Food Contaminants - ACS Publications - American Chemical Society

Chapter 6

Opportunities for Mycotoxin Reduction in Maize Using Biotechnology 1

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B. Hammond, K. Campbell , J. Cea , H. Esin , A. Pietri , G. Piva , T. Pierre , J. Richard , C. Rubinstein , J. Sequeira, and F. Tatli Downloaded by YORK UNIV on July 1, 2012 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch006

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Monsanto Company, Building 03F, 800 North Lindbergh Boulevard, St. Louis, MO 63167 Corn States Hybrid Service, 2505 McKinley Avenue, Des Moines, IA 50321 Mycotoxin Department, Technological Laboratory of Uruguay, Ave Italia, 6201, Montevideo, CP 11500, Uruguay Monsanto Gide ve Tarim Tic, Fahrettin Kerim Gokay Cad. No:22 A Blok Kat: 2, Altunizade, Istanbul, Turkey Istituto di Scienze degli Alimenti e della Nutrizione, Universita Cattolica del Sacro Cuore, Via Emilia Parmense, Piacenza, 84 29100, Italy Monsanto Agriculture France SAS, Hybritech/Asgrow, 1 Rue Jacques Monod, Europarc du Chene, Bron Cedex 69673, France Romer Labs, Inc., 1301 Stylemaster Drive, Union, MO 63084 Monsanto Argentina SAIC, Maipu 1210, 10 Floor, Buenos Aires, C1006 ACT, Argentina Crop Protection Institute of Ministry of Agriculture and Rural Affairs, Koprukoy, Adana, Turkey

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A variety of environmental stress factors increase susceptibility of corn plants to infection with various fungi that produce mycotoxins. These stress factors include insect damage, heat and drought stress, nitrogen deficiency, and genetic susceptibility. For example, insect feeding injures corn kernels, creating ports of entry for fungi that produce ear rot and mycotoxins. Biotechnology is helping to provide season -long protection of corn plants (Bt corn) against corn borer damage through the introduction of insect control proteins derived from Bacillus thuringiensis. Reduction of insect feeding damage in Bt corn has led to lower contamination with © 2008 American Chemical Society In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.

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Downloaded by YORK UNIV on July 1, 2012 | http://pubs.acs.org Publication Date: October 20, 2008 | doi: 10.1021/bk-2008-1001.ch006

fumonisin mycotoxins in most locations where it has been tested around the world. Biotechnology is also being used to develop healthier corn plants by making them less susceptible to drought stress, increasing nitrogen utilization, and protecting plants against a wider variety of insect pests that feed on the ears, stalks and roots. In die future, combining these traits will further improve yield and should reduce corn plant susceptibility to environmental stress factors that contribute to mycotoxin contamination in the field.

Corn (Zea mays L.) can be infected with fungi that produce toxic secondary metabolites called mycotoxins. Fusarium verticillioides (Sacc.) Nirenberg (synonym = F. moniliforme J. Sheld.) and F. proliferatum (T. Matsushima) Nirenberg, which produce fiimonisin mycotoxins, are the most common fungi that infect corn wherever it is grown. Various environmental factors such as insect damage, heat and drought stress, nitrogen deficiency, and genetic susceptibility predispose corn plants to infection with fungi (7,2). Dietary exposure to fumonisins can cause a variety of adverse health effects in farm and laboratory animals (5). Epidemiological studies suggest a link between high dietary intake of fumonisins and elevated rates of liver and/or esophageal cancer in certain regions of Africa, China, Italy, and Brazil (3,4). There are also epidemiological investigations of possible links between fiimonisin exposure in early pregnancy and increased incidence of neural tube defects in newborns (5,6). As a consequence, regulatory agencies have recommended limits for fumonisin contamination of com intended for animal feed and human food use (3). The United States Food and Drug Administration (7) has provided guidance to food and animal feed industries for maximum levels of fumonisin contamination in corn of 2 to 4 ppm (mg/kg) for various corn fractions going into human food. The European Union is proposing setting limits ranging from 0.2 to 2.0 ppm fumonisins in corn intended for use in various human foods, the lowest level being for corn used in baby food (8) Control of insect pest damage to corn can reduce fungal infection since insect feeding provides ports of entry for fungi. Some insect pests also serve as vectors for fungal infection (9). An effective insect pest control strategy has been developed with the introduction of coding sequences for the CrylAb protein derived from B. thuringiensis into corn plants (event MON 810, YieldGard Cornborer®, trademark of Monsanto Technology LLC) (70). The CrylAb protein controls lepidopteran insect pests such as the European corn borer (ECB), Ostrinia nubilalis Hübner, the most important stalk-boring and ear damaging insect pest of corn in the United States Corn Belt (77). The CaMV 35S gene promoter enables constitutive expression of the CrylAb

In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008.


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protein throughout the growing season, thus providing season-long protection against corn borers. The B. thuringiensis-baseá (Bt) microbial pesticides that contain Cry proteins such as CrylAb have been used commercially in agriculture for over 40 years to control larval insect pests (10, 12). They have an exemplary safety record because their insecticidal mode of action is highly specific against target lepidopteran insect pests. The Cry 1 Ab protein has no activity against nontarget organisms such as mammals and birds (10, 12). Munkvold and co-workers were the first to report that corn hybrids protected with the CrylAb protein had significantly lower fumonisin mycotoxin levels in corn (75). This was most evident in corn plants that expressed CrylAb protein constitutively during the growing season. Additional field trials have been conducted in countries that permitted field testing of YieldGard Cornborer hybrids to assess their impact on fumonisin levels under local conditions. Results are presented from field trials in the United States, Italy, France, Germany, Turkey, and Argentina that compared the levels of fumonisins in YieldGard Cornborer varieties with their near-isogenic controls.

Materials and Methods United States For the data presented in Figure 1, and Table II, the materials and methods used are described in a publication (14).

Germany Materials and methods information is summarized in the citation included with the data on Table I.

Turkey The data are presented in Tables I and II. Field trials with YieldGard Cornborer varieties were carried out in Adana province, in the East Mediterranean Cukurova region, where corn is planted as a second crop after wheat. Damage from corn boring insects pests is most severe in the second corn crop. The hybrids used for the study were DK626 Bt, its near-isogenic control (DK626) and a conventional hybrid traditionally grown in the region. The trials were carried out under conditions of natural insect infestation. Trials in 2000 and 2001 were set up using a randomized split plot design with four replicates.



Figure 1. Field trial locations in the US where total fumonisins >2 mg/kgfor control hybrids compared to Bt hybrids grown at the same locations. (Reproduced with permissionfromThe Mycotoxin Factbook, Food and Feed Topics; 2006. Copyright 2006 Wageningen Academic Publishers.)


In Food Contaminants; Siantar, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2008. a

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625 4718 nd 4665 2500 17,500 780 16,750 2460 6290 560 3060

Fumonisin Level

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N o t detected. Mean value for all varieties across 4 locations. Manually infested with European corn borer. (+) Contains C r y l A b protein. (-) Does not contain C r y l A b protein.

Novelis Bt (+) Novelis control) (-) Monumental Bt (+) Monumental (-) DK626 Bt (+) DK626 (-) DK626 Bt (+) DK626 (-) Bt hybrids (+) 3 varieties Near isogenic controls (-) Bt hybrids (+)' 4 Varieties Near isogenic controls (-)

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Germany (mean across 3 sites) Germany (mean across 3 sites) Germany (mean across 3 sites) Germany (mean across 3 sites) Adana, Turkey (2000) Adana, Turkey (2000) Adana, Turkey (2001) Adana, Turkey (2001) Argentina 2000 (mean across 4 sites) Argentina 2000 (mean across 4 sites) Argentina 2001 (mean across 4 sites) Argentina 2001 (mean across 4 sites)

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Table I. Lower Fumonisin Levels in YieldGard Cornborer Hybrids Grown in Different Countries


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Reference


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2000 2001 2001-2002 1997-1999 1999 2000 2001 2000

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USA USA Turkey France Italy Argentina Argentina Argentina 49 125 2 26 30 4 4 57

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Non-Bt Hybrids Mean Fumonisins (mg/kg) 3.1 4.9 17.5 1.0 2.9 6.29 3.06 5.03

Bt Hybrids Mean Fumonisins (mg/kg) 1.4 2.6 2.6 0^03 0.35 2.46 0.56 1.95

55 47 85 97 88 61 82 61

% Reduction in Mean Fumonisins

Table II. % Reduction in Fumonisin Levels Observed in Field Trial Studies with YieldGard Cornborer by Country.


115 Each of the four blocks was divided into two sub-blocks. Four sub-blocks received three applications of the insecticide lambda-cyhalotrine at two to three week intervals, starting the third week of July. The other sub-blocks were not treated with insecticide. Test plots consisted of eight corn rows 20 m in length. At harvest, mycotoxin concentrations were compared across treatments. ELISA test kits (quantitative kit for fumonisin, Veratox, Neogen Corp., Lansing, MI, USA) were used to determine fumonisin levels in accordance with the kit instructions.


Italy The data are presented in Figure 2 and Table II. In 1999, field trials were carried out in northern Italy at 30 different locations with four YieldGard Cornborer varieties of different genotype and their respective near-isogenic controls. The trials were carried out under conditions of natural insect infestation as there is significant ECB infestation in these regions. Approximately 93 samples of corn were randomly collected from one to 10 weeks post-harvest from the various locations and sent to the School of Agriculture, U.C.S.C., Piacenza, Italy, for analysis using published procedures (15).

France The data are presented in Figure 3 and Table II. During 1997 to 1999, the levels of fiimonisin mycotoxins were measured in YieldGard Cornborer varieties and their near-isogenic controls at 25 field trial locations in France. The majority of sites were in the southwest of France where ECB is normally present. The trials were carried out under conditions of natural insect infestation. In nonreplicated field trials, the plot size was a minimum of 20 m x 8 rows. In replicated field trials, there were four randomized blocks in two plots (Bt and non-Bt), in plots 12 m by six rows. For non-replicated trials, corn was collected from 20 consecutive plants in two adjacent rows at four locations in the plot. For replicated trials, corn was collected from 20 consecutive plants in two adjacent rows for each replicate. Corn harvested in 1999 trials was analyzed for fumonisins at INRA, Nantes, France according to published methods (16). Corn samples harvested in 1998 were analyzed for fumonisins at IEEB (European Institute of Environment at Bordeaux) using established methods (75). For corn harvested in 1997, samples were analyzed at AGPM (French Corn Growers Association) using ELISA methods (Diffchamb France, Transía^ plate Fumonisins).



Figure 2. Field trials with YieldGard Cornborer in Italy, 1999. (Reproduced with permission from The Mycotoxin Factbook, Food and Feed Topics; 2006. Copyright 2006 Wageningen Academic Publishers.)



Figure 3. Field trials with YieldGard Cornborer in France, 1997-1999. (Reproduced with permission from The Mycotoxin Factbook, Food and Feed Topics; 2006. Copyright 2006 Wageningen Academic Publishers.)


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Argentina For data in Tables I and II, the material and methods information is summarized in the citation given on the table. For data in Figure 4 and Table II, a YieldGard Cornborer hybrid (DK696) and its near-isogenic control (DK696) were grown in 57 different locations in Buenos Aires province in Argentina during 2000. This province is an important corn growing region for Argentina and corn pests such as Diatraea saccharalis (corn borer) and Helicoverpa zea (ear worm) are frequently found in this region. The field trials were carried out under conditions of natural insect infestation. Corn samples were collected from each of the sites at harvest and submitted for fumonisin analysis at the Laboratorio Tecnológico del Uruguay (LATU) in Montevideo using published methods (77).

Results and Discussion As shown in Table I and Figures 1-4, many of the field locations had lower fiimonisin levels in YieldGard Cornborer hybrids compared to their near isogenic controls. These results confirm that insect feeding is an important contributor to fumonisin contamination of corn under conditions where there is significant corn borer presence. Protecting corn against insect feeding damage can reduce the opportunity for fungi to infect kernels. YieldGard Cornborer hybrids have been reported to have lower fungal contamination based on measurements of ergosterol levels in corn (16, 75). When the levels of fumonisins were averaged across all sites for trials in each country (Table II), the YieldGard Cornborer hybrids had reductions ranging from 47 to 97% compared to their near isogenic controls. At some locations where YieldGard Cornborer was tested, the reduction in fumonisin levels was large enough to make the difference between an unacceptable crop and one that was safe for consumption. For some published field trial studies, the fumonisin levels in Bt hybrids were compared statistically to controls; the statistical procedures used were summarized in the publications. For the large field trials conducted in the US from 2000 to 2001 (Figure 1 and Table II), the fumonisin levels in Bt hybrids were statistically significantly lower (p = 0.0007) than corresponding controls (14). In one of the Argentine trials (21) as summarized in Tables I and II, 3 of 4 different Bt hybrids tested had statistically significantly (p

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