Ten corn hybrids, four stacked-gene, four glyphosate-tolerant and two non-GMO, were purchased commercially in 2011, and were used in both 2011 and 2012 (Table 1). Between planting seasons, remaining seed were preserved in cold storage (4˚C). The research was conducted at two sites, one a Bosket fine sandy loam (fine-loamy, mixed, active, thermic Mollic Hapludalfs), located on the Mississippi State University Delta Branch Research

^†31P41 and 33N56 are conventional hybrids; 1615R, 31P40, 33N55, and DKC 67-22 are single trait glyphosate tolerant only hybrids with no Bt; and 31G96, 31P42, DKC 66-96, and DKC 67-21are classed as Stacked-Gene Hybrids with insect and herbicide resistance traits.

and Extension Center, Stoneville, MS, and the other a Tunica clay (clayey over loamy, smectitic, nonacid, thermic Vertic Haplaquept) on private property 1.5 k north of Elisabeth, MS, U.S.A. Each hybrid was planted in eight row plots (1.4 meter between rows 12 meter long) with a final stand density of approximately 31,000 kernels∙A⁻¹. The experiments were planted 7 April 2011 and 29 March, 2012. All experiments were conducted in conventional tillage plots, furrow irrigation was performed, and all other agronomic measurements as previously described by Bruns [12] . Rows 7 and 8 within each plot were inoculated at the V10 stage of growth with toxigenic Aspergillus flavus K54 (NRRL 58987, isolated from corn kernels in Mississippi), grown on wheat, and applied at a rate of 22.42 kg/ha [35] . The remaining 6 rows were allowed to become naturally infected with disease-producing fungi, including various Fusarium and other Aspergillus spp.

The mature crop was harvested with a two row combine. The non-inoculated rows were harvested first, and then the inoculated rows were harvested. Two different combines were used; one to harvest the inoculated rows (Gleaner K2, AGCO, Duluth, GA) and the other (Kincaid, Haven, KS) to harvest the non-inoculated rows to prevent cross-contamination. Yield and yield components were determined and previously published [12] .

Sub-samples of 2 kg of kernels from each treatment were pooled, mixed, oven dried at 50˚C for 3 to 5 d, and ground (20 mesh) using a Romer mill (Union, MO). A representative 50 g sub-sample was taken and extracted in 250 mL of 70% methanol as previously described [23] . Both aflatoxin and fumonisin levels were determined by High-Pressure Liquid Chromatography (HPLC) with post-column derivations as described below.

Preparation, cleanup and determination of aflatoxin in samples were performed according to protocols as described in detail previously by Abbas et al. [23] . Briefly, aflatoxin levels were analyzed by WATERS HPLC with post-column photochemical derivation (PHRED) (Aura Industries, New York, NY) and fluorescence detection, containing a WATERS 717 Autosampler. Detection limit of this procedure was 0.1 ng/g, and the standard curve was linear up to 200 ng/g.

Fumonison samples extracted in 70% methanol were cleaned up using Bond-Elute SAS columns (Varian, Harbor City, CA) [23] [25] [36] . The columns were washed using 2.5 mL each of 100% methanol and 75% methanol. Next, 2.5 mL of the sample extract was applied to the column, and washed again with 75% methanol and 100% methanol applied to the column. The sample was eluted using 2.5 mL of 2% acetic acid in methanol, dried under nitrogen at 50˚C using a Turbo Vap LV (Biotage, Charlotte, NC), and stored at −20˚C until ready for analysis. The clean samples were reconstituted in 1 mL acetonitrile: water (30:70) and analyzed by HPLC with post-column derivatization according to the method described in [37] , Joerg Stroke, Institute for Reference Materials and Measurements, Belgium (Personal communication)] with little modification. Samples to be analyzed for fumonisin were injected onto an Agilent 1200 system consisting of a binary pump, autosampler, and a fluorescence detector equipped with a 150 mm × 4.6 mm i.d. 5 µm Zorbax Eclipse XDB-C18 column at a temperature of 45˚C. A secondary pump (Waters Reagent Manager) was also attached to the system connected in-line after the column and before the detector. This allowed for mixing of the injected sample and a derivatization solution before going to the detector. The detector was set at 335 nm (excitation) and 440 nm (emission). The mobile phases for this system were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) in a gradient at a flow rate of 1.2 mL/min. for 18 minutes beginning at 68% A and 32% B for 8 minutes, switching to 60% A and 40% B for 3 minutes, and then back to 68% A and 32% B for the rest of the injection. The post-column reaction solution was prepared using sodium carbonate, boric acid, potassium sulfate, N-Acetyl-L-Cysteine, and o-phthaldialdehyde (OPA) and run at a constant flow rate of 0.45 mL/min. Standards for this analysis were prepared using fumonisin B₁ and B₂ (Sigma products F1147 and F3771, respectively) in 30% acetonitrile in water. The 30% acetonitrile was used as blanks for the analysis. The limit of quantitation was 0.025 ng/µL.

The experiment was a randomized complete block design with four replicates for each hybrid. Analyses of variance were conducted using Proc mixed of SAS (9.7) (SAS Institute, Cary, NC). Year, soil, cultivar, and genetic background were considered fixed effects. Replicate (Rep) and Rep (Year) were considered random effects. Residual values shown in Table 5 and Table 6 refer to Restricted Maximum Residual Likelihood (REML), which reflects the total variance of the random parameters in the model.

The levels of aflatoxin in samples from the Tunica clay compared to Bosket FSL are shown in Table 4. Aflatoxin levels observed among samples of the non-inoculated corn of the ten hybrids from the Tunica clay type was variable ranging from 1.4 µg/kg for stacked corn hybrid 31G96 to 88.9 µg/kg for stacked corn hybrid DK67-21. Aflatoxin levels among the inoculated corn of the same ten hybrids were variable as well ranging from 0.1 µg/kg for RR2 corn hybrid 31P40 to 207.4 µg/kg for RR2 corn hybrid DKC67-22. Overall aflatoxin levels in the ten corn hybrids tested were higher from inoculated soils than from non-inoculated soils, and varied significantly (P ≤ 0.05) among the ten corn hybrids tested in the inoculated fields in comparison to the levels of aflatoxin among the same hybrids tested in the non-inoculated soil. Levels of aflatoxin were variable and no one hybrid or group of hybrids was consistent in the levels of aflatoxin contamination when grown in Tunica clay under all experimental conditions.

^*Significance at P ≤ 0.05; ^**Significance at P ≤ 0.01; ^***Significance at P ≤ 0.001.

^†Means of 4 replications and 2 years. ^‡Means are not significantly different. ^§Means within a column followed by the same letter or letters are not significantly different P ≤ 0.05.

Aflatoxin levels of corn grown in Bosket FSL soil were higher ranging from 43.1 µg/kg for stacked corn hybrid DKC67-21 to 335.9 µg/kg for the stacked corn hybrid 31G96 under both non-inoculated and inoculated conditions compared with the overall levels of aflatoxin in Tunica clay soil (Table 4). Although there were significant differences (P ≤ 0.05) in aflatoxin levels among hybrids in Bosket FSL soil, all hybrids had aflatoxins above the regulatory level of 20 µg/kg.

The levels of fumonisin of the combined samples harvested in 2011 and 2012 are shown in Table 5 Fumo- nisin levels were less variable overall than aflatoxin levels ranging from 0.5 to 16.2 mg/kg. Average fumonisin levels were about the same in all hybrids in both non-inoculated and inoculated fields both years (Table 5), and differences were highly significant (P ≤ 0.05) among all hybrids. Fumonisin levels among all hybrids grown in non-inoculated and inoculated Bosket FSL soil type were not significantly different ranging from 1.2 mg/kg to 5.0 mg/kg (Table 5). Observations of fumonisin levels among the hybrids were similar to observations of aflatoxin levels among the hybrids. No single hybrid or group of hybrids stood out as resistant or susceptible.

Aflatoxin levels were quite different between 2011 and 2012, possibly due to temperature and rainfall conditions despite irrigation. Differences in aflatoxin levels were evident between 2011 and 2012 in grain samples from non-inoculated or inoculated treatments in Bosket FSL soil. The aflatoxin levels in the 2011 samples ranged from 237.5 to 339.9 µg/kg for non-inoculated and inoculated Bosket FSL soil, respectively, and were higher than aflatoxin levels from corn samples from the Tunica Clay soil (Table 6). Likewise, aflatoxin levels in the 2012 samples were lower than in 2011 samples and rangedfrom 11.8 µg/ kg to 52.1 µg/kg for the non- inoculated and inoculated field, respectively. The aflatoxin levels in harvested corn samples in 2011 and 2012 were lower in the non-inoculated Tunica Clay field, ranging from 15.2 µg/kg in 2012 to 15.9 µg/kg in 2011.

Fumonisin levels in corn samples harvested from Tunica Clay and Bosket FSL soil types were inconsistentas far as non-inoculated and inoculated fields in 2011 and 2012 (Table 6). Fumonisin levels in samples from both non-inoculated and inoculated soils in the Tunica clay field in 2011 were almost identical at 2.3 and 2.2 mg/kg, respectively. In 2012, the levels were significantly different at 0.3 and 4 mg/kg, respectively (P ≤ 0.05). Samples harvested from the non-inoculated and inoculated Bosket FSL fields were 2.9 and 6.0 mg/kg in 2011 and 2.6 and 0.6 mg/kg in 2012. Yields of the various corn hybrids used in this study were higher for all hybrids in the Tunica clay soil [12] compared to the same hybrids in the sandy loam Bosket FSL. During the course of the experiments, hybrids in the Tunica clay soil exhibited better plant growth and health (Bruns, Unpublished data).

^†Means of 4 replications and 2 years; ^‡Means are not significantly different; ^§Means within a column followed by the same letter or letters are not significantly different P ≤ 0.05.

^†Means of 10 hybrids representing 3 genotypes and 4 replications. ^‡Means are not significantly different. ^§Means within a column are significantly different P ≤ 0.05.

The theory that reducing plant stress during the kernel-filling time will cause reductions in mycotoxin levels was tested in this research by evaluating if weather data during the 2011 and 2012 growing seasons might correlate with aflatoxin and fumonisin levels (Figure 1). Weather comparisons between the 2 years were significantly different in rainfall. For example, the maximum temperature in June, July, and August 2011 were 34.9, 35.4, and 35.4˚C, respectively, compared to 31.7˚C, 33.7˚C, and 34.1˚C in 2012 (Figure 1). Similar trends for rainfall were observed where the precipitation was lower during June, July, and August, resulting in a warmer and drier year in 2011 than in 2012, although the precipitation was higher early in the season in 2011 compared to 2012. These conditions may have created additional stress due to warmer and drier season, especially during the kernel-filling stage and contributed to much higher aflatoxin contamination in sandy soils. Field water holding capacity of sandy soils is lower than that of clay soils, consequently crops grown in sandy soil are more sensitive to drought stress even when the irrigation is used. Fumonisin levels in the corn samples did not consistently relate to weather conditions. Recently it was found that these hybrids did not yield as well in sandy soil, possibly due to drought stress even when they received adequate irrigation, (Bruns, unpublished data) compared to the same hybrids grown in clay soil [12] [25] [39] . In all cases corn grown in sandy loam soil had much higher levels of aflatoxin. None of the hybrids, conventional or stacked-gene, were clearly superior to the others in terms of aflatoxin levels. Some reports of hybrids with a Bt gene showed variable effects on mycotoxin levels. Some authors reported beneficial effects of hybrids, others did not [1] [40] -[43] . A multifaceted strategy to reduce mycotoxins appears to be necessary [44] .