Determination of Mycotoxin Production of Fusarium Species in Genetically Modified Maize Varieties by Quantitative Flow Immunocytometry.

Levels of mycotoxins produced by Fusarium species in genetically modified (GM) and near-isogenic maize, were determined using multi-analyte, microbead-based flow immunocytometry with fluorescence detection, for the parallel quantitative determination of fumonisin B1, deoxynivalenol, zearalenone, T-2, ochratoxin A, and aflatoxin B1. Maize varieties included the genetic events MON 810 and DAS-59122-7, and their isogenic counterparts. Cobs were artificially infested by F. verticillioides and F. proliferatum conidia, and contained F. graminearum and F. sporotrichoides natural infestation. The production of fumonisin B1 and deoxynivalenol was substantially affected in GM maize lines: F. verticillioides, with the addition of F. graminearum and F. sporotrichoides, produced significantly lower levels of fumonisin B1 (~300 mg·kg-1) in DAS-59122-7 than in its isogenic line (~580 mg·kg-1), while F. proliferatum, in addition to F. graminearum and F. sporotrichoides, produced significantly higher levels of deoxynivalenol (~18 mg·kg-1) in MON 810 than in its isogenic line (~5 mg·kg-1). Fusarium verticillioides, with F. graminearum and F. sporotrichoides, produced lower amounts of deoxynivalenol and zearalenone than F. proliferatum, with F. graminearum and F. sporotrichoides. T-2 toxin production remained unchanged when considering the maize variety. The results demonstrate the utility of the Fungi-Plex™ quantitative flow immunocytometry method, applied for the high throughput parallel determination of the target mycotoxins.

Effects of Bt Corn and Egg Density on Western Corn Rootworm (Coleoptera: Chrysomelidae) Adult Emergence and Estimation of Effective Bt Dose.

Since 2003, rootworm-protected transgenic corn has been commercially deployed in the United States as a principal method of control of western corn rootworm, Diabrotica virgifera virgifera LeConte. Durability of this technology depends partly on larval mortality ("dose") exerted by the traits, but density-dependent mortality can confound calculations of dose. Research reported here examined the effects of density-dependent mortality on adult emergence and estimates of trait dose. At sites in Illinois and Indiana, western corn rootworm eggs were infested at four densities on non-Bt corn and at a single density on corn hybrids with transgenic events MON 88017 (VT Triple PRO), DAS-59122-7 (Herculex Insect Protection), and MON 88017 × DAS-59122-7 (SmartStax corn). Beetles were collected weekly in large emergence cages. Density-dependent mortality and the effect of Bt traits were examined using percent survival from egg to adult, sex ratio, and beetle mass. Beetle emergence from Bt treatments was very low, and percent survival from non-Bt treatments was greatest at the lowest egg density (410 eggs per row-meter). Therefore, emergence from the lowest infestation density on non-Bt corn was used to estimate the effective dose of the Bt treatments. Sex ratio and beetle mass were unaffected by density-dependent effects and were not consistently affected by Bt traits. Dose was estimated at 97.4-99.3% for MON 88017, 98.8-99.9% for DAS-59122-7, and 99.7-100.0% for MON 88017 × DAS-59122-7. This study confirms the need to account for density-dependent mortality when estimating dose of corn rootworm protection events even at relatively low egg infestation densities.

Multiple Assays Indicate Varying Levels of Cross Resistance in Cry3Bb1-Selected Field Populations of the Western Corn Rootworm to mCry3A, eCry3.1Ab, and Cry34/35Ab1.

Minnesota populations of Diabrotica virgifera virgifera LeConte, the western corn rootworm, surviving Cry3Bb1-expressing corn in the field and western corn rootworm populations assumed to be susceptible to all Bt proteins were evaluated for susceptibility to Cry3Bb1, mCry3A, eCry3.1Ab, and Cry34/35Ab1 in diet assays and three different plant-based assays. Rootworm populations originating from Cry3Bb1 fields and that consistently experienced greater than expected damage had increased survival and larval growth compared to control populations assayed on Cry3Bb1 as well as mCry3a and eCry3.1Ab. Cross resistance was documented between Cry3Bb1 and both mCry3A and eCry3.1Ab as single toxins. Despite very high resistance ratios in some comparisons, cross resistance was not complete and also varied with the population being evaluated, the trait measured, and the susceptible rootworm population used for comparison. Regardless of resistance and cross resistance, all proteins, even Cry3Bb1, retained some efficacy in terms of either reducing rootworm larval growth, protecting plants from damage, or both, for all rootworm populations evaluated. For one Cry3Bb1-selected population, a resistance ratio of 9.1-fold was found to Cry34/35Ab1 when evaluating EC 50 values relative to a susceptible control population; however, resistance to Cry34/35Ab1 was not evident in all assays in this population. The United States Environmental Protection Agency recently suggested eliminating diet assays as part of the Bt resistance monitoring process. However, given the variability of responses of western corn rootworm populations to different proteins in different assays, both plant and diet assays are needed as options for detecting and fully characterizing resistance.

Quality of laboratory studies assessing effects of Bt-proteins on non-target organisms: minimal criteria for acceptability.

The potential risks that genetically modified plants may pose to non-target organisms and the ecosystem services they contribute to are assessed as part of pre-market risk assessments. This paper reviews the early tier studies testing the hypothesis whether exposure to plant-produced Cry34/35Ab1 proteins as a result of cultivation of maize 59122 is harmful to valued non-target organisms, in particular Arthropoda and Annelida. The available studies were assessed for their scientific quality by considering a set of criteria determining their relevance and reliability. As a case-study, this exercise revealed that when not all quality criteria are met, weighing the robustness of the study and its relevance for risk assessment is not obvious. Applying a worst-case expected environmental concentration of bioactive toxins equivalent to that present in the transgenic crop, confirming exposure of the test species to the test substance, and the use of a negative control were identified as minimum criteria to be met to guarantee sufficiently reliable data. This exercise stresses the importance of conducting studies meeting certain quality standards as this minimises the probability of erroneous or inconclusive results and increases confidence in the results and adds certainty to the conclusions drawn.

Susceptibility to Bt proteins is not required for Agrotis ipsilon aversion to Bt maize.

BACKGROUND: Although Bacillus thuringiensis (Bt) maize has been widely adopted in diverse regions around the world, relatively little is known about the susceptibility and behavioral response of certain insect pests to Bt maize in countries where this maize is not currently cultivated. These are important factors to consider as management plans are developed. These factors were investigated for Agrotis ipsilon, a global pest of maize, with Cry1F and Cry34Ab1/Cry35Ab1 maize. RESULTS: Agrotis ipsilon demonstrated an initial, post-ingestive aversive response to Cry1F maize. Development and mortality were also affected - survival on Cry1F maize tissue was 40% and weight gain of survivors of Cry1F exposure was significantly reduced. A post-ingestive aversive response was also seen for Cry34Ab1/Cry35Ab1 maize; however, longer-term feeding, weight gain and survival were not affected. CONCLUSION: Agrotis ipsilon showed aversion to both Bt treatments. Aversion to Cry34Ab1/Cry35Ab1 maize was unexpected because these proteins have no known insecticidal effect against Lepidoptera; however, results confirm that this aversion was temporary and did not affect growth or development. The Cry1F results suggest that A. ipsilon will abandon Cry1F maize in the field before any selection for resistance. These data support the use of refuge to delay Cry1F resistance development in A. ipsilon populations.