Insect Management to Facilitate Preharvest Mycotoxin Management

Home , Fusarium verticillioides, Peoria (moth)

MARCEL DEKRERJNC • 270 MADISON AVENUE NEW YORK, NY6

Journal of Toxicology TOXIN REVIEWS Vol. 22, Nos. 2 & 3, pp. 327-350, 2003

Insect Management to Facilitate Preharvest Mycotoxin Management#

Patrick F. Dowd

Crop BioProtection Research Unit, U.S.D.A., Agricultural Research Service, National Center for Agricultural Utilization Research, Peoria, Illinois, USA

ABSTRACT

Many species of insects can facilitate the entry of mycotoxin-producing fungi to commodities such as cotton seed, maize, peanuts, and tree nuts. The mycotoxins most commonly associated with insect damage are aflatoxin and fumonisin. Insecticides will likely remain an important management tool, especially as predictive models for forecasting mycotoxigenic fungi or mycotoxins become available. Plants with high levels of resistance to insects that facilitate mycotoxins are likely to assist in mycotoxin management. Several studies now indicate Bt maize hybrids

#The mention of firm names or trade products does not imply that they are endorsed or recommended by the United States Department of Agriculture over other firms or similar products not mentioned. Correspondence: Patrick F. Dowd, Crop BioProtection Research Unit, U.S.D.A., Agricultural Research Service, National Center for Agricultural Utilization Research, 1815 N. University St., Peoria, IL, USA; Fax: 309-681-6686; E-mail: dowdpf@mail. ncaur.usda.gov.

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that express the protein throughout the plant can prevent fumonisin levels rising above guideline levels of 1-2 ppm when European corn borers (Ostrinia nubilalis) are the predominant insect pests.

Key Words: Aspergillus; Fusarium; Aflatoxin; Fumonisin; Deoxynivalenol; Integrated pest management; Bt; Helicoverpa; Ostrinia; Carpophilus; Maize; Cotton; Peanut.

INTRODUCTION

Insects are one of several factors that can influence the levels of mycotoxins in commodities such as maize, peanuts, cottonseed, tree nuts and figs. insects can enhance the fungal infection process by carrying inoculum and causing damage that permits more ready entry of the fungus. The literature dealing with insects and mycotoxins in all of the above mentioned commodities has been reviewed fairly recently (Dowd, 1998), so the present discussion will only briefly cover this prior information. This discussion will concentrate on maize, and also include some information on cotton seed and peanuts. Information on tree nuts will be covered in other articles in this series (see Campbell et al. and Michailides). The present review will concentrate on recent efforts in the major management areas of insecticide control, plant resistance (including Bt plants), and predictive models and management plans not covered in other articles in this series (see also Michailides, Campbell et al., Widstrom and Guo, this issue). Because insect damage frequently results in readily observable losses in yield and quality, controlling insects is an attractive indirect management tool for mycotoxins. The ready adoption of Bt maize and cotton for cost effective yield protection from caterpillar pests in the U.S. and elsewhere has apparently already led to reductions in mycotoxins in these crops.

INTERACTIONS

Considerable variability in the importance of insects in increasing mycotoxins has been reported. The relative importance of insects and other factors needs to be considered in the context of the complex environment in which they interact (see also article by Bruns, this issue). insects contaminated with a sufficiently high level of inoculum (versus airborne inoculum) may be necessary to overwhelm natural plant defenses. Insect damage may be the only way the fungus can enter under some conditions, or the fungus may be able to invade on its own under other conditions. The MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016

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relative importance of insect damage is dependent on the insect population, the degree of resistance of the plant to the fungus, and how the environment favors fungal inoculum and growth, and stresses the plant and its ability to produce resistance mechanisms. However, there are many examples where insect control has greatly reduced the levels of different mycotoxins (Dowd, 1998), indicating the importance of insects in mycotoxin production. Particularly convincing are the numbers of studies that now indicate Bt maize expressing the protein throughout the maize plant can greatly reduce levels of fumonisin when the European corn borer, Ostrinia nubilalis, is the predominant insect pest. Mycotoxins can be toxic not only to people and livestock, but also insects. Mammals may be the inadvertent victims of fungal defenses initially evolutionarily derived against insects (Dowd, 1992a). The interpretation of the importance of mycotoxins and their effects on insects is further complicated by the associated fungal secondary metabolites and proteins, as well as the plant secondary metabolites, proteins, and nutritional status within which these interactions occur. The importance of Aspergillus flavus as an insect pathogen is still being debated. However, recent studies have shown that strains of A. flavus isolated from plant, insect, and human or animal sources were not specialized towards any particular host (St. Leger et al., 2000). Unlike other species of Aspergillus tested, conidia phagocytized by insect hemocytes were still able to germinate (St. Leger et al., 2000). A. flavus propagules may be proliferating in the hindgut of corn earworms, Helicoverpa zea, when consumed (Abel et al., 2002). Conversely, some insect species appear to benefit from the presence of aflatoxin producers (Dowd, 1992a) or mycotoxin-producing Fusarium spp. fungi (Schulthess et al., 2002), possibly in a manner analogous to that reported for fungi that are closely associated with insects (Dowd, 1992b). The activity of fungal metabolites towards insects has been recently reviewed (Dowd, 2002a). Interestingly, fungal resistance to, or attack of insects is comprised of multigenic defenses or pathogenicity factors, respectively, affecting several different biochemical targets. For example, A. flavus produces aflatoxin, which, after conversion to an epoxide, reacts with guanidine residues, ultimately resulting in transcription errors; cyclopia- zonic acid which inhibits calcium transport ATPase and appears to interfere with hydrostatic pressure regulation in insects; kojic acid which inhibits insect detoxifying enzymes, wound healing enzymes and pathogen defense enzymes (Dowd, 1999, 2002a), neuroactive compounds such as fl-aflatrem, and kotanins which inhibit NADH oxidase in insects (Dowd, 2002a). Degradative enzymes produced by A. flavus that can potentially attack the protective exoskeleton of insects include proteases (St. Leger et al., 2000) and lipases (Long et al., 1998). In addition, several other secondary

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Table 1. Insects associated with mycotoxins in different commodities.

Coffee (ochratoxins) coffee berry borer Hypothenemus hampei AM, AF

Cotton (aflatoxins)

boll weevil Anthonomus AM 2 grandis grandis

bollworm Helicoverpa zea AM 2

flea beetle Systena blanda AM 2

fruit fly Drosophila melanogaster AM 2

lygus bug Lygus hesperus AM 2

pink bollworm Pectinophora gossypiella AM 2

predatory beetle Collops vittatis AM 2

sap beetles Carpophilus spp. AM 2

stink bug Chlorochroa sayi AM 2

tarnished plant bug Lygus lineoliris AM 2

Figs (aflatoxins) dried fruit beetle Carpophilus AM hemipterus

fig wasp Blastophaga psenes AM 2

flower thrip Franklinielli tritici AM 2

predatory mite Chelytus sp. AM 2

predatory mite Sejus pomi 2 AM thrip Heliothrips fasciatus AM 2

thrip Liothrips bremneri AM 2

drip Thrips bremneri AM 2

Maize (aflatoxins, fumonisins)

bronze psocid Ectopsocopsis AM 2 cryptomeriae

corn earworm Helicoverpa zea 2 AM corn rootworm adults Diabrotica spp. AM 2

ear borer Mussidia nigrivenella AF 3

European corn borer Ostrinia nubilalis AM, EU 2

fall arrnyworm Spodoptera frugiperda AM 2

maize/rice weevils AM, AF 2,3 Sitophilus spp. mite 2 Caloglyphys rodriguezi AM mold mite Tyrophagus AM 2 put rescentiae pink scavenger Sathrobrota rileyi AM caterpillar MARCEL DEKKER, INC. • 270 MADISON AVENUE . NEW YORK, NY 10016

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Table 1. Continued. (aflatoxins, fumonisins) Maize scarab beetles not listed AM 2

sap beetles Carpophilus spp. AM, AF 2,3

Glischrochilus spp. AM 2 picnic beetles southwestern corn borer Diatrea grandiosella AM 2

stem borer Sesamia calamistis AF 3 stink bugs e.g. AM 2 Euchistus spp. sugarcane borer Eldana saccharina AF 3

western flower thrip Franklinella AM 2 occidentalis

Peanuts (aflatoxins) lesser cornstalk borer Elasmopalpus AM 2 lignosellus mite Caloglyphus sp. AF 2 mite Tyrophagus sp. AF 2 termite Odontotermes badius AF 2 termite Odontotennes latericus AF 2

Small grains - (Claviceps ergot toxins)

beetle Cantharus melanura EU 2

carabid beetle unspecified EU 2

fly Rhagonycha fulva EU 2

fly Melanostoma mellina EU 2

fungus gnat Sciara thomae EU 2

Tree nuts - (aflatoxins) codling moth Cydia pomonella AM 2 naval orange worm Amyelois transitella AM 2 pecan weevil Curculio caryae AM 2 southern green stink bug Nezara viridula AM 2 stink bug Thyanata pallidovirens AM 2

AF = reported from Africa, AM = reported from the Americas, EU = reported from Europe. I, (Vega et al., 1999) 2, (Dowd, 1998); 3, (Setarnou et al., 1998).

metabolites which have presently unknown modes of action are also produced by A. flavus and are active against insects (presumably in defending sclerotia) (Dowd, 2002a; Wicklow et al., 1994). No toxicity of fumonisin BI has been reported at 1000 ppm in 7 day assays to fall armyworms, Spodoptera frugiperda, (Dowd, unpublished data), and only long term exposures at unnatural concentrations (450 ppm) have been MARCEL DEl

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sufficient to produce a 20% growth reduction at 28 days in the mealworm Tenebrio molitor (Abado-Becognee et al., 1998). Thus, the high toxicity of a fumonisin producing strain of F. prolferatum to the aphid Schizaphis graminum (Ganassi et al., 2001) is unlikely. Each crop and geographic location can have a different composition of insects that promote mycotoxins. A summary of insects implicated in the enhancement of mycotoxins is provided in Table 1. The insect species that appear to be more consistently and broadly important in enhancing problems with mycotoxins are typically the more common species that attack the plant structures (nuts, boils, pods or ears) that can become contaminated with mycotoxins. Caterpillars such as the corn earworm, Helicoverpa zea; European corn borer, Ostrinia nubilalis; southwestern corn borer, Diatrea grandiosella; fall armyworm, Spodoptera frugiperda; and beetles such as maize weevils, Sitophilus zeamais; and sap beetles, Carpophilus spp., appear to be the most important insects in promoting mycotoxin problems in the U.S. maize (Dowd, 1998). Corn earworms, Helicoverpa zea; tobacco budworms, Heliothis virescens; pink bollworms, Pectinophora gossipiella; and boll weevils, Anthonomis grandis; are the most important insects in promoting mycotoxin problems in cotton seed in the U.S. The lesser cornstalk borer, Elasmopalpus lignosellus, is most important in peanuts; while codling moths, Cydia pomonella, and navel orangeworms, Amyelois transitella, are the most important insects in promoting mycotoxins in tree nuts in the U.S. (Dowd, 1998).

RECENT BASIC RESEARCH

Several recent basic studies in the Americas have shed further light on the involvement of insects in the mycotoxin problem. A number of these studies have involved looking at the involvement of caterpillars. In studies designed to simulate natural conditions, 0. nubilalis larvae were able to acquire Fusarium nwnilifonne (F. verticillioides) spores from plant surfaces and carry them to ears, thereby causing infection in controlled studies (Sobek and Munkvold, 1999). Increases in symptomless kernel infection by F. moniliforme through the activity of 0. nubilalis larvae was also reported for the first time (Sobek and Munkvold, 1999). However, Gibberella zea infection was not increased by 0. nubilalis presence (Sobek and Munkvold, 1999). This information contrasts with that found for Bt maize studies, when DON (produced by G. zea) was found to be lower in Bt than corresponding nonBt hybrids under naturally occurring high populations of 0. nubilalis (Schaafsma et al., 2002) (see following discussion on Bt hybrids). Kernels which were damaged in milk to soft dough stage or MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016 fli 02002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

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earlier, as indicated by discolored pericarps, could have as much as several hundred ppm of fumonisin and were the predominant source of the fumonisin in ears collected from maize fields under natural conditions (Dowd et al., 1999). Studies in Mexico indicated aflatoxm levels were highly associated with sap beetle presence, primarily Carpophilus freemani (Rodriguez-del-Bosque et al., 1998). Sap beetles carried a commercial strain of the biocompetitor Bacillus subtilis from field placed autoinoculative devices to damaged maize ears, and reduced the percentage of ears having greater than 200 ppm of aflatoxm from 70% to less than 10% on ears subsequently inoculated with A. flavus (Dowd et al., 1998a). Volatile monitoring studies have indicated Fusarium verticillioides produces a complex of alcohols, acetaldehyde, and ethyl acetate that are attractive to the pineapple beetle, Carpophilus humeralis (Bartelt and Wicklow, 1999). Attractive compounds are the same as those previously reported for other Carpophilus sap beetle species (Bartelt et al., 1992; Dowd and Bartelt, 1991). In addition to sap beetles, other beetles and moths were also attracted to corn when it was field inoculated with F. verticillioides in Benin (Cardwell et al., 2000) (see below). A series of recent studies in maize conducted in tropical Africa (Benin) have identified several insect species which can facilitate the presence of both A. flavus and F. verticillioides. The most consistently important insect was the caterpillar ear borer, Mussidia nigrivenella (Lepidoptera: Pyralidae). Countrywide surveys over two years indicated only M. nigrivenella was significantly correlated with aflatoxins in both years (r=0.36 and 0.52) (Setamou et al., 1997). This species was also the major source of maize damage in both years (Setamou et al., 1998). Both the percent incidence and percent grains damaged were significantly correlated with aflatoxin B 1 in both years (Setamou et al., 1998). Other, less common insects that sometimes had significant positive correlations with either A.flavus and/or aflatoxin included the caterpillars, sugar cane borer, Eldana saccharina, and the stem borer, Sesamia calamistis; and the beetles, the maize weevil Sitophilus zeamais, Carpophilus sp. sap beetles, and combined Coleopteran spp. (Setamou et al., 1998). Interestingly, A. flavus had no effect on the survival of M. nigrivenella (Setamou et al., 1998). However, E. saccharina may be more important in promoting aflatoxin where they occur in higher density (Setamou et al., 1998) (as for Fusarium, see below). The cryptic behavior of M. nigrivenella makes it difficult to control with insecticides, so they are not a useful option for aflatoxin management (Setamou et al., 1998). Specific studies with four maize genotypes of different types of derived resistance/yield backgrounds did not indicate any significant effect on aflatoxin or fumonisin production, or insect damage (Cardwell et al., 2000). Interestingly, inoculation with F. verticillioides significantly increased the presence of several insect species, MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016 02002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

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including S. calamistis, E. saccharina, but not M. nigrivenella (Cardwell et al., 2000). In addition, E. saccharina survived better in F. verticillioides inoculated stalks (Cardwell and Schuithess unpub., cited in (Cardwell et al., 2000). In contrast to prior studies in North America (e.g., Munkvold et al., 1997; Sobek and Munkvold, 1999), stem infection by F. verticillioides appeared more important in ultimate ear infection, as opposed to inoculum that landed on the ear (Schulthess et al., 2002). Inoculation with F. verticillioides significantly increased incidence of Eldana saccharina, Cryptophiebia leucotreta, Chilo spp., and pooled beetle species (Schulthess et al., 2002).

INSECTICIDES

Much prior work has focused on frequency and timing of insecticide applications. Unfortunately, in most cases uneconomical numbers of insecticide applications have been needed to reduce mycotoxin contamination to desired levels (Dowd, 1998). For example, six weekly applications of insecticides reduced aflatoxin in corn from 191 ppb to 2.8 ppb in Louisiana (Smith and Riley, 1992). Some recent studies have tested adherent malathion granules prepared from a maize product. The advantages of this formulation included: 1) application at a reduced rate of active ingredient to maize, 2) greater resistance to washoff by rain or dislodgement by scouts or equip- ment, and 3) selectivity by reducing mortality to beneficial insects (which would not feed on the granules). When properly applied, these granules did provide longer lasting, more selective control as good as or better than multiple applications of commercial formulations, and reduced ear mold and mycotoxins, including in commercial fields of specialty corn (Dowd et al., 1998b, 1999, 2000a). However, control efficacy of this formulation is not as good as that seen with maize that expresses the Bacillus thuringiensis crystal protein (Bt maize) throughout the plant, which typically kills all 0. nubilalis that attempt to feed on the plants (see below). The use of insecticides does remain a necessary component of some management plans where economically practical (see below). For example, insecticides are apparently still being used as an important component of aflatoxin management strategies in maize in Mexico.

PLANT RESISTANCE

Plant resistance to insects is now receiving most of the insect-based mycotoxin control effort at various locations. As indicated earlier, because - MARL Dmua, INC. • 270 MADISON AvENuE YORK, NY 10016 02002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

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insects can cause dramatic reductions in yield and quality, and because plant resistance is a more economical way to control insects, especially for lower value crops such as maize, varieties that contain insect resistance as a yield and quality protectant are often readily adopted by growers. In addition, considerable advances have been made in discovering and in- corporating insect resistance traits in tree nuts such as almonds, pistachios and walnuts by conventional breeding and through biotechnology (see articles by Michailides and Campbell et al., this issue). Insect resistant maize varieties are being identified, and mycotoxin resistant varieties are being examined for cross resistance to insects. It is likely that common mechanisms may be involved in both fungal and insect resistance (see following discussion). For example, enhanced peroxidase activity appeared to be an important insect and fungal resistance mechanism in Mp313E, which is also resistant to Aspergillus and aflatoxin production (Dowd, I 994a,b). Studies with transgenic plants that expressed high levels of tobacco anionic peroxidase indicated enhanced resistance to insects in three lines of tobacco (Dowd and Lagrimini, 1997) tomato (Dowd et al., 1998c), sweet gum (Dowd et al., 1998d), and especially maize (Privalle et al., 1999). More recently, strong silk resistance to caterpillars that appears nonmaysin based was discovered in the inbred Tex6 (Dowd and White, 2002). In a study that examined insect and afiatoxin resistance in maize, one maize line, Mp80:04, was found to have both resistance to aflatoxin production and resistance to D. grandiosella (Williams et al., 2002a). Some drought-resistant germplasm that had resistance to A. flavus and aflatoxin production was also insect resistant (Tubijika and Damann, 2001). Genetic markers and controlling factors for the insecticidal maize secondary me- tabolite maysin (a derivative of the flavonoid luteolin), its analogs, and chlorogemc acid have been identified (Guo et al., 2001a,b) (see article in this issue by Widstrom and Guo). Hemicellulose (Williams et al., 1998) and a cysteine protease have been identified as important insect resistance mechanisms of some maize inbreds (Penchan et al., 1999, 2000, 2002). Maize ribosome inactivating protein (RIP), now demonstrated to be antifungal towards A. flavus (Nielsen et al., 2002) has also recently been identified as an insect resistance mechanism, and been validated in transgenic plants (Dowd et al., 1998e, 2000b). Work with Bt maize at several locations has indicated what a tremendous reduction in mycotoxin levels can potentially occur when high levels of insect resistance are available. In small plot studies run under controlled conditions in Iowa using spore contaminated larvae of 0. nubilalis and several different Bt events, overall reductions in fumonisins ranged from less than two fold in some years, to over 10 fold in others (Munkvold et al., 1999). The degree of reduction was dependent on the type of transformation event. For example, MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEw YORK, NY 10016 02002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

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BT 1 and MON810 hybrids, which have the highest and most consistent expression of the Bt protein throughout the plant, also have the greatest and most consistent reductions in fumonisins relative to nonllt hybrids (Munkvold et al., 1999). Overall correlation R values between insect damaged kernels and fumonisin levels R values were 0.50 and 0.69 and highly significant in 1996 and 1997 respectively (Munkvold et al., 1999). Representative isogenic hybrids containing BT11 and MON810 constructs that were grown in Illinois had fumonisin levels up to 40x less than nonBt hybrids in 0.4 ha fields grown under natural conditions, where 0. nubilalis infestation rates ranged 80-90% (Dowd, 2000a). Significant reductions in fumonisin levels for Bt vs. nonBt hybrids were also seen in small plot studies run in the same years at a different location (Dowd, 2000a). Significant reductions in fumonisin for high Bt vs. low or no Bt ears were also noted in commercial fields up to 16 ha, although when H. zea infested more than 20% of the ears, typically no significant differences resulted (Dowd, 2001a). Interestingly, H. zea were noted damaging several kernels along a row without consuming the kernels in the Bt hybrids at significantly higher rates for high Bt compared to low or no Bt maize (Dowd, 2001a). In some hybrids, even without significant reductions in fumonisins, fumonisin levels were more consistently significantly correlated with some hybrid or hybrid pairs than others throughout the years and sites examined in both studies (Dowd, 2000a, 2001 a). This information suggests the genetic ability of the plant to resist the fungus in the absence of insect damage, as well as the plants ability to resist the fungal spread after introduction into insect damaged kernels also plays a role in how beneficial the Bt protein may be in reducing mycotoxins in hybrids with different backgrounds. Although a study in Ontario under natural conditions reported no significant reductions in lumunisin in Bt compared to nonBt hybrids in the presence of 0. nubilalis, mean values were less than 0.3 ppm in all years (Schaafsma et al., 2002). Studies in small plots under controlled conditions with Bt and nonBt hybrids using four hybrid pairs indicated significant reductions only when plants were not inoculated (method not specified) with Fusarium, with both infestations of 0. nubilalis and H. zea (Clements et al., 2002). Interestingly, studies sponsored by Monsanto where infestations were used in the U.S. (2000) have yielded smaller differences (2-3X) in reductions of fumonisin levels compared to 8-30 fold reductions in studies run under natural conditions in Europe (1997-1999, see below) (Hammond et al., 2002). There is also some evidence that Bt hybrids can reduce stalk rot caused by fumonisin-producing species of Fusarium (Gatch and Munkvold, 2002; Gatch et al., 2002). Studies done in Europe under natural conditions have indicated highly significant reductions (greater than lOx) in fumonisins when nonBt hybrids had fumonisin levels above 2 ppm (Bakan et al., 2002; Cahagnier and Melcion, 2000). These reductions in fumonisin are of the same degree as jfl MARCEL DEKKER, INC.. 270 MADISON AVENUE . NEW YORK, NY 10016 02002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

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those that have been reported in the U.S. Studies in Italy also indicated significant reductions in fumonisin from 3 to 6 fold in 1999 (30 locations, 4 genotypes) and 1998 (4 locations, 2 genotypes), respectively, with a non- significant 10 fold reduction in 1997 (3 locations, 2 genotypes) (Pietri and Piva, 2000). However, many environmental effects can mitigate the efficacy of Bt hybrids in protecting against increases in fumonisin related to insect damage (Dowd, 2000a, 2001a). Obviously, if populations of 0. nubilalis are low, differences in fumonisin levels are not as likely to be as great as if populations are high. As mentioned previously, the expression pattern of the gene is important. Event 176 hybrids (which produce only low levels of the Bt protein in silks and kernels) have not proven very effective in indirectly reducing fumonisin under the same insect pressures for MON8I0 and Bt I events (Dowd, 2000a; Munkvold et al., 1999), nor has DBT418 (Munkvold et al., 1999). Correlations between insect damage and fumonisin have been more highly significant in some years than others for the same hybrid in different years, or the same hybrid at different locations in the same year, suggesting environmental factors are influencing the plants ability to defend itself in the absence of insects (i.e. the fungus is able to invade on its own) or else spread after insect damage at different rates (Dowd, 2000a, 2001a). The levels of natural fungal inoculum or type of inoculation method also seems to make a difference. Under natural conditions with relatively high European corn borer levels, significant reductions in fumonisins have been relatively consistent. However, more severe inoculation methods where the ear is damaged may result in less significant reductions. This also appears to be the case for aflatoxin studies (see below). There have also been some reports that Bt hybrids are capable of significantly reducing DON levels in maize. This is somewhat surprising, as insect damage has not been found to significantly increase levels of DON in corn in prior studies (Lew et al., 1991; Sobek and Munkvold, 1999). In studies in France and Spain, DON levels were significantly reduced in three cases, increased in one case, and were unchanged in one other (Bakan et al., 2002; Cahagnier and Melcion, 2000). Interestingly, significant reductions in nivalenol were reported from the same sites that had significant reductions in fumonisin and DON (Bakan et al., 2002; Cahagnier and Melcion, 2000). A significant reduction in zearalenone, but not fumonisin, was reported at the same site where a significant reduction in DON occurred (Bakan et al., 2002; Cahagnier and Melcion, 2000). No significant reductions in DON or zearalenone in Bt compared to nonBt maize occurred in Italy, but overall levels were low (Pietri and Piva, 2000). In some cases, presence of F. graminearum was actually increased in Bt maize stalks compared to nonBt hybrids, but this appeared due to a competitive advantage that occurred when cfJ MARCEL DEKXER, INC. • 270 MADISON AvENUE • NEW YORK, NY 10016 02002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

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potentially competitive F. pro4feratum or F. verticillioides, which are associated with damage by 0. nubilalis, were reduced in the Bt hybrid stalks (Gatch and Munkvold, 2002). However, Bt maize has not been as successful in reducing aflatoxin levels as fumonisin levels, as has been indicated by a few studies in the Midwest and Europe, and several studies in southeastern U.S. and Texas, where aflatoxin is more common (although this may be dependent on the method of inoculation used to some extent). Some studies in the Midwest have seen little effect on aulatoxin reductions under controlled conditions using 0. nubilalis, although aflatoxin levels in general were infrequent and quite low (Dowd, 2000a). More extensive studies in the Midwest under controlled conditions have indicated some significant reductions in aflatoxin for Bt compared to nonBt hybrids that express the protein throughout the plant when nondamaging inoculation techniques are used (Munkvold et al., 2000). In studies where a series of 11 Bt/nonBt hybrid pairs were inoculated using a pinboard, no significant differences in Aspergillus ear rot or aflatoxin were noted (Maupin et al., 2002). No reductions in aflatoxin were demonstrated in Bt vs. nonBt maize in Italy, but overall levels were low (Pietri and Piva, 2000). Results relating to indirect reduction of aflatoxin by Bt maize in the more southern part of the U.S. have initially been more disappointing compared to fumonisin results in the Midwest and Europe. This may be at least partly due to the significant insect damage that is still occurring to Bt ears for most of the transformation events in these areas due to H. zea and S. frugiperda, which are not greatly affected by most existing field maize Bt hybrids. Midwest adapted Bt hybrids still had high levels of aflatoxin when inoculated with A. flavus contaminated cob grits and D. grandiosella, but some reductions were noted with D. grandiosella inoculations alone (Windham et al., 1999). However, newer Bt hybrids that have better protection against H. zea and S. frugiperda have shown better results in studies where more natural inoculation methods are used. When A. flavus infested kernels were distributed among corn rows, the aflatoxin of Cry2Ab hybrids (M0N840) was significantly lower than CrylAb hybrids and nonBt hybrids in 2000 and significantly lower than nonBt hybrids in 2001 (Odvody and Chilcutt, 2002). The M0N840 hybrids had the lowest insect numbers and injury ratings, and ear insect injury rating at harvest was significantly associated with aflatoxin levels (Odvody and Chilcutt, 2002). Unlike methods that damaged kernels, inoculation methods that did not damage ears also showed some significant reduction in aflatoxin levels for some MON8I0 and Btll hybrids compared to nonBt hybrids that were infested with D. grandiosella, despite the presence of significant H. zea (Williams et al., 2002b). Most silk inoculated Bt hybrids also had significantly less aflatoxin than their nonBt counterparts (Williams et al., 2002b). MARCEL DEKKER, INC. • 270 MADISON AVENuE • NEW YORK, NY 10016 02002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

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In addition to work with maize, levels of bright greenish yellow florescence have been significantly reduced in cotton seed in Bt cotton studies, suggesting aflatoxin may also be reduced provided growers harvest immediately after boll opening (Bock and Cotty, 1999; Cotty et al., 1997a,b). Bt walnuts (Dandekar et al., 2000a,b) and peanuts (Ozias-Akins et al., 2000, 2002) for insect resistance and aflatoxin reduction are also being explored. Enhancing insect resistance through biotechnological methods may be an approach worth following for other insects that promote mycotoxins as well. There is a trade-off between desirable specificity of proteins, and broadness of insect species control. It would be impractical to have to introduce a specific gene or gene set for every species of insect that can promote mycotoxin problems. The advantage of targeting insect populations as a strategy for indirectly reducing mycotoxins is that insect resistance can protect yields in an economically practical manner, as has been demonstrated with Bt corn. Resistance to corn earworms comparable to that seen for European corn borers in Bt 11 or Mon810 construct derived maize lines would be highly desirable, and may significantly help with mycotoxin problems in the southeastern U.S., Texas, Mexico, and other areas where aflatoxin is a continual problem from year to year. Maize lines with more than one Bt gene, targeting both the European corn borer and corn earworm, have been developed by Monsanto and were released in 2002 (although these are not approved by the EU). Some Btl 1 hybrids have reduced corn earworm incidence at milk stage maize by 10-fold (Dowd et al., 2002). Unfavorable environmental concerns on insect resistant hybrids produced by biotechnology, such as effects of the Bt gene placed in maize on beneficial insects or nontarget insects such as monarch butterflies noted in laboratory studies have not been demonstrated under field conditions (e.g. Dowd, 2000a,b, 2001a; Sears et al., 2001a). Gene frequency assump- tions for resistance management techniques also appear appropriate based on field studies quantitating resistance alleles (Andow et al., 2000; Tabashnik et al., 2001). Significant reductions of pesticide use have been noted in Bt compared to nonBt fields of maize (Carpenter and Gianessi, 2002) and cotton (Tabashnik et al., 2001). The Bt gene in maize does not adversely affect ethanol production (Dien et al., 2002), cattle growth or milk production (Folmer et al., 2002). However, acceptability of biotechnology enhanced food crops remains a potential problem for both insect and fungal resistant materials, despite the health benefits that have already been demonstrated for these lines through reduction of mycotoxin levels. Continued concerns about the presence of antibiotic selectible marker genes, uncertain copy number and insertion site of transgenes, and potential MARCEL DEKKER, INC. • 270 MADISON AVENUE • NEW YORK, NY 10016 02002 Marcel Dekker, Inc. All rights reserved. This material may not be used or reproduced in any form without the express written permission of Marcel Dekker, Inc.

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allergenic properties of novel proteins (despite in vitro) testing) will remain challenges for this type of plant resistance strategy in the near future. Hybrids with limited Bt expression in seeds (i.e. event 176) have seldom yielded significant reductions in fumonisins (Dowd, 2000a; Munkvold et al., 1999). However, an experimental inbred that contained a pith pro- moter that produced high levels of the protein in silks (unlike event 176) but not seeds, was free of corn borer damage when corn borers were added to ears, compared to levels of infestation of 53% in the corresponding nonBt inbred (Dowd, 2000a). This information suggests that selective expression of insecticidal proteins sufficient to control mycotoxigenic fungi through insect control may indeed be possible without obtaining levels of concern of the transgenic protein in the part of the plant that is consumed by people or animals. As mentioned previously, it is entirely possible that selecting for both fungal and insect resistance involves the same factors, and they can be incorporated at the same time through breeding, biotechnology, or a combination. As discussed previously, there are common antiinsectan and antifungal properties of secondary metabolites, including phenolics, stil- benes, naphthoquinones, terpenes, lignins, flavonoids, N-aromatics, sesqui- terpene lactones and phytoalexins (Dowd, 1992a). The same proteins are also potentially involved in both fungal and insect resistance. For example, as mentioned above, common resistance proteins appear to include peroxidase, lipoxygenase, ribosome inactivating proteins, chitinases, proteinase inhibitors, amylase inhibitors, and lectins (e.g. compare proteins mentioned in Bell, 1981; Dowd, 2002a; Gatehouse and Gatehouse, 2000; Selitrennik- off, 2001). In maize, insect and disease resistance have mapped to the same sites, and appear to be multigenic (e.g. McMullen and Simcox, 1995). It is also potentially important to determine effective combinations of proteins, in order to stabilize resistance in plants, and prevent development of resistance in insects and fungi. Most prevailing considerations have involved using proteins with different modes of action. However, issues such a the ability of the active toxin to penetrate to the target site may actually be the limiting factor. A new strategy was proposed recently, that involved examining proteins or other active compounds for function, and combining compounds of different functional classes (Dowd, 2002b).

INTEGRATED MANAGEMENT PROGRAMS

Integrated management programs involving insect control have been in practice in Mexico since 1992 (CEMCA (Comite Estatal de Modemizacion del Campo del Agua), 1993). In the U.S., development of sophisticated MARCEL DEKXER, INC. • 270 MADISON AVENUE • NEW YORK NY 10016

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programs acceptable to growers involving predictive computer programs have been more of a recent focus. For example, aflatoxin management systems for peanut involve insect management (Wilson, 1995). One important component of insect management is monitoring for insects, so that control measures will only be used when economically practical. Although monitoring tools for pests such as the codling moth exist, improvements have been made in attractants for the codling moth, which have potential for commercialization (Light et al., 2001). Attractants and trapping systems for sap beetles have been developed in order to make them commercially viable (Dowd, 2000b; Bartelt, 1997). The pheromone for the dusky sap beetle, Carpophilus lugubris, the most common sap beetle in midwest corn, was produced commercially starting in 2002 and compared well with prior materials that have been used (Dowd et al., 2002). An IPM program developed for the midwestern U.S. corn belt maize has been tested in central Illinois for two years, and involves participation of a large grower organization, a farm service organization, industry, and extension (Dowd, 2001b). A predictive computer program is crossed checked for validity against fields intensively sampled for insects, mold inoculum, and mycotoxins. Br maize is an important component of this system, but growers are somewhat reluctant to adapt due to the uncertain nature of the market. Insecticide treatment remains an important potential insect management strategy in nonBt maize fields in the presence of high populations of European corn borers, or for other insect pests such as the corn earworm. Predictions of fumonisins for most hybrids in commercial fields have been close to actual levels using data from commercial fields (Dowd et al., in press). Although growers remain interested, adoption and practice remains limited (and probably will be until elevators set fumonisin requirements). A comprehensive program for aflatoxin management in the southeastern U.S. in maize is being developed in Georgia, and involves an economic analysis of components and corresponding choices for both a preplant and growing season phase (Widstrom et al., 2002; Widstrom et al., 2000, article this issue).

CONCLUSIONS

Insects are an important factor in the presence of mycotoxins in several major crops. Targeting insects as a means for indirectly controlling mycotoxins has considerable value as control of insects themselves is typically an economically viable means just to protect yield and quality, which means it is readily adopted. The rapid adoption of Bt maize and cotton, and their associated reductions in mycotoxins in cases of high pressure of target insects, indicates the value of targeting insects for mycotoxin control. MARCEL DEKKER, INC. • 270 MADISON AvENUE • NEW YORK, NY 10016

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Insecticides are still likely to be of value in indirectly controlling mycotoxins in areas where Bt crops, or insects not affected by Bt crops, are planted. If plant resistance to insects is to be pursued as a strategy, a wider range of insect resistance mechanisms, or individual mechanisms with more broad spread efficacy against different insect species, will be needed.

ACKNOWLEDGMENTS

I thank W. P. Williams and C. A. Abel for permission to cite work in press, and anonymous reviewers for useful suggestions. Omission of any additional relevant studies was not intentional.

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