Immunoassay utilizing imaging surface plasmon resonance for the detection of cyclopiazonic acid (CPA) in maize and cheese.

α-Cyclopiazonic acid (CPA) is a tremorgenic mycotoxin produced by Aspergillus and Penicillium fungal species, commonly found on agricultural commodities or fermented food products. A sensitive and rapid imaging surface plasmon resonance (iSPR) assay was developed to detect CPA in maize and cheese by combining an indirect competitive immunoassay and signal amplification based upon a secondary antibody (Ab2) conjugated with gold nanoparticles. Matrix-matched calibration curves were used to determine CPA content in maize and cheese samples. Recoveries, at two spiking levels in maize and cheese, were 89 to 126%, with standard deviations of repeatability (RSDr) of less than 16%. The limits of detection were 17 and 6 µg/kg in maize and cheese, respectively. To separate the CPA-contaminated samples from uncontaminated samples, a cutoff validation level of 40 µg/kg was introduced. The assay was applied to samples of naturally contaminated maize and was compared with competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA). This is the first report to detect CPA using an immuno-biosensor iSPR format.

Monoclonal-Antibody-Based Immunoassays for the Mycotoxins NX-2 and NX-3 in Wheat.

The fungal infestation of crops can cause major economic losses. Toxins produced by the causative fungi (mycotoxins) represent a potential safety hazard to people and livestock consuming them. One such mycotoxin is deoxynivalenol (DON, also known as vomitoxin), a trichothecene associated with Fusarium Head Blight of wheat. DON is commonly found in cereal crops worldwide. A group of trichothecene mycotoxins closely related to DON, the NX toxins, have been reported to occur in the northeastern United States and southern Canada. While many commercial immunoassays are available to detect DON, there are no rapid screening assays for the NX toxins. We describe the development and isolation of three monoclonal antibodies (mAbs) specific towards two NX toxins: NX-2 and NX-3. The mAbs did not recognize DON or several other closely related trichothecenes. One of the mAbs was selected for development of an enzyme-linked immunosorbent assay (ELISA) for NX-2 and NX-3 in wheat. The dynamic ranges for the assay were 7.7 to 127 µg/kg for NX-2 and 59 µg/kg to 1540 µg/kg for NX-3 in wheat. Recoveries from spiked wheat averaged 84.4% for NX-2 and 99.3% for NX-3, with RSDs of 10.4% and 11.3%, respectively (n = 24). The results suggest that this assay can be used to screen for NX toxins in wheat at levels relevant to human food and animal feed safety.

Detection of T-2 Toxin in Wheat and Maize with a Portable Mass Spectrometer.

T-2 toxin is a mycotoxin routinely found as a contaminant of cereal grains worldwide. A portable mass spectrometer was adapted to enable the detection of T-2 toxin in wheat and maize by APCI-MS. In order to facilitate rapid testing, a rapid cleanup was used. The method was able to detect T-2 toxin in soft white wheat, hard red wheat, and yellow dent maize and could be used to screen for T-2 at levels above 0.2 mg/kg. The HT-2 toxin was only detectable at very high levels (>0.9 mg/kg). Based on these results, the sensitivity was not sufficient to allow the application of the screening method to these commodities at levels recommended by the European Commission. With a cut-off level of 0.107 mg/kg, the method correctly classified nine of ten reference samples of wheat and maize. The results suggest that portable MS detection of T-2 toxin is feasible. However, additional research will be needed to develop an application sensitive enough to meet regulatory requirements.

Cyclopiazonic acid in soft-ripened and blue cheeses marketed in the USA.

Strains of Penicillium camemberti and P. roqueforti are used in the production of soft-ripened and blue-veined cheeses. However, some strains can produce toxic secondary metabolites (mycotoxins), including α-cyclopiazonic acid (CPA), a neurotoxin. Data on the levels of CPA in cheeses marketed in the USA are extremely limited. An enzyme-linked immunosorbent assay was adapted for measuring CPA in soft-ripened and blue-veined cheeses. Recoveries from cheese curds were 103 ± 27% (n = 30). A total of 254 samples of soft-ripened, blue and miscellaneous cheeses were examined. CPA was detected in 36/79 (45.6%) of soft-ripened cheeses and in 41/168 (24.4%) of blue-veined cheeses. Median levels in positive samples were 48.5 µg/kg and 30 µg/kg, respectively. The highest levels found were 3,820 µg/kg (in a Brie), 1,250 µg/kg (in a blue) and 7,900 µg/kg (in a Monte Enebro). The implication of such exposures is unknown, as a consensus on acceptable intake remains to be established.

Homogeneous immunoassay for cyclopiazonic acid based upon mimotopes and upconversion-resonance energy transfer.

Strains of Penicillium spp. are used for fungi-ripened cheeses and Aspergillus spp. routinely contaminate maize and other crops. Some of these strains can produce toxic secondary metabolites (mycotoxins), including the neurotoxin α-cyclopiazonic acid (CPA). In this work, we developed a homogeneous upconversion-resonance energy transfer (UC-RET) immunoassay for the detection of CPA using a novel epitope mimicking peptide, or mimotope, selected by phage display. CPA-specific antibody was used to isolate mimotopes from a cyclic 7-mer peptide library in consecutive selection rounds. Enrichment of antibody binding phages was achieved, and the analysis of individual phage clones revealed four different mimotope peptide sequences. The mimotope sequence, ACNWWDLTLC, performed best in phage-based immunoassays, surface plasmon resonance binding analyses, and UC-RET-based immunoassays. To develop a homogeneous assay, upconversion nanoparticles (UCNP, type NaYF4:Yb3+, Er3+) were used as energy donors and coated with streptavidin to anchor the synthetic biotinylated mimotope. Alexa Fluor 555, used as an energy acceptor, was conjugated to the anti-CPA antibody fragment. The homogeneous single-step immunoassay could detect CPA in just 5 min and enabled a limit of detection (LOD) of 30 pg mL-1 (1.5 µg kg-1) and an IC50 value of 0.36 ng mL-1. No significant cross-reactivity was observed with other co-produced mycotoxins. Finally, we applied the novel method for the detection of CPA in spiked maize samples using high-performance liquid chromatography coupled to a diode array detector (HPLC-DAD) as a reference method.

Measurement of Fumonisins in Maize Using a Portable Mass Spectrometer.

Fumonisins are a group of mycotoxins that routinely contaminate maize. Their presence is monitored at multiple stages from harvest to final product. Immunoassays are routinely used to screen commodities in the field while laboratory-based methods, such as mass spectrometry (MS), are used for confirmation. The use of a portable mass spectrometer unlocks the potential to conduct confirmatory analyses outside of traditional laboratories. Herein, a portable mass spectrometer was used to measure fumonisins in maize. Samples were extracted with aqueous methanol, cleaned up on an immunoaffinity column, and tested with the portable MS. The limits of detection were 0.15, 0.19, and 0.28 mg/kg maize for fumonisins B1 (FB1), FB2/FB3, and total fumonisins, respectively. The corresponding limits of quantitation in maize were 0.33, 0.59, and 0.74 mg/kg. Recoveries ranged from 93.6% to 108.6%. However, RSDs ranged from 12.0 to 29.8%. The method was applied to the detection of fumonisins in 64 samples of maize collected as part of the Illinois Department of Agriculture's monitoring program. Good correlations were observed between the portable MS and a laboratory-based LC-MS method (r2 from 0.9132 to 0.9481). Results suggest the portable MS can be applied to the measurement of fumonisins in maize at levels relevant to international regulations.

Volatile Organic Compound Profile Fingerprints Using DART-MS Shows Species-Specific Patterns in Fusarium Mycotoxin Producing Fungi.

Fungal volatile organic compounds (VOCs) are low-molecular weight fungal metabolites that have high vapor pressure at ambient temperatures and can function as airborne signals. Here, we report a VOC study of several different species of Fusarium. Direct analysis in real time mass spectrometry (DART-MS) was applied for non-invasive VOC fingerprinting of Fusarium isolates growing under standardized conditions. A large number of ions were detected from the headspaces of the Fusarium species sampled here. Ions were detected with distinctively high concentrations in some species. While there were few VOCs produced by only one species, the relative concentrations of VOCs differed between species. The methodology has potential for convenient detection and identification of Fusarium contamination in agricultural commodities.

Development and characterisation of a monoclonal antibody to detect the mycotoxin roquefortine C.

Roquefortine, also known as roquefortine C (ROQC) is a fungal secondary metabolite (mycotoxin) that is produced by some of the same Penicillia as the tremorgen penitrem-A (PEN-A). The two mycotoxins have been linked to sporadic cases of toxicosis in dogs, cattle, and humans, leading some to consider ROQC as a biomarker of PEN-A. Reported here are the development of a monoclonal antibody (mAb) and associated competitive enzyme-linked immunosorbent assay (ELISA) for the screening of ROQC in extracts of nuts (nut "milks"), and dog serum. The ELISA was sensitive for ROQC, with a level of 0.117 ng ml-1 inhibiting colour development by 50% (IC50), a limit of detection of 0.026 ng ml-1, and a dynamic range (IC20 to IC80) of 0.038 to 0.289 ng ml-1 in buffer. The assay was tolerant to significant levels of methanol. Recoveries from 4 types of nut milks spiked over the range of 0.25 to 2 ng ml-1 were in the range of 83.5% to 116%. A small survey of commercial nut "milks" and "creamers" indicated 4 of 35 samples contained ROQC at levels so low that they are unlikely to be significant to human health (<0.6 ng ml-1). The assay was also applied to canine serum. Recoveries from serum spiked over the range of 0.2 to 5 ng ml-1 ranged from 98.1% to 123%. The results suggest the ELISA can be applied to the screening of food products, such as nut extracts, as well as for the screening of serum from dogs suspected to be suffering from mycotoxin-induced tremors.

Recent advances in the development of novel materials for mycotoxin analysis.

For sensitive and specific toxin detection, mycotoxin immunoassays depend upon antibodies with high affinity and selectivity. While intact immunoglobulins remain the primary toxin-binding elements used in rapid assays, a number of alternatives have begun appearing in the literature. The alternatives can be broadly classified into those that are obtained by chemical synthesis and those that are obtained by altering biologically derived materials. Examples range from synthetically prepared polymers to recombinant fragments of antibodies, with a wide variety of synthetic and natural materials in-between. To date, obtaining the combination of selectivity and affinity needed for use in sensors has been more readily accomplished with biologically derived materials than with synthetic materials. Despite this, synthetic materials still offer certain potential advantages, such as high binding capacity and the ability to bind in environments that are too harsh for intact antibodies. This review focuses upon recent advances in the development of mycotoxin-binding materials and their potential for application in mycotoxin assays.

Coordination of mycotoxins with lanthanides in luminescent complexes.

The ability of several chelating mycotoxins to form coordination complexes with the lanthanide metals europium and terbium was explored. The mycotoxins examined included ochratoxin A, citrinin, cyclopiazonic acid (CPA), kojic acid, and tenuazonic acid (TeA). Of these compounds, TeA and CPA resulted in the greatest luminescence. Parameters influencing luminescence of TeA were investigated further. These included the type of lanthanide and its concentration, certain environmental factors, and the effect of competing metal cations. Of the two lanthanide metals, the terbium coordination complex (TeA-Tb3+) showed greater luminescence relative to the europium complex (TeA-Eu3+). The effects of solvent type, water content, and pH on the TeA-Tb3+ system suggested that optimal conditions for luminescence were in 90% methanol with 10% aqueous buffer at pH 3. In competitive assays, the luminescence of the TeA-Tb3+ complex decreased as the concentration of competing metal cations increased. Among the cations tested, Cu2+ was the best inhibitor followed by Al3+, Au3+, Fe3+, Co2+, Mn2+, Mg2+, and Ca2+. Two cations, Na+ and K+, showed no significant inhibition. This is the first report to describe the coordination of the metal-chelating mycotoxin TeA with lanthanides and the ability of TeA to serve as an "antenna" for the efficient transfer of energy to the lanthanide with resulting luminescence. Understanding the ability of mycotoxins such as TeA to chelate metals provides insight into how they exert their toxic effects.

Development and Characterization of Monoclonal Antibodies for the Mycotoxin Citreoviridin.

Citreoviridin (CTV) in an inhibitor of mitochondrial ATPase that has been isolated from molded yellow rice and linked to the human disease Shoshin-kakke (acute cardiac beriberi). The disease results from a deficiency of thiamine, however, purified CTV can reproduce the symptoms in experimental animals. The link between CTV and Shoshin-kakke has been difficult to resolve, in part because cases of the disease are rare. In addition to rice, CTV has been found in maize, pecan nuts, and wheat products. A method to screen for CTV and its geometric isomer, iso-CTV, in commodities was developed, based upon the isolation of two novel monoclonal antibodies (mAb). In an antigen-immobilized competitive enzyme-linked immunosorbent assay format (CI-ELISA), the observed IC50s for CTV were 11 ng/mL and 18 ng/mL (mAbs 2-2 and 2-4, respectively). The assays were relatively tolerant to methanol and acetonitrile, which allowed their application to the detection of CTV in spiked polished white rice. For quantification, a standard mixture of CTV and iso-CTV was used, along with matrix matched calibration. The dynamic range of the ELISA using mAb 2-4 was equivalent to 0.23 to 2.22 mg/kg in rice. Recoveries over the range of 0.36 to 7.23 mg/kg averaged 97 ± 10%. The results suggest that the mAb 2-4-based immunoassay can be applied to the screening of white rice for CTV. Both mAbs were also observed to significantly enhance the fluorescence of the toxin.

Fluorescence Polarization Immunoassay for the Determination of T-2 and HT-2 Toxins and Their Glucosides in Wheat.

T-2 and HT-2 toxins and their main modified forms (T-2 glucoside and HT-2 glucoside) may co-occur in cereals and cereal-based products. A fluorescence polarization immunoassay (FPIA) was developed for the simultaneous determination of T-2 toxin, HT-2 toxin and relevant glucosides, expressed as sum. The developed FPIA, using a HT-2-specific antibody, showed high sensitivity (IC50 = 2.0 ng/mL) and high cross-reactivity (100% for T-2 toxin and 80% for T-2 and HT-2 glucosides). The FPIA has been used to develop two rapid and easy-to-use methods using two different extraction protocols, based on the use of organic (methanol/water, 90:10, v/v) and non-organic (water) solvents, for the determination of these toxins in wheat. The two proposed methods showed analytical performances in terms of sensitivity (LOD 10 µg/kg) recovery (92-97%) and precision (relative standard deviations ≤13%), fulfilling the criteria for acceptability of an analytical method for the quantitative determination of T-2 and HT-2 toxins established by the European Union. Furthermore, the methods were then validated in accordance with the harmonized guidelines for the validation of screening methods included in the Regulation (EU) No. 519/2014. The satisfactory analytical performances, in terms of intermediate precision (≤25%), cut-off level (80 and 96 µg/kg for the two methods) and rate of false positives (<0.1%) confirmed the applicability of the proposed methods as screening method for assessing the content of these toxins in wheat at the EU indicative levels reported for T-2 and HT-2 toxins.

Extraction of aflatoxins B1 and G1 from maize by using aqueous sodium dodecyl sulfate.

Aflatoxins are potent carcinogens produced by certain Aspergillus fungi. The aflatoxins were first discovered in the 1960s, and since then have been found to be distributed worldwide in a variety of commodities, foods, and feeds. Many of the early techniques for detecting aflatoxins involved extraction with halogenated solvents. With the increased availability and use of reversed-phase solid-phase extraction cartridges and the availability of immunoaffinity columns, aqueous mixtures of nonhalogenated solvents have been frequently used. To further reduce the need for solvents, we examined the effects of eliminating solvents during the extraction of maize, using aqueous mixtures of the detergent sodium dodecyl sulfate. After extraction and filtration, aflatoxins B1 (AFB1) and G1 (AFG1) were isolated by using commercially available immunoaffinity columns. The isolated AFB1 and AFG1 were derivatized with trifluoroacetic acid before separation by liquid chromatography with fluorescence detection. In spiked maize, the limits of detection were 0.5 and 1 ng/g for AFB1 and AFG1, respectively. Recoveries of AFB1 from maize spiked at 1-20 ng/g averaged 87.5% (range, 76.3-99.0%), with an average repeatability relative standard deviation (RSDr) of 4.0%. Recoveries of AFG1 from maize spiked at 2-20 ng/g averaged 80.4% (range, 70.3-85.8%), with an average RSDr of 3.5%. This is the first reported demonstration of an effective solvent-free extraction of aflatoxins from maize at ambient pressure, and this extraction procedure may serve to help reduce solvent consumption during aflatoxin analysis.

Complexation of the Mycotoxin Cyclopiazonic Acid with Lanthanides Yields Luminescent Products.

Cycopiazonic acid (CPA) is a neurotoxin that acts through inhibition of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). CPA blocks the calcium access channel of the enzyme. The inhibition may involve the binding of CPA with a divalent cation such as Mg2+. The potential for CPA to act as a chelator also has implications for methods to detect this toxin. Certain of the lanthanide metals undergo a dramatic increase in luminescence upon coordination with small molecules that can transfer excitation energy to the metal. This report is the first to describe the coordination of CPA with lanthanide metals, resulting in a substantial enhancement of their luminescence. The luminescence expressed was dependent upon the type of lanthanide, its concentration, and the environment (solvent, water content, pH). Based upon the phenomenon, a competitive assay was also developed wherein terbium (Tb3+) and a series of metal cations competed for binding with CPA. With increasing cation concentration, the luminescence of the CPA/Tb3+ complex was inhibited. The chlorides of ten metals were tested. Inhibition was best with Cu2+, followed by Co2+, Al3+, Fe3+, Mn2+, Au3+, Mg2+, and Ca2+. Two cations in oxidation state one (Na⁺, K⁺) did not inhibit the interaction significantly. The interaction of CPA with lanthanides provides a novel recognition assay for this toxin. It also provides a novel way to probe the binding of CPA to metals, giving insights into CPA’s mechanism of action.

Interaction of zearalenone with bovine serum albumin as determined by fluorescence quenching.

The major aim of this study was to examine the binding of zearalenone (ZEN) to bovine serum albumin (BSA) by measuring the quenching of the intrinsic fluorescence of the protein under aqueous conditions. The results suggest that ZEN has a strong ability to quench the intrinsic fluorescence of BSA through a static mechanism. The hydrophobicity of the microenvironment around the tyrosine (Tyr) residues in BSA was increased in the presence of ZEN. The quenching constants, ratio of protein with ZEN, and thermodynamic parameters were determined. The collaborative action of hydrophobic and electrostatic interactions was involved in the binding process and the formation of the complex was mainly enthalpy-driven. The average binding distance between ZEN and BSA was calculated to be 2.20 nm. This is much closer in magnitude than the distance reported for the binding of most toxins to HSA and most pharmaceuticals to BSA, indicating a strong affinity.

Gold nanoparticle-enhanced multiplexed imaging surface plasmon resonance (iSPR) detection of Fusarium mycotoxins in wheat.

A rapid, sensitive and multiplexed imaging surface plasmon resonance (iSPR) biosensor assay was developed and validated for three Fusarium toxins, deoxynivalenol (DON), zearalenone (ZEA) and T-2 toxin. The iSPR assay was based on a competitive inhibition format with secondary antibodies (Ab2) conjugated to gold nanoparticles (AuNPs) used as a signal amplification tag. Signal was amplified nearly 25-fold for DON, 90-fold for ZEA and 12-fold for T-2 toxin assay using Ab2-AuNPs. Analyses, including steps to regenerate the sensor, took 17.5min. The antigen coated sensor chip was used for more than 46 cycles without affecting signal intensity (< 12%). Matrix matched calibration curves were used to determine Fusarium toxins in wheat. The mean recoveries ranged from 87% to 103% with relative standard deviations of repeatability of less than 5%. The limits of detection were 15µg/kg for DON, 24µg/kg for ZEA and 12µg/kg for T-2 toxin. This provided sufficient sensitivity to monitor contamination of these mycotoxins in wheat in accordance with European Commission (EC) limits. Cut off levels for all three Fusarium toxins were validated using blank wheat and wheat spiked either at the EC regulated levels (100µg/kg for ZEA and T-2 toxin) or at one third of the EC level (for DON: 400µg/kg). The assay was successfully applied and further validated with naturally contaminated wheat samples. This is the first reported AuNP enhanced iSPR assay to detect and classify three agriculturally important Fusarium toxins in wheat.

An Imaging Surface Plasmon Resonance Biosensor Assay for the Detection of T-2 Toxin and Masked T-2 Toxin-3-Glucoside in Wheat.

A sensitive, rapid, and reproducible imaging surface plasmon resonance (iSPR) biosensor assay was developed to detect T-2 toxin and T-2 toxin-3-glucoside (T2-G) in wheat. In this competitive assay, an amplification strategy was used after conjugating a secondary antibody (Ab2) with gold nanoparticles. Wheat samples were extracted with a methanol/water mixture (80:20 v/v), then diluted with an equal volume of primary antibody (Ab1) for analysis. Matrix-matched calibration curves were prepared to determine T-2 toxin and T2-G. Recovery studies were conducted at three spiking levels in blank wheat. Mean recoveries ranged from 86 to 90%, with relative standard deviations for repeatability (RSDr) of less than 6%. Limits of detection were 1.2 ng/mL of T-2 toxin and 0.9 ng/mL of T2-G, equivalent to their levels in wheat, of 48 and 36 µg/kg, respectively. The developed iSPR assay was rapid and provided enough sensitivity for the monitoring of T-2 toxin/T2-G in wheat. This is the first iSPR assay useful for detecting the "masked" T2-G in wheat.

MycoKey Round Table Discussions of Future Directions in Research on Chemical Detection Methods, Genetics and Biodiversity of Mycotoxins.

MycoKey, an EU-funded Horizon 2020 project, includes a series of "Roundtable Discussions" to gather information on trending research areas in the field of mycotoxicology. This paper includes summaries of the Roundtable Discussions on Chemical Detection and Monitoring of mycotoxins and on the role of genetics and biodiversity in mycotoxin production. Discussions were managed by using the nominal group discussion technique, which generates numerous ideas and provides a ranking for those identified as the most important. Four questions were posed for each research area, as well as two questions that were common to both discussions. Test kits, usually antibody based, were one major focus of the discussions at the Chemical Detection and Monitoring roundtable because of their many favorable features, e.g., cost, speed and ease of use. The second area of focus for this roundtable was multi-mycotoxin detection protocols and the challenges still to be met to enable these protocols to become methods of choice for regulated mycotoxins. For the genetic and biodiversity group, both the depth and the breadth of trending research areas were notable. For some areas, e.g., microbiome studies, the suggested research questions were primarily of a descriptive nature. In other areas, multiple experimental approaches, e.g., transcriptomics, proteomics, RNAi and gene deletions, are needed to understand the regulation of toxin production and mechanisms underlying successful biological controls. Answers to the research questions will provide starting points for developing acceptable prevention and remediation processes. Forging a partnership between scientists and appropriately-placed communications experts was recognized by both groups as an essential step to communicating risks, while retaining overall confidence in the safety of the food supply and the integrity of the food production chain.

Capillary electrophoresis of the mycotoxin zearalenone using cyclodextrin-enhanced fluorescence.

Certain of the cyclodextrins are capable of significantly enhancing the native fluorescence of the estrogenic mycotoxin zearalenone (ZEN). Twenty-two cyclodextrins (CDs) were screened for their ability to enhance the fluorescence of ZEN in a capillary electrophoresis-laser induced fluorescence (CE-LIF) format. Of the CDs that were examined heptakis (2,6-di-O-methyl)-beta-CD gave the greatest enhancement. The heptakis (2,6-di-O-methyl)-beta-CD was applied to the development of a CE-LIF method for detection of ZEN in maize. The resulting method was capable of detecting ZEN with a limit of quantitation of 5 ng/g maize. Recoveries of ZEN from maize spiked over the range from 5 ng/g to 500 ng/g averaged 103.1+/-8.5% (n=20). The CE-LIF method will be useful for future studies of ZEN in maize.

Relationships among resistances to fusarium and Aspergillus ear rots and contamination by fumonisin and aflatoxin in maize.

ABSTRACT Fusarium verticillioides, F. proliferatum, and Aspergillus flavus cause ear rots of maize and contaminate the grain with mycotoxins (fumonisin or aflatoxin). The objective of this study was to investigate the relationships between resistance to Fusarium and Aspergillus ear rots and fumonisin and aflatoxin contamination. Based on a previous study of 143 recombinant inbred lines from the cross NC300 x B104, 24 lines with the highest and 24 lines with the lowest mean fumonisin concentration were selected for further evaluation. Paired plots of each line were inoculated with F. verticillioides and F. proliferatum or with A. flavus in replicated trials in 2004 and 2005 in Clayton, NC, and College Station, TX. The low-fumonisin group had significantly lower levels of fumonisin, aflatoxin, and Fusarium and Aspergillus ear rots. Across year-location environments, all four traits were significantly correlated; the genotypic correlation (r(G)) ranged from r(G) = 0.88 (aflatoxin and Aspergillus ear rot) to r(G) = 0.99 (Fusarium and Aspergillus ear rots). Quantitative trait loci (QTLs) were identified and their effects estimated. Two QTLs affected both toxin concentrations, one QTL affected both ear rots, and one QTL affected Aspergillus and Fusarium rots and fumonisin. These results suggest that at least some of the genes involved in resistance to ear rots and mycotoxin contamination are identical or genetically linked.