A real-time PCR assay for the detection of three Chrysodeixis (Lepidoptera: Noctuidae) species of agricultural importance developed from rDNA long-read sequence data

Moths in the genus Chrysodeixis (Noctuidae: Plusiinae) pose a global threat to dozens of important commercial crops. In the United States, three species of Chrysodeixis present identification challenges when intercepted at ports of entry due to morphological similarities. Chrysodeixis includens can be found throughout the U.S. but is also intercepted on commodities from South and Central America. Chrysodeixis chalcites is a prolific pest throughout its native range near the Mediterranean, Africa, and the Middle East and has established invasive populations throughout Europe. It has also been found on field and greenhouse crops in Canada and the northern U. S. Due to the invasive potential and frequent occurrence of this species on imported goods, it poses a significant threat to U.S. agriculture. Chrysodeixis chalcites is all but indistinguishable from C. eriosoma which is native to Asia, Australia, and the South Pacific, including Hawaii. External morphology and genitalic dissection fail to differentiate these two species. We carried out phylogenetic and phylogeographic studies using available CO1 barcodes and newly generated ITS1 sequence data to define the relationship between C. chalcites and C. eriosoma. To find suitable diagnostic regions, we also developed a long-read next generation sequencing approach to generate an rDNA profile for each of these three species from which we created two ITS-based real-time PCR assays. The first assay can identify C. includens and the second can identify C. chalcites and C. eriosoma. When used at ports of entry, these assays are sufficient to determine the quarantine status of an interception.