1999 Progress 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 Period Covered by this Report: November 7, 1998 through November 6, 1999
Project Amount: $393,411
RFA: Exploratory Research - Environmental Biology (1997) RFA Text |  Recipients Lists
Research Category: Aquatic Ecosystems , Biology/Life Sciences

Objective:

Determine the persistence and ecological effects of the insecticidal toxins from various subspecies of Bacillus thuringiensis in soil and other natural habitats, especially when the toxins are bound on surface-active pesticides (e.g., clays and humic substances).

Progress Summary:

Considerable progress has been made on this research project during its second year. One peer-reviewed journal article directly related to this project was published, one is in press, two have been submitted, and four are in preparation. One article, published in Nature, elicited international scientific and lay recognition, with reports on all major wire services; newspapers in England, Italy, Denmark, Sweden, United States, and probably elsewhere; extensive radio and television coverage (e.g., many radio interviews, including National Public Radio, and a Germany TV station sent a crew to interview me); scientific journals (e.g., Chemical Week, Scientist, New Scientist), and various financial publications (e.g., Wall Street Journal, Business News, Bridge). Although Nature does not print acknowledgments to sponsoring agencies, it was made very clear in all interviews that this work was supported by the U.S. Environmental Protection Agency (EPA). (It should be noted that this "notoriety" was neither planned nor desired and consumed more than a week of interviews and explanations.) In addition to these manuscripts, three articles peripherally related to this project and supported by prior grants from the EPA also were published, one is in press, and four are in preparation. A list of these articles is included with this report. Five peer-reviewed abstracts were published, and five presentations (two invited and three voluntary) were made at national and international scientific conferences. In addition, one book is in press. Details on these also are included in this report.

Among the major research achievements were:

? Confirmation with several new batches of corn that the gross metabolic activity (i.e., CO2 evolution) of soil amended with dry, ground biomass of transgenic corn expressing the cry1Ab gene was significantly lower than that of soil amended with the same amounts of biomass of isogenic corn not containing the gene. These differences were not the result of differences in the C:N ratios of the biomass, as leaf and stem tissues, respectively, of a second batch of Bt-corn and non-Bt corn had similar C:N ratios, and changes in the C:N ratios of the soil-biomass system by the addition of glucose, NH4NO3, or glucose plus NH4NO3 did not alter the differences in CO2 evolution between soil amended with biomass of Bt corn or non-Bt corn. These differences also were apparently not the result of the inhibition of the activity of the soil microbiota by the biomass of Bt corn, as the numbers of culturable bacteria and fungi and the activity of enzymes representative of those involved in degradation of plant biomass (i.e., dehydrogenases, proteases, arylsulfatases, and alkaline and acid phosphatases) were not consistently significantly different between soil amended with the biomass of Bt corn or non-Bt corn. These observations confirmed those obtained in vitro, which showed that the toxins from Bacillus thuringiensis subspp. kurstaki and tenebrionis were not toxic to pure cultures of microbes (i.e., gram-positive and gram-negative bacteria; yeasts and filamentous fungi? Zygomycetes, Ascomycetes, and Deuteromycetes; and algae, primarily green and diatoms). A possible explanation for these differences in biodegradability is that the introduction of the cry1Ab gene affects the structure of the plant tissue, as Bt corn showed greater resistance to breakage and maceration. This possibility will be investigated in the coming project year. Similar differences in biodegradability have been observed with Bt rice expressing the cry1Ac gene.

? The demonstration that the Cry1Ab protein is released in root exudates from Bt corn in hydroponic culture, in sterile and nonsterile soil in test tubes, and in natural soil in the field. The presence of the toxin in root exudates was first detected in sterile hydroponic culture by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), where a major band with a molecular mass (Mr) of 66 kDa, the same as that of the Cry1Ab protein, was detected. The presence of the toxin was confirmed by immunological assay with Lateral Flow Quickstix (EnviroLogix, Portland, ME) and verified that it was active in insecticidal assays with the larvae of the tobacco hornworm (Manduca sexta). Although the toxin was detected 7 and 15 days after germination in hydroponic culture, it was not detected after 25 days when the solution was no longer sterile (the 66-kDa band was not detected, there were several new bands of smaller Mr, and the immunological and insecticidal assays were negative), indicating that microbial proteases had hydrolyzed the toxin. In contrast, the toxin was detected 10, 20, 30, and 40 days in both sterile and nonsterile soil amended to 0, 3, 6, 9, or 12 percent (vol vol-1) with montmorillonite or kaolinite in a plant-growth room (26 ? 2 C, 12 hours light-dark cycle) and in the rhizosphere soil of field-grown plants after production of ears of corn and even several months after the death of the plants and after frost. These results indicated that the toxin released in root exudates from Bt corn binds on surface-active particles (i.e., clays and humic substances) in rhizosphere soil, which protects the toxin against biodegradation, as we have previously shown with purified toxin and these surface-active particles. Although we originally assumed that the toxin would be introduced to soil primarily when the biomass of transgenic plants is incorporated after harvest of the crops and, subsequently, based on a recent report, from pollen during tasselling of Bt corn, the results of these studies indicate that the toxin is released throughout the growth of a Bt corn crop. No toxin was detected (i.e., the immunological and insecticidal assays were negative) in hydroponic culture (also, no band with a Mr of 66 kDa was observed), in soil in test tubes, and in soil in the field from non-Bt corn or in soil without plants. The ecological and environmental implications of these observations are not clear: the persistence of the toxin in the rhizosphere may improve the control of insect pests, or it may enhance the selection of toxin-resistant target insects and pose a hazard to nontarget insects and organisms at higher trophic levels. Further investigations are necessary to clarify these possibilities.

? The demonstration that the toxin from B. thuringiensis subsp. kurstaki binds on complexes of montmorillonite-humic acids-Al hydroxypolymers and that the bound toxin becomes recalcitrant to biodegradation and retains insecticidal activity. The results of these studies with organo-mineral complexes, which are more representative of the surface-active components of soil, generally mimicked those obtained separately with clay minerals and humic acids.

Dr. Deepak Saxena joined the project as a postdoctoral fellow on April 19, 1999. Mr. Saul Flores from IVIC in Venezuela again spent several months in the past year in this laboratory on this project, partly supported by funds from the New York University Research Fund. Both investigators made invaluable contributions to the project, as shown by articles published and in preparation. A major advance in the past project year was the discovery (suggested by a colleague at Pioneer-Hybrid) of the commercial availability of a simple Western blot test; i.e., Lateral Flow Quickstix from EnviroLogix in Portland, ME. Although the Quickstix are relatively expensive, they greatly simplify and facilitate the detection of the toxin in soil, plant tissue, etc. (detection limit 10 parts per billion).

During the subsequent project year, studies will continue on the various aspects reported above. A major emphasis will be to determine whether the structure of Bt corn plants differs from that of non-Bt corn plants. This will be done initially by determining the content and susceptibility of the lignin and cellulose of the plants to hydrolysis by ligninase and cellulase and by micromorphological studies. Studies with transgenic biomass of Bt rice will continue, and attempts to obtain plant tissue of other transgenic Bt plants will continue. Similarly, attempts to obtain seeds of other transgenic Bt plant species (and their isogenic non-Bt counterparts) will continue, to determine whether the respective toxins are released in their root exudates. As in the past, we have had no cooperation in these quests from commercial suppliers of seeds of transgenic plants. These studies will be conducted primarily in the Kitchawan soil amended with different concentrations of montmorillonite and kaolinite, to evaluate further the influence of the clay mineralogy and other physicochemical characteristics of soil on the persistence, degradation, and insecticidal activity of the toxins. Another emphasis will be directed towards the toxins of B. thuringiensis subsp. israelensis, i.e., the adsorption and binding of these toxins on clays, humic acids, and organo-mineral complexes (e.g., is there a preferential binding of individual proteins in this toxin complex?) and the effect of binding on the persistence and insecticidal activity (initially against Culex pipiens, the carrier of West Nile disease) of the toxin complex. Large quantities of this toxin complex are being purified.

A minor departure from the original proposal, but a logical one based on the observations of the release of the Cry1Ab protein in root exudates (also a minor departure from the original proposal), is to determine whether the presence of the toxin in soil, either from root exudates or incorporated biomass of Bt corn, has any effect on earthworms, nematodes, and microorganisms. To this end, experiments are in progress in which a standard number of earthworms (Lumbricus terrestris) has been added to soil amended with dry, ground biomass of Bt corn or isogenic non-Bt corn or planted with Bt corn, isogenic non-Bt corn, or no plants. To date, only a few earthworms have come to the surface of the soils and died. Immunological assays of the guts of these few worms have shown that the Cry1Ab protein is present in the intestines of worms from soil amended with biomass of Bt corn or in which Bt corn plants are growing, whereas no Cry1Ab protein is present in worms from soil containing non-Bt biomass or plants and neither biomass nor plants. Similar patterns have been observed in the few worm casts that have been analyzed. Once the experiment is concluded, the time to clear the toxin from the guts of the worms will be evaluated. Baseline values of the numbers of nematodes in these soils will be compared with the numbers of nematodes after the conclusion of the experiment.

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, due in great measure to the contributions of Dr. Saxena and Mr. Flores. Expenditures have been higher than anticipated, primarily because of the extensive use of Quickstix and insecticidal assays, which were necessary to obtain the qualitative and quantitative data on the presence of the toxin in root exudates and, in preliminary studies, in the intestines of earthworms. The results of the studies in this project period have opened numerous new directions of research, and some additional funding for another postdoctoral fellow and supplies (i.e., Quickstix and insecticidal assays) would facilitate progress in both these new directions and those proposed in the original grant proposal. It must be emphasized that this laboratory is probably the only one in the world that is studying the persistence of these toxins and their possible ecological and environmental effects in soil. (There is a field study in Africa, supported by the European Union (EU) and administered in Florence, Italy, the proposal of which I helped put together but from which I cannot obtain funds as a U.S. scientist, although I am an unpaid consultant to this study. Because of political considerations, involving several EU and south African countries, progress in this study has been slow. However, as an unpaid consultant to this project, I eventually will be able to test in the field some of the concepts developed in our studies, once this EU-Africa group gets their "act together." Such opportunities for field studies, especially with B. thuringiensis subsp. israelensis, are not available in the United States.)

Future Activities:

See "Progress Summary/Accomplishments."


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
Type Citation Project Document Sources
Journal Article 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)
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  • Journal Article Dabek-Szreniawska M, Stotzky G, Collins Y, Malicki J. Accuracy in measuring the number of microorganisms in soil. Soil Biology and Biochemistry. R826107 (1998)
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    Journal Article 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)
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    Journal Article 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)
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  • Journal Article Flores S, Saxena D, Stotzky G. Transgenic Bt plants decompose less in soil than non-Bt plants. Soil Biology and Biochemistry 2005;37(6):1073-1082. R826107 (1998)
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  • Journal Article Kasahara Y, Stotzky G, Kobayashi K. Inducible cell lysis system in Bacillus subtilis and transformation by DNA released from lysed cells. Applied and Environmental Microbiology. R826107 (1998)
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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)
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  • Journal Article Lee GH, Stotzky G. Transformation and survival of donor, recipient, and transformants of Bacillus subtilis in vitro and in soil. Soil Biology and Biochemistry 1999;31(11):1499-1508. R826107 (1998)
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  • Journal Article 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)
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  • Journal Article Saxena D, Flores S, Stotzky G. Transgenic plants-Insecticidal toxin in root exudates from Bt corn. Nature 1999;402(6761):480-480. R826107 (1998)
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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)
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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)
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  • Journal Article 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)
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  • Journal Article 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)
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  • Journal Article Vettori C, Calamai L, Yoder M, Stotzky G, Gallori E. Adsorption and binding of AmpliTaq (R) DNA polymerase on the clay minerals, montmorillonite and kaolinite. Soil Biology & Biochemistry 1999;31(4):587-593. R826107 (1998)
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  • Journal Article Vettori C, Stotzky G, Yoder M, Gallori E. Interaction between bacteriophage PBS1 and clay minerals and the transduction of Bacillus subtilis by clay-phage complexes. Environmental Microbiology 1999;1(4):347-355. R826107 (1998)
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  • 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, microbiology, ecological exposure, gene-environment interaction, ecological assessment, insecticides, aquatic ecosystems, assessment methods, bioassay, soil enzyme, ecotoxicological studies, flow cytometry assays, Bacillus thuringiensis, persistence, microbial, transgenic organisms, toxicological assays

    Progress and Final Reports:

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