Barley Disease Screening: A Multiplex Digital Droplet PCR Approach for the Detection of Ramularia collo-cygni, Rhynchosporium graminicola and Pyrenophora teres

Barley is the most widely grown cereal crop in Ireland and faces substantial yield loss annually due to airborne, seedborne and soilborne pathogens. The most prevalent and destructive of these include Ramularia leaf spot, barley leaf scald and net blotch, caused by Ramularia collo-cygni, Rhynchosporium graminicola and Pyrenophora teres, respectively. Infected seeds are considered as the major source of inoculum for these diseases. The absence of varietal resistance for these fungal pathogens suggests the need for better disease management strategies. Accurate pathogen detection is essential for successful disease management. To facilitate such detection in complex samples, such as seed, we demonstrate two digital droplet PCR (ddPCR) assays, with the initial assay capable of detecting R. graminicola, R. collo-cygni and P. teres. For those identified as positive for P. teres samples, a form-specific assay was designed to distinguish between the two economically significant forms of P. teres: P. teres f. maculata (Ptm; spot form of net blotch) and P. teres f. teres (Ptt; net form of net blotch). The assay offers sensitive and specific pathogen detection from barley seeds up to pmol/μL level. Limits of detection for R. graminicola, R. collo-cygni and P. teres in the triplex assay were calculated as 1.67, 1.06 and 4.21 copies/μL, respectively. For the form-specific assay, limits of detection were calculated as 1.44 copies/μL for Ptt and 0.82 copies/μL for Ptm. Finally, the efficacy of the assay was demonstrated by screening a collection of historical barley seeds.