Barley disease screening: a multiplex digital droplet PCR approach for the detection of Ramularia collo-cygni, Rhynchosporium graminicola and Pyrenophora teres

ABSTRACT Barley is the most widely grown cereal crop in Ireland and faces substantial yield loss annually due to airborne, seed-borne, and soil-borne 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 one of the most widely accepted disease management strategies. Here we demonstrate two digital droplet PCR (ddPCR) assays, based on TaqMan probe chemistry, for the detection of the above pathogens. ddPCR is a third generation of PCR, relying on sample partition into thousands of droplets in which each droplet acts as a reaction unit, enabling pathogen detection at extremely low levels. We developed a multiplex assay capable of detecting R. graminicola, R . collo-cygni and P. teres . For P. teres samples, a further assay was designed to distinguish between the two economically significant forms of P. teres i.e. 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 selective pathogen detection from barley seeds to pico moles/µl level. Finally, the efficacy of the assay was demonstrated by screening a collection of historical barley seeds.