Insecticidal toxin from Bacillus thuringiensis is released from roots of transgenic Bt corn in vitro and in situ

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1 FEMS Microbiology Ecology 33 (2000) 35^39 Insecticidal toxin from Bacillus thuringiensis is released from roots of transgenic Bt corn in vitro and in situ D. Saxena, G. Stotzky * Laboratory of Microbial Ecology, Department of Biology, New York University, New York, NY 10003, USA Received 14 January 2000; received in revised form 10 April 2000; accepted 10 April 2000 Abstract The insecticidal toxin encoded by the cry1ab gene from Bacillus thuringiensis was released in root exudates from transgenic Bt corn during 40 days of growth in soil amended to 0, 3, 6, 9, or 12% (v/v) with montmorillonite or kaolinite in a plant growth room and from plants grown to maturity in the field. The presence of the toxin in rhizosphere soil was determined by immunological and larvicidal assays. No toxin was detected in any soils from isogenic non-bt corn or without plants. Persistence of the toxin was apparently the result of its binding on surface-active particles in the soils, which reduced the biodegradation of the toxin. The release of the toxin could enhance the control of insect pests or constitute a hazard to nontarget organisms, including the microbiota of soil, and increase the selection of toxin-resistant target insects. ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Transgenic Bt corn; Bacillus thuringiensis; Insecticidal toxin; Root exudate; Clay mineral; European corn borer 1. Introduction * Corresponding author. Tel.: +1 (212) ; Fax: +1 (212) ; gs5@is2.nyu.edu Bt corn is maize (Zea mays) that has been genetically modi ed to express certain cry genes from Bacillus thuringiensis and produce larvicidal toxins 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 3^7% per borer per plant [1]. The incorporation into plants of genes from B. thuringiensis that encode the production of insecticidal toxins eliminates many problems associated with the use of chemical pesticides, as the toxins are produced continuously in these plants. However, 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 bene- cial insects (e.g., pollinators, predators and parasites of insect pests) [2^10] and other animal classes; and (2) result in the selection and enrichment of toxin-resistant target insects [11^17]. 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 6 30 min, the shortest time studied) on mined clay minerals [montmorillonite (M) and kaolinite (K)], on the clay-size fraction of soil, and on humic acids, indicating that toxins released upon disintegration of transgenic plant and bacterial cells in soil would only brie y be in a free state susceptible to biodegradation [18^28]. Only about 10 and 30% of the toxin from Btk and 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 signi cantly alter the structure of the toxins, as indicated by sodium dodecyl sulfate^polyacrylamide gel electrophoresis (SDS^PAGE) and enzyme-linked immunosorbent assay (ELISA) 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 di raction analyses, and there was no intercalation of K, a nonexpanding clay mineral. Toxins from Btk and Btt added to nonsterile soil were detected by a dot-blot ELISA, with a lower limit of de / 00 / $20.00 ß 2000 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S (00)

2 36 D. Saxena, G. Stotzky / FEMS Microbiology Ecology 33 (2000) 35^39 tection of ca. 3 ng, on the clay-size fraction but not on the silt- and sand-size fractions on which the toxins do not appear to adsorb [21]. The use of ow cytometry as a rapid and sensitive method with which to detect the toxins in soil was also developed [23]. The toxin from Btk or Btt bound on M, K, or the claysize fraction was larvicidal to the tobacco hornworm (Manduca sexta) and the Colorado potato beetle (Leptinotarsa decemlineata) (as well as to a surrogate of the beetle), respectively [22]. When free toxin from Btk was added to nonsterile soils, larvicidal activity was detected after 234 days, the longest time evaluated [24]. 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 [25]. The free toxins were readily utilized as sole 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, slightly as a source of nitrogen, but they did not support growth in the absence of exogenous sources of both available carbon and nitrogen. After exposure of the bound toxins to microbes, both in vitro and in soil, the toxins retained insecticidal activity, even after alternately freezing and thawing or wetting and drying the soil for 40 days. The toxins, free or bound, had no e ect on the growth in vitro of a spectrum of bacteria (both Gram-positive and Gram-negative), fungi (both yeast and lamentous forms ^ e.g., Zygomycetes, Ascomycetes, Deuteromycetes), and algae (primarily green and diatoms) (J. Koskella and G. Stotzky, unpublished data). Similar result were obtained when the toxin from Btk was reacted with humic acids [27] and with complexes of M^humic acids^al hydroxypolymers [28]: 75^85% of the toxin adsorbed at equilibrium was strongly bound; the bound toxin was toxic to larvae of M. sexta; and the bound toxin did not support the growth of a mixed microbial culture from soil, although the free toxin was readily utilized. The Cry1Ab protein was released in root exudates from transgenic Bt corn grown in both sterile hydroponic culture and sterile and nonsterile soil in a plant growth room (Table 1) [29]. The presence of the toxin was indicated by a major band migrating on SDS^PAGE to a position corresponding to a M r of 66 kda, the same as that of the Cry1Ab protein, and con rmed 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 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 bound on surface-active particles in rhizosphere soil, which protected the toxin from hydrolysis, similar to what had been observed with puri ed toxin [20,22,24,25,27,28]. To verify these results and to estimate the importance of the clay mineralogy and other physicochemical characteristics that in uence the activity and ecology of microbes in soil [26] on the persistence of the toxin released in root exudates from Bt corn, studies were done in soil amended with various concentrations of M or K in a plant growth room. Studies were also done with plants grown in a natural soil in the eld. 2. Materials and methods 2.1. Soil and seeds Kitchawan soil, a sandy loam that naturally contains predominantly K, was collected at the Kitchawan Research Laboratory of the Brooklyn Botanical Garden, Ossining, NY. The soil was either not amended [control (C)] or amended to 3, 6, 9, or 12% (v/v) with M (3M, 6M, 9M, and 12M soil) or K (3K, 6K, 9K, and 12K soil). These stable soil^clay mixtures have been used extensively in this laboratory in studies on the e ects of the physicochemical and biological factors of soil on the activity, ecology, and population dynamics of microbes and viruses, on gene transfer among bacteria, on mediating the toxicity of heavy metals and other pollutants, and on the persistence of the toxins from Btk and Btt in soil. Therefore, there is a large data base on these mixtures [22,24,30,31]. Table 1 Presence of toxin in root exudates from corn with (Bt+) and without (Bt3) the cry1ab gene Growth conditions Day assayed after germination of seed a Immunological test b Toxicity (LC 50, Wg) c Immunological test Toxicity (LC 50, Wg) Immunological test Toxicity (LC 50, Wg) Bt3 Bt+ Bt3 Bt+ Bt3 Bt+ Bt3 Bt+ Bt3 Bt+ Bt3 Bt+ Hoagland's solution 3 + NT d NT NT NT Soil 3 + NT NT NT 1.6 a Assayed immunologically and by larvicidal assay (M. sexta) on indicated days after germination of seeds, which was usually 3 days after planting. b Determined with Quickstix; 3 = no toxin detected; + = toxin detected. c Toxicity [concentration necessary to kill 50% of larvae (LC 50 )] expressed in Wg of total protein bioassay per vial; see text for details. d NT = no toxicity.

3 D. Saxena, G. Stotzky / FEMS Microbiology Ecology 33 (2000) 35^39 37 Seeds of Bt corn (NK4640Bt) and of the isogenic strain without the cry1ab gene were planted in test tubes containing 15 g of nonsterile soil amended or not amended with M or K. Seeds were also planted (May, 1999) in a sandy loam soil in East Marion, Long Island, NY. After 10, 20, 30, and 40 days of growth in a plant growth room (26 þ 2³C, 12 h light^dark cycle), soil from randomly selected tubes (two tubes each of Bt corn and non-bt corn for each soil^clay mixture) was analyzed by immunological and larvicidal assays. Rhizosphere soil from the eldgrown plants (nonirrigated, nonfertilized) was similarly analyzed after the production of ears of corn (six plants each of Bt corn and non-bt corn) and several months after the death of the plants and frost Immunological assay Samples of soil (0.5 g) from the rhizosphere were vortexed with 500 Wl of extraction bu er (EnviroLogix, Portland, ME), centrifuged, and the supernatants analyzed by Western blot using Lateral Flow Quickstix (EnviroLogix; detection limit 6 10 ppb) [29] Larvicidal assay Samples of rhizosphere soil (0.5 g) were vortexed with 500 Wl of extraction bu er (EnviroLogix), and the insecticidal activity of the soil suspensions was determined using the larvae of the tobacco hornworm (M. sexta) [22]. Eggs of M. sexta and food medium were obtained from Carolina Biological Supply Company (Burlington, NC). The eggs, dispensed on solidi ed medium in Petri plates, were incubated at 29 þ 1³C under a 40-W lamp for 3^5 days, when the eggs hatched. The medium was dispensed, after microwaving, in 5-ml amounts into vials (3 cm diameter and 6 cm tall) and allowed to solidify [32]. Aliquots (100 Wl) of freshly vortexed soil suspensions were uniformly distributed over the surface of the medium (8.55 cm 2 ) with disposable pipette tips (200-Wl capacity) that had been cut ca. 1.5 cm from the tip, to ensure that all suspended particles of soil were transferred. After air-drying, four second-instar larvae were added to each of duplicate vials prepared from duplicate soil tubes, resulting in 16 larvae for each soil sample. Mortality was determined after 3 and 7 days, and percent mortality was based on mortality after 7 days. Controls consisted of soils amended with M or K but without plants. 3. Results and discussion All samples of rhizosphere soil from plants of Bt corn, grown in a plant growth room in soil amended with various amounts of M or K, were positive 10, 20, 30, and 40 days after germination for the presence of the toxin when assayed immunologically by the Quickstix test. No toxin was detected in any soil with plants of non-bt corn or without plants. All samples of soil from Bt corn were toxic to the larvae of M. sexta, with mortality ranging from 25 to 100% on day 10 and increasing to 88^100% on day 40, whereas there was no mortality with any soil from non-bt plants or without plants. In addition, the size and weight of surviving larvae exposed to soils from Bt corn were signi cantly lower (ca. 50^92% lower) than those exposed to soils from non-bt corn or without plants, and these larvae usually died after an additional 2^3 days (Table 2). 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 signi cantly higher cation exchange capacity and speci c surface area than K, a nonswelling 1:1, Si:Al, clay, bound more toxin in the root exudates than K [19,20,22,26]. Nevertheless, mortality in the M and K soils was essentially the same after 40 days (Table 2), indicating that over a longer time, the persis- Table 2 Larvicidal activity, expressed as % mortality and mean weight (in g) of a single larva þ S.E.M. (in parentheses), of rhizosphere soil from corn with (Bt+) and without (Bt3) the cry1ab gene a Soil c Day assayed after germination of seed b Bt3 Bt+ Bt3 Bt+ Bt3 Bt+ Bt3 Bt+ C 0 (1.0 þ 0.05) 50 þ 10.2 (0.4 þ 0.08) 0 (1.1 þ 0.02) 50 þ 10.2 (0.2 þ 0.02) 0 (1.2 þ 0.02) 63 þ 7.2 (0.1 þ 0.01) 0 (0.9 þ 0.06) 100 þ 0.0 (^) 3K 0 (1.0 þ 0.04) 25 þ 10.2 (0.5 þ 0.07) 0 (1.1 þ 0.03) 38 þ 7.2 (0.2 þ 0.01) 0 (1.2 þ 0.08) 38 þ 7.2 (0.1 þ 0.01) 0 (1.0 þ 0.02) 88 þ 7.2 (0.1 þ 0.02) 6K 0 (1.0 þ 0.03) 25 þ 10.2 (0.4 þ 0.04) 0 (1.1 þ 0.08) 38 þ 7.2 (0.2 þ 0.02) 0 (1.1 þ 0.03) 50 þ 10.2 (0.1 þ 0.02) 0 (0.8 þ.06) 88 þ 7.2 (0.1 þ 0.01) 9K 0 (1.0 þ 0.04) 75 þ 10.2 (0.1 þ 0.04) 0 (1.0 þ 0.02) 75 þ 10.2 (0.2 þ 0.03) 0 (1.2 þ 0.04) 88 þ 7.2 (0.1 þ (0.9 þ 0.02) 100 þ 0.0 (^) 12K 0 (0.8 þ 0.06) 50 þ 10.2 (0.3 þ 0.01) 0 (1.1 þ 0.05) 63 þ 12.5 (0.2 þ 0.02) 0 (1.2 þ 0.03) 88 þ 7.2 (0.1 þ 0.03) 0 (0.9 þ 0.05) 100 þ 0.0 (^) 3M 0 (1.0 þ 0.04) 38 þ 7.2 (0.5 þ 0.02) 0 (1.0 þ 0.04) 50 þ 10.2 (0.2 þ 0.04) 0 (1.2 þ 0.02) 63 þ 12.5 (0.1 þ 0.01) 0 (0.9 þ 0.05) 100 þ 0.0 (^) 6M 0 (0.8 þ 0.01) 100 þ 0.0 (^) 0 (1.1 þ 0.04) 100 þ 0.0 (^) 0 (1.1 þ 0.05) 100 þ 0.0 (^) 0 (0.9 þ 0.03) 100 þ 0.0 (^) 9M 0 (1.0 þ 0.02) 100 þ 0.0 (^) 0 (1.0 þ 0.03) 100 þ 0.0 (^) 0 (1.2 þ 0.01) 100 þ 0.0 (^) 0 (1.0 þ 0.04) 100 þ 0.0 (^) 12M 0 (1.0 þ 0.01) 50 þ 10.2 (0.8 þ 0.04) 0 (1.0 þ 0.05) 100 þ 0.0 (^) 0 (1.0 þ 0.01) 100 þ 0.0 (^) 0 (1.0 þ 0.02) 100 þ 0.0 (^) a Determined with the larvae of the tobacco hornworm (M. sexta); at least 16 larvae per assay; weight data normalized to one larva per treatment. No mortality with soils without plants (weight of a single larva: 0.8^1.3 þ 0.02 g). b Usually 3 days after planting. c Soil amended to 3, 6, 9, or 12% (v/v) with kaolinite (K) or montmorillonite (M) or not amended with clay (C).

4 38 D. Saxena, G. Stotzky / FEMS Microbiology Ecology 33 (2000) 35^39 Table 3 Larvicidal activity, expressed as % mortality and mean weight (in g) of a single larva þ S.E.M. (in parentheses), of rhizosphere soil from corn with (Bt+) and without (Bt3) the cry1ab gene grown in situ under eld conditions a Plant % Mortality b Bt3 Bt+ Before frost F1 0 (0.9 þ 0.01) 38 þ 7.2 (0.3 þ 0.02) F2 0 (0.9 þ 0.03) 100 þ 0.0 (^) F3 0 (1.1 þ 0.03) 38 þ 12.5 (0.5 þ 0.09) F4 0 (1.0 þ 0.02) 100 þ 0.0 (^) F5 0 (1.0 þ 0.03) 38 þ 7.2 (0.6 þ 0.01) F6 0 (1.0 þ 0.02) 38 þ 7.2 (0.5 þ 0.02) After frost F7 0 (1.0 þ 0.07) 75 þ 10.2 (0.1 þ 0.01) F8 ND c 88 þ 12.5 (0.03 þ 0.00) a Immunological tests with Quickstix showed the presence of toxin in the rhizosphere soil of all Bt+ plants but not of Bt3 plants. b Determined with the larvae of the tobacco hornworm (M. sexta); at least 16 larvae per assay; weight data normalized to one larva per treatment. No mortality with soil without plants (weight of a single larva: 0.8^1.3 þ 0.02 g). c Not determined. tence of larvicidal activity appeared to be independent of the clay mineralogy and other physicochemical characteristics of the soils. However, the increase in larvicidal activity between day 10 and day 40 indicated that the toxin in the root exudates was concentrated when adsorbed on surface-active components of the soils. The immunological and larvicidal assays of soil from the rhizosphere of Bt corn grown in the eld were also positive, even in soil collected after the rst frost from plants that had been dead for several months, whereas they were negative for non-bt corn (Table 3). Although the larval mortality in rhizosphere soil from some plants of eld-grown Bt corn was only 38%, the size and weight of the surviving larvae were reduced by 40^50% when compared with soil from non-bt corn or without plants (Table 3), and the larvae died after a few more days. Based on these results and those from previous studies [29], the toxin released from roots of Bt corn could accumulate in soil and retain insecticidal activity, especially when the toxin is bound on surface-active soil particles and 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 discernible root debris when plants were grown for 25 days in hydroponic culture [29]. In 1998, approximately 15 million acres of Bt corn were planted in the USA, just under 20% of the total corn acreage [33]. In addition to the large amount of toxin that will be introduced to soil in plant biomass after harvest and some toxin released in pollen during tasseling [7], these results indicate 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. Because Bt corn contains truncated genes that encode toxins rather than the nontoxic crystalline protoxins produced by B. thuringiensis, this potential hazard is exacerbated, as it is not necessary for an organism that ingests the toxins to have a high gut ph (ca. 10.5) for solubilization of the protoxins and for speci c proteases to cleave the protoxins into toxins. Moreover, receptors for the toxins are present in both target and nontarget insects [34]. Because bene cial nontarget insects and organisms in higher, as well as in lower (e.g., the microbiota of soil), trophic levels could be susceptible to the toxins, the continued large-scale planting of Bt crops should be reevaluated. 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