Original article. Received 2 February 2004; accepted 8 March Available online 07 April 2004

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1 Plant Physiology and Biochemistry 42 (2004) Original article Larvicidal Cry proteins from Bacillus thuringiensis are released in root exudates of transgenic B. thuringiensis corn, potato, and rice but not of B. thuringiensis canola, cotton, and tobacco Deepak Saxena a, C. Neal Stewart b, Illimar Altosaar c, Qingyao Shu d, G. Stotzky a, * a Laboratory of Microbial Ecology, Department of Biology, New York University, New York, NY 10003, USA b Department of Plant Sciences and Landscape Systems, University of Tennessee, Knoxville, TN 37996, USA c Department of Biochemistry, Microbiology, and Immunology, University of Ottawa, Ottawa, Ont., Canada KIN 6N5 d Institute of Nuclear Agricultural Sciences, Zhejiang University, Hua Jia Chi, Hangzhou , China Received 2 February 2004; accepted 8 March 2004 Available online 07 April 2004 Abstract Larvicidal proteins encoded by cry genes from Bacillus thuringiensis were released in root exudates from transgenic B. thuringiensis corn, rice, and potato but not from B. thuringiensis canola, cotton, and tobacco. Nonsterile soil and sterile hydroponic solution in which B. thuringiensis corn, rice, or potato had been grown were immunologically positive for the presence of the Cry proteins; from B. thuringiensis corn and rice, the soil and solution were toxic to the larva of the tobacco hornworm (Manduca sexta), and from potato, to the larva of the Colorado potato beetle (Leptinotarsa decemlineata), representative lepidoptera and coleoptera, respectively. No toxin was detected immunologically or by larvicidal assay in soil or hydroponic solution in which B. thuringiensis canola, cotton, or tobacco, as well as all near-isogenic non-b. thuringiensis plant counterparts or no plants, had been grown. All plant species had the cauliflower mosaic virus (CaMV) 35S promoter, except rice, which had the ubiquitin promoter from maize. The reasons for the differences between species in the exudation from roots of the toxins are not known. The released toxins persisted in soil as the result of their binding on surface-active particles (e.g. clay minerals, humic substances), which reduced their biodegradation. The release of the toxins in root exudates could enhance the control of target insect pests, constitute a hazard to nontarget organisms, and/or increase the selection of toxin-resistant target insects Elsevier SAS. All rights reserved. Keywords: Bacillus thuringiensis; Hydroponics; Insecticidal proteins; Root exudates; Soil; Surface-active particles (e.g. clay minerals, humic substances); Transgenic Bt plants 1. Introduction * Corresponding author. address: gs5@nyu.edu (G. Stotzky). There is concern about some transgenic plants because of potential hazards to the environment from the release and persistence of larvicidal Cry proteins from Bacillus thuringiensis (Bt) in soil [19,20,24]. The toxins could accumulate in the environment to concentrations that may: (a) constitute a hazard to nontarget organisms, such as the soil microbiota, beneficial insects (e.g. pollinators, predators and parasites of insect pests) [1,5,6,8 12,16], and other animal classes; and (b) result in the selection and enrichment of toxin-resistant target insects [2,4,17,28,34]. However, the accumulated toxins could also increase the control of target pests. Accumulation is enhanced when the toxins are bound on surface-active particles in the environment (e.g. clay minerals, humic substances) and, thereby, are rendered less accessible for microbial degradation but still retain their toxic activity [21,27]. The toxins produced by B. thuringiensis subsp. kurstaki (Btk; 66 kda; active against some Lepidoptera), subsp. morrisoni (strain tenebrionis)(btm; 68 kda; active against some Coleoptera), and subsp. israelensis (Bti; 27, 65, 128 and 135 kda; active against some Diptera) adsorbed rapidly (in <30 min, the shortest time studied) on mined clay minerals (montmorillonite and kaolinite), the clay-size fraction of soil, humic acids extracted from soil, and complexes of clay and humic acids, indicating that toxins released upon disintegration of transgenic plant biomass in soil would only briefly be in a free state susceptible to biodegradation [3,13,20,24,29,31,32]. The toxins from Btk, Btm, and Bti 2004 Elsevier SAS. All rights reserved. doi: /j.plaphy

2 384 D. Saxena et al. / Plant Physiology and Biochemistry 42 (2004) bound on these surface-active particles were larvicidal to the tobacco hornworm (Manduca sexta), the Colorado potato beetle (Leptinotarsa decemlineata), and a mosquito (Culex pipiens), respectively [15,29,31]. When purified Cry1Ab protein from Btk was added to nonsterile soils, larvicidal activity was still detected after 234 d, the longest time evaluated [32]. The Cry1Ab protein was released in root exudates from transgenic Bt corn grown in both sterile hydroponic culture and sterile and nonsterile soil [19,20,22]. The presence of the toxin was indicated by a major band migrating on SDS- PAGE to a position corresponding to a molecular mass of 66 kda, the same as that of the Cry1Ab protein, and confirmed by immunological and larvicidal assays. After 25 d, when the hydroponic culture was no longer sterile, the band at 66 kda was not detected and there were several new protein bands of smaller molecular mass, and the immunological and larvicidal assays were negative, indicating that microbial proteases had hydrolyzed the toxin. By contrast, the toxin was detected after more than 25 d 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 purified toxin [3,13,29,31,32]. The toxin was also detected from root exudates in natural soil 180 d (the longest time evaluated) after the growth of Bt corn in a plant-growth room, as well as in the field after harvest and after frost from plants that had been dead for several months [22]. Moreover, the Cry1Ab protein was released in root exudates from all 13 Bt corn hybrids, representing three transformation events (Bt11, MON810, and 176), evaluated, in both the plantgrowth room and the field [20]. These results indicated that in addition to toxin that will be introduced to soil in plant biomass after harvest, as well as some that will be introduced in pollen during tasseling [16,18], toxin will be released to soil from roots during the entire growth of a Bt corn crop. The continual presence of toxin in soil could improve the control of insect pests, enhance the selection of toxin-resistant target insects, and/or constitute a hazard to nontarget organisms. Because of these possibilities and because there appear to be no published data on the release of Cry proteins in the root exudates of other transgenic Bt plants, the release of different Cry proteins in the root exudates of various Bt plants was evaluated. Here we show that Cry1Ab (from corn and rice) and Cry3A (from potato) proteins, but not Cry1Ac (from canola, cotton, and tobacco) protein, are released in root exudates. 2. Results and discussion Soil and hydroponic solution in which Bt corn, rice, or potato had been grown were immunologically positive for the presence of the Cry proteins; from Bt corn and rice, they were toxic to the larva of M. sexta, and from potato, to the larva of L. decemlineata (Table 1). No Cry proteins were detected immunologically or by larvicidal assay in any soil or hydroponic solution in which Bt canola, cotton, or tobacco, as well as all near-isogenic non-bt plant counterparts or no plants, had been grown. The Cry proteins were detected in the tissues of all Bt plants. The size and weight of surviving larvae of M. sexta exposed to soil or hydroponic solution from Bt corn or rice were significantly lower (ca % lower; P < 0.05) than those exposed to soil or solution from other Bt or non-bt plants or without plants (Table 1), and these larvae usually died after an additional 2 3 d. Although some differences in larval weights were apparent among treatments, these differences were not statistically significant when compared with the range of weights of control larvae, which showed an average coefficient of variation of 23.4%. The major criterion of the effects of the toxins was percent mortality, and sublethal effects were evaluated only on the few surviving larvae. There were apparent differences in exudation of the proteins (as evaluated by mortality, weight of surviving larvae, and immunologically) between plant species: exudates from Bt corn were more toxic than those from Bt rice and potato. No green fluorescent protein (GFP) was detected in hydroponic solutions from canola and tobacco genetically modified to express GFP or both GFP and Cry1Ac protein. Based on these results and those from previous studies [19,20,22 25], Cry proteins released in root exudates of Bt corn, rice, and potato accumulated in soil and retained larvicidal activity, probably as the result of the binding of the proteins on surface-active particles (e.g. clays, humic substances) in soil, which rendered them resistant to rapid biodegradation [13,21,27]. Although some Cry proteins were probably released from sloughed and damaged root cells of corn, rice, and potato in soil, the major portion was derived from root exudates, as there was no discernable root debris when plants were grown in hydroponic culture, and no Cry proteins from Bt canola, cotton, or tobacco were detected when grown in soil where some damage probably occurred. In addition to the introduction to soil of Cry proteins in plant biomass and pollen, they will also be released in root exudates during the entire growth of some Bt plants. The presence of the Cry proteins in soil could improve the control of insect pests, enhance the selection of toxin-resistant target insects, and/or constitute a hazard to nontarget organisms [21,27]. The Cry1Ab protein released in root exudates and from biomass of Bt corn had no apparent effects on earthworms, nematodes, protozoa, bacteria, and fungi in soil [24], and it was not taken up by non-bt corn, radish, carrot, and turnip [23]. The reasons for the release of Cry proteins in the root exudates of Bt corn, rice, and potato but not of Bt canola, cotton, and tobacco are not known. The methods of transformation of the cry genes, somaclonal variation, differences in level of protein expression [33] (although all species had the cauliflower mosaic virus (CaMV) 35S promoter, except rice, which had the ubiquitin promoter from maize), or location of

3 D. Saxena et al. / Plant Physiology and Biochemistry 42 (2004) Table 1 Presence of Cry1Ab (corn and rice), Cry1Ac (cotton, canola, and tobacco), and Cry3A (potato) protein from B. thuringiensis in rhizosphere soil and hydroponic solution from plants with (Bt+) and without (Bt ) the cry1ab, cry1ac, and cry3a gene, respectively. Mean ± standard error of the means (S.E.M.) Seeds of transgenic corn (Zea mays L.) and rice (Oryyza sativa L.), potato (Solanum tuberosum L.), and canola (Bras- Plant Plant line Source Soil* Hydroponic solution* Immunological assay Mortality Larval (%) weight (mg) Immunological assay Mortality (%) Larval weight (mg) Corn Bt NK4640 Anonymous ± ± ± 32 Bt+ NK4640Bt Anonymous ± ± ± 12 Rice Bt Xiushu 11 [26] 6.3 ± ± ± 45 Bt+ KMD1; cv. Japonica [26] ± ± ± ± 30 Cotton Bt SG747; Maris Monsanto Co. 6.3 ± ± ± ± 31 Bt+ Coker ; Maris Monsanto Co. 6.3 ± ± ± 50 Canola Bt Binapus; cv. Westar [7] 6.3 ± ± ± ± 31 GFP W45; cv. Westar [7] ± ± 70 Bt+ W45; cv. Westar [7] 12.5 ± ± ± ± 33 GFPBt+ W45; cv. Westar [7] 12.5 ± ± ± ± 38 Tobacco Bt Xanthi [7] 6.3 ± ± ± 92 GFP [7] 6.3 ± ± ± 50 Bt+ Bt9 [7] ± ± ± 34 GFPBt+ [7] 12.5 ± ± ± ± 56 Potato Bt R-Burbank Monsanto Co ± 3.6 ND { 39.5 ± 2.7 ND { Bt+ Newleaf Plus 350 Monsanto Co ± 10.2 ND { ± 2.7 ND { * Days of growth after planting: corn, soil: 40, solution: 35; rice, soil: 50, solution: 60; canola, soil: 55, solution: 40; tobacco, soil: 65, solution: 55; cotton, soil: 55, solution: 40; potato, soil: 65, solution: 35. Cry1Ab and Cry1Ac proteins were determined immunologically with EnviroLogix Lateral Flow Quickstix, and Cry3A protein was determined with Agdia ELISA kit;, no toxin detected; +, toxin detected. Larvicidal activity of Cry1Ab and Cry1Ac proteins was determined with the larva of the tobacco hornworm (M. sexta); at least 16 larvae/assay; expressed as % mortality ± S.E.M.; mean weight, in mg, of a single surviving larva ± S.E.M. is also presented. Larvicidal activity of Cry3A protein was determined with the larva of the Colorado potato beetle (L. decemlineata); at least 80 larvae/assay; expressed as % mortality ± S.E.M. No significant mortality with soils or hydroponic solutions without plants (weight of a single larva: mg). Green fluorescent protein (canola and tobacco were engineered to contain the genes for Cry1Ac protein, GFP, or both). { Not determined the endoplasmic reticulum relative to the plasma membrane (in corn, this appears to be a close relation) may be involved [27]. Although Cry1Ab and Cry1Ac proteins differ in some aspects, the differences are apparently small, as their insect targets are similar and they cross-react with antibodies to each [30]. Nevertheless, the differences may be responsible for release of the Cry1Ab protein and the lack of release of the Cry1Ac protein in root exudates. Further studies by, e.g. plant physiologists and anatomists, are obviously necessary. The relevance of these observation also requires clarification, especially as at least 26 plant species [14], including corn, cotton, potato, canola, rice, broccoli, peanut, eggplant, and other crop species, have been modified to express Cry proteins, and 8.1 million hectares of Bt corn or 26% of total corn acreage, 2.4 million hectares of Bt cotton or 45% of total cotton acreage, and 0.02 million hectares of Bt potato or 3.5% of total potato acreage were planted in the United States alone in 2000 [33]. Moreover, plants into which cry1aa, cry1ba, cry1ca, cry1h, and cry2aa genes, encoding proteins that target lepidopteran larvae, and cry3bb1 and cry6a genes, to control coleopteran larvae, have been introduced are in the developmental stage [14]. 3. Conclusion The reasons why Cry proteins were released in root exudates from only some transgenic B. thuringiensis plants (Cry1Ab from corn and rice and Cry3A from potato) but not from others (Cry1Ac from canola, cotton, and tobacco) are not known, but this disparity indicates that the release of Cry toxins in root exudates must be determined on a case-by-case plant and toxin basis. The relevance of these observations are also not clear, but because the released toxins persist in soil and retain their larvicidal activity, they could enhance the control of target insects, constitute a hazard to nontarget organisms, and/or increase the selection of toxin-resistant target insects. Further studies on the physiology and anatomy of the roots of these transgenic plants, as well as of the environmental effects of these toxins, are obviously necessary. 4. Materials and methods 4.1. Soil and seeds

4 386 D. Saxena et al. / Plant Physiology and Biochemistry 42 (2004) sica napus L.), cotton (Gossypium hirsutum L.), and tobacco (Nicotiana tabacum L.) genetically modified to express cry genes from Bt (Cry1Ab, Cry3A, and Cry1Ac protein, respectively) and of near-isogenic non-bt plant counterparts (Table 1) [7,26] were planted (four seeds/pot and three pots/plant species) in plastic pots (18 cm diam, 21 cm deep) containing ca. 4.5 kg of soil freshly collected from a farm in East Marion, Long Island, New York, USA (ph 5.2; 0.92% and 0.07% carbon and nitrogen; 58%, 41%, and 1% sand, silt, and clay; soil water tension was maintained at ca. 33 kpa, and no water stress was apparent in the plants). Plants were grown in a plant-growth room (26 ± 2 C, 12-h light dark cycle). Transgenic Bt and near-isogenic non-bt plants of each species were the same age when harvested after flowering and production of seeds (rice and cotton were not grown long enough for production of seeds). Randomly selected plants were carefully removed from the pots, and rhizosphere soil was collected by gently shaking the roots to dislodge adhering small clumps of soil. Plants were also grown in sterile hydroponic culture (surface-sterilized seeds were germinated on nutrient agar and plantlets transferred aseptically to glass tubes containing Hoagland s solution) for d in the plant-growth room [19,22] Immunological assay Samples of soil (0.5 g) from the rhizosphere were vortexed with 0.5 ml of extraction buffer (EnviroLogix, Portland, ME), centrifuged, and the supernatants analyzed by Western blot using Lateral Flow Quickstix for Cry1Ab and Cry1Ac proteins (EnviroLogix; detection limit <10 parts 10 9 ) and the DAS ELISA kit (Agdia, Elkhart, IN; detection limit <20 parts 10 9 ) for Cry3A protein [19,20,22,30]. Hydroponic solutions (0.5 ml) were analyzed directly without extraction Fluorescence detection Fluorescence of GFP in samples of hydroponic solutions was determined with a Hitachi Fluorescence Spectrophotometer (model F-2500) with excitation at 490 nm and measurement at 510 nm [7] Insect bioassay The insecticidal activity of the soils and hydroponic solutions was determined with the larvae of the tobacco hornworm (M. sexta) (Cry1Ab and Cry1Ac proteins) or the Colorado potato beetle (L. decemlineata) (Cry3A protein) [31,32]. Eggs of M. sexta and food medium were obtained from Carolina Biological Supply Company (Burlington, NC). Eggs of L. decemlineata were obtained from the New Jersey Department of Agriculture (Trenton, NJ), and food medium was obtained from Bioserve Inc. (Frenchtown, NJ). The eggs, dispensed on solidified medium in Petri plates, were incubated at 29 ±1 Cunder a 40 W lamp for 3 5 d, when the eggs hatched. The media were dispensed, after microwaving for 1 min, in 5-ml amounts into vials (3 cm diam, 6 cm tall) and allowed to solidify. Aliquots (0.1 ml) of freshly vortexed soil suspensions or of hydroponic solutions were uniformly distributed over the surface of the medium (8.55 cm 2 ) with disposable pipette tips (200-µl capacity; Fisher Scientific) that had been cut ca. 1.5 cm from the tip, to ensure that all suspended particles of soil were transferred. After air-drying for 2 h, 4 second-instar larvae (2 3-day-old) of M. sexta were added to each of duplicate vials prepared from duplicate samples of rhizosphere soil or hydroponic solution, resulting in 16 larvae for each treatment. Individual larvae of the Colorado potato beetle were assayed in separate vials: 16 second-instar larvae (2 3-day-old) were added to separate vials prepared from five replicate rhizosphere soil samples or five replicate hydroponic solutions, resulting in 80 larvae for each treatment. Mortality was determined after 3 and 7 d, and percent mortality was based on mortality after 7 d. All surviving larvae of M. sexta were weighed after 7 d, to estimate the extent of sublethal effects. Controls consisted of soil or hydroponic solution in which no plants or nearisogenic non-bt plant counterparts had been grown. The toxicity of the various Cry proteins to the assay larvae, as well as the immunological assays, were confirmed with purified Cry1Ab and Cry1Ac proteins (purified in this laboratory [31]) and purified Cry3A protein provided in the DAS ELISA kit from Agdia Statistics There were at least three replicates of each treatment, and experiments were repeated at least twice. The data are expressed as the mean ± the standard errors (S.E.) of the means. Significance among the data was determined by the paired Student s t-test using SigmaPlot computer software (Jandel Scientific Corporation). 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