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Crop Protection 28 (2009) 302–308

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Crop Protection

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Comparative activity of agrochemical treatments on mycotoxin levels with regardto corn borers and Fusarium mycoflora in maize (Zea mays L.) fields

Laurent Folcher a, Marc Jarry c,g, Alain Weissenberger d, Florence Gerault f, Nathalie Eychenne e,Marc Delos b, Catherine Regnault-Roger a,*

a Universite de Pau et des Pays de l’Adour, UMR CNRS 5254, Institut Pluridisciplinaire Pour l’Environnement et les Materiaux/Equipe Environnement et Microbiologie(IPREM/EEM), IBEAS, BP 1155, F-64013 Pau, Franceb Ministere de l’Agriculture et de la Peche, Service Regional de la Protection des Vegetaux, ‘‘Midi-Pyrenees’’, Bat. E, Boulevard Armand Duportal, F 31074 Toulouse, Francec Universite de Pau et des Pays de l’Adour, UMR 1224 ECOBIOP, IBEAS, BP 1155, F-64013 Pau, Franced Ministere de l’Agriculture et de la Peche, Service Regional de la Protection des Vegetaux « Alsace », Station d’Experimentation, Route de Saverne, F 67370 Wiwersheim, Francee Federation Regionale de Defense contre les Organismes Nuisibles, FREDEC Midi-Pyrenees, Bat 43, 2 Route de Narbonne, B.P. 12267, F 31322 Castanet Tolosan, Francef Ministere de l’Agriculture et de la Peche, Service Regional de la Protection des Vegetaux « Pays de Loire », 10 Rue le Notre, 49044 Angers, Franceg INRA, UMR 1224 ECOBIOP, F-64310 Saint-Pee sur Nivelle, France

a r t i c l e i n f o

Article history:Received 9 May 2008Received in revised form11 November 2008Accepted 12 November 2008

Keywords:Maize (Zea mays L.)Maize/corn borersFusarium mycofloraMycotoxinsAgrochemical treatmentInsecticideFungicide

* Corresponding author. Tel.: þ33 (0)5 59 40 74 79E-mail address: catherine.regnault-roger@univ-pa

0261-2194/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.cropro.2008.11.007

a b s t r a c t

Field trials were carried out in nine areas located in France during 2004, 2005 and 2006 to study thecontrol of Lepidoptera caterpillars by agrochemical treatments and their consequences on Fusarium spp.mycoflora and mycotoxin levels. Treatments involved either an insecticide or an insecticide–fungicideassociation. Two species of maize borers: Ostrinia nubilalis Hubner [Lepidoptera: Crambidae] and Sesamianonagrioides Lefebvre [Lepidoptera: Noctuidae], were monitored. Although the insect populations werecontrolled by agrochemicals, there was no reduction in Fusarium spp. mycoflora. Conversely a significantreduction of mycotoxin (trichothecenes, fumonisins and zearalenone) levels resulted from insecticidetreatment. These experiments and results are discussed regarding the biology of maize borers andrelationships with Fusarium spp.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Among the major pests of maize (Zea mays L.) occurringcurrently in France, the European maize (corn) borer (ECB) Ostrinianubilalis Hubner [Lepidoptera: Crambidae] is the most damaginginsect (Agusti et al., 2005), followed by the maize (corn) stalk borer(CSB) Sesamia nonagrioides Lefebvre [Lepidoptera: Noctuidae](Albajes et al., 2002), way ahead of the other borers Heliothisarmigera Hubner [Lepidoptera: Noctuidae] and Mythimna uni-punctata Haworth [Lepidoptera: Noctuidae]. O. nubilalis occursthroughout the country although S. nonagrioides is essentiallyobserved in the South of France. O. nubilalis is a very cosmopolitaninsect occurring on about 223 plants (Lewis, 1975), with five larval

; fax: þ33 (0)5 59 40 74 94.u.fr (C. Regnault-Roger).

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instars. According to the location, it has one generation per year(monovoltinism) in NorthEastern France, or two generations,exceptionally three (multivoltinism) in the South. S. nonagrioides isconsidered less damaging. It has seven larval instars, but is strictlymultivoltine with two generations per year in SouthWestern France(Delos et al., 2007). These two borers are responsible for 5–27% lossin crops (up to 80% with high proliferation) (Krattiger, 1997).Moreover, these two pests are considered an aggravating factor forear rot infection, because of the presence of mycotoxins at harvest.Maize crop quality was affected qualitatively and quantitatively(Dowd and Munkvold, 1999; Sobek and Munkvold, 1999).

Several studies have established that the control of Lepidopteraborers affected mycotoxin levels within harvested maize. This wasdemonstrated by methods such as prophylaxis (Almaa et al., 2005),biological control with parasitoıds (Dowd, 2000) and geneticcontrol involving GMO Bt technology (Munkvold et al., 1999;Schaafsma et al., 2002; Dowd, 2003). Our work focussed on an

Table 1Trials: geographical locations, maize cultivars and block number.

Area Locality Meteorologicalstation

ZipCode

Cultivar Company

North East 1. Wiwersheim Wiwersheim 67370 Moncada SyngentaSeeds

2. Hurtigheim Wiwersheim 67117 Magistral KWS Saat AG3. Moyenvic Chateau Salins 57630 DK312 Dekalb

Company

Middle East 1. Laloye Tavaux 39380 Pollen MaısAdour2. Charmes Cintrat 03800 DK315 Dekalb

Company3. Saint Genesdu Retz

Cintrat 03380 DK315 DekalbCompany

SouthWestern

1. Castelnaudary Castelnaudary 11400 KWS1393 KWS MaısFrance

2. Thure Marigny Brizay 86380 DK315 DekalbCompany

3. Courpiac Rauzan 33760 DK532 DekalbCompany

L. Folcher et al. / Crop Protection 28 (2009) 302–308 303

evaluation in a comparative study of the efficiency of chemicalpesticide treatments on insect populations, Fusarium spp. myco-flora at harvest and mycotoxin levels within maize kernels.

These experiments involved two kinds of treatments: aninsecticide alone, and an insecticide with a fungicide. The aim of thetreatments was to see if the chemical control of Lepidoptera by aninsecticide decreased mycotoxin levels in the maize, and if theassociation of insecticide plus fungicide was synergistic. Ina previous work on maize it was shown that the fungicide treat-ment alone was inefficient (Weissenberger, unpublished).

2. Materials and methods

2.1. Field trials

Experiments were carried out by the ‘‘Services Regionaux de laProtection des Vegetaux (SRPV)’’ of the French Ministry of Agri-culture and the ‘‘Federations Regionales de Defense contre lesOrganismes Nuisibles (FREDON)’’ in nine fields located all overFrance: Wiwersheim, Hurtigheim and Moyenvic (North EastFrance), Courpiac, Thure and Castelnaudary (South West France),and Laloye, Saint Genes du Retz and Charmes (Middle and EastFrance) (Fig. 1). These trials were located in monovoltine as well asmultivoltine areas. According to the geographic localization and theclimatic conditions, especially temperature average (Table 2), threeareas could be distinguished: North East, Middle East and South-Western including three trial locations in each area (Fig. 1, Table 1).Experimental sites were characterized by an intensive maizeproduction. The maize cultivars involved in these trials are repre-sentative of the cultivars cultivated within the areas chosen for thefield trials (Table 1). The choice of these cultivars was guided by theduration of crop development (earliness factor) in such a way thatthe harvest occurred in all trials simultaneously. Regardless of thelocation of the trials, maize reached maturity in about 150 days.

Field trials were carried out under natural conditions during thesummers of 2004, 2005 and 2006. Fields were seeded over a periodof 20 days beginning April 15, 2004, 2005 and 2006. Temperaturesand recorded rainfall during the bioassays were, respectively,between 16–20 �C, and 246–519 mm (Table 2). The schedule fortreatments was determined according to insect monitoring in thefield. Monitoring within a 30-day period between 15 April and 15May with regard to trial’s latitude, began by collecting maize borerlarvae within the field. This was in order to rear them and toobserve their growth to determine when they were going to changeinto imagos and fly. If the level of infestation in the trial field wasnot significant, the insects were collected in a neighboring fieldlocated in a circle of 2 km diameter, which represented the longest

Fig. 1. Location of the field trials in France.

distance an ECB can fly. The insect rearing was supervised bya specialized network (‘‘reseaux d’Alertes’’�) under the control ofFrench Ministry of Agriculture covering the entire French territory.They counted larvae, pupae and imagos in order to indicate tofarmers the best period for treating the fields.

In monovoltine areas colonized by ECB only, the ECB caterpillarscame out of winter diapause at the end of May–beginning of June,and pupation occurred 2 weeks later under the effect of the longerdaylight and the increase in humidity level in the field (Eychenne,1997). This step took 3 weeks. A temperature of 13 �C is regarded tobe the thermal threshold for insect development (Guennelon,1972). At the end of June and beginning of July, the male imagoshatched first and then the females (protandry) which attracted themales by sexual pheromones. After fertilization they laid synovo-genic eggs (Stengel, 1982). The latter hatched into larvae whichdeveloped from the black head stage to L5 in 40–50 days. Cater-pillars then prepared to enter winter diapause. In multivoltineareas colonized by both ECB and CSB, the ECB caterpillars came outof winter diapause in April–beginning of May and pupationoccurred 2 weeks later (Eychenne, 1997). Over a period of 2 weeks,the imagos hatched at the end of May and beginning of June. Thefemales oviposited and larvae developed until L5. A new pupationtook place and a second generation of imagos emerged from theend of July until middle of August. From these imagos, eggs, thenlarvae, were formed in a second cycle of development until thecaterpillar entered winter diapause (Stengel and Schubert, 1982).The rearing of these insects from the field made possible a precisemonitoring of their reproductive cycle. It allowed the period when50% of imagos emerged (emergence peak) to be determined. It wasat this point that the insecticide should be applied on ECB formaximum efficiency. In the field infested by both ECB and CSB, theECB emergence peak matches with CSB L3 which is the step moresusceptible to insecticide (Eychenne, 1997).

Two kinds of pesticides were used. The insecticide deltamethr-ine (20 g ha�1) was sprayed at the time of each emergence peak inthe trials. The fungicide tebuconazole (250 g ha�1) was sprayed inassociation with the insecticide at the time of maize female flow-ering. Deltamethrine (C22H19Br2NO3, CAS RN 52918-63-5) like allpyrethroids interfered with the sodium channels so that no trans-mission of nerve impulses could take place, whereas tebuconazole(C16H22CIN3O, CAS RN 107534-96-3) is an inhibitor of the biosyn-thesis of sterols (ergosterol) focused on C14-demethylase (Tomlin,2003).

Bioassays were arranged in a randomized block design. Eachassay involved four blocks (Fig. 2) of surface area of 120 m2 for each

Table 2Climatic conditions during experiments: means of temperature (�C), relative humidity and pluviometry for each field trial and the averages for areas (May to October).

Sites Tmoy (�C) Pluviometryin mm

Pluviometry daynumber (>5 mm)

% Of favorable daysfor spore release

Hygrometry daynumber (>90%)

% Of favorable daysfor spore germination

North East 1 16.14 385 23 12.50 27 14.67North East 2 16.08 296 17 9.24 19 10.33North East 3 17.50 393 20 10.93 32 17.49

North East Area 16.57 358 60 10.91 79 14.36

Middle East 1 17.2 321 17 9.24 21 11.41Middle East 2 17.5 289 17 9.14 10 5.38Middle East 3 17.90 519 28 15.22 5 2.72

Middle East Area 17.44 349 62 11.23 36 6.52

SouthWestern 1 19.53 246 12 6.56 32 17.49SouthWestern 2 18.26 359 24 13.41 14 7.82SouthWestern 3 18.35 439 22 11.96 17 9.24

SouthWestern Area 18.72 348 58 10.62 63 11.54

Consequences for spore germination and release.

L. Folcher et al. / Crop Protection 28 (2009) 302–308304

replication. At harvest, caterpillars of the two borers were countedwithin the whole plant (dissection of stalks and ears on20 plants plot�1). Ear rot development was first evaluated in thefield by a rating scale developed by Reid et al. (1992), at harvest. Theears were collected manually and the scale of the intensity of attackwas assessed visually.

These plants (240 field trial�1; 2160 in total for the experiment)were sent to the laboratory for dissection. The identification ofmycoflora and the analysis of mycotoxins were done on twosamples of kernels of 1 kg each for each treatment per field trial.

2.2. Fungal identification

Fungal species were identified by the ‘‘Laboratoire National deProtection des Vegetaux’’ – Unite de Mycologie Agricole et Forest-iere (Nancy zip F-54000), according to the Official Method of FrenchAgriculture Ministry (Ioos et al., 2004). This method used DCPA(Dichloran Chloramphenicol Peptone Agar) and PDA (PotatoDextrose Agar) medium for isolation, SNA (Solution Nutrient Agar)and PDA medium for species determination. Species identificationwas carried out according to the reference keys of Nelson et al.(1983) and Nirenberg (1982).

The following fungal species of the Fusarium genus were iden-tified: Fusarium culmorum, Fusarium graminearum, Fusariumcrookwellense, Fusarium sambucinum, Fusarium avenaceum, Fusa-rium poae, Fusarium sporotrichioides, Fusarium tricinctum, Fusariumequiseti, Fusarium verticillioides, Fusarium proliferatum or stillFusarium subglutinans. The two main species producing the fumo-nisins were F. verticillioides and F. proliferatum and the two mainspecies producing trichothecenes and the zearalenone wereF. culmorum and F. graminearum. Their required temperatures andmoisture for mycotoxin production were the following. Fortemperatures, F. verticillioides needed 25–30 �C with a range of5–40 �C and a temperature for toxinogenesis of 20 �C (Marin et al.,1995; Thibault et al., 1997), F. proliferatum: 25 �C for a 15 �C for

Fig. 2. Experimental field design (surface: 480 m2).

toxinogenesis (Marin et al., 1995; Melcion et al., 1998), F. grami-nearum: 24–26 �C for 15 �C for toxinogenesis (Caldwell et al., 1970)and F. culmorum 20–25 �C for 15 �C for toxinogenesis (Brennanet al., 2005; Magan, 2006). Several rain showers of >5 mm wererequired to release the spores of fungi from the ascospores anda daily RH of up to 90% was required to induce spore germination(Gilbert, 2003).

The number of days with an average moisture of >90% wasconsidered over the 4 months covering the trials (beginning of Juneuntil the end of October) because relative humidity has an influenceon spore germination. The number of days with rain showers of>5 mm (spore release) was considered over the same period. Thepercentage of days suitable for spore release and germination wascalculated for each trial location as well as the averages per area(Table 2).

2.3. Mycotoxin analyses

Mycotoxins (trichothecenes A, B, D; fumonisins B1þ B2; zear-alenone and derivatives) were analyzed by LC–MS–MS by the‘‘Laboratoire de developpement et d’analyses’’ (Ploufragan 22,France). The following mycotoxins were identified: trichothecenesA (T2 toxine, HT2 toxine, DAS Verrucarol, 15 acetoxyscirpenol);trichothecenes B (Nivalenol, Desoxynivalenol (DON), Fusarenone x,150O0ac040 DON, 3 acDON); trichothecenes D (Roridin A, Verru-carin A); fumonisins (Fumonisin B1, Fumonisin B2); and zear-alenone and metabolites (Zearalenone, zearalanol alpha, zearalanolbeta, zearalenol alpha, zearalenol beta).

All reagents, acetonitrile, methanol (SDS and Carlo Erba, Val deReuil 27, France), pure water, and acetic acid LC–MS grade (Fluka,Buchs, Switzerland) were analytical HPLC grade. Standards wereobtained from Biopure and Sigma. Stock solutions were prepared inacetonitrile and were solubilized with 0.01% of acetic acid for LC–MS/MS calibration. Maize samples (1 kg of grain sample�1) wereground and sifted with <0.5 mm particle size filter and stored atambient temperature for analysis. Five grams samples mixed with100 ml of internal standard solutions were extracted by 20 ml ofacetonitrile/water with 2 h of reversed agitations, after which 3 mlof aqueous phase was centrifuged and dried in a rotavapor. The finalproduct was dissolved in a solution of 0.01% acetic acid andmethanol (2/1 v/v) and 50 ml of this solution pipetted with a syringeequipped with a filter, and directly injected in LC–MS/MS. HPLC(Hewlett Packard (Eybens 38, France) 1100 type) analysis wascarried out using a C18 column (VWR (Pessac 33, France),250 mm� 4.6). The mobile phase was ammonium acetate 1 nMand 0.0001% acetic acid/methanol and 1% acetonitrile, with a linear

Table 3Effect of agrochemical treatments on Lepidoptera ECB and CSB (mean (�SE) larvae number/plant) and statistical analyses (ANOVA and Tukey’s HSD test).

Sites Treatment Mean� SE F df P-value

Wiwersheim Control 0.575� 0.111a 8.107 2, 6 0.0197*Insecticide 0.100� 0.041bInsecticide/fungicide 0.150� 0.096b

Moyenvic Control 0.325� 0.052a 35.352 2, 6 0.0005***Insecticide 0.025� 0.014bInsecticide/fungicide 0.013� 0.013b

Hurtigheim Control 0.800� 0.424a 3.000 2, 6 0.1250Insecticide 0.000� 0.000aInsecticide/fungicide 0.100� 0.100a

North East Area Control 0.567� 0.145a 10.978 2, 30 0.0003***Insecticide 0.042� 0.018bInsecticide/fungicide 0.088� 0.045b

Laloye Control 1.313� 0.183a 9.000 2, 6 0.0156*Insecticide 0.938� 0.125aInsecticide/fungicide 0.675� 0.111b

Charmes Control 1.950� 0.310a 12.740 2, 6 0.0069**Insecticide 0.150� 0.050bInsecticide/fungicide 0.900� 0.191ab

Saint Genes du Retz Control 0.850� 0.096a 16.692 2, 6 0.0035**Insecticide 0.050� 0.050bInsecticide/fungicide 0.200� 0.115b

Middle East Area Control 1.371� 0.176a 12.442 2, 30 0.0001***Insecticide 0.379� 0.127bInsecticide/fungicide 0.592� 0.116b

Castelnaudary Control 0.250� 0.096a 1.696 2, 6 0.2608Insecticide 0.050� 0.050aInsecticide/fungicide 0.200� 0.082a

Courpiac Control 0.513� 0.094a 23.043 2, 6 0.0015**Insecticide 0.038� 0.024bInsecticide/fungicide 0.038� 0.024b

Thure Control 1.700� 0.191a 32.392 2, 6 0.0006***Insecticide 0.150� 0.096bInsecticide/fungicide 0.350� 0.096b

SouthWestern Area Control 0.821� 0.203a 9.834 2, 30 0.0005***Insecticide 0.079� 0.037bInsecticide/fungicide 0.196� 0.055b

Nine field trials Control 0.919� 0.114a 25.683 2, 102 <10�4***Insecticide 0.167� 0.050bInsecticide/fungicide 0.292� 0.057b

*P < 0.05, **P < 0.01, ***P < 0.001.

Table 4

L. Folcher et al. / Crop Protection 28 (2009) 302–308 305

gradient over 40 min at a flow rate of 1 ml min�1. The detection wasperformed with quadrupole tandem mass spectrometer API 4000(Applied Biosystems (Foster City, CA, USA)) with Analyst-AppliedBiosystems software for control and data processing. The detectionthreshold was 10 mg kg�1. TURBO Ion Spray� from Applied Bio-systems with positive and negative ionization mode was used. Thedetection conditions were TIS (Interchangeable Turbolon Spray) inpositive and negative mode interface with 500 �C of sourcetemperature and �4500 V and 4500 V for ion spray voltage. Thedetection used Multiple Reaction Monitoring (MRM), and identifi-cation and quantification were carried out on two or three transi-tions for each mycotoxin. The protocol was according to AFNORV03-110.

Effect of agrochemical treatment on Fusarium mycoflora (mean (�SE) percentage ofinfected grains) and statistical analyses (ANOVA and Tukey’s HSD test) P< 0.05.

Response Treatment Mean� SE F df P-value

FuProd Control 24.111� 10.428a 0.195 2, 24 0.8241Insecticide 15.667� 9.152aInsecticide/fungicide 21.556� 9.800a

TriProd Control 12.111� 3.732a 1.460 2, 24 0.3668Insecticide 7.556� 4.083aInsecticide/fungicide 5.444� 1.642a

FuProd: fungi producing fumonisins; TriProd: fungi producing trichothecenes andzearalenone.

2.4. Statistical analysis

The following variables were submitted to statistical analysis:TTRav (O. nubilalisþ S. nonagrioides densities), FuB1B2 (fumonisinB1þ B2 amounts), TriABD (trichothecene A, B, D amounts), ZEA(zearalenone and metabolites), FuProd (percentage of contami-nated grains by fungi producing fumonisins), and TriProd(percentage of contaminated grains by fungi producing trichothe-cenes and zearalenone). The described variables did not follow

a normal distribution except for TTRav, FuProd and TriProd.Consequently we used generalized linear models with gaussianmodel for TTRav (Table 3) and FuProd, TriProd (Table 4), and qua-sipoisson model for the other variables (Venables and Ripley, 2002)(Table 5). ANOVA and Tukey’s HSD test were applied to field trialssite by site and on trials pooled (Table 3), then to Fusarium andmycotoxin levels (P< 0.05) (Tables 4 and 5). Results were analyzedby the software R-project (version 2.5.1 on-line http://www.r-project.org/, June 2007).

Table 5Effect of agrochemical treatment on mycotoxin levels (mean (�SE) in mg kg�1) andstatistical analyses (GLM and Tukey’s HSD test).

Response Treatment Mean� SE F df P-value

FuB1B2 Control 3401.222� 1303.758a <10�4 2, 24 <10�4***Insecticide 341.222� 168.404bInsecticide/fungicide

341.111� 113.361b

TriABD Control 910.111� 274.701a 0.0001 2, 24 <10�4***Insecticide 241.222� 71.950bInsecticide/fungicide

144.111� 47.074b

ZEA Control 50.222� 28.179a 0.020 2, 24 0.0200*Insecticide 7.333� 3.337aInsecticide/fungicide

9.000� 4.137a

*P < 0.05, **P < 0.01, ***P < 0.001. FuB1B2¼ fumonisins B1þ B2; TriABD¼trichothecenes A, B, D; and ZEA¼ zearalenone and metabolites.

L. Folcher et al. / Crop Protection 28 (2009) 302–308306

3. Results

3.1. Lepidoptera control

Taken as a whole, the effect of treatments on the reduction oflevels of populations of O. nubilalis and S. nonagrioides was highlysignificant for insecticide and insecticide plus fungicide(F¼ 25.683; df¼ 2, 102; P< 10�4) for the 3 years (Table 3). Theeffect of the pesticides was statistically observed for all trials exceptat Castelnaudary and Hurtigheim where there were no differencesbetween control and all forms of treatments. In fact, a very shortdelay between the emergence peaks of the insects and pesticidespraying took place at Castelnaudary, and the variability of meansthat resulted did not permit discrimination from the control(F¼ 1.696; df¼ 2, 6; P¼ 0.2608). A detailed analysis of the effectsper site showed that the insecticide treatment was significantlydifferent from the control in all trials except at Laloye in which anoverlap was given by Tukey’s test although the treatment effect waseffective. In a same way, the insecticideþ fungicide treatment wasnot different from control, neither from the insecticide treatment,for the location Charmes (Table 3). In these two places, sprayingtreatments had to be interrupted because of two heavy downpoursof rain. This explained the variability of the results. In all othercases, the control was significantly different from the tests. In thethree previously defined areas (Fig. 1, Table 1), the Tukey testdistinguished the control from the pesticide treatments in all thecases (Table 3). The populations of O. nubilalis and S. nonagrioideswere significantly reduced by both insecticideþ fungicide treat-ments. But no significant difference was noticed between the twokinds of treatments (insecticide alone or in association withfungicide) while Lepidoptera borers ECB and CSB were stronglycontrolled in the three areas: North East area (F¼ 10.978; df¼ 2,30; P¼ 0.0003), Middle East area (F¼ 12.442; df¼ 2, 30;P¼ 0.0001) and SouthWestern area (F¼ 9.834; df¼ 2, 30;P¼ 0.0005).

3.2. Effect on mycoflora

All Fusarium species colonized maize kernels in the nine trialsites. F. verticillioides (previously moniliforme) and F. proliferatumwhich synthesized fumonisins B1 and B2 and moniliformine weremainly identified within SouthWestern areas at Castelnaudary,Thure and Courpiac whereas F. graminearum and F. culmorum whichsynthesized trichothecens B and zearalenone were the mostabundant in EastNorthern and Center (Wiwersheim, Hurtigheim,Moyenvic, Laloye, Charmes and Saint Genes du Retz). Fusarium spp.mycoflora was not significantly affected by treatments (Table 4),although the number of days favorable for release of the spores was

estimated between 6.56 and 15.22, and for spore germinationbetween 2.72 and 17.49 (Table 2). Weather conditions at all siteswere favorable for fungal development. The control had a mean of24.11% of contaminated grains with fungi that produced fumonisins(FuProd). Fungal contamination of 15.67% and 21.56%, respectively,was observed for insecticide and insecticide plus fungicide treat-ments. The differences were not significant (F¼ 0.195; df¼ 2, 24;P¼ 0.8241) indicating that the pesticide treatments did not modifythe infestation by fungal fumonisin producers. The levels ofcontamination of fungi producing trichothecenes (TriProd) were,respectively, 12.11% for control, 7.56% for insecticide treatment and5.44% for insecticide plus fungicide. Again, the treatment effect wasnot statistically different (F¼ 1.460; df¼ 2, 24; P¼ 0.3668) indi-cating that Fusarium spp. mycoflora was not affected by eitherpesticide treatment.

3.3. Effect on mycotoxin levels

The mycotoxin levels for the control (Table 5) were significantlygreater than those for the pesticide treatments for fumonisins(F< 10�4; df¼ 2, 24; P< 10�4), trichothecenes (F¼ 0.0001; df¼ 2,24; P< 10�4) and for zearalenone (F¼ 0.020; df¼ 2, 24;P¼ 0.0200). This reduction could be expressed by a parameter, theefficacy (E) defined by the following ratio:

Eð%Þ ¼�ðControl mycotoxin level� treatment mycotoxin levelÞ

Control mycotoxin level

�� 100

This efficacy was, respectively, evaluated at 89.96% for fumoni-sins with the insecticide treatment and 89.97% with insectici-deþ fungicide. For trichothecenes, the efficacy was 73.50% with theinsecticide treatment and 84.17% with insecticideþ fungicide. Forzearalenone, the efficacy showed a reduction of 85.40% ofcontamination with the insecticide and 82.10% with insectici-deþ fungicide. However, the Tukey test did not discriminate thecontrol from treatments (Table 5).

4. Discussion

Considering the trials as a whole, the agrochemical treatmentsefficiently controlled insects’ larvae in the crops. As expected, thetreatment with an insecticide reduced the maize borer populationsand the insecticideþ fungicide treatment could not be differenti-ated from those obtained with the insecticide. Similarly no differ-ential effect was observed for mycoflora. No synergy effect betweenfungicide and insecticide was observed. This result corroboratedsome previous observations (Weissenberger, unpublished) point-ing out the inefficiency of fungicide treatment alone on maize in theexperimented areas. However, even if the treatments had no effecton the mycoflora levels, the identification of the species of fungicontaminating the grains gave a better idea of the competitionbetween species colonizing the ears of maize (Marın et al., 1998;Velluti et al., 2000). Naef and Defago (2006) identified F. grami-nearum, F. avenaceum and F. proliferatum isolated from maize stalksby multiplex PCR and they underlined the complexity of theinterrelationships within mycoflora for both Bt and non-Bt maizewhich showed, in this case, no consistent difference. Reid et al.(1999) observed by an evaluation of ergosterol, a metabolite bio-synthesized by fungi and considered to be a biomarker of fungalactivity (Bakan et al., 2003) that F. graminearum was more activethan F. verticillioides (Reid et al., 1999). This identification could alsogive relevant information on links between fungal contaminationand maize borer activity. It was established that the level of infes-tation of maize ears by both F. verticillioides and Aspergillus flavusacting competitively could be correlated with insect activity thatdamaged the plant (Cardwell et al., 2000).

L. Folcher et al. / Crop Protection 28 (2009) 302–308 307

In our study, pesticide treatments decreased the amounts offumonisins and trichothecenes within the grains and the controlcould be clearly discriminated from the assays by the Tukey test(Table 5). The differences between agrochemical treatments andcontrols on the levels of insect pests and mycotoxins shouldunderline the link between the damage observed on the plant as theresult of the activity of the insects and the amounts of mycotoxins.Because they bored into the plant tissues, insects opened a door forthe entry of fungal pathogens biosynthesizing mycotoxins.

Several studies with Bt maize hybrids, which geneticallycontrolled Lepidoptera, corroborated this conclusion. Bt maizeappeared essential for preventing contamination by mycotoxins(Wu, 2006). It reduced Fusarium ear rot and symptomless infectionin kernels (Munkvold and Desjardins, 1997; Munkvold et al., 1997).Clements et al. (2003) studying the influence a CryAb protein andhybrid genotype on fumonisin contamination and Fusarium ear rotof maize, hypothesized that the larvae of O. nubilalis would consti-tute a worsening factor in the establishment of Fusarium spp. andconsequently for the production of mycotoxins. Experiments carriedout in central middle Europe concluded that Bt maize hybridsslightly reduced the Fusarium mycotoxin level of maize (Magg et al.,2002, 2003). More recently, it was shown that the MON810 traitreduced the fumonisins B1þ B2 with an efficacy over 90% (Folcheret al., 2008). The use of transgenic maize as a tool is very usefulbecause it indicates that relationships between Lepidoptera, Fusa-rium spp, ear rot disease and mycotoxins are really overlapping andthe links between them not always easy to establish. Even if thepathogen was observed during cultivation (Chuize et al., 1999),the identification of Fusarium spp. at harvest did not indicate theintensity of fungal attack during plant growth. On the other hand,mycotoxins were detected without any fungal detection at harvest(Champeil et al., 2004). Although mycotoxin levels at harvest werethe consequence of Fusarium infesting grains, fungi disappearedfrom plants when environmental conditions were not ideal, but themycotoxin they produced remained. Consequently some methodsinvolving agronomical practices (Bruns, 2003; Mansfield et al.,2005), or reducing the inoculum by residue management or croprotations (Reid et al., 2001; Schmidt and Nitzsche, 2004) were usefulfor controlling Fusarium spp. Biological methods using bacteria likeBacillus subtilis also reduced mycotoxin accumulation (Bacon et al.,2001). Another approach involved genetically tolerant cultivars, butreducing mycotoxin by resistant hybrids remained difficult becauseof the polygenic nature of resistance sources (Porta et al., 2005).Regarding maize borers, results obtained with Bt maize supportedour results. When insects were controlled, mycotoxin amountsdecreased within harvested kernels. This underlined that a linkexisted between insects and mycotoxin levels at harvest, whichaffected the edible acceptability of the crops.

Mycotoxin consumption in a diet induced pathological symp-toms for consumers and affected their health (Ozbek and Ozbek,2003). However, the risk depends on the kind of mycotoxins.Trichothecenes, fumonisins and zearalenone do not develop, withthe same intensity and symptoms. Thus it is important to knowwhich kinds of mycotoxins are decreased by Lepidoptera control.Our results demonstrated that trichothecenes (TriABD) as well asfumonisins (FuB1B2) were significantly reduced by insecticidetreatment at the threshold P< 0.001, but that for zearalenone thevariability of the results observed with the control (SE¼ 28.179) didnot allow any conclusion to be drawn, even if a treatment effect wasnoted. Consequently, further experiments are required to clarifythis result.

5. Conclusion

Our study emphasizes the need to take into account every factorwhich targets plant health. For maize, the control of maize borer

caterpillars, ECB and CSB, by an insecticide treatment is essential forreducing mycotoxin levels biosynthesized by Fusarium spp.although additional fungicide treatment did not have any effect onmycoflora or mycotoxins. Because the number of maize borergenerations per year is linked to climatic conditions, it is necessaryto look at the protection of maize from every angle in order topreserve the quality of the harvest from mycotoxin contamination.

Acknowledgments

These studies were carried out within the Biological RisksMonitoring Network (‘‘reseau de biovigilance’’) which is in chargefor surveying the French national territory. We thank the numerouscolleagues from ‘‘Service de la Protection des Vegetaux’’ and‘‘Federation de Defense contre les Organismes Nuisibles’’ who fol-lowed-up the field trials, Sylvie Rose and Philippe Loevenbruckfrom laboratory of Malzeville for all determinations of fungalspecies, and Eric Marengue and LDA 22 for mycotoxin analyses.

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