Executive Summary
The objectives of this work are to detect and quantify the Bt toxin introduced to soils from Bt corn varieties that release Cry3Bb1, which is toxic to Corn rootworm, and Cry1Ab, which is toxic to European corn borer, and relate these measures to toxicity through bioassays using target larvae. In addition we will characterize the influence of cultural practices (tillage) on persistence of the toxin and investigate the influence of nitrogen status and moisture stress on toxin inputs from corn roots and their exudates. Results to date suggest that neither toxin is detectable using available Elias kits at any time during the growing season even when roots and tissues present in the soil do have detectable levels of the toxin. Our inability to recover toxins released via root exudates and contained in residues may result from their rapid decay, persistence in soils (adsorbed to clays and humic substances) in non-extractable form, or to their loss from soils via leaching. Ongoing research will attempt to quantify the amount of toxin added to soils by BT corn and account for toxin fate under typical cultural scenarios.
Samples were collected during summer 2003 from three experimental locations in Illinois. Samples from field trials conducted in Missouri at the Greenlay experiment station were obtained from Dr. Motavalli where cultivars resistant to rootworm were grown. To detect and quantify Bt toxins we have so far used commercially available Elisa (Enzyme-Linked Immunosorbent Assay) kits. These were developed to qualitatively test toxin in corn seeds. The commercial kit method was modified to test soil extracts and tissue extracts quantitatively with a detection limit of 5~10 ng toxin/mL (0.01ppm toxin) using a standard curve for the toxin in each Elisa plate. Soil, corn root and above ground tissue samples were collected from experimental plots at Urbana, Monmouth, and Dekalb IL producing CRW-corn (Cry3Bb) and suitable controls at three different grow stages. Samples were also collected for ECB-corn (Cry1Ab) and its controls grown at the Urbana site. Using Elisa we did not detect the toxins in soil extracts from IL although root and corn tissues consistently showed presence of active toxins. Toxin contents in plant tissues were highly variable and so did not vary significantly during the season or among sites. Cry3Bb contents in leaves ranged from 1696 to 2452 ng/g fresh weight; in stems from 361 to 982 ng/g fresh wt; in grain from 65 to 261 ng/g fresh wt; in ear husks from 988 to 1513 ng/g fresh wt; kernals from 400 to 1590 ng/g fresh wt; in roots from 98 to 380 ng/g fresh wt. Cry1Ab in plots in Urbana had contents in leaves and stems with an average of 939 ng/g fresh weight; Cobs had an average of 233 ng/g fresh tissue; ear husks had an average of 250 ng/g fresh tissue; kernels of 223 ng/g fresh tissue; and roots an average of 801ng/g fresh tissue. Total toxin loads added by roots and residues during the season are being computed. Soils from Mo. have been extracted but Elisa tests have not been run. In addition, preliminary trials have been run to develop bioassays. This summer, preliminary trials have been conducted to identify exchange membranes that can compete with soil for toxins. The IONICS membranes explored have a low efficiency for recovery.
Research Plans
Work planned for Fall-2004 and the coming year are as follows:
Bt toxins may persist adsorbed to clay and humic substances and remain un-extractable by protein extraction buffers. These toxins may be inert or be degraded. Alternatively, a worst case effect of sustained exposure of target insect larvae to toxins stabilized in soil could be the development of resistance to the toxin. We plan to conduct laboratory bioassay experiments using Tobacco hornworm larvae and Corn rootworm larvae to determine whether stabilized protiens retain ‘toxicity' to larvae. Given the challenges of rearing we have encountered and the large number of bioassays we must run, we are considering working with an insectary to obtain assay organisms. Soil will be mixed directly with the larval diet medium and fed to 2nd to 3rd instar larvae and growth and mortality will be monitored.
Bt toxin released via root exudates and decomposing residues can be lost to leaching through the soil. Anion exchange resins/or membranes suitable to separate large protein molecules are being selected and used in a laboratory study and in the field where transgenic corn is growing and following residue added to soil at end of season, to detect and quantify Bt toxin released through root exudates and residues, and that could be leaching. Resins/membranes will be inserted in to field soil to different depths for different time periods and will be extracted with protein extraction buffers. Extracts will be tested with Elisa and bioassays.
In the coming year we will begin to test influence of nitrogen status and moisture stress on toxin input via root exudates and relate these measures to root growth characteristics in the greenhouse: N fertility levels have shown to influence Bt toxin expression and non-target effects in transgenic crops. Ion-exchange membranes (with high capacity for Bt toxin in exudates)/or anion exchange resins that take up dissolved organic carbon will be placed inside the window of each root box, and CRW corn and its non transgenic isoline will be grown in boxes in 2 soils with differing organic matter contents, under 3 N fertility levels, and two moisture regimes. Root segments and membranes/resin will be sampled over time, by opening the side of the box. The root length also will be photographed and quantified digitally. Toxin extracted from membranes/resin and roots will be quantified by Elisa and bioassay. Relationship between root mass, length and toxin concentration in roots and exudates will be used to estimate toxin output through exudates in the field.
