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Title:   Author  item
                                            BARCENAS, N - WA STATE UNIVitem
                                            Unruh, Thomasitem
                                            Neven, Lisa
  Submitted to: Journal of Economic Entomology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 11/18/2004
                                    Publication  Date: 4/1/2005
                                    Citation: Barcenas, N.M., Unruh, T.R., Neven, L.G. 2005. Dna diagnostics to identify internal fruit feeders (Lepidoptera:Tortricidae) of pome fruits of quarantine importance. Journal of Economic Entomology. 98(2):299-306.Interpretive Summary: Many pest insects are difficult to identify to species by morphological traits and authoritative identifications may be virtually impossible in immature forms.  This problem exists with the four pest moths that as larvae are the internal feeding pests of apple, pear, quince and cherry in North America.  These species are the codling moth, the oriental fruit moth, the lesser apple worm and the cherry fruit worm.  These species are indistinguishable as small larvae, a stage commonly discovered at inspection stations.  Since only one or two of these species represent pests of quarantine significance with many trading partners, more accurate identification methods will allow shipments infested with non-quarantine pests to be accepted. We sequenced a portion of the mitochondrial DNA of these insects collected from various geographic origins and discovered DNA sequences that were characteristic of each species.  We then modified the polymerase chain reaction (PCR) to selectively amplify the DNA of each species and developed a protocol that could be used by importing nations to identify these species fro intercepted immature specimens.
                                Technical Abstract: A diagnostic PCR method is presented for differentiating among the North American internal pome fruit-feeding pests Cydia pomonella (L.), Grapholita molesta (Busck), Grapholita prunivora (Walsh), and Grapholita packardi Zeller. A ca. 470 bp fragment of mitochondrial cytochrome oxidase I (COI) was sequenced in three to six specimens of each species.  Consistent diagnostic differences were observed among the species in two regions of CO1 from which forward and reverse primers were designed to amplify a 112-116 bp of the gene. The primer sets were used to selectively amplify DNA from specimens of diverse geographic origin for each corresponding target species. Protocols were adapted for conventional and quantitative PCR (qPCR), the latter being substantially faster.  The method was validated as a decision making tool for quarantine identifications for Mexico by representatives of their phytosanitary agency (Sanidad Vegetal).   The method can aid in identification of intercepted internal feeding Lepidoptera in apple and pear for many other importing nations.


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