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Digital (d)PCR protocol and tissue sample processing for detection and quantification of live Erwinia amylovora cells in fire blight cankers

Ricardo Santander: Cornell University, Plant Pathology and Plant-Microbe Biology Section

<div>Overwintering fire blight cankers are the main sources of inoculum for disease renewal in spring. Despite their importance in disease epidemiology, <em>E. amylovora</em> (<em>Ea</em>) amount, viability and survival in cankers are less understood. To quantify and distinguish live and total <em>Ea</em> cells in cankers, we optimized a sample processing and combined propidium monoazide (PMA)-dPCR protocol, using previously reported <em>Ea </em>specific Taqman probe and primers. The grinding of <em>Ea</em> inoculated apple branch sections in antioxidant maceration buffer improved <em>Ea</em> recovery on different media. PMA treatment of inoculated plant material allowed differentiation of live and dead cells. Using dPCR, the average <em>Ea </em>cell numbers per canker was determined at 6.7 x 10<sup>4</sup> cp (average number of target DNA copies), out of which 2.7 x 10<sup>4</sup> cp were live cells. We found a radial decline of <em>Ea</em> cell numbers, outwards from the canker edge. Majority of cells were in the first 4 mm of symptomless bark tissue around the canker edge. A total of 115 and 81 cp/mg were detected in 0-2 mm and 2-4 mm sections from the canker edge, respectively. PMA-dPCR of the same samples determined 58 and 22 cp of live cells/mg tissue, respectively. Our results suggest that PMA-dPCR is a valid technique to detect and quantify total and live cells of <em>Ea</em> in cankers. PMA-dPCR has a potential to elucidate unknown aspects of the <em>Ea</em> ecology, as well as the significance of different infection sources in disease epidemiology.</div>