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Neither transgenic Bt maize (MON863) nor teuthrin insecticide adversely affect soil microbial activity or biomass: A 3-year eld analysis
M. Devare1, L.M. Londono-R, J.E. Thies
Department of Crop and Soil Sciences, Cornell University, 719 Bradeld Hall, Ithaca, NY 14853, USA Received 1 August 2006; received in revised form 1 March 2007; accepted 8 March 2007

Abstract Laboratory and greenhouse studies on transgenic Bacillus thuringiensis (Bt) maize have drawn attention to the persistence and activity of the Cry proteins in soil and their potential effects on soil microorganisms, but there have been few eld assessments that evaluate the effects of Bt maize with those of insecticides on soil microbial populations. This study was conducted to determine the effects of Cry3Bb Bt maize with those of the insecticide teuthrin on soil microbial biomass and activity in the eld over a 3-year cropping cycle. The recently commercialized maize variety YieldGards Rootworm (MON863), which produces the Cry3Bb protein, was grown along with a non-Bt isoline with and without teuthrin applied at planting. Microbial biomass, nitrogen (N) mineralization potential, short-term nitrication rate, and respiration rate were measured in rhizosphere and bulk soil samples collected from three replicate eld plots just before planting, at anthesis, and at harvest in each year. There were clear seasonal effects on microbial biomass and activity in the eld soilsas represented by the consistent changes in all measured variables across years and sampling times. Differences in the measured variables were also sometimes observed between bulk and rhizosphere soil. However, there were no adverse effects of either the Bt or non-Bt maize with insecticide applied compared to the non-Bt controls; on the contrary, microbial biomass and soil respiration data suggested a stimulatory effect of the Bt genotype, particularly in comparison to the non-Bt isoline. Although higher does not necessarily mean better, the higher microbial biomass and respiration rates observed in the Bt and insecticide-applied soils compared to non-Bt soils does allay concerns that either the Bt protein or the teuthrin typically used to control the corn rootworm reduce microbial biomass or its respiratory activity in eld soils. Similarly, the higher N mineralization potential and nitrication rates observed in some soil samples from the Bt and teuthrin-treated plots indicate higher activity of N-mineralizing microorganisms, a potentially positive consequence as both ammonium and nitrate are effective N sources for maize during grain lling. Our data suggest that cropping MON863 Bt maize is unlikely to adversely affect soil ecology in the short term. Longer-term monitoring of transgenic cropping systems should assure that the biotic functioning of the soil is maintained as a part of studies on overall ecosystem integrity. r 2007 Elsevier Ltd. All rights reserved.
Keywords: Bt corn; Bt maize; Cry3Bb; Microbial biomass; MON863; N mineralization potential; Short-term nitrication; Soil microbial community; Soil respiration; Transgenic crops

1. Introduction The world market for transgenic crops was estimated to be US$ 6.15 billion in 2006, and is likely to increase to US$ 25 billion by 2010 (James, 1999, 2006). The global area planted to these crops increased more than 60-fold between 1996 and 2006, to about 102 million ha, with plants
Corresponding author. Tel.: +1 607 255 5099; fax: +1 607 255 8615.

E-mail address: jet25@cornell.edu (J.E. Thies). Current address: Bioinformatics and Life Sciences Specialist, Mann Library, Cornell University, Ithaca, NY 14853, USA.
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engineered to express Cry insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) representing about 19% of these crops. An additional 13% of transgenic plants have stacked genes, which confer resistance to multiple insects or are combined with herbicide resistance. These crops were the fastest growing trait group between 2005 and 2006 at 30% growth (James, 2006). Krattiger (1997) estimated that Bt technology products could save farmers about US$ 2.7 billion of the roughly $ 8 billion spent annually on insecticides worldwide. The potential for increased yields and reduced use of insecticides through the planting of insecticidal Bt crops is promising, but

0038-0717/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2007.03.004 Please cite this article as: Devare, M., et al., Neither transgenic Bt maize (MON863) nor teuthrin insecticide adversely affect soil microbial activity or biomass: A 3-year eld analysis, Soil Biology & Biochemistry (2007), doi:10.1016/j.soilbio.2007.03.004

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worldwide public opinion is still reserved regarding their acceptability. The greatest constraints to adoption appear to be the uncertain risks to human and environmental health these crops may pose, even compared to known risks associated with the insecticides normally used to contain pest damage. There are several reports on the effects of Bt technology on certain aspects of environmental health, such as the potential for adverse affects on non-target insects, particularly on benecial arthropods. However, there have been very few studies where the affects of the Bt technology on soil microorganisms or soil ecology have been evaluated and, no studies that have compared the effects in the eld of transgenic Bt maize against those of insecticides commonly used to control the corn rootworm. Since we rely on microbial activity for a myriad of ecosystem services, such as organic matter decomposition and nutrient cycling, changes in the biomass, activity, or diversity of these communities have the potential to impact ecosystem level function. A eld-based risk assessment comparing the effects of Bt maize (hereafter referred to as corn) and a non-transgenic corn isoline grown with and without insecticide is particularly important because farmers in the US will generally rely on insecticides to control the corn rootworm where Bt maize is not grown (Gray et al., 1993; Gray, 2000). The literature contains conicting reports on the effects of transgenic crops on non-target soil microorganisms. Donegan et al. (1995) reported a transient stimulation of soil bacterial and fungal populations by two of three Bt cotton lines. These lines also produced changes in bacterial communities, as measured by Biolog analysis and DNA ngerprinting. Recent eld studies with other transgenic crops have also demonstrated changes in the diversity of soil microbial communities (Di Giovanni et al., 1999; Donegan et al., 1999). However, many reports contend that climatic and edaphic factors are far more important in their inuence on activity or composition of microbial communities than changes due to the presence of transgenic plants (Lukow et al., 2000; Gyam et al., 2002; Blackwood and Buyer, 2004; Wu et al., 2004). Other research has demonstrated that although transgenic crops may produce changes in community composition or physiological proles of the soil microbial community, these effects are either transient in nature or insufcient to affect soil functioning (Grifths et al., 2000; Cowgill et al., 2002). Stotzky (1999) and Flores et al. (2005) reported that CO2 evolved from soil amended with ground Bt (Cry1Ab) plant biomass from six different crop species was signicantly lower than that from soil amended with unmodied, nearisogenic plant biomass. While no mechanism by which the presence of the Cry1Ab protein in plant biomass might depress soil respiratory activity was suggested by these authors, it is unlikely to be due to toxicity of the protein to microorganisms, although trophic level-mediated effects may be a possibility. However, it is more likely that genotypic differences related to the various hybrids may be

affecting the degradability of their residues. Saxena and Stotzky (2001) observed a signicantly higher lignin content in Bt corn expressing Cry1Ab than in the non-Bt isoline, but there was no difference in lignin content between the other ve Bt crops they examined and their non-transgenic counterparts. Despite their data on soil respiration and Cry protein persistence and activity, these authors reported that there was no effect of Cry1Ab Bt corn on culturable bacteria or fungi, or numbers of protozoa or nematodes in laboratory trials. Similarly, Blackwood and Buyer (2004) demonstrated that PLFA and Biolog proles of microbial populations in soils sampled from Bt and non-Bt corn plots that were incubated in a growth chamber were not signicantly different. These authors also showed that community structure of the b-Proteobacteria, as assessed by paramagnetic beadenabled T-RFLP, did not vary between treatments. Reports on the effects of teuthrin, the pyrethroid insecticide used in this study, on soil microbiota are scarce, although there seems to be little doubt that a variety of synthetic pyrethrins can be metabolized by bacteria in pure or mixed cultures (Maloney et al., 1988, 1993; Topp and Akhtar, 1990, 1991; Maloney et al., 1997; Grant et al., 2002; Liu et al., 2004) or by microorganisms in soil (Sakata et al., 1992). Data on persistence of the insecticide indicate that it has a half-life of 17 d at 30 1C (24 d at 20 1C), and is not expected to affect soil microora at normal application rates (Tomlin, 1997). In their work with two other pyrethroid insecticides, Taiwo and Oso (1997) reported that microbial populations were signicantly reduced after an initial rise. Carbon (C), nitrogen (N) and potassium (K) contents, phosphorus (P) solubilization, and pH were also reduced in insecticide-treated soil compared to controls. In contrast, several studies have demonstrated that eld application rates of numerous synthetic pyrethrins had little or no effect on soil enzyme activities, bacterial growth, or protozoan survival (Rangaswamy et al., 1994; Antonious, 2003; Boucard et al., 2004). Similarly, a laboratory study with teuthrin also demonstrated that soil bacterial and fungal populations initially depressed by applying the insecticide recovered after 2 weeks, as did nitrication activity (Tu, 1980). Sulfur (S) oxidation and soil respiration were also not affected in the Tu (1980) study. We conducted this study to determine the comparative effects of transgenic Bt corn versus use of the insecticide teuthrin on soil microbial ecology and nutrient dynamics in the eld over a 3-year cropping cycle. The recently commercialized corn variety, MON863, which produces the Cry3Bb protein, and its non-transgenic isoline with and without insecticide applied were grown. Microbial biomass, N mineralization potential, short-term nitrication potential, and respiratory activity of soil biota in the rhizosphere and bulk soil were measured three times over each crop cycle. We hypothesized that these microbial indicators may be adversely affected, not necessarily by direct toxicity of the Cry3Bb protein released by plant roots and decaying plant tissue, but by trophic level

Please cite this article as: Devare, M., et al., Neither transgenic Bt maize (MON863) nor teuthrin insecticide adversely affect soil microbial activity or biomass: A 3-year eld analysis, Soil Biology & Biochemistry (2007), doi:10.1016/j.soilbio.2007.03.004

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interactions or by other hybrid differences, such as root exudation patterns or the relative proportions of structural C compounds in the corn biomass. 2. Materials and methods 2.1. Study site

A portion of bulk and rhizosphere soil samples were separated after sieving and immediately frozen at 20 1C and the remainder stored at 4 1C for microbial biomass and activity measurements, which were begun within a day of sampling. Rainfall and temperature data were obtained from the on-farm weather station. 2.2. Microbial biomass

A eld trial was established in May 2001, in a farmers eld that had been previously cropped to alfalfa adjacent to Cornell Universitys Homer C. Thompson Vegetable Farm in Freeville, NY. The eld soil is a Howard gravelly loam (mesic Glossic Hapludalf; 45.4% sand, 42.2% silt, 12.4% clay; 5.4% organic matter, pH 6.9, 10 mg kg1 available nitrate). Corn ears were harvested and residues chopped and returned to plots after harvest in each year. Trial plots were replanted to the same treatments in 2002 and 2003. Crop treatments consisted of four replicated plots of transgenic Bt corn resistant to the corn rootworm (Bt; YieldGards Rootworm, MON863), and three replicated plots each of the non-transgenic isoline (non-Bt) without and with (non-Bt+I) the insecticide teuthrin (Force G; Dow Elanco, St. Louis, MO) applied to the soil at planting. MON863 Bt corn has been shown to express 49 86 mg protein g1 fresh weight of grain, 3093 mg protein g1 fresh weight of pollen, and 3.266 mg protein g1 fresh weight of roots. Average above-ground production of the protein in the whole plant was 1354 mg g1 (United States Environmental Protection Agency, 2003). All plots were sown with seed treated with the fungicide Captan (Drexel Chemical Co., Memphis, TN) and the insecticide Gaucho (Bayer AG, Germany). Gaucho protects seeds from a variety of insect pests including wireworms, seed corn maggot, and re ants, and protects seedlings from damage by ea beetles and white grubs. All seeds were supplied by Monsanto Co. (St. Louis, MO). Seeds were planted at 76 cm spacing into 50 m 46 m plots. Soil samples from each plot were taken three times during each of the three growing seasons, at planting (May 2022), anthesis (August 2021), and harvest (November 1525). A total of 10 bulk and 10 rhizosphere soil samples to 15 cm depth were taken along two 68 m transects across each plot. These samples were composited into one bulk and one rhizosphere soil sample per plot. Bulb planters were used to collect samples in 2001; soil was immediately sieved (2 mm) and roots with adhering soil were removed from this bulk sample and sieved again (1 mm) to separate rhizosphere soil from the roots. This method did not yield sufcient rhizosphere soil for all measurements, and soil samples collected at anthesis in 2002 were too dry to permit separation of roots from bulk soil, so an alternative method was employed to collect rhizosphere soil in 2002 and 2003. Whole plants were uprooted and soil that fell off the root mass when gently shaken was considered to be bulk soil. Rhizosphere soil was that which adhered tightly to the root mass and could be dislodged only by knocking the root mass several times against a collection bucket.

Microbial biomass N was determined by the chloroform fumigationextraction method using 10 g oven dry weight (ODW) equivalent of soil and 40 ml of 0.5 M K2SO4 (Brookes et al., 1985; Vance et al., 1987) followed by spectrophotometric measurement of ninhydrin-positive compounds in the K2SO4 extracts at 520 nm, and biomass N used to estimate biomass C (Joergensen and Brookes, 1990). Biomass C was calculated using the empirical relationship: biomass C Bnin 20.6, where Bnin Nnin extracted from fumigated soilNnin extracted from unfumigated soil. 2.3. Microbial activity Nitrogen mineralization potential and soil respiration rate were measured as indicators of microbial activity. Nitrogen mineralization potential was assessed through the component processes of ammonication potential and short-term nitrication rate. Ammonication potential was measured as described by Waring and Bremner (1964). Briey, 5.0 g ODW equivalent of soil was weighed into 20 ml screw-cap test tubes, 12.5 ml distilled water was added so that the soil would be waterlogged and most of the N would be mineralized as NH+. Test tubes were 4 incubated at 40 1C for 7 d, while replicate test tubes were incubated at 4 1C. After incubation, the contents of each tube were washed into round-bottom distillation asks with 2 N KCl and about 0.2 g of MgO added to the asks prior to steam distillation. Distillate was collected in 25 ml Erlenmeyer asks with 5 ml of boric acid indicator added, and the distillate titrated against 0.005 M H2SO4 to estimate NH+-N in the samples (Keeney and Nelson, 4 1982). The short-term nitrication rate was determined according to the method of Berg and Rosswall (1985) using 5 g ODW equivalent of each soil sample and an incubation time of 5 h at 25 1C. Control soil samples were incubated in the freezer at 20 1C. This method employs NaOCl3 to inhibit the oxidation of nitrite to nitrate. The resulting NO-N produced in each sample was determined spectro2 photometrically, measuring colour intensity at 520 nm using a reagent made by dissolving sulphanilamide and napthyl ethylenediamine dihydrochloride in water and adding phosphoric acid. Soil respiration rate was measured using 20 g ODW equivalent of soil weighed into 250 ml Ball canning jars (Isermeyer, 1952; Anderson, 1982). Carbon dioxide (CO2) evolved from the soil was trapped in vials containing 10 ml

Please cite this article as: Devare, M., et al., Neither transgenic Bt maize (MON863) nor teuthrin insecticide adversely affect soil microbial activity or biomass: A 3-year eld analysis, Soil Biology & Biochemistry (2007), doi:10.1016/j.soilbio.2007.03.004

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of 0.5 M NaOH placed in the jars; these vials were sampled every 710 d and 10 ml of 0.5 M NaOH was replaced in the trap after each measurement. The CO2 in the sampled NaOH was precipitated using 8 ml of 0.5 M BaCl2 and the amount of remaining NaOH determined by titration against 0.5 M standard HCl. Cumulative evolved CO2 measurements were plotted and the slopes of the resultant curves used to compare treatments. 2.4. Data analysis Analysis of variance was conducted on microbial biomass and activity data using the general linear model (GLM) procedure and Tukeys pairwise comparison of the means in MINITAB Statistical Software (v. 13.1). All results are reported at p 0.05. 3. Results Average daily rainfall (mm) and air temperature (1C) starting at the time of the pre-plant soil sampling and ending at harvest soil sampling for each year of the study are presented in Fig. 1. The time of anthesis sampling for each year is indicated by an arrow in each graph. These data show that although there were four rain episodes in the 6 days preceding anthesis in 2002, the period prior to that was signicantly drier than in either of the other years. Ten days preceding sampling in 2002, the corn had rolled,
120 100 80 60 40 20 0 120 Rainfall (mm) 100 80 60 40 20 0 120 100 80 60 40 20 0 140 160 180 200 220 240 260 Julian Day 280 300 320 Anthesis 10 0 -10 Anthesis 10 0 -10 40 30 20 Anthesis 10 0 -10 40 Air Temperature (C) 30 20 2001 40 30 20

almost-erect leaves, indicating the plants were under severe water stress. 3.1. Microbial biomass Microbial biomass measured in bulk and rhizosphere soils for all samples is presented in Fig. 2, while the data trends across seasons and years are shown in Fig. 5. There was signicant variation from one year to the next, and to a lesser extent, from one sampling point to another within a growing season. No signicant differences were observed between bulk and rhizosphere samples, or between the Bt and two non-Bt treatments, with the exception of samples from the non-Bt plots at harvest in 2001 and 2003. Microbial biomass in non-Bt samples at harvest was 19% lower than in non-Bt+I samples in 2001, and 33% lower than in Bt and non-Bt+I samples in 2003. Biomass C was signicantly higher at all sample points in 2001 compared to other study years, and at harvest in 2003 compared to 2002. Microbial biomass prior to planting was 26% higher than at anthesis and 19% higher than at harvest in 2001.

900 800 700 600 500 400 c 300 Biomass C (g C g-1 soil) 200 100 0 900 800 700 600 c 500 400 300 200 100 0 PP Anth Harvest 2001 PP Anth Harvest 2002 PP Anth Harvest 2003 fff c c a Bulk soil a b b d dc c c c ee ce g c c cg ee e h c d dd aa b b b a cc cc Rhizosphere Bt NoBt NoBt + I a a c a

2002

2003

Fig. 1. Rainfall and air temperature in the eld plots during the growing season in 2001, 2002, and 2003. Each graph begins on the day of the preplant soil sampling, and ends at the harvest sampling.

Fig. 2. Microbial biomass carbon in Bt, non-Bt, and non-Bt+I plots measured in rhizosphere and bulk soil at pre-plant, anthesis, and harvest in 2001, 2002, and 2003. Error bars represent standard error of the mean. Different letters associated with values indicate signicant differences at (p 0.05).

Please cite this article as: Devare, M., et al., Neither transgenic Bt maize (MON863) nor teuthrin insecticide adversely affect soil microbial activity or biomass: A 3-year eld analysis, Soil Biology & Biochemistry (2007), doi:10.1016/j.soilbio.2007.03.004

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Microbial biomass in 2002 pre-plant and anthesis samples did not differ, but did drop precipitously to about a fourth of this biomass in harvest samples. Microbial biomass was comparable for all sample points in 2003, with the exception of the non-Bt harvest samples, which were signicantly lower than the pre-plant and anthesis samples. 3.2. N mineralization potential Nitrogen mineralization potential was between 20% and 23% higher in rhizosphere soil than in bulk soil for the 2002 and 2003 samples (Fig. 3, trends in Fig. 5). The only signicant crop treatment effects were observed at harvest in 2003, when the non-Bt soil exhibited 18% and 20% lower N mineralization potential than Bt and non-Bt+I bulk and rhizosphere soil, respectively. A greater effect of sampling time was noted; N mineralization potential was lower in 2003 pre-plant samples compared to pre-plant samples in other years. Yearly differences also existed for the harvest samples, where N mineralization potential was about a third higher
100 90 80 70 60 50 N mineralization potential (g N g-1 soil) 40 30 20 10 0 100 90 80 70 60 50 40 30 aa 20 10 0 PP Anth Harvest 2001 PP Anth Harvest 2002 PP Anth Harvest 2003 a b ab a ccc b b b cc c Bulk soil Bt NoBt NoBt + I d d e f b b a e d a b ab a a bb bb b d d d Rhizosphere c c

in bulk and rhizosphere harvest soil in 2002 than 2003 and there was an 83% increase in N mineralization potential in 2002 compared to 2001 bulk harvest samples. With the exception of the 2001 samples, harvest samples exhibited signicantly higher N mineralization potential than anthesis or pre-plant samples, and N mineralization potential in all anthesis samples was slightly higher than in pre-plant samples. With the exception of the 2001 samples, harvest samples exhibited signicantly higher N mineralization potential than anthesis or pre-plant samples, and anthesis samples in all 3 years showed greater N mineralization potential than pre-plant samples. 3.3. Short-term nitrication rate Short-term nitrication rate in rhizosphere soil was 38% lower than in bulk soil in 2002, but did not differ signicantly in 2003 between sampling locations (Fig. 4 and trends in Fig. 5). There was a crop treatment effect similar to that observed for the microbial biomass and N mineralization measurements: nitrication rate in non-Bt pre-plant samples was 20% and 15% lower than in Bt samples for 2002 and 2003, respectively.
2.5 2.25 2 1.75 1.5 1.25 Nitrification rate (g NO2 g-1 soil d-1) 1 0.75 0.5 0.25 0 2.5 2.25 2 1.75 1.5 1.25 1 0.75 0.5 0.25 0 PP Anth Harvest 2001 PP Anth Harvest 2002 PP Anth Harvest 2003 b bb gg g dd d hh h Bulk soil a b e e f c aa a b bb c cc b b b NoBt + I Rhizosphere Bt NoBt

a a a

Fig. 3. Nitrogen mineralization potential in Bt, non-Bt, and non-Bt+I plots measured in rhizosphere and bulk soil at pre-plant, anthesis, and harvest in 2001, 2002, and 2003. Error bars represent standard error of the mean. Different letters associated with values indicate signicant differences at (p 0.05).

Fig. 4. Short-term nitrication rate in Bt, non-Bt, and non-Bt+I plots measured in rhizosphere and bulk soil at pre-plant, anthesis, and harvest in 2002 and 2003. Error bars represent standard error of the mean. Different letters associated with values indicate signicant differences at (p 0.05).

Please cite this article as: Devare, M., et al., Neither transgenic Bt maize (MON863) nor teuthrin insecticide adversely affect soil microbial activity or biomass: A 3-year eld analysis, Soil Biology & Biochemistry (2007), doi:10.1016/j.soilbio.2007.03.004

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1000 Rhizosphere 800 600 N mineralization potential (g N g-1 soil) 2001 400 Biomass (g C g-1 soil) 200 2002 0 1000 Bulk soil 800 600 400 200 2002 0 Pre-plant Anthesis Year 2001 2002 2003 Harvest Bt NonBt 2001 2003 2003

100 Rhizosphere 80 60 40 20 0 100 Bulk soil 80 2002 60 40 20 2001 0 Pre-plant NonBt+ I Anthesis Harvest 2003 2002

2.5 Rhizosphere 2 1.5 Nitrification rate (g NO2 g-1 soil d-1) 1 0.5 0 2.5 Bulk soil 2 1.5 2002 1 2003 0.5 0 2003 2002

2003

Pre-plant

Anthesis

Harvest

Fig. 5. Trends in microbial biomass, N mineralization potential and short-term nitrication rate in Bt, non-Bt, and non-Bt+I plots measured in rhizosphere and bulk soil at pre-plant, anthesis, and harvest in 2001, 2002, and 2003.

Consistent with the other measurements, signicant effects of sampling time were observed. Nitrication rate did not differ between treatments at anthesis, but was signicantly higher at pre-plant and harvest in 2002 compared with 2003 soil samples from all eld plots. Ammonium in pre-plant and harvest bulk soil from 2002 was converted to NO2 at 88% and 56% faster than at anthesis, respectively, and 83% and 33% faster in anthesis samples taken in 2003. 3.4. Soil respiration rate The cumulative soil respiration rate was always signicantly higher in rhizosphere than in bulk soil (Fig. 6). Bt corn cropping did not reduce soil respiration; on the contrary, respiration measured in Bt plots was signicantly higher in the 2002 and 2003 bulk soil samples at harvest compared to the non-Bt samples, and in the 2002 rhizosphere anthesis soil compared to the non-Bt+I soil. Respiration rate at pre-plant and at anthesis in bulk and rhizosphere soil was highest in 2003; however, it was markedly higher at harvest in 2003 compared with any other time in the study. Respiration rate at harvest was always greater than at anthesis or before planting.

Although no differences were observed between pre-plant and anthesis bulk soil in 2002, the rate of soil respiration in the following years anthesis samples was signicantly higher than at the pre-plant sample point. 4. Discussion The debate surrounding the use of transgenic crops has been highly polarizedwith proponents and opponents often arguing their cases very emotionally, publicly, and without the backing of robust, long-term eld research data. When we began our studies, there were few published reports on the effects of Bt corn on non-target soil microorganisms. There were some data, however, on the effects of other transgenic crops on bacterial and fungal community proles, and on protozoa and nematodes (Donegan et al., 1995, 1999; Di Giovanni et al., 1999; Grifths et al., 2000; Lukow et al., 2000). We evaluated Monsantos Cry3Bb (Yieldgards Rootworm, MON863) corn under typical eld conditions, within large plots and in comparison to a non-transgenic isoline with and without the use of a common anti-corn rootworm insecticide (teuthrin). Soil microbial activity and biomass in the eld plots were assessed as indicators of the potential for

Please cite this article as: Devare, M., et al., Neither transgenic Bt maize (MON863) nor teuthrin insecticide adversely affect soil microbial activity or biomass: A 3-year eld analysis, Soil Biology & Biochemistry (2007), doi:10.1016/j.soilbio.2007.03.004

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1400 1200 1000 800 600 400 200 0 Cumulative respiration (g Cg-1 soil) 1400

2001

2002 1200 1000 800 600 400 200 0 1400 2003 1200 1000 800 600 400 200 0 0 7 14 28 35 42 49 Time (d) 57 65 82

Bulk soil Pre-plant Anthesis Harvest

Bt NonBt +I Rhizosphere Anthesis Harvest

Fig. 6. Cumulative soil respiration rate from pre-plant, anthesis, and harvest samples in 2001, 2002, and 2003. Solid and dotted lines indicate respiration in bulk and rhizosphere soil, respectively. Error bars represent standard errors of the means.

transgenic Bt corn to affect the ecosystem level processes of nutrient cycling and organic matter decomposition. None of the variables we measured were adversely affected in either Bt or non-Bt+I plots compared to nonBt controls; on the contrary, the data sometimes indicated a stimulatory effect of Cry3Bb Bt corn, particularly in comparison to the non-Bt crop treatment, in which measurements were often signicantly lower than in both of the other treatments (Figs. 25). While it is unlikely that the Cry3Bb protein itself stimulated microbial activity, it is

possible that other genotypic differences between MON863 and the non-Bt isoline tested have resulted in inter-hybrid differences in root exudation, as has been suggested by Siciliano et al. (1998), Di Giovanni et al. (1999), and Donegan et al. (1999). Our eld-based results are consistent with greenhouse and laboratory experiments conducted with Cry1Ab Bt corn without insecticide applied by Blackwood and Buyer (2004). Although they did not measure the same variables as reported here, their study indicated that the transgenic corn had no discernable effect

Please cite this article as: Devare, M., et al., Neither transgenic Bt maize (MON863) nor teuthrin insecticide adversely affect soil microbial activity or biomass: A 3-year eld analysis, Soil Biology & Biochemistry (2007), doi:10.1016/j.soilbio.2007.03.004

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on the bacterial community. Further, research conducted by Wu et al. (2004) on Bt rice (Cry1A) in China suggested that Bt rice had no negative effect on a range of soil microbial indicators. In contrast, Duneld and Germida (2001, 2003) reported that bacterial communities associated with the roots of one of four transgenic canola lines tested were distinct from those of non-transgenic varieties, but noted that the effect of plant variety on root-associated microbial communities could be substantially modied by the effect of eld site location. Of more interest are the observations that indicate microbial biomass and N mineralization potential in several samples were signicantly lower in the non-Bt plots than in the other treatments (Figs. 2 and 3). Several authors have assessed the effects of low intensity of nematode infestation and grazing on white clover roots on microbial biomass and activity, and reported a stimulatory effect of such below-ground herbivory on root exudation and microbial biomass and activity (Yeates et al., 1998; Denton et al., 1999; Yeates et al., 1999). Based on these reports, we anticipated that microbial biomass and activity might be elevated in the non-Bt plots, but this was infrequently observed. Moderate to high levels of corn rootworm infestation were observed in the eld plots (L. Allee and J. Losey, pers. comm.). All authors of the herbivory studies cited previously specied that infestation levels were low in sampled soils and the reported stimulatory effect on microbial biomass and activity may not hold true once infestation and grazing pressure increase beyond a given threshold. We did observe some stimulatory rhizosphere effects on measured variables as compared to bulk soil. It is no surprise that N mineralization potential was higher in the rhizosphere compared with bulk soil, as the amount of mineralizable material is almost certainly higher, given the presence of root exudates, a highly labile organic matter input. The nding that the rate of nitrication was lower in the rhizosphere than in bulk soil at least in 1 of the 2 years measured may reect reduced oxygen availability near the root surface due to higher respiratory activity. Higher soil respiration rates were observed in the rhizosphere, despite the fact that microbial biomass C did not differ between rhizosphere and bulk soil (Fig. 6). These ndings are in keeping with previous work demonstrating that increases in plant root exudation result in increased microbial activity and the selection of specic microbial communities (Swinnen, 1994; Lemanceau et al., 1995; Latour et al., 1996; Vance and Chapin, 2001; Falchini et al., 2003). Vance and Chapin (2001) reported further that microbial respiration in a taiga forest soil showed a stronger response to substrate addition than did microbial biomass. Thus, increased activity does not necessarily translate into increases in population density, as was observed in our study. There were clear seasonal effects on microbial biomass and activity in our eld plotsas represented by the consistent changes in all measured variables across years

and sampling times. Microbial biomass in all samples was higher in 2001 than any other year of the study (Figs. 2 and 5). Biomass C was highest at pre-plant in this year, and lowest at harvest in 2002. The high biomass values we observed during our rst study year might be explained, in part, by the fact that the eld had been cropped to alfalfa in the previous year. There are many studies that have reported the stimulatory effects of various legumes on microbial biomass (Mazzarino et al., 1991; Haynes and Beare, 1997; Spehn et al., 2000; Salamon et al., 2004). Biological and environmental variables are important determinants of ecosystem processes as they strongly affect elemental cycles. Drought-mediated decreases in microbial biomass C have been reported (Willson et al., 2001; Li et al., 2004), similar to the large decrease in microbial biomass from pre-plant and anthesis to harvest in 2002, which we postulate was a result of the droughty conditions in the days leading up to anthesis in that year (Fig. 1). Average rainfall and temperature and soil moisture measurements at harvest for all 3 years are similar, making it unlikely that microbial populations at the harvest 2002 sample point were adversely affected by soil moisture or temperature. The high N mineralization potential measured in the harvest 2002 and 2003 samples likely reects a larger amount of mineralizable material present in soil after the senescence of the corn root system. In contrast to N mineralization potential, short-term nitrication rate at anthesis was extremely low in both 2002 and 2003 (Figs. 4 and 5). Short-term nitrication rate was always signicantly higher in pre-plant and harvest samples than at anthesis (Figs. 3 and 5), indicating a temporal effect of the sort that has been previously reported by Wheatley et al. (1991, 1997, 2003). This is likely due to the presence of actively growing corn plants at anthesis, which compete for available NH4-N, and the absence of plants at the preplant sampling point. For the same reason, nitrication, as well as soil respiration at harvest, is also likely to be higher than at anthesis, since at harvest plant uptake of NH+ has 4 ceased as the plants have senesced. Clearly, caution must be exercised in interpreting results from experiments such as these; higher does not necessarily mean better. However, the higher microbial biomass and soil respiration rates observed in Bt and insecticide-applied plots compared to non-Bt plots does reduce concerns that either the Bt protein or the teuthrin used to control the corn rootworm in its absence may adversely affect microbial biomass. Similarly, the higher N mineralization potential and short-term nitrication rates observed in some soil samples from the Bt and teuthrin treatments indicate higher populations of N-mineralizing microorganisms, a potentially positive consequence as both ammonium and nitrate are effective N sources for corn during the grain lling period. In conclusion, our 3-year comparative eld assessment of Cry3Bb Bt corn vs non-Bt corn grown with and without the insecticide teuthrin demonstrates that neither the Bt corn nor the insecticide had adverse effects on microbial

Please cite this article as: Devare, M., et al., Neither transgenic Bt maize (MON863) nor teuthrin insecticide adversely affect soil microbial activity or biomass: A 3-year eld analysis, Soil Biology & Biochemistry (2007), doi:10.1016/j.soilbio.2007.03.004

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biomass, N mineralization potential, or nitrication and respiration rates. Rhizosphere effects and seasonal changes in the variables measured were consistently observed throughout the study period, indicating that their inuence on microbial biomass and activity is likely to outweigh any more subtle effects due to crop treatment. While issues of gene ow and economicsparticularly in developing countriesmay continue to be of concern for Bt corn, this transgenic technology does not appear to adversely impact soil microbial activity or abundance. Acknowledgments We gratefully acknowledge Chris Jones, Daniel Clune, Steven Culman, Allison Hornor, Brendan ONeill, John Reilly, and Angelika Rumberger for eld and laboratory assistance at various times over the 3 years of this eld study. This work was supported by grants from Cornell University Agricultural Experiment Station Federal Formula Funds, the Cooperative State Research, Education and Extension Service (CSREES) of the USDA, the USDA CSREES Program on Biotechnology Risk Assessment, and the USDA Agriculture Ecosystems Program. Any opinions, ndings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reect the view of the USDA. References
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Please cite this article as: Devare, M., et al., Neither transgenic Bt maize (MON863) nor teuthrin insecticide adversely affect soil microbial activity or biomass: A 3-year eld analysis, Soil Biology & Biochemistry (2007), doi:10.1016/j.soilbio.2007.03.004