Susanne Vogelgsang

Fusarium mycotoxins: overlooked aquatic micropollutants?

Deoxynivalenol and zearalenone are among the most prevalent toxins produced by Fusarium spp. They have been investigated in food and feed products for decades but rarely in the environment. We therefore established solid-phase extraction and liquid chromatography-mass spectrometry (LC-MS) methods to quantify these mycotoxins at trace concentrations in aqueous natural samples. In a model emission study, we inoculated a winter wheat field with Fusarium graminearum and subsequently monitored deoxynivalenol and zearalenone in its drainage water. Before during and after harvest in June and July 2007, these toxins were emitted in concentrations from 23 ng/L to 4.9 microg/L for deoxynivalenol and from not detected to 35 ng/L for zearalenone. Simultaneously, in July and August 2007, deoxynivalenol was also detected in a number of Swiss rivers in concentrations up to 22 ng/L and zearalenone was present in several river samples below the method quantification limit. Other mycotoxins might be emitted from Fusarium-infected fields as well, because some of them are produced in similar amounts as deoxynivalenol and zearalenone and exhibit similar or even higher water solubility than deoxynivalenol. The ecotoxicological consequences of the presence of mycotoxins in surface waters remain to be elucidated.

Effect of fungal strain and cereal substrate on in vitro mycotoxin production by Fusarium poae and Fusarium avenaceum

Single conidia strains of Fusarium poae and Fusarium avenaceum were investigated for their ability to synthesise mycotoxins in vitro. In a first experiment, rice was inoculated with three strains of F. poae and three strains of F. avenaceum. In a second experiment, the same strains were cultivated on four different cereal substrates. For both experiments, the colonised substrates were analysed using liquid chromatography coupled with tandem mass spectrometric detection (LC-MS/MS) for the content of Fusarium mycotoxins. On rice, a strong effect of strains on mycotoxin content was found. With the analyses of different cereal types, substantial substrate effects were observed. For F. poae, these effects were highly dependent on the strain. The results of this study are of benefit for risk assessment concerning naturally infected grain samples since substrate effects could also be triggered by different cereal species or varieties, and thus could lead to variable mycotoxin contamination in cereal products intended for human consumption.

Incidence of Fusarium Species and Mycotoxins in Silage Maize

Maize is frequently infected by the Fusarium species producing mycotoxins. Numerous investigations have focused on grain maize, but little is known about the Fusarium species in the entire plant used for silage. Furthermore, mycotoxins persist during the ensiling process and thus endanger feed safety. In the current study, we analyzed 20 Swiss silage maize samples from growers’ fields for the incidence of Fusarium species and mycotoxins. The species spectrum was analyzed morphologically and mycotoxins were measured by LC-MS/MS. A pre-harvest visual disease rating showed few disease symptoms. In contrast, the infection rate of two-thirds of the harvest samples ranged from 25 to 75% and twelve different Fusarium species were isolated. The prevailing species were F. sporotrichioides, F. verticillioides and F. graminearum. No infection specificity for certain plant parts was observed. The trichothecene deoxynivalenol (DON) was found in each sample (ranging from 780 to 2990 µg kg−1). Other toxins detected in descending order were zearalenone, further trichothecenes (nivalenol, HT-2 and T-2 toxin, acetylated DON) and fumonisins. A generalized linear regression model containing the three cropping factors harvest date, pre-precrop and seed treatment was established, to explain DON contamination of silage maize. Based on these findings, we suggest a European-wide survey on silage maize.

On-farm experiments over 5 years in a grain maize/winter wheat rotation: effect of maize residue treatments on Fusarium graminearum infection and deoxynivalenol contamination in wheat

Over the course of 5 years, different maize residue treatments were conducted on 14 zero tillage on-farm sites in Switzerland to evaluate their effect on the development of Fusarium head blight (FHB) and the contamination with the mycotoxin deoxynivalenol (DON) in winter wheat grains and wheat straw following grain maize. Two experimental series with three and five different treatments were carried out, respectively. Fusarium graminearum (Schwabe) was the predominant FHB-causing species with an overall incidence of 15% infected wheat grains. A significant correlation between symptoms in the field, F. graminearum incidence and DON content in wheat grains and wheat straw was observed. The average DON content in both wheat grains and wheat straw was approximately 5,000 μg/kg and thus several times higher than the European maximum limit of 1,250 μg/kg for unprocessed small-grain cereals for human consumption. Of all grain samples, 74% were above the maximum limit. Pooled over both experimental series, the average reduction of DON in grains through treatments of the maize residue compared with a control treatment ranged between 21 and 38%. The effect of various other factors, including the year, the wheat variety, the site, the maize hybrid and the production system was evaluated as well. The year and the wheat variety were the most important FHB influencing factors. Over all treatments, the variety Levis showed a fivefold higher average DON content compared with the variety Titlis. From different categories of maize residue particles, intact pieces of 5–15 cm length were strongly correlated with F. graminearum incidence and DON content in grains. During the time course of this study, the recommendation from a preliminary version of the internet-based DON forecasting system FusaProg to apply or to omit a fungicide treatment was correct in 32 out of 42 cases. The results are currently being used to optimise the FusaProg models. This study has shown that in a grain maize/winter wheat rotation, the DON content in wheat grains frequently exceeded the European maximum limit, even with a thorough treatment of maize residues and less susceptible wheat varieties. Hence, in order to reduce the contamination risk in a zero tillage system, the crop rotation needs to be modified.

Characterisation of novel Fusarium graminearum microsatellite markers in different Fusarium species from various countries

Fifteen novel microsatellite markers were isolated from Fusarium graminearum. The level of polymorphism at these novel and 13 previously published microsatellite markers was analysed in 33 F. graminearum strains from Europe, North America, and Nepal. The number of alleles for each of the novel markers ranged from 4 to 20 and gene diversity from 0.417 to 0.962. In comparison with the previously published markers, the resolution for distinguishing among different strains was slightly increased. Twenty-seven markers were also detectable in three F. culmorum strains and one F. crookwellense strain. None of the markers was detected in three F. avenaceum and four F. poae strains, underlining the potential use of these microsatellite markers for species differentiation.

Fusarium Head Blight Control and Prevention of Mycotoxin Contamination in Wheat with Botanicals and Tannic Acid

Suspensions or solutions with 1% of Chinese galls (Galla chinensis, GC) or 1% of tannic acid (TA), inhibited germination of conidia or mycelium growth of Fusarium graminearum (FG) by 98%–100% or by 75%–80%, respectively, whereas dried bark from buckthorn (Frangula alnus, FA) showed no effect at this concentration. In climate chamber experiments where the wheat variety “Apogee” was artificially inoculated with FG and F. crookwellense (FCr) and treated with 5% suspensions of TA, GC and FA, the deoxynivalenol (DON) content in grains was reduced by 81%, 67% and 33%, respectively. In field experiments with two commercial wheat varieties and artificial or semi-natural inoculations, mean DON reductions of 66% (TA) and 58% (FA), respectively, were obtained. Antifungal toxicity can explain the high efficacies of TA and GC but not those of FA. The Fusarium head blight (FHB) and mycotoxin reducing effect of FA is probably due to elicitation of resistance in wheat plants. With semi-natural inoculation, a single FA application in the first half of the flowering period performed best. However, we assume that applications of FA at the end of ear emergence and a treatment, triggered by an infection period, with TA or GC during flowering, might perform better than synthetic fungicides.

Fusarium and mycotoxin spectra in Swiss barley are affected by various cropping techniques.

ABSTRACT Fusarium head blight is one of the most important cereal diseases worldwide. Cereals differ in terms of the main occurring Fusarium species and the infection is influenced by various factors, such as weather and cropping measures. Little is known about Fusarium species in barley in Switzerland, hence harvest samples from growers were collected in 2013 and 2014, along with information on respective cropping factors. The incidence of different Fusarium species was obtained by using a seed health test and mycotoxins were quantified by LC-MS/MS. With these techniques, the most dominant species, F. graminearum, and the most prominent mycotoxin, deoxynivalenol (DON), were identified. Between the three main Swiss cropping systems, Organic, Extenso and Proof of ecological performance, we observed differences with the lowest incidence and toxin accumulation in organically cultivated barley. Hence, we hypothesise that this finding was based on an array of growing techniques within a given cropping system. We observed that barley samples from fields with maize as previous crop had a substantially higher F. graminearum incidence and elevated DON accumulation compared with other previous crops. Furthermore, the use of reduced tillage led to a higher disease incidence and toxin content compared with samples from ploughed fields. Further factors increasing Fusarium infection were high nitrogen fertilisation as well as the application of fungicides and growth regulators. Results from the current study can be used to develop optimised cropping systems that reduce the risks of mycotoxin contamination.

Fusarium Mycotoxins in Swiss Wheat: A Survey of Growers’ Samples between 2007 and 2014 Shows Strong Year and Minor Geographic Effects

To assess the occurrence of Fusarium toxins in wheat in Switzerland, an eight-year survey was conducted by analysing a total of 686 harvest samples from growers using LC-MS/MS. Between 2007 and 2010, 527 samples were obtained from 17 cantons. Between 2011 and 2014, 159 samples were collected from the canton Berne. The most frequent toxins detected were deoxynivalenol (DON), zearalenone (ZEA) and nivalenol (NIV). The overall mean DON content in all samples was 607 µg/kg, and 11% exceeded the European limit for unprocessed cereals for foodstuffs (1250 µg/kg). For ZEA (mean 39 µg/kg), 7% exceeded the respective limit (100 µg/kg), and the mean content of NIV (no limit established) was 15 µg/kg. Between the years, the ratio of mycotoxin-contaminated samples ranged between 52% and 98% for DON, 9% and 43% for ZEA and 0% and 49% for NIV. The yearly mean contents varied substantially between 68 and 1310 µg/kg for DON, 5 and 56 µg/kg for ZEA and 6 and 29 µg/kg for NIV. The geographic origin showed a significant effect on DON and ZEA contamination, but was inconsistent between the years. This study has shown that the majority of Swiss-produced wheat is, in terms of Fusarium toxins, fit for human consumption and feed purposes. Nevertheless, depending on the year, high toxin contents can be expected, an issue that growers, cereal collection centres and the food industry have to deal with to ensure food and feed safety.

Responses of Oat Grains to Fusarium poae and F. langsethiae Infections and Mycotoxin Contaminations

Recent increases of Fusarium head blight (FHB) disease caused by infections with F. poae (FP) and F. langsethiae (FL) have been observed in oats. These pathogens are producers of nivalenol (NIV) and T-2/HT-2 toxin (T-2/HT-2), respectively, which are now considered major issues for cereal food and feed safety. To date, the impact of FP and FL on oat grains has not yet been identified, and little is known about oat resistance elements against these pathogens. In the present study, the impact of FL and FP on oat grains was assessed under different environmental conditions in field experiments with artificial inoculations. The severity of FP and FL infection on grains were compared across three field sites, and the resistance against NIV and T-2/HT2 accumulation was assessed for seven oat genotypes. Grain weight, β-glucan content, and protein content were compared between infected and non-infected grains. Analyses of grain infection showed that FL was able to cause infection on the grain only in the field site with the highest relative humidity, whereas FP infected grains in all field sites. The FP infection of grains resulted in NIV contamination (between 30–500 μg/kg). The concentration of NIV in grains was not conditioned by environmental conditions. FL provoked an average contamination of grains with T-2/HT-2 (between 15–132 μg/kg). None of the genotypes was able to fully avoid toxin accumulation. The general resistance of oat grains against toxin accumulation was weak, and resistance against NIV accumulation was strongly impacted by the interaction between the genotype and the environment. Only the genotype with hull-less grains showed partial resistance to both NIV and T-2/HT-2 contamination. FP and FL infections could change the β-glucan content in grains, depending on the genotypes and environmental conditions. FP and FL did not have a significant impact on the thousand kernel weight (TKW) and protein content. Hence, resistance against toxin accumulation remains the only indicator of FHB resistance in oat. Our results highlight the need for new oat genotypes with enhanced resistance against both NIV and T-2/HT-2 to ensure food and feed safety.

Resistance against Fusarium graminearum and the relationship to β-glucan content in barley grains

Fusarium head blight (FHB) caused by Fusarium graminearum (FG) is a destructive disease impacting barley worldwide. The disease reduces the grain yield and contaminates grains with mycotoxins, such as the trichothecene deoxynivalenol (DON). Although the infection mainly affects the grain yield, little is known about its impact on grain structural and biochemical properties. Yet, such information is instrumental to characterize the facets of resistance in the grains. After artificial inoculation of six barley cultivars with FG in a 2 years field test, different levels of symptoms on spikes, of colonisation of grains and of DON content were observed. The infections caused a reduction in grain weight and an average decrease of 10% of the β-glucan content in grains, indicating alterations of grain filling, composition and structure. According to our results, we postulate the presence of two distinct resistance mechanisms in the grain, tolerance to grain filling despite infection as well as the inhibition of mycotoxin accumulation. Differently to wheat, in barley, type IV resistance (tolerance of the grain to infection) is directly linked with type III resistance (resistance against kernel infection). The resistance against toxin accumulation (named type V resistance in wheat) appeared to be independent to all other resistance types. Generally, the resistance was significantly influenced by the environment and by genotype x environment interactions explaining the generally weak stability of resistance in barley. Interestingly, a significant and inverse relationship between DON contamination and β-glucan content in grains suggests that high β-glucan content in grains contributes to type V resistance.