| Autor: |
Dhar BC; Alson H. Smith Jr. Agricultural Research and Extension Center, School of Plant and Environmental Sciences, Virginia Polytechnic Institute and State University, 595 Laurel Grove Rd, Winchester, VA 22602, USA., Delgado Santander R; Irrigated Agriculture Research and Extension Center, College of Agricultural, Human and Natural Resource Sciences, Washington State University, Prosser, WA 99350, USA., Aćimović SG; Alson H. Smith Jr. Agricultural Research and Extension Center, School of Plant and Environmental Sciences, Virginia Polytechnic Institute and State University, 595 Laurel Grove Rd, Winchester, VA 22602, USA. |
| Jazyk: |
angličtina |
| Zdroj: |
Microorganisms [Microorganisms] 2024 Feb 12; Vol. 12 (2). Date of Electronic Publication: 2024 Feb 12. |
| DOI: |
10.3390/microorganisms12020376 |
| Abstrakt: |
The bacterium Erwinia amylovora causes fire blight and continues to threaten global commercial apple and pear production. Conventional microbiology techniques cannot accurately determine the presence of live pathogen cells in fire blight cankers. Several factors may prevent E. amylovora from growing on solid culture media, including competing microbiota and the release of bacterial-growth-inhibitory compounds by plant material during sample processing. We previously developed a canker processing methodology and a chip-based viability digital PCR (v-dPCR) assay using propidium monoazide (PMA) to bypass these obstacles. However, sample analysis was still time-consuming and physically demanding. In this work, we improved the previous protocol using an automatic tissue homogenizer and transferred the chip-based v-dPCR to the BioRad QX200 droplet dPCR (ddPCR) platform. The improved sample processing method allowed the simultaneous, fast, and effortless processing of up to six samples. Moreover, the transferred v-ddPCR protocol was compatible with the same PMA treatment and showed a similar dynamic range, from 7.2 × 10 2 to 7.6 × 10 7 cells mL -1 , as the previous v-dPCR. Finally, the improved protocol allowed, for the first time, the detection of E. amylovora viable but nonculturable (VBNC) cells in cankers and bark tissues surrounding cankers. Our v-ddPCR assay will enable new ways to evaluate resistant pome fruit tree germplasm, further dissect the E. amylovora life cycle, and elucidate E. amylovora physiology, epidemiology, and new options for canker management. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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