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.

Fusarium head blight (FHB) remains one of the most difficult cereal diseases to manage because disease severity and mycotoxin contamination are shaped by pathogen diversity, weather conditions, host susceptibility, crop residues, and the narrow timing window for fungicide application. Endophytic fungi have been proposed as complementary tools within integrated FHB management because they may suppress Fusarium spp. directly, reduce mycotoxin accumulation, compete for ecological niches, or prime host defence responses. This review evaluates the evidence supporting endophyte-mediated FHB control in cereals, distinguishing mechanisms demonstrated in cereal-Fusarium systems from those inferred from broader plant-endophyte studies. We compare application strategies including residue treatment, spike application, seed or rhizosphere inoculation, and postharvest approaches, with emphasis on their practical advantages, limitations, and current level of validation. Available studies indicate that selected fungal endophytes and endophyte-associated fungi can reduce FHB symptoms and, in some cases, deoxynivalenol accumulation under controlled or field-oriented conditions; however, field consistency, formulation, delivery, ecological safety, compatibility with existing management practices, and regulatory requirements remain major barriers to adoption. We conclude that endophytes are unlikely to replace fungicides or host resistance in the near term, but they represent promising components of integrated FHB management when supported by strain-specific evidence, quantitative disease and mycotoxin data, and multi-environment validation.

The relatively recently described family Anteagloniaceae has four genera-Anteaglonium, Flammeascoma, Purpureofaciens and a newly defined monotypic genus Neolophiotrema. The newly described species Flammeascoma ryskae sp. nov. is the first species of this genus found in Europe so far. It was found during an ecological study of fungal diversity in floodplain forests at selected locations in the Czech Republic. As one of the four species of this family so far known, this new species is characterized by the production of a reddish pigment in the agar medium. In this study, in addition to the description of the new species, the current knowledge about the family Anteagloniaceae is summarized, including a discussion of their secondary metabolism.

In recent years, recurrent droughts have significantly affected spring barley production, reducing the quantity and quality of grain. This study aims to identify genotype-specific traits and the drought resilience of six different Hordeum vulgare L. (spring barley) genotypes, while also examining the potential of potassium application and fungal endophyte Serendipita indica inoculation to mitigate the negative effects of dry periods during the growing season. Field experiments were conducted over a three-year period from 2020 to 2022, measuring physiological, growth, and yield parameters. To get insight into the physiological state of the plants, we measured the soluble sugars content and the ratio of stable carbon isotopes in the flag leaf tissue, which reflects conditions during its formation. The dominant factors that influenced the measured parameters were the genotypes and seasons, as well as their interaction, rather than other experimental factors. The results showed that the Spitfire and Accordine varieties were the best performing in both the 2020 and 2021 seasons, as indicated by their yield. However, in the drier 2022 season, the yield of these two varieties decreased significantly (to 55% for Spitfire and to 69% for Accordine of their yield in 2021), while for the arid-region genotypes, it remained at the same level as the previous year. This study sheds light on the potential of various genotypes to withstand periods of drought and the effectiveness of using potassium application and S. indica inoculation as mitigation approaches.

Microdochium bolleyi is a fungal endophyte of cereals and grasses proposed as an ideal model organism for studying plant-endophyte interactions. A qPCR-based diagnostic assay was developed to detect M. bolleyi in wheat and Brachypodium distachyon tissues using the species-specific primers MbqITS derived from the ITS of the ribosomal gene. Specificity was tested against 20 fungal organisms associated with barley and wheat. Colonization dynamics, endophyte distribution in the plant, and potential of the seed transmission were analyzed in the wheat and model plant B. distachyon. The colonization of plants by endophyte starts from the germinating seed, where the seed coats are first strongly colonized, then the endophyte spreads to the adjacent parts, crown, roots near the crown, and basal parts of the stem. While in the lower distal parts of roots, the concentration of M. bolleyi DNA did not change significantly in successive samplings (30, 60, 90, 120, and 150 days after inoculation), there was a significant increase over time in the roots 1 cm under crown, crowns and stem bases. The endophyte reaches the higher parts of the base (2-4 cm above the crown) 90 days after sowing in wheat and 150 days in B. distachyon. The endophyte does not reach both host species' leaves, peduncles, and ears. Regarding the potential for seed transmission, endophyte was not detected in harvested grains of plants with heavily colonized roots. Plants grown from seeds derived from parental plants heavily colonized by endophyte did not exhibit any presence of the endophyte, so transmission by seeds was not confirmed. The course of colonization dynamics and distribution in the plant was similar for both hosts tested, with two differences: the base of the wheat stem was colonized earlier, but B. distachyon was occupied more intensively and abundantly than wheat. Thus, the designed species-specific primers could detect and quantify the endophyte in planta.

This study brings new knowledge about the taxonomy of some fungal species that originally belong to the polyphyletic genus Leptosphaeria. Molecular-phylogeny and morphology methods were carried out for taxonomic revision of some taxa in the genus Leptosphaeria (L. clavispora, L. barriae, L. rubicunda and its similar species) and defined new taxonomic combinations of these species, namely Ophiobolus clavisporus (J. H.Mill. & Burton) Mlčoch, Chaetosphaeronema barriae (Shoemaker) Mlčoch and Paraphoma rubicunda (Rehm ex G. Winter) Mlčoch. In the genus Chaetosphaeronema, Ch. misuriniensis sp. nov. was described, and in the genus Paraphoma, Paraphoma betonicicola sp. nov. and P. moravica sp. nov. were described. Furthermore, the synoptic keys of species from Paraphoma rubicunda (Rehm ex Winter) Mlčoch comb. nov. morphological species group, and dichotomous keys for phragmosporous species of the order Pleosporales, which are known from dead stems of Lamiaceae and phragmosporous taxa associated with Lupinus (Fabaceae), are provided.

Brachypodium distachyon is a useful model organism for studying interaction of cereals with phytopathogenic fungi. The present study tested the possibility of a compatible interaction of B. distachyon with the endophytic fungus Microdochium bolleyi originated from wheat roots. There was evaluated the effect of this endophytic fungus on the intensity of the attack by pathogen Fusarium culmorum in B. distachyon and wheat, and also changes in expression of genes (in B. distachyon: BdChitinase1, BdPR1-5, BdLOX3, BdPAL, BdEIN3, and BdAOS; and in wheat: TaB2H2(chitinase), TaPR1.1, TaLOX, TaPAL, TaEIN2, and TaAOS) involved in defence against pathogens. Using light microscopy and newly developed specific primers was found to be root colonization of B. distachyon by the endophyte M. bolleyi. B. distachyon plants, as well as wheat inoculated with M. bolleyi showed significantly weaker symptoms on leaves from infection by fungus F. culmorum than did plants without the endophyte. Expression of genes BdPR1-5, BdChitinase1, and BdLOX3 in B. distachyon and of TaPR1.1 and TaB2H2 in wheat was upregulated after infection with F. culmorum. M. bolleyi-mediated resistance in B. distachyon was independent of the expression of the most tested genes. Taken together, the results of the present study show that B. distachyon can be used as a model host system for endophytic fungus M. bolleyi.

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.

Superabsorbent polymers (SAPs) applied to soil have been recognized as water reservoirs that allow plants to cope with periods of drought. Their application as a seed coat makes water available directly to the seeds during their germination and early growth phase, but on the other hand, it can affect the efficiency of plant protection substances used in seed dressing. In our experiments, we evaluated the effect of seed coating with SAP on fungicide leaching and changes in their effectiveness in suppressing Fusarium culmorum infestation. Leaching of fungicide from wheat seeds coated with SAP after fungicide dressing, as measured by the inhibition test of mycelium growth under in vitro conditions, was reduced by 14.2–15.8% compared to seeds without SAP coating. Germination of maize seeds and growth of juvenile plants in artificially infected soil did not differ significantly between seeds dressed with fungicide alone and seeds treated with SAP and fungicide. In addition, plants from the seeds coated with SAP alone grew significantly better compared to untreated seeds. Real-time PCR also confirmed this trend by measuring the amount of pathogen DNA in plant tissue. Winter wheat was less tolerant to F. culmorum infection and without fungicide dressing, the seeds were unable to germinate under strong pathogen attack. In the case of milder infection, similar results were observed as in the case of maize seeds.

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.

The use of biological control is becoming a common practice in plant production. One overlooked group of organisms potentially suitable for biological control are Rhizoctonia-like (Rh-like) fungi. Some of them are capable of forming endophytic associations with a large group of higher plants as well as mycorrhizal symbioses. Various benefits of endophytic associations were proved, including amelioration of devastating effects of pathogens such as Fusarium culmorum. The advantage of Rh-like endophytes over strictly biotrophic mycorrhizal organisms is the possibility of their cultivation on organic substrates, which makes their use more suitable for production. We focused on abilities of five Rh-like fungi isolated from orchid mycorrhizas, endophytic fungi Serendipita indica, Microdochium bolleyi and pathogenic Ceratobasidium cereale to inhibit the growth of pathogenic F. culmorum or Pyrenophora teres in vitro. We also analysed their suppressive effect on wheat infection by F. culmorum in a growth chamber, as well as an effect on barley under field conditions. Some of the Rh-like fungi affected the growth of plant pathogens in vitro, then the interaction with plants was tested. Beneficial effect was especially noted in the pot experiments, where wheat plants were negatively influenced by F. culmorum. Inoculation with S. indica caused higher dry shoot biomass in comparison to plants treated with fungicide. Prospective for future work are the effects of these endophytes on plant signalling pathways, factors affecting the level of colonization and surviving of infectious particles.

A set of 92 genetic resources of spring barley split into groups according to the areas of origin was studied in terms of grain malting quality. The following malting parameters were monitored in this study: nitrogen content in barley grain, malt extract, relative extract at 45 °C, Kolbach index, diastatic power, apparent final attenuation, friability, β-glucans in wort, haze of wort measured at the angles of 15° and 90°. In the studied set, the largest variability was found in the haze of wort (90°), the smallest in the apparent final attenuation. The optimal values of the extract content in the malt dry matter according to the malting quality index were only detected in the genotypes originating from Europe. The group of the genetic resources from South America and Australia, together with the genotypes from the Middle East and Africa, differed statistically significantly in the values of the Kolbach index from other groups. The two-row genotypes differed statistically significantly in the values of the malt extract content, relative extract at 45 °C, Kolbach index, diastatic power, friability and β-glucan content in wort. Although the European and non-European groups differed in a number of traits, the non-European groups also included genetic resources that in their malting parameters approached or equaled the European malting varieties (such as Psaknon). In this study, several significant correlations were found; the strongest correlations were between friability and malt extract (r = 0.85), friability and Kolbach index (r = 0.84).

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.

Susceptibility of common pollen beetle (Brassicogethes aeneus Fabricius) to four insecticides was monitored at 14 locations in the Czech Republic during 2013-2017 on oilseed rape. Seventy local pollen beetle populations were tested for resistance to lambda-cyhalothrin, etofenprox, chlorpyrifos-ethyl, and thiacloprid. Resistance to lambda-cyhalothrin and etofenprox was determined with the recommended rates of 7.5 g a.i. ha(-1) and 40 g a.i. ha(-1), respectively. Resistance increased significantly over time only for lambda-cyhalothrin. Chlorpyrifos-ethyl at the recommended rate of 187.5 g a.i. ha(-1) exceeded by 4 times the mean 90% lethal dose (LD90) value of 44.4 g a.i. ha(-1), and mortality was 100% in all years. Thiacloprid had widely dispersed LD50 and LD90 values, as did lambda-cyhalothrin and etofenprox. Thiacloprid's recommended rate of 72 g a.i. ha(-1) was substantially higher than mean LD90 values. Several subpopulations with reduced susceptibility to thiacloprid were, however, found in this study. No cross-resistance among lambda-cyhalothrin, etofenprox, chlorpyrifos, and thiacloprid was determined.

A facultative parasite of cereals, Fusarium culmorum is a soil-, air- and seed-borne fungus causing foot and root rot, fusarium seedling blight, and especially Fusarium head blight, a spike disease leading to decreased yield and mycotoxin contamination of grain. In the present study, we tested changes in expression of wheat genes (B2H2, ICS, PAL, and PR2) involved in defence against diseases. We first compared expression of the analysed genes in seedlings of non-inoculated and artificially inoculated wheat (variety Bohemia). The second part of the experiment compared expression of these genes in seedlings grown under various treatment conditions. These treatments were chosen to determine the effects of prochloraz, sodium bicarbonate, ergosterol, aescin and potassium iodide on expression of the analysed defence genes. In addition to the inoculated and non-inoculated cultivar Bohemia, we additionally examined two other varieties of wheat with contrasting resistance to Fusarium sp. infection. These were the blue aleurone layer variety Scorpion that is susceptible to Fusarium sp. infection and variety V2-49-17 with yellow endosperm and partial resistance to Fusarium sp. infection. In this manner, we were able to compare potential effects of inductors upon defence gene expression among three varieties with different susceptibility to infection but also between inoculated and non-inoculated seedlings of a single variety. The lowest infection levels were detected in the sodium bicarbonate treatment. Sodium bicarbonate had not only negative influence on Fusarium growth but also positively affected expression of plant defence genes. Expression of the four marker genes shown to be important in plant defence was significantly affected by the treatments. The greatest upregulation in comparison to the water control was identified under all treatments for the B2H2 gene. Only expression of PAL under the ergosterol and prochloraz treatments were not statistically significant.

Due to the increasing sewage sludge production in the world and problems with its disposal, an application of sludge to the soil appears to be a suitable solution considering its fertilizer properties and ability to improve the soil physical conditions. On the other hand, the sludge may also contain undesirable and toxic substances. Since soil microorganisms are sensitive to environmental changes, they can be used as indicators of soil quality. In this study, we used sewage sludge (SS) from two municipal wastewater treatment plants (SS-A and SS-B) in the dose of 5 t/ha and 15 t/ha in order to determine possible changes in the fungal community diversity, especially arbuscular mycorrhizal fungi (AMF), in the rhizosphere of Arundo donax L. Rhizosphere samples were collected in summer and autumn for two consecutive years and the fungal diversity was examined using terminal restriction fragment length polymorphism and 18S rDNA sequencing. Fungal alpha diversity was more affected by SS-A than SS-B probably due to the higher heavy metal content. However, based on principal component analysis and ANOSIM, significant changes in overall fungal diversity were not observed. Simultaneously, 18S rDNA sequencing showed that more various fungal taxa were detected in the sample with sewage sludge than in the control. Glomus sp. as a representative of AMF was the most represented. Moreover, Funneliformis in both samples and Rhizophagus in control with Septoglomus in the sludge sample were other representatives of AMF. Our results indicate that the short-term sewage sludge application into the soil does not cause a shift in the fungal community composition.

Being natural plant antimicrobials, saponins have potential for use as biopesticides. Nevertheless, their activity in plant-pathogen interaction is poorly understood. We performed a comparative study of saponins' antifungal activities on important crop pathogens based on their effective dose (EC50) values. Among those saponins tested, aescin showed itself to be the strongest antifungal agent. The antifungal effect of aescin could be reversed by ergosterol, thus suggesting that aescin interferes with fungal sterols. We tested the effect of aescin on plant-pathogen interaction in two different pathosystems: Brassica napus versus (fungus) Leptosphaeria maculans and Arabidopsis thaliana versus (bacterium) Pseudomonas syringae pv tomato DC3000 (Pst DC3000). We analyzed resistance assays, defense gene transcription, phytohormonal production, and reactive oxygen species production. Aescin activated B. napus defense through induction of the salicylic acid pathway and oxidative burst. This defense response led finally to highly efficient plant protection against L. maculans that was comparable to the effect of fungicides. Aescin also inhibited colonization of A. thaliana by Pst DC3000, the effect being based on active elicitation of salicylic acid (SA)-dependent immune mechanisms and without any direct antibacterial effect detected. Therefore, this study brings the first report on the ability of saponins to trigger plant immune responses. Taken together, aescin in addition to its antifungal properties activates plant immunity in two different plant species and provides SA-dependent resistance against both fungal and bacterial pathogens.

Ramularia collo-cygni is a devastating plant pathogen of barley with an increasing importance in Europe and several other barley growing regions worldwide. R. collo-cygni is a pathogen with potential to adapt rapidly to environmental changes and is classified as a "high-risk" phytopathogen with respect to the evolution of fungicide resistance. Quinone-outside inhibitors (QoIs), demethylation inhibitors (DMIs) and succinate dehydrogenase inhibitors (SDHIs) represent three major fungicide classes that are frequently used to control a broad range of relevant cereal pathogens. However, in recent years R. collo-cygni has acquired resistance to QoIs; in addition reduced efficacy of SDHI- and DMI-containing products has been observed. In this study, we have investigated the frequency of SDHI- and DMI-adapted isolates in a European population of R. collo-cygni and evaluated the underlying resistance mechanisms towards both fungicide classes. Several mutations in the target genes of SDHIs were detected in the population of R. collo-cygni under investigation (B-H266Y/R, B-T267I, B-I268V, C-N87S, C-H146R, C-H153R and some others) with increasing frequencies since 2014. SDHI resistance in R. collo-cygni is mainly driven by both the presence of mutations and the high frequency of such mutations in the population. Additionally, DMI-adapted isolates of R. collo-cygni were found at a high frequency in the countries under investigation. Fifteen different Cyp51 haplotypes were detected in the set of isolates from 2009 to 2017. The most frequent haplotype in 2017 was C1 haplotype, which comprises three mutations in Cyp51 and shows a substantial increase in EC50 values to DMIs compared with other isolates. As R. collo-cygni has adapted to several groups of fungicides in many regions in Western Europe, future control of this highly diverse and adaptable pathogen must focus on new molecules, choosing resistant varieties and the improvement in seed hygiene standards.

Microdochium nivale and Microdochium majus cause brown foot rot and snow mould in cereals. The aim of the present study was to evaluate the population genetic structure of Microdochium spp. associated with foot rot of wheat in the Czech Republic and the reaction of that population to the succinate dehydrogenase inhibitor (SDHI) fungicide penthiopyrad. Using amplified fragment length polymorphism (AFLP) analysis to examine genetic structure, agar dilution to measure inhibitory effect, and various statistical methods to analyse populations, two genetic populations were found corresponding to the species Microdochium majus and M. nivale and restricted gene flow in populations between years was determined. This study demonstrates for the first time the occurrence of SDHI-insensitive reaction in populations of M. nivale and M. majus . Low sensitivity was identified in both species, but it was observed more frequently in M. majus populations.

The reaction of ten winter wheat cultivars grown in the Czech Republic to inoculation with Oculimacula yallundae and Oculimacula acuformis was evaluated in a small plot trial. In a parallel field trial the natural occurrence of stem-base disease complex in six of the tested cultivars was assessed. Lower severity of eyespot (and/or stem-base diseases) was observed in cultivars possessing the resistance gene Pch1 (cvs Hermann, Annie, Princeps, Manager, and Rebell) in plots inoculated with Oculimacula spp. as well as in natural field conditions. A total of 468 wheat stem bases from the Czech Republic was screened by PCR to study the frequency of eyespot causal agents. The plants were colonised significantly more often by both species Oculimacula yallundae and O. acuformis together than separately.