Mycotoxin content in 244 samples of wheat ears randomly collected during 2014 and 2015 from various localities in the Czech Republic was analysed using liquid chromatography coupled to mass spectrometry (LC-MS). Mean mycotoxin concentration in 2014 was highest for deoxynivalenol (DON; 760 μg/kg), followed by zearalenone (ZEA; 115 μg/kg), 3-acetyldeoxynivalenol (3-ADON; 88 μg/kg), deoxynivalenol-3-glucoside (83 μg/kg), and enniatins (ENNs; 102 μg/kg). In 2015, DON (66 μg/kg) also had the highest concentration level, followed by ENNs (35 μg/kg), nivalenol (2 μg/kg), and beauvericin (2 μg/kg). The maximum limit for DON in the European Union (1,250 μg/kg) was exceeded in 2% of samples, and the maximum limit for ZEA (100 μg/kg) was exceeded in 0.8% of samples.Fusarium species causing head blight were identified using PCR assays. During 2014-2015,Fusarium poae considerably dominated (48.7% average value of occurrence in the samples). Other species were detected in much lower frequencies in both years:Fusarium graminearum (average frequency of occurrence 13.7%),Fusarium avenaceum (11.9%),Fusarium culmorum (4.2%), andFusarium equiseti (2.9%).Fusarium langsethiae was identified only in 2015, at a frequency of 10.2%, andFusarium sporotrichioides was present only sporadically in 2014.

The PAV strain of barley yellow dwarf virus (BYDV) is one of the causal agents of yellow dwarf disease in cereals. The use of germplasm resistant to BYDV is generally regarded as the most effective means of controlling damage caused by this pathogen. In field trials, response to infection with a barley yellow dwarf virus of selected wheat cultivars registered in the Czech Republic was compared with that of control cultivars. Although a good level of resistance to BYDV-PAV was found in cultivar Athlon and yield loss was low, symptoms were more severe than on the moderately resistant control cultivar Sparta. Several other cultivars, such as Nordika, Julie, and Replik, also had slightly less than a 30% reduction in grain weight per spike, even though symptoms were more severe on Sparta or Athlon. Our results showed that, in the case of approximately 60% of wheat plants with BYDV-PAV symptoms, the yield reductions under optimal agronomic conditions reached approximately 17% for moderately resistant cultivars and 30% for moderately susceptible cultivars. The application of N fertilizer significantly reduced yield losses in BYDV-PAV-infected wheat cultivars, particularly in the moderately resistant cultivars. Even when infected with BYDV-PAV, the yield of moderately resistant cultivars, including those of spring wheat, was still acceptable. However, the re-cultivation costs of spring wheat in replacing damaged winter wheat lead to a total economic loss per hectare that is much greater than that for BYDV-infected wheat cultivars (moderately resistant and/or moderately susceptible ones). Furthermore, the economic loss is much lower when a moderately resistant cultivar is used. Hence, even with a high level of disease symptoms in winter wheat, the re-cultivation of spring wheat is not economically feasible.

Crop Wild Relatives (CWRs), as potential sources of new genetic variants, are being extensively studied to identify genotypes that will be able to confer resistance to biotic stresses. In this study, a collection of barley wild relatives was assessed in the field, and their phenotypic variability was evaluated using a Barley Description List, reflecting the identified ecosites. Overall, the CWRs showed significant field resistance to various fungal diseases. To further investigate their resistance, greenhouse tests were performed, revealing that several CWRs exhibited resistance against Fusarium culmorum, Pyrenophora teres, and Puccinia hordei G.H. Otth. Additionally, to characterize the genetic diversity within the collection, DNA polymorphisms at 21 loci were examined. We successfully employed barley-specific SSR markers, confirming their suitability for identifying H. spontaneum and even H. marinum, i.e., perennial species. The SSR markers efficiently clustered the investigated collection according to species and ecotypes, similarly to the phenotypic assessment. Moreover, SSR markers associated with disease resistance revealed different alleles in comparison to those found in resistant barley cultivars. Overall, our findings highlight that this evaluated collection of CWRs represents a valuable reservoir of genetic variability and resistance genes that can be effectively utilized in breeding programs.

Fusarium species are one of the most studied fungal pathogens of cereals. They cause Fusarium head blight or stem base diseases that are risky not only in terms of yield losses but also due to mycotoxins production. Several strategies are used to prevent Fusarium colonisation of plants, including fungicide treatment. However, according to the EU Biodiversity Strategy for 2030, the use of fungicides needs to be reduced, and therefore, alternative seed treatments should be sought. One possible method is the use of a pulsed electric field (PEF) that could kill or remove the fungi from the grains surface. The sensitivity of fungi to PEF treatment was first investigated. Spore suspensions of four Fusarium species (F. culmorum, F. graminearum, F. poae, F. sporotrichioides) were treated in a continuous PEF system (OMNIPEF; VITAVE, Czech Republic), and spore viability was subsequently assessed on an artificial medium. Different species of the genus Fusarium showed statistically significant differences in their response to PEF. As the frequency increased from 450 to 900 Hz, the number of viable spores gradually decreased for all Fusarium species tested. Fusarium poae responded already at the lowest frequency of 450 Hz with a strong reduction in spore viability (only 9.4% of spores survived). At the same frequency, 15.3% of F. graminearum spores, 28.9% of F. sporotrichioides spores, and 53.4% of F. culmorum spores remained viable.

Eyespot, caused by the related fungal pathogens Oculimacula acuformis and O. yallundae, is an important cereal stem-base disease in temperate parts of the world. Both species are dispersed mainly by splash-dispersed conidia but are also known to undergo sexual reproduction, yielding apothecia containing ascospores. Field diagnosis of eyespot can be challenging, with other pathogens causing similar symptoms, which complicates eyespot management strategies. Differences between O. acuformis and O. yallundae (e.g., host pathogenicity and fungicide sensitivity) require that both be targeted for effective disease management. Here, we develop and apply two molecular methods for species-specific and mating-type (MAT1-1 or MAT1-2) discrimination of O. acuformis and O. yallundae isolates. First, a multiplex PCR-based diagnostic assay targeting the MAT idiomorph region was developed, allowing simultaneous determination of both species and mating type. This multiplex PCR assay was successfully applied to type a global collection of isolates. Second, the development of loop-mediated isothermal amplification (LAMP) assays targeting β-tubulin sequences, which allow fast (<9 min) species-specific discrimination of global O. acuformis and O. yallundae isolates, is described. The LAMP assay can detect very small amounts of target DNA (1 pg) and was successfully applied in planta. In addition, mating-type-specific LAMP assays were also developed for rapid (<12 min) genotyping of O. acuformis and O. yallundae isolates. Finally, the multiplex PCR-based diagnostic was applied, in conjunction with spore trapping in field experiments, to provide evidence of the wind dispersal of ascospores from a diseased crop. The results indicate an important role of the sexual cycle in the dispersal of eyespot.

Fusarium fungi are widespread pathogens of food crops, primarily associated with the formation of mycotoxins. Therefore, effective mitigation strategies for these toxicogenic microorganisms are required. In this study, the potential of pulsed electric field (PEF) as an advanced technology of increasing use in the food processing industry was investigated to minimize the viability of Fusarium pathogens and to characterize the PEF-induced changes at the metabolomic level. Spores of four Fusarium species (Fusarium culmorum, Fusarium graminearum, Fusarium poae, and Fusarium sporotrichioides) were treated with PEF and cultured on potato dextrose agar (PDA) plates. The viability of the Fusarium species was assessed by counting the colony-forming units, and changes in the mycotoxin content and metabolomic fingerprints were evaluated by using UHPLC-HRMS/MS instrumental analysis. For metabolomic data processing and compound identification, the MS-DIAL (v. 4.80)–MS-CleanR–MS-Finder (v. 3.52) software platform was used. As we found out, both fungal viability and the ability to produce mycotoxins significantly decreased after the PEF treatment for all of the species tested. The metabolomes of the treated and untreated fungi showed statistically significant differences, and PEF-associated biomarkers from the classes oxidized fatty acid derivatives, cyclic hexapeptides, macrolides, pyranocoumarins, carbazoles, and guanidines were identified.