Glyphosate Resistance Technology Has Minimal or No Effect on

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Agricultural and Environmental Chemistry

Glyphosate Resistance Technology Has Minimal or No Effect on Maize Mineral Content and Yield Krishna N Reddy, James V. Cizdziel, Martin Williams, Jude Maul, Agnes M. Rimando, and Stephen O. Duke J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01655 • Publication Date (Web): 11 Sep 2018 Downloaded from http://pubs.acs.org on September 14, 2018

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Journal of Agricultural and Food Chemistry

Reddy et al. - 1

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Glyphosate Resistance Technology Has Minimal or No Effect on Maize

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Mineral Content and Yield

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Krishna N. Reddy,1 James V. Cizdziel,2 Martin M. Williams, II,3 Jude E. Maul,4 Agnes M.

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Rimando5 and Stephen O. Duke,*5

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38776, United States

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2

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University, Mississippi 38677, United States

Crop Production Systems Research Unit, USDA-ARS, P.O. Box 350, Stoneville, Mississippi

Department of Chemistry and Biochemistry, University of Mississippi, 222 Coulter Hall,

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3

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Illinois 61801, United States

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4

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Maryland 20705, United States

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5

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Mississippi 38677, United States

Global Change and Photosynthesis Research unit, USDA-ARS, 1102 S. Goodwin Ave., Urbana,

Sustainable Agricultural Systems Laboratory, USDA-ARS, 10300 Baltimore Ave., Beltsville,

Natural Products Utilization Research Unit, USDA-ARS, P.O. Box 1848, University,

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ACS Paragon Plus Environment


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Reddy et al. - 2 17

ABSTRACT

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Controversy continues to exist regarding whether the transgene for glyphosate resistance (GR)

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and/or glyphosate applied to GR crops adversely affect plant mineral content. Field studies were

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conducted in 2013 and 2014 in Stoneville, MS and Urbana, IL to examine this issue. At each

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location, the experiment was conducted in fields with no history of glyphosate application and

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fields with several years of glyphosate use preceding the study. Neither glyphosate nor the GR

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transgene affected yield or mineral content of leaves or seed, except for occasional (< 5%)

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significant effects that were inconsistent across minerals, treatments, and environments.

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Glyphosate and AMPA (aminomethylphosphonic acid), a main degradation product of

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glyphosate, were found in leaves from treated plants, but little or no glyphosate and no AMPA

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was found in maize seeds. These results show that the GR transgene and glyphosate application,

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whether used for a single year or several years, have no deleterious effect on mineral nutrition or

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yield of GR maize.

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KEYWORDS: aminomethylphosphonic acid, glyphosate, glyphosate resistance, mineral content,

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transgenic crop, Zea mays

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Reddy et al. - 3 34

INTRODUCTION

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Worldwide, glyphosate (N-(phosphonomethyl)glycine) is used more than any other herbicide,1

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due in large part to the widespread adoption of transgenic, glyphosate-resistant (GR) crops.1-3

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GR crops include alfalfa, canola, cotton, maize, soybean, and sugarbeet, with maize and

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soybean accounting for most GR hectares. All GR crops are made resistant to glyphosate by a

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transgene encoding a GR form of 5-enolpyruvylshikimic acid-3-phosphate synthase (EPSPS),

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the enzyme target of glyphosate. In the majority of cases, the transgene encodes a resistant

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EPSPS from the soil microbe Agrobacterium sp. (cp4 epsps). One exception is that some

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maize cultivars contain the modified maize GR GA21 EPSPS.4 GR crops are about 50-fold

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more resistant to glyphosate than non-transgenic crops.5

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Some researchers have claimed glyphosate causes mineral deficiencies in GR crops that can

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lead to increased plant disease, impaired physiology, and reduced yield.6-17 Others have not

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found negative effects of glyphosate on mineral content, plant disease, or yield of GR crops.18-28

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In a recent literature review,29 the authors concluded glyphosate does not adversely affect

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mineral nutrition, disease, or yield of GR crops. Since then, additional studies have found no

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adverse effects of glyphosate on GR crops.30-32 In one of these papers, the effects of glyphosate

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on mineral content of GR soybean was examined over three years in five US states and one

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Canadian province.31 There were no effects of glyphosate treatment on Mn content at any site in

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any year. Potential effects of glyphosate on Mn content have been the focus of several papers

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claiming adverse effects on mineral nutrition of GR crops.e.g.,8,10,17 A few inconsistent effects on

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Fe, P and Zn occurred in this study.31 For example, the only significant effect on Zn content was

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higher levels in glyphosate treatments than in treatments with other herbicides in Ontario in one

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year of the study.



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Previous studies were mostly on GR soybean. The GR trait is equally important in maize;2,3

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however, there are only two studies of glyphosate effects on mineral content of GR maize, and

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they were both limited in scope (one year and one research site).33, 34 More definitive studies of

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possible effects of glyphosate on mineral nutrition in GR maize are needed. In this paper, we

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investigate whether the GR transgene or glyphosate adversely affects plant mineral nutrition and

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yield of maize. The study was conducted over two growing seasons at two geographically

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distinct regions in fields both with and without a history of continuous glyphosate use. Our

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results provide further evidence for the null hypothesis that GR crop technology, whether used

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over one or several years, has no significant effect on mineral nutrition or yield of maize.

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MATERIALS AND METHODS Field Experiments. Field trials were conducted in the Southern and Midwestern U.S. maize production regions

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over two years. Maize grown for grain was investigated in the Southern region at Stoneville,

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MS. Maize grown for sweet corn was investigated in the Midwestern region at Urbana, IL. At

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each location, trials were conducted in two field types: 1) fields with a history of glyphosate use

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(hereafter called ‘glyphosate history’ fields), and 2) fields with no recent history of glyphosate

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use (hereafter called ‘no-history’ fields).

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Stoneville. Field experiments were conducted in 2013 and 2014 at the USDA-ARS Crop

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Production Systems Research farm, Stoneville, Mississippi, USA. The glyphosate history field

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had grown either GR soybean or GR cotton the previous 15 years. The no-history field had

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cogongrass [Imperata cylindrica (L.) Beauv.] grown without herbicides the previous 12 years.

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In 2012, cogongrass was killed with repeated tillage, and then non-GR soybean and non-GR


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maize were planted in alternate rows to prepare the land. Maize and soybean were grown until

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maturity and flail mowed. Fields were prepared by disking and bedding in the fall of 2012 and

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2013. The soil in both fields was a Dundee silt loam (fine-silty, mixed, active, thermic Typic

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Endoqualf) with pH 6.7, 1.1% organic carbon, and a cation exchange capacity of 15 meq 100

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g−1. At planting, soil samples from the top 15-cm depth were collected by taking four random

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cores (7.5-cm diameter) from all plots. Soil samples were analyzed for mineral content as

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before.32

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The treatments were a non-GR cultivar without glyphosate, a GR cultivar without

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glyphosate, and a GR cultivar with glyphosate (application details below). The experimental

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design was a randomized complete block with four replications. Each plot consisted of four

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rows spaced 102 cm apart and 15.2 m long. Maize isogenic cultivars, DKC65-17 RR2 (GR,

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containing the cp4 epsps transgene) and DKC65-18 (non-GR) were planted at 70,000 plants/ha

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on April 8, 2013 and April 2, 2014 and grown using standard production practices under

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irrigation. Herbicides, (pendimethalin and S-metolachlor), plus hand weeding were used to keep

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the experimental area weed-free during the experiment.

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A commercial formulation of potassium salt of glyphosate (Roundup WeatherMAX,

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Monsanto Agricultural Co., St. Louis, MO) was used in the treatments. Consistent with label

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recommendations, the first glyphosate application (0.87 kg ae/ha) was applied over the top and

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the second application (0.87 kg ae/ha) was applied as post directed to the base of the maize

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plants. Glyphosate was applied with a tractor-mounted sprayer with TeeJet 8004 standard flat

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fan nozzles (TeeJet Spraying Systems Co., Wheaton, IL) delivering 187 L/ha spray volume at

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179 kPa.



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At R2 (blister stage, the silks are beginning to dry and darken to a brownish color) maize

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growth stage (~ 4 weeks after second glyphosate application and 11 weeks after planting), 40

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flag (uppermost) leaves were sampled randomly from the middle two rows of each plot. At

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physiological maturity, 20 ears were sampled randomly from the middle two rows, and seed

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were collected from all the ears for chemical analysis. Grain from all four rows of each plot was

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harvested using a combine, and grain yield was adjusted to 15% moisture. Leaf and seed

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samples were stored in sealed plastic bags at 4°C and room temperature, respectively.

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Urbana. Similar field experiments were conducted in 2013 and 2014 at two fields at the

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University of Illinois Crop Sciences Research and Education Center near Urbana, IL, USA. A

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GR soybean crop had been grown regularly on the glyphosate history field the previous 15-years,

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while the no-history field had been used as a perennial grass pasture for decades. Soil samples

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were analyzed by the Agricultural Analytical Services Laboratory, Pennsylvania State

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University. P, K, Mg, and Ca are Mehlich 3 extractable, and all other metals are total sorbed

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metals by the EPA 3050 method. The Urbana location differed from Stoneville in terms of

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isogenic maize cultivars (glyphosate-sensitive sweet corn ‘Passion’ and GR sweet corn ‘Passion

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II’), glyphosate rate (a single application of 1.68 kg ae/ha glyphosate applied at the V4-5 growth

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stage), and timing of yield measurements (R3 growth stage). Additional details are published in

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the plant pathology results from this same study which addressed the influence of glyphosate and

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the GR transgene on Goss’s wilt incidence and yield in maize.35

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Mineral Analyses

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Sample Preparation. Leaves were dried at 60 ℃ for 24 h to constant weight prior to digestion.

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Seeds were digested without drying. Samples of each treatment were triplicated. Samples were

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digested and prepared for analysis as before.12, 32


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ICP-MS Analysis. The tissue concentrations of 26 minerals (Al, As, Ba, Be, Ca, Cd, Co, Cr,

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Cs, Cu, Fe, Ga, K, Li, Mg, Mn, Na, Ni, Pb, Rb, Se, Sr, Tl, U, V, Zn) were determined by sector

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field inductively coupled plasma mass spectrometry (SF-ICPMS) using a Thermo Fisher

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Element-XR as before.32 Instrumental and data acquisition parameters are provided in Table S1.

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Leaf sample data are provided on a dry-weight basis and seed data are on a wet-weight (fresh)

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basis. Reference material (NIST SRM 1547, Peach Leaves) recoveries generally ranged from

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80-120%. Standard deviations between samples of the same treatment were generally less than

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10%.

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Analysis of glyphosate and AMPA

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Samples were prepared as before.36 GC-MS analyses were performed as before36 with

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modifications.32

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Statistical Analyses. Since different types of maize were grown at Stoneville and Urbana, crop

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responses were analyzed separately by location. Within each location, glyphosate history and

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no-history fields were each represented once due to the difficulty of identifying fields without a

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recent history of glyphosate use. Therefore, data from glyphosate history and no-history fields

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were analyzed separately.

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Response variables were individually analyzed by ANOVA using the Proc Mixed procedure

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of SAS, version 9.4 (SAS Institute, Cary, North Carolina). Years and replicates were considered

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random effects. The three treatments (i.e. non-GR cultivar without glyphosate, GR cultivar

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without glyphosate, and GR cultivar with glyphosate) were considered fixed effects. In a few

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instances, some minerals could not be quantified in one of the two years. In such cases, year was

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removed as a random effect in the mixed model. For all data, when the treatment effect was

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considered significant (P < 0.05), treatment means were compared using the Bonferroni-



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corrected multiple comparisons procedure. Yield data were subjected to analysis of variance

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using SAS PROC GLM and treatment means were separated at the 5% level of significance

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using Fisher’s least significant difference test.

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RESULTS

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At planting, there were no differences in soil samples for pH, cation exchange capacity, organic

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matter, or content of 20 minerals among plots assigned to the three treatments at the Mississippi

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location (Table S2). There were no differences in soil composition between glyphosate history

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and no-history fields (Table S2). Soil mineral analyses at the Illinois location were similar

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(Table S3), one exception being lower Mn in soil of the no-history fields that were to be treated

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with glyphosate.

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Mineral content of leaves and seed.

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Stoneville. There were few treatment differences in leaf mineral content 4 weeks after

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glyphosate application, and none were consistent across both glyphosate history and no-history

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fields. Neither the GR transgene nor glyphosate had an effect on leaf content of 24 of 26

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minerals (Tables 1 and 2). In the glyphosate history field, leaf Mg and Mn content in the GR

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with glyphosate treatment were significantly (10 and 20%, respectively) less than the non-GR,

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no-glyphosate treatment (Table 1). However, leaf Mg and Mn content were unaffected by

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treatments in the no-history field.

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Similarly, few treatment differences in seed mineral content were observed and none

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were consistent across both glyphosate history and no-history fields. The transgene and

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glyphosate application had no effect on seed content of 21 of 26 minerals (Tables 3 and 4).

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Significant treatment effects were observed for Cd seed content; however, the pattern of


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treatment responses was inconsistent across fields, with increases above non-GR technology

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plant levels with glyphosate treatment of GR plants in glyphosate history fields and decreases

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below non-sprayed GR plants with glyphosate treatment of GR plants in no-history fields

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(Table 3). Similarly, there were small treatment differences in Be, Li, Rb, and Tl seed content,

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but these effects were inconsistent across fields (Table 4).

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Across leaf and seed samples, when treatment differences were observed, glyphosate or

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the GR transgene appeared to equally increase or decrease mineral content relative to the non-

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GR, no-glyphosate treatment. In the instances where treatment differences were observed, the

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range of differences was 10 to 20%.

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Urbana. With one exception, there were no effects of glyphosate or the transgene on leaf

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mineral content 5 weeks after glyphosate was applied (Table 5). Glyphosate application to GR

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sweet corn in the no-history field reduced leaf Mg content 17% compared to the non-GR no-

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glyphosate treatment. This effect was not observed in the glyphosate history field.

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Neither glyphosate nor the GR transgene affected content of the 26 minerals measured

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in sweet corn seed (Tables 6 and 7).

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Glyphosate and AMPA content of leaves and seed.

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Stoneville. Both glyphosate and AMPA were found in leaves of plants that were treated with

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glyphosate (Figure 1). Neither glyphosate nor AMPA were found in leaves or seed of plants

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not treated with glyphosate. The levels of AMPA were much lower than those of glyphosate.

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Both glyphosate and AMPA levels were similar in both years with the same treatments, but

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measured amounts were higher in glyphosate history field than the no-history field both years,

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although statistical design did not allow a rigorous comparison. Neither glyphosate nor AMPA

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was detected in maize seed in either year.



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Urbana. Glyphosate levels were approximately one order of magnitude lower in the leaves of

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glyphosate-treated plants in Urbana than in Stoneville (Figure 1). No AMPA was detected in

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these samples. As with the Stoneville experiment, in 2013 neither glyphosate nor AMPA was

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detected in seed of treated plants. In 2014, there were 24.9 ± 4.4 and 40.9 ± 2.4 ng g-1

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glyphosate in the seed from the no glyphosate history and glyphosate history fields,

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respectively.

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Yield

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Stoneville yields were unaffected by the transgene or glyphosate (Figure 2). Sweet corn yield

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results of the experiment in Urbana, IL (data provided in Williams et al.35) were similar, in that

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glyphosate use in GR maize did not reduce yield, but was associated with a significant yield

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increase (8.3%) that was not related to weed control.35

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DISCUSSION

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As with soybean,18, 32 few significant effects of GR technology on mineral content in maize

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leaves or harvested seed were observed. The small number of significant differences appeared

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random, because they represented less

Glyphosate Resistance Technology Has Minimal or No Effect on

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