Development of a portable real-time PCR and digital droplet PCR assay for Cercospora beticola detection on beet

Cercospora beticola, the causal agent of Cercospora leaf spot, is one of the most destructive pathogens affecting leaf beet (Beta vulgaris). The pathogen spreads rapidly under warm and humid conditions and frequently coexists in mixed infections with Phoma betae. Disease management is challenging, particularly due to the emergence and spread of fungicide resistance in the pathogen populations. Rapid and accurate pathogen detections is essential for the development of effective and sustainable strategies of disease control. This study aimed at developing new sensitive and specific TaqMan probe-based assays for rapid detection and quantification of C. beticola using portable real-time qPCR systems for in-field diagnostic tests, and digital droplet PCR (ddPCR) for lab tests. The specificity of both qPCR and ddPCR was confirmed against a panel of fungal and bacterial species commonly associated with beet crops. Sensitivity was calculated as the mean of three independent experiments. The limit of detection for the ddPCR assay was 10 pg of DNA, while qPCR was 100-fold more sensitive, quantifying up to 0,1 pg of fungal DNA. To enable in-field detection, different protocols for rapid DNA extraction from fresh leaves, crop residues, and seeds using commercial kits (Biomeme Sample Homogenization and M1 Sample Prep Cartridge kit for DNA; Sigma-Aldrich REDExtract-N-Amp Plant PCR Kit; and BN QuickPick Plant DNA) were tested and compared in qPCR assays. The developed methods for early detection and quantification of C. beticola provide valuable tools for enhancing epidemiological studies that can contribute to design appropriate strategies for the disease management.