Effects of Bread Making and Wheat Germ Addition on the Natural Deoxynivalenol Content in Bread

Deoxynivalenol (DON, vomitoxin) is a type-B trichothecene mycotoxin produced by several field fungi such as Fusarium graminearum and Fusarium culmorum and known to have various toxic effects. This study investigated the effect of the bread making process on the stability of DON in common bread and wheat germ-enriched bread using naturally contaminated ingredients at the level of 560 µg/kg. The concentration of DON and its evolution during bread making were determined by immunoaffinity column cleanup followed by liquid chromatography with diode array detection (HPLC-DAD). During the bread making process, DON was reduced by 2.1% after fermentation and dropped by 7.1% after baking, reaching a maximum reduction of 19.8% in the crust as compared with a decrease of 5.6% in the crumb. The addition of 15% wheat germ to the dough did not affect DON stability during bread making, showing an apparent increase of 3.5% after fermentation and a reduction by 10.2% after baking.     

Deoxynivalenol and Oxidative Stress Indicators in Winter Wheat Inoculated with Fusarium graminearum

This study comprises analyses of contents of mycotoxins, such as deoxynivalenol and zearalenone, as well as the level of oxidative stress in ears of a susceptible wheat cultivar Hanseat and cv. Arina, resistant to a pathogenic fungus Fusarium graminearum. Starting from 48 h after inoculation, a marked increase was observed in the contents of these mycotoxins in ears of wheat; however, the greatest accumulation was recorded in the late period after inoculation, i.e., during development of disease. Up to 120 h after inoculation, in ears of both wheat cultivars, the level of deoxynivalenol was higher than that of zearalenone. The susceptible cultivar was characterized by a much greater accumulation of deoxynivalenol than the resistant cultivar. At the same time, in this cultivar, in the time from 0 to 72 h after inoculation, a marked post-infection increase was observed in the generation of the superoxide radical (O₂^•−). Additionally, its level, at all the time points after inoculation, was higher than in the control. In wheat cv. Arina, a markedly higher level of O₂^•− generation in relation to the control was found up to two hours after inoculation and, next, at a later time after inoculation. In turn, the level of semiquinone radicals detected by electron paramagnetic resonance (EPR) increased at later culture times, both in cv. Hanseat and Arina; however, in infested ears of wheat, it was generally lower than in the control. Analysis of disease symptoms revealed the presence of more extensive lesions in ears of a susceptible wheat cv. Hanseat than resistant cv. Arina. Additionally, ergosterol level as a fungal growth indicator was higher in ears of susceptible wheat than in the resistant cultivar.     

Combination of Bacillus velezensis RC218 and Chitosan to Control Fusarium Head Blight on Bread and Durum Wheat under Greenhouse and Field Conditions

Fusarium graminearum sensu stricto is, worldwide, the main causal agent of Fusarium head blight in small cereal crops such as wheat, barley, and oat. The pathogen causes not only reductions in yield and grain quality but also contamination with type-B trichothecenes such as deoxynivalenol. Prevention strategies include the use of less susceptible cultivars through breeding programs, cultural practices, crop rotation, fungicide application, or a combination of them through an integrated pest management. Additionally, the use of more eco-friendly strategies by the evaluation of microorganisms and natural products is increasing. The effect of combining Bacillus velezensis RC218 and chitosan on Fusarium Head Blight (FHB) and deoxynivalenol accumulation under greenhouse and field conditions in bread and durum wheat was evaluated. Under greenhouse conditions, both B. velezensis RC218 and chitosan (0.1%) demonstrated FHB control, diminishing the severity by 38 and 27%, respectively, while the combined treatment resulted in an increased reduction of 54% on bread wheat. Field trials on bread wheat showed a biocontrol reduction in FHB by 18 to 53%, and chitosan was effective only during the first year (48% reduction); surprisingly, the combination of these active principles allowed the control of FHB disease severity by 39 and 36.7% during the two harvest seasons evaluated (2017/18, 2018/19). On durum wheat, the combined treatment showed a 54.3% disease severity reduction. A reduction in DON accumulation in harvested grains was observed for either bacteria, chitosan, or their combination, with reductions of 50.3, 68, and 64.5%, respectively, versus the control.     

Transformation of Selected Fusarium Toxins and Their Masked Forms during Malting of Various Cultivars of Wheat

This study investigated the impact of malting of six wheat cultivars inoculated with Fusarium culmorum on the dynamics of content changes of selected Fusarium toxins. The grains of all the tested cultivars showed a high content of deoxynivalenol (DON), zearalenone (ZEN), and their derivatives, whereas nivalenol (NIV) and its glucoside were found only in the Legenda cultivar. Our experiments confirmed that the malting process of wheat grain enables the secondary growth of Fusarium, and mycotoxin biosynthesis. The levels of toxins in malt were few-fold higher than those in grain; an especially high increase was noted in the case of ZEN and its sulfate as the optimal temperature and pH conditions for the biosynthesis of these toxins by the pathogen are similar to those used in the grain malting process. This is the first paper reporting that during the malting process, biosynthesis of ZEN sulfate occurs, instead of glycosylation, which is a typical modification of mycotoxins by plant detoxication enzymes.     

Relationship of Deoxynivalenol Content in Grain,Chaff, and Straw with Fusarium Head Blight Severity in Wheat Varieties with Various Levels of Resistance

A total of 122 wheat varieties obtained from the Nordic Genetic Resource Center were infected artificially with an aggressive Fusariumasiaticum strain in a field experiment. We calculated the severity of Fusarium head blight (FHB) and determined the deoxynivalenol (DON) content of wheat grain, straw and glumes. We found DON contamination levels to be highest in the glumes, intermediate in the straw, and lowest in the grain in most samples. The DON contamination levels did not increase consistently with increased FHB incidence. The DON levels in the wheat varieties with high FHB resistance were not necessarily low, and those in the wheat varieties with high FHB sensitivity were not necessarily high. We selected 50 wheat genotypes with reduced DON content for future research. This study will be helpful in breeding new wheat varieties with low levels of DON accumulation.     

Incentives to Stimulate European Wheat Farmers to Adapt Their Fusarium Species Mycotoxin Management

Fusarium species infection in wheat can lead to Fusarium Head Blight (FHB) and contamination with mycotoxins. To fully exploit more recent insights into FHB and mycotoxin management, farmers might need to adapt their agronomic management, which can be stimulated through incentives. This study aimed to identify incentives to stimulate European farmers to adapt their agronomic management to reduce FHB and related mycotoxins in wheat. A questionnaire was distributed among 224 wheat farmers from Italy, the Netherlands, Serbia, and the United Kingdom. Using the respondents’ data, Bayesian Network modelling was applied to estimate the probability that farmers would adapt their current agronomic management under eight different incentives given the conditions set by their farm and farmer characteristics. Results show that most farmers would adapt their current agronomic management under the incentives “paid extra when wheat contains low levels of mycotoxins” and “wheat is tested for the presence of mycotoxins for free”. The most effective incentive depended on farm and farmer characteristics, such as country, crop type, size of arable land, soil type, education, and mycotoxin knowledge. Insights into the farmer characteristics related to incentives can help stakeholders in the wheat supply chain, such as farmer cooperatives and the government, to design tailor-made incentive plans.     

Pydiflumetofen Co-Formulated with Prothioconazole: A Novel Fungicide for Fusarium Head Blight and Deoxynivalenol Control

Fusarium head blight (FHB) is an important disease of small grain cereals worldwide, resulting in reduced yield and quality as well as the contamination of harvested grains with mycotoxins. The key mycotoxin of concern is deoxynivalenol (DON), which has legislative and advisory limits in numerous countries. Cereal growers have a number of control options for FHB including rotation, cultivation, and varietal resistance; however, growers are still reliant on fungicides applied at flowering as part of an IPM program. Fungicides currently available to control FHB are largely restricted to triazole chemistry. This study conducted three field experiments to compare a new co-formulation of pydiflumetofen (a succinate dehydrogenase inhibitor (SDHI) with the tradename ADEPIDYN^™) and prothioconazole (a triazole) against current standard fungicides at various timings (flag leaf fully emerged, mid-head emergence, early flowering, and late flowering) for the control of FHB and DON. Overall, the co-formulation showed greater efficacy compared to either pydiflumetofen alone or current fungicide chemistry. This greater activity was demonstrated over a wide range of spray timings (flag leaf fully emerged to late flowering). The availability of an SDHI with good activity against FHB and the resulting DON contamination of harvested grain will give growers an additional tool within an IPM program that will provide a greater flexibility of spray application windows and reduce fungicide resistance selection pressure.     

Bioprospecting Phenols as Inhibitors of Trichothecene-Producing Fusarium: Sustainable Approaches to the Management of Wheat Pathogens

Fusarium spp. are ubiquitous fungi able to cause Fusarium head blight and Fusarium foot and root rot on wheat. Among relevant pathogenic species, Fusarium graminearum and Fusarium culmorum cause significant yield and quality loss and result in contamination of the grain with mycotoxins, mainly type B trichothecenes, which are a major health concern for humans and animals. Phenolic compounds of natural origin are being increasingly explored as fungicides on those pathogens. This review summarizes recent research activities related to the antifungal and anti-mycotoxigenic activity of natural phenolic compounds against Fusarium, including studies into the mechanisms of action of major exogenous phenolic inhibitors, their structure-activity interaction, and the combined effect of these compounds with other natural products or with conventional fungicides in mycotoxin modulation. The role of high-throughput analysis tools to decipher key signaling molecules able to modulate the production of mycotoxins and the development of sustainable formulations enhancing potential inhibitors’ efficacy are also discussed.     

Rapid Analysis of Deoxynivalenol in Durum Wheat by FT-NIR Spectroscopy

Fourier-transform-near infrared (FT-NIR) spectroscopy has been used to develop quantitative and classification models for the prediction of deoxynivalenol (DON) levels in durum wheat samples. Partial least-squares (PLS) regression analysis was used to determine DON in wheat samples in the range of <50–16,000 µg/kg DON. The model displayed a large root mean square error of prediction value (1,977 µg/kg) as compared to the EU maximum limit for DON in unprocessed durum wheat (i.e., 1,750 µg/kg), thus making the PLS approach unsuitable for quantitative prediction of DON in durum wheat. Linear discriminant analysis (LDA) was successfully used to differentiate wheat samples based on their DON content. A first approach used LDA to group wheat samples into three classes: A (DON ≤ 1,000 µg/kg), B (1,000 < DON ≤ 2,500 µg/kg), and C (DON > 2,500 µg/kg) (LDA I). A second approach was used to discriminate highly contaminated wheat samples based on three different cut-off limits, namely 1,000 (LDA II), 1,200 (LDA III) and 1,400 µg/kg DON (LDA IV). The overall classification and false compliant rates for the three models were 75%–90% and 3%–7%, respectively, with model LDA IV using a cut-off of 1,400 µg/kg fulfilling the requirement of the European official guidelines for screening methods. These findings confirmed the suitability of FT-NIR to screen a large number of wheat samples for DON contamination and to verify the compliance with EU regulation.     

Sequence Divergence of the Enniatin Synthase Gene in Relation to Production of Beauvericin and Enniatins in Fusarium Species

Beauvericin (BEA) and enniatins (ENNs) are cyclic peptide mycotoxins produced by a wide range of fungal species, including pathogenic Fusaria. Amounts of BEA and ENNs were quantified in individual rice cultures of 58 Fusarium strains belonging to 20 species, originating from different host plant species and different geographical localities. The species identification of all strains was done on the basis of the tef-1α gene sequence. The main aim of this study was to analyze the variability of the esyn1 gene encoding the enniatin synthase, the essential enzyme of this metabolic pathway, among the BEA- and ENNs-producing genotypes. The phylogenetic analysis based on the partial sequence of the esyn1 gene clearly discriminates species producing exclusively BEA from those synthesizing mainly enniatin analogues.     

The Effects of Low Doses of Two Fusarium Toxins,Zearalenone and Deoxynivalenol,on the Pig Jejunum. A Light and Electron Microscopic Study

Immature gilts were administered per os with zearalenone (ZEN) at 40 μg/kg BW (group Z, n = 9), deoxynivalenol (DON) at 12 μg/kg BW (group D, n = 9), a mixture of ZEN and DON (group M, n = 9) or a placebo (group C, n = 9) over a period of six weeks. The pigs were sacrificed after one, three, or six weeks of the treatment (12 pigs per each time-point). Histological investigations revealed an increase in the mucosal thickness and the crypt depth as well as a decrease in the ratio of the villus height to the crypt depth in groups D and M after six weeks of exposure to the mycotoxins. The number of goblet cells in the villus epithelium was elevated in groups Z and M after one week and in group D after three weeks. The administration of ZEN increased the lymphocyte number in the villus epithelium after 1 week and the plasma cell quantity in the lamina propria after one, three, and six weeks of the experiment. DON treatment resulted in an increase in the lymphocyte number in the villus epithelium and the lamina propria after six weeks, and in the plasma cell quantity in the lamina propria after one, three, and six weeks of exposure. In group M, lymphocyte counts in the epithelium and the lamina propria increased significantly after six weeks. Neither mycotoxin induced significant adverse changes in the ultrastructure of the mucosal epithelium and the lamina propria or in the intestinal barrier permeability. Our results indicate that immune cells are the principal target of low doses of ZEN and DON.     

Biocontrol of Fusarium graminearum,a Causal Agent of Fusarium Head Blight of Wheat,and Deoxynivalenol Accumulation: From In Vitro to In Planta

Crop diseases caused by Fusarium graminearum threaten crop production in both commercial and smallholder farming. F. graminearum produces deoxynivalenol mycotoxin, which is stable during food and feed processing. Therefore, the best way to prevent the sporulation of pathogens is to develop new prevention strategies. Plant-based pesticides, i.e., natural fungicides, have recently gained interest in crop protection as alternatives to synthetic fungicides. Herein we show that treatment with the methanolic extract of medicinal plant Zanthoxylum bungeanum (M20 extract), decreased F. graminearum growth and abrogated DON production. The F. graminearum DNA levels were monitored by a quantitative TaqMan real-time PCR, while DON accumulation was assessed by HPLC quantification. This M20 extract was mainly composed of four flavonoids: quercetin, epicatechin, kaempferol-3-O-rhamnoside, and hyperoside. The in vitro bioassay, which measured the percent inhibition of fungal growth, showed that co-inoculation of four F. graminearum strains with the M20 extract inhibited the fungal growth up to 48.5%. After biocontrol treatments, F. graminearum DNA level was reduced up to 85.5% compared to that of wheat heads, which received F. graminearum mixture only. Moreover, DON production was decreased in wheat heads by 73% after biocontrol treatment; meanwhile in wheat heads inoculated with F. graminearum conidia, an average of 2.263 ± 0.8 mg/kg DON was detected. Overall, this study is a successful case from in vitro research to in planta, giving useful information for wheat protection against F. graminearum responsible for Fusarium Head Blight and DON accumulation in grains. Further studies are needed to study the mechanism by which M20 extract inhibited the DON production and what changes happened to the DON biosynthetic pathway genes.     

Systemic Growth of F. graminearum in Wheat Plants and Related Accumulation of Deoxynivalenol

Fusarium head blight (FHB) is an important disease of wheat worldwide caused mainly by Fusarium graminearum (syn. Gibberella zeae). This fungus can be highly aggressive and can produce several mycotoxins such as deoxynivalenol (DON), a well known harmful metabolite for humans, animals, and plants. The fungus can survive overwinter on wheat residues and on the soil, and can usually attack the wheat plant at their point of flowering, being able to infect the heads and to contaminate the kernels at the maturity. Contaminated kernels can be sometimes used as seeds for the cultivation of the following year. Poor knowledge on the ability of the strains of F. graminearum occurring on wheat seeds to be transmitted to the plant and to contribute to the final DON contamination of kernels is available. Therefore, this study had the goals of evaluating: (a) the capability of F. graminearum causing FHB of wheat to be transmitted from the seeds or soil to the kernels at maturity and the progress of the fungus within the plant at different growth stages; (b) the levels of DON contamination in both plant tissues and kernels. The study has been carried out for two years in a climatic chamber. The F. gramineraum strain selected for the inoculation was followed within the plant by using Vegetative Compatibility technique, and quantified by Real-Time PCR. Chemical analyses of DON were carried out by using immunoaffinity cleanup and HPLC/UV/DAD. The study showed that F. graminearum originated from seeds or soil can grow systemically in the plant tissues, with the exception of kernels and heads. There seems to be a barrier that inhibits the colonization of the heads by the fungus. High levels of DON and F. graminearum were found in crowns, stems, and straw, whereas low levels of DON and no detectable levels of F. graminearum were found in both heads and kernels. Finally, in all parts of the plant (heads, crowns, and stems at milk and vitreous ripening stages, and straw at vitreous ripening), also the accumulation of significant quantities of DON-3-glucoside (DON-3G), a product of DON glycosylation, was detected, with decreasing levels in straw, crown, stems and kernels. The presence of DON and DON-3G in heads and kernels without the occurrence of F. graminearum may be explained by their water solubility that could facilitate their translocation from stem to heads and kernels. The presence of DON-3G at levels 23 times higher than DON in the heads at milk stage without the occurrence of F. graminearum may indicate that an active glycosylation of DON also occurs in the head tissues. Finally, the high levels of DON accumulated in straws are worrisome since they represent additional sources of mycotoxin for livestock.     

Survey of Freshly Harvested Oat Grains from Southern Brazil Reveals High Incidence of Type B Trichothecenes and Associated Fusarium Species

The current study investigated the fungal diversity in freshly harvested oat samples from the two largest production regions in Brazil, Paraná (PR) and Rio Grande do Sul (RS), focusing primarily on the Fusarium genus and the presence of type B trichothecenes. The majority of the isolates belonged to the Fusarium sambucinum species complex, and were identified as F. graminearum sensu stricto (s.s.), F. meridionale, and F. poae. In the RS region, F. poae was the most frequent fungus, while F. graminearum s.s. was the most frequent in the PR region. The F. graminearum s.s. isolates were 15-ADON genotype, while F. meridionale and F. poae were NIV genotype. Mycotoxin analysis revealed that 92% and 100% of the samples from PR and RS were contaminated with type B trichothecenes, respectively. Oat grains from PR were predominantly contaminated with DON, whereas NIV was predominant in oats from RS. Twenty-four percent of the samples were contaminated with DON at levels higher than Brazilian regulations. Co-contamination of DON, its derivatives, and NIV was observed in 84% and 57.7% of the samples from PR and RS, respectively. The results provide new information on Fusarium contamination in Brazilian oats, highlighting the importance of further studies on mycotoxins.     

Deoxynivalenol: Toxicology,Degradation by Bacteria,and Phylogenetic Analysis

Deoxynivalenol (DON) is a toxic secondary metabolite produced by fungi that contaminates many crops, mainly wheat, maize, and barley. It affects animal health, causing intestinal barrier impairment and immunostimulatory effect in low doses and emesis, reduction in feed conversion rate, and immunosuppression in high doses. As it is very hard to completely avoid DON’s production in the field, mitigatory methods have been developed. Biodegradation has become a promising method as new microorganisms are studied and new enzymatic routes are described. Understanding the common root of bacteria with DON degradation capability and the relationship with their place of isolation may bring insights for more effective ways to find DON-degrading microorganisms. The purpose of this review is to bring an overview of the occurrence, regulation, metabolism, and toxicology of DON as addressed in recent publications focusing on animal production, as well as to explore the enzymatic routes described for DON’s degradation by microorganisms and the phylogenetic relationship among them.     

Occurrence of Deoxynivalenol and Deoxynivalenol-3-glucoside in Hard Red Spring Wheat Grown in the USA

Deoxynivalenol (DON) is a mycotoxin found in wheat that is infected with Fusarium fungus. DON may also be converted to a type of “masked mycotoxin”, named deoxynivalenol-3-glucoside (D3G), as a result of detoxification of the plant. In this study, DON and D3G were measured using gas chromatographic (GC) and liquid chromatography-mass spectrometry (LC-MS) in wheat samples collected during 2011 and 2012 in the USA. Results indicate that the growing region had a significant effect on the DON and D3G (p < 0.0001). There was a positive correlation between both methods (GC and LC-MS) used for determination of DON content. DON showed a significant and positive correlation with D3G during 2011. Overall, DON production had an effect on D3G content and kernel damage, and was dependent on environmental conditions during Fusarium infection.     

Transformation of Selected Trichothecenes during the Wheat Malting Production

The transformation of deoxynivalenol (DON), nivalenol (NIV), and their glucosides (DON3G and NIV3G) during the malting of grains of two wheat varieties was studied. The concentration of DON3G and NIV3G started to increase significantly before the concentration of DON and NIV increased. This may reflect the transformation of the parent mycotoxin forms into their glucosides due to xenobiotic detoxification reactions. After a sharp rise during the last 2 days of the process (day 6 and 7), the DON concentration reached 3010 ± 338 µg/kg in the Legenda wheat-based malt and 4678 ± 963 µg/kg in the Pokusa wheat-based malt. The NIV concentration, at 691 ± 65 µg/kg, remained the same as that in the dry grain. The concentration of DON3G in the Legenda and Pokusa wheat-based malt was five and three times higher, respectively, than that in the steeped grain. The concentration of NIV3G in the Legenda wheat-based malt was more than twice as high as that in the steeped grain. The sharp increase in the concentration of DON at the end of the malting process reflected the high pathogen activity. We set aside some samples to study a batch that was left undisturbed without turning and aeration, for the entire period of malting. The concentration of DON in the malt produced from the latter batch was 135% and 337% higher, for Legenda and Pokusa, respectively, than that in the malt produced from the batch that was turned and aerated. The NIV concentration was 22% higher in the latter batch.     

Naturally Occurring Fusarium Species and Mycotoxins in Oat Grains from Manitoba,Canada

Fusarium head blight (FHB) can lead to dramatic yield losses and mycotoxin contamination in small grain cereals in Canada. To assess the extent and severity of FHB in oat, samples collected from 168 commercial oat fields in the province of Manitoba, Canada, during 2016–2018 were analyzed for the occurrence of Fusarium head blight and associated mycotoxins. Through morphological and molecular analysis, F. poae was found to be the predominant Fusarium species affecting oat, followed by F. graminearum, F. sporotrichioides, F. avenaceum, and F. culmorum. Deoxynivalenol (DON) and nivalenol (NIV), type B trichothecenes, were the two most abundant Fusarium mycotoxins detected in oat. Beauvericin (BEA) was also frequently detected, though at lower concentrations. Close clustering of F. poae and NIV/BEA, F. graminearum and DON, and F. sporotrichioides and HT2/T2 (type A trichothecenes) was detected in the principal component analysis. Sampling location and crop rotation significantly impacted the concentrations of Fusarium mycotoxins in oat. A phylogenetic analysis of 95 F. poae strains from Manitoba was conducted using the concatenated nucleotide sequences of Tef-1α, Tri1, and Tri8 genes. The results indicated that all F. poae strains belong to a monophyletic lineage. Four subgroups of F. poae strains were identified; however, no correlations were observed between the grouping of F. poae strains and sample locations/crop rotations.     

Trichothecene Genotype Profiling of Wheat Fusarium graminearum Species Complex in Paraguay

Paraguay is a non-traditional wheat-producing country in one of the warmest regions in South America. Fusarium Head Blight (FHB) is a critical disease affecting this crop, caused by the Fusarium graminearum species complex (FGSC). A variety of these species produce trichothecenes, including deoxynivalenol (DON) and its acetylated forms (3-ADON and 15-ADON) or nivalenol (NIV). This study characterized the phylogenetic relationships, and chemotype diversity of 28 strains within FGSC collected from wheat fields across different country regions. Phylogenetic analysis based on the sequence of elongation factor-1α gene (EF-1α) from 28 strains revealed the presence of four species in the FGSC: F. graminearum sensu stricto, F. asiaticum, F. meridionale and F. cortaderiae. Ten strains selected for further analysis revealed that all F. graminearum strains were 15-ADON chemotype, while the two strains of F. meridionale and one strain of F. asiaticum were NIV chemotype. Thus, the 15-ADON chemotype of F. graminearum sensu stricto was predominant within the Fusarium strains isolated in the country. This work is the first report of phylogenetic relationships and chemotype diversity among Fusarium strains which will help understand the population diversity of this pathogen in Paraguay.