The Influence of Fusarium culmorum on the Technological Value of Winter Wheat Cultivars

Abstract

The research hypothesis assumes that Fusarium culmorum infection affects the baking value of wheat. The aim of the research was to determine the effect of the cultivar on the rheological properties of wheat dough in response to Fusarium culmorum infection of wheat. A two-factor experiment conducted during the 2018–2020 growing seasons in Osiny, Poland, was set up using the completely randomized block design with three replications. The first factor was winter wheat cultivars (six cultivars), while the second factor was inoculation (two levels—Fusarium culmorum and distilled water—control). The immunoenzymatic ELISA method was used to determine the content of deoxynivalenol (DON) in grain. The DON content in the grain varied between cultivars. Fusarium culmorum inoculation resulted in an increase in protein, ash content, and flour water absorption, changes in dough rheological properties, and a decrease in the sedimentation index. Inoculation also caused negative changes in starch properties. The observed interaction between Fusarium culmorum inoculation and cultivars in shaping the qualitative parameters and rheological properties of the dough indicates that there are wheat cultivars less susceptible to Fusarium infection, which do not show any significant changes as a result of infection.

1. Introduction

Wheat is one of the most important crops in the world, ranking as the third largest in terms of production, after sugar cane and maize. In 2022, global wheat production reached 808 million tons, cultivated on an area of 219.2 million hectares [1]. The data indicate that over 50% of wheat harvests are used by food enterprises for production of flour, bread, breakfast cereals, pasta, and others [2]. Wheat intended for bread production must meet specific quality indicators that determine its suitability for flour production and baking. For this purpose, the grain undergoes various analyses, which provide information on the quantity and quality of gluten, the quality of the protein complex (sedimentation index), the activity of amylolytic enzymes (falling number), and the rheological properties of the dough [3].

The raw material delivered to flour mills must not only meet the requirements for baking quality but also be completely safe for human health. The quality of the raw material, collected from the field, is influenced by the presence of physical, chemical, or biological contaminants. Chemical contaminations, such as pesticide residues, heavy metals, and nitrites, are not acceptable in final food products and feed. Grain infection by Fusarium spp. causing Fusarium head blight (FHB) may be indicated by high levels of mycotoxins.

Mycotoxins are a group of harmful compounds, which are secondary metabolites of filamentous fungi. These substances can have strong toxic, mutagenic, or teratogenic effects. Secondary metabolites are organic compounds produced by organisms, such as plants, fungi, and bacteria, that are not directly involved in their normal growth, development, or reproduction. The presence of mycotoxins in food poses a potential threat to human health. It is estimated that about 25% of global cereal production is contaminated with these metabolites, leading to a deterioration in the health quality of grain and its elimination from consumption and feed [4]. To ensure consumer safety, the European Commission has established unified standards and legal regulations regarding the maximum levels of some of the most important mycotoxins in cereal grains and products. The maximum level of DON is set at 1000 µg kg⁻¹ for unprocessed cereals, except for durum wheat with a limit of 1500 µg kg⁻¹ and oats with a limit up to 1750 µg kg⁻¹ [5]. For zearalenone (ZEN), the maximum level for these products is 100 µg kg⁻¹. The maximum levels of mycotoxins in cereal-based products are lower. The DON level in flour, pasta, and bran must not exceed 600 µg·kg⁻¹, in bread 400 µg·kg⁻¹, and in products for children 150 µg·kg⁻¹. Similarly, the ZEN level in flour and bran must not exceed 75 µg·kg⁻¹, and in bread it must not exceed 50 µg·kg⁻¹ [6].

Wheat cultivation is primarily aimed at producing grain for consumption, especially for flour and bread production. Wheat grain is rich in various components that differ in molecular structure, physicochemical, and functionality. Its dry mass consists of 60–70% starch, 10–17% proteins, 3–6% sugars, 2–2.5% lipids, vitamin E, B group vitamins (riboflavin, niacin, thiamine), dietary fiber, 1.4–2.3% minerals (Cu, Mg, Zn, P, and Fe), and other phytochemical compounds [7,8]. The chemical composition of wheat grain is influenced by environmental conditions, agricultural practices, and genotype [7].

Fusarium fungal infection of grain can lead to biochemical changes and structural alteration in the grain [9]. The extent of these changes depends on the level of fungal invasion [10]. Infected grain may have fungal spores on its surface, and its internal structure may contain mycotoxins. Infested grain shows a reduction in the thickness of the aleurone layer, altering the proportion of seed coat to endosperm, which results in a decrease in thousand kernel weight and an increase in ash content in whole-grain flour [11,12,13]. A positive correlation between the degree of Fusarium culmorum infection and the ash content, which includes potassium and calcium salts in wheat grain, was previously reported [14,15].

Increased activity of amylase, protease, and other enzymes responsible for the degradation of non-starch polysaccharides in infected samples was also observed [16,17,18,19]. Fungal proteases produced by Fusarium culmorum are active in a wide range of temperatures (10–100 °C) and pH values (4.5–8.5), potentially impairing dough functionality and bread quality throughout processing. Kreuzberger et al. [17], in an in vitro study, observed a clear effect of Fusarium graminearum proteases on glutenin and gliadin fractions, with a higher preference for glutenins, which are responsible for dough elasticity and viscosity. Fusarium-infected ears, during kernel growth, may show little visible damage or macromolecules inside the kernel but still contain high levels of DON [20]. The fungal infection may destroy the cellular structures of the starchy endosperm, damaging starch granules. These compositional may reduce flour quality, resulting in a low falling number [21]. The presence of Fusarium leads to starch degradation, which correlates with the activity of α-amylase in the grain and a decrease in the falling number value [9,22,23].

Starch damage caused by Fusarium may contribute to increased water absorption in the samples [18]. The quality of wheat flour primarily depends on gluten proteins, which play an important role in carbon dioxide retention, dough development, and baking quality [24]. Fusarium fungi and their toxins can affect both protein quantity and disturb the mutual proportions of individual protein fractions, thereby impacting the quality of the raw material and the final product [25]. In some cultivars (Greina, Lona, and Brusino), the sedimentation index decreases after Fusarium infection, indicating an effect on the protein quantity and quality, while no such negative changes were observed in others. Studies indicate that Fusarium decreases gluten quantity and quality, as evidenced by the reduction in the Zeleny sedimentation index in Fusarium-infected grain compared to healthy seeds [14,21]. The reduction in the quality of gluten proteins due to Fusarium inoculation also affected the rheological properties of wheat flour dough. The increase in protease activity found in wheat flour infected by Fusarium had a significant effect on the increase in dough softening and decrease in dough stability of flour [13,14,18]. However, the Fusarium infection did not influence the water absorption of flour [14,18]. The deterioration of qualitative characteristics and rheological properties of the dough made by infected flour was also noted previously. Fusarium infection also negatively impacted dough energy, maximum tensile strength, and the strength to extensibility ratio, reducing these parameters significantly. Additionally, some authors observed a 20-fold increase in the gliadin to glutenin ratio in flour samples with a DON content of 3.98 mg·kg⁻¹, resulting in reduced bread volume [13,26]. Contrary to the abovementioned results, other authors noted an increase of loaf volume under Fusarium infection regarding the dose of Fusarium infection and different bread production technologies [18,27]. Limited studies using Mixolab, which allows for the assessment of the protein–starch complex in a single test, showed that Fusarium spp. infection of the grain led to a reduction in the rheological properties of dough. Other research showed that an increasing intensity of Fusarium spp. contamination had negative effects on protein and mainly on the starch part of grain which was noted on the Mixolab curves [13]. The authors of [13,28] suggested that the cause may be the degree of starch damage granules in the inoculated samples, which affects the dough characteristics, making it stickier and potentially resulting in poor baking quality.

The research hypothesis posits that Fusarium infection and mycotoxins in grain and flour affect wheat’s baking quality, causing changes in both starch and protein structure. These effects are cultivar-dependent. Therefore, the aim of this research is to determine the impact of cultivar on the rheological quality of wheat dough in response to stress caused by Fusarium infection of wheat.

2. Materials and Methods

A two-factor experiment was established using the completely randomized block method in three replications. The factors were winter wheat cultivars and spray. In the experiment, six winter wheat cultivars were used, with each cultivar being treated with either Fusarium culmorum inoculation or distilled water (as a control sample).

Plant material. The research work concerned winter wheat cultivars representing two technological groups approved by the Polish National List of Agricultural Plant Varieties issued yearly by COBORU (the Research Centre for Variety Testing). The individual cultivars differed in morphological structure, utility features, genotype and place of cultivation (

Table 1. Characteristics of winter wheat cultivars.

Cultivar	Breeding Place	Resistance to FHB *	Height (cm)	Wheat Quality Group	Flowering, Maturing (Days from 1st January)
KWS Ozon	KWS Lochow GmbH	7.1	83	B	151 203
Legenda	Poznańska Plant Breeding	7.9	115	A	151 203
Muszelka	DANKO Plant Breeding	6.5	80	B	150 202
Pokusa	Strzelce Plant Breeding	7.9	97	A	150 202
Tonacja	Strzelce Plant Breeding	7.8	104	A	152 204
Sailor	DANKO Plant Breeding	7.7	101	A	150 203

* FHB—Fusarium head blight.

). Group A comprises quality bread cultivars, namely: ‘Legenda’, ‘Pokusa’, ‘Tonacja’, and ‘Sailor’. Group B comprises the following bread cultivars: ‘Muszelka’ and ‘KWS Ozon’.

The cultivars differed in earliness and the earing stage; the fastest were cv. ‘Muszelka’, ‘Pokusa’, ‘Sailor’, and the latest was cv. ‘Tonacja’. The maturity stage was reached the fastest by cv. ‘Muszelka’ and ‘Pokusa’ (202 days from 1 January), and the latest by: cv. ‘KWS Ozon’ and ‘Sailor’ (203 days from 1 January). The cultivars differed in resistance to Fusarium head blight (FHB). Resistance is estimated by the Central Research Centre for Cultivar Testing on a nine-point scale, where 1 means susceptible and 9 completely resistant. The cv. ‘Legenda’ was characterized by the highest resistance (7.9) and the cv. ‘Muszelka’ (6.5) showed the highest susceptibility. Moreover, the cultivars differed in height. The height difference was 35 cm. The tallest cultivar was cv. ‘Legenda’ (115 cm) and the shortest was cv. ‘Muszelka’ (80 cm) (Table 1).

Field trials. The field experiment was conducted in 2018/2019 and 2019/2020 growing seasons at the Experimental Station in Osiny (51°35′, 21°55′), belonging to the Institute of Soil Science and Plant Cultivation—State Research Institute (IUNG-PIB) Puławy, Poland. The experiment was established on pseudopodzolic soil, class IIIb, agricultural soil suitability complex—very good rye complex, with a pH of 6.1. The mineral content in the soil was: extractable phosphorus (P: 12.6 mg kg⁻¹), exchangeable potassium (K: 15.3 mg kg⁻¹), magnesium (Mg 4.9 mg kg⁻¹). The forecrop was winter rape. The plot size was 24 m², 11.25 m² was designed for harvest. In autumn, after harvesting the forecrop, pre-sowing of crops was carried out—cultivation with a disc cultivator, ploughing and cultivation with a seed drill. Wheat sowing was performed on optimal dates in subsequent years of the experiment: 2 October 2018 and 7 October 2019, respectively. Sowing density was 450 grains per m². The plots were fertilized with: N (160 kg ha⁻¹), P (70 kg ha⁻¹), and K (105 kg ha⁻¹). Nitrogen fertilizer was applied three times in individual vegetation stages: beginning of vegetation (BBCH 23)—70 kg N ha⁻¹, stem shooting (BBCH 32)—60 kg N ha⁻¹, full earing (BBCH 53)—30 kg N ha⁻¹. Plants were protected against weeds, diseases, and pests using Bizon 400 L (active substance: tralkoxydim) 1.0 l·ha⁻¹; Mustang Forte 195 SE (active substance: florasulam, aminopyralid, 2.4D) 0.5 l ha⁻¹; Unix 75WG (active substance: cyprodinil) 0.6 kg ha⁻¹; Fury 100 EC (active substance: zeta-cypermethrin) 0.1 L ha⁻¹. Harvesting was carried out at full maturity on 20 July 2019 and 1 August 2020.

Weather conditions. The weather conditions during the wheat growing period, including the heading (May), flowering (June), and maturity (July) stages, varied across different growing seasons (

Table 2. Weather conditions in the growing seasons.

Month	Temperature (°C)		Precipitation (mm)
	Growing Season
	2019	2020	2019	2020
March	5.5	4.5	22.7	25.3
April	9.6	8.5	35.5	11.9
May	12.9	11.1	86.1	112.7
June	21.7	18.4	38.7	189.5
July	18.6	18.6	33.9	49.8

). In 2019, air temperature was higher in May and June by 1.8 °C and 3.3 °C, respectively compared to the 2020 harvest year. Precipitation in the mentioned months of 2019 was lower by 26.6 mm, 150.8 mm, and 15.9 mm, respectively, compared to 2020. The high air temperature in June and July, together with very low precipitation, led to the occurrence of drought. These were unfavorable conditions for the growth and development of Fusarium culmorum.

Preparation of Fusarium culmorum inoculum and inoculation in the field. The inoculation suspension was obtained from grain infected with Fusarium culmorum spores, prepared at the Plant Breeding and Acclimatization Institute (IHAR)—National Research Institute in Radzików, Poland. To produce the inoculum, a Fusarium culmorum isolate forming deoxynivalenol was prepared according to the methodology followed in [29]. The isolate was incubated on autoclaved wheat grain in glass flasks for approximately 4 weeks and then irradiated with continuous UV light for 4 to 7 days at a temperature of about 18 °C. Then the grain infected with Fusarium culmorum was dried and stored at 4 °C until the inoculates were prepared for spraying. On the day of inoculation, grain with mycelium and conidial spores of Fusarium culmorum was soaked in sterile tap water for two hours and then filtered through gauze to obtain a spore suspension without mycelium fragments. After thoroughly mixing the spore suspension, the number of colony-forming units (CFU) was determined using a Thom counting chamber. The field experiment used artificial inoculation by spraying with Fusarium culmorum. It was performed during the flowering phase of the wheat cultivars (BBCH 65). The plants were sprayed manually with a suspension of spores prepared in the laboratory at a concentration of 5 × 10⁵ CFU·mL⁻¹ at 100 mL per 1 m². The inoculation method used imitated the natural conditions of ear infection by Fusarium and allowed for determining type I resistance (resistance to infections) and type II resistance (resistance to the spread of the pathogen in tissues). Inoculations were performed in the late afternoon hours, when the temperature decreased and the relative air humidity increased. The experiment included a control, on which spraying with distilled water was used during the flowering period in order to maintain similar humidity conditions to the inoculated field.

Determination of deoxynivalenol in wheat grain. The immunoenzymatic ELISA method was used to determine the content of DON in wheat grains, using the AgraQuant test from RomerLabs^® (Getzersdorf, Austria). The optical density of the samples was measured using a Stat Fax 303 Plus microplate reader (photometer) (Awareness Technology, Inc., Palm City, FL, USA) at a wavelength of 650 nm. According to the recommendation, the results were read within 20 min of adding the Red Stop solution. The standard curve was prepared by using a standard (optical density of mycotoxin concentration). For each analysis the correlation coefficient R (dependence of absorbance on A/c concentration) was obtained. According to recommendations, the results are considered reliable when the value of the R coefficient is not less than 0.996. Representative samples of grain were ground in a Laboratory Mill 3100 by Perten and stored at 2–8 °C until extraction. After adding the extractant (distilled water), the samples were shaken vigorously for 3 min. The extracts were filtered by passing at least 5 mL through a Whatman 1 filter. The estimation was carried out in accordance with AgraQuant standards in duplicate. The contents of DON in inoculated and uninoculated samples (2019) and the control samples (2020) were below the detection limit (LOD < 200 µg kg⁻¹), therefore, these values are not presented in the manuscript.

Technological quality analysis. The quality analysis was determined after harvest. The physical and chemical properties were tested in the samples obtained only from the 2020 harvest year. In 2019 Fusarium infection was not observed at a level that would affect the technological properties of the grain, therefore the date from this year was abandoned. The thousand kernel weight was determined according to ISO 520:2010 [30]. A grinder (FN 3100, Perten Instruments AB, Hägersten, Sweden) was used for preparation of whole-grain flours, and next the following quality parameters were marked: moisture content (determined according to ISO 712:2009) [31], protein content (ISO 20483:2013) [32], gluten quantity (ISO 21415-2:2015) [33], Zeleny sedimentation index (ISO 5529:2007) [34], and ash content (ISO 2171:2023) [35] were determined in order to evaluate the technological quality of tested wheat cultivars. Wheat flour samples for the Zeleny sedimentation test were prepared by using a Sedimat laboratory mill (Brabender GmbH & Co. KG, Duisburg, Germany).

Rheological properties of a dough. Mixolab ChopinWheat+ Protocol according to ISO 17718:2013 [36] (KPM Analytics, Villeneuve-la-Garenne, France) was used to analyze the rheological properties of a dough. Grain was ground by an FN 3100 grinder (Perten InstrumentsAB, Hägersten, Sweden) to obtain whole-grain flour to perform the Mixolab test. The whole-grain flour samples (50 g) based on 14% moisture content were used to determine the water absorption of flour at the consistency of 1.1 ± 0.05 Nm. The mixing speed was 80 rpm, and bowl temperature was set at 30 °C. Protein properties related to water absorption, dough development time, stability, and weakening (C2, C1–C2, slope α) were determined. The starch properties correspond to starch gelatinization during the increase in the temperature from 30 to 90 °C (C3, C3–C2, slope β) and enzymatic activities (C4, C3–C4, slope γ) and retrogradation (C5, C5–C4) were also evaluated. The following Mixolab Profiler indexes were evaluated to better characterize the baking quality potential of tested cultivars: water absorption, mixing, gluten+, viscosity, amylase, and retrogradation.

Statistical analyses. The results were statistically analyzed by the one-way analysis of variance (ANOVA) with a subsequent Tukey’s HSD test with the significance level of p < 0.05 to compare the means of control treatments and inoculations with Fusarium culmorum. The second factor was the wheat cultivar used. Principal component analysis (PCA) was performed to reduce the dimensionality of the data and represent the samples in a new coordinate system. All results were analyzed using Statistica software (v. 13.3, StatSoft, Tulsa, OK, USA).

3. Results

Wheat grain samples from the 2020 harvest year showed significant infection by Fusarium spp. and were also characterized by relatively high DON content. Variation in DON levels and basic quality parameters were observed. These characteristics are essential for assessing their suitability for grain processing for consumption. The results of the analysis of variance (

Table 3. Significance of effect in ANOVA for wheat samples from the 2020 harvest.

Parameter/Factor	A (Cultivar)	B (Inoculation)	A × B	V (%)
Thousand kernel weight	***	***	*	6.28
Protein content	***	**	***	3.29
Gluten content	***	n.s.	***	4.43
Zeleny sedimentation index	***	***	***	4.24
Ash content	***	***	***	7.55
Water absorption	***	***	***	3.49
Time T1	***	***	***	4.45
Stability	***	***	***	5.36
Slope α	***	***	***	3.85
Protein weakening, C2	**	***	**	5.63
C1–C2	***	***	***	4.40
Slope β	***	***	***	4.02
Slope γ	***	***	***	7.92
C3	***	***	***	3.44
C4	***	***	***	2.54
C5	***	***	***	4.83
C3–C2	***	***	***	3.47
C3–C4	***	***	***	2.54
C5–C4	***	***	***	3.12

Abbreviation: *—significant at p < 0.05; **—significant at p < 0.01; ***—significant at p < 0.001; n.s.—non-significant; V—coefficient of variation.

) indicated that both the cultivars and Fusarium inoculation significantly affected the measured technological parameters of wheat grain. However, Fusarium inoculation had no significant effect on the gluten content. Table 3 does not include the results of wheat cultivars harvested in 2019 because no ear infection was observed, which was confirmed by DON determinations below the LOD. Additionally, no difference was found in the amount of gluten and the Zeleny sedimentation index between inoculated and control samples.

3.1. Deoxynivalenol Content in Wheat Grain

The deoxynivalenol (DON) content of individual grain wheat cultivars varied across different years. Grain samples harvested in 2019 were characterized by a DON level below the limit of detection (LOD) of 200 µg·kg⁻¹. In 2020, the grain of the tested wheat cultivars contained higher levels of DON compared to 2019. The lowest amounts of DON were accumulated in the cv. ‘Sailor’ (6847 µg·kg⁻¹), cv. ‘Pokusa’ (7477 µg·kg⁻¹), and cv. ‘Legenda’ (8790 µg·kg⁻¹), while the highest in cv. ‘Muszelka’ (11,021 µg·kg⁻¹), cv. ‘KWS Ozon’ (11,033 µg·kg⁻¹), and cv. ‘Tonacja’ (11,513 µg·kg⁻¹) (Figure 1). In 2020, in uninoculated samples (control), DON content was below limit of detection (200 µg·kg⁻¹).

3.2. Quality Characteristics of Wheat Cultivars

The effect of Fusarium culmorum inoculation on technological features of grain is presented in

Table 4. The quality characteristics of winter wheat cultivars control and F. culmorum-infected samples.

Parameter	Inoculation	Cultivar						Mean
		Sailor	Legenda	KWS Ozon	Pokusa	Muszelka	Tonacja
Thousand kernel weight (g)	Control	42.15 a	38.93 a	47.51 a	46.92 a	46.84 a	41.67 a	44.00 B
	Fusarium	38.52 b	33.24 b	43.52 a	43.73 b	42.15 b	36.17 b	39.56 A
	Average	40.3 AB	36.08 B	45.51 A	45.32 A	44.49 A	38.92 B
Protein content (%)	Control	13.9 b	12.9 b	13.1 a	13.4 b	13.3 b	11.9 a	13.1 B
	Fusarium	14.8 a	16.4 a	13.2 a	14.3 a	14.1 a	12.4 a	14.2 A
	Average	14.4 A	14.6 A	13.1 D	13.9 B	13.6 C	12.1 E
Gluten content (%)	Control	29.4 b	35.6 a	28.6 a	29.6 b	28.5 a	28.8 a	30.08 A
	Fusarium	30.5 a	34.2 b	27.4 a	31.8 a	28.7 a	30.0 a	30.43 A
	Average	30.0 A	34.9 A	28.0 B	30.7 A	28.6 B	29.4 A
Zeleny sedimentation index (cm3)	Control	48 a	45 a	52 a	55 a	47 a	46	48.8 A
	Fusarium	45 a	51 a	34 b	46 b	42 a	44 a	43.6 B
	Average	47.0 A	48.0 A	43.0 A	50.7 A	44.5 A	45.5 A
Ash content (%)	Control	1.42 b	1.48 a	1.51 b	1.27 b	1.41 b	1.45 b	1.42 B
	Fusarium	1.56 a	1.59 a	1.91 a	1.71 a	1.71 a	1.58 a	1.68 A
	Average	1.49 B	1.53 B	1.71 A	1.49 B	1.56 B	1.52 B

^a,b,A−E—homogeneous groups, statistically significant differences at p = 0.05.

. Thousand kernel weight (TKW) is related to grain yield, milling efficiency, and seedling vigor. TKW differed significantly between control samples of the cultivars, ranging from 38.93 g (cv. ‘Legenda’) to 47.51 g (cv. ‘KWS Ozon’). Fusarium inoculation caused a significant reduction in TKW in all cultivars, except for cv. ‘KWS Ozon’ (Table 4).

The protein content significantly depended on the cultivar. An interaction between Fusarium culmorum inoculation and cultivar was observed for protein content. Wheat samples inoculated with Fusarium had protein content in the range of 12.4% (cv. ‘Tonacja’) to 16.4% (cv. ‘Legenda’). The protein content in non-inoculated samples ranged from 11.9 (cv. ‘Tonacja’) to 13.9 (cv. ‘Sailor’). Fusarium inoculation caused a significant increase in protein content in grain, except for cv. ‘KWS Ozon’, where no effect was observed. The greatest increase in protein content was found in cv. ‘Legenda’, where the difference between inoculated and control samples was 3.5 percentage points. The difference for cv. ‘Sailor’, ‘Pokusa’, and ‘Muszelka’ ranged from 0.8 to 0.9 percentage points.

The gluten content in control wheat samples ranged from 28.6% (cv. ‘Muszelka’) to 35.6% (cv. ‘Legenda’) (Table 3). Fusarium inoculation caused a significant increase in gluten content in cv. ‘Sailor’ and cv. ‘Pokusa’ by 1.1, and 2.2 percentage points, respectively. In cv. ‘Muszelka’, ‘Tonacja’, and ‘KWS Ozon’, no significant differences were observed compared to the control samples.

The Zeleny sedimentation index values for flour from wheat grain samples ranged from 45 cm³ (cv. ‘Legenda’) to 55 cm³ (cv. ‘Pokusa’) (Table 3). Fusarium inoculation reduced the sedimentation index value on average by 5.2 cm³ across the cultivars. The highest reduction was observed in cv. ‘KWS Ozon’ (18 cm³) and cv. ‘Pokusa’ (9 cm³), while in cv. ‘Legenda’, there was an increase of 6 cm³.

Ash content in grain was influenced by both the cultivar and Fusarium inoculation (Table 3). Wheat samples were characterized by ash content in the range of 1.27% (cv. ‘Pokusa’) to 1.51% (cv. ‘KWS Ozon’). Infected grain showed significantly higher ash content compared to the control samples, with differences ranging from 0.11 percentage points (cv. ‘Legenda’) to 0.44 percentage points (cv. ‘Pokusa’).

3.3. Rheological Properties of the Protein Complex of Tested Wheat Cultivars

The rheological properties of whole-grain flour dough were studied using the ChopinWheat+ protocol of Mixolab (Chopin Technologies), which allows for the determination of both protein and starch properties of wheat flour in a single test.

Determination of flour water absorption is an important criterion for wheat flour, as it provides information about baking absorption, which is related to bread yield and quality of bakery products. In the presented studies, the water absorption of the flour samples from the cultivars ranged from 60.5% (cv. ‘Sailor’) to 62.9% (cv. ‘Tonacja’). A significant increase in this indicator was observed in all inoculated cultivars compared to the control samples. Flour obtained from grain infected with Fusarium showed higher water absorption, on average by 3.6 percentage points, compared to control grain samples (

Table 5. The rheological properties of dough obtained from winter wheat cultivar control and F. culmorum-infected samples.

Parameter	Inoculation	Cultivar						Mean
		Sailor	Legenda	KWS Ozon	Pokusa	Muszelka	Tonacja
Water absorption (%)	Control	60.5 b	61.4 b	62.6 b	62.1 a	61.8 b	62.9 b	61.9 B
	Fusarium	64.6 a	63.4 a	66.4 a	66.7 b	66.8 a	64.9 a	65.5 A
	Average	62.6 A	62.4 A	64.5 A	64.4 A	64.3 A	63.9 A
Time T1 (min)	Control	3.37 b	4.4 a	5.0 a	4.1 a	5.4 a	8.2 a	5.0 A
	Fusarium	4.0 a	2.6 b	1.9 b	3.9 a	1.2 b	3.2 b	2.8 B
	Average	3.66 B	3.50 B	3.47 C	4.00 B	3.25 D	5.65 A
Stability (min)	Control	11.0 a	9.0 a	10.3 a	8.4 a	9.8 a	11.8 a	10.1 A
	Fusarium	7.7 b	8.2 b	7.0 b	6.2 b	5.3 b	5.2 b	6.6 B
	Average	9.3 A	8.6 A	8.6 A	7.3 B	7.6 B	8.5 A
Slope α (Nm min−1)	Control	−0.102 b	−0.088 a	−0.101 b	−0.083 b	−0.090 a	−0.091 b	−0.092 B
	Fusarium	−0.082 a	−0.091 a	−0.091 a	−0.070 a	−0.090 a	−0.060 a	−0.080 A
	Average	−0.092 B	−0.090 B	−0.096 B	−0.076 A	−0.090 B	−0.075 A
Protein weakening, C2 (Nm)	Control	0.53 a	0.42 a	0.46 a	0.40 a	0.47 a	0.55 a	0.47 A
	Fusarium	0.21 b	0.22 b	0.17 b	0.18 b	0.15 b	0.13 b	0.18 B
	Average	0.36 A	0.32 B	0.31 B	0.29 C	0.31 B	0.33 B
C1–C2 (Nm)	Control	0.58 b	0.66 b	0.63 b	0.67 b	0.62 b	0.55 b	0.62 B
	Fusarium	0.91 a	0.89 a	0.94 a	0.95 a	0.97 a	0.97 a	0.94 A
	Average	0.74 B	0.77 B	0.78 A	0.81 A	0.79 A	0.76 AB

^a,b,A−D—homogeneous groups, statistically significant differences at p = 0.05.

). The highest increases in this parameter were observed for cv. ‘Pokusa’ and cv. ‘Muszelka’ (4.6 and 5 percentage points, respectively).

The protein characteristics of whole-meal flour samples were determined based on time T1 (dough development time), stability, and protein weakening (C2, C1–C2, slope α). Fusarium inoculation significantly influenced the protein characteristics of the tested winter wheat cultivars.

Dough development time (T1), which is primarily related to protein content, varied between cultivars and was significantly affected by Fusarium inoculation (Figure 2, Figure 3 and Figure 4). Time T1 for the cultivar samples ranged from 3.3 min (cv. ‘Sailor’) to 8.2 min (cv. ‘Tonacja’) (Table 5). However, the influence of Fusarium inoculation varied between cultivars. A reduction in time T1 was observed for four of the six tested cultivars. The highest difference was stated for cv. ‘Tonacja’ (on average 5 min), while for cv. ‘Pokusa’, there was no change in this parameter, and for cv. ‘Sailor’, T1 increased by 0.7 min.

Dough stability, measured as the time until the upper frame of the Mixolab curve decreases by 11% from the point C1, corresponds to the dough strength. The value of this parameter significantly depended on the experimental factors (Table 4). Among the control samples of wheat cultivars, the highest stability during dough mixing was found for the cv. ‘Tonacja’ (11.8 min), while the lowest value was observed for cv. ‘Pokusa’ (8.4 min). Inoculation with Fusarium fungi caused a significant decrease in dough stability for all tested cultivars, by an average reduction of 3.5 min. The greatest decrease was observed for cv. ‘Tonacja’ (6.6 min), while the lowest for cv. ‘Legenda’ (0.8 min), which was characterized by one of the lowest stability values among the control cultivars.

Protein weakening, as measured by Mixolab, was determined based on the change in dough consistency during temperature increases (C2, C1–C2, and slope α). The C2 parameter for the tested wheat flours obtained from control samples was within an appropriate level for baking, varying between cultivars. The lowest protein weakening (C2) was observed in cv. ‘Pokusa’ (0.40 Nm), while the highest was found in cv. ‘Tonacja’ (0.55 Nm). Fusarium inoculation caused a significant decrease in the C2 value (on average 0.30 Nm). The largest decrease was found for cv. ‘Tonacja’ (0.42 N m) and the lowest for cv. ‘Legenda’ (0.20 Nm). However, the C2 parameter for all tested samples ranged from 0.13 to 0.22 Nm, which corresponds to weak dough and potential issues during mechanical processing in dough preparation.

To better characterize the changes in dough properties, and referring to the studies [13,37,38], the differences in dough resistance measured at points C1 and C2, as well as C3 and C2, C3 and C4, and C5 and C4, were determined. The low quality of gluten proteins in samples inoculated with Fusarium culmorum was confirmed by increased differences in torque values between points C1–C2 (0.62 Nm for control samples vs. 0.94 for inoculated samples) (Table 4). The difference in dough resistance at points C1 and C2 (C1–C2) indicates changes in the structure of gluten proteins due to heating [13]. According to Koksel et al. [38], this corresponds to dough resistance to mixing. The greatest increase in the C1–C2 parameter was observed in cv. ‘Tonacja’ (0.42 Nm), which had the lowest C2 value. The smallest increase was stated for cv. ‘Legenda’ and ‘Pokusa’ (0.23 and 0.28 Nm, respectively). Winter wheat cultivars inoculated with Fusarium culmorum also exhibited a higher rate of protein weakening under heat, as measured by slope α (−0.080 Nm min⁻¹ for inoculated grain samples vs. −0.092 Nm min⁻¹ for control samples).

3.4. Rheological Properties of the Starch Complex of Tested Wheat Cultivars

The properties of starch and the activity of amylolytic enzymes are crucial for the dough fermentation process and for obtaining high-quality wheat bread. The following Mixolab parameters were used to determine the starch complex of wheat flour: starch gelatinization (C3), amylase activity (C4), and retrogradation (C5). Starch gelatinization speed and enzyme degradation speed are determined as slope β and slope γ, respectively.

The β index, which characterizes the increase in dough resistance due to the swelling of starch granules as the temperature rises from 30 to 90 °C, ranged from 0.587 Nm min⁻¹ (cv. ‘KWS Ozon’) to 0.782 Nm min⁻¹ (cv. ‘Muszelka’). Inoculation of ears with the fungus caused a significant decrease in this parameter in all analyzed wheat cultivars (

Table 6. The effect of Fusarium culmorum contamination of grain on the rheological properties of dough from winter wheat cultivars determined using Mixolab, which characterizes the properties of starch.

Parameter	Inoculation	Cultivar						Mean
		Sailor	Legenda	KWS Ozon	Pokusa	Muszelka	Tonacja
Slope β (Nm/min)	Control	0.658 a	0.602 a	0.587 a	0.673 a	0.782 a	0.699 a	0.667 A
	Fusarium	0.583 b	0.563 b	0.087 b	0.419 b	0.518 b	0.602 b	0.462 B
	Average	0.621 B	0.583 C	0.337 E	0.546 D	0.650 A	0.651 A
Slope γ (Nm/min)	Control	0.129 b	−0.138 b	−0.111 b	−0.111 b	−0.089 a	−0.071 a	−0.108 B
	Fusarium	−0.089 a	−0.111 a	−0.499 a	−0.069 a	−0.052 a	−0.089 a	−0.152 A
	Average	−0.109 B	−0.125 B	−0.305 A	−0.090 C	−0.071 C	−0.080 C
C3 (Nm)	Control	2.02 a	1.99 a	1.81 a	1.84 a	2.06 a	2.09 a	1.97 A
	Fusarium	1.50 b	1.43 b	0.47 b	1.47 b	1.43 b	1.20 b	1.25 B
	Average	1.76 A	1.71 A	1.14 C	1.66 B	1.74 A	1.64 B
C4 (Nm)	Control	1.56 a	1.320 a	0.72 a	0.97 a	1.50 a	1.79 a	1.31 A
	Fusarium	0.88 b	0.72 b	0.20 b	0.86 a	0.73 b	0.41 b	0.63 B
	Average	1.22 A	1.02 D	0.46 F	0.92 E	1.12 B	1.10 C
C5 (Nm)	Control	2.67 a	1.97 a	1.10 a	1.61 a	2.56 a	2.91 a	2.14 A
	Fusarium	1.47 b	1.20 b	0.01 b	1.28 b	1.05 b	0.62 b	0.94 B
	Average	2.07 A	1.59 C	0.55 E	1.44 D	1.81 B	1.77 B
C3–C2 (Nm)	Control	1.50 a	1.58 a	1.35 a	1.43 a	1.59 a	1.54 a	1.50 A
	Fusarium	1.29 b	1.20 b	0.30 a	1.29 a	1.28 b	1.07 b	1.07 B
	Average	1.39 A	1.39 A	0.82 B	1.36 A	1.43 A	1.30 A
C3–C4 (Nm)	Control	0.46 b	0.68 a	1.09 a	0.87 a	0.56 b	0.29 b	0.66 A
	Fusarium	0.62 a	0.71 a	0.27 b	0.61 b	0.70 a	0.79 a	0.62 B
	Average	0.54 D	0.69 B	0.68 B	0.74 A	0.63 C	0.54 D
C5–C4 (Nm)	Control	1.11 a	0.65 a	0.39 a	0.63 a	1.06 a	1.11 a	0.83 A
	Fusarium	0.59 b	0.48 a	−0.20 b	0.42 a	0.32 b	0.21 b	0.30 B
	Average	0.851 A	0.569 C	0.097 D	0.525 C	0.690 B	0.663 B

^a,b,A−F—homogeneous groups, statistically significant differences at p = 0.05.

). The greatest decrease in slope β was observed in cv. ‘KWS Ozon’ which also had the lowest value (0.087 Nm min⁻¹) among the cultivars.

The values of slope γ, which characterize the rate of enzymatic starch decomposition, ranged from −0.111 Nm min⁻¹ (cv. ‘KWS Ozon’ and ‘Pokusa’) to −0.071 Nm min⁻¹ (cv. ‘Tonacja’). Inoculation with Fusarium culmorum significantly affected the values of slope γ in four cultivars, causing an increase in this parameter. However, for the cv. ‘KWS Ozon’ and cv. ‘Tonacja’, the value of slope γ decreased by −0.388 and −0.018 Nm min⁻¹, respectively (Table 6).

Regarding starch gelatinization (C3), control wheat flours exhibited medium to low α-amylase activity, with values ranging from 1.81 Nm (cv. ‘KWS Ozon’) to 2.09 Nm (cv. ‘Tonacja’). Fusarium culmorum inoculation caused significant reductions in C3 values (from 1.92 Nm for control samples to 1.25 Nm for inoculated samples). The greatest reduction was stated for cv. ‘KWS Ozon’ (−1.34 Nm to 0.47 Nm) (Figure 2) compared to other cultivars, where the reduction was on average −0.6 Nm. The C3 value for cv. ‘KWS Ozon’, with the lowest C3 in inoculated samples, was characterized by flour from sprouted grain with very high α-amylase activity.

The control winter wheat samples were more variable in terms of amylase activity (C4) and retrogradation (C5) than starch gelatinization (C3). C4 values for control samples ranged from 0.72 Nm (cv. ‘KWS Ozon’) to 1.79 Nm (cv. ‘Tonacja’) (Table 6). Fusarium inoculation caused a significant decrease in amylase activity (C4) (from 1.31 Nm in control samples to 0.63 Nm in inoculated samples). The highest drop was found for cv. ‘Tonacja’ (1.79 Nm to 0.41 Nm), and the lowest for cv. ‘Pokusa’ (0.97 Nm to 0.86 Nm). The lowest value of C4 was stated for cv. ‘KWS Ozon’ (0.20 Nm), confirming the stickiness of the dough.

Starch retrogradation in the cooling stage of Mixolab analysis varied between control winter wheat samples. The highest C5 value was recorded for cv. ‘Tonacja’ (2.91 Nm), compared to the very low value for cv. ‘KWS Ozon’ (1.10 Nm). Similar to other starch complex characteristics, starch retrogradation was strongly influenced by Fusarium culmorum inoculation (on average 2.14 vs. 0.94 Nm, respectively). The largest difference was found for cv. ‘Tonacja’ (a drop of 2.29 Nm) and the smallest for cv. ‘Pokusa’ (a drop of 0.33 Nm).

In partial agreement with the previous evaluation of the Mixolab curve, the differences between C3–C2, C3–C4, and C5–C4 were also analyzed. Dough properties related to starch gelatinization are determined at point C3 of the graph, and the difference in dough resistance at points C3 and C2 (C3–C2) are determined [39]. The value of the C3–C2 parameter was influenced by the cultivar and Fusarium inoculation. Inoculation caused a significant decrease in the value of this parameter. Flour from control cv. ‘Muszelka’ exhibited the highest C3–C2 value (1.59 Nm), while cv. ‘KWS Ozon’ showed the lowest (1.35 Nm). The greatest decrease in C3–C2 was observed in cv. ‘KWS Ozon’ (a drop of 1.05 Nm), whereas the reduction for inoculated cv. ‘Pokusa’ was only 0.14 Nm (Table 6).

Control samples of cv. ‘KWS Ozon’ were characterized by the highest differences between C3–C4 (1.09 Nm), indicating the highest α-amylase activity or less resistance to shear thinning. In contrast, cv. ‘Tonacja’ had the lowest C3–C4 difference (0.29 Nm), corresponding to low α-amylase activity. Fusarium inoculation caused an increase in C3–C4 difference in four of the six cultivars. In the case of cv. ‘KWS Ozon’, the dough became sticky at points C3 and C4, making it difficult to analyze, which explains the opposite change in this parameter.

The difference in dough resistance between points C5 and C4 (C5–C4) reflects the susceptibility of bread to staling and may indicate its shelf life, as described by Papouškova et al. [13]. The smallest difference was observed in the flour of cv. ‘Pokusa’ and the largest in cv. ‘Tonacja’ and cv. ‘Sailor’. Fusarium inoculation significantly affected the values of this parameter (Table 6). Among the cultivars after inoculation, cv. ‘KWS Ozon’ was assessed as the most favorable, as it exhibited the lowest C5–C4 value, suggesting the potential for the longest shelf life of the obtained bread. On the other hand, cv. ‘Sailor’ and cv. ‘Tonacja’ were considered the least favorable. For cv. ‘KWS Ozon’, negative values of the C5–C4 parameter in the flour after grain inoculation indicated very high amylolytic activity and technological problems in bread production.

3.5. Mixolab Wheat Flour Profiles

The Mixolab profiler allows the evaluation of dough consistency over time and determines, in the same assay, both the mixing and pasting characteristics of flour during a steady increase in temperature [40]. The Mixolab profiler provides users with the ability to classify the quality of tested flours based on six quality criteria: water absorption, mixing, gluten+, viscosity, amylase, and retrogradation, with values ranging from 0 to 9. This quality control tool, based on both protein and starch characteristics, helps to better screen and detect differences between tested samples and identify underperforming flours.

The Mixolab profilers obtained in our research significantly differed between the control cultivar samples and those inoculated with Fusarium culmorum (Figure 5, Figure 6 and Figure 7). Differences were also noted between the tested cultivars.

The water absorption index, which corresponds to the ability of flour to absorb water, was in the range of 4 (cv. ‘Sailor’) to 7 (other cultivars) among the control samples. Fusarium inoculation significantly increased the water absorption index to 8 in each cultivar.

In contrast, other parameters showed the opposite effect. The mixing index, which corresponds to the stability of flour dough during kneading, is higher for stronger dough. Control samples of wheat cultivars had a mixing index ranging from 5 (medium quality—cv. ‘Legenda’, cv. ‘Pokusa’) to 8 (strong gluten structure—cv. ‘Tonacja’). Fusarium inoculation weakened the gluten structure, lowering the mixing index (an average reduction of 2 units). However, different effects were observed across cultivars. The greatest reduction in this parameter was seen in cv. ‘Tonacja’ (Figure 7), while cv. ‘Sailor’ and cv. ‘Legenda’ showed no effect (Figure 6).

The gluten+ index describes the resistance of gluten to heat and the changes in the quality of gluten proteins under the influence of an increase in temperature from 30 °C to 60 °C. In non-inoculated samples, this index ranged from 2 to 8, with the highest value observed in the control samples of cv. ‘Sailor’ and cv. ‘Tonacja’ and the lowest in cv. ‘Pokusa’. Fusarium inoculation significantly reduced the heat-induced weakening of gluten proteins, lowering the gluten+ index by an average of 5 units to 0 (cv. ‘Sailor’, ‘Legenda’, ‘KWS Ozon’, ‘Pokusa’) and 1 (cv. ‘Muszelka’ and cv. ‘Tonacja’), indicating a reduction in stability to heat.

The next index with significant differences observed was viscosity, which corresponds to changes in dough characteristics as the temperature increases from 60 °C to 80 °C. This index is influenced by both amylase activity and starch quality, ranging from 4 (cv. ‘KWS Ozon’) to 8 (cv. ‘Sailor’ and cv. ‘Tonacja’). Fusarium inoculation significantly lowered the viscosity index to 0 (cv. ‘KWS Ozon’) and 1 (the rest of the cultivars). The average drop of 6 units was the highest among the other Mixolab Profiler indexes.

The amylase index, reflecting changes in dough properties due to the α-amylase enzyme, ranged from 1 (cv. ‘KWS Ozon’) to 8 (cv. ‘Sailor’ and cv. ‘Tonacja’). Fusarium inoculation caused a significant decrease in this index in cv. ‘Muszelka’ (Figure 5), cv. ‘Sailor’ (Figure 6), and cv. ‘Tonacja’ (Figure 7) compared to the control samples. However, an increase in the viscocity index was observed in cultivars with the lowest amylase index in the control samples: cv. ‘KWS Ozon’ (from 1 to 4) and cv. ‘Pokusa’ (from 3 to 4).

The retrogradation index, which depends on the characteristics of the starch and its hydrolysis during the test, ranged from 3 (cv. ‘KWS Ozon’) to 8 (cv. ‘Sailor’, cv. ‘Muszelka’, cv. ‘Tonacja’) in non-inoculated cultivars. In flour samples from grain contaminated with Fusarium culmorum, a significant reduction in this index was observed (on average a drop of 3 units).

3.6. Principal Component Analyses (PCAs)

Almost all investigated factors were presented in a two-dimensional coordinate system (Figure 8). Based on the results, we identified four distinct clusters of samples. The first group consisted of KWSOI (blue color), the second group included TC, MC, and SC (red color), the third group comprised PI, MI, SI, TI, and LI (green color), and the fourth group included LC, KWSOC, and PC (black color). Notably, opposite properties were observed between the first and fourth groups of samples. A similar trend was noted between the second and third clusters. The KWSOI sample was characterized by a high ash content and water absorption. In contrast, the LC, KWSOC, and PC samples exhibited higher rheological parameters (C3 and C3–C2), sedimentation index, and gluten content. The samples PI, MI, SI, TI, and LI stood out due to their total protein content and specific rheological parameters (C1–C2 and C3–C4). On the other hand, the TC, MC, and SC samples were distinguished by TKW, sedimentation time, and other rheological parameters (T1, C2, C4, C5, and C5–C4). It is worth noting that all inoculated samples were positioned on the left side of the score plot, while the control samples were located on the right side. This distribution indicates a negative interdependence between the different plant treatments.

4. Discussion

The literature review and the results of our studies indicate that Fusarium culmorum is pathogenic to winter wheat and produces the mycotoxin deoxynivalenol (DON) in the grain [21,41,42]. The results indicate differences in the response of wheat cultivars to stress caused by Fusarium culmorum inoculation. This is confirmed by changes in the properties of the starch and protein complex induced by Fusarium culmorum inoculation. The research hypothesis was tested through conducting a field experiment and laboratory tests. They included the inoculation of Fusarium culmorum, which, according to the literature, is the species that most commonly infects wheat in Polish weather conditions and produces the largest amounts of mycotoxins [29,43].

4.1. Mycotoxin Content in Grain of Winter Wheat Cultivars

The presented studies focused on the inoculation of Fusarium culmorum as it is a predominant species in Polish conditions and produces the most mycotoxins [29,44,45]. Bryła et al. [44] examined the levels of 26 mycotoxins in cereal grains in Poland and found the presence of DON in all tested wheat samples. The studies described different levels of DON depending on the crop year. For the infection of wheat by Fusarium ssp., the most crucial factors are the weather conditions during the flowering period and the initial ripeness of wheat (May and June). As shown in Table 2, the weather conditions in 2019 were not favorable for the development of Fusarium spp. The air temperature was higher in May and June, by 1.8 and 3.3 °C, respectively, compared to 2020. There were very large differences in the amount of precipitation—2019 was a very dry year. The amount of rainfall in 2020 in May and June was 26.6 mm and 150.8 mm higher, respectively, than in 2019. During the flowering period, it was too dry for the fungi to develop in the wheat grains even after artificial inoculation. Weather conditions caused the DON level to be very low in 2019, therefore only seeds from 2020 were used in our experiment.

The studies found different levels of DON depending on the cultivars. The lowest content of this mycotoxin was found in cv. ‘Sailor’, while the highest was in cv. ‘Muszelka’ and cv. ‘Legenda’. Selection of genotypes resistant to Fusarium head blight (FHB) is one of the methods used in wheat cultivation to reduce the occurrence of mycotoxins in wheat grain. Resistance to FHB is controlled by major and minor genes located on all wheat chromosomes, except for chromosome 7D [46]. The presented research results confirm the literature data indicating differences in the amounts of Fusarium mycotoxins in individual wheat cultivars grown under the same weather conditions [29,42,44]. The differences in the amount of mycotoxins in wheat grain may result from different types of resistance [47,48,49]. Therefore, there is not always a highly significant relationship between resistance to the Fusarium disease complex and the accumulation of mycotoxins. This was confirmed in our studies, as it was shown that cultivars with high resistance to FHB (cv. ‘Legenda’) had higher levels of mycotoxins than cultivars characterized by lower resistance (cv. ‘Sailor’). Most likely, these cultivars are characterized by resistance to the accumulation of Fusarium toxins in the grain. Studies by Siou et al. [50] report that the cropping of cultivars with a short vegetation period or early flowering allows for the avoidance or significant reduction of infection with fungi of the genus Fusarium and the accumulation of toxins. This relationship was not confirmed in the presented studies. It may have resulted from the fact that the difference in the earliness of cultivars was only 5 days. Another factor that is most likely the reason for the varying amounts of toxin accumulation is the structure and composition of grain. Walter et al. show that cell walls and the aleurone layer are significant physical barriers to the penetration of the fungus [51]. Literature data also indicate that other morphological features such as grain color, hulls, wax coating, and ferulic acid concentration affect the levels of mycotoxins in wheat grain [42].

The wheat microbiome plays a crucial role in limiting infections caused by Fusarium spp., which are responsible for diseases such as Fusarium head blight and root rot. The plant microbiota can influence plant health through various mechanisms, including competition with pathogens, induction of plant immunity, and the production of antimicrobial substances. Microorganisms in the wheat microbiome, particularly bacteria and fungi, can produce a range of antimicrobial compounds that inhibit pathogen growth. Some Bacillus strains produce lipopeptides that act as antimicrobial agents, inhibiting the development of Fusarium [52]. Other bacteria produce enzymes, such as chitinases, which can degrade the cell walls of Fusarium fungi, limiting their growth and increasing biodiversity within the microbiome. The biodiversity of the wheat microbiome may also play a key role in protecting against Fusarium infections. Studies have shown that plant microbiomes with higher biodiversity can improve plant health and enhance resistance to diseases caused by Fusarium [53,54]. The biodiversity of the wheat microbiome may also play a key role in protecting against Fusarium infections. Diverse microbial communities in the rhizosphere can collaborate to more effectively suppress pathogens.

4.2. Influence of Fusarium culmorum Infection on the Grain Quality and Rheological Properties of Dough

The technological value of wheat depends on the basic quality indicators, including, among others, the total protein content and its quality, the quantity and quality of gluten, the Zeleny sedimentation index, and the degree of starch damage [7,55]. To assess wheat flour quality more precisely, tests can be extended to include the rheological properties of the dough, which are performed using devices such as the farinograph, extensograph, and alveograph. Based on the data obtained from these tests, changes in the dough’s consistency and extensibility can be assessed. The viscosity of the flour suspension in water during heating is determined using an amylograph [56]. A comprehensive assessment of rheological properties requires the use of several devices. In the studies discussed, Mixolab was used to assess the quality of whole-grain wheat flour. This device allows for the simultaneous evaluation of both protein and starch properties [40,56].

The impact of wheat infection by fungi of the Fusarium genus and grain contamination with mycotoxins on technological value has been analyzed in only a few studies. Typically, the effect of Fusarium on individual quality features, such as nutrient content, protein content, gluten quantity and quality, Zeleny sedimentation index, or dough rheological properties, has been taken into account [21,57,58]. According to the literature, Fusarium infection of grains causes changes in the proportion of seed coat to endosperm, which results in an increase in ash content [11,12,13]. These relationships were confirmed in the present study and were visualized in PCA charts. In all cultivars, inoculation with Fusarium culmorum led to an increase in ash content. The highest increase was recorded in the cv. ‘Pokusa’ (0.44%), while the lowest was in the cv. ‘Tonacja’ (0.13%) and cv. ‘Legenda’ (0.11%).

Another aspect of the study was to identify wheat genotypes less susceptible to the reduction of thousand kernel weight (TKW) due to Fusarium infection. This is of significant importance for both yield size and quality, as TKW affects the milling and baking value of flour [15]. Cultivars with low TKW are characterized by low flour extraction due to disturbed proportions of endosperm to fruit-seed coat. While they have higher protein content, these proteins are not accumulated in the endosperm but in the fruit-seed coat, which is irrelevant for flour production. In the present study, the greatest reduction in TKW due to Fusarium inoculation was observed in the cv. ‘Legenda’, ‘Muszelka’, and ‘Tonacja’, which was proved in PCA charts. The results of these studies are consistent with those of Packa et al. [59], who found a reduction in TKW under Fusarium infection, with an average of 13% over several years.

Proteins stored in wheat grain are an important source of nutrients for humans and play a major role in determining bread baking quality [7]. The present research showed an increase in protein content after Fusarium culmorum inoculation, with an average increase of 1.1% percentage point across the cultivars (PCA chart). The highest increase, 3.5% compared to the control, was observed in the cv. ‘Legenda’, while the lowest increases were seen in the ‘Pokusa’ and ‘Muszelka’ cultivars. In the cv. ‘KWS Ozon’, the difference was not significant. The results suggest a significant change in one of the most important quality indicators [7]. These findings align with the literature, which indicates an increase in protein content due to the Fusarium contamination, as well as showing varietal differences [14,25]. The increase in protein content in the grain may be a response to stress, with plant cells synthesizing specific proteins and potentially altering their fractional and amino acid composition [60,61]. These proteins help mitigate the effects of stress by neutralizing its impact and protecting essential cellular structures and metabolic processes [62].

From the perspective of flour suitability for baking, both the quantity and quality of protein are important. The distinguishing factor determining both values is the sedimentation index. In the present studies, the sedimentation index was found to decrease by an average of 10.7% in cultivars subjected to Fusarium inoculation (PCA chart). Similar results were obtained by El Chami et al. [21]. Research by Gärtner et al. indicates varietal differences in this index, a finding confirmed in this work [14]. Kreuzberger et al. and Wang et al. obtained different results, suggesting no effect of Fusarium mycotoxins on the sedimentation index [17,18]. This discrepancy may be attributed to the selection of cultivars.

Based on the results, cultivars were classified into those where the sedimentation index was significantly reduced (cv. ‘KW Ozon’, cv. ‘Pokusa’), those with an increase (cv. ‘Legenda’), and those that showed no effect of Fusarium on the sedimentation index (cv. ‘Muszelka’ and cv. ‘Tonacja’). Changes in the sedimentation index reflect the impact of Fusarium on the balance between gliadins and glutenins that form gluten. Therefore, it can be concluded that, among the cultivars compared, ‘Muszelka’ and ‘Tonacja’ were more resistant to changes in protein quality due to Fusarium infection, as evidenced by the negligible change in gluten content in these cultivars.

In addition to determining the effect of Fusarium culmorum inoculation on the qualitative characteristics of grain, the effect on the quality of starch–protein complex was also examined. Laboratory tests were performed using Mixolab, which allowed for a comprehensive assessment of the experimental factors’ impact on the baking value and dough quality. It is worth noting that the quality was assessed based on the quality parameters of the starch and protein complex.

According to Gärtner et al., flour obtained from wheat cultivars containing mycotoxins, compared to flours from uncontaminated cultivars, is characterized by reduced baking quality parameters such as water absorption and dough softening [14]. Similar results were obtained by Horvat et al., who compared the rheological properties of dough from contaminated and uncontaminated flours [26]. They found a reduction in water absorption by 1.6%, dough development time by 17%, and quality number by 19.9%. The authors also showed that Fusarium contamination reduced dough energy, maximum tensile strength, and the strength-to-extensibility ratio by 57.7%. Additionally, they observed a negative effect on the specific volume of bread. Similarly, the presented studies demonstrated a negative effect of Fusarium on flour and dough properties. Water absorption of flours from inoculated cultivars increased by an average of 3.6% compared to uninfected grain, and dough development time was shortened by an average of 44.5%. A decrease in dough stability was also observed, with an average reduction of 34.4%, alongside changes in dough resistance to kneading depending on the cultivars. These changes were likely caused by proteolytic enzymes of Fusarium, which remain inactive in the grain but can be activated during dough kneading [18,19]. In the research by Peršić et al. [63], FHB infection did not affect the water absorption of wheat flour from either location. However, FHB infection caused the most significant increase in dough softening, by over 400% compared to control samples in one of the locations, suggesting significant genotypic variability.

The results also indicate a negative effect of Fusarium on starch properties, particularly the swelling of starch granules and the rate of enzymatic degradation. Changes in starch properties under the influence of Fusarium have been confirmed in the literature [20,21,22]. The presented study found that the effect of Fusarium on starch degradation varied depending on the genotype. The β index, which characterizes the increase in dough resistance due to starch granule swelling, decreased significantly in all analyzed wheat cultivars. In contrast, the γ index, indicating the high amylolytic enzyme activity, varied between the cultivars. The greatest decrease in the γ index, indicating high amylolytic enzyme activity, was found in cv. ‘KWS Ozon’, the cultivar with the highest cumulative level of DON. As a result, the β index, indicating a reduction in dough resistance, was also reduced. Similar conclusions were drawn by Gärtner et al. and Wang et al. [14,18].

5. Conclusions

The effect of Fusarium culmorum inoculation on the grain quality, starch properties, and protein complex was investigated, resulting in changes in flour properties and dough rheological characteristics. Fusarium culmorum inoculation led to an increase in protein, gluten, and ash content, a decrease in the sedimentation index, an increase in flour water absorption, as well as changes in dough rheological properties: a shorter dough development time, reduced dough stability time, decreased dough resistance to mixing, and reduced dough consistency. Inoculation caused changes in starch properties, as indicated by the determination of the β slope parameter in Mixolab (the rate of enzymatic starch decomposition) and the γ slope (starch gelatinization). It also led to a weakening of dough consistency. The observed interaction between Fusarium culmorum inoculation and the wheat cultivars in shaping the qualitative parameters and rheological properties of the dough indicates that some wheat cultivars include less susceptible genotypes, which do not exhibit any changes as a result of Fusarium infection.

Analyses of the technological value of grain showed that there were no significant differences between cv. ‘KWS Ozon’, cv. ‘Tonacja’, and cv. ‘Muszelka’ in terms of protein content, gluten content, and Zeleny sedimentation index. It was shown that, in the case of ‘Pokusa’ cv., the effect of Fusarium inoculation was not significant in contrast to ‘KWS Ozon’ cv., where a significant effect on starch–protein complex was observed.

The studies have shown that, under the same environmental and cultivation conditions, different cultivars accumulate various amounts of mycotoxins. From a scientific perspective, it is important to further investigation the underlying causes of this variability. The ‘Pokusa’ and ‘Sailor’ cultivars may serve as a valuable source of resistance to mycotoxin accumulation in grain, as they exhibited the lowest contamination with DON among all tested cultivars.

A review of the literature suggests that the cultivar’s microbiome may play a key role in this process, therefore, future research should consider this aspect.