Wheat (Triticum aestivum L.) is an economically important cereal crop. Its genetic diversity is a prerequisite for successful breeding for resistance to leaf and stem rust, as well as for other important traits. Therefore, the main objective of the study was to determine the level of genetic diversity of a file of 185 wheat accessions chosen to represent a wide range of resistance levels to leaf and stem rusts. They were evaluated at two locations in three years (2023–2025) for leaf and stem rust resistance by means of visual evaluation and fungus content using real-time PCR. BLUP values were then computed from these multi-environmental data. In addition, data on other traits such as heading time, plant height, and grain quality were collected. In the PCA, grain quality variables represented the first factor, explaining 41% of the total variability. Rust resistance was mainly associated with the second and the third factor. Population analysis revealed primary two (K = 2) and secondary six (K = 6) clusters, computed by using 13,274 DArT markers. The correlation between genotype and phenotype data was high (R = 0.89) when all traits were analyzed, and lower (R = 0.58) when only leaf and stem rust resistance variables were considered. Genetic diversity was also studied through an association analysis between markers and phenotypic traits using GLMs and MLMs. As a result, 300 statistically significant marker-trait associations (MTAs) were identified (p ≤ 3.77 × 10⁻⁶). Of these MTAs, 180 were associated with rust resistance variables and 120 with other traits. A BLAST search of rust resistance MTAs identified 128 putative genes; 47 of them can be taken for candidate genes related to fungal resistance. The results show that association analysis can help to explain the genetic diversity of selected wheat accessions, and that leaf and stem rust resistance are complex traits involving many non-specific resistance genes.

Eyespot is one of the most important fungal diseases of wheat in the Czech Republic. As part of a long-term study (2015–2024), the occurrence, population structure, and pathogenic variability of Oculimacula yallundae and Oculimacula acuformis were investigated. In total, 356 O. yallundae, 24 O. acuformis, and 33 mixed cultures were collected and identified using PCR. The study also included small-plot inoculation trials (2022–2023) to assess the response of widely grown winter wheat cultivars. Disease severity was evaluated visually, pathogen DNA was quantified using qPCR, and the presence of the resistance gene Pch1 was determined with the STS marker Xorw1. In addition to these analyses, monitoring of fungicide resistance to two commonly used fungicides (fluxapyroxad and prothioconazole) was performed. The results showed significant differences among cultivars and seasons. Genotypes carrying Pch1—including Annie, Campesino, Illusion, KWS Donovan, LG Absalon, and Pallas—exhibited the lowest levels of infection, whereas Mercedes and Dagmar were the most susceptible. The qPCR reliably detected and distinguished both pathogens, with O. yallundae occurring at higher concentrations. Fungicide sensitivity testing revealed EC₅₀ values (mean ± SD) of 0.09 ± 0.13 μg·mL⁻¹ for fluxapyroxad and 0.30 ± 0.22 μg·mL⁻¹ for prothioconazole, indicating that eyespot pathogens remain largely sensitive, with only minor signs of reduced sensitivity.