Short review: Metabolism of theFusarium mycotoxins deoxynivalenol and zearalenone in plants

Plants have a high capacity to transform and thereby detoxify deleterious or poisonous compounds, like mycotoxins. The formation of glucose conjugates has a central role in this process. Mammals, however, are able to (partly) release the precursor substances during digestion, reactivating the mycotoxins. This short review provides a brief summary about the metabolism of theFusarium mycotoxins deoxynivalenol and zearalenone in plants. Two examples are discussed in greater detail. First, the formation of deoxynivalenol-3-glucoside in wheat is linked to a quantitative trait locus that is often used forFusarium head blight resistance breeding. Secondly, the metabolism of zearalenone inArabidopsis thaliana results in at least 17 different metabolites, all of which are potentially hazardous for humans and animals. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006 Financial support: Christian Doppler Society, the Austrian Genome Research Initiative GEN-AU, the Lower Austrian government, the Austrian Science Fund FWF     

Deoxynivalenol (DON)- und Zearalenon (ZEA)-Gehalte in Weizen, Roggen und Triticale: Ergebnisse aus der Besonderen Ernteermittlung (BEE) 2001 und 2002

Deoxynivalenol and Zearalenone contents were determined in wheat, rye and triticale samples which were representative of the 2001 and 2002 harvests in Germany. The frequency of DON and ZEA detection and the mean levels of these toxins were higher in wheat, rye and triticale in 2002 in comparison to 2001. The influence of varieties and preceding crop on DON contents in wheat and triticale is presented.

Presented at the 25th Mykotoxin-Workshop in Giessen, Germany, May 19–21, 2003     

Effects of a diet containing fusarium toxins on the fertility of gilts and on bulbourethral gland weight in barrows

Nine gilts weighing 80 kg at the beginning of the trial were fed a mycotoxin contaminated diet containing 2 mg deoxynivalenol (DON) and 0.4 mg zearalenone (ZON) per kg (Diet M). Their daily weight gain until 103 kg BW was reduced in comparison to the nine control animals fed an uncontaminated diet (Diet C) (763 vs. 912 g; p = 0.02). There was no treatment effect on the age at first observed oestrus. Seven and eight gilts receiving Diet M and C, respectively, became pregnant after being mated once or being again mated three weeks later. The examination of the uteri of gilts slaughtered 35–61 days after mating showed that the exposure to DON and ZON had no effect on the number of foetuses per gilt (p = 0.54), but increased their growth rate (p = 0.003). Thus, low dietary DON and ZON levels had no negative effects on the reproductive parameters examined. The hypothesis that the bulbourethral gland weight of barrows can be used for the bioassay of low dietary ZON levels was rejected since feeding Diet M from 80–103 kg BW did not increase the weight of that accessory sex gland (p = 0.51).     

玉米赤霉烯酮对膨胀青萍G_3生长与发育的影响

玉米赤霉烯酮(Zearalenone,以下简称ZEN)被证明具有动物雌性激素的作用(Mirocha等 1967),并且是某些真菌的一种性激素(Mirocha等1968)。李季伦等首次报道ZEN与高等植物的生长与发育有关(1980)。现已证实ZEN与植物的春化作用(孟繁静等1986)、短日光周期诱导(韩玉珍和孟繁静1990)、以及花器官的发生、分化乃至开花和受精等(阙月美等1990)     

A survey on the occurrence of citrinin in feeds and their ingredients in Russia

More than 1700 samples of forage grain, sunflower and soy-bean oil-seed meal and cakes, gluten, and compounded feeds were analyzed for citrinin by indirect ELISA with a minimum detectable level of 10 ppb. Out of 829 compounded feeds (rations and concentrates) 8.8 per cent samples were positive and the amount of citrinin ranged between 12 and 182 ppb. Only 4.5% of wheat and 3.6% of barley contained citrinin at 50–998 ppb. 1.9% of maize grain samples were positive at levels of 218–953 ppb. Among the other ingredients the highest incidence (28.9%) at the levels of 14–397 ppb was found for sunflower oil-seed meal and cakes. Three positive cases of 148 processed soy-bean samples contained citrinin in a range of 14–30 ppb. Presented at the IV Russian Congress on Medical Mycology, Moscow, Russia, March 2006     

Corn as a source of mycotoxins in Indonesian poultry feeds and the effectiveness of visual examination methods for detecting contamination

Every truck load of corn (n=52) entering and every batch of poultry feed (n=290) leaving a Bogor feedmill over one year was analysed for aflatoxins, zearalenone, ochratoxin A and sterigmatocystin. Fifty loads of corn and 274 of the batches of chicken feed contained aflatoxins. Zearalenone was detected in 11 corn samples but was not found in the formulated feed. Ochratoxin A was detected in one corn sample, but not in feed. Corn can account for all of the aflatoxin in the feed since levels were always lower in the finished product. There was no quantitative association between the proportion of bright green-yellow fluorescent, purple or mouldy kernels and the mycotoxin contents of the composite samples. Nevertheless, the absence of abnormal kernels indicates higher quality corn since the highest levels of mycotoxins occurred in the abnormal kernels.     

Validation of an HPLC Analytical Method Coupled to a Multifunctional Clean-up Column for the Determination of Deoxynivalenol

To evaluate a method using a multifunctional clean-up column coupled with high performance liquid chromatography as an official analytical method for the determination of deoxynivalenol in wheat used as food or feed, an inter-laboratory study was performed in 12 laboratories using four naturally contaminated wheat samples and one spiked sample. The relative standard deviations for repeatability (RSD_r) and reproducibility (RSD_R) of naturally contaminated wheat were in the range 5.8–11.3% and 12.0–20.7%, respectively. The HORRAT was less than 1.0 in each sample. From the spiking test, the recovery rate, RSD_r, RSD_R and HORRAT value were 100.0%, 11.2%, 10.3% and 0.5, respectively. The limit of quantification is 0.10 mg/kg from the range obtained in a linear calibration. Thus, it should be useful as a sensitive and validated analytical method for the determination of deoxynivalenol in wheat intended for use in food and feed.     

Capping proteins regulate fungal development,DON-toxisome formation and virulence in Fusarium graminearum

Deoxynivalenol (DON) is an important trichothecene mycotoxin produced by the cereal pathogen Fusarium graminearum. DON is synthesized in organized endoplasmic reticulum structures called toxisomes. However, the mechanism for toxisome formation and the components of toxisomes are not yet fully understood. In a previous study, we found that myosin I (FgMyo1)-actin cytoskeleton participated in toxisome formation. In the current study, we identified two new components of toxisomes, the actin capping proteins (CAPs) FgCapA and FgCapB. These two CAPs form a heterodimer in F. graminearum, and physically interact with FgMyo1 and Tri1. The deletion mutants ΔFgcapA and ΔFgcapB and the double deletion mutant ΔΔFgcapA/B dramatically reduced hyphal growth, asexual and sexual reproduction and endocytosis. More importantly, the deletion mutants markedly disrupted toxisome formation and DON production, and attenuated virulence in planta. Collectively, these results suggest that the actin CAPs are associated with toxisome formation and contribute to the virulence and development of F. graminearum.     

Reduction of Deoxynivalenol in Barley by Treatment with Aqueous Sodium Carbonate and Heat

Naturally contaminated lots of Canadian barley containing either 18.4 or 4.3 μg/g deoxynivalenol (DON) were heated at 80 °C, with small amounts of water or 1 M sodium carbonate solution to study the rate of DON reduction. Samples were heated in sealed polypropylene containers for periods of up to 8 days. In the 18.4 μg/g DON barley, rapid reductions were observed: with no solutions added, DON declined to 14.7 μg/g after 1 day, and to 4.9 μg/g after 8 days solely due to heat; with water at 10 mL/100 g barley, DON levels reached 3.7 μg/g after 8 days; with 1 M sodium carbonate solution added at 10 mL/100 g barley, DON declined to 4.7 μg/g after 1 day, and to 0.4 μg/g after 8 days; with 20 mL/100 g barley, DON declined to 1.4 μg/g after 1 day and to near-zero levels after 8 days. In the 4.3 μg/g DON barley, more gradual reductions were evident: with no solutions added, DON declined to 2.9 μg/g after 8 days solely due to heat; with water at 10 mL/100 g barley, DON levels reached 2.3 μg/g after 8 days; with 1 M sodium carbonate solution added at 10 mL/100 g barley, DON declined to 2.7 μg/g after 1 day, and to near-zero levels after 8 days; with 20 mL/100 g barley, DON declined to 1.4 μg/g after 1 day and to near-zero levels after 3, 5 and 8 days.     

Zearalenone and flower bud formation in thin-cell layers of Nicotiana tabacum L.

Three lines of evidence indicated a connectionbetween zearalenone (ZEN) and flower bud formationin thin cell layer (TCL) explants of Nicotianatabacum L. cv. Samsun. (1) There were two peaks inthe endogenous ZEN level during the formation offlower buds. (2) The specific inhibitor of ZENbiosynthesis, malathion (MAL), inhibited thebiosynthesis of endogenous ZEN and at the same timeflower bud neoformation. (3) Exogenous ZEN inducedflower bud neoformation.     

Effect of Cooking Process on the Deoxynivalenol Content and Its Subsequent Cytotoxicity in Wheat Products

The retention of deoxynivalenol in noodles and bread made from naturally-contaminated flour was examined by a chemical analysis (HPLC) and bioassays. The retention level of deoxynivalenol obtained from both assays was reduced by boiling process, although only the bioassays showed it to have been reduced by baking. This study is the first to estimate the exposure to deoxynivalenol from the consumption of the final products of wheat flour in Japan.     

Mycobiota in poultry feeds and natural occurrence of aflatoxins, fumonisins and zearalenone in the Rio de Janeiro State, Brazil

The intake of mycotoxin-contaminated feeds can lead to nutrient losses and may have adverse effects on animal health and on productivity. The aims of this study were (1) to determine the mycobiota present in poultry feed samples, and (2) to evaluate the natural occurrence of aflatoxin B₁, fumonisin B₁ and zearalenone. Fungal counts were similar between all culture media tested (10³ CFU g⁻¹). The most frequent genus isolated was Penicillium spp. (41.26%) followed by Aspergillus spp. (33.33%) and Fusarium spp. (20.63%). High precision liquid chromatography was applied to quantify aflatoxin B₁ and fumonisin B₁. Thin layer chromatography was used to determine zearalenone levels. Aflatoxin B₁ values ranged between 1.2 and 17.5 μg kg⁻¹. Fumonisin B₁ levels ranged between 1.5 and 5.5 μg g⁻¹. Zearalenone levels ranged between 0.1 and 7 μg g⁻¹. The present study shows the simultaneous occurrence of two carcinogenic mycotoxins, aflatoxin B₁ and fumonisin B₁, together with another Fusarium mycotoxin (zearalenone) in␣feed intended for poultry consumption. Many samples contained AFB₁ levels near the permissible maximum and it could affect young animals. A synergistic toxic response is possible in animals under simultaneous exposure.     

Fusarium spp. and storage fungi in suboptimally stored wheat: Mycotoxins and influence on wheat gluten proteins

When wheat is stored under suboptimal conditions, a further mycotoxin increase of deoxynivalenol (DON), but especially of mycotoxins produced by storage fungi, e.g. ochratoxin A, is possible, lowering wheat quality and food safety. Different storage trials were conducted under suboptimal storage conditions.Fusarium survival during suboptimal storage was monitored by cultural technique and multiplex-PCR and set into relation to DON contents. Furthermore, XANES spectroscopy was applied on a selected storage trial in order to characterize sulfur speciation in low molecular weight (LMW) subunits of glutenin isolated from suboptimally stored wheat samples highly infected withFusarium and from wheat infected withAspergillus andPenicillium. Distinct changes in sulfur speciation were observed in grains infected with storage fungi, especially a significant increase of higher oxidation states (sulfoxide state, sulfonate state). Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

in Vitro Effect of Deoxynivalenol on the Differentiation of Human Colonic Cell Lines Caco-2 and t84

The effects of low concentrations of deoxynivalenol (DON) on structural and functional characteristics of human colonic adenocarcinoma cell lines Caco-2 and T84 were examined. Scanning electron microscopic (SEM) analysis of the apical surfaces of Caco-2 cells revealed reduction or abnormal formation of brush borders in the presence of 50, 100 and 200 ng/ml of DON. Monolayer integrity of Caco-2 and T84 cells was studied using cells which were cultured on permeable membranes. The transepithelial electrical resistance (TEER) of Caco-2 cells was significantly reduced at 50, 100 and 200 ng/ml of DON, significant increase in lucifer yellow (LY) permeability was also observed in these cells at 100 ng/ml of DON. The TEER of T84 cells was significantly reduced at 100 and 200 ng/ml of DON. LY permeability significantly increased at 200 ng/ml of DON in T84 cells. Enzyme activities in Caco-2 cells were also examined. Alkaline phosphatase activity was reduced from the 6th to 15th day of culture in the presense of 100 or 200 ng/ml of DON, whereas sucrase- isomaltase activity was significantly decreased by adding 50 or 100 ng/ml of DON for 15 or 20 days. Protein content was attenuated only by treatment with 200 ng/ml of DON thoughout the experimental period. The results indicate that DON interferes with structural and functional characteristics of differentiation in enterocytes at low doses. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fusarium toxin contents of maize and maize products purchased in the years 2000 and 2001 in Germany

Samples (n=106) of maize and maize products were analysed for 13 trichothecene toxins and zearalenone (ZON). All 14 toxins examined were detected, although with varying frequency. Cooccurrence of two or more toxins was observed in 96% of samples. The toxins of the scirpenol group scirpentriol, 15-monoacetoxyscirpenol and diacetoxyscirpenol were detected in 14, 27 and 3% of the samples analysed, the toxins of the T-2 group T-2 toxin, HT-2 toxin, T-2 triol und T-2 tetraol were found in 33, 66, 2 and 7%. Toxin content was higher in feeds than in foods (semolina and flour). In food samples, the German regulatory level for DON (500 μg/kg) was not exceeded, three samples of maize flour contained ZON above the regulatory level (50 μg/kg). Presented at the 26^th Mykotoxin-Workshop in Herrching, Germany, May17–19, 2004     

Analysis of Fusarium toxins in maize and wheat using thin layer chromatography

Thin layer chromatography (TLC) methods for identifying and quantifying deoxynivalenol (DON), fumonisin B1 (FB1) and zearalenone in grain samples were compared to immunoassay (ELISA) and high performance liquid chromatography (HPLC) methods to determine the reliability of the less expensive TLC. There was a very good agreement between levels of DON measured by TLC and competitive-direct ELISA, and between levels of fumonisin B1 measured by TLC and HPLC, over a wide range of concentrations. Correlation coefficients (Pearson's) were 0.978, 0.914 and 0.953 for DON in maize, DON in wheat and FB1 in maize respectively. A lower correlation coefficient (r = 0.672) was obtained when zearalenone was quantified by TLC and HPLC. Possible reasons for this are discussed. A cost comparison of the various methods revealed that TLC was the least expensive for sample analysis. It is recommended that researchers choose which analytical method to use based upon individual considerations of cost and precision. This revised version was published online in June 2006 with corrections to the Cover Date.     

A survey on the occurrence of mycotoxins in wheat and maize from western Romania

Samples of wheat (n = 25) and maize (n = 30) for animal consumption, collected in 1997 after harvest from western Romania, were analyzed by enzyme immunoassays for mycotoxin contamination. Toxins analyses included deoxynivalenol (DON), 3-acetylDON, 15- acetylDON, fusarenone X (FX), T-2 Toxin (T-2), diacetoxyscirpenol (DAS), zearalenone (ZEA), fumonisin B1 (FB1), aflatoxin B1 (AFB1), ochratoxin A (OA), and citrinin (CT). DON and acetylDONs were the major contaminants in wheat (100%) and maize (46%). Median values for DON, 3-acetylDON, and 15-acetylDON were 880 μg kg-1, 66 μg kg- 1, and 150 μg kg-1 in wheat, and 890 μg kg-1, 180 μg kg-1, and 620 μg kg- 1 in maize, respectively. Additionally, 3,15-diacetylDON was detected in some samples by HPLC-EIA analysis. All samples were negative for FX (<150 μg kg-1). T-2 was found in wheat (n = 6) and maize (n = 1) at levels between 13 and 63 μg kg- 1. DAS (2.6 μg kg-1) was found in one maize sample. ZEA occurred in all wheat and in four maize samples, median values were 10 μg kg-1 and 250 μg kg-1, respectively. One maize sample contained FB1 (140 μg kg-1). All samples were AFB1-negative (<4 μg kg-1). OA was found in one wheat sample (37 μg kg- 1), CT was found in one maize sample (580 μg kg- 1). This first reported natural occurrence of a range of mycotoxins in Romanian feeding stuff shows that DON and acetyl DONs may be present at levels which may affect animal production. This revised version was published online in June 2006 with corrections to the Cover Date.     

Sample clean-up methods,immunoaffinity chromatography and solid phase extraction,for determination of deoxynivalenol and deepoxy deoxynivalenol in swine serum

Concentrations of deoxynivalenol (DON) and deepoxy deoxynivalenol (DOM-1) in animal blood are important parameters for studies in toxicology and biological detoxification of DON. Clean-up methods, using either immunoaffinity chromatography (IAC) or solid phase extraction (SPE), were compared in order to determine the free form of DON or DOM-1 and the sum amount (free form plus glucuronide conjugated form of DON or DOM-1), respectively, in swine serum. Detection was achieved by high performance liquid chromatography with ultraviolet detection (HPLC-UV). Compared with the SPE-HPLC method, the IAC-HPLC method provided lower quantitation limit (DON: 18 vs 42 ng/ml; DOM-1: 21 vs 30 ng/ml) and higher recoveries (DON: 93.4–102.7% vs 63.7–85.3%; DOM-1: 85.5–91.1% vs 68.0–82.6%). Compared with previously published methods, the developed IAC-HPLC method removed analytical interferences from swine serum in one quick and easy step, and eliminated steps of extraction with organic solvent and/or pre-purification using SPE cartridges. This IAC-HPLC method was used to analyze swine serum samples collected from pigs that were evaluated in a feeding trial of a microbiological detoxification of DON. No DON or DOM-1 were detected in serum samples from pigs given a toxin-free diet or a microbial control diet. In serum samples from pigs given a DON diet (5 mg/kg of DON), free form DON and sum free DON + conjugated DON were 38.8 ± 13.7 and 49.8 ± 14.1 ng/ml, respectively. In serum samples from those given a detoxified-DON diet (DON was transformed to DOM-1), free form DOM-1 was detected but not quantified, and the sum DOM-1 was found as 47.5 ± 6.3 ng/ml.     

Analysis of herbicides: demonstration of the utility of enzyme immunoassay verification by HPLC

Evidence has accumulated that herbicides in the environment present a significant health hazard to the population. Therefore, the levels of heavily used substances such as atrazine and simazine and their metabolites need to be regularly assessed. The objective was to develop a rapid and simple tube ELISA procedure suitable for use in field studies and non-specialized laboratories. The antisera used were polyclonal antibodies raised in sheep against atrazine or simazine amido caproic acid conjugated to bovine serum albumin. The antibodies were first used to construct a two-step competitive ELISA procedure in 96-well microtitre plates. The 96-well format was then adapted to a coated-tube enzyme immunoassay, by immobilization of hapten-gelatine conjugates on polystyrene tubes. This enabled the colour to be read using a basic spectrophotometer. Soil samples were collected from agricultural and non-agricultural sites in Poland. Atrazine and simazine were extracted by liquid extraction from soil and assayed by tube ELISA. In addition, the samples were extracted by solid-phase extraction before analysis by HPLC. The immunoassays and chemical analysis were carried out by different individuals who were unaware of each other's results, which were then compared at the end of the study. Correlation of the two methods was excellent, with R=98.7 and 81.3 for atrazine and simazine, respectively. The immunoassay yielded the same order of results without having to perform solid-phase extraction before analysis. The study has demonstrated that the simple antigen-coated tube assay provides a cost-effective and valuable screening test. Comparison with the more elaborate, heavily labour-intensive HPLC analysis demonstrated that the results obtained by the simpler enzyme-immunoassay tests were within the same order.     

Knowing your enemies: Integrating molecular and ecological methods to assess the impact of arthropod predators on crop pests

The importance of natural enemies as the foundation of integrated pest management (IPM) is widely accepted, but few studies conduct the manipulative field experiments necessary to directly quantify their impact on pest populations in this context. This is particularly true for predators. Studying arthropod predator–prey interactions is inherently difficult: prey items are often completely consumed, individual predator–prey interactions are ephemeral (rendering their detection difficult) and the typically fluid or soft‐bodied meals cannot be easily identified visually within predator guts. Serological techniques have long been used in arthropod predator gut‐contents analysis, and current enzyme linked immunosorbent assays (ELISA) are highly specific and sensitive. Recently, polymerase chain reaction (PCR) methods for gut‐contents analysis have developed rapidly and they now dominate the diagnostic methods used for gut‐contents analysis in field‐based research. This work has identified trophic linkages within food webs, determined predator diet breadth and preference, demonstrated the importance of cannibalism and intraguild predation within and between certain taxa, and confirmed the benefits (predator persistence) and potential disadvantages (reduced feeding on pest species) of the availability of alternative nonpest prey. Despite considerable efforts to calibrate gut‐contents assays, these methods remain qualitative. Available techniques for predator gut‐contents analysis can provide rapid, accurate, cost‐effective identification of predation events. As such, they perfectly compliment the ecological methods developed to directly assess predator impacts on prey populations but which are imperfect at identifying the key predators. These diagnostic methods for gut‐contents analysis are underexploited in agricultural research and they are almost never applied in unison with the critical field experiments to measure predator impact. This paper stresses the need for a combined approach and suggests a framework that would make this possible, so that appropriate natural enemies can be targeted in conservation biological control.