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.

Phenotypic response to twig canker and shoot blight varies substantially across almond germplasm. We hypothesized that genetically diverse almond accessions grown in a common orchard environment would exhibit genotype-dependent differences in symptom severity under natural disease pressure. Thirty-three almond accessions of different geographic origins were evaluated under field conditions in southern Italy. Disease incidence and severity were assessed, and symptomatic tissues were subjected to mycological and molecular analyses. Significant differences among accessions were detected for both disease incidence (I) and McKinney’s Disease Index (McK). ‘Merced’, ‘Nonpareil’, and ‘Genco’ exhibited high levels of disease incidence and severity, characterized by twig canker, shoot blight, and shoot clustering, with incidence values of 50% or higher, whereas local or less widely cultivated genotypes such as ‘Vesta’ and ‘Rana Gentile’ showed low disease incidence and severity. The Diaporthe complex represented the predominant fungal group isolated from symptomatic tissues, with D. foeniculina accounting for more than 80% of the identified Diaporthe isolates, whereas D. rudis was detected at lower frequencies. Other fungal taxa, particularly Botryosphaeriaceae, were recovered at lower frequencies. The observed variability supports the use of almond germplasm collections to identify genotypes with reduced symptom expression that may support breeding programs aimed at improving disease resilience.