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Title:   Author  item
                                            GAGNON, M - Canadian Food Inspection Agencyitem
                                            BERGERON, M - Natural Resources Canadaitem
                                            HAMELIN, R - Natural Resources Canadaitem
                                            Grunwald, Niklaus

                                                         - *Nik*item
                                            BILODEAU, G - Canadian Food Inspection Agency
  Submitted to: Canadian Journal of Plant Pathology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 8/7/2014
                                    Publication  Date: 8/18/2014
                                    Citation: Gagnon, M.C., Bergeron, M.J., Hamelin, R.C., Grunwald, N.J., Bilodeau, G.J. 2014. Real-time PCR assay to distinguish the four Phytophthora ramorum lineages using cellulose binding elicitor lectin (CBEL) locus. Canadian Journal of Plant Pathology. 36(3):367-376.Interpretive Summary: Phytophthora ramorum is the organism that causes sudden oak death in the Western United States. This pathogen has so far caused extensive mortality of oak and tanoak in California and of Japanese larch in the United Kingdom. Scientists currently  recognize four distinct genetic clones of the pathogen named EU1, NA1 and NA2 and EU2. We developed a rapid method to identify and distinguish these clones using a DNA based assay. Blind tests performed on a panel of representative samples revealed diagnostic profiles unique to each clone. This assay facilitates the rapid typing of the clone of the sudden oak death pathogen.
                                Technical Abstract: Phytophthora ramorum is a pathogenic oomycete responsible for causing sudden oak death in the Western United States and sudden larch death in the United Kingdom. This pathogen has so far caused extensive mortality of oak and tanoak in California and of Japanese larch in the United Kingdom. Until recently, three genetically divergent clonal lineages of this microorganism were recognized (EU1, NA1 and NA2), each named according to the continent on which they were first detected. In 2009, a fourth lineage named EU2 was discovered in the United Kingdom. Sequencing and microsatellite genotyping revealed that the EU2 lineage is genetically distinct from all other lineages. Having a rapid method to identify and distinguish these lineages is important because of their different characteristics and geographic distributions. The objectives of our study were to 1) develop allele-specific oligonucleotide-PCR (ASO-PCR) assays using real-time PCR allowing for the identification of the new EU2 lineage and 2) validate a combination of ASO-PCR assays from this study and others targeting the cellulose binding elicitor lectin (CBEL) locus to rapidly identify all four P. ramorum lineages. Sequencing of the CBEL locus revealed eight single nucleotide polymorphisms (SNPs) distinguishing EU2 from the other three lineages. Using two of the eight SNPs revealed by this study, two ASO-PCR assays were developed providing the ability to rapidly genotype EU2 individuals relative to EU1, NA1 and NA2 individuals. These new assays were combined with two existing assays targeting the same locus to allow for rapid and simple identification of all four lineages. Blind tests performed on a panel of representative samples revealed diagnostic profiles unique to each lineage. These markers can be used with field samples, making them well suited for routine diagnostic procedures in regulatory laboratories.


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