Enhancement of trichothecene production in Fusarium graminearum by cobalt chloride

The effects of cobalt chloride on the production of trichothecene and ergosterol in Fusarium graminearum were examined. Incorporation experiments with (13)C-labeled acetate and leucine confirmed that both 3-acetyldeoxynivalenol and ergosterol were biosynthesized via a mevalonate pathway by the fungus, although hydroxymethyl-glutaryl CoA (HMG-CoA) from intact leucine was able to be partially used for ergosterol production. Addition of cobalt chloride at concentrations of 3-30 μM into liquid culture strongly enhanced 3-acetyldeoxynivalenol production by the fungus, whereas the amount of ergosterol and the mycelial weight of the fungus did not change. The mRNA levels of genes encoding trichothecene biosynthetic proteins (TRI4 and TRI6), ergosterol biosynthetic enzymes (ERG3 and ERG25), and enzymes involved in the mevalonate pathway (HMG-CoA synthase (HMGS) and HMG-CoA reductase (HMGR)) were all strongly up-regulated in the presence of cobalt chloride. Precocene II, a specific trichothecene production inhibitor, suppressed the effects of cobalt chloride on Tri4, Tri6, HMGS, and HMGR, but did not affect erg3 and erg25. These results indicate that cobalt chloride is useful for investigating regulatory mechanisms of trichothecene and ergosterol production in F. graminearum.     

Determination of triazine and chloroacetanilide herbicides in soils by microwave-assisted extraction (MAE) coupled to gas chromatographic analysis with either GC-NPD or GC-MS

A simple and rapid method based on microwave-assisted extraction (MAE) coupled to gas chromatographic analysis was developed for the analysis of triazine (atrazine, cyanazine, metribuzine, simazine and deethylatrazine, and deisopropylatrazine) and chloroacetanilide (acetochlor, alachlor, and metolachlor) herbicide residues in soils. Soil samples are processed by MAE for 5 min at 80 degrees C in the presence of acetonitrile (20 mL/sample). Mean recovery values of most solutes are >80% in the 10 to 500 microg/kg fortification range with respective RSDs (relative standard deviations) < 20%. The limits of quantification (LOQ) and limits of detection (LOD) are 10 and 1 to 5 microg/kg, respectively. The method was validated with two types of soils containing 1.5 and 3.0% organic matter content, respectively; no statistically significant differences were found between solute recovery values from the two types of soils. The solute mean recovery values from freshly spiked (24 h aging) and spiked samples stored refrigerated for one week before processed were also not statistically different. Residue levels determined in field weathered soils were higher when soils were processed by MAE than with a comparison method based on flask-shaking of soil suspensions overnight. Extracts were analyzed by a gas chromatographic system equipped either with a thermionic (GC-NPD) or a mass spectrometric detector (GC-MS).     

Fusarium graminearum TRI14 is required for high virulence and DON production on wheat but not for DON synthesis in vitro

Fusarium head blight (FHB) of wheat (Triticum aestivum L.), caused by the fungus Fusarium graminearum, is a major concern worldwide. FHB grain is reduced in yield, may fail to germinate, and is often contaminated with deoxynivalenol, a trichothecene mycotoxin linked to a variety of animal diseases and feed refusals. Annual losses in the tens of millions of dollars due to FHB underscore the need to develop improved methods of disease control and prevention. Previous research has identified deoxynivalenol biosynthesis as a virulence factor on wheat. Recently, we found that the TRI14 gene of F. sporotrichioides, closely related to F. graminearum, was not required for synthesis of a related trichothecene, T-2 toxin. TRI14 does not share similarity with any previously described genes in the databases. In this study, we examined the role that F. graminearum TRI14 may play in both deoxynivalenol synthesis and in virulence on wheat. TRI14 deletion mutants synthesize deoxynivalenol on cracked maize kernel medium and exhibit wild-type colony morphology and growth rate on complex and minimal agar media. However, FHB assays on greenhouse-grown wheat indicate that FgDeltaTri14 mutants cause 50-80% less disease than wild type and do not produce a detectable quantity of deoxynivalenol on plants. We discuss a number of possible roles that TRI14 may play in the disease process.     

Rapid detection of kernel rots and mycotoxins in maize by near-infrared reflectance spectroscopy

Near-infrared (NIR) spectroscopy is a practical spectroscopic procedure for the detection of organic compounds in matter. It is particularly useful because of its nondestructiveness, accuracy, rapid response, and easy operation. This work assesses the applicability of NIR for the rapid identification of micotoxigenic fungi and their toxic metabolites produced in naturally and artificially contaminated products. Two hundred and eighty maize samples were collected both from naturally contaminated maize crops grown in 16 areas in north-central Italy and from ears artificially inoculated with Fusarium verticillioides. All samples were analyzed for fungi infection, ergosterol, and fumonisin B1 content. The results obtained indicated that NIR could accurately predict the incidence of kernels infected by fungi, and by F. verticillioides in particular, as well as the quantity of ergosterol and fumonisin B1 in the meal. The statistics of the calibration and of the cross-validation for mold infection and for ergosterol and fumonisin B1 contents were significant. The best predictive ability for the percentage of global fungal infection and F. verticillioides was obtained using a calibration model utilizing maize kernels (r2 = 0.75 and SECV = 7.43) and maize meals (r2 = 0.79 and SECV = 10.95), respectively. This predictive performance was confirmed by the scatter plot of measured F. verticillioides infection versus NIR-predicted values in maize kernel samples (r2 = 0.80). The NIR methodology can be applied for monitoring mold contamination in postharvest maize, in particular F. verticilliodes and fumonisin presence, to distinguish contaminated lots from clean ones, and to avoid cross-contamination with other material during storage and may become a powerful tool for monitoring the safety of the food supply.     

Possible role of plant phenolics in the production of trichothecenes by Fusarium graminearum strains on different fractions of maize kernels

Four trichothecene-producing strains of Fusarium graminearum were grown on three maize grain fractions, whole grain, degermed grain, and the germ, to determine the effect of natural substrates on mycotoxin production. Monitoring the ergosterol content after 25 days of incubation indicated that fungal growth on all grain fractions was comparable. Trichothecene (TCT) production was highest on degermed grain, less on whole grain, and very low or nondetectable on the germ; similar results were found with four different strains. It was concluded that inhibitor(s) of TCT biosynthesis were present in maize germ. The presence of phenolic compounds was investigated in the different fractions. The hydroxamate 4-acetylbenzoxazolin-2-one (4-ABOA), a known inhibitor of mycotoxin production, was found in the degermed and whole grain fractions but not in the germ. Therefore, the TCT inhibition observed on the maize germ fraction used in our study is clearly not linked to 4-ABOA. Other soluble phenolic compounds were found at a much higher concentration in the germ than in the two other fractions. The inhibition property of the soluble ester-bound extracts was tested in liquid culture. A possible role for these compounds is discussed.     

Impact of competitive fungi on Trichothecene production by Fusarium graminearum

Bioassays were used to determine the production of the trichothecene mycotoxin, deoxynivalenol (DON), by two isolates of Fusarium graminearum when grown in association with potentially competitive fungi and an antifungal chemical, 6-pentyl-alpha-pyrone (6PAP). The presence of 6PAP in the culture medium reduced DON production by as much as 80%, but this effect was reduced for the F. graminearum isolate that most efficiently metabolized the added 6PAP. A 6PAP-producing Trichoderma isolate grown in a competition assay system with the F. graminearum isolates was also able to substantially reduce DON production. When Fusarium isolates (F. crookwellense, F. culmorum, F. subglutinans, F. poae, F. equiseti, F. avenaceum, and F. sambucinum), which co-occur with F. graminearum in New Zealand maize plants (Zea mays), were grown in competition assays, the effect on DON production was variable. However, all isolates of F. subglutinans tested were shown to cause reductions in DON production (by 13-76%, mean = 62%). F. subglutinans frequently co-occurs with F. graminearum, but its presence can vary with location and time of the season. When the competitive fungus tested was also a trichothecene producer (e.g., of nivalenol), both toxins were produced in the assay medium. The results indicate that mycotoxin production by F. graminearum can be affected by the presence of particular competitive fungi. These results have implications for an ecological understanding of pathogenicity and of mycotoxin accumulation in plants. Early establishment of F. subglutinans, for example, may act as a biological control mechanism providing a temporary protection against invasion by more commonly toxigenic fusaria such as F. graminearum.     

Near-Infrared Spectroscopy for Estimation of Ergosterol Content in Barley: A Comparison Between Reflectance and Transmittance Techniques

The fungal-specific lipid ergosterol correlates with fungal biomass and often also with the degree of mycotoxin contamination of cereals. We compared the ability of a near-infrared reflectance (NIR) instrument with a broad wavelength range (400–2500 nm) and a near-infrared transmittance (NIT) instrument with a narrower wavelength range (850–1050 nm) to predict the ergosterol content of naturally infected barley samples. The two instruments were equally good at predicting ergosterol content in Swedish samples (r² = 0.81 and 0.83 for NIT and NIR, respectively). The NIT instrument was then used for samples from three countries (Sweden, Ireland, UK). This model had about the same root mean-squared error (≈5 mg of ergosterol/kg, db, of grain) as the dataset with only Swedish samples, although the r² value was lower (0.58). The investigation has shown that it is possible to predict ergosterol content in whole barley samples using NIR or NIT instrumentation, and acceptable models can be obtained using different barley cultivars and samples from different countries and harvest years. This should make it possible to routinely predict the fungal biomass at grain terminals.     

Collaborative study for the comparison of two methods for the determination of total cholesterol in multicomponent foods.

The gas chromatographic method for determining total cholesterol in multicomponent foods, collaboratively studied by the AOAC in 1974 (Method 1), has been evaluated by 9 collaborating laboratories and compared with the Interim Methodology Instructions No. 2 modified method (Method 2). The 5 samples selected for collaboration were deviled ham sandwich spread, vegetable beef stew, frozen chicken pot pie, frozen fish sticks, and mayonnaise. The recovery data were obtained from a sample of wheat germ oil spiked with 0.297% cholesterol as cholesteryl palmitate. Collaborators performed 2 replicate analyses on all samples by both methods. The statistical evaluation of the results showed that Method 1 is superior to Method 2. Average recoveries from the spiked wheat germ oil samples were 91.4% (9 laboratories) and 85.8% (7 laboratories) with coefficients of variation of 12.5 and 14.4%, respectively. Based on the collaborative results and statistical evaluation, Method 1 has been adopted as official first action.     

Ergosterol Levels and Mould Colony Forming Units in Swedish Grains of Food and Feed Grade

Abstract

Although ergosterol is considered to be a suitable indicator of mould growth in cereal grains, there are few reference values available for Scandinavian conditions. We have determined the ergosterol levels in Swedish grain of different origins: cleaned food-grade wheat from a commercial mill, feed-grade cereals (oats and barley) with different odours and cereals (winter wheat, “American wheat”, triticale and rye) from various field trials conducted in south-central Sweden in 1990. Specific objectives were to elucidate the relationships between ergosterol levels and numbers of mould colony forming units (CFU) and between ergosterol and grain odour.

Ergosterol levels in the food-grade wheat ranged between 2.4 and 2.8 μg/g DW, and between 3.0 and 5.6 μg/g DW in the field trial cereals, while values in most of the feed grain samples ranged from 8–15 μg/g DW. The levels agree with other published data for European grains.

A positive correlation was found between numbers of colony-forming units and ergosterol concentration. The degree of correlation was higher when numbers of CFU were determined on dichloran-glycerol 18% agar with a low water activity (aw = 0.95) than on malt extract agar (aw = 0.99). There was no agreement between ergosterol levels and grain odour, since even samples described as having a fresh smell had high ergosterol levels. However, the highest level (33 μg/g DW) was found in a sample with a pronounced musty odour, and the lowest (1.1 μg/g DW) in a sample that smelled as if it had been heat damaged.     

An improved method for the purification of the trichothecene deoxynivalenol (vomitoxin) from Fusarium graminearum culture

It was hypothesized that a simplified and efficient strategy could be developed for large-scale production and purification of the mycotoxin deoxynivalenol from Fusarium graminearum rice cultures for toxicological studies. F. graminearum R6576 was cultured on rice and extracted with methanol, and the extract was concentrated and subjected to silica gel low-pressure liquid chromatography (LPLC) under a hexane-acetone gradient system. Deoxynivalenol isolation was monitored by thin-layer chromatography, and fractions containing deoxynivalenol were pooled, concentrated, and applied to a second LPLC column under the same conditions. An enriched deoxynivalenol fraction was obtained, which yielded a crystalline material. Repeated crystallization yielded spectroscopically pure deoxynivalenol. The identity of this compound was confirmed by HPLC comparison to an authentic deoxynivalenol standard, FABMS analysis, and comparison of the (1)H and (13)C NMR spectra with published data. This simplified purification scheme eliminated many laborious steps and equipment previously required to obtain gram quantities of crystalline deoxynivalenol for biological testing in animal models.     

Simple determination of 40 organophosphate pesticides in raw wool using microwave-assisted extraction and GC-FPD analysis

A validated analytical method for the multiresidue analysis of 40 organophosphate pesticides (OPs) and conversion products in raw wool has been developed. The method is based on the selective microwave-assisted extraction (MAE) of raw wool with acetonitrile and analysis of extracts by gas chromatography-flame photometric detector. The optimum MAE conditions were 20 min duration at 80 °C with 30 mL of acetonitrile per gram of wool. A validation study was performed according to the European SANCO guidelines 10684/2009. Limits of detection and quantification for all pesticides tested were from 0.01 to 0.2 mg/kg and from 0.2 to 1.0 mg/kg, respectively. The average recoveries of pesticides spiked at different levels were in the range of 70-120% with relative standard deviations of ≤ 20%. The extraction performance was compared to the one obtained with a reference Soxhlet extraction. The method was also applied in the analysis of real wool (after field application) samples.     

秸秆降解过程中拮抗链霉菌对禾谷镰刀菌消长动态的影响

由禾谷镰刀菌(Fusarium graminearum)引起的小麦赤霉病是导致小麦产量和品质降低的主要因素，其在田间生长的基质主要为秸秆。基于各种拮抗菌的生物防治是控制小麦赤霉病的主要措施之一，但秸秆降解过程中拮抗菌的拮抗效应是否与平板对峙获取的拮抗效应一致尚不清楚。本研究首先通过平板对峙试验，筛选出对禾谷镰刀菌CGMCC3.4598有潜在拮抗作用的3株菌：吸水链霉菌ACCC41648 (Streptomyces hygroscopicus)、吸水链霉菌井冈变种ACCC40051 (S. hygroscopicus var. jingganggensis)和娄彻氏链霉菌ACCC41594(S.rochei)，其抑菌带平均宽度依次为2.67、1.67和1.12mm。基于室内70d培育试验，发现秸秆降解7、42和70 d后，吸水链霉菌ACCC41648及其井冈变种ACCC40051未表现抑制秸秆中禾谷镰刀菌生长的效应，而娄彻氏链霉菌ACCC41594在秸秆降解7 d和42 d后显著降低禾谷镰刀菌丰度分别达30.91%和69.28%。与对照相比，娄彻氏链霉菌ACCC41594显著促进秸秆降解...     

Mycoflora and fumonisin mycotoxins associated with cowpea [Vigna unguiculata (L.) Walp] seeds

Cowpea seed samples from South Africa and Benin were analyzed for seed mycoflora. Fusariumspecies detected were F. equiseti, F. chlamydosporum, F. graminearum, F. proliferatum, F. sambucinum, F. semitectum, and F. subglutinans. Cowpea seed from South Africa and Benin and F. proliferatum isolates from Benin, inoculated onto maize patty medium, were analyzed for fumonisin production. Samples were extracted with methanol/water and cleaned up on strong anion exchange solid phase extraction cartridges. HPLC with precolumn derivatization using o-phthaldialdehyde was used for the detection and quantification of fumonisins. Cowpea cultivars from South Africa showed the presence of fumonisin B(1) at concentrations ranging between 0.12 and 0.61 microg/g, whereas those from Benin showed no fumonisins. This is believed to be the first report of the natural occurrence of FB(1) on cowpea seed. Fumonisin B(1), B(2), and B(3) were produced by all F. proliferatum isolates. Total fumonisin concentrations were between 0.8 and 25.30 microg/g, and the highest level of FB(1) detected was 16.86 microg/g.     

Gas chromatographic determination of deoxynivalenol in wheat with electron capture detection: collaborative study

Ten laboratories participated in a collaborative study of a method for the determination of deoxynivalenol in wheat by gas chromatography with electron capture detection. Each laboratory analyzed 6 samples in duplicate. Each collaborator received samples spiked at the 100.3, 501.3, and 1002.6 ng/g levels; a control sample; and 2 naturally contaminated samples. The average recovery (outliers excluded) for the spiked samples was 92.2%. The mean repeatability and reproducibility, respectively, were 32.2 and 41.3% for the spiked samples and 30.9 and 47.6% for the naturally contaminated samples. The method was adopted official first action.     

Usefulness of near-infrared spectroscopy to determine biological and chemical soil properties: Importance of sample pre-treatment

Near-infrared reflectance spectroscopy (NIRS) is known for its inexpensiveness, rapidity and accuracy and may become a useful tool for the assessment of soil quality. Objectives were (i) to evaluate the ability of NIRS to predict several chemical and biological properties of organically managed arable soils as well as the properties of grain yield from winter cereals for a closed population and (ii) to test whether the use of field-moist and pre-treated (quick-freezing followed by freeze-drying and grinding) samples will generate similar results. One hundred and sixteen soil samples from nine organically managed farms from Germany sampled in 2005 and 2006 were used for this investigation. Spectra of the near-infrared region (including the visible range, 400–2500 nm) from field-moist (<2 mm) or pre-treated soil samples were recorded. A modified partial least-square regression method and cross-validation were used to develop an equation over the whole spectrum (first–third derivation). For the pre-treated soils, good predictions were obtained for pH, contents of organic C, total N, plant-available P (Olsen) and exchangeable K (calcium-acetate-lactate (CAL)), contents of microbial biomass C and N (C_mic and N_mic) and ergosterol, basal respiration, metabolic quotient, the ratio of organic C/total N, the grain yield of winter cereals and grain nitrogen uptake. The RSC (the ratio of standard deviation of laboratory results to standard error of cross-validation) was greater than 2.0, the correlation coefficients (r) of a linear regression (measured against predicted values) were greater than or equal to 0.9 and the regression coefficients (a) ranged from 0.9 to 1.1. Similar good predictions were obtained if field-moist samples were used, with the exception of P (Olsen), K (CAL), metabolic quotient, grain yield of winter cereals and grain nitrogen uptake (satisfactory predictions) and ergosterol content (unsatisfactory prediction). Good predictions of the contents of Mg (CaCl₂) and microbial biomass P (P_mic) were achieved for field-moist but not for pre-treated samples. Despite sample preparation, only satisfactory predictions were obtained for the ratios of C_mic/N_mic and ergosterol/C_mic and grain nitrogen content (1.4RSC2.0, r0.8 and 0.8a1.2). However, unsatisfactory predictions for field-moist and pre-treated samples were achieved for the content of P (CAL), the nitrogen mineralisation rate and the ratios of C_mic/P_mic and basal respiration/nitrogen mineralisation rate. Our results demonstrate that biological soil properties can be predicted with NIRS for closed populations in both sample states. The pre-treatment should be used if samples have to be stored prior to infrared measurements for periods longer than a month.     

Determination of ergosterol levels in barley and malt varieties in the Czech Republic via HPLC

Ergosterol is considered to be a suitable indicator of mold infestation in barley and malt. In this study ergosterol levels in different varieties of barley and malt produced in the Czech Republic were determined. A modified high-performance liquid chromatography (HPLC) method was statistically processed, validated (Effivalidation program), and applied to 124 samples of barley and malt. Ergosterol was isolated by extraction and saponification, and the quantification was performed using HPLC with diode array detection. The content of ergosterol ranged between the limit of detection (LOD) and 36.3 mg/kg in barley and between the LOD and 131.1 mg/kg in malt. Ergosterol is presumably connected with metabolites generated when barley grain is attacked by pathogens, and such barley often shows a high overfoaming (gushing) value. However, it was found that the content of ergosterol does not correlate with the degree of beer gushing.     

Solid phase extraction and liquid chromatography-tandem mass spectrometry for the evaluation of 4-hydroxy-2-nonenal in pork products

This research was aimed at setting up an analytical method for the determination in pork products of 4-hydroxy-2-nonenal (4-HNE), an aldehyde produced from the oxidation of omega-6-polyunsaturated fatty acids. Such a compound mediates various biological effects, but it is considered to be very toxic to mammalian cells at levels higher than physiological ones. The methods used for the determination of 4-HNE in biological fluids, such as blood, were found to be unsuitable for meat samples because both the repeatability and the recovery in spiked samples were unsatisfactory. A new method, based on solid phase extraction and HPLC-MS/MS, was therefore developed and validated. The limit of detection of 4-HNE in spiked samples was 0.043 mg/kg, and the recovery was approximately 60% depending on the concentration. Good linearity was observed in the range of 0.1-10 mg/kg, and repeatability and interday and intraday precision expressed as relative standard deviation were <10%. The method has been successfully applied to the determination of the aldehyde in samples of various pork products. 4-HNE was present in some products, especially the smoked and/or cooked ones, at levels that might not be a real risk for human health.     

Occurrence of Fusarium species and mycotoxins in nepalese maize and wheat and the effect of traditional processing methods on mycotoxin levels

Maize (Zea mays) and wheat (Triticum aestivum) collected in the foothills of the Nepal Himalaya Mountains were analyzed for Fusarium species and mycotoxins: fumonisins, nivalenol (NIV), and deoxynivalenol (DON). Predominant species were Gibberella fujikuroi mating population A (F. moniliforme) in maize and F. graminearum in maize and wheat; G. fujikuroi mating population D (F. proliferatum), F. acuminatum, F. avenaceum, F. chlamydosporum, F. equiseti, F. oxysporum, F. semitectum, and F. torulosum were also present. Strains of G. fujikuroi mating population A produced fumonisins, and strains of F. graminearum produced NIV or DON. By immunoassay or high-performance liquid chromatography, fumonisins were >1000 ng/g in 22% of 74 maize samples. By immunoassay or fluorometry, NIV and DON were >1000 ng/g in 16% of maize samples but were not detected in wheat. Fumonisins and DON were not eliminated by traditional fermentation for producing maize beer, but Nepalese rural and urban women were able to detoxify contaminated maize by hand-sorting visibly diseased kernels.     

Assessment of extraction procedures in the analysis of naturally contaminated grain products for deoxynivalenol (vomitoxin)

A comparison of 2 extraction solvent systems (acetonitrile-water, 21 + 4 and methanol-water, 1 + 1) and 3 mixing apparatus (high-speed blender, wrist-action shaker, and mechanical stirrer) was carried out for different extraction time periods. Methods were evaluated using uncontaminated corn spiked with pure deoxynivalenol (DON), field-inoculated (Fusarium graminearum) corn, and uncontaminated and naturally infected wheat in swine diets. After sample extraction, aliquots were passed through alumina-charcoal cleanup columns, evaporated to dryness, dissolved in 8% aqueous methanol, and injected onto the liquid chromatograph. Results confirm published reports of recoveries from DON-spiked samples; however, longer extraction times (less than or equal to 120 min) were required for naturally contaminated samples. Use of the high-speed blender resulted in faster extractions, but in our laboratory more samples could be more conveniently extracted simultaneously with the wrist-action shaker or mechanical stirrer. Less carryover (co-extraction) of interfering contaminants was observed when acetonitrile-water was used vs methanol-water. Results emphasize the importance of careful evaluation of extraction procedures with not only spiked samples but also naturally contaminated samples to establish extraction times required for maximum deoxynivalenol recoveries.     

Development of a polyclonal antibody-based sensitive enzyme-linked immunosorbent assay for fumonisin B(4)

Polyclonal antibodies (PAb) against fumonisin B(4) (FmB(4)), which have good cross-reactivity with four major fumonisins, were produced by immunizing a rabbit with FmB(4)-keyhole limpet hemocyanin conjugate. A sensitive competitive direct enzyme-linked immunosorbent assay (CD-ELISA) for fumonisins was developed. Because of the limited supply of FmB(4), both FmB(1)-horseradish peroxidase conjugate (HRP) and FmB(3)-HRP were tested as the toxin-enzyme markers in the CD-ELISA. In the FmB(1)-HRP-based CD-ELISA, the concentrations of FmB(1), FmB(2), FmB(3), and FmB(4) causing 50% inhibition of binding of enzyme marker (IC(50)) were 9.0, 2.1, 9.0, and 6.5 ng/mL (or the relative cross-reactivities toward FmB(1), FmB(2), FmB(3), and FmB(4) were 58.5, 309.5, 58.5, and 100%), respectively. In the FmB(3)-HRP-based CD-ELISA, the IC(50) values for FmB(1), FmB(2), FmB(3), and FmB(4) were 7.1, 1.9, 7.6, and 5.3 ng/mL (or the relative cross-reactivities toward FmB(1), FmB(2), FmB(3), and FmB(4) were 74, 280, 70, and 100%), respectively. The FmB(3)-HRP-based CD-ELISA was then used in a series of analytical recovery experiments using Fusarium moniliforme corn culture material spiked with FmB(1) and with clean corn spiked with a FmB(3)/FmB(4)-containing extract. The overall recovery of FmB(1) from culture material in the range of 10-100 ppm was 65%. The detection limit for FmB(1) with clean corn as matrix was between 100 and 500 ppb. F. moniliforme cultures were analyzed with the developed CD-ELISA and a well-established FmB(1) antibody-based ELISA, which is not sensitive to FmB(4). Differences in the fumonisin levels found by the two assays were used as an indication of the presence of FmB(4) in the culture material and, therefore, as a method to identify FmB(4)-producing strains. Using ELISA in combination with HPLC individual B-series fumonisins were quantified. The ELISA developed in the present study would be a useful supplement to FmB(1) antibody-based ELISA for screening of Fusarium strains for the production of major fumonisins.