A newly developed ddPCR-based system for potato pathogen detection applied to study temperature-dependent dynamics of Alternaria spp.

Abstract Potato leaf diseases are the greatest threat to potato yields worldwide. A novel system for detection of spores was designed, combining a rotation spore trap with droplet digital PCR (ddPCR). This novel system was then evaluated for disease signalling and to study the dynamics of leaf pathogens. The spore traps were placed in six representative potato fields within Czechia’s primary potato growing regions. Up to 24 spores’ specimens were collected per locality in three-day intervals from May to September 2024. The DNA extraction procedures were optimised and new species-specific combinations of primers and TaqMan probes were designed, verified, and multiplexed to detect Alternaria alternata and A. solani based on polymorphisms in beta-tubulin gene, and P. infestans based on unique sequences in Avr4 effector protein gene. The optimised ddPCR protocol worked consistently in both singleplex and multiplex. Reliable detection limits were measured to be approximately one copy of target DNA per µL of PCR mix. Field testing detected the presence of Alternaria spp. spores only. Although Phytophthora infestans were present, its sporangia levels remained below the detection threshold; likely due to the prevailing climatic conditions that year. Single Alternaria species generally differed by the dynamics of seasonal conditions, and these differences were consistent amongst all localities. The progression pattern of A. alternata strongly correlated with five-day moving sums of temperatures exceeding 25 °C (growing degree hours - GDH), suggesting a suitable indicator for signalling plant protection responses.