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Abstract
Early detection provides the best way to prevent introduction and establishment of alien plant pathogens. Amplification ofDNAbyPCRhas revolutionized the detection and monitoring of plant pathogens. Most of those assays rely on the amplification of a fraction of the genome of the targeted species. With the availability of whole genomes for a growing number of fungi and oomycetes it is becoming possible to compare genomes and discover regions that are unique to a target organism. This study has applied this pipeline to develop a set of hierarchical TaqMan real‐timePCRdetection assays targetingDNAof all fourPhytophthora ramorumlineages, and a closely related species,P. lateralis. Nine assays were generated: three targetingDNAof allP. ramorumlineages, one for each lineage ofP. ramorum, one forP. lateralisand one targetingDNAofP. ramorumandP. lateralis. These assays were very accurate and sensitive, ranging from 98.7% to 100% detection accuracy of 2–10 gene copies of the targeted taxa from pure cultures or inoculated tissues. This level of sensitivity is within the lowest theoretical limit of detection ofDNA. It is expected that these assays will be useful because of their high level of specificity and the ease with which they can be multiplexed because of the inherent flexibility in primer and probe design afforded by their lack of conservation in non‐target species.
Highlights
Human-mediated movement of plants and plant products is undoubtedly recognized as one of the major forces driving the spread of invasive plant pathogens, threatening the health and sustainability of ecosystems and causing serious economic and social costs (Chapman et al, 2017)
The genome-enhanced detection and identification (GEDI) pipeline described by Feau et al (2018) was applied to identify genes and genome regions that were only found at three hierarchical levels: (i) group: genes conserved in the sister species P. ramorum and P. lateralis but absent in other phytophthoras; (ii) species: genes conserved in all lineages of P. ramorum but absent in other phytophthoras and genes present in the North American lineage of P. lateralis but absent from all other phytophthoras; and (iii) lineages: unique genes found in each of the currently recognized lineages of P. ramorum (NA1, NA2, EU1 and EU2)
A subset was selected consisting of 28 primer pairs located on different scaffolds in P. ramorum that were tested in a first round of in vitro screening, to eliminate those that generated amplicons with DNA from non-targeted species and/or those that failed to generate an amplicon with DNA from the targeted species
Summary
Human-mediated movement of plants and plant products is undoubtedly recognized as one of the major forces driving the spread of invasive plant pathogens, threatening the health and sustainability of ecosystems and causing serious economic and social costs (Chapman et al, 2017). Phytophthora ramorum emerged in the early 1990s in Europe and on the West Coast of North America and has since expanded to a large number of hosts and ecosystems (Gru€nwald et al, 2012). Four genetically distinct clonal lineages are currently recognized within P. ramorum These lineages are believed to have diverged prior to the current outbreak and have probably emerged in their current range as a result of distinct migration events (Ivors et al, 2006). Plant Pathology published by John Wiley & Sons Ltd on behalf of British Society for Plant Pathology.
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