Fly Density Does Not Limit <i>Ceratocystis fimbriata</i> Transmission to Sweetpotato by <i>Drosophila hydei</i> , Revealed Through Molecular Diagnostics

Insect vectors are increasingly recognized as overlooked drivers of postharvest disease spread. <i>Ceratocystis fimbriata</i>, the causal agent of sweetpotato black rot, can spread rapidly in postharvest environments. Previous work established that <i>Drosophila hydei</i> can acquire viable <i>C. fimbriata</i> propagules both externally and internally, identifying this fly as a potential vector in storage facilities. Here, we expand on that finding by testing whether vector density influences disease transmission and by developing molecular diagnostic assays to improve pathogen detection. Transmission assays were conducted with four fly densities (10, 30, 50, and 80 flies) exposed to inoculum sources and then transferred to clean targets (sterile agar and uninfected sweetpotatoes, with and without wounds). Transmission occurred regardless of fly density, indicating that even small populations are sufficient to spread the inoculum, although incidence was significantly higher in wounded roots. To complement these assays, we standardized qPCR assays using dual-quencher probes targeting two <i>C. fimbriata</i>-specific markers (T3G9 and T5G26). Pathogen DNA was detected in both flies and asymptomatic roots, with the more sensitive marker identifying latent infections that were not visible through symptoms. Together, these results demonstrate that <i>D. hydei</i> vectors <i>C. fimbriata</i> in a density-independent manner, that wounding increases the success of infection, and that qPCR diagnostics can detect transmission events that are overlooked by visual assessment. These findings provide new epidemiological insight into postharvest black rot and support integrated management strategies that combine vector suppression with molecular surveillance.