A major component of managing economic and emerging pests is the ability to accurately identify the target and similar species; this is even more difficult when those species are not consistently observed within the environment. This is the case when differentiating two co-occurring stem borers (the sugarcane borer [SCB] and the Mexican rice borer [MRB]) in the U.S. sugarcane cropping system. Both species have overlapping geographic and host ranges and create relatively similar-looking stalk tunnels. Currently, borer plant damage is assessed by counting the number of borer holes in stalks at the end of the season when most of the larvae and pupae used for identification are gone. However, eDNA left within the tunnels may allow us to attribute the damage to either species. To identify each species, we designed qPCR primer pairs and assessed primer specificity using non-target arthropods common to the system. We then determined the potential for PCR inhibition when samples contained caterpillar frass. Finally, to evaluate primer use with eDNA collected from frass left in larval tunnels, we assessed DNA degradation with time and field-relevant temperatures. One primer set was successfully developed and optimized for each species. Dilution of samples was required for SCB but not MRB primers. These protocols can be used to comparably estimate plant damage by both borer species for the first time, which, in turn, will be used to validate other identification methods, inform mapping of the newly invasive MRB in Louisiana, and allow for more accurate species-specific management decisions.
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