Exploring Wild Hordeum spontaneum and Hordeum marinum Accessions as Genetic Resources for Fungal Resistance.

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.

The Use of Real-Time PCR for the Pathogen Quantification in Breeding Winter Wheat Varieties Resistant to Eyespot.

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.

Resistance of Winter Spelt Wheat [Triticum aestivum subsp. spelta (L.) Thell.] to Fusarium Head Blight.

Resistance to Fusarium head blight (FHB) of spelt wheat was investigated in field trials carried out at three European locations between 2016 and 2018. Resistance was assessed after artificial inoculation by visual scoring of symptoms and the determination of the contamination of grains and glumes with the mycotoxin deoxynivalenol (DON). It was found that typical spelt traits such as tall plant height, lax spikes, and tough glumes play a role as passive resistance factors. Across all test environments, modern spelt varieties with a significantly reduced plant height showed a significantly higher susceptibility to FHB and a higher contamination of the grains with DON compared to old landraces/varieties and plant genetic resources. Similarly, the lowest mycotoxin levels in grains were found only in old landraces and varieties, while the highest DON concentration was observed mainly in modern varieties. The results obtained can be used for the selection of suitable parental material for breeding spelt with improved FHB resistance.

Analysis of Small RNAs of Barley Genotypes Associated with Resistance to Barley Yellow Dwarf Virus.

Barley yellow dwarf virus (BYDV) causes an often-devastating disease of cereals that is most effectively controlled by using plant genotypes that are resistant or tolerant to the virus. New barley lines Vir8:3 and Vir13:8, with pyramided resistance genes against different pathogens and resistance gene Ryd2 against BYDV, are currently being tested. Because microRNAs (miRNAs) are associated with antiviral plant defense, here we compared the miRNA profiles in these lines and in cultivar Wysor (carrying one resistance gene, Ryd2), with and without BYDV infection and after feeding by virus-free aphids, to determine whether the miRNA profile in the resistant variety bear similarities with the newly developed lines. The BYDV titer for each group was also determined and compared to the titer in sensitive cultivar Graciosa. Among 746 miRNAs identified in barley, 66 were known miRNAs, and 680 were novel. The expression of 73 miRNAs differed significantly after BYDV infection, including the strong, specific upregulation of novel miRNA10778 that was conserved across all the barley genotypes. This miRNA belongs to the H box and ACA box (H/ACA) snoR14 family of RNAs (Rf01280) and is associated with pseudourydilation. The expression of 48 miRNAs also differed depending on the barley genotype. The profile of miRNAs expressed in Vir8:3 and Vir13:8 in response to BYDV was similar and differed from that of Wysor. Insights into the expression patterns of miRNAs in response to BYDV in barley provided here will benefit further studies toward understanding the resistance mechanisms and developing novel strategies against virus infections.

Untargeted metabolomics based on ultra-high-performance liquid chromatography-high-resolution mass spectrometry merged with chemometrics: A new predictable tool for an early detection of mycotoxins.

In order to explore the early detection of mycotoxins in wheat three standardized approaches (Fusarium disease severity, PCR assays for Fusarium spp. identification and mycotoxin quantification) and a novel untargeted metabolomics strategy were jointly assessed. In the first phase of this research, standardized approaches were able to quantify mycotoxins and identify Fusarium spp. Then, an UHPLC-QTOF metabolic fingerprinting method was developed to investigate plant-pathogen cross-talk. At the same time, chemometrics analysis demonstrated to be a powerful tool in order to distinguish low and strong infection levels. Combining these results, the cross-talk plant pathogen related to the early detection of mycotoxins was discovered. As a rapid response to fungal infection an overexpression of phosphatidic acids was discovered. By contrast, when the infection became stronger an increase of oxylipins and diacylglycerols was revealed.

The effect of Fusarium culmorum infection and deoxynivalenol (DON) application on proteome response in barley cultivars Chevron and Pedant.

Fusarium head blight (FHB) disease adversely affects grain quality and final yield in small-grain cereals including barley. In the present study, the effect of an artificial infection with Fusarium culmorum and an application of deoxynivalenol (DON) on barley spikes of cultivars Chevron and Pedant during flowering was investigated at grain mid-dough stage (BBCH 73) 10days after pathogen inoculation (10 dai). Proteomic analysis using a two-dimensional differential gel electrophoresis (2D-DIGE) technique coupled with LC-MS/MS investigated 98 protein spots revealing quantitative or qualitative differences between the experimental variants. Protein functional annotation of 93 identified protein spots revealed that most affected functional groups represent storage proteins (globulins, hordeins), followed by proteins involved in carbohydrate metabolism (α-amylase inhibitor, ß-amylase, glycolytic enzymes), amino acid metabolism (aminotransferases), defence response (chitinase, xylanase inhibitor, serpins, SGT1, universal stress protein USP), protein folding (chaperones, chaperonins), redox metabolism (ascorbate-glutathione cycle), and proteasome-dependent protein degradation. The obtained results indicate adverse effects of infection on plant proteome as well as an active plant response to pathogen as shown by enhanced levels of several inhibitors of pathogen-produced degradation enzymes (α-amylase inhibitor, xylanase inhibitor, serpins), chaperones, and other stress-related proteins (SGT1, USP). Genotypic differences were found in hordein abundance between Chevron and Pedant.

A European Database of Fusarium graminearum and F. culmorum Trichothecene Genotypes.

Fusarium species, particularly Fusarium graminearum and F. culmorum, are the main cause of trichothecene type B contamination in cereals. Data on the distribution of Fusarium trichothecene genotypes in cereals in Europe are scattered in time and space. Furthermore, a common core set of related variables (sampling method, host cultivar, previous crop, etc.) that would allow more effective analysis of factors influencing the spatial and temporal population distribution, is lacking. Consequently, based on the available data, it is difficult to identify factors influencing chemotype distribution and spread at the European level. Here we describe the results of a collaborative integrated work which aims (1) to characterize the trichothecene genotypes of strains from three Fusarium species, collected over the period 2000-2013 and (2) to enhance the standardization of epidemiological data collection. Information on host plant, country of origin, sampling location, year of sampling and previous crop of 1147 F. graminearum, 479 F. culmorum, and 3 F. cortaderiae strains obtained from 17 European countries was compiled and a map of trichothecene type B genotype distribution was plotted for each species. All information on the strains was collected in a freely accessible and updatable database (www.catalogueeu.luxmcc.lu), which will serve as a starting point for epidemiological analysis of potential spatial and temporal trichothecene genotype shifts in Europe. The analysis of the currently available European dataset showed that in F. graminearum, the predominant genotype was 15-acetyldeoxynivalenol (15-ADON) (82.9%), followed by 3-acetyldeoxynivalenol (3-ADON) (13.6%), and nivalenol (NIV) (3.5%). In F. culmorum, the prevalent genotype was 3-ADON (59.9%), while the NIV genotype accounted for the remaining 40.1%. Both, geographical and temporal patterns of trichothecene genotypes distribution were identified.

The development of frost tolerance and DHN5 protein accumulation in barley (Hordeum vulgare) doubled haploid lines derived from Atlas 68 x Igri cross during cold acclimation.

The dynamics of a long-term cold acclimation (CA) was studied in spring barley cultivar Atlas 68, winter barley cultivar Igri and a set of doubled haploid (DH) lines derived from an Atlas 68xIgri cross. The aim was to evaluate the effect of plant development on the ability to induce frost tolerance (FT) and to accumulate dehydrin 5 (DHN5) during CA. The plant developmental stage was evaluated by phenological development of the shoot apex and by determination of days to heading after a certain period of CA. FT was determined by direct frost tests. Plant winter survival was also determined. DHN5 was evaluated by densitometric analysis of protein gel blots. Cold led to the induction of increased FT and to the accumulation of DHN5 in both spring and winter lines. However, with the progression of CA, differences between the growth habits occurred as the winter lines were able to maintain increased FT and DHN5 levels for a significantly longer period of time than the spring lines. After vegetative/reproductive transition, a significant decrease in DHN5 accumulation was found in all lines; however, a discrepancy between the acquired FT level and DHN5 accumulation in vernalized winter barley plants was found. A correlation between DHN5 accumulation and plant winter survival was found when the studied lines were differentiated according to their developmental stage and DHN5 level. Possible explanations for these phenomena are provided.