Hydrogen peroxide induced by the fungicide prothioconazole triggers deoxynivalenol (DON) production by <Emphasis Type="Italic">Fusarium graminearum</Emphasis>

Background  

Fusarium head blight is a very important disease of small grain cereals with F. graminearum as one of the most important causal agents. It not only causes reduction in yield and quality but from a human and animal healthcare point of view, it produces mycotoxins such as deoxynivalenol (DON) which can accumulate to toxic levels. Little is known about external triggers influencing DON production.     

Isolates of Fusarium graminearum collected 40–320?meters above ground level cause Fusarium head blight in wheat and produce trichothecene mycotoxins

The aerobiology of fungi in the genus Fusarium is poorly understood. Many species of Fusarium are important pathogens of plants and animals and some produce dangerous secondary metabolites known as mycotoxins. In 2006 and 2007, autonomous unmanned aerial vehicles (UAVs) were used to collect Fusarium 40–320 m above the ground at the Kentland Farm in Blacksburg, Virginia. Eleven single-spored isolates of Fusarium graminearum (sexual stage Gibberella zeae) collected with autonomous UAVs during fall, winter, spring, and summer months caused Fusarium head blight on a susceptible cultivar of spring wheat. Trichothecene genotypes were determined for all 11 of the isolates; nine isolates were DON/15ADON, one isolate was DON/3ADON, and one isolate was NIV. All of the isolates produced trichothecene mycotoxins in planta consistent with their trichothecene genotypes. To our knowledge, this is the first report of a NIV isolate of F. graminearum in Virginia, and DON/3ADON genotypes are rare in populations of the fungus recovered from infected wheat plants in the eastern United States. Our data are considered in the context of a new aerobiological framework based on atmospheric transport barriers, which are Lagrangian coherent structures present in the mesoscale atmospheric flow. This framework aims to improve our understanding of population shifts of F. graminearum and develop new paradigms that may link field and atmospheric populations of toxigenic Fusarium spp. in the future.     

Quantification of Trichothecene-Producing Fusarium Species in Harvested Grain by Competitive PCR To Determine Efficacies of Fungicides against Fusarium Head Blight of Winter Wheat

We developed a PCR-based assay to quantify trichothecene-producing Fusarium based on primers derived from the trichodiene synthase gene (Tri5). The primers were tested against a range of fusarium head blight (FHB) (also known as scab) pathogens and found to amplify specifically a 260-bp product from 25 isolates belonging to six trichothecene-producing Fusarium species. Amounts of the trichothecene-producing Fusarium and the trichothecene mycotoxin deoxynivalenol (DON) in harvested grain from a field trial designed to test the efficacies of the fungicides metconazole, azoxystrobin, and tebuconazole to control FHB were quantified. No correlation was found between FHB severity and DON in harvested grain, but a good correlation existed between the amount of trichothecene-producing Fusarium and DON present within grain. Azoxystrobin did not affect levels of trichothecene-producing Fusarium compared with those of untreated controls. Metconazole and tebuconazole significantly reduced the amount of trichothecene-producing Fusarium in harvested grain. We hypothesize that the fungicides affected the relationship between FHB severity and the amount of DON in harvested grain by altering the proportion of trichothecene-producing Fusarium within the FHB disease complex and not by altering the rate of DON production. The Tri5 quantitative PCR assay will aid research directed towards reducing amounts of trichothecene mycotoxins in food and animal feed.     

A review on comparative data concerning <Emphasis Type="Italic">Fusarium</Emphasis> mycotoxins in Bt maize and non-Bt isogenic maize

The European corn borer reportedly promotes the infection of maize by Fusarium spp. Stalk and ear rots caused by Fusarium spp. are often related to mycotoxin accumulation in maize kernels. As a result, food and animal feed from maize are more severely contaminated with Fusarium mycotoxins: e.g. fumonisins (FUM), deoxynivalenol (DON) and zearalenone (ZEA). Bt maize is primarily an important potential tool for insect pest protection, both in the European Union and in other countries. Bt maize carrying the Bt genes is highly resistant to European corn borer larval feeding due to Bt toxin (δ toxin) production. Effective measures to combat pests therefore often have a positive side-effect in that they also reduce mycotoxin levels. Comparative analysis was used to the evaluation of the studies dealing with the reduction of Fusarium mycotoxins in Bt maize. Nineteen out of 23 studies on Bt maize came to the conclusion that Bt maize is less contaminated with mycotoxins (FUM, DON, ZEA) than the conventional control variety in each case.     

Survey and risk assessment of the mycotoxins deoxynivalenol,zearalenone, fumonisins,ochratoxin A,and aflatoxins in commercial dry dog food

The aim of the present study was to investigate the occurrence of mycotoxins in commercial dog food, as a basis to estimate the risk of adverse effects. Seventy-six dry dog food samples from 27 producers were purchased from retail shops, supermarkets, and specialized pet food shops in Vienna, Austria. The frequency and levels of deoxynivalenol (DON), zearalenone (ZEA), fumonisins (FUM), ochratoxin A (OTA). and aflatoxins (AF) in dry dog food were determined. Mycotoxin analysis were performed by commercial enzyme-linked immunosorbent assay (ELISA) test kits. Confirmatory analyses were done for DON, ZEA, and FUM by high performance liquid chromatography (HPLC) after extract clean-up with immunoaffinity columns. The correlations between ELISA and HPLC results for DON and ZEA were acceptable and indicated that ELISA could be a simple, low cost, and sensitive screening tool for mycotoxins detection, contributing to quality and safety of pet food. DON was the mycotoxin most frequently found (83% positives; median 308 μg/kg, maximum 1,390 μg/kg). ZEA (47% positives, median 51 μg/kg and maximum 298 μg/kg) and FUM (42% positives, median 122 μg/kg and maximum 568 μg/kg) were also frequently detected in dog food. OTA was less frequently found (5%, median 3.6 μg/kg, maximum 4.7 μg/kg. AF were not detected (<0.5 μg/kg) in any sample. The results show that dry dog food marketed in Vienna are frequently contaminated with mycotoxins (DON > ZEA > FUM > OTA) in low concentrations, but do not contain AF. The high frequency of Fusarium toxins DON, ZEA, and FUM indicates the need for intensive control measures to prevent mycotoxins in dog foods. The mycotoxin levels found in dry dog food are considered as safe in aspects of acute mycotoxicoses. However, repeated and long-time exposure of dogs to low levels of mycotoxins may pose a health risk.     

(−)-Epigallocatechin gallate suppresses the cytotoxicity induced by trichothecene mycotoxins in mouse cultural macrophages

Trichothecene mycotoxins are toxic secondary metabolites produced by a number of fungi including Fusarium species, which adversely affect lymphocytes. Deoxynivalenol (DON) and HT-2 toxin (HT-2) belong to the trichothecene group of mycotoxins and the occurrence of cereals and foodstuffs with these compounds are serious health problems. The aim of this study was to examine the effect of (−)-epigallocatechin gallate (EGCG), one of the main components in green tea catechins, on DON- or HT-2-induced cytotoxicity in mouse macrophages. EGCG had protective effects against the trichothecene-induced cytotoxicities of both mycotoxins. Additionally, EGCG suppressed the DON-induced activation of caspase-3/7, which is an indicator of apoptosis. These results indicate that EGCG might be useful in protection against DON- or HT-2-induced cell death, suggesting that EGCG could contribute to reducing the toxicities of trichothecenes.     

Structural and functional characterization of TRI3 trichothecene 15‐O‐acetyltransferase from Fusarium sporotrichioides

Fusarium head blight is a devastating disease of cereal crops whose worldwide incidence is increasing and at present there is no satisfactory way of combating this pathogen or its associated toxins. There is a wide variety of trichothecene mycotoxins and they all contain a 12,13‐epoxytrichothecene skeleton but differ in their substitutions. Indeed, there is considerable variation in the toxin profile across the numerous Fusarium species that has been ascribed to differences in the presence or absence of biosynthetic enzymes and their relative activity. This article addresses the source of differences in acetylation at the C15 position of the trichothecene molecule. Here, we present the in vitro structural and biochemical characterization of TRI3, a 15‐O‐trichothecene acetyltransferase isolated from F. sporotrichioides and the “in vivo” characterization of Δtri3 mutants of deoxynivalenol (DON) producing F. graminearum strains. A kinetic analysis shows that TRI3 is an efficient enzyme with the native substrate, 15‐decalonectrin, but is inactive with either DON or nivalenol. The structure of TRI3 complexed with 15‐decalonectrin provides an explanation for this specificity and shows that Tri3 and Tri101 (3‐O‐trichothecene acetyltransferase) are evolutionarily related. The active site residues are conserved across all sequences for TRI3 orthologs, suggesting that differences in acetylation at C15 are not due to differences in Tri3. The tri3 deletion mutant shows that acetylation at C15 is required for DON biosynthesis even though DON lacks a C15 acetyl group. The enzyme(s) responsible for deacetylation at the 15 position of the trichothecene mycotoxins have not been identified.     

Evaluation of an Oral Subchronic Exposure of Deoxynivalenol on the Composition of Human Gut Microbiota in a Model of Human Microbiota-Associated Rats

Background

Deoxynivalenol (DON), a mycotoxin produced by Fusarium species, is one of the most prevalent mycotoxins present in cereal crops worldwide. Due to its toxic properties, high stability and prevalence, the presence of DON in the food chain represents a health risk for both humans and animals. The gastrointestinal microbiota represents potentially the first target for these food contaminants. Thus, the effects of mycotoxins on the human gut microbiota is clearly an issue that needs to be addressed in further detail. Using a human microbiota-associated rat model, the aim of the present study was to evaluate the impact of a chronic exposure of DON on the composition of human gut microbiota.

Methodology/Principal Findings

Four groups of 5 germ free male rats each, housed in 4 sterile isolators, were inoculated with a different fresh human fecal flora. Rats were then fed daily by gavage with a solution of DON at 100 µg/kg bw for 4 weeks. Fecal samples were collected at day 0 before the beginning of the treatment; days 7, 16, 21, and 27 during the treatment; and 10 days after the end of the treatment at day 37. DON effect was assessed by real-time PCR quantification of dominant and subdominant bacterial groups in feces. Despite a different intestinal microbiota in each isolator, similar trends were generally observed. During oral DON exposure, a significant increase of 0.5 log10 was observed for the Bacteroides/Prevotella group during the first 3 weeks of administration. Concentration levels for Escherichia coli decreased at day 27. This significant decrease (0.9 log10 CFU/g) remained stable until the end of the experiment.

Conclusions/Significance

We have demonstrated an impact of oral DON exposure on the human gut microbiota composition. These findings can serve as a template for risk assessment studies of food contaminants on the human gut microbiota.     

Intracellular Expression of a Single Domain Antibody Reduces Cytotoxicity of 15-Acetyldeoxynivalenol in Yeast

15-Acetyldeoxynivalenol (15-AcDON) is a low molecular weight sesquiterpenoid trichothecene mycotoxin associated with Fusarium ear rot of maize and Fusarium head blight of small grain cereals. The accumulation of mycotoxins such as deoxynivalenol (DON) and 15-AcDON within harvested grain is subject to stringent regulation as both toxins pose dietary health risks to humans and animals. These toxins inhibit peptidyltransferase activity, which in turn limits eukaryotic protein synthesis. To assess the ability of intracellular antibodies (intrabodies) to modulate mycotoxin-specific cytotoxocity, a gene encoding a camelid single domain antibody fragment (V_HH) with specificity and affinity for 15-AcDON was expressed in the methylotropic yeast Pichia pastoris. Cytotoxicity and V_HH immunomodulation were assessed by continuous measurement of cellular growth. At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris than was DON. In turn, DON was orders of magnitude more toxic than 3-acetyldeoxynivalenol. Intracellular expression of a mycotoxin-specific V_HH within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to 100 μg·ml⁻¹. We also documented a biochemical transformation of DON to 15-AcDON to account for the attenuation of DON cytotoxicity at 100 and 200 μg·ml⁻¹. The proof of concept established within this eukaryotic system suggests that in planta V_HH expression may lead to enhanced tolerance to mycotoxins and thereby limit Fusarium infection of commercial agricultural crops.     

First survey on the natural occurrence of Fusarium mycotoxins in Bulgarian wheat

Wheat for human consumption (140 samples) was collected after harvest from all regions of Bulgaria. The 1995 crop year was characterized by heavy rainfall in the spring and summer months. The internal mycoflora of wheat samples was dominated by Fusarium spp. and Alternaria spp., and storage fungi were rarely present. The samples were analysed for contamination with Fusarium mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), and zearalenone (ZEA), using enzyme immunoassay methods. DON and ZEA were the predominant toxins, with a contamination frequency of 67% and 69%, respectively. The average levels of these toxins in positive samples were 180 g/kg (DON) and 17 g/kg (ZEA), maximum concentrations were 1800 g kg^–1 and 120 g kg^–1, respectively. Acetyl derivatives of DON, namely 3-AcDON and 15-AcDON, were found in 2.1 % and 0.7% of the samples, at at maximum level of about 100 g kg^–1. Only one sample was positive for T-2 (55 g/kg), DAS was not detected. This is the first report about the natural occurrence of a range of Fusarium mycotoxins in wheat for human consumption in Bulgaria.Abbreviations 3-AcDON 3-acetyldeoxynivalenol - 15-AcDON 15-acetyldeoxynivalenol - DAS diacetoxyscirpenol - DON deoxynivalenol - EIA enzyme immunoassay - T-2 T-2 toxin - ZEA zearalenone     

Development and evaluation of multiplex PCR for detection of T-2 and zearalenone producing Fusarium spp

The study aimed to develop and evaluate a multiplex polymerase chain reaction assay (mPCR) for the concurrent detection of three major mycotoxin metabolic pathway genes, namely tri8 (T-2 toxin), tri6 (trichothecene) and pks4 (zearalenone), along with competitive internal amplification control. Specific primers for each of the aforementioned genes were optimized and validated using 14 reference strains and 10 pure culture isolates. The optimized mPCR assay detected the three metabolic pathway genes in artificially contaminated maize samples with a sensitivity of 2 × 10³ CFU per g for tri6 and pks4 positive Fusarium strains, whereas 2 × 10⁴ CFU per g for tri8 positive Fusarium strains. Application of the developed mPCR assay to 30 cereal and 20 feed samples revealed 24% (12 of 50) contamination with either one or more mycotoxins. The results of mPCR assay were further evaluated with high performance liquid chromatography (HPLC), and both methods provided unequivocal results. This mPCR assay might be a supplementary tool to conventional mycotoxin analytical techniques like thin-layer chromatography, HPLC, etc. The current mPCR assay is a rapid and reliable tool for simultaneous, sensitive and specific detection of T-2, zearalenone and trichothecene producing Fusarium spp. from naturally contaminated foods and to monitor them during the processing steps of food and feed commodities.     

Simultaneous detection of Fusarium culmorum and F. graminearum in plant material by duplex PCR with melting curve analysis

Background  

Fusarium head blight (FHB) is a disease of cereal crops, which has a severe impact on wheat and barley production worldwide. Apart from reducing the yield and impairing grain quality, FHB leads to contamination of grain with toxic secondary metabolites (mycotoxins), which pose a health risk to humans and livestock. The Fusarium species primarily involved in FHB are F. graminearum and F. culmorum. A key prerequisite for a reduction in the incidence of FHB is an understanding of its epidemiology.     

A case of sporotrichosis caused by two genetically differentSporothrix schenckii strains

A survey for the natural occurrence of Fusarium mycotoxins, deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN), in Dutch cereals (totaling 29 samples) harvested in 1984/1985, showed that 90%, 79% and 62% of samples were contaminated with DON, NIV and ZEN, respectively. Average contents (ng/g) in the total of positive samples were 221 (DON), 123 (NIV) and 61 (ZEN). Among the cereals examined, the highest concentrations (ng/g) was 3198 (DON), 1875 (NIV) and 677 (ZEN) in a yellow corn sample for animal feed. The results of this survey show that Dutch cereals were relatively significantly contaminated with Fusarium mycotoxins.     

Thioredoxin-1 contributes to protection against DON-induced oxidative damage in HepG2 cells

Leucocytes are susceptible to the toxic effects of deoxynivalenol (DON), which is a trichothecene mycotoxin produced by a number of fungi including Fusarium species. One mechanism of action is mediated by reactive oxygen species (ROS). The liver is an important target for toxicity caused by foreign compounds including mycotoxins. On the other hand, little is known about the influence of the redox state on hepatocytes treated with DON. The present study investigated the effect of DON on the cytosolic redox state and antioxidative system in the human hepatoma cell line HepG2. The cell viability of human monocyte cell line THP-1 or leukemia cell line KU812 treated with 2.5 and 5???mol/l DON were significantly reduced. However, HepG2 cells showed no toxic effects under the same conditions and did not exhibit an increased oxidative state. Further experiments showed that thioredoxin-1 (Trx-1) protein levels but not glutathione increased in the cells treated with 10???mol/l DON. In addition, the enhancement of Trx-1 content was repressed by antioxidants. These results suggest that DON-induced accumulation of Trx-1 in HepG2 cells plays one of the key roles in protection against cytotoxicity caused by DON and that the mechanism may be mediated by the antioxidant properties of Trx-1.     

Contribution of the endogeic earthworm species <Emphasis Type="Italic">Aporrectodea caliginosa</Emphasis> to the degradation of deoxynivalenol and <Emphasis Type="Italic">Fusarium</Emphasis> biomass in wheat straw

In arable fields managed by conservation tillage combined with crop residue mulching, plant pathogen repression is an important ecosystem service to prevent cultivated plants from fungal diseases and mycotoxin contamination. A laboratory microcosm study was conducted to investigate the contribution of the endogeic, geophagous earthworm species Aporrectodea caliginosa as a secondary decomposer to the reduction of the phytopathogenic fungus Fusarium culmorum and its mycotoxin deoxynivalenol (DON) in wheat straw residues. After 5 weeks experimental time, the Fusarium biomass and the DON concentration in aboveground straw were reduced considerably to the same extent both in presence and absence of A. caliginosa. Another substantial reduction of Fusarium biomass and DON concentration was found in belowground straw, which A. caliginosa had buried into the soil. Thus, we conclude that the particular contribution of secondary decomposers like A. caliginosa to the degradation of phytopathogenic fungi like Fusarium species and their mycotoxins like DON in the soil systems has to be assessed as minor.     

Fusarium species and their mycotoxins in infected corn in Italy

Surveys of corn (infected plants and commercial kernels) forFusarium species and their mycotoxins were carried out on samples collected all over Italy and from some European and mediterranean countries.Investigations on samples of corn stalk and ear rot standing in the field, mainly collected in southern Italy, proved to be contaminated with zearalenone (ZON), zearalenols (ZOL), and deoxynivalenol (DON). TheFusarium species most frequently isolated, and their recorded toxigenic capability (in parentheses), were:F. moniliforme;F. culmorum (ZON, ZOL, DON, 3AcDON);F. equiseti (ZON, ZOL); andF. proliferatum (MF). Along with these species,F. graminearum group 2 (ZON, DON and/or 3AcDON or 15AcDON);F. chlamydosporum;F. acuminatum (type-A trichothecene derivatives); andF. semitectum were often found to be associated.F. heterosporum (ZON, ZOL);F. solani;F. crookwellense (ZON, ZOL, FUS, NIV);F. oxysporum (MF);F. avenaceum (MF);F. sporotrichioides (T-2 toxin and derivatives); andF. poae (DAS, MAS) were occasionally isolated.     

Molecular identification and determination of the trichothecene chemotypes of fungi causing crown and root in different growth stages of wheat in north of Iran

In order to determine the crown and root agents and their mycotoxins produced in different growth stages of wheat including seedling, tillering and heading, sampling was done in north of Iran, during 2011–2012. From 160 isolates of Fusarium, eight species were obtained including F. graminearum, F. culmorum, F. equiseti, F. nygamai, F. semitectum, F. solani, F. acuminatum and F. oxysporum. Sampling at different growth stages showed that F. graminearum was the predominant causal agent of crown and root at the heading stage, whereas other species of Fusarium were mostly observed at the seedling and tillering stages. Moreover, identification of pathogenic species was confirmed using species-specific primers pairs. In F. graminearum isolates, presence of Tri13 gene, responsible for nivalenol (NIV) and deoxynivalenol (DON) mycotoxins biosynthesis, was detected using specific PCR primers. Finally, the ability of trichothecene production of five F. graminearum isolates was confirmed with high-performance liquid chromatography.     

Early activation of wheat polyamine biosynthesis during Fusarium head blight implicates putrescine as an inducer of trichothecene mycotoxin production

Background  

The fungal pathogen Fusarium graminearum causes Fusarium Head Blight (FHB) disease on wheat which can lead to trichothecene mycotoxin (e.g. deoxynivalenol, DON) contamination of grain, harmful to mammalian health. DON is produced at low levels under standard culture conditions when compared to plant infection but specific polyamines (e.g. putrescine and agmatine) and amino acids (e.g. arginine and ornithine) are potent inducers of DON by F. graminearum in axenic culture. Currently, host factors that promote mycotoxin synthesis during FHB are unknown, but plant derived polyamines could contribute to DON induction in infected heads. However, the temporal and spatial accumulation of polyamines and amino acids in relation to that of DON has not been studied.     

The Human Fecal Microbiota Metabolizes Deoxynivalenol and Deoxynivalenol-3-Glucoside and May Be Responsible for Urinary Deepoxy-Deoxynivalenol

Deoxynivalenol (DON) is a potent mycotoxin produced by Fusarium molds and affects intestinal nutrient absorption and barrier function in experimental and farm animals. Free DON and the plant metabolite DON-3-β-d-glucoside (D3G) are frequently found in wheat and maize. D3G is stable in the upper human gut, but some human intestinal bacteria release DON from D3G in vitro. Furthermore, some bacteria derived from animal digestive systems degrade DON to a less toxic metabolite, deepoxy-deoxynivalenol (DOM-1). The metabolism of D3G and DON by the human microbiota has not been fully assessed. We therefore conducted in vitro batch culture experiments assessing the activity of the human fecal microbiota to release DON from D3G. We also studied detoxification of DON to DOM-1 by the microbiota and its potential effect on urinary DON excretion in humans. Fecal slurry from five volunteers was spiked with DON or D3G and incubated anaerobically (from 1 h to 7 days), and mycotoxins were extracted into acetonitrile. Mycotoxins were detected in fecal extracts and urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The fecal microbiota released DON from D3G very efficiently, with hydrolysis peaking after 4 to 6 h. The fecal microbiota from one volunteer transformed DON to DOM-1. Urine from the same volunteer also contained DOM-1 (4.7% of DON), whereas DOM-1 was not detectable in urine from other volunteers. Our results confirm that the fecal microbiota releases DON from its glycosylated form, hence increasing the toxic burden in exposed individuals. Furthermore, this is first evidence that the human fecal microbiota of one volunteer detoxifies DON, resulting in the appearance of DOM-1 in urine.