Grantee Research Project Results
Final Report: Toxins of Bacillus thuringiensis in Transgenic Organisms: Persistence and Ecological Effects
EPA Grant Number: R826107Title: Toxins of Bacillus thuringiensis in Transgenic Organisms: Persistence and Ecological Effects
Investigators: Stotzky, Guenther
Institution: New York University
EPA Project Officer: Hahn, Intaek
Project Period: November 7, 1997 through November 6, 2000 (Extended to May 5, 2002)
Project Amount: $393,411
RFA: Exploratory Research - Environmental Biology (1997) RFA Text | Recipients Lists
Research Category: Aquatic Ecosystems , Biology/Life Sciences
Objective:
The objective of this research project was to study the release, persistence, and biological activity of the larvicidal proteins from Bacillus thuringiensis in transgenic plants in soil.
Summary/Accomplishments (Outputs/Outcomes):
The findings are reported below under two categories. Presented first are the findings from the summary of interaction of purified Bt toxins with surface-active particles. Presented second are findings from the summary of fate and effects of Bt toxins in root exudates and biomass of transgenic plants.
Summary of Interactions of Purified Bt Toxins With Surface-Active Particles: Effects on Persistence and Larvicidal Activity
- Larvicidal proteins from Bacillus thuringiensis subspp. kurstaki (Btk; antilepidopteran), tenebrionis (Btt; anticoleopteran), and israelensis (Bti; antidipteran) bound rapidly and tightly on clays, humic acids, and complexes of clay-humic acid-Al hydroxypolymers; binding was pH dependent and greatest near the isoelectric point (pI) of the proteins; binding of the toxin from Btk was greater than binding of the toxin from Btt, even though the Mr of both was similar (66 and 68 kDa, respectively).
- Bound toxins retained their structure, antigenicity, and insecticidal activity.
- Intercalation of clays by the toxins was minimal.
- Biodegradation of the toxins was reduced when bound; microbial utilization of the toxins as a source of carbon was reduced significantly more than use as a source of nitrogen.
- Larvicidal activity of bound toxins was retained.
- Larvicidal activity of the toxin from Btk was detected 234 days after addition to nonsterile soils (longest time studied).
- Persistence of larvicidal activity was greater in acidic soils, most likely, because microbial activity was lower than in less acid soils; persistence was reduced when the pH of acidic soils was raised to about 7.0 with CaCO3.
- Persistence was similar under aerobic and anaerobic conditions, and when soil was alternately wetted and dried or frozen and thawed, this indicated tight binding.
- Persistence in soil was demonstrated by dot-blot ELISA, flow cytometry, Western blots, and larvicidal assays.
- Toxins from Btk, Btt, and Bti had no microbiostatic or microbicidal effect against a spectrum of bacteria (gram positive and negative), fungi (filamentous and yeast), and algae, neither in pure nor in mixed cultures.
Summary of Fate and Effects of Bt Toxins in Root Exudates and Biomass of Transgenic Plants
- Biodegradation of biomass of transgenic Bt corn, measured by CO2 evolution, was significantly lower than that of isogenic non-Bt corn.
- No consistent statistically significant differences in the numbers of culturable bacteria, fungi, and the activity of representative enzymes between soil amended with Bt or non-Bt corn or not amended.
- Reduced metabolic activity of soil amended with Bt corn may have been the result of significantly higher lignin content in Bt than in non-Bt corn.
- Biodegradation of biomass of Bt rice, cotton, canola, tobacco, and potato also was significantly lower than that of biomass of isogenic non-Bt plants; however, the lignin content of these plant species, which was significantly lower than that of corn, was not significantly different between Bt and non-Bt biomass.
- CrylAb protein was released in root exudates of Bt corn (13 hybrids representing three transformation events) and persisted in rhizosphere soil in vitro and in situ; protein accumulated more in soil amended (3 to 12 percent) with montmorillonite than with kaolinite.
- CrylAb protein released in root exudates or from biomass of Bt corn appeared to have no effect on numbers of earthworms, nematodes, protozoa, bacteria, and fungi in soil.
- CrylAc protein was not released in root exudates of Bt canola, tobacco, and cotton. Cry1Ab protein was released in root exudates of rice, and Cry3A protein was released in root exudates of Bt potato.
- CrylAb protein released in root exudates and from biomass of Bt corn was not taken up from nonsterile soil or sterile hydroponic culture by non-Bt corn, carrot, radish, and turnip, even though the toxin persisted for at least 180 days in soil (the longest time studied).
- CrylAb protein-purified, in root exudates, and from biomass of Bt corn moved through soil during leaching with water. Movement was less in soils amended with montmorillonite than with kaolinite and it decreased as the concentration of added clays increased.
- Toxins from Bt could persist, accumulate, and remain insecticidal in soil as the result of binding on clays and humic substances and, therefore, pose a hazard to nontarget organisms, enhance selection of toxin-resistant target species, or enhance control of insect pests.
Journal Articles on this Report : 16 Displayed | Download in RIS Format
Other project views: | All 96 publications | 39 publications in selected types | All 32 journal articles |
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Crecchio C, Stotzky G. Insecticidal activity and biodegradation of the toxin from Bacillus thuringiensis subsp. kurstaki bound to humic acids from soil. Soil Biology & Biochemistry 1998;30(4):463-470. |
R826107 (1998) R826107 (2000) R826107 (Final) |
Exit |
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Crecchio C, Stotzky G. Biodegradation and insecticidal activity of the toxin from Bacillus thuringiensis subsp. kurstaki bound on complexes of montmorillonite-humic acids-Al hydroxypolymers. Soil Biology & Biochemistry 2001;33(4-5):573-581. |
R826107 (Final) |
Exit |
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Koskella J, Stotzky G. Larvicidal toxins from Bacillus thuringiensis subspp. kurstaki, morrisoni (strain tenebrionis), and israelensis have no microbicidal or microbiostatic activity against selected bacteria, fungi, and algae in vitro. Canadian Journal of Microbiology 2002;48(3):262-267. |
R826107 (1998) R826107 (1999) R826107 (2000) R826107 (Final) |
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Lee L, Saxena D, Stotzky G. Activity of free and clay-bound insecticidal proteins from Bacillus thuringiensis subsp. israelensis against the mosquito, Culex pipiens. Applied and Environmental Microbiology 2003;69(7):4111-4115. |
R826107 (Final) |
Exit Exit Exit |
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Saxena D, Stotzky G. Insecticidal toxin from Bacillus thuringiensis is released from roots of transgenic Bt corn in vitro and in situ. FEMS Microbiology Ecology 2000;33(1):35-39. |
R826107 (1998) R826107 (1999) R826107 (2000) R826107 (Final) |
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Saxena D, Stotzky G. Bacillus thuringiensis (Bt) toxin released from root exudates and biomass of Bt corn has no apparent effect on earthworms, nematodes, protozoa, bacteria, and fungi in soil. Soil Biology & Biochemistry 2001;33(9):1225-1230. |
R826107 (2000) R826107 (Final) |
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Saxena D, Stotzky G. Bt toxin uptake from soil by plants. Nature Biotechnology 2001;19(3):199. |
R826107 (Final) |
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Saxena D, Stotzky G. Fate and effects of the insecticidal toxins from Bacillus thuringiensis in soil. ISB News Report 2001:5-7. |
R826107 (Final) |
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Saxena D, Stotzky G. Bt corn has a higher lignin content than non-Bt corn. American Journal of Botany 2001;88(9):1704-1706. |
R826107 (2000) R826107 (Final) |
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Saxena D, Stotzky G. Bt toxin is not taken up from soil or hydroponic culture by corn, carrot, radish, or turnip. Plant and Soil 2002;239(2):165-172. |
R826107 (2000) R826107 (Final) |
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Saxena D, Flores S, Stotzky G. Bt toxin is released in root exudates from 12 transgenic corn hybrids representing three transformation events. Soil Biology & Biochemistry 2002;34(1):133-137. |
R826107 (2000) R826107 (Final) |
Exit Exit |
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Saxena D, Flores S, Stotzky G. Vertical movement in soil of insecticidal Cry1Ab protein from Bacillus thuringiensis. Soil Biology & Biochemistry 2002;34(1):111-120. |
R826107 (2000) R826107 (Final) |
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Saxena D, Stotzky G. Fate and effects in soil of the insecticidal toxins from Bacillus thuringiensis in transgenic plants. Collection of Biosafety Reviews 2003;1:9-85. |
R826107 (Final) |
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Stotzky G. Persistence and biological activity in soil of insecticidal proteins from Bacillus thuringiensis and of bacterial DNA bound on clays and humic acids. Journal of Environmental Quality 2000;29(3):691-705. |
R826107 (1998) R826107 (1999) R826107 (2000) R826107 (Final) |
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Tapp H, Stotzky G. Persistence of the insecticidal toxin from Bacillus thuringiensis subsp. kurstaki in soil. Soil Biology & Biochemistry 1998;30(4):471-476. |
R826107 (Final) |
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Vettori C, Paffetti D, Saxena D, Stotzky G, Giannini R. Persistence of toxins and cells of Bacillus thuringiensis subsp. kurstaki introduced in sprays to Sardinia soils. Soil Biology and Biochemistry 2003;35(12):1635-1642. |
R826107 (2000) R826107 (Final) |
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Supplemental Keywords:
Bacillus thuringiensis, toxin, transgenic organism, ecology, persistence, larvicidal protein;, RFA, Scientific Discipline, Waste, Ecosystem Protection/Environmental Exposure & Risk, Bioavailability, Ecosystem/Assessment/Indicators, Ecosystem Protection, Ecological Effects - Environmental Exposure & Risk, Environmental Microbiology, Biochemistry, Ecology and Ecosystems, Ecological Risk Assessment, Ecological Indicators, ecological effects, ecological exposure, microbiology, gene-environment interaction, ecological assessment, insecticides, aquatic ecosystems, assessment methods, bioassay, ecotoxicological studies, soil enzyme, flow cytometry assays, Bacillus thuringiensis, persistence, microbial, transgenic organisms, toxin resistant target insectsProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.