Soil microbial properties in Bt (Bacillus thuringiensis) corn cropping systems

Applied Soil Ecology 63 (2013) 127–133

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Soil microbial properties in Bt (Bacillus thuringiensis) corn cropping systems Newton Z. Lupwayi ∗ , Robert E. Blackshaw Agriculture & Agri-Food Canada, Lethbridge Research Centre, Box 3000, Lethbridge, Alberta, T1J 4B1 Canada

a r t i c l e

i n f o

Article history: Received 23 May 2012 Received in revised form 20 August 2012 Accepted 7 September 2012 Keywords: Crop rotation Genetically modified (GM) crops Soil biological quality Soil microbiology

a b s t r a c t Growing Bt crops reduces the use of insecticides applied to them, but these crops could affect soil microorganisms and their activities. We evaluated the effects of Bt (Cry1Ab) corn (Zea mays L.) and deltamethrin ([S]-␣-cyno-3-phenoxybenzyl [1R, 3R]-3-[2,2-dibromovinyl]-2,2-dimethylcyclopropane-1-carboxylate) insecticide application on soil microbial biomass C (MBC), ␤-glucosidase enzyme activity (final season only), bacterial functional diversity, and bacterial community-level physiological profiles (CLPPs) in corn monoculture in five seasons. We also determined if growing Bt corn in crop rotation would alter these effects. Statistical analysis of pooled data across seasons did not show any effects of Bt technology, insecticide application or crop rotation on soil microbial biomass or diversity even though differences between seasons and between the rhizosphere and bulk soil were observed. Annual analyses of results also showed that neither the Bt technology nor insecticide application affected soil MBC, enzyme activity, or functional diversity of bacteria in corn rhizosphere, but shifts in bacterial CLPPs due to Bt trait were observed in one year. Crop rotation effects on soil microbial properties were not observed in most cases. Where effects were observed, Bt corn grown in rotation resulted in greater MBC, enzyme activity and functional diversity than Bt corn grown in monoculture or conventional corn grown in rotation, and these effects were observed only in bulk soil. Therefore, the Bt technology is safe with respect to the non-target effects measured in this study. However, the effects of repeated use of Bt crops over many years on the soil environment should continue to be monitored. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.

1. Introduction Bacillus thuringiensis (Bt) is a soil bacterium that produces insecticidal crystal (Cry) proteins (toxins). The genes that encode Bt toxins have been engineered into many crops as inherent bioinsecticides, and these crops are called Bt crops (e.g., Bt corn). Use of such genetically modified (GM) Bt crops reduces the use of insecticides applied to them and presents an opportunity to replace them. However, growing Bt crops could have non-target effects on soil microorganisms and their activities due to the presence of Cry proteins in soil (Saxena et al., 1999), alteration of the amounts and composition of root exudates (Lynch et al., 2004), horizontal transfer of transgenic DNA and the associated antibiotic genes that are used as markers (Ma et al., 2011), and alteration of the biochemical composition of decomposing crop residues (Fang et al., 2007). The insecticidal Cry toxins released from root exudates and biomass of Bt crops have been shown accumulate in soil (Saxena and Stotzky, 2001; Saxena et al., 1999, 2002), but the duration of their persistence is variable. However, these toxins usually do not affect soil microorganisms (Saxena and Stotzky, 2001; Griffiths et al., 2007; Icoz et al., 2008) or their processes (Cortet et al., 2006;

∗ Corresponding author. Tel.: +1 403 317 3315. E-mail address: [email protected] (N.Z. Lupwayi).

Lawhorn et al., 2009) although some negative effects were reported after four years of Bt cotton (Gossypium hirsutum L.) monoculture (Chen et al., 2011). The neomycin phosphotranferase (nptII) gene, an antibiotic-resistance gene commonly used as a marker in GM plants, was detected in soil throughout the season in Bt (Cry3Bb1) corn grown in monoculture for three years (Zhu et al., 2010). However, there was no evidence of transfer of this gene to soil bacteria (Ma et al., 2011). Residues of Bt corn have been shown to have higher lignin concentrations (Saxena and Stotzky, 2001) or C/N ratios (Flores et al., 2005) than conventional corn although it is not always the case (Tarkalson et al., 2008; Yanni et al., 2011). But in most studies (reviewed by Yanni et al., 2010), the rate and extent of decomposition of Bt crop residues is not different from that of non-Bt crop residues. The studies reviewed above explain why most investigations on the effects of Bt cropping systems on soil microorganisms have reported no effects (Oliveira et al., 2008; Liu et al., 2008), minor and transient effects (Devare et al., 2007), or inconsistent effects (Griffiths et al., 2005). When assessing the effects of Bt technology on the soil environment, it is important to weigh the effects of the Bt trait vs. the effects of applying the insecticides (the best management practice) that the Bt technology replaces, i.e., to check if the effects of using a Bt crop are worse than those of applying insecticides to a non-Bt crop. The experimental designs of most studies do not allow for this comparison. The few studies that

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Table 1 Treatments and assignment of contrasts. Treatment

1. Btb corn 2. Conventional corn, insecticide appliedc 3. Conventional corn, no insecticide applied 4. Bt corn, Rotd 1 (GfRe canola – GRf corn – GfR canola – Bt corn) 5. Bt corn, Rot 2 (GR canola – GR corn – GR canola – Bt corn) 6. Conventional corn, Rot 1 (Canola – Corn – Canola – Corn) 7. Conventional corn, Rot 2 (Canola – Corn – Canola – Corn)

Contrasta A

B

C

D

1 −1 0 0 0 0 0

0 1 −1 0 0 0 0

0 0 0 1 1 −1 −1

−2 0 0 1 1 0 0

a Constrasts: (A) Bt corn vs. conventional corn with insecticide application, i.e., Bt technology vs. current best management practice. (B) Insecticide application vs. no insecticide application. (C) Bt corn in rotation vs. conventional corn in rotation and (D) Bt corn in rotation vs. Bt corn in monoculture. b Bt, Bacillus thurigiensis. c The insecticide applied was deltamethrin at 15 g a.i. per hectare. d Rot, rotation. e GfR, glufosinate ammonium resistant. f GR, glyphosate resistant.

have compared the effects of the two factors on soil microorganisms have reported no significant effects of either the Bt crop or insecticide application (Griffiths et al., 2006; Devare et al., 2004, 2007; Liu et al., 2008). In a greenhouse study with Bt (Cry1Ab) corn, Griffiths et al. (2006) found no Bt trait or deltamethrin insecticide (against European corn borer – Ostrinia nubilalis H.) effects on soil microbial community structures measured as phospholipid fatty acid (PLFA) profiles or community-level physiological profiles (CLPPs). In a field trial, Devare et al. (2004) found similar results for Bt (Cry3Bb) corn and tefluthrin [2,3,5,6-tetrafluoro4-methylbenzyle (Z)-(1RS)-cis-3-(2-chloro-3,3,3-trifluoroprop-1enyl)-2,2-dimethylcyclopropanecarboxylate] insecticide (against corn rootworm – Diabrotica spp.) effects on soil microbial community structures as determined by terminal restriction length polymorphism (T-RFLP). In an extension of the same study, Devare et al. (2007) also found no effects of Bt corn or tefluthrin insecticide on soil microbial biomass and activity. In a field trial with Bt (Cry1Ab) rice, Liu et al. (2008) found no effects of the Bt trait or application of triazophos [3-(o,o-dimethyl)-1phenyl thiophosphryl-1,2,4-triazol] insecticide (a broad-spectrum organo-phosphate insecticide and acaricide with some nematicidal properties) on soil enzyme activities and microbial composition determined by denaturing-gradient gel electrophoresis (DGGE) or T-RLFP. In all these studies, other factors like soil type and time of year (seasonal variation) affected soil microbial properties more than the Bt trait or insecticide application. Since growers normally grow these crops in rotation with other crops rather than in monoculture, the crop rotation effect also needs to be evaluated to ensure that soil biological quality is maintained in cropping systems with Bt crops. The effects of Bt corn on soil microbiological properties have not been conducted in the soils, environmental conditions and management systems and practices of western Canada. In this paper, we (a) evaluate the separate effects of Bt corn and deltamethrin insecticide application on soil MBC, activity of ␤-glucosidase enzyme, functional diversity and CLPPs, and (b) determine if growing Bt corn in rotation with other crops would alter these effects.

contained 20 g organic C, 370 g sand, 300 g silt, and 330 g clay kg−1 in the top 15 cm. Seven of 19 treatments were selected for the study (Table 1). The other treatments were on glyphosateresistant corn, the results of which have already been published (Lupwayi and Blackshaw, 2012). In the first treatment, Bt corn (variety Dekalb DKC26-82) was grown in monoculture without applying insecticide. In Treatments 2 and 3, conventional corn (variety DKC26-75) was grown in monoculture with or without deltamethrin insecticide application at 15 g a.i. per hectare. The aim of these three treatments was to compare Bt trait effects with the effects of applying the insecticide that the Bt technology replaces. Corn was grown in monoculture in these treatments to increase the selection pressure exhibited by the Bt trait to increase the chances of detecting potential treatment differences within a short time. In the last four treatments, Bt corn was grown in rotation with glufosinate (2-amino-4-(hydroxymethylphosphinyl)butanoic acid) ammonium-resistant (GfR) canola (variety Invigor 5020) and glyphosate (N-(phosphonomethyl)glycine)-resistant (GR) canola (variety Pioneer 45H21) (Treatments 4 and 5), and equivalent rotations consisting of conventional crops (canola variety Pioneer 46H02 and corn variety DKC26-75) were included as controls (Treatments 6 and 7). The difference between Treatments 6 and 7 was the phase of the rotation in which the corn was sampled (underlined in Table 1). The 4-year rotations of these treatments represent the way farmers may grow these crops. All phases of the rotations were present each year. The treatments were replicated four times in a randomized complete block design (RCBD), and each experimental plot was 15 m wide and 35 m long. Prior to seeding, weeds were controlled by disc cultivation in all plots except GR corn and GR canola, which were sprayed with glyphosate. All corn was seeded at 75,000 plants ha−1 in rows 75 cm apart in mid to late May and irrigated as needed during the season. Further details about field operations can be obtained from Floate et al. (2007) and Bourassa et al. (2010). Monthly rainfall and air temperature were recorded during each season (Fig. 1).

2.2. Soil sampling 2. Materials and methods 2.1. Treatments This study was conducted from 2002 to 2007 on a dark brown Chernozem (Typic Haplustoll) soil at Lethbridge, Alberta, Canada (49◦ 41 N, 112◦ 40 W) in a field trial that was conducted from 2000 to 2007 examining environmental impacts of GM corn and canola (Brassica napus L.). The soil had a pH of 8.0 and

From 2002 to 2007 (except 2005), soil samples were collected at the tasselling stage of corn, usually in midsummer (July). Samples were not collected in 2005 because the plots were flooded due to excessive rainfall. Soils were sampled once per season because earlier studies had shown that treatment (tillage and crop rotation) differences in soil microbial biomass, diversity and activity were similar throughout the season, but were clearest in July (Lupwayi et al., 1998, 1999). Corn plants were excavated from four random 0.5-m lengths of row, i.e., four samples per plot. Loose soil was

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Table 2 Analysis of variance results for all factors from 2002 to 2007 (except 2005, when data were not collected due to flooding). Sampling location refers to rhizosphere or bulk soil. Factor

Treatment (A) Sampling location (B) A×B Year (C) A×C B×C A×B×C

Probability Microbial biomass C

Shannon index of diversity (H )

0.0937 0.0000 0.6634 0.0000 0.9671 0.0000 0.9925

0.3695 0.0000 0.2780 0.0000 0.2947 0.0167 0.3704

Multi-Variate Statistical Package (MVSP) software (Kovach, 1999): H  = −˙pi (ln pi ) Fig. 1. Monthly rainfall and mean air temperatures during the corn growing seasons from 2002 to 2007, except in 2005 because plots were flooded. Soil samples for microbiological analysis were taken in July each year.

where pi is the ratio of activity (i.e., optical density reading) on the ith substrate to the sum of activities on all 31 substrates. 2.4. Statistical analysis

removed from the roots and the remaining soil that was strongly adhered to the roots was recovered as rhizosphere soil. Bulk soil (0–7.5 cm depth) was sampled from the middle of two adjacent crop rows at four locations per plot. The four bulk or rhizosphere soil samples from each plot were combined, passed through a 2-mm sieve and stored at 4 ◦ C until analysis.

2.3. Soil analysis Soil MBC was measured using the substrate-induced respiration method (Horwath and Paul, 1994), in which 300 mg of glucose was dissolved in 4.5–6.0 mL water and added to 50 g soil to bring it to 50% water-holding capacity. The exact amount of water added depended on the pre-determined water content and water-holding capacity of the soil. After stir-mixing, the soil was incubated in a 1 L jar for 3 h at 22 ◦ C and the amount of CO2 that accumulated in the head space was measured by gas chromatography. In 2007 samples, the activity of ␤-glucosidase enzyme was measured by colorimetrically determining p-nitrophenol released by the enzyme after incubating 1 g soil with buffered (pH 6.0) pnitrophenyl-␤-d-glucoside (Dick et al., 1996). Community-level physiological profiles of soil bacteria were evaluated using the Biolog® method (Zak et al., 1994), which tests the ability of a bacterial community to utilize different C substrates contained in a microplate (Eco-plate® ) (Biolog Inc., Hayward, California). The procedure was adapted by colorimetrically standardizing inoculum densities in 1 g soil samples to about 103 cells mL−1 . Aliquots of 150 ␮L of the soil suspension were added to Biolog Ecoplate® microplates containing 31 substrates and a water control (Insam, 1997). The plates were incubated at 28 ◦ C without shaking. Optical densities in the wells were read with an enzyme-linked immunosorbent assay (ELISA) plate reader at 590 nm after 48 h of incubation. Preliminary tests showed that treatment differences in substrate utilization profiles read every 12 h up to 96 h of incubation were similar throughout the incubation period, but were clearest at 48 h. The optical density readings were corrected for the water controls in subsequent analyses. Negative readings after the correction were adjusted to zero. On the basis of CLPPs, functional diversity was evaluated by calculating Shannon index (H ) following the method of Zak et al. (1994), using

A combined analysis of variance (ANOVA) using Repeated Measures in RCBD was used to examine the effects of all factors (treatment, sampling location, i.e., rhizosphere or bulk soil, and year) on MBC, ␤-glucosidase enzyme activity, and H . In addition, these microbial data in the rhizosphere or bulk soil in each year were subjected to analysis of variance according to the RCBD of the trial. These separate ANOVAs were done because the main objective of the trial was to compare the effects of Bt trait, deltamethrin insecticide, and crop rotation on soil microbial characteristics in different environments (years and sampling locations). In all cases, differences were considered significant at 0.05 probability level and means were separated by the least significant difference (LSD) test. Groups of treatments were also compared using four orthogonal contrasts (Table 1): Contrast A: Bt corn vs. conventional corn with insecticide application, i.e., Bt corn technology vs. current best practice. Contrast B: Insecticide application vs. no insecticide application. Contrast C: Bt corn in rotation vs. conventional corn in rotation. Contrast D: Bt corn in rotation vs. Bt corn in monoculture. To compare the physiological structures of soil bacteria, principal component analysis was used to classify treatments according to their CLPPs (Pielou, 1984) using MVSP software (Kovach, 1999). A covariance matrix of the substrate utilization data was used in PCA analysis. After classification of treatments with PCA, the substrates that accounted for differences between classes of treatments in substrate utilization were identified by correlating principal component scores with optical density readings for individual substrates. These key substrates are described in the text because biplot figures were not easy to read due to congestion of data points and vector lines. 3. Results 3.1. Soil microbial biomass C (MBC) In ANOVA of all factors, there were no treatment effects on MBC (Table 2). However, the effects of sampling location (rhizosphere vs. bulk soil), year and the interaction of these two factors were significant. Corn rhizosphere had more MBC than

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Table 3 Microbial biomass C (MBC) in the bulk soil of Bt (Bacillus thurigiensis) corn and conventional corn plots from 2002 to 2007. Data were not collected in 2005 due to flooding. Treatment

MBC (mg kg−1 soil) 2002

2003

2004

2006

2007

Mean

1. Bt corn 2. Conventional corn, insecticide 3. Conventional corn, no insecticide 4. Bt corn, Rotation 1 5. Bt corn, Rotation 2 6. Conventional corn, Rotation 1 7. Conventional corn, Rotation 2 Standard error (mean)

526a 591a 633a 637a 629a 724a 580a 59.8

398a 424a 373a 488a 418a 493a 414a 52.1

515a 563a 567a 625a 705a 666a 572a 43.5

480d 534cd 568bcd 721ab 672abc 750a 583bcd 51.6

623bc 618bc 569c 714ab 728ab 845a 709b 44.1

508a 546a 542a 637a 630a 695a 571a 41.4

Contrasts A. Bt corn vs. Conventional corn B. Insecticide vs. No insecticide C. Bt corn in rotation vs. Conventional corn in rotation D. Bt corn in rotation vs. Bt corn in monoculture

NSa NS NS NS

NS NS NS NS

NS NS

NS NS

NS NS

*,+

** , +

NS

NS

NS NS NS NS

** , +

NS

Means followed by the same letter in a column are not significantly different at 5% significance level. a NS, Not significant at 5% level. * Significant at 5% level. ** Significant at 1% level. + The treatment on the first side of the comparison had greater MBC than the treatment on the second side.

bulk soil (761 vs. 590 mg kg−1 soil), and this pattern existed every year except 2002. The 2002 season had more than 2× normal rainfall in June, the month just before soil samples were taken in July (Fig. 1). The annual MBC contents were in the order: 2007 > 2006 > 2004 > 2002 > 2003 (903, 750, 674, 589, and 463 mg kg−1 soil, in that order). The 2007 season was the hottest of all seasons in July, when soils were sampled (Fig. 1). In separate ANOVAs each year in corn rhizosphere, no treatment effects on MBC were observed (rhizosphere data not presented). Contrast analysis did not show significant differences either. The grand MBC means in corn rhizosphere were 560 mg kg−1 soil in 2002, 496 mg kg−1 soil in 2003, 746 mg kg−1 soil in 2004, 885 mg kg−1 soil in 2006, and 1119 mg kg−1 soil in 2007. In bulk soil in 2006, conventional corn grown in rotation with GfR canola and GR canola (Rotation 1) had numerically the greatest MBC, and the least MBC was observed in Bt corn grown in monoculture (Table 3). In 2007, conventional corn grown in Rotation 1 also had numerically the greatest MBC, but conventional corn grown in monoculture without insecticide application had the least MBC. Contrast analysis in 2004, 2006 and 2004–2007 showed that Bt corn grown in rotation had greater MBC than conventional corn grown in rotation (Table 3, Contrast C).

In separate ANOVAs each year in corn rhizosphere, no treatment effects were found, and contrast analysis also showed no differences, in any year (rhizosphere data not presented). The grand means of H in corn rhizosphere were 2.83 in 2002, 2.42 in 2003, 2.70 in 2004, 2.61 in 2006, and 2.70 in 2007. In bulk soil, H was greatest for Bt corn grown in Rotation 1 and conventional corn grown in Rotation 1, and it was lowest in Bt corn grown in monoculture in 2002 (Table 5). Contrast analysis in that year also showed greater H in Bt corn grown in rotation than Bt corn in monoculture (Contrast D). 3.4. Functional community structures of soil bacteria Principal component analysis of soil bacterial CLPPs in different treatments usually did not show treatment effects on bacterial community structures, but some patterns were observed. The effect of Bt trait was observed in 2003 in corn rhizosphere, where the Table 4 ␤-Glucosidase enzyme activity in the rhizosphere and bulk soil of Bt (Bacillus thurigiensis) corn and conventional (conv.) corn 2007. Enzyme activity was measured only in 2007. Treatment

3.2. ˇ-Glucosidase enzyme activity ␤-Glucosidase enzyme activity was determined only in 2007. In corn rhizosphere, neither ANOVA nor contrast analysis revealed significant treatment differences in enzyme activity (Table 4). In bulk soil, the highest enzyme activity was observed in conventional corn grown in Rotation 1, and the lowest in conventional corn grown in monoculture without insecticide. Contrast analysis showed that Bt corn grown in rotation had greater enzyme activity than Bt corn grown in monoculture (Contrast D). 3.3. Functional diversity of soil bacteria In ANOVA of all factors, there were no treatment effects on the functional diversity of soil bacteria (Shannon index, H ) (Table 2). Only the effects of sampling location (rhizosphere vs. bulk soil) and its interaction with year were significant. Corn rhizosphere had greater H (2.65) than bulk soil (2.13), and this was a consistent pattern in all years although it was less pronounced in 2006 than in other years.

1. Bt corn 2. Conventional corn, insecticide 3. Conventional corn, no insecticide 4. Bt corn, Rotation 1 5. Bt corn, Rotation 2 6. Conventional corn, Rotation 1 7. Conventional corn, Rotation 2 Standard error (mean) Contrasts A. Bt corn vs. conventional corn B. Insecticide vs. no insecticide C. Bt corn in rotation vs. conventional corn in rotation D. Bt corn in rotation vs. Bt corn in monoculture

␤-Glucosidase enzyme activity (mg nitrophenol kg−1 soil−1 h−1 ) Rhizosphere

Bulk soil

625a 568a 595a 663a 647a 619a 632a 45.5

786 cd 847bcd 716d 1061ab 1017abc 1175a 1062ab 79.5

NSa NS NS

NS NS NS

NS

*,+

Means followed by the same letter in a column are not significantly different at 5% significance level. a NS, not significant at 5% level. * Significant at 5% level. + The treatment on the first side of the comparison had greater enzyme activity than the treatment on the second side.

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Table 5 Functional diversity (Shannon index, H ) of bacteria in the bulk soil of Bt (Bacillus thurigiensis) corn and conventional (conv.) corn plots from 2002 to 2007. Data were not collected in 2005 due to flooding. Treatment

H 2002

2003

2004

2006

2007

Mean

1. Bt corn 2. Conventional corn, insecticide 3. Conventional corn, no insecticide 4. Bt corn, Rotation 1 5. Bt corn, Rotation 2 6. Conventional corn, Rotation 1 7. Conventional corn, Rotation 2 Standard error (mean)

1.55c 1.98bc 2.00bc 2.58a 2.29ab 2.58a 2.39ab 0.156

1.71a 1.92a 1.96a 2.03a 2.12a 2.08a 1.47a 0.364

2.35a 1.80a 2.28a 2.03a 2.33a 2.40a 2.05a 0.197

2.49a 2.61a 2.55a 2.21a 2.14a 2.23a 2.44a 0.182

1.84a 1.83a 1.90a 2.35a 2.14a 2.05a 2.09a 0.170

1.99a 2.03a 2.14a 2.74a 2.20a 2.27a 2.09a 0.094

Contrasts A. Bt corn vs. conventional corn B. Insecticide vs. no insecticide C. Bt corn in rotation vs. conventional corn in rotation D. Bt corn in rotation vs. Bt corn in monoculture

NSa NS NS

NS NS NS NS

NS NS NS NS

NS NS NS NS

NS NS NS NS

NS NS NS NS

** , +

Means followed by the same letter in a column are not significantly different at 5% significance level. a NS, Not significant at 5% level. ** Significant at 1% level. + The treatment on the first side of the comparison had greater functional diversity than the treatment on the second side.

bacterial community structures in the three treatments where Bt corn was grown were different from the community structures in the other four treatments where conventional corn was grown (separation along PC 1, left to right, Fig. 2a). In the Biolog assay, the soil bacterial communities where Bt corn had been grown utilized more d-cellobiose (a carbohydrate) but less substrates like Tween 40 and Tween 80 (both carbohydrates) than bacterial communities where conventional corn had been grown (biplots not shown). Similar, but less pronounced, grouping of bacterial community structures with respect to Bt corn was also observed in 2007 in corn bulk soil (separation along PC2, top–bottom, Fig. 2b). In this case, soil bacterial communities where Bt corn had been grown utilized more d-galacturonic acid (a carboxylic acid) but less substrates like N-acetyl-d-glucosamine (a carbohydrate) and l-phenylalanine (an amino acid) than bacterial communities where conventional corn had been grown. Fig. 2b also shows that bacterial communities in the bulk soil of Bt corn grown in Rotations 1 and 2 had different functional structures from those in the other treatments (separation along PC1). 4. Discussion

bacteria were observed in Bt corn rhizosphere in 2003 and, to a lesser extent, in bulk soil in 2007. Fang et al. (2007) also observed similar shifts after soil-incorporation of Bt corn residues with higher lignin concentration (12%) and lignin/N ratio (9.9/1) compared to the non-Bt near-isoline (10% lignin and 8.6/1 lignin/N ratio). 4.2. Insecticide effects Application of deltamethrin had no effects on the measured soil microbiological characteristics, either in corn rhizosphere or bulk soil. Other studies have also reported no or stimulatory effects of this insecticide on soil microorganisms (Germida et al., 1987; Vig et al., 2008; Munoz-Leoz et al., 2009) although an inhibitory effect on soil respiration was observed under anaerobic conditions in a wetland soil (Munoz-Leoz et al., 2009). Griffiths et al. (2006) also found no effects of Bt corn or deltamethrin in a study that was designed similarly to our study to compare the effects of Bt trait with those of the insecticide that would otherwise be applied. Similar results have been obtained when other insecticides have been used, e.g., tefluthrin (Devare et al., 2004, 2007) and triazophos (Liu et al., 2008).

4.1. Bt trait effects 4.3. Crop rotation effects One way that growing GM plants can affect soil microorganisms is the possible release of transgenic DNA into soil, where it could be acquired by indigenous bacterial cells. In Bt (Cry3Bb1) corn grown in monoculture for three years in Ontario, Canada, Zhu et al. (2010) detected the neomycin phosphotranferase (nptII) gene, an antibiotic-resistance gene commonly used as a marker in GM plants, in soil throughout the year. However, there was no evidence of transfer of this gene to soil bacteria (Ma et al., 2011). The insecticidal Cry toxins released from root exudates and biomass of Bt crops have been shown to accumulate in soil (Saxena and Stotzky, 2001; Saxena et al., 1999, 2002). However, these toxins do not seem to affect soil microorganisms (Saxena and Stotzky, 2001; Griffiths et al., 2007; Icoz et al., 2008) or their processes (Cortet et al., 2006; Lawhorn et al., 2009). Such observations may explain why, in our study, the Bt (Cry1Ab) trait had no effects on MBC, ␤glucosidase enzyme activity, or bacterial functional diversity in any year when Bt corn was grown in monoculture. Similar results, or small and inconsistent effects, have also been reported in other field studies (Fang et al., 2005; Griffiths et al., 2005; Oliveira et al., 2008). However, shifts in the functional community structures of

In crop rotations, Bt corn did not affect soil MBC, ␤-glucosidase enzyme activity or functional diversity in the majority of cases. This is not a surprising result considering that neither the Bt trait nor application of deltamethrin insecticide affected these soil microbial properties. When crop rotation effects were significant, growing GR corn in rotation always increased MBC, ␤-glucosidase enzyme activity and functional diversity in bulk soil. Therefore, if there had been any negative effects of growing Bt corn on biological soil quality, they would have been mitigated by growing the crops in rotations. Crop rotations would have this effect by reducing the frequency at which GM (or any other) crops are grown on the same piece of land in a given time period and, therefore, reducing the selection pressure exhibited by the GM (or any other) technology. Considering that the beneficial effects of crop rotations occurred only in bulk soil, they were probably related to crop residues rather than to root exudates. Thus, crop rotations probably increased soil microbial diversity by providing soil microorganisms with a diverse suite of substrates from different crop residues. ␤-Glucosidase is an important enzyme in decomposition of organic compounds in soil,

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a

0.19 0.16

Bt-Rot1-03R

0.12 Conv-Rot1-03R

PC2 (28%)

0.08

Conv-NoI-03R

0.04 Conv-I-03R

0.00 -0.04

Conv-Rot2-03R

-0.08 Bt-NoI-03R Bt-Rot2-03R

-0.12 -0.16 -0.19 -0.19

-0.16

-0.12

-0.08

-0.04

0.00

0.04

0.08

0.12

0.16

0.19

PC1 (37%)

b

0.15

Conv-I-07NR

0.12

PC2 (26%)

0.09 0.06

Conv-Rot1-07NR

0.03 0.00 Conv-Rot2-07NR

-0.03

Bt-Rot2-07NR

Conv-NoI-07NR Bt-Rot1-07NR

-0.06

Bt-NoI-07NR

-0.09 -0.12 -0.12

-0.09

-0.06

-0.03

0.00

0.03

0.06

0.09

0.12

0.15

PC1 (42%) Fig. 2. Ordination by principal component analysis (PCA) of community-level physiological profiles (CLPPs) of soil bacteria in different treatments in corn rhizosphere in 2003 (a) and in bulk soil in 2007 (b). The percentage of total variance explained by each axis is shown. The first part of treatment labels indicates corn type (Conv, conventional corn; Bt, Bt corn). The middle part of the label indicates other experimental treatments of the corn (Rot1, grown in Rotation 1; Rot2, grown in Rotation 2; I, insecticide applied in monoculture; noI, no insecticide applied in monoculture). The last part of the label indicates the sampling year (03 = 2003, 07 = 2007) and sampling location (NR, bulk soil; R, rhizosphere). The main Biolog substrates whose utilization accounted for the separation of treatments are listed in the text. Each data point is a mean of 4 replicates.

and these results mean that the Bt technology did not have negative effects on the capacity of the soil to break down soil organic materials (including crop residues) and release the nutrients that they contained. Many other studies, reviewed by Lynch et al. (2004), Icoz and Stotzky (2008) and Mocali (2010), have shown that other factors associated with growing Bt crops have greater effects on soil microorganisms than the Bt technology itself. These factors include conservation tillage, soil type and crop rotations. 5. Conclusions The overall statistical analysis of results across years did not show any treatment (Bt technology, insecticide application or crop rotation) effects on soil microbial biomass or diversity even though differences between sampling location (rhizosphere vs. bulk soil)

and years were observed. Annual analyses of results also showed that neither the Bt technology nor insecticide application affected soil MBC, ␤-glucosidase enzyme activity, or functional diversity of bacteria in corn rhizosphere, but shifts in the functional structures of soil bacteria associated with the Bt trait were observed in one year. The crop rotation effect on the measured soil microbial properties was also not observed in most cases. Where effects were observed, Bt corn grown in crop rotation resulted in greater MBC, enzyme activity or functional diversity than Bt corn grown in monoculture or conventional corn grown rotation, and these effects were observed only in bulk soil. Therefore, the Bt technology is safe with respect to the non-target effects measured in this study. However, the effects of repeated use of Bt crops over many years on the soil environment should continue to be monitored.

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