Development of an early detection assay for monitoring the impact of the fungal biocontrol agent <i>Ramularia crupinae</i> on the noxious weed <i>Crupina vulgaris</i>

The fungal plant pathogen Ramularia crupinae is the first biological control agent approved for the management of the federally-listed noxious weed Crupina vulgaris (common crupina) in the United States. Widespread common crupina infestations threaten western U.S. rangelands and pastures by decreasing biodiversity and agricultural productivity through the displacement of native and beneficial plant species. This study reports the development of a sensitive and species-specific quantitative PCR (qPCR) diagnostic assay designed for tracking R. crupinae infections and monitoring impact following a field release. A unique group I intron located within the 18S ribosomal RNA region permitted the development of a specific and sensitive diagnostic assay capable of detecting R. crupinae in both symptomatic and asymptomatic common crupina tissue. Species-specificity was validated with no cross-reactivity against the closely related species R. acroptili and 47 common crupina fungal endophyte cultures collected from field samples prior to R. crupinae release. Serially diluted R. crupinae DNA was used to demonstrate a qPCR detection limit of 47 fg. This R. crupinae diagnostic assay is highly accurate and specific, does not require post-amplification visualization, and supports high-throughput processing of field samples, making it well suited for tracking R. crupinae establishment and spread. Monitoring R. crupinae movement is critical for studying the impact and epidemiology of this introduced biological control agent.