Genetic Fusarium chemotyping as a useful tool for predicting nivalenol contamination in winter wheat

International Journal of Food Microbiology 137 (2010) 246–253

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International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

Genetic Fusarium chemotyping as a useful tool for predicting nivalenol contamination in winter wheat M. Pasquali ⁎, F. Giraud, C. Brochot, E. Cocco, L. Hoffmann, T. Bohn Centre de Recherche Public-Gabriel Lippmann, Department Environment and Agro-Biotechnologies, 41, rue du Brill, L-4422 Belvaux, Luxembourg

a r t i c l e

i n f o

Article history: Received 6 August 2009 Received in revised form 5 November 2009 Accepted 11 November 2009 Keywords: Prevention Fusarium head blight Grains Trichothecene LC-MS/MS PCR 3-Acetyl Deoxynivalenol 15-Acetyl Deoxynivalenol F. graminearum F. culmorum

a b s t r a c t Fusarium graminearum [teleomorph Gibberella zeae] and Fusarium culmorum together with Fusarium poae are the main species known to produce nivalenol (NIV). The NIV content in wheat (Triticum aestivum L.) harvested in Luxembourg was investigated in 2007 and 2008 at 17 different locations. Species determination and genetic chemotyping of F. graminearum and F. culmorum were used to understand the spatial distribution of NIV producers in wheat from Luxembourg. Three hundred thirteen F. graminearum, 175 F. culmorum and 117 F. poae strains respectively were isolated. Chemotypes of the first two species were determined by PCR and confirmed on a sub-sample of single isolates by LC-MS/MS analysis. The 15-acetylated DON chemotype of F. graminearum was dominant in both years representing 94.2% of the population while the NIV chemotype represented 5.8%. The F. culmorum chemotypes were rather evenly distributed, with 3-acetylated DON and NIV profiles present with similar abundances (53.2% and 46.8%, respectively). NIV presence in wheat flour obtained from the 17 sites was correlated with the number of F. culmorum (NIV chemotype) isolated from 100 seeds, suggesting its primary role in NIV production on grains. The predictive power for identifying NIV contamination in grains based on NIV chemotype presence was confirmed by coupling the isolation procedure with a cut-off value, resulting in the successful identification (100%, p = 0.008) of NIV contamination in grains collected from 9 additional experimental sites. In conclusion, the results highlight the importance of chemotyping for improved prediction of toxin contamination in wheat. © 2009 Elsevier B.V. All rights reserved.

1. Introduction Fusarium head blight (FHB) is a widespread disease of small grains and one of the major diseases of wheat worldwide, including Luxembourg. Fusarium species affecting wheat are the cause of trichothecene accumulation such as deoxynivalenol (DON), its acetylated derivatives (3acetylated DON (3-ADON) and 15-acetylated DON (15-ADON)), and nivalenol (NIV), as well as zearalenone, moniliformin and other toxins (Desjardins, 2006). In Luxembourg, the main species associated with FHB disease are Fusarium graminearum, F. culmorum, F. poae, F. avenaceum and Microdochium nivale (Giraud et al., 2008). Among these species, the first three are known to produce NIV (Desjardins, 2006). The F. graminearum species complex is composed of at least twelve lineages (O'Donnell et al., 2008). It is the most commonly associated pathogen with FHB and the major producer of DON in wheat (Goswami and Kistler, 2004). F. culmorum is abundant in cold-temperate climates and is associated with FHB in Europe, Canada and China (Bottalico and Perrone, 2002; Tóth et al., 2004). However, its prevalence in FHB infected fields in Europe has diminished during the last years (Wagacha

⁎ Corresponding author. Tel.: +352 470261436; fax: +352 470264. E-mail address: [email protected] (M. Pasquali). 0168-1605/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2009.11.009

and Muthomi, 2007). Fusarium poae is widespread in cold climates and associated to head blight symptoms. It is capable of producing both type A and type B (Nivalenol) trichothecenes (Stenglein, 2009). Chemotype characterization has been extensively used to characterize F. graminearum and F. culmorum for their toxigenic potential. Different chemotyping methods have been developed during the last 10 years (Chandler et al., 2003; Lee et al., 2002; Li et al., 2005; Quarta et al., 2006; Starkey et al., 2007; Wang et al., 2008; Ward et al., 2008). All methods are based on polymorphism on coding genes and introns (such as tri3, tri7, tri12, tri13) of the TRI cluster, the main region containing genes involved in trichothecene synthesis. Interestingly, this region has a parallel and independent evolution from the rest of the genome – originating from ancestral independent acquisition events in the genome (Ward et al., 2002) – and may be useful to give further insight into the structure of a field population. For example, chemotyping was used to discriminate lineages in F. graminearum and to identify population structures in infected fields (Lee et al., 2009). Previous reports in North America (Burlakoti et al., 2008; Gale et al., 2007), China (Zhang et al., 2007), Europe (Szecsi et al., 2005; Stepień et al., 2008), Iran (Haratian et al., 2008), Japan (Suga et al., 2008), and Argentina (Pinto et al., 2008) have shown differences over space and time of chemotype distribution of F. graminearum and F. culmorum species, suggesting that this technique is a valuable approach to map a population and to identify

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population changes in the field (Karugia et al., 2009). No comprehensive data, however, are available on the Fusarium population from Luxembourg. Among the trichothecenes, NIV is probably the most neglected toxin because it is often found at lower level in wheat (Yazar and Omurtag, 2008), despite its higher toxicity as compared to DON, causing, for instance, inhibition of DNA synthesis, that may account for various toxic phenomena such as induction of cell death (Poapolathep et al., 2002). NIV was also reported to be more toxic than DON for human blood cells (Minervini et al., 2004). Most importantly, it has been shown that combinations of NIV with T2, diacetoxyscirpenol (DAS) or DON resulted in additive toxicity in the lymphocyte proliferation test (Thuvander et al., 1999). Therefore, developing models for predicting the presence of NIV in grains and co-occurrence of NIV and DON, would be beneficial for a comprehensive food safety approach. The aims of the present investigation were thus to:

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2.3. PCR species identification and chemotyping

1. Characterize the chemotypes of F. culmorum and F. graminearum found in Luxembourg during the years 2007 and 2008; 2. Measure the toxigenic potential of Fusarium species from Luxembourg; 3. Evaluate the usefulness of fungal population studies coupled with genetic chemotyping determination to predict nivalenol presence in grains.

Specific primers (Appendix A) for species confirmation of the four main Fusarium species that can be found in Luxembourg were used (Demeke et al., 2005). A multiplex PCR protocol was optimized for its use on a Biometra UNO II (Goettingen, Germany) thermalcycler using a Finnzyme phusion master mix (Finnzyme, Espoo, Finland). In particular, phusion taq master mix and 500 µM of each primer in a total volume of 10 µl were used to distinguish F. poae, F. culmorum, F. graminearum and F. avenaceum in the same reaction mix. The following program was used: 98 °C for 2 min, followed by 35 cycles of denaturation at 98 °C for 10 s, annealing at 60 °C for 20 s, extension at 72 °C for 45 s and one cycle of final extension at 72 °C for 5 min and 4 °C until gel loading. PCRs for chemotype characterization using Ward et al. (2002) primers (Table 1) were carried out using 10–50 ng of DNA obtained as described above. Reaction volume was 10 µl and PCR conditions were the following: 98 °C for 2 min, 30 cycles of 98 °C for 10 s, 56 °C for 20 s, 72 °C for 40 s, followed by 5 min at 72 °C and 4 °C until gel loading. All PCR products were loaded on 3% BIORAD agarose gels (Bio-Rad Laboratories, Hercules, CA, USA). PCR fragments were separated by applying a tension of 100 V for 40 min. Electrophoretic runs were visualized using Syngene Genius Image analyzer (Cambridge, UK).

2. Materials and methods

2.4. Chemical analyses of toxins in vitro

2.1. Field locations and strain isolations

In order to standardize the procedure of toxin induction, a highly concentrated liquid saccharose media was used for inducing toxin synthesis (Jiao et al., 2008). Single-spore strains were grown on V8 medium for 5 d and mycelium was collected from 1/3 of the plate and deposited in triplicate in 10 ml toxin inducing media (1 g K2HPO4, 0.5 g KCl, 0.5 g MgSO4 7H2O, 2 g L-glutamic acid, 10 g sucrose in a liter of solution). Tubes were shaken at 200 rpm in the dark for 10 d. Fungal biomass was then filtered and weighted after drying using a freeze drier for 48 h, and the remaining media kept for further extraction. The medium was filtered through a 0.2 µm GHP membrane filter (PAL, MI, USA) and diluted in methanol (extract/methanol, 9/1, V/V) in order to be in the appropriate solvent ratio for chromatographic analysis.

During a FHB survey (years 2007 and 2008) carried out on different cereal growing areas in Luxembourg, soft wheat grains (500 g) were harvested from 17 fields (Fig. 1). The samples were packed in plastic bags, dried at 60 °C for 72 h in order to standardize humidity content, and stored at 4 ± 1 °C prior to mycobiota and mycotoxin analysis. For further analyses, unless stated otherwise, all chemicals were obtained from Sigma (Saint Louis, Missouri, USA), and were of analytical grade or superior. Fifty ml of grains were surface sterilized using 0.37% NaOCl and 0.1% Tween20®, for 10 min and then dried on sterile filter paper under a laminar flow hood. One hundred grains were then plated on modified dichloran-chloramphenicol-peptone agar (DCPA) with crystal violet (Ioos et al., 2004) and incubated for 12 d at 22 ± 2 °C with a 12 h light period. The Fusarium spp. were subsequently transferred to potato dextrose agar (PDA, Difco Laboratories, Detroit, MI, USA), incubated at 22 ± 2 °C for 6–10 d, and isolates were then morphologically identified according to Nelson et al. (1983). Monoconidial isolates were obtained by dilution and needle selection under the microscope on water agar (2% agar). Monoconidial isolates were maintained in PDA fragments at − 80 °C in 15% glycerol. 2.2. DNA extraction Fungal strains were grown in 5 ml potato dextrose broth (PDB, Gibco, USA) shaking the culture at 150 rpm at 22 °C for 6 d. Mycelium was then freeze-dried and DNA was extracted using a Qiagen Plant DNA extraction kit according to the manufacturer's protocol (Qiagen, Crawley, West Sussex). DNA was quantified by using a Nanodrop 1000 (Thermo, Waltham, MA, USA) and dilutions (from 1 to 500 ng/µl) were performed for PCR optimization. On a subsample of isolates, a quick DNA extraction method was also developed. Briefly, 1 cm2 area of aerial mycelium from a 4 d old V8 (1 L includes 200 ml of V8 juice, 2 g of CaCO3, 18 g of agar and H2O to the volume) plate was collected, mixed with 50 µl TE (Tris 1 M, EDTA 10 mM) and vortexed for 1 min. Five minutes of microwaving at 800 W was applied to the mixture followed by 30 s of centrifugation at 8000 g. Five µl of the upper phase were then used for the multiplex PCRs carried out as described below with a volume modification (a total of 50 µl PCR volume was used to reduce effects of PCR inhibitors).

2.5. Chemical analyses of toxins in wheat samples Toxin identification and quantification in wheat samples were performed selecting 500 g of wheat grains from each field studied. After drying for 24 h at 30 °C, 200 g were milled with a Cyclotec™ 1093 (Foss, Belgium). An aliquot of 5 g of flour and 15 ml of acetonitrile/ water (80/20, v/v) were homogenized by vortex and sonicated (Elma Transonic TS540, Germany) for 15 min. After centrifugation (720 g, 20 °C, Sigma 3 K12, Bioblock Scientific), the supernatant was filtered through a 0.2 µm GHP membrane filter and diluted 10 times with water to reduce matrix effects during LC-MS/MS analyses. 2.6. Quantification of toxins by LC-MS/MS Mycotoxin separation and detection were achieved by LC coupled to tandem mass spectrometry (LC-MS/MS, Dionex Ultimate 3000; Applied Biosystems API 3200, Foster City, CA, USA) in multiple reaction monitoring (MRM) in negative mode for the following toxins analysed: NIV, DON, 3-ADON, 15-ADON. For separation, an Alltima HP RP-C18 column (Grace Davison, IL, USA) was used (150 × 2.1 mm; 3 µm) with a mobile phase consisting of methanol and water with 2.5 mM of ammonium acetate in a linear gradient. Quantification was based on external standards (Biopure, Tulln, AT). The differentiation of 3-ADON and 15-ADON was obtained by calculating the ratio of two different selected fragment ions (397 to N337 and 397 to N307). The detection and quantification limits obtained by the extraction of

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Fig. 1. Luxembourg map indicating location of sampled fields identified by numbers (1 to 17), the presence of nivalenol chemotype and nivalenol detection in wheat grains. Circles indicate grain contamination of NIV; squares indicate NIV chemotype in at least one isolated strain of F. culmorum or F. graminearum; black and white squares indicating year 2007; black indicating year 2008.

5 g of wheat were 35 and 70 ng/g for the trichothecenes. Analyses were repeated twice and average values are reported. The detection and quantification limits obtained for the in vitro analysis were 1100 and 2780 ng/g dry biomass for the trichothecenes. The analyses were done in triplicate and average values are reported. 2.7. Statistical analysis All statistical analyses were carried out using SPSS (v16) (SPSS Inc., Chicago, IL, USA). Correlations were determined using Spearman coefficients, and were deemed significant for p b 0.01. Linear mixed models were created with the total number of NIV isolates and NIV

content as the dependent variable, and preceding crop, location and year and the number of Fusarium poae isolates per 100 seeds, number of Fusarium graminearum NIV chemotypes per 100 seeds and number of Fusarium culmorum NIV chemotypes per 100 seeds as explanatory variables, using the entire dataset. A p-value b0.05 was chosen to indicate significant differences (2-sided). The linear mixed models were followed by post-hoc tests (Bonferroni) given an overall significant Fisher F-value. Cut-off levels were determined based on the number of isolates and the percentage of NIV positive genotypes of F. culmorum, based on 34 field observations (Table 1A). The obtained cut-off levels were then applied to 9 additional sites for verification of the model (Table 1B). A

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Table 1 Field name, year of isolation, localization of the field, NIV content in grains, DON content in grains (from Giraud et al., in press), number of Fusaria obtained from 100 seeds (includes also Michrodochium spp.), number of Fusarium poae, Fusarium culmorum NIV chemotype, Fusarium culmorum 3-ADON chemotype, F. graminearum NIV chemotype, Fusarium graminearum 15-ADON chemotype, % of Fusarium culmorum NIV chemotype calculated on the number of total isolates obtained from 100 seeds. In bold data are those reaching the cut-off values for F. culmorum NIV+ and percentages of F. culmorum NIV+ chemotype. A (data obtained from the original survey); B (data obtained from experimental sites used to validate the model). Field

Table A LT-01 LT-02 LT-03 LT-04b LT-05 LT-06 lt17bisb LT-07 LT-08 LT-09 LT-10 LT-11 LT-12 LT-13 LT-14 LT-15 LT-16 LT-17

Table B CHR 8A 0 T CHR 8B 1 T CHR 8C 3 T BUR 13 0 T BUR 13 1 T BUR 13 3 T REU T5 REU 2T5 REU 3T5

Year

Location

DON contenta

NIV content

ng/g

ng/g

Total Fusaria

Fusarium poae

Fusarium culmorum NIV+

Fusarium culmorum 3ADON+

Fusarium graminearum NIV+

Fusarium graminearum 15ADON+

% F. culmorum NIV+

0 30 12.5 7.7 0 0 0 9.5 0 0 0 34.6 5.4 0 33.3 0 2.5 28.6 0 0 10.2 0 0 0 7 0 0 0 0 0 0 0 0 0

33.3 14.7 46.1 8.6 3.1 0 0 0 8.3

2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008 2007 2008

Centre Centre Centre Centre North North North North North North North North Centre Centre Centre Centre Centre Centre South South Centre Centre South South South South South South South South North North North North

277.92a 220.42a 249.14a 301.00a 139.41a 0.00a 88.03a 927.87a 88.92a 290.99a 0.00a 268.00 1213.00a 1014.29a 501.03a 204.49a 1933.01a 0.00a 657.01a 0.00a 4505.54a 8111.00a 381.86a 100.57a 1189.01a 0.00a 365.02a 0.00a 0.00a 462.81a 0.00a 486.89a 146.55a 680.70a

0 241 0 0 0 0 0 0 0 0 0 341 0 0 293 0 0 0 0 0 236 0 0 0 0 0 0 0 0 0 0 0 0 0

48 10 8 13 40 3 25 21 5 9 39 26 92 18 39 4 81 7 42 4 88 57 26 1 43 1 24 7 18 2 4 10 19 14

17 2 2 2 14 2 1 4 1 4 22 2 0 1 3 0 9 1 3 2 0 0 12 0 0 0 11 2 8 2 0 6 5 5

0 3 1 1 0 0 0 2 0 0 0 9 5 0 13 0 2 2 0 0 9 0 0 0 3 0 0 0 0 0 0 0 0 0

0 1 0 2 5 0 0 0 0 0 0 0 3 0 4 0 1 0 7 0 0 0 0 0 24 1 1 0 0 0 0 0 0 1

0 0 1 0 0 0 0 0 0 0 0 3 1 0 1 0 0 0 0 0 6 0 0 0 0 0 0 0 0 0 1 0 0

5 1 0 1 0 0 0 9 0 4 0 1 46 13 9 4 44 1 7 1 58 50 1 0 2 0 4 0 0 0 0 2 0 0

2007 2007 2007 2007 2007 2007 2007 2007 2007

Centre Centre Centre South South South North North North

968.00 489.00 1215.00 2487.00 991.00 1903.00 285.00 516.00 842.00

532 202 462 125 0 0 0 0 0

24 34 26 35 32 10 27 24 24

1 4 0 0 0 0 0 4 0

8 5 12 3 1 0 0 0 2

3 4 1 11 13 3 3 2 3

1 1 0 0 0 1 2 0 0

6 8 3 2 2 5 5 0 1

a

DON contents are from Giraud et al. (in press). Field LT6 was not sampled in 2008 because wheat was not cultivated anymore and it was therefore substituted by the nearest field sown with wheat that was site LT17bis (the site is located in between site LT17 and site LT6). b

permutation test was applied to calculate the exact corresponding p-value based on the used cut-off criteria to correctly detect all sites with detectable NIV. 3. Results 3.1. Species and chemotype determination In total, 1084 isolates were analysed. F. graminearum was the major species found in Luxembourg (28.9%) for the two years. A total of 488 isolates obtained from 100 seeds collected in each location in 2007 and 2008 belonging to F. graminearum (148 added to the previously 165 characterized strains by Pasquali et al., 2009) and F. culmorum (175) were analysed for their chemotype. Genetic chemotyping confirmed previous surveys carried out on a subsample of isolates (Pasquali et al.,

2009), showing that 15-ADON chemotype was the major population (94.3%) and NIV chemotype a rather sporadic one, representing only 5.8% of the total population. 3-ADON F. graminearum chemotype was not detected. F. culmorum was less frequently associated with wheat grains during the two years of sampling: the population of F. culmorum represented 16.1% of the total Fusarium population. F. culmorum chemotypes were more equally distributed between 3-ADON and NIV. The 3-ADON chemotype represented 53.2% of the population while the NIV chemotype 46.8% of the total population. One hundred fifty two strains (14%) belonging to F. poae were obtained. The frequency of isolation was relatively low but the species was quite homogeneously spread. The presence of the NIV chemotype in Luxembourg was found to be inhomogeneous. NIV isolates could be detected sporadically in the

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south while they were more frequently observed in the north and centre of the country (Fig. 1). In order to identify which factors may explain NIV chemotype distribution in Luxembourg, the effect of location, year and preceding crop was assessed by a linear mixed model (thus taking into account the effects of climate and of cultural practices). A significant impact on the amount of NIV chemotype (F. culmorum + F. graminearum) was observed for maize as preceding crop (p = 0.032) while neither location nor year had any significant impact.

isolates with the NIV chemotype (R = 0.748, p b 0.001). The influence of the different parameters on NIV content in grains was tested with a regression analysis indicating the only significant factor for NIV accumulation in grains to be F. culmorum NIV+ population (p b 0.001). All sites containing NIV in grains satisfied the two following criteria: presence of at least 3 NIV F. culmorum isolates per 100 seeds and the presence of more than 8% of NIV F. culmorum isolates of the total number of Fusarium strains isolated from 100 seeds. These two criteria were chosen as simultaneous cut-off values necessary to identify detectable measures of NIV in wheat (Table 1A).

3.2. Toxin analysis 3.4. Verification of the criteria for identifying NIV in grains 3.2.1. Toxin production by single isolates in toxin inducing media Genetic analysis was confirmed by the results obtained by chemical analysis on a random subsample of isolates belonging to the two species (Table 2). Trichothecene B production (sum of acetylated DON, NIV and DON) was variable among species and chemotypes, ranging from below detection limit to 624 μg/g for the 8 isolates of F. graminearum analysed and from below detection limit to 640 μg/g for the 10 isolates of F. culmorum (Table 2). Isolates producing no toxin (below detection limit) were identified in both species (2 isolates belonging to F. culmorum NIV chemotype, one isolate belonging to F. culmorum 3-ADON chemotype, 2 isolates belonging to F. graminearum 15-ADON chemotype). 3.2.2. Quantification of NIV in grains Grains obtained from the 17 fields for two consecutive years were examined for their NIV content. During both years nivalenol was only found in four samples (Fig. 1). In 2007, two sites had detectable levels of nivalenol (site LT08 with 293 μg/kg and site LT11 with 236 μg/kg), while in 2008 only grains obtained from sites LT01 and LT17bis contained nivalenol (241 μg/kg and 341 μg/kg, respectively). 3.3. Determination of factors influencing NIV production Correlations coefficients were calculated between NIV content in grains and (1) the percentages or (2) the number of isolates per 100 seeds belonging to F. culmorum NIV chemotype, F. graminearum NIV chemotype, and F. poae (the three NIV producers). Correlation with F. poae was very low and non-significant. Correlation was found with F. gramineaurm NIV chemotype presence (R = 0.553, p b 0.001), and between the content of NIV in grains and the number of F. culmorum

To confirm the validity of the cut-off values for predicting NIV content based on the NIV chemotype, these cut-off values were verified by investigating grains from 9 additional experimental sites — representing North, Centre and South of Luxembourg from 2007 (Table 1B). Population from each grain lot was analysed, chemotype was determined and nivalenol content was measured. Four fields contained nivalenol ranging from 125 to 532 μg/kg while the other 5 did not contain measurable quantities of nivalenol. The four positive sites were used for validating the efficacy of the predictive model and the cut-off values while the 5 fields without measurable nivalenol were used to test the probability of a false positive identification. The application of the isolation method and of the cut-off criteria on the nine additional sites allowed for successful prediction of NIV contaminated sites for all cases (p = 0.008, exact permutation test) without any false positive identification. 3.5. Fusarium culmorum as the main cause of NIV accumulation in grains To further elucidate the role that F. culmorum NIV chemotype may play as determinant of NIV accumulation in grains, a linear mixed model was developed using as dependent factors the amount of F. poae, F. graminearum NIV chemotype, F. culmorum NIV chemotype (the three Fusaria able to produce nivalenol), preceding crop, location, year, and NIV concentration as the observed variable. The model showed that the only significant factor contributing to NIV accumulation in grains was the amount of F. culmorum with NIV chemotype (p b 0.001). On the contrary, F. poae and F. graminearum NIV chemotype did not contribute to NIV accumulation, as did the other factors. A tendency could be observed for maize as preceding crop but did not reach

Table 2 Isolate ID number, species, field of collection, NIV, DON, 3-ADON, 15-ADON and total trichothecene type B amount produced in toxin-inducing media (Jiao et al., 2008) measured in ng of toxin per grams of dried mycelium (ng/g). Isolate

Speciesa

Field

NIV μg/g

60 82 183 233 357 439 572 601 640 708 5 210 401 444 453 630 713 734

F.c. F.c. F.c. F.c. F.c. F.c. F.c. F.c. F.c. F.c. F.g. F.g. F.g. F.g. F.g. F.g. F.g. F.g.

LT03 LT03 LT07 LT07 LT10 LT11 REU T5 BUR130T LT13 LT08 LT01 LT07 LT11 LT11 LT11 BUR130T LT08 LT08

N.D. N.D. 18.1 N.D. N.D. 3.2 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 624.8 N.D. N.D. N.D.

N.D. means not detectable (N.D. is below limit of detection). a F.c. = Fusarium culmorum; F.g. = Fusarium graminearum.

DON (± SD)

1.5

0.6

52

3ADON

15ADON

μg/g

(± SD)

μg/g

(± SD)

μg/g

8.7 241.7 N.D. N.D. N.D. N.D. 67.9 49.9 31.7 N.D. 21.1 61.3 N.D. 79.5 N.D. N.D. 123.6 252.7

0.9 106.7

16.1 398.7 N.D. N.D. N.D. N.D. 126.7 69.6 66.3 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D.

3.6 95.1

N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 31.7 N.D. 17.6 N.D. N.D. 12.3 83.1

36.2 10.1 13.9 5.3 12 9

25 27

54.8 13.8 32.6

Total (± SD)

8.1 N.D. 2.1

7.2 12.7

μg/g 24.8 640.4 18.1 N.D. N.D. 3.2 194.7 119.5 98.1 N.D. 21.0 93.1 N.D. 97.1 624.8 N.D. 136.0 335.7

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significance (p = 0.064). The model confirmed that NIV accumulation in grains is mainly due to the presence of the NIV + F. culmorum chemotype. 4. Discussion It has been suggested that knowledge of Fusarium chemotype distribution may help tailoring forecasting schemes for disease development and mycotoxin contamination on a regional basis (Jennings et al., 2004b; Scoz et al., 2009). Indeed, it was postulated that the difference in chemotype distribution may result in the shift in toxin accumulation in grains, as suggested for DON content in grains in the case of the presence of the F. graminearum 3-ADON population in US and Canada (Foroud et al., 2008; Ward et al., 2008), but no definitive proof on the correlation of chemotype detection and grain contamination has been found to date. Here we showed, for the first time according to the authors' knowledge, the usefulness of chemotyping F. culmorum population for predicting the possible presence of a toxin (NIV) in wheat samples. The first aim of the paper was to fully characterize the chemotype of F. graminearum and F. culmorum from Luxembourg, during the years 2007 and 2008, in order to understand population distribution patterns of two of the main causal agents of head blight on winter wheat. Because carefulness concerning the use of genetic chemotyping has often been suggested, highlighting the variability of toxin production among strains (Desjardins, 2008), we combined genetic chemotyping with chemical analysis of toxins production in a liquid medium. Large differences in the toxin concentrations produced in vitro by single isolates were found, as have been already reported (Quarta et al., 2005; Szecsi et al., 2005). The chemical analysis corresponded to the results obtained by genetic chemotyping, as already verified by Burlakoti et al. (2008), suggesting the validity of a large-scale approach based on genetic chemotyping. Few isolates did not produce enough toxins to be detected in the inducing medium. This may be due to the efficiency of toxin stimulation by the medium (Jiao et al., 2008) and therefore other inducing substances may be tested in the future (Gardiner et al., 2009). Our enlarged set of data for F. graminearum confirmed previous observations from Luxembourg (Pasquali et al., 2009) showing the presence of two populations. Similar as in the Netherlands, Brazil, UK and China, the 15-ADON population was the predominant one (Jennings et al., 2004b; Ji et al., 2007; Scoz et al., 2009; Waalwijk et al., 2003), with a NIV population rarely present. No 3-ADON strains were detected. Thus, Luxembourg has not been reached by this expanding population that seems to have spread recently both in North America and China (Ward et al., 2008; Guo et al., 2008; Yang et al., 2008), and that may pose a serious threat to food safety due to its higher toxigenic potential (Ward et al., 2008) as well as its higher aggressiveness on certain wheat cultivars (Foroud et al., 2008). This paper represents also the first report of chemotype characterization of F. culmorum in Luxembourg. Previous works elsewhere obtained heterogeneous results on the chemotype composition within the species. While studies conducted in the Netherlands showed a higher proportion of NIV isolates (Waalwijk et al., 2003), other studies in England and Wales (Jennings et al., 2004a) and in many European countries together (Quarta et al., 2005) showed a predominance of 3-ADON population. In Luxembourg, an equal distribution of the two existing chemotypes (3-ADON and NIV) was observed, despite the inhomogeneous distribution of the isolates within the Luxembourg territory. To explain chemotype distribution in different geographic areas, hypotheses based on wheat seed shipment and long-distance spore transportation (Guo et al., 2008) were proposed for F. graminearum, while environmental favorable conditions (Jennings et al., 2004a) were suggested in the case of F. culmorum. Our data suggested that, in Luxembourg, maize played a significant role for the presence of the NIV chemotype for both species. This can be due to the fact that NIV is a

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pathogenicity factor useful for maize colonization (Maier et al., 2006), therefore the plant probably represents an ecological niche for hosting the NIV chemotype. Thus maize as preceding crop, being an important source of inoculum, is not only influencing positively the presence of DON and Fusarium spp. on wheat (Dill-Macky and Jones, 2000; Osborne and Stein, 2007), but also seems to favor the NIV chemotype. The isolation method coupled with species and chemotype determination allowed for investigating which Fusarium species and which chemotype was responsible for NIV accumulation in grains. In addition to F. graminearum and F. culmorum, F. poae is able to produce NIV (Stenglein, 2009), and it was identified as the cause of NIV production in wheat from Japan (Sugiura et al., 1993). A correlation between NIV content and the amount of F. poae DNA was reported for Northern Europe (Yli-Mattila et al., 2008). In Luxembourg, the F. poae presence in grains was not predictive of NIV contamination in the grains. This confirms a recent report from Poland that found no correlation between F. poae and NIV accumulation (Kulik and Jestoi, 2009). Correlation between NIV content in grains and presence of F. culmorum (without taking into account the chemotype) was less strong (Spearman correlation coefficient = 0.61) than using the chemotyping information, highlighting therefore that chemotyping is a powerful technique with predictive value for indicating toxin accumulation in grains. The F. graminearum NIV chemotype presence in grains was slightly correlated to the amount of NIV in grains, but the effect of the species was not significant (as shown by the linear mixed model) as F. graminearum co-occurred with the F. culmorum NIV chemotype. The current study further demonstrates that prevalence of a certain toxigenic species is not predictive of the accumulation of toxins in grains. Here, for example F. poae represented 14.0% of the total Fusarium population obtained from wheat sampling, while F. culmorum with NIV chemotype represented only 7.5% of the isolates, but was, however, mostly responsible for toxin accumulation. The causal heterogeneity of toxin accumulation in grains in different geographic locations requires further epidemiological surveys to adapt specific preventive practices to the agro-environmental situation of the area under scrutiny. Despite NIV content in feed being not currently regulated by international laws, the potential synergistic toxic effects of the presence of more than one trichothecene in the same product and the specific high toxicity of NIV (Bony et al., 2007; Gutleb et al., 2002; Marzocco et al., 2009), call for models able to predict the presence of more than a single toxin. For example, grains from two experimental sites (CHR 8A 0 T and CHR 8C 3 T) reached values of NIV + DON near the limit established for DON only in the EU-regulation1881/2006 for unprocessed white winter wheat (b1.75 μg/kg). It would seem therefore prudent to identify NIV contaminated fields and possibly to take into account NIV co-occurrence for safety levels. A preventive tool for identifying NIV contaminated fields would be especially important because NIV presence in grains cannot be inferred by the amount of DON. Therefore, the cut-off values for predicting the NIV content in grains (based on number and percentage of F. culmorum NIV+ isolates per 100 seeds) were established based on the contamination levels of grains obtained from 2 years, representing indeed different infection levels for FHB. The obtained cut-off threshold successfully discriminated fields containing and not-containing NIV in additional fields in Luxembourg. The robustness of the sampling method and of the cut-off criteria for prediction will have to be further tested on a larger number of samples from different years. Moreover, as already done for predicting DON content by quantifying the fungal biomass of DON producers (Burlakoti et al., 2007; Fredlund et al., 2008; Schnerr et al., 2002; Waalwijk et al., 2004), a real-time PCR quantification method for F. culmorum NIV chemotype seems a promising tool to be developed. Acknowledgements We would like to thank Kerry O'Donnell and Virgilio Balmas for providing reference strains used as controls for chemotype determination

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