to KG.
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Santander, R.D., Gašić, K., Aćimović, S.G. (2022). Selective Quantification of Erwinia amylovora Live Cells in Pome Fruit Tree Cankers by Viability Digital PCR. In: Luchi, N. (eds) Plant Pathology. Methods in Molecular Biology, vol 2536. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2517-0_14
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DOI: https://doi.org/10.1007/978-1-0716-2517-0_14
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