The incorporation into plants of genes from Bacillus thuringiensis (Bt) that code for the production of insecticidal toxins (Cry proteins) reduces many problems associated with the use of chemical pesticides, as the toxins are produced continuously within these plants. Impressive control of Bt-susceptible insect pests has been obtained with such plants in the laboratory and in the field. However, there is concern that genetically engineered crops may pose risks to natural and agricultural ecosystems.(1) If production exceeds consumption and inactivation by insect larvae, degradation by the microbiota, and abiotic inactivation, the toxins could accumulate in the environment to concentrations that may: 1) 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; 2) result in the selection and enrichment of toxin-resistant target insects; and 3) enhance the control of target insects. Accumulation is enhanced when the toxins are bound on surface-active particles in the environment (e.g., clays and humic substances) and, thereby, are rendered less accessible for microbial degradation but still retain their toxic activity.

The toxins produced by B. thuringiensis subsp. kurstaki (Btk; 66 kDa; active against Lepidoptera) and subsp. tenebrionis (Btt; 68 kDa; active against Coleoptera) adsorbed rapidly (in less than 30 min, the shortest time studied) on mined clay minerals [montmorillonite (M) and kaolinite (K)], on the clay-size fraction of soil, on humic acids, and on clay-humic acid complexes.(1,2) Only about 10 and 30% of the toxin from Btk or Btt, respectively, adsorbed at equilibrium, was desorbed by one or two washes with water, and additional washing desorbed no more toxins, indicating that the toxins were tightly bound on the clays. Interaction of the toxins with the clays did not alter significantly the structure of the toxins, as indicated by ELISA examination of the equilibrium supernatants and desorption washes and by Fourier-transform infrared analyses and insect bioassays of the bound toxins. The toxins only partially intercalated M, as determined by X-ray diffraction analyses, and there was no intercalation of K, a nonexpanding clay mineral.

The toxin from Btk or Btt bound on M, K, or the clay-size fraction was larvicidal to the tobacco hornworm (Manduca sexta) or the Colorado potato beetle (Leptinotarsa decemlineata), respectively. When free toxin from Btk was added to nonsterile soils, larvicidal activity was detected after 234 days, the longest time evaluated.(2)

The binding of the toxins from Btk and Btt on clays reduced their availability to microbes, which is probably responsible for their persistence in soil. The free toxins were readily utilized, both in vitro and in soil, as sources of carbon and/or nitrogen by pure and mixed cultures of microbes, including soil suspensions, whereas the bound toxins were not utilized as a source of carbon, and utilized slightly as a source of nitrogen, but they did not support growth in the absence of exogenous sources of both available carbon and nitrogen. The toxins, free or bound, had no effect on the growth in vitro of a spectrum of bacteria (both gram-positive and gram-negative), fungi (both yeast and filamentous forms), and algae (primarily green and diatoms).(1)

The toxin was released in root exudates from Bt corn (NK4640Bt) grown in sterile hydroponic culture and in sterile and nonsterile soil in a plant-growth room.(3,4) Bt corn is maize (Zea mays L.) that has been genetically modified to express the cry1Ab gene from B. thuringiensis to kill lepidopteran pests, especially the European corn borer (Ostrinia nubilalis), a major pest in Europe and North America that can reduce yields of corn by 3 to 7% per borer per plant. The presence of the toxin was indicated by a major band migrating on SDS-PAGE to a position corresponding to a molecular mass (M sub r) of 66 kDa, the same as that of the Cry1Ab protein, and was confirmed by immunological and larvicidal assays. After 25 days, when the hydroponic culture was no longer sterile, the band at 66 kDa was not detected (there were several new protein bands of smaller M sub r) and the immunological and larvicidal assays were negative, indicating that microbial proteases had hydrolyzed the toxin. By contrast, the toxin was detected after 25 days in both sterile and nonsterile soil, indicating that the released toxin was bound on surface-active particles in rhizosphere soil, which protected the toxin from hydrolysis, similar to results observed with purified toxins.

To estimate the importance of the clay mineralogy and other physicochemical characteristics, which influence the activity and ecology of microbes in soil, on the persistence of the toxin released in root exudates from Bt corn, studies were done in a sandy loam soil amended with various concentrations (3-12%) of M or K in a plant-growth room. Rhizosphere soil from plants of Bt corn were positive 10-40 days after germination for the presence of the toxin when assayed immunologically with Lateral Flow Quickstix.(4) No toxin was detected in any soil with plants of non-Bt corn or without plants. All samples of soil in which Bt corn was grown were toxic to the larvae of M. sexta, with mortality ranging from 25-100% on day 10 and increasing to 88-100% on day 40, whereas there was no mortality with any soil from non-Bt plants and with soil without plants. In addition, the size and weight of surviving larvae exposed to soils from Bt corn were significantly lower (ca. 50-92% lower) than those exposed to soil from non-Bt corn or to soil without plants. The larvicidal activity was generally higher in soil amended with M than with K, probably because M, a swelling 2:1, Si:Al, clay mineral with a significantly higher cation-exchange capacity and specific surface area than K, a nonswelling 1:1, Si:Al, clay, bound more toxin in the root exudates than did K, as has been observed with pure toxin. Nevertheless, mortality in the M and K soils was essentially the same after 40 days, indicating that over a longer time, the persistence of larvicidal activity is independent of the clay mineralogy and other physicochemical characteristics of the soils. The increase in larvicidal activity between 10 and 40 days indicated that the toxin in the root exudates was concentrated when adsorbed on surface-active components of the soils.

The toxin was also released in root exudates of field-grown Bt corn plants, and it remained larvicidal for months after their death and subsequent frost.(4) Although the larval mortality in rhizosphere soil from plants of field-grown Bt corn ranged from 38-100% and the coefficients of variation were large, the size and weight of the surviving larvae were reduced by 40-50% when compared with soil from non-Bt corn or from soil without plants.

To determine whether the release of the Cry1Ab protein is a common phenomenon with transgenic Bt corn, the release of the Cry1Ab protein in the exudates of 13 Bt corn hybrids, representing three different transformation events (Bt11, MON810, and 176), and of their isogenic nontransgenic counterparts was studied. All samples of rhizosphere soil from the 13 hybrids, whether grown in the plant-growth room for 40 days or in the field (harvested after the production of ears of corn), were positive for the presence of the toxin when assayed immunologically, and all samples were toxic to the larvae of M. sexta, with mortality ranging from 38-100%. No toxin was detected immunologically or by larvicidal assay in any soil in which plants of non-Bt corn or no plants had been grown. In addition, the weight of surviving larvae exposed to soils from Bt corn was significantly lower (80-90%) than those exposed to soils from non-Bt corn or without plants.

These results indicated that the release of the toxin in exudates from roots of Bt corn is a common phenomenon and that the released toxin could accumulate in soil and retain insecticidal activity, especially when the toxin is bound on surface-active soil particles and, thus, becomes resistant to degradation by microorganisms. Although some toxin was probably released from sloughed and damaged root cells, the major portion was derived from exudates, as there was no discernable root debris after centrifugation of the Hoagland's solution when plants were grown for 25 days in hydroponic culture.

In addition to the large amount of toxin that will be introduced to soil in plant biomass after harvest and some that will be introduced in pollen released during tasseling, these results indicated that the toxin will also be released to soil from roots during the entire growth of a Bt corn crop. The presence of the toxin in soil could improve the control of insect pests, or the persistence of the toxin in soil could enhance the selection of toxin-resistant target insects and constitute a hazard to nontarget organisms, as receptors for the toxins are present in both target and nontarget insects. Consequently, nontarget insects and organisms in higher, and perhaps also in lower trophic levels, could be susceptible to the toxins.

To determine the effects of the Cry1Ab toxin released in root exudates and from biomass of Bt corn on various organisms in soil, 20 medium-size earthworms (Lumbricus terrestris) were introduced into soil planted with Bt or non-Bt corn or amended with 1% biomass of Bt or non-Bt corn and soil not planted or amended. After incubation, the numbers of earthworms were counted and their weight determined. Representative worms were dissected, and soil from the guts, as well as from the casts, was analyzed for the presence of the Cry1Ab protein by immunological and larvicidal assays.(5)

There were no significant differences in the percent mortality and weight of earthworms after 40 days in soil planted with Bt or non-Bt corn or not planted or after 45 days in soil amended with biomass of Bt or non-Bt corn or not amended. However, the toxin was present in both the casts and guts of worms in soil planted with Bt corn or amended with biomass of Bt corn, whereas it was absent in casts and guts of worms in soil planted with non-Bt corn or not planted and in soil amended with biomass of non-Bt corn or not amended. When worms from soil with Bt corn or amended with biomass of Bt corn were transferred to fresh soil, the toxin was cleared from the guts in one to two days. All samples of soil amended with biomass of Bt corn and from the rhizosphere of Bt corn were positive for the presence of the toxin and were lethal to the larvae of M. sexta after 45 and 40 days, respectively, whereas there was no mortality in soil amended with biomass of non-Bt corn, in rhizosphere soil of non-Bt corn, or in soil with no plants or not amended, which were also negative in the immunological assays. There were no statistically significant differences (P>0.5) in the total numbers of nematodes and culturable protozoa, bacteria (including actinomycetes), and fungi between rhizosphere soil of Bt and non-Bt corn or between soils amended with Bt or non-Bt biomass.

These results suggest that despite its persistence in soil, the toxin released in root exudates of Bt corn or from the degradation of the biomass of Bt corn is not toxic to a variety of organisms in soil. The toxin was detected in the guts and casts of earthworms grown with Bt corn and in soil amended with biomass of Bt corn, indicating again that the released toxin bound on surface-active particles in soil, which protected the toxin from biodegradation, similar to what has been observed with purified toxins. Because only one species of earthworms and only total culturable microorganisms and nematodes were evaluated, more detailed studies, including studies using techniques of molecular biology, on the composition and diversity of these groups of organisms are necessary to confirm the absence of effects of the Cry1Ab toxin on biodiversity in soil.

Sources

1. Stotzky G. 2000. 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 29: 691-705.

2. Tapp H and Stotzky G. 1998. Persistence of the insecticidal toxin from Bacillus thuringiensis subsp. kurstaki in soil. Soil Biology & Biochemistry 30: 471-476.

3. Saxena D, Flores S, and Stotzky G. 1999. Insecticidal toxin in root exudates from Bt corn. Nature 402: 480.

4. Saxena D and Stotzky G. 2000. Insecticidal toxin from Bacillus thuringiensis is released from roots of transgenic Bt corn in vitro and in situ. FEMS Microbiology Ecology 33: 35-39.

5. Saxena D and Stotzky G. 2001. 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 (in press).