Artificial Infection of Oats with Fusarium Species in Relation to (1-3)(1-4)-β-D-Glucan Content in the Grain

Abstract

Oats are increasingly recognized for their nutritional and industrial significance. Among various bioactive compounds in the oat grain, (1-3)(1-4)-β-D-glucan is a key functional component providing industrial, nutritional, and health advantages. This study investigates the correlation between oat (Avena sativa L.) cultivars’ resistance to Fusarium artificial infection and the concentration of β-D-glucan in the grain. Five oat cultivars, including hulled and naked varieties, were artificially inoculated with Fusarium graminearum (FG) and Fusarium culmorum (FC) strains. β-D-glucan content and pathogenic DNA accumulation were analyzed pre- and post-infection. The results show that β-D-glucan content in uninfected grains ranges from 1.97% to 2.53%, with naked varieties generally containing higher levels (2.30%) in comparison with hulled varieties (2.08%). Fusarium infection reduced the concentration of β-D-glucan by 10.60% (FG) and 16.05% (FC). Naked varieties demonstrated greater resilience to infection-induced β-D-glucan loss. Pathogen DNA analysis confirmed higher virulence of FC compared to FG. Our findings suggest β-D-glucan’s dual role as a potential defense mechanism and a pathogen source, emphasizing its complexity in plant–pathogen interactions.

1. Introduction

In recent years, oats (Avena sativa L.) have been gaining prominence as an increasingly popular crop, recognized for their versatile cultivation, use in animal and human nutrition, minimal input requirements, and high nutritional value. Oats can be classified as ‘functional food’ with a high content of bioactive compounds important for both physical and mental health. In the current era of increased stress and pressure, the development of healthy and functional foods is becoming paramount [1,2]. Therefore, oats, with their exceptional nutritional properties and value-added phytochemical content, represent a suitable food to address the current nutritional needs of the population and health status. Among the wide range of active compounds in oats, (1-3)(1-4)-β-D-glucan has gained considerable attention due to its numerous health benefits for both human health and plant resistance. In human nutrition, β-D-glucan is known for its cholesterol-lowering effects, gut health support, and ability to regulate blood glucose levels [3,4]. In plants, this cell wall polysaccharide not only contributes to structural integrity but also functions as a dynamic molecule involved in stress responses and defense against pathogens. These multifaceted roles underline the importance of β-D-glucan in food and agricultural research [5].

β-D-glucan is a polysaccharide composed of glucose monomers linked by β-(1-3), β-(1-4) or β-(1-6) glycosidic bonds. β-D-glucan represents an essential component of the cell wall of plants, algae, fungi, and bacteria [1,2]. Specifically, oat β-D-glucan represents a so-called mixed linkage glucan (MGL), with a predominance of β-(1-3) and β-(1-4) linkages. Structurally, it is a linear polysaccharide consisting of long stretches of β-(1-4)-linked glucose residues (about 70%) separated by simple β-(1-3) linkages (the remaining 30%). The ratio of β-(1-3) to β-(1-4)-bonds serves as a good indicator of the fine structure of β-D-glucan, determining its solubility and physicochemical properties [2,5,6,7].

The molecular and structural properties of β-D-glucan determine its physicochemical properties, including its rheological behavior. These properties play a key role in its various functions within plants. The unique binding and structure of β-D-glucan define its basic properties, specifically, solubility in aqueous solutions and the ability to form viscous and gel-like environments. These rheological properties contribute significantly to the plant’s structural integrity and defense mechanisms [1].

The solubility of β-glucan is one of its most important parameters, as it determines functional properties such as stability, emulsification properties, and membrane characteristics [8]. Moreover, it influences the extraction of β-glucan and its potential practical applications [9]. Under appropriate conditions, β-D-glucan increases the viscosity of solutions and forms a gel-like environment [2]. These rheological properties directly depend on the molecular weight and concentration of the β-D-glucan molecule in solution. The structure of β-D-glucan, especially the DP3:DP4 ratio (ratio of cellotriose units to cellotetraose units), is an important determinant for physicochemical properties, particularly affecting viscosity and solubility [1].

β-D-glucan is localized in the cell walls of the grass and cereal family (Poaceae), which includes commercially important cereals: oats (Avena sativa L.), barley (Hordeum vulgare L.), rye (Secale cereale L.), and wheat (Triticum aestivum L.). Within these plants, the grain is the primary site of β-D-glucan accumulation. The distribution of this polysaccharide within the grain is not uniform; the highest concentration is found in the starchy endosperm (75%), followed by the aleurone layer (26%) surrounding the endosperm [2,10,11]. Additionally, β-D-glucan has been observed, albeit in lower concentrations, in the vegetative organs of the plant, including the root, coleoptile, stem, and leaf [12].

The concentration of β-D-glucan in oat grains can vary. Studies have shown that its content ranges from 2% to 6%, or from 2.5% to 6.5% of the dry weight of oats [13,14], with some genotypes reaching even higher concentrations. The content of this metabolite in grain is influenced not only by genotype but also by the environment [15,16]. Specifically, temperature, water availability, soil quality, and nutrients are the main environmental factors affecting the final β-D-glucan content [17].

β-D-glucan plays a crucial role in the structural integrity of plant cells and developmental processes such as seed germination and seedling growth. During plant growth and development, the amount of β-D-glucan in the cell wall increases proportionally to the rate of cell elongation, reaching its maximum during the fastest phase of cell growth. Oat β-D-glucan is primarily localized in elongating cells distinct from meristematic cells. Conversely, β-D-glucan is largely absent in cells of mature tissues where growth has stopped [2].

Regarding the plant defense, the plant cell wall is a dynamic structure providing mechanical support and protection from stress factors [5]. β-D-glucan contributes to cell wall reinforcement through its accumulation, along with β-(1-3) glucan (callose) and proteins, between the cell wall and the cell membrane [18]. This hardening can significantly reduce the success rate of pathogen penetration. The rheological properties of β-D-glucan lead to the formation of a gel layer that acts as a defensive barrier against fungal invasion. The gel matrix is closely linked with cellulose and xylan, which provide cell wall reinforcement and help to orient cellulose microfibrils during grain development [19,20]. Furthermore, this layer can also serve as a potential signaling system alerting the plant of ongoing attacks [21].

In addition to its structural role, β-D-glucan contributes to plant defense through molecular recognition and signaling. Studies suggest that β-D-glucan may assist in the recognition of pathogens as danger-associated molecular patterns (DAMPs) or as microbe-associated molecular patterns (MAMPs) in monocotyledonous and dicotyledonous plants, respectively. These molecules are recognized by pattern recognition receptors (PRRs) located in the plasma membrane, including receptor-like kinases (RLKs) and receptor-like proteins (RLPs). This recognition triggers a signaling cascade that leads to the activation of pattern-triggered immunity (PTI), an essential component of plant defense against a wide range of pathogens [5,22,23,24].

Carbohydrates, including β-D-glucan, play a crucial role as signaling biomolecules in various plant developmental processes and stress responses, significantly contributing to plant defense mechanisms. The importance of sugars present in plant cell walls during pathogen invasion has led to the concept of “sweet immunity”, introduced by Bolouri-Moghaddam and Van den Ende [25], which defines carbohydrates as key components in enhancing plant resistance to both abiotic and biotic stresses [26]. Research suggests that several structural carbohydrates, including pectin-derived oligosaccharides, cellobiose, β-1,3-glucans, chitin, and its deacetylated derivative chitosan, effectively enhance plant resistance in various plant–pathogen interactions through exogenous application. Some of these carbohydrates may function as microbe-associated molecular patterns (MAMPs) or damage-associated molecular patterns (DAMPs) in the pathogen recognition process [27].

During pathogen attacks, β-D-glucan fragments can be released, acting as elicitors that trigger further defense responses in the plant. This process aligns with the concept of so-called sweet immunity, where carbohydrate metabolism and signaling influence the plant immune network [27]. Carbohydrates, including β-D-glucan, are thought to function as the primary molecules responsible for pathogen-associated molecular pattern recognition (PAMP), leading to PAMP-induced immunity [28].

Considering these complex interactions, our research aims to observe the relationship between β-D-glucan content in grains of oats (Avena sativa L.) and the effect of artificial infection caused by the genus Fusarium. We have subjected five oat varieties with varying β-D-glucan contents to artificial inoculation with the fungal pathogens Fusarium graminearum (FG) and Fusarium culmorum (FC), to observe both metabolomic and molecular-biological manifestations of infection. By investigating these processes, we aim to contribute to a broader understanding of plant defense mechanisms and strategies for producing high-quality and safe primary food materials.

2. Materials and Methods

This study utilized seed samples from five varieties of oats (Avena sativa L.), namely Hucul, Tatran, Vaclav, Auron, and Pushkinskij. These varieties were selected based on specific traits such as the presence or absence of hulls (hulled vs. naked grains) and their average β-D-glucan content in grain. Hucul, Vaclav, and Auron varieties produce hulled grains, while Pushkinskij and Tatran produce naked grains. In addition, the varieties also differ in the year of registration and the country of origin and qualitative parameters of the grain (

Table 1. Basic characterization of oat varieties used in the experiment. The results of the content of the selected quality parameters of the grain are from a field experiment (Vígľaš-Pstruša, Detva, central Slovakia) carried out in 2019 in three biological repetitions.

Oat Variety	Year of Registration	Country of Origin	Type of Grain	β-D-Glucan ± Stdev (%)	Starch ± Stdev (%)	Proteins ± Stdev (%)	Lipids ± Stdev (%)
Hucul	2017	Slovakia	Hulled—black hulled	4.68 ± 0.04	58.19 ± 0.01	16.16 ± 0.10	5.39 ± 0.07
Vaclav	2013	Slovakia	Hulled—yellow hulled	4.93 ± 0.01	59.85 ± 0.01	16.61 ± 0.05	5.33 ± 0.10
Auron	1991	Czechia	Hulled—yellow hulled	4.88 ± 0.01	60.27 ± 0.03	16.55 ± 0.04	5.80 ± 0.11
Pushkinskij		Russia	Naked	5.25 ± 0.14	50.87 ± 0.04	14.66 ± 0.01	6.81 ± 0.53
Tatran	2010	Slovakia	Naked	5.51 ± 0.12	53.08 ± 0.01	16.56 ± 0.01	7.08 ± 0.02

).

The plant material selection was based on various criteria, including the β-D-glucan content, determined from samples grown from April to August 2019 at Vígľaš-Pstruša (Detva, central Slovakia), as part of a larger analysis involving 100 oat varieties. From this dataset, five representative varieties were chosen for further research of factors beyond β-D-glucan content, such as potential applications in agricultural practices such as plant breeding and food processing.

Grains for the artificial infection experiment were obtained from the Gene Bank of the Slovak Republic, which falls under the National Agricultural and Food Centre—Research Institute of Plant Production (NPPC—VURV) in Piešťany (Slovakia). The selected varieties were grown in a container experiment from April to July 2022 in the unheated greenhouse of the NPPC—VURV in Piešťany.

The experiment involved sowing five oat varieties in container experiments, with each variety planted in three variants: control, artificial infection with FC, and artificial infection with FG. Each variant had three replications, with three separate pots per replicate. This resulted in a total of 45 pots (5 varieties × 3 variants × 3 replications), ensuring a robust and systematic approach for subsequent analyses.

For artificial infection, we used FG and FC isolates conserved in the microorganism collection at NPPC—VURV, originating from wheat fields in Slovakia. Cultivation of FG and FC colonies was performed on potato-dextrose agar (PDA, Difco Lab., Detroit, MI, USA) at 25 °C for 21 days [29]. Agar plates with fungal colonies were covered with sterile distilled water and then gently scraped with the sporulated aerial mycelium to prepare the inoculum. We adjusted the inoculum concentration to approximately 5 × 10⁶ conidia per ml. Each oat panicle was inoculated with 1 milliliter of the conidial suspension.

Artificial infection was performed using a combination of the spray method and covering the plants with a polythene bag [30]. Using the spray approach, we artificially infected oat panicles at the flowering stage for 48 h with two separate FG and FC inoculums. The control group was sprayed with distilled water without covering. Plants were allowed to mature under controlled conditions, and we harvested 15 panicles from each treatment and replicated them. The grains were manually dehulled and used for further analysis. Before analysis, we ground the grains to a thickness of 0.5 mm and determined the dry weight using an automatic analyzer at 110 °C.

DNA isolation was performed using the DNeasy Plant Maxi Kit (Qiagen, Germany) for both plant tissue and fungal mycelia. For plant samples, 1 g of finely ground oat grains (both infected and control) was used. For fungal samples, FC and FG mycelia grown on Petri dishes were scraped and homogenized to a fine powder using liquid nitrogen. The homogenized sample was transferred to a 15 mL tube and mixed with 5 mL of preheated (65 °C) AP1 buffer (pH = 5.5) and 10 μL of RNase A stock solution (100 mg/mL). Following vortexing, the mixture was periodically mixed while being incubated at 65 °C for 15 min. Next, 1.8 mL of AP2 buffer was added, blended, and allowed to sit on ice for 10 min to cause the proteins, polysaccharides, and detergents to separate. In the following step, the lysate was centrifuged at 5000× g for 5 min at room temperature. The resulting supernatant was processed through a QIAshredder Maxi Spin Column, followed by the addition of 1.5 volumes of AW1 buffer. This mixture was transferred to a DNeasy Maxi Spin Column and centrifuged. DNA elution was performed twice using 0.6 mL of preheated (65 °C) AE buffer (pH = 9), with a 5 min incubation at room temperature before each centrifugation step. A QubitTM Flex Fluorometer (InvitrogenTM, Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to quantify total DNA, and a NanoDrop1000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to measure the concentration and quality of extracted DNA. All DNA samples obtained from oats were diluted to a concentration of 25 ng/μL. The final DNA samples were transferred to labeled microtubes and frozen for subsequent evaluation of pathogenic DNA presence.

Real-time PCR on ABI PRISM^® 7000 was used to analyze the amount of DNA in oat grains. For quantification of pathogenic FC DNA, we used the TaqManTM FC92s1 probe with the primer pair FC92s1 forward and FC92s1 reverse, generating an amplicon size of 92 bp [31]. DNA FG quantification was performed with the TaqManTM TMFg12p probe and the primer pair TMFg12 forward and TMFg12 reverse, with a reference size of 103 bp [32]. The PCR reaction mix contained 25 µL of TaqMan Universal PCR Master Mix, 300 nM of each primer, 200 nM of TaqManTM probe, and 2 µL of DNA (50 ng). PCR conditions were 95 °C for 10 min, 40 cycles: 95 °C for 15 s, 60 °C for 1 min. For standard curves, we used five dilutions of pathogen DNA isolated from FC mycelia and FG mycelia (1; 10; 100; 1000; and 10,000 pg/uL) in three replications. ABI PRISM^® 7000 software was then used to quantify unknown samples by interpolating Ct values. Pathogen DNA content was expressed as ng DNA/g grain and the percentage of infection was calculated as (Fusarium DNA/total DNA) × 100.

The β-D-glucan content of plant samples was determined using the analytical kit “Mixed-linkage β-glucan assay procedure” (KBGLU, BBG 5/03). We weighed 100 mg of ground sample into a 30 mL centrifuge tube. We added 200 μL of 50% (v/v) ethanol and 4 mL of 20 mmol·dm⁻³ sodium phosphate buffer (pH 6.5). The sample prepared was mixed thoroughly in the tubes and transferred to a boiling water bath, where it was incubated for 1 min. After incubation, the samples were again mixed and incubated for 2 min at 100 °C. Subsequently, we lowered the temperature of the water bath to 50 °C and tempered the samples for 5 min. We added 200 μL of lichenase (10 U) and mixed thoroughly. This was followed by incubation of the tubes for 60 min at 50 °C, mixing the entire contents of the tubes repeatedly every 15 min. Following this step, 200 mmol·dm⁻³ (pH 4.0) of sodium acetate buffer (5000 μL) was added to the tubes. The prepared samples were allowed to settle for 5 to 10 min at room temperature and subsequently centrifuged for 10 min at 8990 rpm and then 100 μL of supernatant was collected in 3 tubes. To 2 of these tubes, we added 100 μL of β-glucosidase solution (0.2 U), and, to the third tube (blank), we added 100 μL of 50 mmol·dm⁻³ sodium acetate buffer (pH 4.0). The prepared tubes were incubated for 10 min at 50 °C. Subsequently, we prepared a separate sample with standard glucose content (GS). We also prepared a glucose blank (GB). Finally, we added 3000 μL of the enzyme reagent GOPOD (glucose oxidase (>12,000 U), peroxidase (>650 U), 4-aminoantipyrine (80 mg)) to all incubated tubes. Samples were then incubated in a water bath for 20 min at 50 °C. Finally, we spectrophotometrically measured the extinction of the samples and the extinction of GS versus GB at a wavelength of 510 nm. We calculated the percentage of β-D-glucan in the sample based on the following formula:

β-D-glucan (%) = ∆E · F · 94 · 1/1000 · 100/W · 162/180 = ∆E · F/W · 8.46

The value of F is calculated as the mass of glucose (100 μg) divided by the absorbance of glucose (100 μg). The load, W, is expressed in mg. The change in extinction (∆E) was obtained by subtracting the extinction value of the sample (Er), which is the average of two measurements, from the blank extinction value (Eb).

We used several software and procedures for statistical evaluation of the data. Differences between the control and the respective infected oat cultivar in β-D-glucan content were tested by t-test at 95% significance level. The relative differences between the control, FC-infected, and FG-infected cultivars in β-D-glucan content and amount of pathogenic DNA were evaluated by analysis of variance (ANOVA), and, when p ≤ 0.05, the Post Hoc LSD test was used to determine significance between cultivars. Correlation analysis between β-D-glucan content and the amount of pathogenic DNA in infected cultivars was performed using Pearson’s correlation coefficient at 95% significance level. Calculations of these statistics, including t-test, ANOVA, LSD test, and correlation analysis, were performed using Statgraphics XVII-X64 software. For Principal Component Analysis (PCA) to assess the effect of β-D-glucan content and the amount of pathogenic DNA, we used Past4, version 4.17 (PAleontological STatistics) software [33].

3. Results

3.1. β-D-Glucan Content in Oat Varieties

We analyzed five varieties of sown oats, selected based on their nakedness, origin, year of registration, and initial β-D-glucan content from previous experiments. The β-D-glucan content ranged from 1.97% (Vaclav) to 2.53% (Tatran), with a mean of 2.17% (Figure 1). Generally, naked varieties exhibited a higher concentration of β-D-glucan compared to hulled varieties, with averages of 2.30% and 2.08%, respectively. Statistical analysis revealed that the Tatran variety, a naked oat from Slovakia, had significantly higher β-D-glucan content (2.53%) compared to other varieties. On the contrary, the lowest content was observed in Vaclav (1.97%), a hulled variety from Slovakia. Other varieties included Pushkinskij (2.08%, naked, Russian origin), Auron (2.03%, hulled, Czech origin), and Hucul (2.24%, hulled, Slovak origin with distinctive black panicles).

The Hucul variety, a black-hull oat, exhibited distinctive traits compared to other varieties. The black color of the hull represents a significant phenotypic difference from other varieties colored in yellow. The country of origin of this variety is the Slovak Republic and it is a relatively new variety of black oats (year of registration 2017). We found that the Hucul variety has the highest average β-D-glucan content in grain among all the hulled varieties we analyzed, showing the second highest content (after the naked variety Tatran) among all the analyzed samples.

In the context of investigating the effect of β-D-glucan on stress, we subjected all oat cultivars to artificial infection with two common cereal pathogens: FG and FC. Subsequently, we focused on β-D-glucan content and pathogen DNA concentration in the infected grains.

3.2. Effect of Fusarium Infection on β-D-Glucan Content in the Analyzed Set of Oats

We investigated the effect of artificial infection with FC and FG on β-D-glucan content in oat grains. In the control samples, the average β-D-glucan content was 2.17%. After infection, β-D-glucan decreased to 1.82% for FC and 1.93% for FG, indicating that fungal infection generally reduced β-D-glucan content. The impact of infection varied between Fusarium species and oat varieties. Infection with both FC and FG resulted in decreased β-D-glucan content across all varieties. FC caused a more substantial average decrease (16.05%) compared to FG (10.60%). Naked oat cultivars showed greater resistance, losing less β-D-glucan after infection compared to hulled cultivars. Specifically, naked cultivars lost an average of 4.71% β-D-glucan after FG and 8.16% after FC infection, while hulled cultivars lost 14.94% and 15.05%, respectively.

Subsequently, we analyzed the β-D-glucan content of the oat cultivars before and after artificial infection with FC. Our findings show a consistent decrease in the amount of β-D-glucan after infection in all varieties observed (Figure 2).

Figure 2 shows that β-D-glucan levels were demonstrably higher in the control grains before artificial infection compared to the levels after artificial infection with FC. Specifically, a statistically demonstrable decrease in β-D-glucan was observed after artificial infection with FC in Auron, Vaclav, and Hucul cultivars. The highest change in β-D-glucan abundance in grain before and after the infection was observed in Auron (from 2.03% to 1.51%) and Vaclav (from 1.97% to 1.56%), in which total β-D-glucan content decreased by 25.52% and 20.71%, respectively. The third most significant decrease was observed in the black-hulled variety, Hucul (1.80%), with a decrease of 19.60% compared to the control variants. The lowest percentage decrease was shown by Pushkinskij (2.03%) with a minimal decrease in the studied polysaccharide by 2.03%. Tatran (2.19%), with a difference of 13.20% representing the second variety with the lowest loss of β-D-glucan after artificial infection with FC.

Evaluating the results presented in Figure 2, we can conclude that the highest β-D-glucan values observed after artificial infection with FC, and thus the lowest losses of this polysaccharide were observed in varieties classified as naked oats, which are generally characterized by a higher β-D-glucan content. We can therefore assume that the presence of β-D-glucan in the cell wall may protect the plant against the pathogen, to some extent.

Secondly, we analyzed the β-D-glucan content of oat cultivars subjected to artificial infection with FG and compared the content of the polysaccharide in control samples before and after artificial infection (Figure 3). We found a percentage decrease in β-D-glucan present in all the varieties studied after artificial infection.

The greatest percentage difference between controls and samples after infection with FG was shown for the hulled varieties Vaclav (−19.66%), Hucul (−13.30%), and Auron (−12.16%). On the contrary, the lowest loss of the studied metabolite after the infection was observed in naked grains. The average value of β-D-glucan in the variety Pushkinskij (1.95% after artificial infection with FG) decreased by 6.25% compared to the control (2.08%), while the lowest decrease was observed in the Tatran variety (2.43% after artificial infection), only by 3.57% compared to the control (2.52%). Similar to FC infection, we observed lower to minimal loss of β-D-glucan after artificial infection with FG in naked cultivars, leading to the assumption of a stronger pathogen attack and subsequent reduction in β-D-glucan. Consequently, we can conclude that the presence of β-D-glucan act as a protective factor for the plant against the pathogen.

3.3. Pathogen DNA Analysis

We evaluated the presence of pathogenic DNA in host oat samples as a manifestation of artificial infection (Figure 4 and Figure 5). We also assessed the presence of pathogenic DNA in control (uninfected) samples subjected to DNA extraction, isolation, and analysis. The control samples did not show the presence of pathogenic DNA, indicating that protocols were followed correctly during DNA extraction and isolation, thus ensuring accurate analysis and reliability of the results.

For FC, the naked varieties, Pushkinskij and Tatran, showed the highest levels of pathogenic DNA (514.42 and 513.49 ng/g flour, respectively). Hulled varieties showed comparatively lower amounts: Hucul (408.71 ng/g), Vaclav (173.22 ng/g), and Auron (74.11 ng/g).

In addition to assessing the presence of FC DNA in infected oat cultivars, we assessed its presence in control samples which were subjected to extraction, isolation, and analysis. The control samples did not show any statistically significant presence of pathogenic DNA, indicating accurate analysis and reliability of the results.

For FG, DNA levels were consistently lower compared to FC in all cultivars. Hucul showed the highest concentration (186.62 ng/g), followed by Tatran (127.77 ng/g) and Auron (86.94 ng/g). Pushkinskij and Vaclav showed very low levels (4.55 and 4.37 ng/g, respectively).

Notably, Pushkinskij’s reaction to FG (4.55 ng/g) differed significantly from that of FC (514.42 ng/g). When faced with a milder stressor like FG, Pushkinskij was able to protect itself effectively, but it was less successful when faced with a tougher aggressor like FC.

The manifestation of infection by FC and FG shows significant differences in the response of the oat varieties used in our experiment. On average, FC showed higher pathogenic DNA content (336.79 ng/g) compared to FG (82.05 ng/g). For FC, we observed a significant decrease—16.05% on average of β-D-glucan in infected grains compared to the control. Additionally, FG caused an average decrease in β-D-glucan in infected oat grains by 10.6%. Consequently, our findings suggest that FC may be a more aggressive pathogen.

3.4. Statistical Evaluation

Correlation analysis revealed a statistically significant positive correlation between β-D-glucan content and the amount of pathogenic FC DNA in infected plants (r = 0.7374, p = 0.0017). These findings reveal a positive trend between β-D-glucan content and FC. That is, the higher the content of the polysaccharide under study, the higher the content of pathogenic DNA after infection with FC. This suggests that higher β-D-glucan content may be associated with increased susceptibility to FC infection, contrary to our initial hypothesis.

The results of the correlation analysis showed a weak positive correlation between β-D-glucan and the amount of pathogenic FG DNA in infected plants. Although at first glance a positive trend can be observed, the result of the correlation analysis clearly showed that the relationship between β-D-glucan and FG is not statistically significant (r = 0.3949, p = 0.1452).

Principal Component Analysis (PCA) revealed distinct clustering patterns for different oat varieties in response to infection. Tatran, Hucul, and Pushkinskij varieties showed the highest β-D-glucan content in both control and infected samples, as well as a high presence of pathogenic DNA. Their expression was consistent in all the parameters studied, leading to their significant influence on the result of correlation. On the contrary, the cultivars Auron and Vaclav reached the lowest values of the polysaccharide under study and low presence of pathogenic DNA of FC, which is why their influence was not significant (Figure 6).

After PCA, a clustering of specific oat varieties after artificial infection with FG can be observed. Similar to FC, the singled-out groups were Auron and Vaclav. However, after an inoculation caused by the phytopathogen FG, these two varieties were joined by the variety Pushkinskij, all showing low β-D-glucan content and minimal pathogenic DNA accumulation, justifying their separation from varieties Hucul and Tatran (Figure 7).

4. Discussion

The β-D-glucan content observed in our study aligns with previous reports of 2–6% of β-D-glucan in mature oat grains [13,14]. The higher content in naked varieties could be attributed to genetic factors [10]. However, the literature emphasizes that the content of this metabolite in mature grain is variable, and the β-D-glucan content is influenced not only by the genotype but also by the environment [15,16]. For example, in Hucul we detected the average content of β-D-glucan 2.24%; however, in our previous research, it was 5% in the mature grain (Havrlentová, unpublished data) or 3.15% [34]. The Hucul variety, a black-hull oat, exhibited unique biochemical parameters potentially influenced by its black panicles. Čertík et al. [35] suggested that the color of panicles may influence biochemical parameters (protein, β-D-glucan, and lipid accumulation) in oat grains, potentially affecting their nutritional and functional properties; thanks to the presence of pigments and their potential antioxidant activities, it can also protect the grain from stress factors in the external environment.

The naked and hulled varieties of Avena sativa L. differ from each other not only morphologically but also in terms of β-D-glucan content. Tiwari and Cummins [10] reported an average β-D-glucan content of 3.5% in hulled grains, while, in naked oats, the average content was 4.25%. Similarly, Havrlentová et al. [13] reported an average β-D-glucan content of 3.20% in hulled oat grains and 4.75% in naked grains, or for naked genotypes, the range from 38.5 to 43.1 g·kg⁻¹ and husked oat 24.9 to 35.2 g·kg⁻¹ in the work of Zute et al. [36]. Our results confirm these findings: naked oat varieties generally dispose of higher β-D-glucan content in comparison with the hulled varieties. We observed an average β-D-glucan content of 2.30% in naked oat varieties and 2.08% in hulled varieties, consistent with values in the literature. Compared to the literature, we generally observed lower content of β-D-glucan in analyzed oat samples, which might be subjected to the environmental factors playing a crucial role in determining final β-D-glucan content in mature grains. Our experiment was carried out in laboratory conditions, in containers. The presented results of other authors are based on analysis of plants grown in field conditions. Additionally, environmental factors, such as soil quality and nitrogen levels or soil pH, temperature, or precipitation influence β-D-glucan content significantly [12,15,17,37]. Also, the changing weather conditions [38] and dry and warm climates have been associated with increased β-D-glucan content in cereals [16]. The ontogenetic stage of the plant also influences the accumulation of β-D-glucan in the oat grain, and, therefore, capturing the correct developmental stage of the plant and comparing the results of several studies in this regard is justified [12].

It is generally accepted that oats, as a good natural source of β-D-glucan in the grain, are successfully used in the food industry in the preparation of so-called functional foods [1,2]. In human nutrition, β-D-glucan is known for its biological activity [3,4]. On the other hand, this cell wall polysaccharide plays an important role in the response of plants to various environmental factors. Studies in the scientific literature suggest that the localization of β-D-glucan in the outer epidermal layer may indicate that the metabolism of this cell wall polysaccharide is linked to plant responses to environmental stressors [39,40]. Therefore, our work aimed to determine the relationship between the content of β-D-glucan and the manifestation of the Fusarium infection.

Fusarium species negatively affect the growth and quality of grains due to the high mycotoxin production. These species cause diseases in different plant parts, ranging from seedling infection, leaf spot, and root rot to Fusarium Head Blight (FHB), one of the most serious cereal diseases [41]. These infections and subsequent diseases caused by these pathogens can lead to up to 50% crop yield loss [42]. In our study, β-D-glucan decreased in all oat varieties after the pathogenic infection. This decline could be attributed to two mechanisms: (1) fungal consumption of β-D-glucan for its nutrition and growth [43] and (2) plant utilization of β-D-glucan to activate defense responses, a concept known as “sweet immunity” [25].

We observed a difference in the concentration of the pathogenic DNA (both FG and FC) between naked and hulled varieties of oats. Contrary to reports in the literature of lower pathogen DNA in naked oats [44,45], our results showed a higher percentage of pathogenic DNA in naked grains (2.30%), compared to hulled ones (2.07%). Havrlentova et al. [44] demonstrated a lower percentage of pathogen DNA (0.39%) observed in naked compared to dehulled oat grains, where the pathogen DNA level was significantly higher (2.09%). A similar result was described by Martin et al. [45] for infection of sown barley with the pathogen FG. The variation in our study may be due to the complex interaction of oat cultivars, β-D-glucan content, and their response to the artificial inoculation. PCA revealed that FC acts as a stronger stress factor, explaining the observed DNA variation and significant β-D-glucan reductions in hulled cultivars, suggesting that FC is a more aggressive pathogen.

Our findings align with studies suggesting that FC is more aggressive than FG [46,47]. The manifestation of FC’s higher invasiveness underscores the need to consider pathogen-specific responses when evaluating oat cultivars. We hypothesized that higher β-D-glucan content might protect against mild stressors, a theory supported by previous work stating that the Poaceae family’s β-D-glucan metabolism may be largely responsible for plants’ responses to environmental signals in the moderate, physiological range [13,39,40,44,48]. However, our results indicate that β-D-glucan levels in our experiments were insufficient as a protective agent to counteract the severe stress induced by Fusarium strains.

In our experiment, the oat variety Pushkinskij deviates from other varieties in response to artificial infection. For example, in the concentration of pathogenic DNA, in this variety, it was 4.55 ng·g⁻¹ for FG, compared to 514.42 ng·g⁻¹ for FC. One explanation can be the composition of the grain. Vargach [49] found that this Russian variety showed high antioxidant activity of the water extract of the grain, indicating the presence of secondary metabolites with antioxidant capacity that could protect the grain against some forms of stressors. Furthermore, Loskutov [50] emphasizes that the Pushkinskij variety is suitable for a celiac diet, which indicates its other grain composition compared to varieties of oats. These factors may explain why the variety Pushkinskij effectively defended itself against the mild stress induced by the pathogen F. graminearum, while its response to the more aggressive F. culmorum was less effective.

5. Conclusions

This study investigated the relationship between artificial infection by FC and FG and β-D-glucan content in oat grains and molecular, biological, and metabolomic manifestations of this infection. Our results reveal several significant implications for understanding oat resistance to fungal pathogens and potential strategies for crop improvement.

Our analysis of β-D-glucan content in selected oat cultivars revealed variability, ranging from 1.97% (Vaclav) to 2.53% (Tatran), with an average of 2.17%. Naked oat varieties (Tatran and Pushkinskij) had higher β-D-glucan content (2.30%) compared to hulled varieties (Auron, Vaclav, and Hucul) at 2.08%. Fusarium infection notably reduced β-D-glucan content, with mean decreases of 10.60% for FG and 16.05% for FC. Hulled varieties showed significant declines post-infection, while naked varieties exhibited smaller, non-significant decreases, suggesting a potential protective effect of higher β-D-glucan content in naked varieties. FC was more virulent than FG, with an average pathogenic DNA accumulation of 336.79 ng·g⁻¹ flour compared to 82.05 ng·g⁻¹ for FG. Tatran and Pushkinskij (naked varieties) had the highest FC DNA concentration, but Pushkinskij had the lowest FG DNA accumulation, indicating complex interactions between variety traits and pathogen susceptibility. PCA revealed distinct clustering, with Auron and Vaclav consistently showing lower β-D-glucan and pathogenic DNA levels post-infection. Additionally, Pushkinskij, with Auron and Vaclav, was selected in response to FG infection. Correlation analysis showed a significant positive correlation between β-D-glucan content and FC DNA (r = 0.7374, p = 0.0017), but only a weak, non-significant correlation with FG DNA (r = 0.3949, p = 0.1452). This suggests that the relationship between β-D-glucan content and pathogen resistance is more complex than initially assumed. Our findings suggest that, under mild stress, such as FG infection, increased β-D-glucan content may protect against pathogen invasion, while, under severe stress (e.g., FC infection), β-D-glucan alone is insufficient as a defense mechanism. These results have important implications for future research and strategies to improve oat resistance to pathogens.

The content of β-D-glucan in oat grains was monitored in the presented work after artificial inoculation with Fusarium in controlled laboratory conditions. The results indicate the different responses of hulled and naked oat varieties to the infection. The naked cultivars probably used higher β-D-glucan content as a defense tool against milder stresses such as FG infection. However, after FC infection, β-D-glucan alone was not sufficient to protect the grain, which was shown by a higher accumulation of pathogenic DNA. The results indicate that the hulled varieties are more sensitive to infections caused by more aggressive pathogens. These findings are important for the strategy of breeding oat cultivars resistant to Fusarium.

Further research could be directed towards clarifying the threshold at which β-D-glucan ceases to be an effective protective tool. In the future, it would be appropriate to investigate also other metabolites and their synergistic effects with β-D-glucan in plant defense mechanisms, e.g., mono- and disaccharides and other molecules involved in MAMPs and PAMPs.