Portable qPCR and ddPCR Diagnostics for Cercospora Leaf Spot: Integrating Field and Laboratory Detection of Cercospora beticola in Beet

Abstract

Cercospora beticola, the causal agent of Cercospora leaf spot (CLS), is a major pathogen affecting beet (Beta vulgaris) worldwide. Rapid and accurate detection is critical for effective disease control. This study validated a diagnostic framework combining portable quantitative PCR (qPCR) and laboratory-based digital droplet PCR (ddPCR) assays to detect and quantify C. beticola in leaves, seeds, and soil-associated crop residues. We used two portable qPCR systems (Biomeme Franklin® Three9 and Hyris bCUBE™) for in-field diagnostics, and ddPCR (Bio-Rad Laboratories) for laboratory analysis. Two rapid DNA extraction protocols, the Sigma-Aldrich REDExtract-N-Amp Plant PCR Kit (brief incubation at 95 °C) and the BN QuickPick Plant DNA Kit (magnetic-bead-based), were evaluated. The optimized portable qPCR assays achieved detection limits of 1 pg μL−1, PCR efficiencies of 96–102%, and R2 > 0.98. The ddPCR assay enabled accurate and absolute quantification even at low target concentrations and was resilient to common PCR inhibitors. Both methods proved robust in complex environmental matrices, with a detection threshold of 1% in infected residue soil. The framework’s sensitivity, specificity, and portability support timely on-site CLS diagnosis and informed management. This integrated approach advances portable molecular diagnostics and provides a platform for future field-ready disease surveillance and sustainable crop protection.