G. Stotzky

Insecticidal toxin in root exudates from Bt corn

Bt corn is corn (Zea mays) that has been genetically modified to express insecticidal toxins derived from the bacterium Bacillus thuringiensis to kill lepidopteran pests feeding on these plants. Here we show that Bt toxin is released into the rhizosphere soil in root exudates from Bt corn.

Bt corn has a higher lignin content than non-Bt corn

Bt corn has been genetically modified to express the Cry1Ab protein of Bacillus thuringiensis to kill lepidopteran pests. Fluorescence microscopy and staining with toluidine blue indicated a higher content of lignin in the vascular bundle sheaths and in the sclerenchyma cells surrounding the vascular bundle in all ten Bt corn hybrids, representing three different transformation events, studied than of their respective non-Bt isolines. Chemical analysis confirmed that the lignin content of all hybrids of Bt corn, whether grown in a plant growth room or in the field, was significantly higher (33-97% higher) than that of their respective non-Bt isolines. As lignin is a major structural component of plant cells, modifications in lignin content may have ecological implications.

Bt toxin is released in root exudates from 12 transgenic corn hybrids representing three transformation events

Abstract The anti-lepidopteran toxin (Cry1Ab protein) encoded by truncated genes from Bacillus thuringiensis was released in the root exudates from all hybrids of Bt corn studied and which represented three transformation events (Bt11, MON810, and 176). In vitro and in situ studies indicated that the toxin released in root exudates accumulates in soil, as it adsorbs and binds rapidly on surface-active particles (e.g. clays and humic substances), and retains insecticidal activity for at least 180xa0d, the longest time studied. The results indicated that the release of the Cry1Ab protein by roots is a common phenomenon with transgenic Bt corn and is not restricted to only the one Bt corn hybrid (NK4640Bt) and tranformation event (Bt11) studied initially.

Persistence and biological activity in soil of the insecticidal proteins from Bacillus thuringiensis, especially from transgenic plants

Insecticidal proteins produced by various subspecies (kurstaki, tenebrionis, and israelensis) of Bacillus thuringiensis (Bt) bound rapidly and tightly on clays, both pure mined clay minerals and soil clays, on humic acids extracted from soil, and on complexes of clay and humic acids. Binding reduced susceptibility of the proteins to microbial degradation. However, bound proteins retained biological activity. Purified Cry1Ab protein and protein released from biomass of transgenic Bt corn and in root exudates of growing Bt corn (13 hybrids representing three transformation events) exhibited binding and persistence in soil. Insecticidal protein was also released in root exudates of Bt potato (Cry3A protein) and rice (Cry1Ab protein) but not in root exudates of Bt canola, cotton, and tobacco (Cry1Ac protein). Vertical movement of Cry1Ab protein, either purified or in root exudates or biomass of Bt corn, decreased as the concentration of the clay minerals, kaolinite or montmorillonite, in soil increased. Biomass of transgenic Bt corn decomposed less in soil than biomass of near-isogenic non-Bt corn, possibly because biomass of Bt corn had a significantly higher content of lignin than biomass of non-Bt corn. Biomass of Bt canola, cotton, potato, rice, and tobacco also decomposed less than biomass of the respective near-isogenic non-Bt plants. However, the lignin content of these Bt plants, which was significantly less than that of Bt corn, was not significantly different from that of their near-isogenic non-Bt counterparts, although it was consistently higher. The Cry1Ab protein had no consistent effects on organisms (earthworms, nematodes, protozoa, bacteria, fungi) in soil or in vitro. The Cry1Ab protein was not taken up from soil by non-Bt corn, carrot, radish, or turnip grown in soil in which Bt corn had been grown or into which biomass of Bt corn had been incorporated.

Vertical movement in soil of insecticidal Cry1Ab protein from Bacillus thuringiensis

Abstract The effects of montmorillonite (M) or kaolinite (K) on the vertical movement of the insecticidal Cry1Ab protein of Bacillus thuringiensis subsp. kurstaki (Bt) were studied in repacked soil columns. The protein was added to the columns either in a purified form, as root exudates from growing plants of Bt corn, or within the biomass of residues of Bt corn. The soil was amended to 0, 3, 6, 9, or 12% (volxa0vol −1 ) with the clays. Vertical movement of the protein generally decreased as the content of either clay was increased, and the amount of protein recovered in leachates increased as the concentration of purified protein added was increased. The largest amount of purified protein (ca. 75%) was leached from soil not amended with clay, whereas the lowest amount (ca. 16%) was recovered from columns containing soil amended to 12% with M or K. The Cry1Ab protein was also present in leachates from soil columns in which various hybrids of Bt corn were grown or to which biomass of Bt corn had been added, whereas it was absent in leachates from columns in which the respective isolines of non-Bt corn were grown or to which biomass of non-Bt corn had been added. The Cry1Ab protein exhibited stronger binding and higher persistence, as well as remaining nearer the soil surface, in soil that contained the higher clay concentrations (i.e. had a higher cation-exchange capacity and specific surface area), indicating that it could be transported to surface waters via runoff and erosion. In contrast, the protein was more readily leached through soil with lower clay concentrations, indicating that it could contaminate groundwater.

Activity of Free and Clay-Bound Insecticidal Proteins from Bacillus thuringiensis subsp. israelensis against the Mosquito Culex pipiens

ABSTRACT Bacillus thuringiensis subsp. israelensis produces parasporal insecticidal crystal proteins (ICPs) that have larvicidal activity against some members of the order Diptera, such as blackflies and mosquitoes. Hydrolysis of the ICPs in the larval gut results in four major proteins with a molecular mass of 27, 65, 128, and 135 kDa. Toxicity is caused by synergistic interaction between the 25-kDa protein (proteolytic product of the 27-kDa protein) and one or more of the higher-molecular-mass proteins. Equilibrium adsorption of the proteins on the clay minerals montmorillonite and kaolinite, which are homoionic to various cations, was rapid (<30 min for maximal adsorption), increased with protein concentration and then reached a plateau (68 to 96% of the proteins was adsorbed), was significantly lower on kaolinite than on montmorillonite, and was not significantly affected by the valence of the cation to which the clays were homoionic. Binding of the toxins decreased as the pH was increased from 6 to 11, and there was 35 to 66% more binding in phosphate buffer at pH 6 than in distilled water at pH 6 or 7.2. Only 2 to 12% of the adsorbed proteins was desorbed by two washes with water; additional washings desorbed no more toxins, indicating that they were tightly bound. Formation of clay-toxin complexes did not alter the structure of the proteins, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the equilibrium supernatants and desorption washes and by dot blot enzyme-linked immunosorbent assay of the complexes, which was confirmed by enhanced chemiluminescence Western blot analysis. Free and clay-bound toxins resulted in 85 to 100% mortality of the mosquito Culex pipiens. Persistence of the bound toxins in nonsterile water after 45 days was significantly greater (mortality of 63% ± 12.7%) than that of the free toxins (mortality of 25% ± 12.5%).

Bt toxin is not taken up from soil or hydroponic culture by corn, carrot, radish, or turnip

The culture of transgenic Bt corn (Zea mays L.) has resulted in concern about the uptake of the Cry1Ab protein toxin by crops subsequently grown in soils in which Bt corn has been grown. The toxin released to soil in root exudates of Bt corn, from the degradation of the biomass of Bt corn, or as purified toxin, was not taken up from soil, where the toxin is bound on surface-active particles (e.g. clays and humic substances), or from hydroponic culture, where the toxin is not bound on particles, by non-Bt corn, carrot (Daucus carota L.), radish (Raphanus sativus L.), and turnip (Brassica rapa L.). The persistence of the toxin in soil for 90 days after its addition in purified form or for 120–180 days after its release in exudates or from biomass, the longest times evaluated, confirmed that the toxin was bound on surface-active particles in soil, which protected the toxin from biodegradation. The greater toxicity of the toxin in soil amended with 9% montmorillonite or kaolinite than in soil amended with 3% of these clay minerals indicated that the binding and persistence of the toxin increased as the clay concentration was increased.

Fate and Effects in Soil of Cry Proteins from Bacillus thuringiensis: Influence of Physicochemical and Biological Characteristics of Soil

Bacillus thuringiensis (Bt) is a useful alternative or supplement to synthetic chemical pesticides in agriculture, forest management, and control of mosquitoes and some other biting insects. When modified Bt cry genes are inserted into a plant species (e.g., corn, cotton, potato, canola, rice), the plant expresses active larvicidal proteins in its tissues. The toxins continue to be synthesized during growth of the plants, making the plant toxic to various insect pests throughout their life or as biomass incorporated into soil. If production exceeds consumption, inactivation, and degradation, the toxins could accumulate to concentrations that may enhance the control of target pests or constitute a hazard to nontarget organisms, such as the soil microbiota, beneficial insects (e.g., pollinators, predators and parasites of insect pests), and other animal classes. The accumulation and persistence of the toxins could also result in the selection and enrichment of toxin-resistant target insects. Persistence is enhanced when the toxins are bound on surface-active particles in the environment (e.g., clays and humic substances) and, thereby, rendered more resistant to biodegradation while retaining toxic activity. Moreover, major problem we face today is of Molecular pharming that utilizes transgenic plants and animals for production of pharmaceuticals and chemicals for their use in human beings and industries respectively. Their release to the environment, especially to soil and potentially to waters of the pharmaceutical and industrial products of transgenic plant and animal pharms could pose a hazard to the environment. In contrast to the products of most transgenic plants currently available commercially (e.g., the insecticidal proteins from subspecies of Bt) that primarily target insects and other pests. These pharms are being genetically engineered to express products for use primarily in human beings. Consequently, these products constitute a class of compounds that is seldom found in natural habitats and that primarily target higher level eukaryotes. Hence, they are xenobiotics with respect to the environment, and their persistence in and effects on the environment have not been adequately studied and sober risk assessments on a case-by- case basis must be made before major releases of such transgenic organisms.

Is the Cry1Ab protein from Bacillus thuringiensis (Bt) taken up by plants from soils previously planted with Bt corn and by carrot from hydroponic culture

The uptake of the insecticidal Cry1Ab protein from Bacillusthuringiensis (Bt) by various crops from soils on which Bt corn had previously grown was determined. In 2005, the Cry1Ab protein was detected by Western blot in tissues (leaves plus stems) of basil, carrot, kale, lettuce, okra, parsnip, radish, snap bean, and soybean but not in tissues of beet and spinach and was estimated by enzyme-linked immunosorbent assay (ELISA) to be 0.05xa0±xa00.003xa0ngxa0g−1 of fresh plant tissue in basil, 0.02xa0±xa00.014xa0ngxa0g−1 in okra, and 0.34xa0±xa00.176xa0ngxa0g−1 in snap bean. However, the protein was not detected by ELISA in carrot, kale, lettuce, parsnip, radish, and soybean or in the soils by Western blot. In 2006, the Cry1Ab protein was detected by Western blot in tissues of basil, carrot, kale, radish, snap bean, and soybean from soils on which Bt corn had been grown the previous year and was estimated by ELISA to be 0.02xa0±xa00.014xa0ngxa0g−1 of fresh plant tissue in basil, 0.19xa0±xa00.060xa0ngxa0g−1 in carrot, 0.05xa0±xa00.018xa0ngxa0g−1 in kale, 0.04xa0±xa00.022xa0ngxa0g−1 in radish, 0.53xa0±xa00.170xa0ngxa0g−1 in snap bean, and 0.15xa0±xa00.071xa0ngxa0g−1 in soybean. The Cry1Ab protein was also detected by Western blot in tissues of basil, carrot, kale, radish, and snap bean but not of soybean grown in soil on which Bt corn had not been grown since 2002; the concentration was estimated by ELISA to be 0.03xa0±xa00.021xa0ngxa0g−1 in basil, 0.02xa0±xa00.008xa0ngxa0g−1 in carrot, 0.04xa0±xa00.017xa0ngxa0g−1 in kale, 0.02xa0±xa00.012xa0ngxa0g−1 in radish, 0.05xa0±xa00.004xa0ngxa0g−1 in snap bean, and 0.09xa0±xa00.015xa0ngxa0g−1 in soybean. The protein was detected by Western blot in 2006 in most soils on which Bt corn had or had not been grown since 2002. The Cry1Ab protein was detected by Western blot in leaves plus stems and in roots of carrot after 56xa0days of growth in sterile hydroponic culture to which purified Cry1Ab protein had been added and was estimated by ELISA to be 0.08xa0±xa00.021 and 0.60xa0±xa00.148xa0ngxa0g−1 of fresh leaves plus stems and roots, respectively. No Cry1Ab protein was detected in the tissues of carrot grown in hydroponic culture to which no Cry1Ab protein had been added. Because of the different results obtained with different commercial Western blot (i.e., from Envirologix and Agdia) and ELISA kits (i.e., from Envirologix, Agdia, and Abraxis), it is not clear whether the presence of the Cry1Ab protein in the tissues of some plants under field condition and in carrot in sterile hydroponic culture was the result of the uptake of the protein by the plants or of the accuracy and sensitivity of the different commercial kits used. More detailed studies with additional techniques are obviously needed to confirm the uptake of Cry proteins from soil by plants subsequently planted after a Bt crop.