The impact of Bacillus thuringiensis technology on the occurrence of fumonisins and other mycotoxins in maize

In many developing countries, maize is both a staple food crop and a widely-used animal feed. However, adventitious colonisation or damage caused by insect pests allows fungi to penetrate the vegetative parts of the plant and the kernels, the latter resulting in mycotoxin contamination. Maize seeds contaminated with fumonisins and other mycotoxins pose a serious threat to both humans and livestock. However, numerous studies have reported a significant reduction in pest damage, disease symptoms and fumonisin levels in maize hybrids expressing the Bacillus thuringiensis (Bt) gene cry1Ab, particularly in areas where the European corn borer is prevalent. When other pests are also present, the cry1Ab gene alone offers insufficient protection, and combinations of insecticidal genes are required to reduce damage to plants caused by insects. The combination of Cry1Ab protein with other Cry proteins (such as Cry1F) or Vip proteins has reduced the incidence of pests and, indirectly, mycotoxin levels. Maize hybrids expressing multiple Bt genes, such as SmartStax®, are less susceptible to damage by insects, but mycotoxin levels are not routinely and consistently compared in these crops. Bt maize has a greater economic impact on Fusarium toxins than aflatoxins. The main factors that determine the effectiveness of Bt hybrids are the type of pest and the environmental conditions, but the different fungal infection pathways must also be considered. An alternative strategy to reduce mycotoxin levels in crops is the development of transgenic plants expressing genes that protect against fungal infection or reduce mycotoxin levels by in situ detoxification. In this review article, we summarise what is known about the relationship between the cultivation of Bt maize hybrids and contamination levels with different types of mycotoxins.


Introduction
Maize (Zea mays L.) is the most common source of fumonisins in human and animal diets.Maize seeds are often contaminated with fumonisins produced primarily by Fusarium verticillioides and Fusarium proliferatum.These fungi can infect the seeds, or the silks can be contaminated by airborne or waterborne conidia, systemic infections can be caused by contamination of the roots, or pest insects can injure the plants allowing fungal penetration (Munkvold and Desjardins, 1997).Other mycotoxins may be present alone or together with fumonisins, including aflatoxins, deoxynivalenol (DON), zearalenone (ZEA), and some recently-discovered Fusarium metabolites collectively known as emergent mycotoxins, such as moniliformin (MON), beauvericin, enniatins and fusaproliferin (Desjardins, 2006;Marín et al., 2013).Aflatoxins are mainly produced by Aspergillus flavus, which synthesises type B aflatoxins as well as cyclopiazonic acid (CPA) depending on the strain, and by Aspergillus parasiticus, which synthesises both type B and type G aflatoxins, but not CPA.DON and ZEA are mainly produced by Fusarium graminearum and Fusarium culmorum and they are often found as cocontaminants.Among the emergent mycotoxins, MON is the most prevalent and can be produced by several Fusarium species, including Fusarium avenaceum, Fusarium The impact of Bacillus thuringiensis technology on the occurrence of fumonisins and other mycotoxins in maize J. Díaz-Gómez 1 , S. Marín 1 , T. Capell 2 , V. Sanchis 1 and A.J. Ramos 1* tricinctum, F. proliferatum, Fusarium subglutinans and F. verticillioides (Marín et al., 2013).
The consumption of mycotoxin-contaminated kernels is associated with a range of diseases and disorders in humans and domestic animals, including cancer, immune system dysfunction and metabolic disorders (Marasas, 2001;Marín et al., 2013;Sobrova et al., 2010;Williams et al., 2004).Fumonisins and aflatoxins are carcinogens (aflatoxin B 1 (AFB 1 ) is the most carcinogenic natural compound known), and this creates a strong impetus to restrict the exposure of human and animal populations as far as possible (IARC, 1993(IARC, , 2002(IARC, , 2012)).In 2001, several countries submitted information on the concentration of fumonisins in maize and maize-derived foods, and fumonisins were detected in more than 60% of all food products (JECFA, 2001).These data are supported by a European Union report on the exposure of the EU population to Fusarium toxins.Among samples of raw maize material, 67% were positive for fumonisin B 1 (FB 1 ) (total=801) and a 51% were positive for fumonisin B 2 (FB 2 ) (total=544) (SCOOP, 2003).
The European Commission has set maximum levels for mycotoxins in maize and maize products.When such products are intended for human consumption, these values are currently 200-1000 µg/kg for fumonisins, 750 µg/kg for DON, 100 µg/kg for ZEA, 3 µg/kg for ochratoxin A (OTA), 5 µg/kg for AFB 1 , and 10 µg/kg for total aflatoxins.Recently, indicative levels of 100 µg/kg in maize were also established for the total content of the trichothecene mycotoxins T-2 and HT-2 toxin (EC, 2006b(EC, , 2007(EC, , 2010(EC, , 2012(EC, , 2013a)).In animal feed, only the level of AFB 1 is currently regulated in the EU (maximum 0.005-0.02mg/kg, depending on the type of feed).Other important mycotoxins in feed are limited by guidance values that vary with the species of livestock: 0.9-12 mg/kg for DON, 0.1-3.0mg/kg for ZEA, 0.05-0.25 mg/kg for OTA and 5-60 mg/kg for FB 1 +FB 2 .Recently, indicative levels of 250-2000 µg/kg in cereal products for feed and compound feed have been specified for the total content of T-2 and HT-2 toxin, with the exception of feed for cats, for which the guidance value is 50 µg/kg (EC, 2002(EC, , 2003b(EC, , 2006a(EC, , 2013a,b),b).
In contrast, the United States Food and Drug Administration (FDA) has proposed a guideline for total fumonisin levels in food of 2-4 mg/kg (depending on the product), and total aflatoxin levels of 20 µg/kg in maize and maize products for human consumption.For animal feed, the levels vary from 5 to 100 mg/kg for fumonisins and from 20 to 300 µg/kg for aflatoxins, depending on the animal species (FDA, 2000(FDA, , 2001)).

Factors that affect mycotoxin occurrence
The presence of mycotoxins in maize results from the interaction of several factors, including temperature and humidity, nutrient availability, the presence of other fungi, stress, and physical damage caused by pest insects.Before harvest, important factors include the weather (temperature, humidity and rainfall), exposure to insect pests, fungi and other pathogens, planting dates, the maize genotype and cropping system.Fumonisin contamination in maize is directly associated with Fusarium pink ear rot (mainly produced by F. verticilliodes) and its incidence depends on both environmental conditions and pest damage.Kernel damage caused by insects exposes the kernels to fungal spores, although there are several additional infection pathways.Fungal growth and mycotoxin accumulation can also be stimulated post-harvest by poor storage conditions such as high humidity and the presence of other pests (Marín et al., 2004;Miller, 2001).
Fungal growth and mycotoxin production are affected by multiple ecophysiological factors.The main factors that control fumonisin production in grain are temperature and water activity (a w ).F. verticillioides and F. proliferatum germinate at 5-37 °C when a w exceeds 0.88, although the growth range fluctuates in the range 7-37 °C when a w exceeds 0.90.The optimum conditions for fumonisin production by F. verticillioides are 30 °C at 0.97 a w and for F. proliferatum the corresponding values are 15 °C at 0.97 a w .Physicochemical and nutritional factors such as pH and carbon/nitrogen ratio can also affect fumonisin production.The presence of other fungi, such as A. flavus and Aspergillus niger, can affect the growth of Fusarium species, which is most competitive at 15 °C and 0.98 a w .At high a w values, fumonisin production can be stimulated by A. niger and other species (Marín et al., 1999;Picot et al., 2010;Sanchis et al., 2006).

Suppression of insect pests using Bt technology
Injuries caused by insects are common sites of fungal infection on maize ears and stalks.The fungi can be airborne or may be suspended in water droplets that splash the wound, but insects can also act as vectors.One of the most prevalent examples is the European corn borer (ECB) (Ostrinia nubilalis Hübner), a maize pest that not only injures plants and exposes them to infection, but also vectors fungal spores, particularly F. verticillioides and F. proliferatum.ECB therefore promotes Fusarium infection of maize kernels and stalks, and may reduce yields by increasing the incidence of stalk rot (Munkvold and Desjardins, 1997;Munkvold et al., 1997;Sobek and Munkvold, 1999).F. verticillioides is the most prevalent fungal pathogen of maize but fungicides are only partially http://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2015.1960-Reference Specialist <arspurchase@infotrieve.com> -Monday, July 11, 2016 1:03:16 PM -IP Address:24.38.73.2 effective, with efficacy depending on the pathogen strain and the fungicide mechanism of action (Falcão et al., 2011).Therefore, pest insects are more appropriate targets than fungi for the development of strategies to reduce mycotoxin levels in maize.
In many parts of the world, the management of ECB now relies on transgenic hybrid maize lines expressing the cry1Ab gene from the Gram-positive soil bacterium Bacillus thuringiensis (Bt).This gene encodes a potent pro-toxin that is activated in the alkaline environment of the insect gut and is highly specific towards particular insect species.These insecticidal crystal proteins are also named δ-endotoxins or Cry proteins.B. thuringiensis has been used since 1938 to produce an insecticidal spray, but Bt transgenic plants resistant to ECB larvae were first made available in the USA in 1996 and in the EU in 1998.Different strains of the bacterium express different cry genes producing different pro-toxins that can protect plants against many different pests, including the corn rootworm complex (Diabotrica virgifera) (EPA, 2011;Höfte and Whiteley, 1989;Koziel et al., 1993;Schnepf et al., 1998).Therefore, alternative Bt genes (such as cry1F) have also been expressed in maize to protect against further lepidopteran pests (Abbas et al., 2013;Bowers et al., 2013;Koziel et al., 1993).Economically-important maize pests that can be partially controlled using Bt hybrids include the corn earworm (CEW; Helicoverpa zea), common stalk borer (Papiapema nebris), southwestern corn borer (SWCB; Diatraea grandiosella) and western bean cutworm (WBC; Striacosta albicosta), whereas this strategy has proven less efficient against the fall armyworm (FAW; Spodoptera frugiperda) and black cutworm (Agrotis ipsilon) (Bowers et al., 2013(Bowers et al., , 2014;;Dowd, 2000;Munkvold and Hellmich, 1999;Williams et al., 2002Williams et al., , 2005Williams et al., , 2006)).Table 1 lists the Bt events targeting lepidopteran pests and the corn rootworm complex that are currently commercially available in the USA.
Since its adoption in the USA, Bt maize has become the second most widely cultivated genetically modified (GM) crop worldwide, after herbicide-tolerant soybean.About 30% of global maize production in 2014 (184 million ha) was represented by GM varieties (55.2 million ha) (James, 2014).However, the EU has a strict, complex and contradictory legislative framework for GM crops, with only the Mon810 maize event currently authorised for cultivation (EC, 1998(EC, , 2003a(EC, , 2008)).

Fumonisin contamination in Bt and non-Bt maize
The ability of Bt genes to protect maize against ECB and other lepidopteran pests means that Bt maize tends to suffer a lower frequency of fungal infections and the infections that occur are often less severe or even symptomless.In the case of fumonisins, there is a large body of evidence to support the benefits of Bt maize, as shown by the comparison of fumonisin concentrations in Bt and non-Bt maize hybrids in different field locations (Supplementary Table S1).Munkvold et al. (1999) published a fundamental study concerning the effect of Bt maize on disease management and concluded that transgenic hybrids expressing cry1Ab were less susceptible to ECB, suffered less from Fusarium ear rot and had lower fumonisin levels than their nontransgenic counterparts.However, if other insect pests were present alone or concurrent with ECB then the levels of fumonisins remained high (Clements et al., 2003;Dowd, 2000;Hammond et al., 2004;Papst et al., 2005).
Importantly, Bowers et al. (2013) confirmed that lower levels of fumonisins were present in Bt hybrids exposed to ECB, but found that a cry1Ab × vip3Aa hybrid was more resistant to ECB, CEW and WBC than the cry1Ab hybrid and the non-Bt hybrid in all of the years covered by the study.The vegetative insecticidal protein Vip3Aa can therefore be combined with other toxins, such as Cry1Ab to target additional lepidopteran pests.Unlike the cry genes, which are expressed during sporulation, vip genes are expressed during the B. thuringiensis vegetative growth phase and they do not share sequence homology with cry genes (Lee et al., 2003;Schnepf et al., 1998).The cry1Ab × vip3Aa hybrid also showed a lower level of pest damage, a lower incidence of Fusarium ear rot and lower levels of fumonisins when infested with ECB, whereas cry1F hybrids were better protected against WCB because cry1F specifically targets this pest (Bowers et al., 2014).
A comparison of eight commercially available Bt hybrids expressing multiple genes found no significant differences in the content of fumonisins among the hybrids (Abbas et al., 2013).Recent studies of SmartStax® maize, which produces Cry1F, Cry1A.105+Cry2Ab2,Cry34Ab1/Cry35Ab1 and Cry3Bb1 to protect against common lepidopteran pests and the corn rootworm complex, reported less pest damage compared with single Bt hybrids and non-Bt hybrids, but did not consider mycotoxin levels (Head et al., 2014;Rule et al., 2014).

Aflatoxin contamination in Bt and non-Bt maize
Whereas the link between Bt maize and lower fumonisin levels is clearly established, the data for aflatoxin contamination are more contentious (Ostry et al., 2015).Windham et al. (1999) showed a significant correlation between fungal and insect exposure, inoculation or infestation dates and aflatoxin contamination.They found that Bt hybrids suffered less damage from insects and had lower aflatoxin levels than the other hybrids studied (A.flavus resistant and A. flavus susceptible hybrids and a non-Bt isogenic hybrid) when manually infested with SWCB.More recent studies have focused on the inoculation technique, showing that inoculation with A. flavus by kernel wounding, which facilitates fungal penetration, results in high-level aflatoxin contamination regardless of the hybrids used.There were no differences among the hybrids when infected with fungi alone because lower aflatoxin levels in the Bt hybrids reflected the reduction in insect damage, which indirectly reduced fungal contamination.In contrast, a non-wounding inoculation technique combined with SWCB infestation resulted in significantly lower levels of aflatoxin in the Bt hybrids (Williams et al., 2002(Williams et al., , 2005;;2006).A testcross involving aflatoxin-resistant and aflatoxin-susceptible lines crossed with Bt and non-Bt maize revealed lower aflatoxin levels in the Bt testcrosses, but the difference for individual lines was significant in only two of 10 lines investigated.The low insect pressure during the experiment could explain these results, because the higher the insect pressure, the greater the differences between hybrids (Williams et al., 2010).
In a 3-year study, Wiatrak et al. (2005) observed significantly lower aflatoxin levels in Bt compared to non-Bt hybrids during the first year of the experiment, but there was no difference with a tropical non-Bt hybrid.In the second year, significantly lower levels of aflatoxins were detected in the Bt hybrids than the tropical non-Bt hybrid, but there were no differences compared to the non-Bt hybrids.In the final year, there were no differences in aflatoxin contamination among the hybrids.Another 3-year study reported lower levels of aflatoxins in Bt than non-Bt hybrids, but only in one of the years (Abbas et al., 2006;2007;Bruns and Abbas, 2006).Nevertheless, a subsequent study showed that aflatoxin contamination was significantly reduced in the Bt hybrid compared to its non-Bt isoline (Abbas et al., 2008).The authors continued these field trials until 2009, reporting lower mycotoxin levels in Bt maize, but the difference was not significant, perhaps due to the continuous cultivation (Abbas et al., 2013).
In the USA, Odvody et al. (2000) observed less insect damage in Bt hybrids but aflatoxin levels were not consistent.In a subsequent study of different Bt hybrids, the lowest level of insect damage was observed in the Mon840 event (cry2Ab) correlating with significantly lower aflatoxin levels compared to non-Bt and cry1Ab hybrids in 2000, but only compared to the non-Bt hybrid in 2001 (Odvody and Chilcutt, 2002).Different cryAb events were also evaluated, revealing less insect damage in the Bt hybrids Mon810 and Bt11 compared to non-Bt hybrids, but aflatoxin levels were also inconsistent in this experiment (Odvody and Chilcutt, 2003).Similarly, Maupin et al. (2001) did not find significant differences in the levels of ear rot and aflatoxin accumulation when comparing Bt and non-Bt hybrids inoculated with A. flavus.Buntin et al. (2001) found no significant differences in aflatoxin levels between Bt and non-Bt maize, but the Bt hybrids suffered less severe FAW infestations.These data indicate that insect damage is strongly correlated with fumonisin levels but not aflatoxin levels, suggesting that other factors such as drought stress and individual hybrid vulnerability may play a more dominant role than insect damage in the determination of aflatoxin levels.The field experiments concerning aflatoxin levels in Bt and non-Bt hybrids are summarised in Supplementary Table S2.

Contamination with other mycotoxins in Bt and non-Bt maize
The results obtained with other mycotoxins are also controversial.Significantly lower levels of DON were observed in Bt compared to non-Bt hybrids in some studies (Magg et al., 2002;Schaafsma et al., 2002;Selwet, 2011;Valenta et al., 2001), whereas in other cases the mycotoxin levels appeared to be location-dependent (Bakan et al., 2002;Papst et al., 2005;Pinson et al., 2002) or there was no difference between Bt and non-Bt hybrids (Barros et al., 2009).A few studies have even found evidence for slightly higher DON levels in Bt hybrids, although the location was an important confounding effect (Folcher et al., 2010;Bakan et al. 2002).Schaafsma et al. (2002) analysed 102 commercial maize fields in Canada, reporting a reduction in DON levels in Bt hybrids depending on the severity of ECB infestation in each field.This was supported by a study carried out in Germany showing a reduction in DON levels in Bt compared to non-Bt hybrids (Valenta et al., 2001).In another study, also in Germany, Magg et al. (2002) found significantly lower concentrations of DON in Bt maize in one of the two years of the experiment.Pinson et al. (2002) described differences in DON and ZEA levels between plots in two different fields in south and central France.Lower levels were observed in two Bt hybrids, but two others contained significantly higher levels of both DON and ZEA compared to the corresponding non-Bt cultivars.Bakan et al. (2002) reported low ZEA levels in their study, but significantly higher concentrations were observed in a traditional cultivar in France.In all the studies, the Bt hybrids expressed cry1Ab (Supplementary Table S3).
Only one study has investigated the impact of Bt on MON levels, and the Bt hybrids showed significantly lower levels of MON than non-Bt hybrids (isogenic and commercial hybrids) when infested with ECB.MON levels were significantly higher following the manual infestation of unprotected plants (296 µg/kg) compared to those treated with insecticide (66.2 µg/kg).In the infested plots, the MON concentrations were 153.5, 336.7 and 266.1 µg/kg for the transgenic, isogenic and commercial hybrids, respectively.In contrast, in the protected plots, the MON concentrations were 49.1, 99.3 and 42.9 µg/kg for the transgenic, isogenic and commercial hybrids, respectively (Magg et al., 2003).

Economic impact of mycotoxin reduction in Bt maize
The main goal of Bt technology is to reduce pest damage and promote higher yields.However, indirect benefits, such as the reduction of fumonisin levels, also increase the percentage of maize grain that meets US and/or EU regulatory limits, which can have a significant economic impact and may also reduce the prevalence and severity of human and animal diseases (Bowers et al., 2013;Folcher et al., 2010;Hammond et al., 2004;Magg et al., 2002Magg et al., , 2003;;Munkvold et al., 1997Munkvold et al., , 1999;;Pinson et al., 2002).
Estimations for the cost of crop losses due to mycotoxin contamination in the USA range from US$ 500,000 to US$ 1.5 billion, reflecting variations in contamination levels, regulatory limits, price variations and production outputs (CAST, 2003).However, in the USA most losses are regulatory in nature (i.e. based on the rejection of grain based on quality) rather than actual harvest losses, and maize grains rejected for food and feed use may still be suitable for industrial processes, such as biofuel production.
On the other hand, the economic benefit of Bt maize in the USA has been specifically valued at US$ 8.8 million in terms of preventing losses caused by fumonisins, and similarly US$ 8.1 million for DON and US$ 14 million for aflatoxins, even though the aflatoxin levels depend on the predominant pest species (Wu, 2006(Wu, , 2007;;2014;Wu et al., 2004).These data based on studies carried out over a decade ago do not take into account the increased adoption of Bt maize, which has risen from 30% in 2005 to more than 80% in 2015 (USDA-ERS, 2015).A recent study of the Thailand maize market estimated the economic losses due to aflatoxins.A loss of US$ ~6.9 million per annum was estimated assuming low levels of aflatoxin contamination (data from harvest and dried maize supplied by a pet company) but this increased to US$ ~100 million per annum assuming higher aflatoxin levels (data from retail markets).The rejection of aflatoxincontaminated maize by the livestock sector is the most influential factor contributing to economic losses.Thus, the selection of high-quality maize (by the pet company) reflects lower levels of aflatoxin contamination, and lower economic losses (Lubulwa et al., 2015).Bt technology can improve the quality of maize by reducing mycotoxin levels as an indirect consequence of preventing infestations with insect pests.This technology also reduces the need for chemical insecticides, resulting in lower levels of pesticide residues in food and water and less environmental impact (Brookes and Barfoot, 2013;Qaim, 2009).

Alternative strategies to reduce mycotoxin levels in maize
Although targeting pest insects can help to reduce opportunistic fungal infections of maize, other transgenic approaches have emerged more recently in which the fungus itself is the target.For example, Kant et al. (2012) reported a field study of transgenic maize expressing a modified rice Rp13 gene encoding ribosomal protein L3, a primary target of DON.They developed two transgenic maize lines expressing the Rp13 gene, one using the constitutive CaMV 35S promoter and the other using the silk-specific ZmGRP5 promoter.Both plants were less susceptible to F. graminearum than wild-type plants, and those containing the silk-specific promoter were the most tolerant, with the mildest symptoms under field conditions (DON levels were not evaluated).The difference in efficacy may http://www.wageningenacademic.com/doi/pdf/10.3920/WMJ2015.1960-Reference Specialist <arspurchase@infotrieve.com> -Monday, July 11, 2016 1:03:16 PM -IP Address:24.38.73.2 reflect the broader activity of the silk promoter in the seed pericarp tissue.Maize silks are the primary route used by F. graminearum to infect the kernels.Expression of the modified Rpl3 gene in silk tissue may therefore help to reduce Gibberella ear rot and hence DON levels.
Maize plants expressing the α-amylase inhibitor protein from Lablab purpurea (AILP) can also be used to reduce fungal infection.Fungal amylases liberate fermentable sugars from kernel starch which are essential for mycotoxin production.Kernel screening assays in AILP-transgenic maize plants revealed aflatoxin levels 56% lower than controls.AILP expression therefore appears to reduce both fungal growth on the kernels and aflatoxin accumulation (Chen et al., 2015).
Another promising approach to reduce mycotoxin contamination is enzymatic mycotoxin detoxification in situ, which converts the mycotoxins into less toxic compounds.
The ZEA lactone ring is sensitive to hydrolysis by the fungus Clonostachys rosea, which synthesises an alkaline lactonohydrolase responsible for detoxification (Kimura et al., 2006;Takahashi-Ando et al., 2002).The corresponding gene (zhd101) was able to reduce ZEA levels in vitro and in field-grown plants compared to non-transgenic controls, even when infected with F. graminearum.The ability of transgenic seeds to degrade ZEA was evaluated by immersing the seeds in 50 µg/ml ZEA for 48 h.The kernel tissues were then analysed by HPLC, showing that wild-type seeds contained 24.6±1.7 µg ZEA/g, whereas the transgenic seeds contained only 1.6±0.4µg ZEA/g.The detoxification of ZEA in Fusarium-infected transgenic kernels was evaluated after inoculation with F. graminearum.The wildtype seeds contained 15.4±3.7 ng ZEA/g, whereas the level of ZEA in non-inoculated seeds and inoculated transgenic seeds was below the detection threshold (Igawa et al., 2007).
Similarly, the yeasts Exophiala spinifera and Rhinocladiella atrovirens, and the Gram-negative bacterium ATCC 55552, can produce enzymes that metabolise fumonisins.Duvick et al. (2003) patented a fumonisin esterase produced by these yeasts which can hydrolyse the tricarballylate esters of FB 1 , and this is active in transgenic maize.The esterase gene reduced fumonisin levels in Fusarium-infected grain from 1.522 mg/kg without the enzyme to 0.379 mg/kg in esterase positive plants.Aflatoxin detoxification in transgenic plants has yet to be reported (Duvick, 2001;Hartinger and Moll, 2011;Jard et al., 2011).

Concluding remarks
Several transgenic strategies can be used to reduce mycotoxin contamination in food and feed but Bt maize hybrids are widely grown and several studies have confirmed the reduction in pest damage, disease symptoms and fumonisin levels, particularly when ECB is the predominant pest.This is because fumonisin levels are reduced in Bt hybrids if the Fusarium population is dominated by species whose colonisation of the plant is promoted by ECB damage (Miller, 2001).
Among the commercial Bt hybrids, Mon810 and Bt11 (which express cry1Ab) were associated with a reduction in the occurrence of Fusarium ear rot and fumonisins due to the lower level of kernel damage caused by susceptible lepidopteran pests (Dowd, 2000(Dowd, , 2001;;Magg et al., 2001;Munkvold and Hellmich, 1999;Papst et al., 2005).Conversely, no difference has been reported between Bt 176 hybrids (Bt event 176 was withdrawn in 2001) and non-Bt maize (Magg et al., 2002;Schaafsma et al., 2002), and it has even been proposed that resistance against ECB and Fusarium ear rot may be inherited independently (Magg et al., 2002;Miller, 2001).
The pest species and its abundance are key determinants of mycotoxin levels, particularly if the main pest is CEW which is unaffected by Cry1Ab (Clements et al., 2003;Dowd, 2000;Hammond et al., 2004).Lower levels of aflatoxins occur in Bt maize if the main pest is SWCB or CEW, but there is no difference between Bt and non-Bt maize if the pest is FAW (Buntin et al., 2001;Williams et al., 2002Williams et al., , 2005Williams et al., , 2006Williams et al., , 2010)).When other pests are present, hybrids expressing Cry1Ab are inefficient and must be combined with further Cry proteins (such as Cry1F) or Vip proteins to increase the level of protection (Bowers et al., 2013;2014).Thus, if Bt events are not selected by taking into account prevalent insect pests and environmental conditions in each field, Bt technology will not be effective.Hence the importance of Bt hybrids expressing multiple genes, whose performance in the presence of different pests and climate conditions has yet to be studied in detail.
The impact of Bt maize on the accumulation of aflatoxins, DON and ZEA is inconclusive because the extent of contamination depends on many interrelated factors.The different fungal infection pathways must be considered to understand the effect of Bt hybrids.The most common route used by F. verticillioides to infect the kernel is through the silks or through wounds caused by insect pests, whereas F. graminearum primarily reaches the kernels via the silks (Munkvold, 2003;Munkvold and Desjardins, 1997).
A. flavus can infect maize kernels through the silks too.This may explain why Bt maize hybrids that are resistant to insect pests have less Fusarium ear rot and lower fumonisin levels, whereas the relationship with DON, ZEA and aflatoxin levels is not so clear cut.Further studies are necessary to determine the interaction between Bt maize and the different fungal species that produce these toxins.
Mycotoxin contamination is frequently linked with drought, heat stress and insects.Drought favours the accumulation of fumonisins more than heat stress (Miller, 2001).A 2-year study was carried out by Traore et al. (2000) to characterise the effect of drought stress on Bt maize.Water deficit during the vegetative period delayed leaf emergence, reduced the leaf area and caused stunted growth in both Bt and non-Bt hybrids.It also reduced grain and biomass yields and kernel number per ear during both years.However, the Bt hybrids had greater biomass in 1997 and greater grain yields in 1998 because they were not so severely infected by secondgeneration ECB.Therefore, Bt maize continues to play an important role in insect resistance under drought stress.
The first commercially available drought-tolerant GM maize variety (MON87460) expresses a bacterial cold shock protein B (CspB), a molecular chaperone derived from Bacillus subtilis, which may provide a yield advantage under limited water availability.A recent field study confirmed the higher grain yield of MON87460 under drought conditions compared to a conventional hybrid (Nemali et al., 2015).The combination of Bt and drought-tolerant maize should therefore achieve even higher yields and lower mycotoxin levels because it will be protected against two major environmental stress factors.
Studies that considered aflatoxins and fumonisins simultaneously reported variable results, suggesting that diverse environmental conditions may prevent the control of both mycotoxins in the same crops (Abbas et al., 2002(Abbas et al., , 2006(Abbas et al., , 2007(Abbas et al., , 2008;;Bruns and Abbas, 2006).More studies are needed to determine whether aflatoxin resistance traits can be crossed into Bt hybrids.Aflatoxin-resistant germplasm tends to possess undesirable agronomic traits such as tight husk coverage and late maturity.Breeding programs aiming to achieve the introgression of aflatoxin resistance into Bt hybrids could remove these undesirable characteristics while reducing aflatoxin contamination (Williams et al., 2008(Williams et al., , 2010)).Bt maize has a greater economic impact on Fusarium mycotoxins than aflatoxins (Wu, 2006(Wu, , 2007;;Wu et al., 2004).Further studies are needed to evaluate the effect of both Bt hybrids expressing multiple genes and Bt hybrids combined with maize lines that are resistant to the accumulation of other mycotoxins, especially aflatoxins.
A potential risk that must be borne in mind comes in the form of mycotoxin derivatives (modified mycotoxins) that escape routine analytical techniques but may be digested by animals triggering toxic effects comparable to free mycotoxins.These derivatives should be included in the total mycotoxin allowances, and future legislation must consider their presence even though this would increase the stringency of testing and rejection, resulting in further economic loss.This highlights the benefits of Bt maize, which would reduce the levels of mycotoxins and potentially their derivatives, although the impact of Bt hybrids on the accumulation of modified mycotoxins needs to be addressed in more detail (De Boevre et al., 2012, 2014;De Saeger and Van Egmond, 2012;Wu, 2006).Finally, the development of transgenic plants expressing genes that protect against fungal infection (e.g. the modified Rp13 gene and the AILP transgene) or reduce mycotoxin levels by in situ detoxification (e.g.zhd101 and fumonisin esterase) could provide an additional strategy to control mycotoxins (Chen et al., 2015;Duvick, 2001;Duvick et al., 2003;Igawa et al., 2007;Kant et al., 2012) and could be combined with Bt hybrids to provide additive or even synergistic protection against mycotoxin-producing fungal pathogens.