Grantee Research Project Results
2000 Progress 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 Period Covered by this Report: November 7, 1999 through November 6, 2000
Project Amount: $393,411
RFA: Exploratory Research - Environmental Biology (1997) RFA Text | Recipients Lists
Research Category: Aquatic Ecosystems , Biology/Life Sciences
Objective:
The objectives of the project are to: (1) determine the persistence in soil of the insecticidal toxins produced by subspecies of Bacillus thuringiensis; (2) elucidate the mechanisms responsible for persistence; (3) compare the insecticidal activity of free and particle-bound toxins, as well as their toxicity to microorganisms; and (4) evaluate the effects of the toxins, both purified and in genetically-modified plants, on microbial populations and processes in soil.Progress Summary:
Among the major research achievements were:
- The demonstration that the Cry1Ab protein is released in the root exudates from 12 other Bt corn hybrids, representing three transformation events (Bt11, MON810, and 176), in addition to the initial Bt hybrid studied (see last year's report). These results, obtained both in the plant-growth room and in the field, indicate that the release of the Bt toxin in the root exudates of Bt corn is a phenomenon common to all Bt corn hybrids. It also raises again the question of how the 66-kDa protein is released from roots. Apparently, corn does not need a signal peptide for the exudation of macromolecules because of the interaction of the endoplasmic reticulum with the cytoplasmic membrane. Studies on the release of the toxin from other plant species may help to resolve this question (see below). The detection of the toxin in soil 180 days (the longest time studied) after the harvest of Bt corn plants confirms again that the toxin persists in soil, probably as the result of its binding on surface-active particles (i.e., clays and humic substances) in soil (see below).
- Confirmation with other batches (different hybrids) of corn that Bt corn is degraded less than isogenic non-Bt corn. Although no consistent differences in numbers of culturable bacteria and fungi or of enzymes involved in biodegradation of plant biomass were again observed (see last year's report), the addition of purified Cry1Ab protein (the toxin) to biomass of Bt and non-Bt corn reduced biodegradation, suggesting that the toxin either has an effect on organisms involved in biodegradation or on some other, as yet undefined, processes involved in degradation. Molecular studies on the composition of the degradative microbiota may resolve this apparent paradox (see below).
- As one of the reasons for the lower biodegradation of Bt corn may be structural (see last year's report), the lignin content (as determined cytochemically and chemically) of 10 hybrids of Bt and non-Bt corn, grown in the plant growth-room and in the field, was compared. The lignin content of all Bt corn hybrids was significantly higher (33 to 97 percent higher) than the lignin content of their non-Bt isolines. The lignin content of hybrids with the Bt11 transformation event was significantly higher than that of hybrids with the MON810 transformation event, and the one available sample of the hybrid with the 176 transformation event was significantly lower than either. These results indicated that the insertion of the cry1Ab gene into the genome of corn may cause pleiotropic effects and that the lower degradation of Bt corn in soil may be a function of the higher lignin content.
- The Cry1Ab protein released in root exudates or from biomass of Bt corn appears to have no effect on the numbers of culturable bacteria and fungi, on protozoa and nematodes, and on earthworms in soil in the plant growth room. Earthworms (Lumbricus terrestis) were placed in soil in which Bt or non-Bt corn was grown for 40 days and for 45 days in soil amended with ground biomass of Bt or non-Bt corn. The numbers of earthworms and their weight were not significantly different between soil with Bt corn or non-Bt corn, either as plants or as biomass, or soil in which no plants were grown and no corn biomass was added. Similar results were obtained with the numbers of bacteria (including actinomycetes) and fungi (determined by serial dilution plate techniques), protozoa (determined by a most-probable-number method), and nematodes (determined by the Baermann technique), with the exception that the numbers of culturable bacteria were higher in soil without biomass or in which no plants were grown. The reasons for this exception are being studied. Moreover, techniques of molecular biology will be used to "fine-tune" the effects of the Bt toxin on bacterial diversity (see below).
- The Cry1Ab protein is not taken up by plants (i.e., non-Bt corn, carrot, radish, turnip) grown in soil in which Bt corn has been grown or into which biomass of Bt corn has been incorporated. This was not unexpected, as it is difficult to understand how a 66-kDa molecule can be taken up intact by roots (it is somewhat easier to understand how such a molecule can be released from roots). These studies were done because a major food chain in the United Kingdom indicated that it will not sell crops that have been grown in soils in which Bt crops had previously been grown. These studies also showed that the toxin released in root exudates and biomass from Bt corn was easily detectable (by both immunological and larvicidal assays; see last year's report) 180 days (the longest time studied) after the harvest of Bt corn or incorporation of biomass of Bt corn. These long periods of persistence confirm results found previously in this laboratory (see previous years' reports) and contrast with periods of persistence reported by "industry" and others, which erroneously report "persistence" as half-life values, even though their data do not conform to the first-order kinetics necessary to calculate half-life.
- The vertical movement of the Cry1Ab proteins?either purified, from root exudates of growing plants, or from incorporated biomass of Bt corn?during leaching of soil amended with different concentrations of the clay minerals, montmorillonite and kaolinite, decreased as the cation-exchange capacity and specific surface area of the soil-clay mixtures increased. These preliminary studies have relevance to the movement of the Bt toxins through soil to surface and ground waters. Moreover, the toxin bound at different depths in the soil columns retained larvicidal activity.
- The Cry4 proteins from B. thuringiensis subsp. israelensis have been purified and, most importantly, stabilized in solution after purification. This major breakthrough will facilitate studies on the adsorption and binding of these proteins on clay minerals and humic acids and their subsequent persistence and biological activity in soil and sediment (see last year's report and below).
- Participation in the EU field studies in Africa (see last year's report) was minimal, principally because of time constraints. Although I was invited to visit field sites in Africa in September, prior commitments prevented me from doing so. However, I am still informally "consulting" on these studies. Moreover, a new cooperative program with people in Florence, Italy, has been established on the persistence of the toxin in sprays in soils of Sardinia and on the horizontal transfer of the cry genes in soil (see publications below).
The contributions of Dr. Deepak Saxena and Mr. Saul Flores, who again spent several months in the laboratory this summer, were invaluable to the progress made on this project. Several undergraduate and graduate students, as well as a high school student, also contributed significantly and, in turn, received valuable exposure to and experience in experimental science.
Future Activities:
During the subsequent project year (a no-cost, one-year extension of this project has been approved), studies will continue on various aspects discussed above, and several new aspects will be initiated. Some of these new initiatives are the result of the recent interest in cooperation and collaborative research that some industries have expressed.- The release of the Cry1Ab protein from other transgenic Bt plants will be studied. Plants of Bt canola, cotton, potato, and tobacco (which also contains the gene for green fluorescent protein) will be grown in nonsterile soil and in sterile hydroponic culture (with the exception of potato, as it will be difficult to sterilize "eyes"), and the release of the protein in root exudates will be determined by the immunological and larvicidal assays now used routinely in this laboratory. These studies should indicate whether the release of the 66-kDa protein is unique to Bt corn or occurs with other Bt plants. If it occurs with all the studied Bt plants, then the mechanisms of release of such a large protein by both mono- and dicotyledonous plants need to be considered. Such considerations are beyond the scope of this project, but they could form the basis for future studies by either this or some other laboratory.
- Adapt the techniques of molecular biology (e.g., single-strand conformation polymorphism, denaturing and temperature gradient gel electrophoresis for bacteria), as well as of other techniques (e.g., the BIOLOG method for bacteria, nutritional groupings for protozoa and nematodes, colony development from hyphae and species identification for fungi), to determine the effects of Cry proteins, whether derived from root exudates, plant residues, or purified, on microbial species diversity in soil.
- Continue degradation studies (i.e., CO2 evolution and microbial counts and enzyme activities) with biomass of Bt and isogenic non-Bt plants of different species. Central to these studies will be the effects on biodegradation of the addition of purified Cry1Ab toxin to soils amended with Bt and non-Bt biomass.
- Studies on root exudation, degradation, and lignin content of Bt corn expressing the Cry9C (Aventis) and Cry1F (Dow) proteins will be initiated using the techniques described above for Bt corn expressing the Cry1Ab protein. Activity of these Cry proteins will be determined only by larvicidal assays until appropriate immunological assays become available.
- Leaching studies will continue using more soil-clay mixtures, different concentrations of humic substances, different leaching solutions (e.g., soil extract), larger columns, and longer growth of Bt plants for longer release of root exudates.
- Perfusion studies on the effect of the Cry toxins (purified, in root exudates, and in biomass) on nitrogen transformations in soil (NH2+ -> NH4+ -> NO2- -> NO3-) will be continued. Modifications have been made in the perfusion system that eliminate many of the problems in the previous system, such as collapse of the soil columns.
- More field studies will be initiated, especially in collaboration with some industries. However, most field studies will, hopefully, be conducted with new grant funds.
- Studies with the Cry4 proteins will continue, especially as we have developed methods to stabilize these proteins. These studies will involve primarily the adsorption and binding of the proteins on clays, humic substances, and soils, and the persistence and biological activity of the bound toxins (see last year's report).
The quality of the data is being assured by generally following the QA/QC procedures outlined in the original grant proposal. The publication of papers in peer-reviewed journals is indicative that these procedures are being adequately followed.
In summary, progress on this project in the past year has been excellent, and significant new data and concepts have been produced. Moreover, the activities of the past years will be continued and expanded in the next year. One measure of the progress on this project is the large number of invitations for lectures, workshops, panels, and review papers that the PI has received. This progress could not have been achieved without the support of the EPA.
Journal Articles on this Report : 19 Displayed | Download in RIS Format
Other project views: | All 96 publications | 39 publications in selected types | All 32 journal articles |
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Calamai L, Lozzi I, Stotzky G, Fusi P, Ristori GG. Interaction of catalase with montmorillonite homoionic to cations with different hydrophobicity: effect on enzyme activity and microbial utilization. Soil Biology and Biochemistry 2000;32(6):815-823. |
R826107 (1998) R826107 (1999) R826107 (2000) |
Exit |
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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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Findley LA, Stotzky G. Inactivation and utilization by bacteria of coliphage T1 as a source of carbon and nitrogen for growth. Applied and Environmental Microbiology 2000. |
R826107 (1998) R826107 (1999) R826107 (2000) |
not available |
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Flores S, Herrera G, Stotzky G. Rapid method of extraction (Expresso) for determining available nitrogen in soil. Soil Biology and Biochemistry 1999;16:65–79 |
R826107 (1998) R826107 (1999) R826107 (2000) |
Exit |
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Flores S, Stotzky G. Biomass from transgenic corn expressing the insecticidal toxin from Bacillus thuringiensis subsp. kurstaki affects some microbe-mediated processes in soil. Soil Biology and Biochemistry 2000;35(12):1635-1642 |
R826107 (2000) |
Exit |
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Flores S, Saxena D, Stotzky G. Bt corn decomposes less in soil than non-Bt corn. Science. |
R826107 (2000) |
not available |
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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) |
Exit |
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Lozzi I, Calamai L, Fusi P, Bosetto M, Stotzky G. Interaction of horseradish peroxidase with montmorillonite homoionic to Na+ and Ca2+: effects on enzyme activity and microbial degradation. Soil Biology and Biochemistry 2001;33(7-8):1021-1028. |
R826107 (1998) R826107 (1999) R826107 (2000) |
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) |
Exit Exit Exit |
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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) |
Exit |
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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) |
Exit Exit |
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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) |
Exit |
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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) |
Exit Exit |
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Stotzky G. Persistence and biological activity in soil of the insecticidal proteins from Bacillus thuringiensis, especially from transgenic plants. Plant and Soil 2004; 266(1-2):77-89. |
R826107 (1998) R826107 (1999) R826107 (2000) |
Exit |
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Vettori, C., Gallori, E., and Stotzky, G. Clay minerals protect bacteriophage PBS1 of Bacillus subtilis against inactivation and loss of transducing ability by UV radiation. Canadian Journal of Microbiology 2000;46(8):770-773. |
R826107 (1998) R826107 (1999) R826107 (2000) |
Exit |
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Vettori C, Paffetti D, Stotzky G, Giannini R. Genetic exchange between Bacillus thuringiensis subsp. kurstaki and indigenous bacteria in soils of Sardinia. Environmental Microbiology 2000. |
R826107 (2000) |
not available |
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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) |
Exit |
Supplemental Keywords:
soil, risk assessment, ecological effects, bioavailability, vulnerability, sensitive populations, animal, chemicals, toxics, organics, ecosystem protection, terrestrial, environmental, biology, ecology, genetics, pathology, zoology, agriculture., RFA, Scientific Discipline, Waste, Ecosystem Protection/Environmental Exposure & Risk, Bioavailability, Ecosystem/Assessment/Indicators, Ecosystem Protection, Ecological Effects - Environmental Exposure & Risk, Environmental Microbiology, Biochemistry, Ecological Risk Assessment, Ecology and Ecosystems, 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.