Microbial Populations and Enzyme Activities in Soil In Situ under

TECHNICAL REPORTS: ECOLOGICAL RISK ASSESSMENT

Microbial Populations and Enzyme Activities in Soil In Situ under Transgenic Corn Expressing Cry Proteins from Bacillus thuringiensis I. Icoz and D. Saxena New York University D. A. Andow and C. Zwahlen University of Minnesota G. Stotzky* New York University Transgenic Bt crops produce insecticidal Cry proteins that are released to soil in plant residues, root exudates, and pollen and that may affect soil microorganisms. As a continuation of studies in the laboratory and a plant-growth room, a field study was conducted at the Rosemount Experiment Station of the University of Minnesota. Three Bt corn varieties that express the Cry1Ab protein, which is toxic to the European corn borer (Ostrinia nubilalis Hübner), and one Bt corn variety that expresses the Cry3Bb1 protein, which is toxic to the corn rootworm complex (Diabrotica spp.), and their near-isogenic non-Bt varieties were evaluated for their effects on microbial diversity by classical dilution plating and molecular (polymerase chain reaction-denaturing gradient gel electrophoresis) techniques and for the activities of some enzymes (arylsulfatases, acid and alkaline phosphatases, dehydrogenases, and proteases) involved in the degradation of plant biomass. After 4 consecutive years of corn cultivation (2003–2006), there were, in general, no consistent statistically significant differences in the numbers of different groups of microorganisms, the activities of the enzymes, and the pH between soils planted with Bt and nonBt corn. Numbers and types of microorganisms and enzyme activities differed with season and with the varieties of corn, but these differences were not related to the presence of the Cry proteins in soil. The Cry1Ab protein of Bt corn (events Bt11 and MON810) was detected in most soils during the 4 yr, whereas the Cry3Bb1 protein was not detected in soils of Bt corn (event MON863) expressing the cry3Bb1 gene.

Copyright © 2008 by the American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America. All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Published in J. Environ. Qual. 37:647–662 (2008). doi:10.2134/jeq2007.0352 Received 4 July 2007. *Corresponding author ([email protected]). © ASA, CSSA, SSSA 677 S. Segoe Rd., Madison, WI 53711 USA

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ransgenic Bt corn is corn (Zea mays L.) into which cry genes from subspecies of the bacterium, Bacillus thuringiensis (Bt), that encode larvicidal proteins (Cry proteins), have been inserted. These proteins are the active ingredients of commercial Bt insecticidal sprays that have been used for many years (e.g., Dipel, Xentari, Javlin, Foray, M-One, VIP). When modified cry genes are inserted into a plant, the plant expresses active larvicidal proteins in its tissues, making the plant toxic to various insect pests. Many plant species (e.g., corn, cotton, potato, canola, rice) are now genetically modified (GM) to express the Cry proteins (e.g., Saxena et al., 2004). However, the large-scale release of these Bt crops remains a concern because of their presumed potential ecological and environmental risks. The Cry proteins can enter soil via root exudation throughout the growth of the plant (Saxena et al., 1999; Saxena and Stotzky, 2000; Saxena et al., 2002b; Icoz and Stotzky, 2007), in pollen released during tasseling (Losey et al., 1999; Obrycki et al., 2001), from crop residues after harvest (Zwahlen et al., 2003a; Stotzky, 2002, 2004), and from feces of animals feeding on Bt plant material (Wandeler et al., 2002; Einspanier et al., 2004; Pont and Nentwig, 2005). The released Cry proteins are rapidly adsorbed and bound on clay minerals and humic substances, which renders them less available for biodegradation, and their insecticidal activity is retained (e.g., Venkateswerlu and Stotzky, 1992; Tapp et al., 1994; Tapp and Stotzky, 1995a,b, 1998; Koskella and Stotzky, 1997; Crecchio and Stotzky, 1998, 2001; Lee et al., 2003). Moreover, repeated and large-scale use of Bt crop plants could lead to accumulation and persistence of plant-produced Cry proteins in soil (e.g., Tabashnik, 1994; Crecchio and Stotzky, 1998; Tapp and Stotzky, 1998; Saxena and Stotzky, 2001a,b; Saxena et al., 2002a,b; Zwahlen et al., 2003a; Muchaonyerwa et al., 2004; Stotzky, 2004). When assessing the ecological risks of transgenic plants, their impact on soil microbes, which are essential for biogeochemical cycles and soil fertility, should be considered because plants release into

I. Icoz and G. Stotzky, Lab. of Microbial Ecology, Dep. of Biology, New York Univ., New York, NY 10003. D. Saxena, Basic Science, College of Dentistry, New York Univ., New York, NY 10010. D.A. Andow and C. Zwahlen, Dep. of Entomology, Univ. of Minnesota, St. Paul, MN 55108. Abbreviations: ARISA, automated ribosomal intergenic spacer analysis; Bt, Bacillus thuringiensis; CLPP, community level physiological profiles; MANOVA, multivariate analysis of variance; MPN, most probable numbers; PCR-DGGE, polymerase chain reaction–denaturing gradient gel electrophoresis; PNP, p-nitrophenol; SPRM-ANOVA, split-plot repeated measure ANOVA.

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rhizosphere soil as much as 20% of their assimilates as root exudates, and residues from transgenic crops are usually incorporated into soil (Whipps, 1990; Saxena et al., 1999). Organisms in soil will come into contact with transgenic Cry proteins when the proteins are released from Bt corn and other Bt crops in root exudates or from decomposing plant tissue (e.g., Palm et al., 1996; Zwahlen et al., 2003a; Saxena et al., 2004; Icoz and Stotzky, 2007, 2008), thus posing a potential risk for nontarget organisms, including microorganisms. Zwahlen et al. (2003a) reported that Cry proteins are present in soil as long as Bt plant material is present and that soil organisms feeding on plant residues are continuously exposed to Cry proteins (e.g., Zwahlen and Andow, 2005), particularly if the field is repeatedly planted with a transgenic Bt crop. Most studies have suggested that Bt plants that have been released cause only minor changes in the structure of the microbial community of soil and that the changes are often transient (e.g., Donegan et al., 1995, 1996; Blackwood and Buyer, 2004; Brusetti et al., 2004; Griffiths et al., 2006), whereas other studies have shown no apparent deleterious effects of Cry proteins released by Bt plants in root exudates or from biomass incorporated into soil on microbial communities (e.g., Saxena and Stotzky, 2001b; Koskella and Stotzky, 2002; Devare et al., 2004; Wu et al., 2004a,b; Flores et al., 2005; Naef et al., 2006; Shen et al., 2006) or some enzymes (e.g., Flores et al., 2005; Shen et al., 2006). Studies using culture-independent methods reported minor or no Bt-specific effects on soil microorganisms, and the age and type of plant and the type and texture of soil seemed to be the major factors affecting bacterial diversity (Blackwood and Buyer, 2004; Griffiths et al., 2005; Baumgarte and Tebbe, 2005; Fang et al., 2005). Few or no effects of Bt plants expressing Cry proteins were found on nontarget invertebrates in soil, such as earthworms (Saxena and Stotzky, 2001b; Zwahlen et al., 2003b; Clark and Coats, 2006), Collembola (Sims and Martin, 1997; Yu et al., 1997; Cowgill et al., 2002; Al-Deeb et al., 2003; Clark and Coats, 2006), isopods (Escher et al., 2000; Wandeler et al., 2002; Pont and Nentwig, 2005), mites (Yu et al., 1997; Cowgill and Atkinson, 2003), nematodes (Saxena and Stotzky, 2001b; Manachini and Lozzia, 2002), and snails (de Vaufleury et al., 2007; Kramarz et al., 2007). In contrast, several studies have indicated that Bt plants affect microbial communities (Turrini et al., 2004; Castaldini et al., 2005; Rui et al., 2005), the activities of some enzymes (Wu et al., 2004a,b; Sun et al., 2007), and some microbe-mediated processes and functions in soil (Dinel et al., 2003; Castaldini et al., 2005; Flores et al., 2005). Microbial activity, assessed by measuring soil respiration (i.e., CO2 evolution), has also been reported to be affected by the presence of Bt crops in soil. For example, evolution of CO2 was lower in soils under Bt corn or amended with Bt corn biomass than in soils with non-Bt corn (Dinel et al., 2003; Castaldini et al., 2005). Flores et al. (2005) reported that the biomass of various transgenic Bt plants decomposed less in soil, as measured by CO2 evolution, than the biomass of near-isogenic non-Bt plants, which they attributed to a higher lignin content in the biomass of the Bt plants. Hopkins and Gregorich (2003), however, did not observe any differences in the decomposition of plant material from Bt and non-Bt corn, as determined by CO2 648

evolution. The reasons for these contradictory results of the effects of Bt and other transgenic plants on microbes in soil may be the result of the different techniques, plants, and proteins used. In this study, the effects in soil of genetically modified Bt corn expressing Cry1Ab (events Bt11 and MON810) or Cry3Bb1 (event MON863) proteins were evaluated during 4 consecutive years of corn cultivation under field conditions on (i) microbial diversity, using techniques of dilution-plating, most probable numbers (MPN), and polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE); (ii) the activity of some enzymes involved in the degradation of plant biomass; and (iii) the persistence and larvicidal activity of the proteins.

Materials and Methods Experimental Design A field study was conducted from 2003 through 2006 at the Rosemount Experiment Station of the University of Minnesota. Four transgenic Bt corn varieties (Novartis N45-A6, Novartis Attribute GSS-0966, and Pioneer 38A25 that express the Cry1Ab protein, which is toxic to the European corn borer [Ostrinia nubilalis Hübner], and DeKalb DKC46-24 that expresses the Cry3Bb1 protein, which is toxic to the corn rootworm complex [Diabrotica spp.]) were compared with their near-isogenic, non-Bt varieties (N45-T6, PrimePlus, 38A24, and DKC46-28, respectively). DKC46-23 (Cry3Bb1) and DKC46-26 (near-isogenic) varieties were planted in 2003 but were unavailable in subsequent years. DKC46-24 was commercially available only with a seed-applied insecticide (Poncho 1250), which was, therefore, applied to the seed of the DKC46-28 control before planting. Sweet corn varieties (Attribute GSS-0966 and Prime Plus) were not planted in 2005 and 2006, but soil planted to these sweet corn varieties in previous years was analyzed before (April 2005) and after (November 2005) planting with non-Bt corn to estimate the persistence in soil of the Cry1Ab protein. In 2006, Pioneer 38A26 and 38A81 were used because 38A25 was no longer in production. Pioneer 38A26 has a genetic background similar to 38A25. These eight treatments were randomized in a complete block design with four replicates each on 40 × 40 m adjacent plots separated by 20 m of non-Bt corn for a total of 32 plots. The varieties were planted in the same plots in all 4 yr. Some of the physicochemical properties of the soils, which belong predominantly to the Dakota and Waukegan series, are shown in Table 1. Mineralogically, the soils contain quartz, feldspars, amphiboles, some well crystallized mica-illite, low amounts of chlorite and kaolinite, and no smectites or other swelling clays.

Soil Sampling Soils were collected randomly from a depth of 3 to 15 cm from the inner 20 × 20 m of each plot with a shovel and immediately placed on dry ice in August 2003, August 2004, three times in 2005 (before planting [April], midseason [August], and after harvest [November]), and two times in 2006 (before planting [April] and after harvest [October]) from each of the 32 plots and sent to New York University on dry ice. Soils adhering to the root systems of decomposing corn plants were sampled before tillage (April), from growing plants (August, except in August 2003, when bulk

Journal of Environmental Quality • Volume 37 • March–April 2008

rather than rhizosphere soil was sampled), or from harvested plants (October and November). Two samples were taken from each plot; each sample was a subsample of pooled soil from three corn root systems separated by >2 m from each other. Soil samples were immediately stored at –20°C on receipt and analyzed for microbial populations, enzyme activities, pH, water content, presence of the Cry1Ab and Cry3Bb1 proteins, and larvicidal activity of the Cry1Ab protein. Because no consistent significant differences in the numbers of microorganisms and activities of enzymes between soils of Bt and non-Bt corn were observed and funds and personnel were limited, only two soils (Bt and non-Bt) from each block were analyzed for pH, total bacteria and fungi, and activities of dehydrogenases and proteases in October 2006.

Analysis of Culturable Soil Microbes Serial dilutions (10-fold) of the soil samples were prepared with sterile distilled water. Counts of colonies of total culturable aerobic bacteria, gram-negative bacteria, chitin- and celluloseutilizing organisms, and fungi were determined at 25 ± 2°C using the spread-plate technique to inoculate four replicate agar plates per dilution. Colony-forming units of bacteria (including actinomycetes) were estimated on soil extract agar after 10 d of incubation. The colony-forming units of gram-negative bacteria were estimated on MacConkey Agar after incubation for 4 d. Chitin-utilizing organisms were estimated on Chitin Agar, and cellulose-utilizing organisms were estimated on cellulose agar after incubation for 10 d. Fungi were estimated on Rose BengalStreptomycin agar after incubation for 4 d (Stotzky et al., 1993). Nitrifying organisms were evaluated by the MPN method in 96-well plates. Ammonium oxidizers were determined in ammonium-oxidizer broth, and nitrite-oxidizers were determined in nitrite-oxidizer broth after incubation for 42 d. Denitrifying organisms (nitrate reducers and denitrifyers) were estimated in nitrate broth after incubation for 14 d. The appropriate Bray’s powder was used for each group of nitrifying and denitrifying organisms (Stotzky et al., 1993). Numbers of culturable protozoa were estimated by the MPN method using hay infusion broth: 270 μL of broth was aseptically dispensed into each well of sterile 96-well plates, and 30 μL of the 10−1, 10−2, and 10−3 dilution was added to each well, with five replicates of each dilution. The plates were incubated for 7 d at 25 ± 2°C, when each well was examined under low-power magnification (×100) for the presence of protozoa (Stotzky et al., 1993).

Polymerase Chain Reaction–Denaturing Gradient Gel Electrophoresis Isolation of Soil Bacterial DNA Total soil DNA was extracted using soil DNA isolation kits according to the manufacturer’s instructions (PowerMax; MoBio Laboratories Inc., Carlsbad, CA). The quality and quantity of the DNA were measured at 260 nm and 280 nm (DU640; Beckman Instruments, Inc., Fullerton, CA). The final concentration of each DNA sample was adjusted to 10 ng μL−1 for all PCR. Extracted DNA was stored at −20°C until use.

Table 1. Some properties of the soils used†. Property

Soil A Soil B 56.0 57.0 Organic matter (g kg−1) 29.7 28.2 Total carbon (g kg−1) 0.00 0.00 Inorganic carbon (g kg−1) 29.7 28.2 Total organic carbon (g kg−1) 2.8 2.8 Total nitrogen (g kg−1) pH 6.5 6.5 8.5 7.9 NO3−–N (mg kg−1) 27 20 P (mg kg−1) 195 116 K (mg kg−1) 2387 2193 Ca (mg kg−1) 475 420 Mg (mg kg−1) Sand (%) 22.5 22.5 Silt (%) 55.4 54.6 Clay (%) 22.1 22.9 † Two composite soil samples from different parts of the field were analyzed by the Soil Testing and Research Analytical Laboratory of the University of Minnesota.

PCR for Amplification of Bacterial 16S rDNA Gene Fragments All PCR were performed with the GeneAmp PCR System 9700 (PE Applied Biosystems, Foster, CA). Nested PCR was used to amplify the targeted gene fragments. Initially, the complete 16S rDNA gene locus (?1500 bp) was preamplified for all DNA extracts with a set of universal 16S rDNA gene sequence primers (Lane, 1991). Each PCR mixture (a total volume of 50 μL) contained 100 ng of genomic DNA, 200 μM of each deoxynucleotide triphosphate, 50 pmole of the universal primers 16S-8f and 16S-1492r, 1.5 mM MgCl2, 5 μL of 10× PCR buffer II, and 2.5 U of Taq DNA polymerase. Polymerase chain reaction conditions were initial denaturation at 95°C for 4 min; 30 cycles consisting of 1 min at 95°C, 1 min at 55°C, and 2 min at 72°C; and extension at 72°C for 5 min. In the second nested PCR reaction, the universal bacterial 16S rDNA gene primers, prbac1 and prbac2 (Rupf et al., 1999), were used with a 40-nucleotide guanine-cytosine (GC)-clamp, which was added to the 5′ end of primer prbac1 to prevent the dissociation of the 16S rDNA gene duplexes during denaturation electrophoresis (Sheffield et al., 1989; Muyzer et al., 1993; Zoetendal et al., 1998). Each PCR reaction mixture (a total volume of 50 μL) contained 5 μL of the preamplified genomic DNA sample, 200 μM of each deoxynucleotide triphosphate, 40 pmole each of forward and reverse primer, 1.5 mM MgCl2, 5 μL of 10× PCR buffer II, and 2.5 U of Taq DNA polymerase. Polymerase chain reaction conditions were initial denaturation at 94°C for 2 min; 45 cycles consisting of 30 s at 93°C, 1 min at 40°C, and 1 min at 72°C; and extension at 72°C for 7 min (Li et al., 2007). The PCR products were evaluated by electrophoresis in 1.0 to 2.0% agarose gels, and the size of all amplicons was determined with a molecular size standard.

Denaturing Gradient Gel Electrophoresis Assay A 40 to 60% linear DNA denaturing gradient (100% denaturant was 7 mol L−1 of urea and 40% deionized formamide) was formed in an 8% (w v−1) polyacrylamide gel, 20 μL of each PCRamplified product was loaded in each lane, and electrophoresis was performed at a constant 60 V at 58°C for 16 h in 1× Tris-acetate-

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EDTA (TAE) buffer (pH 8.5) (Li et al., 2005, 2006, 2007). After electrophoresis, the gels were rinsed and stained for 15 min in an ethidium bromide solution (0.5 mg L−1), followed by 15 min of destaining in water. The DGGE profile images were digitally captured and recorded (Alpha Innotech Corporation, San Leandro, CA) and normalized by means of Fingerprinting II Informatix Software (Bio-Rad). Denaturing gradient gel electrophoresis profiles were determined by measuring the migration distances and the intensities of the bands within each lane. The results were transferred into a microbial database, which enabled cross-comparing multiple DGGE profiles simultaneously (Li et al., 2005, 2007).

Analysis of Soil Enzymes The activity of soil enzymes representative of those involved in the degradation of plant biomass was determined by the methods described by Stotzky et al. (1993), with two replicates of each soil. The activity of acid and alkaline phosphatases was measured at 37°C with p-nitrophenyl phosphate as the substrate in a modified universal buffer solution at pH 6.5 and 11.0, respectively, using p-nitrophenol (PNP) as the standard. Activities are expressed as mg of PNP kg−1 of oven-dry soil. The activity of arylsulfatases was determined at 37°C using p-nitrophenyl sulfate as the substrate, and activity is expressed as mg of PNP kg−1 of oven-dry soil. Dehydrogenase activity was measured by the reduction of 3,5,-triphenyltetrazolium chloride to 2,3,5,-triphenylformazan and expressed as mg of 2,3,5,-triphenylformazan kg−1 of ovendry soil. The activity of proteases was determined by the modified Anson’s method of Yang and Huang (1994). Briefly, the reaction mixture, containing 1 mL of 1% casein in phosphate buffer (0.1 M Na2HPO4 and 0.1 M NaH2PO4 [pH 7]) and 1 g of soil, was incubated for 60 min. The reaction was stopped with 3 mL of 10% trichloroacetic acid, the suspension was filtered through Whatman No. 2 filter paper, and the absorbance of the liberated tyrosine in the filtrate was measured at 280 nm. Protease activity is expressed as mg of tyrosine kg−1 of oven-dry soil.

Soil pH and Water Content Water content was measured by drying the soils overnight at 105°C and expressed using the formula (Gardner, 1965): %H2O = (wt of wet soil – wt of dry soil) × 100 ÷ wt of dry soil. The pH of the 10−1 dilution used for the microbial analyses was measured with a pH meter (McLean, 1982).

Immunological Assay The presence of the Cry1Ab and Cry3Bb1 proteins in soils of Bt and non-Bt corn was determined by Western blot with ImmunoStrips (Agdia, Elkhart, IN). Soil (0.5 g) was vortexed for 1.5 min in a 1.5-mL Eppendorf microcentrifuge tube with 0.5 mL of extraction buffer (SEB4 buffer, Agdia) and centrifuged at 10,500 × g for 5 min. The supernatant was analyzed with an ImmunoStrip following the recommended protocol of the manufacturer.

Larvicidal Assay The larvicidal activity of Cry1Ab protein was determined with the larvae of the tobacco hornworm (Manduca sexta) (Lepidoptera:

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Sphingidae) (Tapp and Stotzky, 1998). Eggs of M. sexta and food medium were obtained from Carolina Biological Supply Company (Burlington, NC). The eggs, dispensed on solidified medium in Petri plates, were incubated at 29 ± 1°C under a 40-W lamp for 3 to 5 d. The medium was dispensed, after microwaving, in 5-mL amounts into vials (3 cm in diameter and 6 cm tall) and allowed to solidify. Aliquots (100 μL) of freshly vortexed suspensions of soil, prepared as for the immunological assay (0.5 g of soil was vortexed with 0.5 mL of SEB4 buffer) but without centrifugation, were uniformly distributed over the surface of the medium (8.55 cm2) with disposable pipette tips (200-μL capacity) that had been cut ?1.5 cm from the tip to ensure that all suspended soil particles were transferred. After air-drying, four second-instar larvae were added to each of duplicate vials prepared from duplicate soil samples, resulting in 16 larvae for each soil sample. Mortality was determined after 3 and 7 d, and percent mortality was based on mortality after 7 d. Control vials contained only 100 μL of extraction buffer dispensed over the surface of the medium. No larvicidal assay of the Cry3Bb1 protein was done because of the absence of a readily available assay organism.

Statistical Analyses The data are expressed as means ± SEM. Analysis of variance was used to examine significance between treatments (Bt vs. non-Bt) and between plant varieties (field corn vs. sweet corn) for each microbial group and the activity of each enzyme individually for each year, and p < 0.05 was considered significant. Bt corn varieties expressing the Cry1Ab or Cry3Bb1 protein were evaluated statistically against their near-isogenic, non-Bt corn counterparts to determine whether there was a significant difference in effects between Bt and non-Bt corn. Because there was more than one dependent variable and where the dependent variables could simply be combined, multivariate analysis of variance (MANOVA), which enables a single analysis of all variables and all sample times, was also conducted to confirm the main effect of treatments (Bt vs. non-Bt) on microbial groups and the activity of enzymes during the 4 yr of corn cultivation. Each microbial group and enzyme activity was also analyzed using a splitplot repeated measure ANOVA (SPRM-ANOVA), which evaluated all years together, to determine the main effect of treatments and interactions between sampling time and plant variety. The data were analyzed using the SAS statistical package for MANOVA, which included the general linear model procedure.

Results Water Content and pH of Soils In general, the water content and pH did not differ significantly or consistently between Bt and non-Bt soils. The pH of the soils ranged between 6.1 and 6.7 during the 4 yr (Fig. 1). There was no apparent relation between changes in the pH and water content and changes in microbial diversity, enzyme activities, and persistence of the proteins.


Soil Microbes Total Culturable Bacteria Individual ANOVA showed no consistent significant differences in the numbers of total culturable bacteria between soils of Bt and non-Bt corn during the 4 yr (Fig. 2A). The only significant difference in the numbers of bacteria was in August 2005, with significantly (p < 0.05) lower numbers in soils of Bt corn expressing the Cry3Bb1 protein than in soils of its near-isogenic variety of non-Bt corn (Fig. 2A). However, significant differences in the numbers of bacteria were sometimes observed between varieties of corn expressing the Cry1Ab protein (e.g., the numbers of bacteria were higher [p < 0.01] in soil planted with the sweet corn varieties [Novartis Attr. GSS-0966 and Prime Plus] than with the field corn varieties [Pioneer 38A25 and Pioneer 38A24; Novartis 45-A6 and Novartis 45-T6] in April 2005 and in November 2005) (Fig. 2A). Split-plot repeated measure ANOVA showed no significant differences in the numbers of total bacteria between soils of Bt and non-Bt corn. However, significant (p < 0.0001) seasonal differences in the numbers of total bacteria were observed. There was also a significant interaction (p < 0.05) between Bt and non-Bt sweet corn varieties and season on the numbers of total bacteria.

Gram-Negative Bacteria The numbers of gram-negative bacteria were significantly (p < 0.01) higher in individual ANOVA only in August 2005 in soils of Bt corn, both expressing the Cry1Ab or Cry3Bb1 protein, than in soils of their near-isogenic non-Bt corn counterparts (Fig. 2B). No other significant differences in the numbers of gramnegative bacteria between soils of Bt corn and non-Bt corn were observed. The variety of corn significantly affected the numbers of gram-negative bacteria in April, August, and November 2005: Numbers were higher (p < 0.01) in soils planted with the sweet corn varieties than in soils with the varieties of field corn in April and November 2005 and higher (p < 0.05) in soils with Pioneer than with Novartis in August 2005 (Fig. 2B). No significant differences in the numbers of gram-negative bacteria between soils of Bt and non-Bt corn, or any interaction between plant varieties and season, were observed by SPRM-ANOVA. However, seasonal differences in the numbers of gram-negative bacteria were significant (p < 0.0001).

Chitin-Utilizing Organisms Significant differences in the numbers of chitin-utilizing organisms between soils of Bt and non-Bt corn were observed in April 2005, August 2005, and April 2006. The numbers of chitinutilizing organisms were significantly (p < 0.01) higher in soils of Bt corn expressing the Cry1Ab protein than in soils of their nearisogenic non-Bt corn counterparts in April 2005 and 2006, and they were significantly (p < 0.05) higher in soils of Bt corn expressing the Cry3Bb1 protein than in soils of its near-isogenic non-Bt corn in August 2005 (Fig. 2C). Some significant differences in the numbers of chitin-utilizing organisms were also observed in ANOVA between varieties expressing the Cry1Ab protein: Numbers were higher (p < 0.01) in soils with the field corn variety Novartis than with the field corn variety Pioneer and the varieties of sweet corn in April and November 2005 (Fig. 2C).

Fig. 1. Water content (%) and pH of soils of Bt and non-Bt corn. The data are expressed as the means of four replicates ± SEM. DKC46-23 (Bt) and DKC46-26 (near-isogenic non-Bt) rather than DKC46-24 (Bt) and DKC46-28 (non-Bt) corn varieties were planted in August 2003. No sweet corn (Attribute GSS-0966 and Prime Plus) was planted in August 2005 and subsequently. In 2006, Pioneer 38A26 and 38A81 were used because 38A25 was no longer in production. Only two soils from each block were analyzed in October 2006.

Split-plot repeated measure ANOVA also showed significant (p < 0.05) differences in the numbers of chitin-utilizing organisms between soils of Bt field corn expressing the Cry1Ab protein and their near-isogenic non-Bt field corn. Seasonal differences in the numbers of organisms (p < 0.0001) and interaction between sweet corn varieties and sampling time (p < 0.05) were also significant.

Cellulose-Utilizing Organisms No significant differences in the numbers of celluloseutilizing organisms between soils of Bt and non-Bt corn were observed in August 2004, the only sampling date on which this group of organisms was measured (data not shown).

Fungi The numbers of fungal propagules did not differ significantly between soils of Bt and non-Bt corn in 2004, 2005, and 2006 (Fig. 2D). However, in August 2003, the numbers of fungi were significantly (p < 0.05) lower in soils of Bt corn expressing the Cry3Bb1 protein than in soils of its near-isogenic non-Bt corn variety. Some significant differences in the number of fungi were


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Fig. 2. Numbers of total culturable aerobic bacteria (including actinomycetes) (A), gram-negative bacteria (B), chitin-utilizing organisms (C), fungi (D), and protozoa (E) in soils planted to different varieties of corn with or without the genes expressing Cry1Ab or Cry3Bb1 protein. The data are expressed as the means of four replicates ± SEM (ods, oven-dry soil). See legend to Fig. 1 for more details.

observed between varieties: The numbers in August 2003 were higher (p < 0.01) in soils with sweet corn than in soils with the varieties of field corn, and in November 2005, they were higher (p < 0.01) in soils with field corn than with sweet corn (Fig. 2D). No significant differences in the numbers of fungi between soils of Bt and non-Bt corn were observed by SPRM-ANOVA. However, the numbers were significantly (p < 0.0001) different with season. No significant interaction between plant varieties and season was observed on the numbers of fungi.

Protozoa The only significant (p < 0.05) difference in the numbers of protozoa between soils of Bt and non-Bt corn was observed in November 2005, with lower numbers in soils of Bt corn expressing the Cry1Ab protein than in soils of their near-isogenic

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non-Bt varieties (Fig. 2E). Significant differences in the numbers of protozoa were observed between varieties only in August 2005: The numbers were significantly (p < 0.05) higher in soils planted with Novartis than with Pioneer (Fig. 2E). Split-plot repeated measure ANOVA confirmed that there were no significant differences in the numbers of protozoa between soils of Bt and non-Bt corn, whereas seasonal differences in the numbers were significant (p < 0.0001). No significant interaction between plant varieties and season was observed on the numbers of protozoa.

Ammonium- and Nitrite-Oxidizing Bacteria Significantly (p < 0.05) lower numbers of ammoniumoxidizing bacteria were observed in soils of Bt corn expressing the Cry1Ab protein than in soils of their near-isogenic non-Bt


Fig. 3. Numbers of NH4+–oxidizers (A) and NO2−–oxidizers (B) in soils planted to different varieties of corn with or without the genes expressing Cry1Ab or Cry3Bb1 protein. The data are expressed as the means of four replicates ± SEM (ods, oven-dry soil). See legend to Fig. 1 for more details.

Fig. 4. Numbers of NO3−–reducers (A) and denitrifyers (B) in soils planted to different varieties of corn with or without the genes expressing Cry1Ab or Cry3Bb1 protein. The data are expressed as the means of four replicates ± SEM (ods, oven-dry soil). See legend to Fig. 1 for more details.

varieties in August 2004, and there were significantly (p < 0.01) higher numbers of ammonium-oxidizing bacteria in soils of Bt corn expressing the Cry3Bb1 protein than in soils of its nearisogenic non-Bt corn in August 2005 (Fig. 3A). By contrast, no consistent significant differences were observed in the numbers of nitrite-oxidizing bacteria between soils of Bt corn expressing the Cry1Ab or Cry3Bb1 protein and their near-isogenic non-Bt corn counterparts during the 4 yr (Fig. 3B). Some significant differences in the numbers of nitrifying bacteria were observed between varieties: Soils planted with Pioneer had significantly (p < 0.01) higher numbers of ammonium-oxidizing bacteria than soils planted with Novartis in August 2005, and soils planted with sweet corn had significantly (p < 0.01) higher numbers of ammonium-oxidizing bacteria than soils planted with field corn in November 2005 (Fig. 3A). The numbers of nitrite-oxidizing bacteria were higher (p < 0.01) in soils planted with sweet corn than in soils planted with field corn in August 2003 (Fig. 3B). No significant differences in the numbers of ammoniumand nitrite-oxidizing bacteria between soils of Bt and non-Bt corn were observed by SPRM-ANOVA. However, seasonal differences in the numbers were significant (p < 0.0001).

Nitrate-Reducing and Denitrifying Bacteria There were no consistent significant differences in ANOVA in the numbers of nitrate-reducing bacteria between soils of Bt corn and non-Bt corn (Fig. 4A). The numbers were significantly (p < 0.01) higher in soils of Bt corn expressing the Cry1Ab protein in August 2003 and significantly higher in August 2005 in soils of Bt corn expressing the Cry1Ab (p < 0.01) or Cry3Bb1 (p < 0.05) protein than in soils with their near-isogenic non-Bt corn counterparts (Fig. 4A). There were also no consistent significant differences in the numbers of denitrifying bacteria between soils of Bt corn and non-Bt corn during the 4 yr. The only significant (p < 0.01) difference was observed in August 2005, with higher numbers of denitrifying bacteria in soils of Bt corn expressing the Cry3Bb1 protein than in soils of its near-isogenic non-Bt corn variety (Fig. 4B). Some significant (p < 0.05) differences in the numbers of nitrate-reducing and denitrifying bacteria were observed between varieties: Soils with sweet corn had higher numbers of nitratereducing bacteria than soils with field corn in April 2005 (Fig. 4A), and the numbers of denitrifying bacteria were higher in soils with Novartis than with Pioneer or sweet corn in November 2005 (Fig. 4B). However, these differences in the numbers of nitrate-reducing


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Fig. 5. Denaturing gradient gel electrophoresis band profiles of PCR-amplified bacterial 16S rDNA gene fragments from the total genomic DNA of the soils of Bt and non-Bt corn collected in October 2006 (lanes 8, 4, 7, 13, 11, 9, 1, 3, 19, 26, 17, 23, 27, 30, 29, and 25 are Bt soils; lanes 14, 15, 6, 5, 2, 16, 12, 10, 21, 20, 22, 18, 28, 24, 32, and 31 are non-Bt soils; lanes M1 and M2 are markers).

and denitrifying bacteria between Bt and non-Bt corn or between varieties were not consistent during the 4 yr. There were no significant differences in the numbers of nitrate-reducing bacteria between soils of Bt and non-Bt corn, whereas there were significant differences (p < 0.05) in the numbers of denitrifying bacteria between soils of Bt corn expressing the Cry3Bb1 protein and its near-isogenic non-Bt corn by SPRM-ANOVA. Seasonal differences (p < 0.0001) and interaction between sweet corn varieties and season on the numbers of denitrifying bacteria were also significant (p < 0.05).

PCR-DGGE 16S rDNA fragments amplified from DNA extracted from the soils of Bt and non-Bt corn were compared on the same denaturing gradient gels. Denaturing gradient gel electrophoresis band profiles of PCR-amplified 16S rDNA fragments from the soils of Bt and non-Bt corn showed a high degree of similarity when each pair of lanes (i.e., from soil with Bt corn and its nearisogenic non-Bt counterpart) within the gels was compared (e.g., in October 2006) (Fig. 5). No differences in the relative distribution and in the intensity profiles of representative lanes of the DGGE gels were observed in August 2004, April and November 2005, and October 2006 (Fig. 6). The DGGE band profiles indicated that bacterial diversity did not differ between soils of Bt and non-Bt corn. However, there were marked seasonal differences in the bacterial community structure between the samples of August 2004, April and November 2005, and October 2006 (Fig. 6).

Enzyme Activities In general, there were no consistent significant differences in the activities of arylsulfatases, acid and alkaline phosphatases, dehydrogenases, and proteases between soils of Bt corn expressing the Cry1Ab or Cry3Bb1 protein and soils of their near-isogenic

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non-Bt corn counterparts during the 4 yr (Fig. 7). Some significant differences in the activity of these enzymes were observed between soils planted to different varieties, but they were also not consistent. Although there were no significant differences in the activity of arylsulfatases between soils of Bt and near-isogenic non-Bt corn in individual ANOVA, there were significant difference (p < 0.05) between soils of Bt corn expressing the Cry3Bb1 protein and its near-isogenic non-Bt corn variety by SPRM-ANOVA. Some significant differences between varieties were observed in April 2005: Activities were significantly (p < 0.01) higher in soils with sweet corn than with the varieties of field corn (Fig. 7A). Seasonal differences in the numbers were also significant (p < 0.0001) by SPRM-ANOVA. There was a significant difference (p < 0.01) in the activity of acid phosphatases only in April 2005, with higher activity in soils with Bt corn expressing the Cry1Ab protein than in soils with their near-isogenic non-Bt counterparts. There were significant differences in the activity between varieties only in November 2005, when the activity in soils with sweet corn was significantly (p < 0.01) higher than in soils with varieties of field corn (Fig. 7B). Analysis of all groups by SPRM-ANOVA showed significant differences (p < 0.05) only between soils of Bt sweet corn expressing the Cry1Ab protein and its near-isogenic non-Bt corn. The activity of alkaline phosphatases did not differ significantly between soils of Bt and non-Bt corn during the 4 yr, and there was a significant difference in activity between varieties only in August 2004, when the activity with Pioneer was significantly (p < 0.01) higher than with Novartis and sweet corn (Fig. 7C). No significant differences in the activity of alkaline phosphatases between treatments (Bt vs. non-Bt corn) were observed by SPRMANOVA. However, significant (p < 0.0001) seasonal differences and significant (p < 0.05) interactions between plant varieties (Bt corn expressing the Cry3Bb1 protein and its non-Bt isogenic


Fig. 6. Standard graphs generated by the Diversity Database software for comparison of the relative distributions and the intensity profiles of representative lanes from denaturing gradient gel electrophoresis images of DNA extracted from soils of Bt (Bt+) and non-Bt (Bt-) corn collected in August 2004, April 2005, November 2005, and October 2006.

counterpart) and sampling time on the activity of alkaline phosphatases were observed by SPRM-ANOVA. Some significant differences were observed in the activity of dehydrogenases between soils of Bt and non-Bt corn in August 2004 and April 2005, but these differences were also not consistent (Fig. 7D). In August 2004, dehydrogenase activity in soils of Bt corn expressing the Cry1Ab protein was significantly (p < 0.05) higher than in soils of their near-isogenic non-Bt varieties, whereas there were no significant differences between soils of Bt corn expressing the Cry3Bb1 protein and its near-isogenic non-Bt corn counterpart. In April 2005, there were no significant differences between the soils of Bt corn expressing the Cry1Ab protein and of their near-isogenic non-Bt varieties, whereas significant (p < 0.05) differences occurred between soils of Bt corn expressing the Cry3Bb1 protein and its near-isogenic non-Bt corn, with higher activity in soils with non-Bt corn. The variety of corn did not affect the activity of dehydrogenases in soils during the 4 yr (Fig. 7D). No significant differences in the activity of dehydrogenases between soils of Bt and non-Bt corn were found by SPRM-ANOVA, whereas there were significant (p < 0.0001) seasonal differences and a significant

(p < 0.05) interaction between plant varieties (Bt corn expressing the Cry3Bb1 protein and its non-Bt counterpart) and season. There were no significant differences by ANOVA or SPRM-ANOVA in the activity of proteases between soils of Bt corn expressing the Cry1Ab or Cry3Bb protein and their near-isogenic non-Bt counterparts, with the exception of August 2004, when the activity in soils of Bt corn expressing the Cry3Bb1 protein was significantly (p < 0.01) lower than in soils of its near-isogenic non-Bt corn. However, some significant differences were observed between varieties in August 2003 and 2004: The activity was significantly higher in soils with sweet corn than with the varieties of field corn in 2003 (p < 0.05) and 2004 (p < 0.01) (Fig. 7E).

Persistence of Cry Proteins in Soil The Cry1Ab protein was detected by Western blots in most soils of Bt corn expressing the Cry1Ab protein, except it was not detected in the bulk soils collected in August 2003, which may be explained by the fact that these were bulk rather than rhizosphere soil samples. The Cry3Bb1 protein was


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Fig. 7. Activity of arylsulfatases (A), acid phosphatases (B), alkaline phosphatases (C), dehydrogenases (D), and proteases (E) in soils planted to different varieties of corn with or without the genes expressing Cry1Ab or Cry3Bb1 protein. The data are expressed as the means of four replicates ± SEMs (ods, oven-dry soil). See legend to Fig. 1 for more details.

not detected in soils of Bt corn expressing the Cry3Bb1 protein throughout the 4 yr (Table 2). The Cry1Ab protein was also detected in some non-Bt soils (14 out of 80 non-Bt soils tested), possibly as a result of contamination from adjacent plots of Bt corn or of false positives in the Western blots.

Larval Mortality Statistically significant differences were observed in larval mortality between soils of Bt corn expressing the Cry1Ab protein and soils of their respective near-isogenic non-Bt counterparts in August 2004 (p < 0.05), April 2005 (p < 0.01), and April 2006 (p < 0.05) (Table 3). The mortality with non-Bt soils was essentially equivalent to the normal background mortality (i.e., mortality in control vials to which only extraction buffer had been added). In August 2004,

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larval mortality was 15.6 ± 4.49, 14.1 ± 3.93, and 10.9 ± 3.20% in soils of Bt corn expressing the Cry1Ab protein (N45-A6, Attr. GSS-0966, and 38A25, respectively) and 9.4 ± 3.13, 3.1 ± 2.13, and 7.8 ± 2.99% in soils planted with their near-isogenic non-Bt counterparts (N45-T6, Prime Plus, and 38A24, respectively). In April 2005, larval mortality was 22.9 ± 2.09, 29.2 ± 5.52, and 33.3 ± 5.52% in soils of these Bt corn varieties and 6.3 ± 3.61, 6.3 ± 3.61, and 8.3 ± 2.09% in soils of their respective near-isogenic non-Bt varieties. In April 2006, larval mortality was 20.3 ± 4.69% and 21.9 ± 5.04% in soils of Bt corn (N45-A6 and 38A25, respectively) and 10.9 ± 3.20% and 7.8 ± 3.76% in soils of their near-isogenic non-Bt corn (N45-T6 and 38A24, respectively) (Table 3). No significant differences in larval mortality among Bt or non-Bt plant varieties were observed during the 4 yr.


Table 2. Presence of Cry proteins in soils planted to different varieties of corn with or without the genes expressing Cry1Ab or Cry3Bb1 protein. Presence of the Cry proteins was determined with appropriate Western blots with ImmunoStrips (Agdia, Elkhart, IN). The data are expressed as the numbers of positives per four replicates. Gene Aug. Plant Variety Company Type transformed Trans. event† 2003 Bt+ N45-A6 Novartis Field cry1Ab Bt11 0/4§ Bt− N45-T6 Novartis Field – – 0/4 Bt+ Attr.Gss. 0966 Novartis Sweet cry1Ab Bt11 0/4 Bt− Prime Plus Novartis Sweet – – 0/4 Bt+ Pioneer 38A25 Pioneer Field cry1Ab MON810 0/4 Bt− Pioneer 38A24 Pioneer Field – – 0/4 Bt+ DKC 46-24 DeKalb Field cry3Bb1 MON863 0/4# Bt− DKC 46-28 DeKalb Field – – 0/4# † Transformation event: insertion of the cry1Ab or cry3Bb1 gene by transformation. ‡ Western blot with ImmunoStrips (Agdia). § The presence of protein in four replicates of the soil. ¶ ND, no data. # DKC 46-23 and DKC 46-26 corn varieties were planted in August 2003.

Discussion Effects of Cry Proteins Different effects, ranging from no effects to minor and statistically significant effects, of Bt plants on microbial communities in soil have been reported (e.g., Donegan et al., 1995, 1996; Saxena and Stotzky, 2001b; Koskella and Stotzky, 2002; Devare et al., 2004; Blackwood and Buyer, 2004; Brusetti et al., 2004; Turrini et al., 2004; Wu et al., 2004a,b; Castaldini et al., 2005; Flores et al., 2005; Rui et al., 2005; Shen et al., 2006; Icoz and Stotzky, 2008). These conflicting results were probably the result of differences in the type of Cry protein, plant variety, experimental methods used, soil type, and environmental factors. The present study showed no consistent statistically significant differences between soils planted with Bt corn expressing the Cry1Ab or Cry3Bb1 protein and with their near-isogenic non-Bt corn counterparts in the numbers of culturable aerobic bacteria (including actinomycetes), gram-negative bacteria, cellulose- (evaluated only in 2004) and chitin-utilizing organisms, nitrifying and denitrifying bacteria, fungi, and protozoa, determined by classical culturing techniques, and in bacterial community structure and diversity, determined by nonculture techniques (i.e., PCR-DGGE), during 4 consecutive years of corn cultivation. Some differences between Bt and non-Bt

Aug. 2004 3/4 1/4 2/4 0/4 2/4 0/4 0/4 0/4

Presence of Cry proteins‡ Apr. Aug. Nov. 2005 2005 2005 3/4 2/4 2/4 2/4 1/4 2/4 4/4 ND¶ 2/4 1/4 ND 1/4 3/4 1/4 2/4 1/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4 0/4

Apr. 2006 4/4 1/4 4/4 0/4 4/4 1/4 0/4 0/4

Oct. 2006 4/4 2/4 4/4 1/4 4/4 0/4 0/4 0/4

soils in the numbers of the various group of microorganisms were observed at some sampling times, but these differences were not consistent from one season to the next. Analysis of the data using MANOVA and SPRM-ANOVA supported all of the conclusions for the main effect of treatments (Bt vs. non-Bt corn) on microbial groups and the activity of some enzymes. Similar results have been obtained in other studies with Bt corn and other Bt plants. Saxena and Stotzky (2001b) and Flores et al. (2005) found no significant effects of the Cry1Ab protein released to soil from Bt corn in root exudates or biomass on culturable bacteria, fungi, and protozoa. The addition of purified Cry1Ab and Cry1Ac proteins to soil did not result in any consistent detectable effects on soil bacteria, fungi, and protozoa when compared with the control soil (Donegan et al., 1995), and the insecticidal proteins from B. thuringiensis subsp. kurstaki, morrisoni (strain tenebrionis), and israelensis did not affect the growth of a variety of bacteria, fungi, and algae in pure or mixed culture (Koskella and Stotzky, 2002). Devare et al. (2004) detected no deleterious effects of growing Bt corn expressing the Cry3Bb1 protein for two consecutive seasons in the field on microbial biomass and activity or on bacterial community structure, as determined by terminal restriction fragment length polymorphism analysis. In a continuation of this study, a 3-yr field assessment of Bt corn expressing the

Table 3. Larvicidal activity of soils planted to different varieties of corn with or without the gene expressing Cry1Ab protein. % Mortality‡ Gene Trans. Plant Variety Company Type transformed event† Aug. 2003 Aug. 2004* Apr. 2005** Aug. 2005 Nov. 2005 Apr. 2006* Bt+ N45-A6 Novartis Field cry1Ab Bt11 3.1 ± 2.13 15.6 ± 4.49 22.9 ± 2.09 4.7 ± 2.52 9.4 ± 3.87 20.3 ± 4.69 Bt− N45-T6 Novartis Field – – 3.1 ± 2.13 9.4 ± 3.13 6.3 ± 3.61 3.1 ± 2.13 7.8 ± 3.76 10.9 ± 3.20 Bt+ Attr.Gss. 0966 Novartis Sweet cry1Ab Bt11 9.4 ± 3.87 14.1 ± 3.93 29.2 ± 5.52 ND§ 7.8 ± 2.99 17.2 ± 3.76 Bt− Prime Plus Novartis Sweet – – 6.3 ± 3.61 3.1 ± 2.13 6.3 ± 3.61 ND 4.7 ± 2.52 20.3 ± 5.21 Bt+ Pioneer 38A25 Pioneer Field cry1Ab MON810 6.3 ± 3.61 10.9 ± 3.20 33.3 ± 5.52 4.7 ± 3.40 7.8 ± 3.76 21.9 ± 5.04 Bt− Pioneer 38A24 Pioneer Field – – 7.8 ± 2.99 7.8 ± 2.99 8.3 ± 2.09 6.3 ± 2.80 3.1 ± 2.13 7.8 ± 3.76 * Significant differences (p < 0.05) were observed between Bt and non-Bt soils. ** Significant differences (p < 0.01) were observed between Bt and non-Bt soils. † Transformation event: insertion of the cry1Ab gene by transformation. ‡ Larvicidal activity was determined with the larvae of the tobacco hornworm (Manduca sexta). Each treatment had four replicates, and at least 16 larvae were assayed in each treatment. Data are expressed as the means of 64 larvae ± SEM. § ND, no data.


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Cry3Bb1 protein vs. near-isogenic non-Bt corn grown with and without the insecticide tefluthrin demonstrated that neither the Bt corn nor the insecticide had adverse effects on microbial biomass, N mineralization potential, or rates of nitrification and respiration. However, effects of the rhizosphere and seasonal changes were consistently observed throughout the study, indicating that their influence on microbial biomass and activity was probably greater than any subtle effects resulting from crop treatment (Devare et al., 2007). Brusetti et al. (2004) compared the bacterial community of the rhizosphere of Bt and near-isogenic non-Bt corn using several techniques, including viable counts; community-level catabolic profiling; and PCR-based, automated ribosomal intergenic spacer analysis (ARISA). Viable counts and community-level catabolic profiling did not show any differences between Bt and non-Bt corn, but ARISA showed that the community structure differed between Bt and non-Bt corn and with the age of the plants, suggesting that root exudates could select different bacterial communities. No significant effects of the Cry1Ab and Cry1F proteins from Bt corn on bacterial and fungal phospholipids fatty acid profiles and only a few significant effects of the Cry proteins on microbial community level physiological profiles (CLPP) were reported by Blackwood and Buyer (2004), and these effects were the result primarily of differences in soil type. Studies with Cry proteins and products of other transgenic plants also showed no or minor differences in microbial community structure of soils when compared with their appropriate control soils. A significant but transient increase in the populations of culturable bacteria and fungi was found in soil microcosms with leaves of Bt cotton (Gossypium hirsutum L.) expressing the Cry1Ac protein (Donegan et al., 1995); minimal differences were observed in the populations of culturable aerobic bacteria and fungi in soil with transgenic Bt potato (Solanum tuberosum L.) expressing the Cry3A protein (Donegan et al., 1996); populations of nematodes were higher in soil with transgenic tobacco (Nicotiana tabacum L.) expressing protease inhibitor I than with nonmodified tobacco (Donegan et al., 1997); and significantly higher levels of culturable aerobic spore-forming and celluloseutilizing bacteria and lower activity of enzymes (dehydrogenases and alkaline phosphatases) were found in soils with transgenic alfalfa (Medicago sativa L.) expressing α-amylase or lignin peroxidase (Donegan et al., 1999). Wu et al. (2004a,b) found that decomposing straw of Bt rice containing the Cry1Ab protein was not toxic to a variety of culturable microorganisms in a flooded paddy soil under laboratory conditions. Rui et al. (2005) found lower numbers of culturable functional bacteria (potassiumdissolving bacteria, inorganic phosphate-dissolving bacteria, and nitrogen-fixing bacteria) in the rhizosphere of Bt cotton than of near-isogenic non-Bt cotton only during the early and middle growth stages but found no significant differences after the growing season. Shen et al. (2006) reported that the functional diversity of microbial communities were not different in rhizosphere soils of Bt and non-Bt cotton. Sabharwal et al. (2007) reported no significant differences in the number of various groups of microorganisms and in the activities of some enzymes between rhizosphere soil of transgenic tobacco expressing human serum albumin and of nonmodified tobacco.

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By contrast, other studies have reported significant effects of Bt corn on microbial community structure in soil. Root exudates of Bt corn (event Bt176) significantly reduced presymbiotic hyphal growth of the arbuscular mycorrhizal fungus Glomus mosseae compared with root exudates of another Bt corn hybrid (event Bt11) and non-Bt corn hybrids (Turrini et al., 2004). Castaldini et al. (2005) reported changes in total and metabolically active 16S rRNA fractions of culturable rhizosphere bacteria by DGGE between Bt corn expressing the Cry1Ab protein and near-isogenic non-Bt corn and a significantly lower level of G. mosseae in roots of Bt corn. Another approach for evaluating the effects of transgenic plants on microorganisms is to monitor microbe-mediated processes rather than population levels or taxonomic groups. Because soil enzymes are important for the growth of microorganisms (e.g., decomposition of organic residues, cycling of nutrients, formation of organic matter and soil structure), they are an indicator of overall microbial activity in soil (Dick, 1994). The present study showed no consistent significant differences in the activities of some representative enzymes involved in the biodegradation of plant biomass between soils with Bt corn or their near-isogenic non-Bt corn counterparts during the 4 yr. These results were consistent with those of Flores et al. (2005), who reported that the activities of these enzymes were not consistently different in soil amended or unamended with biomass of Bt or non-Bt corn. Similarly, Shen et al. (2006) found no consistent significant differences in the activities of some enzymes (urease, alkaline phosphatases, dehydrogenases, phenol oxidase, and proteases) between soils of Bt and non-Bt cotton. In contrast, Wu et al. (2004a,b) found increased activities in soil of phosphatases and dehydrogenases, as well as increased methanogenesis, after the addition of transgenic Bt-rice straw to flooded soil. Sun et al. (2007) also found that the addition of leaves and stems of two Bt cotton varieties to soil stimulated the activities of soil urease, acid phosphomonoesterase, invertase, and cellulase, whereas the activity of soil arylsulfatase was inhibited.

Effects of Plant Variety Plant variety had a significant, albeit transient, effect on the numbers of microorganisms and the activities of enzymes in the present study. In general, soils with Bt and non-Bt sweet corn varieties (Attr. GSS-0966 and Prime Plus, respectively) had significantly higher numbers of microorganisms and higher enzyme activities than soils with the Bt and non-Bt varieties of field corn (Novartis 45-A6 and Novartis 45-T6; Pioneer 38A25 and Pioneer 38A24) as determined by ANOVA. These differences between varieties (sweet and field) were temporary and transient. Plants can alter the composition and diversity of soil microbial communities in a selective manner (Nehl et al., 1997). The type of microbial community that results from plant-selective pressure differs with plant species (e.g., Germida et al., 1998; Grayston et al., 1998; Smalla et al., 2001), indicating that plant type and their root exudates influence the microorganisms that colonize their rhizosphere. Donegan et al. (1995) found that the Cry1Ab and Cry1Ac proteins, both purified and expressed in transgenic plants, did not have a direct effect on soil microorganisms and that the effects observed, which


were related to the plant varieties used, may have been caused by unexpected changes in plant characteristics that resulted from genetic manipulation or tissue culturing of the engineered plants. Rengel et al. (1998) suggested that the selective effect of plants on the rhizosphere community could occur even at the cultivar level. Siciliano et al. (1998) and Siciliano and Germida (1999) reported that the composition of the root-associated microbial community differed between transgenic and nontransgenic canola cultivars, as determined by CLPP and fatty acid methyl ester analyses. Field and greenhouse trials with corn expressing the Cry1Ab protein showed that changes in microbial and microfaunal (protozoa and nematodes) communities due to the Bt trait were small and less than changes that resulted from different non-Bt corn cultivars, different crops, soil type, and stage of plant growth (Griffiths et al., 2005, 2006). Naef et al. (2006) reported no direct effect of the Cry1Ab protein in corn residues on the pathogen Fusarium graminearum and on the biocontrol agent Trichoderma atroviride and showed that some Bt hybrids and their near-isogenic non-Bt corn counterparts differed more in the chemical composition of the corn tissue as a result of different environmental conditions (e.g., drought-stress) than from the Cry protein content alone, which could affect the saprophytic growth of fungi on crop residues. Saxena and Stotzky (2001a) found a higher lignin content in 10 Bt corn hybrids, representing three different transformation events (Bt11, MON810, 176), than in their respective non-Bt isolines. High lignin content decreases the rate of decomposition of crop residues (Parton et al., 1996; Hopkins et al., 2001) and therefore may influence the residue-associated microbial population. Soil bacterial communities are usually influenced more by plant species and cultivars than by other environmental factors, such as soil type and agricultural practices (e.g., Gomes et al., 2001; Heuer et al., 2002).

Seasonal and other Non-Bt Effects The numbers of microorganisms and the activity of some enzymes differed significantly with season, as determined by SPRM-ANOVA, probably as the result of differences in the water content of the soils, ambient temperatures, and stage of growth of the plants at the times of sampling. These seasonal differences were independent of the presence of the Cry proteins in the plants and, subsequently, in the soils. Although there were some statistically significant interactions between plant varieties and season, they were temporary, transient, and varied among the 4 yr with no discernable trends. These results were in agreement with other studies that also showed that the effects of transgenic plants on microbial communities in soil are subject to seasonal variations. For example, Lottmann et al. (1999, 2000) reported that transgenic potato expressing T4 lysozyme influenced the composition of root-associated bacterial antagonists; however, this was dependent on the year and the time of sampling. The rhizosphere microbial community associated with two transgenic potato varieties that produced the lectins Galanthus nivalis agglutinin and concanavalin A had different CLPP than the nontrangenic variety, but the profiles were also subject to seasonal variation, and the differences did not persist from one season to the next (Griffiths et al., 2000). Heuer et al. (2002) found significant differences between the community struc-

ture of the rhizosphere of transgenic and nontransgenic potatoes that were dependent on environmental factors but independent of the expression of T4 lysozyme by the transgenic plants. Dunfield and Germida (2003) found significant seasonal differences in the microbial community associated with the rhizosphere of a transgenic and a nontransgenic variety of canola, but these differences did not persist into the next season. The bacterial community structure in the rhizosphere of Bt corn expressing Cry1Ab protein was less affected by the protein than by other environmental factors, such as the age of the plant or heterogeneities in the field (Baumgarte and Tebbe, 2005). Fang et al. (2005) reported that bacterial diversity in the rhizosphere of transgenic and nontransgenic corn was affected more by soil texture than by cultivation of transgenic varieties. Denaturing gradient gel electrophoresis analysis on the same gel of 16S rDNA fragments amplified from DNA extracted from soils of Bt and non-Bt corn enabled a careful comparison of the microbial communities in these soils. The similarity of the DGGE band profiles indicated that there was no difference in the bacterial communities between the soils of Bt and non-Bt corn. However, there were differences in bacterial patterns between years, indicating that the bacterial communities in these soils were influenced more by season than by the presence of the Cry proteins. These results confirmed those obtained from microbial plate counts, which also showed no consistent differences in the numbers of culturable microbes between soils with Bt or non-Bt corn but showed differences in bacterial patterns with season.

Persistence of Cry Proteins in Soil The Cry1Ab protein was detected in most soils with Bt corn expressing the cry1Ab gene during the 4 yr, whereas the Cry3Bb1 protein was not detected in soils of Bt corn expressing the cry3Bb1 gene, confirming other studies (e.g., Ahmad et al., 2005; Icoz and Stotzky, 2007) that reported that the Cry3Bb1 protein does not persist as long as the Cry1Ab protein in soil. Ahmad et al. (2005) found no detectable Cry3Bb1 protein in soil planted with Bt corn and its nearisogenic non-Bt corn for three consecutive seasons under field conditions in Manhattan, KS, and concluded that the Cry3Bb1 protein released in root exudates or from decaying plant residues does not persist and is rapidly broken down in soil. Several laboratory studies in soil microcosms reported that the proteins from Bt corn, Bt cotton, and Bt potato (Cry1Ab or Cry3Bb1, Cry1Ac, and Cry3Aa protein, respectively) did not persist and were generally degraded with a half-life of 20 d or less (Ream et al., 1994; Palm et al., 1996; Sims and Holden, 1996; Icoz and Stotzky, 2007). Sims and Holden (1996) reported a half-life (50% dissipation time) of 1.6 d and 15 d for 90% dissipation in the insecticidal activity of Cry1Ab protein in soil and suggested that the protein in corn plant tissue would be unstable under field conditions and likely to degrade rapidly under normal cultivation practices. Similarly, Wang et al. (2006) reported that the Cry1Ab protein from biomass of Bt rice degraded with a half-life of 11.5 d in alkaline soil and with a half-life of 34.3 d in acidic soil. No Cry3Bb1 protein was detected after 21 d in montmorillo-


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nite-amended and after 40 d in kaolinite-amended soil microcosms to which different amounts of Bt corn residues (event MON863) had been added (Icoz and Stotzky, 2007). Some field studies on the persistence of Cry proteins released by transgenic plants were in good agreement with laboratory studies and also showed that Cry proteins do not persist and degrade rapidly in soil, although a small fraction may be protected from biodegradation in plant matrix or bound on surface-active particles. For example, Head et al. (2002) found no detectable levels of Cry1Ac protein by ELISA and insect bioassay in soils collected from fields of Bt cotton that had been grown and the biomass incorporated into soil for 3 to 6 consecutive years. Hopkins and Gregorich (2003) reported that much of the Bt δ-endotoxin in corn residues is highly labile and quickly decomposes in soils in the field but that a small fraction may be protected from degradation in relatively recalcitrant residues. Dubelman et al. (2005) found no evidence of persistence or accumulation of Cry1Ab protein in soils from fields planted for at least 3 consecutive years with Bt corn. In contrast, Tapp and Stotzky (1995a,b, 1998) showed that the insecticidal activity of purified Cry proteins was retained in soil as the result of their adsorption and binding on clay particles, which protected the proteins from microbial degradation for at least 234 d. The Cry2A protein from Bt cotton was detectable after 120 d in the field (Sims and Ream, 1997). Similarly, Saxena and Stotzky (2002) and Stotzky (2002, 2004) reported that the Cry1Ab protein released in root exudates and from biomass of Bt corn persisted in soil microcosms for at least 180 d and 3 yr, respectively. Zwahlen et al. (2003a) found that the Cry1Ab protein in plant residues was stable and degraded only as the plant material degraded. In the present study, the Cry1Ab protein was detected in most soils, with Bt corn expressing the cry1Ab gene during the 4 consecutive years of corn cultivation. Persistence apparently differs between soil in the field and in laboratory microcosms, and these differences in persistence and in rates of degradation of Cry proteins and organic residues, in general, are probably the result of differences in plant species, Cry protein, soil type, pH, microbial activity, temperature, and method used. Moreover, the binding of Cry proteins on clays and humic acids reduced their availability to microbes, which is probably responsible for the persistence of the proteins in soil (Koskella and Stotzky, 1997; Crecchio and Stotzky, 1998, 2001; Lee et al., 2003; Stotzky, 2004; Fiorito et al., 2007).

Conclusions Based on published data (see review by Icoz and Stotzky [2008]) and on the results of the present study, the effects of transgenic plants on microbial communities in soil seem to depend more on the species, variety, and age of the plant and on environmental factors than on the transgenes and their products. The Cry1Ab and Cry3Bb1 proteins in root exudates and plant residues of Bt corn had no consistent significant effects on a spectrum of microorganisms and their enzymatic activities in soil over 4 consecutive years of corn cultivation. The occasional significant differences observed in the numbers of some microbial groups and in the activities of some enzymes between soils with Bt and non-Bt corn were not consistent and did not persist.

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Acknowledgments These studies were supported, in part, by grant 2003-35107-13776 from the U.S. Dep. of Agriculture (USDA) and grant N5238 from the New York Univ. Research Challenge Fund (NYURCF). The opinions expressed herein are not necessarily those of the USDA and the NYURCF. We thank Dawn M. Kochanek, Nok Chhun, Jason Chan, Jennie Zhu, Dr. Nidhi Sabharwal, Dr. Yhiong Li, and Yao Ge for their help in the laboratory and Shannon McCrindle, Tim Stodola, Keegan Hasselmann, Matthew Korthauer, Yang Hu, and numerous undergraduate assistants for their help in the field.

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