The effect of nitrogen rate on volunteer corn Bt protein expression

Volunteer corn expressing herbicide resistance is a problematic weed.  This issue is partially due to the increasing prevalence of stacking both herbicide and insect-resistant (mainly Bt) traits into the same genetically-modified plant.  Previous research indicates that the Bt toxin concentrations in nitrogen deficient volunteer corn roots may be less than that of volunteer corn plants with sufficient nitrogen. Because all current Bt toxins are crystalline proteins, we hypothesized that in-field factors such as soil nutrient levels (nitrogen, sulfur, etc.) could affect the expression levels of these proteins by corn plants. Our objectives were to quantify the concentration of Bt expressed in volunteer and hybrid corn root tissue in various nitrogen fertility environments. We conducted two sets of experiments (field and greenhouse) to accomplish these objectives.  Cry3Bb1 toxin levels in roots were determined using quantitative ELISA. In the field, we planted three corn varieties (DKC 61-19 VT3 Cry3Bb1-positive hybrid corn, DKC 61-22 Cry3Bb1-negative hybrid corn, and an F₂ of DKC 61-19 VT3 Cry3Bb1-positive volunteer corn), and applied 5 rates of nitrogen (0, 45, 90, 180, and ≈ 360 kg N ha^-1). Expression of Cry3Bb1 protein in root tissues from the field experiment was highly variable, but there was no difference in the overall concentration of Cry3Bb1 expressed in the root tissue between Cry3Bb1-positive volunteer corn (18.7 ± 2.70 ppm) and Cry3Bb1-positive hybrid corn (13.2 ± 2.93 ppm) at the V6 to V9 growth stage.  Nitrogen rate did affect Cry3Bb1 expression in the field; lower rates of nitrogen resulted in decreased Cry3Bb1 expression. We also conducted a greenhouse trial to quantify Cry3Bb1 expression in hybrid and volunteer corn growing in 5 nitrogen fertility environments (0, 25, 50, 100, and 200 mg N L^-1). DKC 61-19 was used as the Cry3Bb1-positive hybrid corn and the F₂ of DKC 61-19 was used as the Cry3Bb1-positive volunteer corn. All plants were harvested at the V6 growth stage. Root and shoot biomass was quantified after drying, and root tissue samples were collected at harvest to quantify Bt expression.  As with the field experiment, there was no difference in Cry protein expression between volunteer corn (6.94 ± 1.24 ppm) and hybrid corn (9.32 ± 1.23 ppm).  As in the field experiment, there was an effect of nitrogen on Cry3Bb1 expression.  When nitrogen was not applied there was less Cry3Bb1 expressed in volunteer corn plants.  Interestingly, this effect was not observed in hybrid corn.  These data illustrate that reduced volunteer corn Cry toxin expression may be more typically observed in soybean fields where nitrogen is not applied, and volunteer corn plants would be nitrogen deficient.  In addition to being a troublesome weed, volunteer corn may be problematic for insect resistance management plans by exposing the target insects to sublethal doses of the toxin. Recent data has illustrated that the acetyl-CoA carboxylase inhibitor, clethodim, (a commonly used herbicide for POST volunteer corn control in soybean) also has reduced efficacy on nitrogen deficient volunteer corn. Thus, in order to control Cry3Bb1-positive volunteer corn, and decrease the likelihood of insects feeding on sublethal doses of the toxin, it is advisable to control volunteer corn in soybean early in the season and follow recommended herbicide use rates.