Final Report: Toxins of Bacillus thuringiensis in Transgenic Organisms: Persistence and Ecological Effects

EPA Grant Number: R826107
Title: 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: 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 : 21 Displayed | Download in RIS Format

Other project views: All 96 publications 39 publications in selected types All 32 journal articles
Type Citation Project Document Sources
Book Bitton G, ed. Encyclopedia of Environmental Microbiology, 6 Volume Set. Hoboken, NJ: Wiley, 2002, 3551 pp. R826107 (Final)
  • Abstract: Wiley - Abstract
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  • Book Goodman RM, ed. Encyclopedia of Plant and Crop Science. New York, NY: Marcel Dekker, 2004, pp. 1360. R826107 (Final)
    R829479C020 (2005)
  • Abstract: Amazon - Abstract
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  • Book Chapter Stotzky G. Release, persistence, and biological activity in soil of insecticidal proteins from Bacillus thuringiensis. In: Letourneau DK, Burrows BE, eds. Genetically Engineered Organisms: Assessing Environmental and Human Health Effects. Boca Raton, FL: CRC Press, September 26, 2002, Chapter 8, pp. 187-222. R826107 (2000)
    R826107 (Final)
  • Other: CRC - Book Description
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  • Book Chapter Stotzky G. Clays and humic acids affect persistence and biological activity of insecticidal proteins from Bacillus thuringiensis in soil. In: Violante A, Huang PM, Bollag J-M, Gianfreda L, eds. Soil Mineral-Organic Matter-Microorganism Interactions and Ecosystem Health (Developments in Soil Science). Amsterdam, The Netherlands: Elsevier, 2002, Volume 28, Part 2, pp. 1-16. R826107 (Final)
  • Abstract: Science Direct - Abstract
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  • Other: Science Direct - Book Table of Contents
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  • Book Chapter Stotzky G. Larvicidal proteins from Bacillus thuringiensis in soil: release, persistence, and effects. In: Goodman RM, ed. Encyclopedia of Plant and Crop Science. New York, NY: Marcel Dekker, December 12, 2007. R826107 (Final)
  • Other: Taylor & Francis - Book Table of Contents
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  • Journal Article 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)
  • Abstract: Science Direct - Abstract
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  • Journal Article 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)
  • Abstract: Science Direct - Abstract
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  • Journal Article 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)
  • Abstract from PubMed
  • Abstract: Canadian Journal of Microbiology - Abstract
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  • Journal Article 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)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: Applied and Environmental Microbiology - Full text
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  • Abstract: Applied and Environmental Microbiology - Abstract
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  • Other: Applied and Environmental Microbiology - PDF
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  • Journal Article 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)
  • Abstract from PubMed
  • Full-text: Wiley - Full text
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  • Abstract: Wiley - Abstract
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  • Other: Wiley - PDF
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  • Journal Article 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)
  • Abstract: Science Direct - Abstract
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  • Journal Article Saxena D, Stotzky G. Bt toxin uptake from soil by plants. Nature Biotechnology 2001;19(3):199. R826107 (Final)
  • Associated PubMed link
  • Abstract: Nature - Abstract
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  • Journal Article Saxena D, Stotzky G. Fate and effects of the insecticidal toxins from Bacillus thuringiensis in soil. ISB News Report 2001:5-7. R826107 (Final)
  • Other: ISB - PDF
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  • Journal Article 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)
  • Full-text: American Journal of Botany - Full text
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  • Abstract: American Journal of Botany - Abstract
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  • Other: American Journal of Botany - PDF
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  • Journal Article 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)
  • Abstract: Springer - Abstract
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  • Journal Article 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)
  • Full-text: StopOGM - PDF
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  • Abstract: Science Direct - Abstract
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  • Journal Article 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)
  • Abstract: Science Direct - Abstract
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  • Journal Article 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)
  • Other: International Centre for Genetic Engineering and Biotechnology (ICGEB) - PDF
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  • Journal Article 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)
  • Abstract: Journal of Environmental Quality - Abstract
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  • Journal Article 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)
  • Abstract: Science Direct - Abstract
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  • Journal Article 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)
  • Abstract: Science Direct - Abstract
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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 insects

    Progress and Final Reports:

    Original Abstract
  • 1998 Progress Report
  • 1999 Progress Report
  • 2000 Progress Report
  • 2001