单端孢霉烯族毒素的产生及分子调控机制

禾谷镰刀菌复合种(Fusarium graminearum species complex,FGSC)引起的赤霉病是小麦生产上危害最为严重的病害之一。赤霉病除了造成减产外,感病籽粒中含有多种镰刀菌毒素,如单端孢霉烯族的呕吐毒素,可引起人畜中毒和重大疾病,给食品安全构成严重威胁。过去20年,随着禾谷镰刀菌全基因组序列的公布和遗传转化体系的成熟,禾谷镰刀菌Fusarium graminearum的功能基因组学的研究取得了较大进展,单端孢霉烯族毒素的产生、调控机制及网络研究成为热点。本文综述国内外单端孢霉烯族毒素的生物合成和分子调控机制,包括合成基因簇及决定不同产毒化学型的基因、产毒调控元件、环境因子调控产毒的分子机制,可为小麦抗赤霉病的育种提供新思路,为新型药剂的研发提供分子靶标,为赤霉病的持续防控和毒素污染的有效治理提供理论依据。     

Development of a generic PCR detection of deoxynivalenol- and nivalenol-chemotypes of Fusarium graminearum

Based on the intergenic sequences of Tri5-Tri6 genes involved in the mycotoxin pathways of Fusarium species, a generic PCR assay was developed to detect a 300 bp fragment of deoxynivalenol (DON)-chemotypes and a 360 bp sequence of nivalenol (NIV)- chemotypes of Fusarium graminearum. Mycotoxin chemotypes identified by the PCR assays were confirmed by the chemical analyses of HPLC or GC/MS. Further analysis of 364 F. graminearum isolates from 12 provinces of China showed that 310 were DON-chemotypes and 54 were NIV-chemotypes. Sequence analyses revealed that DON-chemotypes display more variations than NIV-chemotypes. This PCR assay could be used to detect mycotoxin-producing Fusarium-species and may thus help to develop strategies to avoid or reduce mycotoxin contamination of cereals. Also this assay may provide useful alternatives to antibody-based mycotoxin tests.     

Mycotoxins in organic and conventional cereals and cereal products grown and marketed in Croatia

In this study, the levels of aflatoxin B₁ (AFB₁), ochratoxin A (OTA), zearalenone (ZEN), deoxynivalenol (DON) and fumonisins (FUM) in unprocessed cereals (n = 189) and cereal-based products (n = 61) were determined using validated ELISA methods. All samples originated from either conventional or organic production corresponded to the 2015 harvest in Croatia. Based on the mean mycotoxin concentrations, the risk for the consumer to exceed the tolerable daily intake (TDI) for these toxins by the consumption of both types of cereals and cereal-based products was assessed. Mycotoxin contamination of organic cereals and organic cereal-based products was not significantly different (p > 0.05). Given that the exposure assessment resulted in a small fraction of the TDI (maximum: DON, 12% of TDI), the levels of the investigated mycotoxins in both types of cereals and cereal-based products from the 2015 harvest did not pose a human health hazard.     

Immunochemical assessment of mycotoxins in 1989 grain foods: evidence for deoxynivalenol (vomitoxin) contamination.

To assess the potential for mycotoxin contamination of the human food supply following the 1988 U.S. drought, 92 grain food samples were purchased from retail outlets in the summer of 1989 and surveyed for aflatoxin B1, zearalenone, and deoxynivalenol (DON [vomitoxin]) by monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Only one sample (buckwheat flour) was found to contain aflatoxin B1 (12 ng/g), whereas zearalenone was found in 26% of the samples at a mean concentration of 19 ng/g. In contrast, the DON ELISA was positive in 50% of the samples at a detection level of 1.0 micrograms/g. Between 63 and 88% of corn cereals, wheat flour/muffin mixes, rice cereals, and corn meal/muffin mixes yielded positive results for DON, whereas 25 to 50% of oat cereals, wheat- and oat-based cookies/crackers, corn chips, popcorn, and mixed-grain cereals were positive for DON. The mean DON content of the positive samples was 4.0 micrograms/g, and the minimum and maximum levels were 1.2 and 19 micrograms/g, respectively. When positive ELISA samples were also analyzed by high-performance liquid chromatography, a strong correlation between the two methods was found. The presence of DON in the two highest samples, corn meal and mixed-grain cereal, which contained 19 and 16 micrograms/g, respectively, was quantitatively confirmed by gas chromatography-mass spectrometry. The results indicated that DON was present in 1989 retail food products at concentrations that exceeded those found in previous market surveys and that have been experimentally associated with impaired animal health.     

Immunochemical assessment of mycotoxins in 1989 grain foods: evidence for deoxynivalenol (vomitoxin) contamination

To assess the potential for mycotoxin contamination of the human food supply following the 1988 U.S. drought, 92 grain food samples were purchased from retail outlets in the summer of 1989 and surveyed for aflatoxin B1, zearalenone, and deoxynivalenol (DON [vomitoxin]) by monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Only one sample (buckwheat flour) was found to contain aflatoxin B1 (12 ng/g), whereas zearalenone was found in 26% of the samples at a mean concentration of 19 ng/g. In contrast, the DON ELISA was positive in 50% of the samples at a detection level of 1.0 micrograms/g. Between 63 and 88% of corn cereals, wheat flour/muffin mixes, rice cereals, and corn meal/muffin mixes yielded positive results for DON, whereas 25 to 50% of oat cereals, wheat- and oat-based cookies/crackers, corn chips, popcorn, and mixed-grain cereals were positive for DON. The mean DON content of the positive samples was 4.0 micrograms/g, and the minimum and maximum levels were 1.2 and 19 micrograms/g, respectively. When positive ELISA samples were also analyzed by high-performance liquid chromatography, a strong correlation between the two methods was found. The presence of DON in the two highest samples, corn meal and mixed-grain cereal, which contained 19 and 16 micrograms/g, respectively, was quantitatively confirmed by gas chromatography-mass spectrometry. The results indicated that DON was present in 1989 retail food products at concentrations that exceeded those found in previous market surveys and that have been experimentally associated with impaired animal health.     

A Model Transgenic Cereal Plant with Detoxification Activity for the Estrogenic Mycotoxin Zearalenone

Zearalenone (ZEN) is an estrogenic mycotoxin produced by the necrotrophic cereal pathogen Fusarium graminearum. This mycotoxin is detoxified by ZHD101, a lactonohydrolase from Clonostachys rosea, or EGFP:ZHD101, its fusion to the C-terminus of an enhanced green fluorescence protein. We previously showed that egfp:zhd101 is efficiently expressed in T₀ leaves of rice. In this study, we assessed the feasibility of in planta detoxification of the mycotoxin using progeny. When protein extract from T₁ leaves was incubated with ZEN, the amount of the toxin decreased significantly as measured by HPLC. ZEN degradation activity was also detected in vivo in transgenic T₂ seeds. These results suggest that zhd101 can be exploited as an efficient and cost-effective system for protection of important cereals that are more susceptible to the pathogen (e.g., wheat and maize) from contamination with the estrogenic mycotoxin.     

The cereals root eelworm,Heterodera major (O. Schmidt) Franklin,in North Wales

The cereals root eelworm Heterodera major (O. Schmidt) is shown to be locally distributed in North Wales, the most heavily infested centres being in the arable districts of the eastern counties of the province. The most severe attacks occurred on oats where the crop had been grown too frequently; wheat and barley were apparently unaffected at the cyst populations encountered.     

Comparison of Canadian Fusarium graminearum isolates for aggressiveness,vegetative compatibility,and production of ergosterol and mycotoxins

Fusarium graminearum is the predominant pathogen causing fusarium head blight of cereals in North America. Fifteen Canadian isolates of Fusarium graminearum were highly diverse in terms of vegetative compatibility grouping (VCG) and varied for production of ergosterol and mycotoxin production in rice culture. Aggressiveness was assessed by scoring the disease severity incited in wheat spikes by each isolate. Two inoculation methods, single-floret injection and spray of entire spikes, were used to screen 4 wheat varieties for reaction to the F. graminearum isolates. All isolates were of broadly similar aggressiveness, with disease severity ranging from 17.2 to 39.1 for single floret injection, and 39.1 to 69.0 for spray inoculation. Disease severity, ergosterol production, and mycotoxin development were not correlated. Using nitrate non-utilizing mutants the 15 isolates were grouped into 14 VCGs. Deoxynivalenol (DON) was produced by all isolates in rice culture, at levels between 0.2 and 249 ppm. 15-acetyldeoxynivalenol was produced by 14 of the 15 isolates at levels between 0.4 and 44.6 ppm. These results reveal a high level of diversity for several characteristics among F. graminearum isolates from Canada. This revised version was published online in June 2006 with corrections to the Cover Date.     

Fusarium cerealis Associated with Barley Seeds in Argentina

Fusarium head blight is a fungal disease caused by a complex of Fusarium species on cereals, such as barley and wheat. It has economic impacts due to yield reductions and mycotoxin contamination. As barley production has increased considerably in the last 5 years in Argentina, a survey was conducted for identifying Fusarium species associated with barley grains. Fusarium cerealis was isolated and identified based on morphological and molecular analysis. The potential production of nivalenol and zearalenone was assessed using specific PCR assays. Koch′s postulates were carried out to confirm the pathogenicity of the fungus.     

An electrochemical competitive biosensor for ochratoxin A based on a DNA biotinylated aptamer

Ochratoxin A (OTA) is one of the most important mycotoxin contaminants of foods, particularly cereals and cereal products, with strict low regulatory levels (of ppb) in many countries worldwide. An electrochemical competitive aptamer-based biosensor for OTA is described. Paramagnetic microparticle beads (MBs) were functionalized with an aptamer specific to OTA, and were allowed to compete with a solution of the mycotoxin conjugated to the enzyme horseradish peroxidase (OTA-HRP) and free OTA. After separation and washing steps helped with magnetic separations, the modified MBs were localized on disposable screen-printed carbon electrodes (SPCEs) under a magnetic field, and the product of the enzymatic reaction with the substrate was detected with differential-pulse voltammetry. In addition to magnetic separation assays, other competitive schemes (direct/indirect aptasensors performed on the SPCEs surface or using gold nanoparticles functionalized with the aptamer) were preliminary tested, optimized and compared. The magnetic aptasensor showed a linear response to OTA in the range 0.78-8.74 ng mL(-1) and a limit of detection of 0.07±0.01 ng mL(-1), and was accurately applied to extracts of certified and spiked wheat samples with an RSD lower than about 8%.     

A practical method for measuring deoxynivalenol, nivalenol, and T-2 + HT-2 toxin in foods by an enzyme-linked immunosorbent assay using monoclonal antibodies

We have developed and tested an enzyme-linked immunosorbent assay system for individual measurement of deoxynivalenol, nivalenol, and T-2 + HT-2 toxin using monoclonal antibodies for 3,4,15-triacetyl-nivalenol, for both 3,4,15-triacetyl-nivalenol and 3,15-diacetyl-deoxynivalenol, and for acetyl-T-2 toxin. The assay system comprised three kits (desinated the DON + NIV kit, the NIV kit, and the T-2 + HT-2 kit). The practical performance of the enzyme-linked immunosorbent assay system was assessed by assaying trichothecene mycotoxins in wheat kernels. The enzyme-linked immunosorbent assay system meets all the requirements for use in a routine assay in terms of sensitivity (detection limit: deoxynivalenol 80 ng/g, nivalenol 80 ng/g, T-2 toxin 30 ng/g), reproducibility (total coefficient of variation: 1.9-6.2%), accuracy (recovery: 93.8-112.0%), simplicity and rapidity (time required: <2 h), mass handling (>42 samples/assay), and a good correlation with gas chromatography-mass spectrometry (r=0.9146-0.9991). Components derived from the wheat extract did not interfere with the assay kits. The enzyme-linked immunosorbent assay system is a useful alternative method to gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, or liquid chromatography-ultraviolet absorption for screening cereals and foods for trichothecene mycotoxin contamination.     

Correlation between DNA of trichothecene-producing Fusarium species and deoxynivalenol concentrations in wheat-samples

AIMS: Correlations between DNA content of trichothecene-producing Fusarium spp. and concentration of the key mycotoxin deoxynivalenol (DON) in cereal samples. METHODS AND RESULTS: A LightCycler PCR-based assay was used to quantify the DNA from trichothecene-producing Fusarium spp. in 300 wheat samples. DNA concentrations ranged from not detectable to 16.3 mg kg-1 whereas DON concentrations (GC/MS data) varied from not detectable to 34.3 mg kg-1. Data analysis revealed a coefficient of correlation r=0.9557 between DON concentrations and DNA-amounts over all samples. An interval of confidence for P=95% was calculated based on samples with DON concentrations < or = 1.5 mg kg-1 (n=234). CONCLUSIONS: Quantification of 32 samples of Fusarium-contaminated wheat was performed within 45 min. Data analysis allowed estimation of DON contamination from quantitative PCR data in the wheat samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The method described is useful for the screening of cereals in industrial quality control.     

Fusarium langsethiae – a HT‐2 and T‐2 Toxins Producer that Needs More Attention

Fusarium langsethiae is a toxigenic fungus that was formally described as a new species in 2004. This fungus was first detailed in the 1990s but was initially referred to as ‘powdery Fusarium poae’ having a spore morphology similar to F. poae but a mycotoxin profile like that of Fusarium sporotrichioides. The species has been isolated from infected oat, wheat and barley grains but has been reported as more problematic in the former crop rather than the latter two. Whilst the epidemiology of F. langsethiae remains unclear, the fungus has been shown to produce high levels of type‐A trichothecenes HT‐2 and T‐2 toxins in small‐grain cereals. HT‐2 and T‐2 toxins are two of the most potent trichothecenes capable of inhibiting protein synthesis in eukaryotes. In this regard, mycotoxin contamination caused by F. langsethiae is clearly a food and feed safety hazard. With the European Commission considering legislation of HT‐2 and T‐2 toxins, more information is required not only on the producer and conditions favouring mycotoxin production, but also on reliable methods of pathogen detection and reduction of cereal contamination. This review describes recent research concerning the known epidemiology of F. langsethiae and suggestions of what needs to be known about the fungus in order to be able to understand and employ measures for preventing its infection and contamination of cereals with HT‐2 and T‐2 toxins.     

Natural occurrence of the mycotoxin fusarochromanone, a metabolite of Fusarium equiseti, in cereal feed associated with tibial dyschondroplasia

The mycotoxin fusarochromanone, a metabolite of Fusarium fungi, is able to induce tibial dyschondroplasia (TD) in chickens under experimental conditions. On the basis of health surveillance data on TD, two broiler farms with TD prevalence rates of up to 56% were identified. In the corresponding pelleted feed samples, fusarochromanone was detected in all 12 samples analyzed by column purification and TLC, with concentrations 4 to 59 micrograms/kg. No Fusarium fungi were available from the feed because of the pelleting process, but seven Fusarium equiseti strains previously isolated from Danish cereals were checked for fusarochromanone production, and all produced fusarochromanone at 57 to 1,435 mg/kg. Thus, the potential for fusarochromanone production by F. equiseti is considerable. The identification of fusarochromanone from feed and F. equiseti was confirmed by mass, infrared, and nuclear magnetic resonance spectral analyses. This is the first report of fusarochromanone as a naturally occurring contaminant.     

Content of the <Emphasis Type="Italic">Alternaria</Emphasis> mycotoxin tenuazonic acid in food commodities determined by a stable isotope dilution assay

The Alternaria mycotoxin tenuazonic acid (TA) was quantified in fruit juices (n = 50), cereals (n = 12) and spices (n = 38) using a recently developed stable isotope dilution assay (SIDA). [¹³ C₆,¹⁵ N]-TA was used as the internal standard. Method validation revealed low limits of detection (LODs) of 0.15 μg/kg (fruit juices), 1.0 μg/kg (cereals) and 17 μg/kg (spices). The respective limits of quantitation were about three times higher. Recovery was about 100% for all matrices. The precision (relative standard deviation of replicate analyses of naturally contaminated samples) was 4.2% (grape juice; 1.7 μg/kg), 3.5% (whole wheat flour; 36 μg/kg) and 0.9% (curry powder; 215 μg/kg). The median content of TA in the analyzed samples was 1.8 μg/kg (fruit juices), 16 μg/kg (cereals) and 500 μg/kg (spices). Positive samples amounted to 86% (fruit juices), 92% (cereals) and 87% (spices).     

Natural occurrence of the mycotoxin fusarochromanone, a metabolite of Fusarium equiseti, in cereal feed associated with tibial dyschondroplasia.

The mycotoxin fusarochromanone, a metabolite of Fusarium fungi, is able to induce tibial dyschondroplasia (TD) in chickens under experimental conditions. On the basis of health surveillance data on TD, two broiler farms with TD prevalence rates of up to 56% were identified. In the corresponding pelleted feed samples, fusarochromanone was detected in all 12 samples analyzed by column purification and TLC, with concentrations 4 to 59 micrograms/kg. No Fusarium fungi were available from the feed because of the pelleting process, but seven Fusarium equiseti strains previously isolated from Danish cereals were checked for fusarochromanone production, and all produced fusarochromanone at 57 to 1,435 mg/kg. Thus, the potential for fusarochromanone production by F. equiseti is considerable. The identification of fusarochromanone from feed and F. equiseti was confirmed by mass, infrared, and nuclear magnetic resonance spectral analyses. This is the first report of fusarochromanone as a naturally occurring contaminant.     

High prevalence of theNBN gene mutation c.657-661del5 in Southeast Germany

Nijmegen breakage syndrome (NBS), a rare autosomal recessive chromosomal instability disorder, is caused by mutations in theNBN gene. Most patients known so far are of Slavic origin and carry the major founder mutation c.657-661del5. Due to an unexpectedly high incidence of NBS patients (homozygous for the c.657-661del5 mutation) in a Northeast Bavarian region in Southeast Germany, we estimated the prevalence of this mutation in this area and compared it to another German region. We found a high carrier frequency of 1/176 for the c.657-661del5 mutation among newborns in Northeast Bavaria, while the frequency of the mutation in Berlin was 1/990. We further studied families from a Slavic population isolate, the Sorbs, in the Lusatian region in Northeast Saxony, and revealed a prevalence of the c.657-661del5 mutation of 1/34. Whereas the Slavic origin of the Sorbs has been known, we attribute the surprisingly high frequencies of c.657-661del5 mutation in Bavaria (similar to frequencies of this mutation in various Eastern European countries) to a high percentage of people of Slavic origin in Northeast Bavaria.     

Standardization of an indirect PTA-ELISA for detection ofFusarium spp. in infected grains

Plant pathogenic fungi of the genusFusarium cause agriculturally important diseases of small grain cereals and maize. Especially the contamination of grains with the mycotoxin deoxynivalenol (DON) is harmful for animals and humans. For quantification of the severity ofFusarium head blight (FHB) in resistance evaluation and selection of cereals a fast, economical and reliable method is essential. Immunological methods appear to be particularly suitable for such an assessment. The results demonstrate that a selected antiserum was appropriate for the detection ofFusarium-exoantigens (ExoAg) in cereal grains by an indirect ELISA-format, although a discrimination between variousFusarium-species was not possible. The polyclonal antibody detection system was optimized for different parameters and a standard test protocol was elaborated. Presented at the 26^th Mykotoxin-Workshop in Herrsching, Germany, May 17–19, 2004     

Occurrence of<Emphasis Type="Italic">Fusarium</Emphasis> toxins in the 1999’s harvest

Continuing the monitoring ofFusarium toxins in cereals, we investigated 245 samples of wheat, barley, triticale and oats in 1999. 84 samples out of 100 analysed forFusarium could be found to be infected. The most prominentFusarium species detected whereF. avenaceum, F. poae, F. detected whereF. avenaceum, F. poae, F. graminearum, andF. sporotrichoides. The level of mycotoxin contamination of the samples varied depending on their origins and was in general very low in comparison with the result obtained in samples of the previous year. There where only some wheat samples with deoxynivalenol (DON) concentrations beyond existing advisory levels. The average DON concentration of all samples was 0.35 mg/kg with a median of 0.007 mg/kg. 3-Acetyldeoxynivalenol and zearalenone (ZEA) could only be detected at minor concentrations (below 0.1 and 0.05 mg/kg, respectively) in less than 10% of the samples. The analysis of commercial cereal flour reflects this situation. Flour bought in the first quarter of 1999, which was suspected to contain a high portion of the 1998 harvest, was contaminated by DON to a higher extent than those purchased in 2000. The average DON concentration in the flour samples of 1999 and 2000 was 0.35 mg/kg and 0.23 mg/kg, respectively. Although the general mycotoxin level in the 1999 harvest was lower as in 1998 there were some highly contaminated samples that had mainly been grown in fields with either maize or other cereals as previous crop and reduced tillage. The combination of maize as previous crop and non-tillage could be stated as most unsuitable, which promotes enhanced mycotoxin contamination, and should therefore be avoided.     

Combined metabonomic and quantitative real-time PCR analyses reveal systems metabolic changes of Fusarium graminearum induced by Tri5 gene deletion

Fusarium graminearum (FG) is a serious plant pathogen causing huge losses in global production of wheat and other cereals. Tri5-gene encoded trichodiene synthase is the first key enzyme for biosynthesis of trichothecene mycotoxins in FG. To further our understandings of FG metabolism which is essential for developing novel strategies for controlling FG, we conducted a comprehensive investigation on the metabolic changes caused by Tri5-deletion by comparing metabolic differences between the wild-type FG5035 and an FG strain, Tri5(-), with Tri5 deleted. NMR methods identified more than 50 assigned fungal metabolites. Combined metabonomic and quantitative RT-PCR (qRT-PCR) analyses revealed that Tri5 deletion caused significant and comprehensive metabolic changes for FG apart from mycotoxin biosynthesis. These changes involved both carbon and nitrogen metabolisms including alterations in GABA shunt, TCA cycle, shikimate pathway, and metabolisms of lipids, amino acids, inositol, choline, pyrimidine, and purine. The hexose transporter has also been affected. These findings have shown that Tri5 gene deletion induces widespread changes in FG primary metabolism and demonstrated the combination of NMR-based metabonomics and qRT-PCR analyses as a useful way to understand the systems metabolic changes resulting from a single specific gene knockout in an eukaryotic genome and thus Tri5 gene functions.