Introduction

More than 50% of global daily caloric intake is derived directly from cereal crop consumption, especially rice and wheat (Awika 2011). Wheat is the most important source of protein and food calories at the global level, and it is part of many food products, such as bread, pasta, pastries, noodles, semolina, bulgur or couscous. It is also the food crop that covers the largest share of the global crop area (about 14%) and has the largest share in global food trade. With a production of 752 Mt in the base period (OECD/FAO 2020), wheat is the second most produced cereal after maize.

Fusarium head blight (FHB) belongs to the most damaging diseases of cereals, particularly in years with intensive rainfall. In Europe, Fusarium graminearum Schwabe, F. culmorum (W.G. Smith) Sacc., F. poae (Peck) Wollenw, and F. avenaceum (Fr.) Sacc. are the most common fungal species responsible for FHB, but the proportional species composition depends on the country and period investigated (Covarelli et al. 2015). The fungus infects the spikes during and shortly after anthesis and this can lead to significant reduction in grain yield in the major wheat production regions (Bai and Shaner 2004). Fusarium fungi can infect the inflorescence structure of small grain cereals under favorable environmental conditions, such as high humidity. Both Fusarium ascospores and macroconidia can cause FHB, penetrating the cell wall within 1–2 days post-germination. Over the course of infection, the fungus produces trichothecene toxins that are secreted from the mycelial tip (Kang and Buchanauer 1999). These toxins are involved in causing necrosis of infected tissues, and have been identified as important factors of aggressiveness (Maier et al. 2006; Proctor et al. 1995). Crop losses due to FHB represent a significant problem worldwide (Mesterházy et al. 2020). FHB reduces yield and quality of grains, as it is manifested in their weight loss, carbohydrate and protein composition changes and the presence of fungal toxins (Magliano and Kikot 2013). It can destroy starch granules, storage proteins, and grain cell wall and subsequently affect the quality of wheat flour (Dexter et al. 2003). Additional losses of wheat production can also be attributed to the contamination of grains by deoxynivalenol (DON) and other trichothecenes produced by these species (Shah et al. 2018).

Use of resistant cultivars, from an economical point of view, is the most effective control method for plant diseases (Španić et al. 2017). FHB resistance is polygenic and its expression is highly influenced by the environment. The following components (types) of head blight resistance were distinguished by Mesterházy (1995): I. Resistance to invasion, II. Resistance to spreading, III. Resistance to kernel infection, IV. Tolerance, V. Resistance to toxin accumulation, VI. Resistance to late blighting, and VII. Resistance to head death above infection site. The FHB resistance associated with plant physiological and morphological characteristics is also called passive resistance (Mesterházy 1995). Plant defences consist of physical barriers such as the cell wall and its modifications as well as chemical defence mechanisms that are induced in response to external stimuli. Many naturally occurring secondary metabolites in plants are involved in resistance mechanisms against FHB. The majority of these are phenolic compounds with antioxidant properties. Wheat contains significant amount of phenolic acids in free, conjugated, and bound forms such as flavonoids, alkylresorcinols, benzoxazinoids, phytohormones, and volatile organic compounds (Chrpová et al. 2021a, b).

These substances are accumulated in aleurone, pericarp or endosperm of wheat and may influence the colour of the grain (blue, purple, and yellow, respectively). Purple colour is restricted to the pericarp of grain whereas blue colour to the aleurone. The yellow colour is caused by carotenoids and blue and purple colour is produced by anthocyanins (Garg et al. 2016). Anthocyanins represent the major red, purple, and blue pigments in many flowers, fruits, vegetables, and cereals (Cappellini et al. 2021). There are several reviews of epidemiological and preclinical intervention studies reporting the beneficial effects of anthocyanins on health (He and Giusti 2010; Kozłowska and Dzierżanowski 2021; Li et al. 2017; Mattioli et al. 2020; Pojer et al. 2013; Salehi et al. 2020; Tsuda et al. 2006; Wallace 2014; Wallace and Giusti 2015).

Red grain colour is common in most American and European wheats. It is controlled by one to three alleles of the three dominant R-1 homologous genes located on the long arms of chromosomes 3A (R-A1), 3B (R-B1), and 3D (R-D1). The degree of red colour is additive, the intensity depending on the number of R alleles, the alleles conferring red colour are designated R-A1b, R-B1b, and R-D1b (McIntosh et al. 1998). By contrast, white grain colour is determined by the combination of three recessive alleles at R-A1a, R-B1a, and R-D1a (Himi and Noda 2005; Sherman et al. 2008). The red pigment of the grain coat is formed from catechins and proanthocyanidins, which are synthesized via the flavonoid biosynthetic pathway (Wang et al. 2016).

The best strategy for controlling FHB is to breed new cultivars with reduced susceptibility. Coloured-grain wheats are a potentially great alimentary source of other health enhancing compounds such as carotenoids, phlobaphenes, phenolic acids, anthocyanins, and other polyphenols. However, information on the response of wheats with coloured-grains to Fusarium infection is still scant. The aim of this research was to study the effect of high infection pressure of Fusarium culmorum on wheat cultivars with different grain colour.

Materials and methods

Plant material

Material for this study comprised 19 spring conventional red cultivars (CR) that were registered in the Czech Republic during 2004–2018 and commercially utilized and 25 coloured-grain wheat cultivars and lines (Table 1, Supplement Table 1.): 10 × blue aleurone (BA), 6 × purple pericarp (PP), 7 × red grain (RG), 2 × white grain (WG). They are potential sources of nutritional quality that are part of a collection of coloured wheats from different origins. Description of registered cultivars is available in National List & Plant Variety Rights Database (2023). The Chinese cultivar Sumai 3 globally used as a source of FHB Type II resistance was utilized as resistant check (Del Blanco et al. 2003; Gunnaiah and Kushalappa 2014; Ittu et al. 2008).

Table 1 List of the tested conventional red wheat cultivars and the tested cultivars and lines of coloured-grain wheat
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Field experiment

The field experiment was performed at the Crop Research Institute in Prague in the Czech Republic (50°05′03.3″ N, 14°18′15.4″ E). Each genotype was grown in hill plots in two variants (infected and control) in three replications. To determine resistance, three-year experiments with three biological repetitions were used (2016–2018). To minimize year/location effects on results, it was necessary in these conditions to suppurt disease development by irrigation of plots. The meteorological conditions during the growing season were monitored with the meteorological station of Crop Research Institute in Czech Republic and the data are presented in Fig. 1.

Fig. 1
figure 1

Average air temperature (A) and photosynthetically active radiation (B) during anthesis in years 2016, 2017 and 2018

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Evaluation of resistance against FHB

The inoculation of spikes with highly pathogenic isolate B of Fusarium culmorum (Chrpová et al. 2007) was performed at the phase of full flowering. Inoculum (conidial suspension 0.8 × 107 ml−1) was applied by spraying onto bunches of 10 flowering spikes randomly selected within hill plots. Inoculated spikes were then kept for 24 h in polyethene bags to achieve 100% humidity during 24 h after inoculation (Mesterházy 1997). Head blight symptoms were evaluated in three moments (usually 14th, 21st and 28th day after inoculation) on a 1–9 scale, where 1 < 5%, 2 = 5–17%, 3 = 18–30%, 4 = 31–43%, 5 = 44–56%, 6 = 57–69%, 7 = 70–82%, 8 = 83–95% and 9 > 95% of the spikelets with FHB symptoms (Chrpová et al. 2013). Visual symptom score (VSS) screened 28 days after infection was used for the final evaluation. Fusarium damaged (scabby) kernels (FDK) were calculated as percentage by total seed number. Tolerance to the disease (reduction of grain weight per spike GWS-R) was expressed as percent reduction from non-inoculated control in the grain weight per spike.

Plant height was evaluated according to the Methodology of Wheat Utility Value Tests, which is used by Central Institute for Supervising and Testing in Agriculture in the Czech Republic. Plants are measured at points representing the average length of the plants, at a distance of at least 1 m from the front of the experimental plot, at the time after flowering, from the base of the plant to the top of the ear (without awns). In non-stunted stands, one measurement shall be taken for each repetition. In flattened and uneven stands, the plants should be straightened and the length measured on three to five plants.

Determination of DON

Seeds from infected spikes were analysed for DON (deoxynivalenol) content determined by ELISA with the use of RIDASCREEN® FAST DON kits from R- Biopharm GmbH, Darmstadt, Germany. The detailed description of methods for the determination of DON content was provided in Chrpová et al. (2007).

Statistical analyses

The data were analysed using the statistical software STATISTICA 14.0.0.15 (RRID:SCR_014213). A general factorial ANOVA (analysis of variance) for DON, VSS, FDK and GWS-R with two independent factors (grain colour and year) was performed. The interaction between grain colour and year was included in the statistical model. If the interaction of grain colour by year was significant (p-value is lower than 0.05) and the effect of grain colour was also significant, multiple comparisons (Scheffé's test) were carried out between the coloured cultivar groups individually for each year. If neither the interaction between grain colour and year nor grain colour is significant (p-value higher than 0.05), multiple comparisons are carried out between groups of varieties on the basis of their means. The Scheffé's test for multiple comparisons was used because it is appropriate when comparing groups of different ranges, as is the case with the data. The significantly different mean values were represented by different letters. The relationship between DON, plant height, VSS, FDK, and GWS-R was described by Spearman’s rank correlation coefficient.

Results

Classification of cultivar resistance on the basis of four resistance traits

Wheat cultivars/lines with different grain colour/origin were screened for response to inoculation with Fusarium culmorum. Great attention has been paid to the DON content, which is considered to be the most important character. These studies also involved the importance of determination of symptoms (VSS), percentage of Fusarium damaged kernels (FDK) and reduction of grain weight per spike (GWS-R). The lowest mean level of DON content was found in the resistant check cultivar Sumai 3 and in the cultivars/lines Rufia, Vánek, ANBW 6A, ANK-28A etc. (Table 2). The highest mean level of DON content was found in the line ANDW20A/4A and in the conventional red cultivars (CR) Kitri, Kabot, Cornetto, KWS Mairra, Libertina etc.

Table 2 Mean values for DON (deoxynivalenol) content, visual symptom scores (VSS), % of Fusarium damaged (scabby) kernels (FDK), and reduction of grain weight per spike (GWS-R)
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A general factorial ANOVA results showed statistically significant interactions at 5% p-value between grain colour and year for all the variables: DON, VSS, FDK, GWS-R (Table 3). Therefore, the data were also analysed separately for each year (Table 3). The coloured cultivar/line groups differed significantly in DON content in the years 2016 and 2018, but not in 2017 (Tables 3, 4). Red grain materials (RG) were found to have the lowest DON levels of all the groups studied in 2016 (13.241 mg kg−1) and 2017 (21.209 mg kg−1). However, in 2018, the DON level of red cultivars/lines increased to 34.871 mg kg−1, which was the highest among the colour groups compared to the conventional red cultivars, which had the highest DON level (53.438 mg kg−1). The cultivars/lines with purple pericarp (PP) had the most constant DON level in all three years (19.126–24.999 mg kg−1). DON value is considered as the most important trait evaluated; the values of all traits are given in the Table 4. There were found no significant differences in the reduction of grain weight per spike (GWS-R) among coloured groups.

Table 3 The ANOVA results for the effect of the year and different traits between groups of spring wheat cultivars/lines with different grain colour inoculated with Fusarium culmorum
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Table 4 Multiple comparisons (Scheffé's test) between groups of spring wheat cultivars/lines with different grain colour inoculated with Fusarium culmorum for different traits (2016–2018)
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Relationship between the examined traits

Spearman’s rank correlation between individual traits was statistically significant for all trait combinations except VSS × heigh and GWS-R × heigh (Table 5). The closest relationship was detected between DON content and FDK (0.835). The negative significant correlation was found out for DON x plant height (−0.222) and FDK × plant height (−0.278).

Table 5 Correlation analysis of following parametres, plant height, visual symptom score, Fusarium damaged kernels, deoxynivalenol and reduction of grain weight per spike
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However, certain differences in the type of resistance were indicated. Some genotypes (e.g. Vánek) showing relatively lower accumulation of DON expressed a higher resistance level also in all other traits, but the cultivars Rufia or ANK-1E, producing low amounts of DON, displayed relatively high yield reduction. On the contrary, the cultivar Kabot showed susceptibility to accumulation of DON and rather medium yield tolerance to disease.

Discussion

Impacts of grain colour

Several studies have reported the effects of secondary metabolites in cereals. Among the secondary metabolites of cereal kernels with antioxidant properties, carotenoids, peptides, and especially phenolic compounds have been studied for their efficiency to reduce mycotoxin biosynthesis (Chen et al. 2006; Norton 1997). Anthocyanins have a protective role under extreme conditions, as they prevent lipid oxidation and protect the plasma membrane from damage (Pervaiz et al. 2017; Shoeva et al. 2017). Anthocyanins are present in common wheat in a certain amount as a result of multiple and independent gene transfers from other Triticum species or wild relatives (Lachman et al. 2017). Guo et al. (2011) stated that coloured-grain wheat is one kind of new germplasm resource in cereal crops, some of which are rich in beneficial anthocyanins. It has been reported that high contents of total anthocyanins are present especially in wheat with purple pericarp (Gozzi et al. 2023; Syed Jaafar et al. 2013).

The finding of the significant interaction between grain colour and year means that the benefits of the grain colour effect are different in each year. Žofajová et al. (2017) found that anthocyanin content in 17 colour winter wheat varieties and genotypes was influenced by weather conditions of experimental years and their interaction. The higher temperature and unequal distribution of precipitation caused reduction in anthocyanin content.

Martini et al. (2015) reported a study carried out to investigate the effects of genetic and environmental factors on the total antioxidant activity and on the occurrence of certain antioxidant compounds in durum wheat. The authors highlighted that the occurrence of antioxidant compounds and thereby the level of the total antioxidant activity are significantly affected by genotype, growing area and crop year. The results of the study noted that the yellow coloured pigments and the level of total antioxidant capacity are mainly affected by genetic factors, differently from the content of phenolic acids and of total phenolics, which appears to be mostly affected by the environment and secondly by the genotype, to an extent which varies for the 3 forms. The effect of genotype is more prominent in the bound form of both the content of phenolic acids and of total phenolics and much less in the conjugated and free forms.

The statistically significant interactions between grain colour and year were also found in our study for all the variables evaluated. Wheat cultivars and lines with purple pericarp showed higher resistance to FHB similar to genotypes with red grain that were of Japanese or Russian origin. However, none of these genotypes with coloured-grain reached the level of the control variety Sumai 3 either in resistance to DON accumulation or in overall resistance to FHB. It is not without interest that anthocyanins are also deemed as resistance-related metabolites in wheat variety Sumai-3 (Bolina et al. 2011; Gunnaiah et al. 2014). The anthocyanin composition of blue aleurone wheat is reported to be relatively simple in relation to the number of different anthocyanins while wheat with purple pericarp had more different anthocyanin glycosides (Abdel-Aal et al. 2016). In our study, the group of genotypes with blue grain showed a higher DON content and % FDK compared to the other groups of genotypes with coloured-grain, and the ANDW 20A/4A line with blue aleurone and reduced height had the highest DON content of the entire set of genotypes. This finding is in accordance with our previous results, when the cultivars of winter wheat with blue aleurone layer Skorpion (Martinek et al. 2013) and AF Oxana (Chrpová—personal communication) were found to be susceptible to FHB. Also the study of Gozzi (2023) highlighted a remarkable susceptibility of blue-pigmented wheat cultivars and breeding lines to mycotoxin contamination, compared to genotypes with different anthocyanin content and histological placement in the grain. These authors bring forward a possible explanation that in blue-grained wheats, anthocyanins are accumulated in the inner aleurone layer, whereas in purple types, these secondary metabolites are concentrated directly in the pericarp that is the outer layer directly exposed to the first contact with Fusarium.

It is also possible that the intensity of FHB in genotypes with blue grain aleurone is also dependent on the individual Ba alleles. For the Ba1 allele, which comes from the donor UC66049, a whole chromosome arm translocation from Thinopyrum ponticum (Podp.), replacing the long arm of chromosome 4B (Qualset et al. 2005). Zeller (1991) found that the pair of chromosome 4A in blue-grained T. aestivum replaced with chromosome 4Am of Triticum boeoticum, in several European common wheat strains. This gene for blue grained wheat line is designated as Ba2. T. aestivum Xiao Yan, the donor of Ba3, is a translocation line from Lophopyrum ponticum (Pop.) replacing the long arm of chromosome 4D and a part of short arm of chromosome 4D (Burešová et al. 2014; Jeewani et al. 2021). It should be noted that Lophopyrum ponticum (Pop.) is a synonym of Thinopurum ponticum (Pod.).

From the results obtained (correlation, ANOVA), it is clear that, among the observed characteristics associated with resistance to Fusarium head blight (FHB), it is mainly the content of DON and FDK that are related to the colour of the grain. FHB resistance is polygenic and its expression is strongly influenced by the environment. For example, Czaban et al. (2011) confirmed that the spread of FHB and the infestation of winter wheat grains by fungi of the genus Fusarium are mainly dependent on weather factors. Therefore, the effect of individual years was evaluated separately.

In our study, the coloured cultivars showed a high degree of instability in the traits studied in each year, but it can still be said that they have a high potential. It makes sense to learn more about grain colour cultivars/lines and many genotypes have advantages over conventional cultivars. Purple and red materials seem to be the most promising, but it is always better to evaluate each genotype separately.

The influence of other morphological characters

It should be stated that large differences in plant height were found between evaluated varieties and lines. Plant height can play a role in “disease escape” from natural infection by Fusarium spores from the ground (Mesterházy 1995), the effect of plant height is known to occur also in artificial infection experiments (Buerstmayr and Buerstmayr 2016; He et al. 2016). A number of authors, e.g. Jones et al. (2018), Tessmann and Van Sanford (2019), Chrpová et al. (2021a, b) conclude a direct or indirect effect on FHB severity of plant height per se via morphological and structural differences (e.g., reduced peduncle length) and thereby changes in the canopy microclimate. Another possible reason explaining why shorter plants tend to be more susceptible to FHB is that genes reducing plant height could be positioned in a proximity of those conferring a susceptibility to the disease. This possibility is based on findings showing that several QTLs conferring FHB resistances have been co-located with reduced height (Rht) loci (Gervais et al. 2003; Paillard et al. 2004; Schmolke et al. 2005; Srinivasachary et al. 2009). The correlation between DON content and plant height was found also in our study to be statistically significant. Therefore, this influence on the manifestation of resistance of some cultivars or lines cannot be excluded.

Impact of climatic conditions

Several studies have investigated the effect of environmental conditions on the development of FHB and DON contamination. Mycotoxin production is genetically regulated in response to environmental conditions (Holliger and Ekperigin 1999). Temperature and water activity (aW) are the primary environmental factors that influence growth and mycotoxin production by several Fusarium species. In our study, the highest average DON content was found in 2018 (38.6 mg kg−1) and then in 2017 (29.0 mg kg−1). On the contrary, in 2016 the DON content was only 24.4 mg kg−1. In the case of our study, where irrigation is used, the temperature and the intensity of solar radiation apparently played a role. In the years with more suitable conditions for the development of infection, the DON content even exceeded 100 mg kg−1 in susceptible varieties such as Kabot, Corneto, etc. On the contrary, resistant varieties such as Sumai 3, Vánek and Rufia maintained low DON values even under conditions that stimulated the pathogen and promoted the accumulation of mycotoxins.

Coloured-grain wheat as a potential dietary source?

Coloured wheat also represents an alternative to the cultivation of common wheat because of its nutritional value. Coloured-grain wheats are a potentially dietary source of other health enhancing compounds such as anthocyanins (Lachman et al. 2017), tocols (Lachman et al. 2018), and phenolic acids (Paznocht et al. 2020). Whole-wheat flour is a good source of dietary fibre and antioxidants, which can promote health benefits toward several chronic diseases usually associated with oxidative stress (Yu 2008). However, the presence of natural contaminants (mycotoxins) in the most external layers poses a risk for consumer safety and need to be taken into serious consideration (Zanoletti et al. 2017). Anthocyanins occur mainly in the surface layers of grain (pericarp), therefore it seems advantageous that some genotypes with coloured grain show lower DON accumulation and therefore whole grain milling may be recommended. Encouragingly, a spring wheat cultivar Rufia with purple pericarp was registered in 2021.

Even among conventional red wheat cultivars, materials with a higher level of resistance were found (Table 2). Resistance against FHB was confirmed in a standard cultivar Vánek used as moderately resistant check in FHB testing of spring wheat in the Czech Republic. Cultivar Vánek with elite (E) bread quality is adapted to environmental conditions of Centrale Europe and possesses many other desirable characteristics (Chrpová et al. 2013).

Wheat breeders are currently attempting to develop new types of coloured-grain wheat with improved properties including quality, yield and higher pigment contents with potential beneficial effects on human nutrition and health (Martinek et al. 2013). Several authors (Etzerodt et al. 2016; Španić et al. 2017) reported that differences in the antioxidant response of wheat cultivars or metabolite profiling can be a valuable marker for the selection of FHB resistance.

Conclusion

Results of our study provide interesting data that could be used to select cultivars for genetic studies aimed at finding genetic associations between grain colour and Fusarium head blight resistance or to select cultivars as resistance sources in breeding. Statistically significant interactions were found between grain colour and year for DON, considered to be the most important character, and also for other evaluated variables (VSS, FDK, GWS-R). Red grain materials had the lowest DON levels in 2016 and 2017, but not in 2018. Cultivars/lines with purple pericarp had the most constant and second lowest DON levels in all three years. Rufia and ANK-28A (materials with purple pericarp) with ANK-1E and ANK-1B (materials with red grain) had low levels of DON. However, relatively high resistance to FHB was also found in some conventional red cultivars (e.g. cultivar Vánek). Further studies are needed to elucidate the potential role of secondary metabolites with antioxidant properties in resistance to FHB.