High-Throughput, Quantitative Detection of Pseudoperonospora cubensis Sporangia in Cucumber by Flow Cytometry: A Tool for Early Disease Diagnosis

Cucumber downy mildew, caused by the obligate parasitic oomycete Pseudoperonospora cubensis [(Berkeley & M. A. Curtis) Rostovzev], is a major threat to global cucumber production. Effective disease management relies on rapid and accurate pathogen detection. However, due to the specialized parasitic nature of P. cubensis, conventional methods are often laborious, low-throughput and inadequate, necessitating the development of a new approach for high-throughput sporangia counting. To address this limitation, we developed a rapid, high-throughput flow cytometry (FCM) assay for the direct quantification of P. cubensis sporangia. The optimal staining protocol involved adding 30 µL of 1000× diluted SYBR Green I to 500 µL of sporangial suspension and incubating at room temperature for 20 min. The flow cytometry parameters were set to a high sample loading speed with a 30-s acquisition time. Instrumental settings included an FL1 (green fluorescence) threshold of 8 × 104 and an SSC (side scatter) threshold of 3 × 105, with low gain. Validation against hemocytometer counts revealed a strong positive correlation (r = 0.8352). The assay demonstrated high reproducibility, with relative standard deviations (RSDs) ranging from 1.96–9.84%, and a detection limit of 1–10 sporangia/µL. Operator-dependent variability ranged from 8.85% to 18.79%. These results confirm that the established flow cytometry assay is a reliable and efficient tool for P. cubensis quantification, offering considerable potential for improving cucumber downy mildew monitoring and control strategies.