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The reaction of twelve winter wheat cultivars frequently grown in the Czech Republic and twenty-five new breeding lines to inoculation with Oculimacula yallundae and Oculimacula acuformis was evaluated in small plot trials from 2017–2018. The assessment was carried out visually by symptoms and by a quantitative real-time polymerase chain reaction (qPCR). The aims of the study were to compare the results of both methods, to evaluate the effect of the resistance gene Pch1 to eyespot, and to select new breeding lines resistant to eyespot. The relationship between the eyespot symptoms and the pathogen DNA content in plant tissues followed a moderate linear regression. Low levels of eyespot were observed in the cultivars/lines possessing the resistance gene Pch1 (Annie, Hermann, Rebell, SG-S1215-14, SG-S1825-14, SG-S791-13) and also in the line SG-SU630-15. The qPCR method was able to detect low levels of the pathogens in the plant tissue and to distinguish two eyespot pathogens. O. acuformis was detected in very low concentrations in the inoculated plants compared with O. yallundae. The eyespot infection rate was significantly higher in 2017 than in the next agricultural season due to extremely dry and warm spring weather in 2018.

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The reaction of twenty-five winter wheat cultivars frequently grown in the Czech Republic to inoculation with Oculimacula yallundae and Oculimacula acuformis was evaluated in small plot trials from 2019 to 2021. The eyespot infection assessment was carried out visually using symptoms on stem bases and quantitative real-time polymerase chain reaction (qPCR). The cultivars were also tested for the presence of the resistance gene Pch1 using the STS marker Xorw1. Statistical differences were found between cultivars and between years. The lowest mean level of eyespot infection (2019–2021) was visually observed in cultivar Annie, which possessed resistance gene Pch1, and in cultivar Julie. Cultivars Turandot and RGT Sacramento were the most susceptible to eyespot. The method qPCR was able to distinguish two eyespot pathogens. O. yallundae was detected in higher concentrations in inoculated plants compared with O. acuformis. The relationship between the eyespot symptoms and the pathogen’s DNA content in plant tissues followed a moderate linear regression only in 2021. The highest eyespot infection rate was in 2020 due to weather conditions suitable for the development of the disease.