Real-Time PCR and Droplet Digital PCR Are Accurate and Reliable Methods To Quantify <i>Pseudomonas syringae</i> pv. <i>actinidiae</i> Biovar 3 in Kiwifruit Infected Plantlets.

Show simple item record Barrett-Manako, Kelvina Andersen, Mark Martínez-Sánchez, Marcela Jenkins, Heather Hunter, Shannon Reese-George, Jonathan Montefiori, Mirco Wohlers, Mark Rikkerink, Erik Templeton, Matt Nardozza, Simona
dc.coverage.spatial United States 2022-07-26T03:02:28Z 2022-07-26T03:02:28Z 2021-06
dc.identifier.citation (2021). Plant Disease: an international journal of applied plant pathology, 105(6), 1748-1757.
dc.identifier.issn 0191-2917
dc.description.abstract <i>Pseudomonas syringae</i> pv. <i>actinidiae</i> is the etiological agent of kiwifruit canker disease, causing severe economic losses in kiwifruit production areas around the world. Rapid diagnosis, understanding of bacterial virulence, and rate of infection in kiwifruit cultivars are important in applying effective measures of disease control. <i>P. syringae</i> pv. <i>actinidiae</i> load in kiwifruit is currently determined by a labor-intense colony counting method with no high-throughput and specific quantification method being validated. In this work, we used three alternative <i>P. syringae</i> pv. <i>actinidiae</i> quantification methods in two infected kiwifruit cultivars: start of growth time, quantitative PCR (qPCR), and droplet digital PCR (ddPCR). Method performance in each case was compared with the colony counting method. Methods were validated using calibration curves obtained with serial dilutions of <i>P. syringae</i> pv. <i>actinidiae</i> biovar 3 (Psa3) inoculum and standard growth curves obtained from kiwifruit samples infected with Psa3 inoculum. All three alternative methods showed high correlation (<i>r</i> > 0.85) with the colony counting method. qPCR and ddPCR were very specific, sensitive (5 × 10<sup>2</sup> CFU/cm<sup>2</sup>), highly correlated to each other (<i>r</i> = 0.955), and flexible, allowing for sample storage. The inclusion of a kiwifruit biomass marker increased the methods' accuracy. The qPCR method was efficient and allowed for high-throughput processing, and the ddPCR method showed highly accurate results but was more expensive and time consuming. While not ideal for high-throughput processing, ddPCR was useful in developing accurate standard curves for the qPCR method. The combination of the two methods is high-throughput, specific for Psa3 quantification, and useful for research studies (e.g., disease phenotyping and host-pathogen interactions).
dc.format.medium Print-Electronic
dc.language eng
dc.publisher Scientific Societies
dc.relation.ispartofseries Plant disease
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher.
dc.subject Pseudomonas syringae
dc.subject Actinidia
dc.subject Fruit
dc.subject Plant Diseases
dc.subject Real-Time Polymerase Chain Reaction
dc.subject Actinidia chinensis var. chinensis
dc.subject Pseudomonas syringae pv. actinidiae
dc.subject bacterial quantification
dc.subject colony counting
dc.subject cultivar/resistance
dc.subject ddPCR
dc.subject disease management
dc.subject droplet digital PCR
dc.subject kiwifruit
dc.subject prokaryotes
dc.subject qPCR
dc.subject start of growth time
dc.subject techniques
dc.subject ‘Hort16A’
dc.subject ‘Zesy002’
dc.subject Infection
dc.subject Science & Technology
dc.subject Life Sciences & Biomedicine
dc.subject Plant Sciences
dc.subject 'Hort16A'
dc.subject 'Zesy002'
dc.subject RESISTANCE
dc.subject TOLERANCE
dc.subject GERMPLASM
dc.subject SPREAD
dc.subject 0605 Microbiology
dc.subject 0607 Plant Biology
dc.subject 0703 Crop and Pasture Production
dc.title Real-Time PCR and Droplet Digital PCR Are Accurate and Reliable Methods To Quantify <i>Pseudomonas syringae</i> pv. <i>actinidiae</i> Biovar 3 in Kiwifruit Infected Plantlets.
dc.type Journal Article
dc.identifier.doi 10.1094/pdis-08-20-1703-re
pubs.issue 6
pubs.begin-page 1748
pubs.volume 105 2022-06-15T01:58:01Z
dc.rights.holder Copyright: The author en
dc.identifier.pmid 33206018 (pubmed)
pubs.end-page 1757
pubs.publication-status Published
dc.rights.accessrights en
pubs.subtype Journal Article
pubs.elements-id 851650 Science Biological Sciences
dc.identifier.eissn 1943-7692
pubs.record-created-at-source-date 2022-06-15 2021-06

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