Correlation of Deoxynivalenol Accumulation in Fusarium-Infected

May 19, 2016 - Fusarium infection in wheat causes Fusarium head blight, resulting in yield losses and contamination of grains with trichothecenes. Som...
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Correlation of Deoxynivalenol Accumulation in Fusarium-Infected Winter and Spring Wheat Cultivars with Secondary Metabolites at Different Growth Stages Thomas Etzerodt,*,† Rene Gislum,† Bente B. Laursen,† Kirsten Heinrichson,† Per L. Gregersen,‡ Lise N. Jørgensen,† and Inge S. Fomsgaard† †

Department of Agroecology and ‡Department of Molecular Biology and Genetics, Aarhus University, Forsøgsvej 1, 4200 Slagelse, Denmark S Supporting Information *

ABSTRACT: Fusarium infection in wheat causes Fusarium head blight, resulting in yield losses and contamination of grains with trichothecenes. Some plant secondary metabolites inhibit accumulation of trichothecenes. Eighteen Fusarium infected wheat cultivars were harvested at five time points and analyzed for the trichothecene deoxynivalenol (DON) and 38 wheat secondary metabolites (benzoxazinoids, phenolic acids, carotenoids, and flavonoids). Multivariate analysis showed that harvest time strongly impacted the content of secondary metabolites, more distinctly for winter wheat than spring wheat. The benzoxazinoid 2-βglucopyranoside-2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-glc), α-tocopherol, and the flavonoids homoorientin and orientin were identified as potential inhibitors of DON accumulation. Several phenolic acids, lutein and β-carotene also affected DON accumulation, but the effect varied for the two wheat types. The results could form a basis for choosing wheat cultivars using metabolite profiling as a marker for selecting wheat cultivars with improved resistance against Fusarium head blight and accumulation of trichothecene toxins in wheat heads. KEYWORDS: FHB resistance, principal component analysis, wheat secondary metabolites, benzoxazinoids, phenolic acids, flavonoids, tocopherols

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INTRODUCTION Fusarium head blight (FHB) is a devastating disease in wheat caused by members of the Fusarium genus, of which Fusarium graminearum and F. culmorum are found to be of major importance. During 1998 to 2000 economic losses due to wheat infection by Fusarium in the Northern and Central United States alone were estimated to be approximately $2.7 billion.1 FHB is caused by accumulation of trichothecene mycotoxins, predominantly DON, in wheat grains resulting in reduced yields. Furthermore, trichothecenes accumulated in wheat grains are stable against food and feed processing2 and thus are transferred to consumers and livestock if not controlled. Management of FHB involves crop rotation, avoiding maize as a precrop to wheat, employing resistant cultivars, tillage methods, application of fungicides, and possibly biological control.3,4 According to Mesterházy,5 cultivar resistance against FHB can be divided into five classes: (I) resistance against initial infection, (II) resistance against disease spread, (III) resistance against kernel infection, (IV) tolerance against FHB and trichothecenes, and (V) resistance to trichothecene accumulation. Class V is further divided into two subclasses by Boutigny et al.,6 resistance (a) by chemical modification of trichothecenes (e.g., glycosylation) and (b) by inhibition of trichothecene biosynthesis. Inhibition of trichothecene biosynthesis (type V-b resistance) has been shown for different secondary metabolites naturally present in cereals.7−9 Benzoxazinoids are a group of secondary metabolites with allelopathic and antifungal properties present in graminaceous plants10 and surrounding soil11 FHB resistance was correlated © XXXX American Chemical Society

with some benzoxazinoids in wheat heads by Søltoft et al. using principal component analysis12 and the benzoxazinoid 2,4dihydroxy-7-methoxy-benzoxazin-3-one (DIMBOA) inhibited trichothecene accumulation in liquid cultures.9 Phenolic acids are ubiquitous throughout the plant kingdom as part of the lignified plant cell walls. They are well-known for their antioxidant activity and have been studied for their regulatory effects on trichothecene accumulation, though with varying results depending on the type of study and conditions.8,9,13,14 Flavonoids are compounds with antioxidant activity and are identified from many plants. Some were found to suppress trichothecene biosynthesis,15,16 and transgenic flax plants expressing higher levels of flavonoid glucosides exhibited increased resistance against FHB.17 Carotenoid and tocopherol effects on FHB and trichothecene accumulation are less investigated. The carotenoid lutein stimulates accumulation of DON and DON-3-glucoside in some wheat cultivars,18 whereas some carotenoids inhibit aflatoxin accumulation.19 The aim of this project was to elucidate potential correlations between concentrations of 38 wheat secondary metabolites and the trichothecene DON in a field experiment employing nine spring wheat and nine winter wheat cultivars with varying degrees of resistance against FHB. Heads of Fusarium infected wheat cultivars were harvested at five time points (from Received: March 12, 2016 Revised: May 17, 2016 Accepted: May 19, 2016

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Figure 1. Chemical structures for compounds quantitated in this study. Me denotes methyl group, MeO denotes methoxy group, and glc denotes glucosyl group. distributed throughout the plots using 100 g of infected seeds/m2 at growth stage BBCH 37 along with 4 maize stalks/m2 to mimic natural infection with Fusarium. BBCH is a scale used to describe different developmental stages of different plant types.22 The plots were mist irrigated twice during 5 min/d from heading to BBCH 75 to ensure good infection conditions. Susceptibility to FHB was assessed at BBCH 75−77, where 100 heads per cultivar and replicate were assessed for numbers of attacked heads with FHB (incidence) as well as the average degree of severity in the heads. Based on these data, the FHB index (percentage infected spikelets times percentage average severity per 100 wheat heads) was calculated for each cultivar. Five wheat heads were harvested for each cultivar at five time points: (1) At heading of wheat heads (BBCH 55−59), (2) at anthesis (BBCH 65), (3) at first FHB symptoms (BBCH 71−75), (4) at FHB index assessment (BBCH 75−77), and (5) at grain maturity (BBCH 89). The harvested wheat heads were frozen in liquid nitrogen immediately after harvesting and stored at −20 °C until lyophilization. The lyophilized samples were blended and extracted and analyzed as described below. Wheat secondary metabolites were quantitated at all five harvest times, but DON was only quantitated for harvest time 3− 5, since the wheat heads are most susceptible to Fusarium infection at time point 2 and consequently DON will only accumulate hereafter. The absence of DON prior to harvest time 3 was confirmed by analysis of samples randomly selected for harvest time 1 and 2. Extraction and Analysis of Wheat Heads. Chemical structures for all analytes quantitated in this study are shown in Figure 1. Carotenoids and Tocopherols. Extraction and Analysis of Carotenoids and Tocopherols. Two hundred milligrams of ground wheat material was extracted in the dark with 2 mL of MTBE + 0.1% butylated hydroxytoluene. Samples were shaken overnight at room temperature and centrifuged in at 8300g at room temperature for 10 min. The supernatant was filtered and stored at −20 °C until analysis (10 μL) by liquid chromatography at 30 °C interfaced with diode array detection (DAD) and mass spectrometry (MS) on an HP1100 system (Agilent, Glostrup, Denmark). Chromatography was done with eluents (A) 95% acetonitrile, 5 mM ammonium acetate, and (B) 85% methyl tert-butyl ether, 11% methanol, 4% water, with a flow rate of 0.4 mL/ min and gradient elution as follows: 0−17 min at 0% B; 17−35 min at 0−50% B; 35−38 min at 50% B, 38−39 min at 50−0% B, and 39−45 min at 0% B. The column was a 250 mm × 3.0 mm i.d., 3 μm Acclaim C30 (Thermo Scientific) equipped with a 10 mm × 3.0 mm, i.d., 5 μm guard column of the same material. Carotenoids were detected at λ =

heading to maturity), and correlation between secondary metabolites and DON was determined using principal component analysis and Pearsson correlation coefficient analysis to obtain a thorough understanding the wheat secondary metabolite profile and its possible influence on the accumulation of DON.

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MATERIALS AND METHODS

Chemicals and Equipment. HPLC grade solvents acetonitrile, methyl-tert-butyl ether (MTBE), and methanol were purchased from Rathburn Chemicals (Walkerburn, Scotland). Chloroform (CHCl3) was obtained from Merck (Darmstadt, Germany). In the following, the term “glc” refers to glucoside, that is, a glucosyl derivative of the corresponding aglucone. 2-β-Glucopyranoside-2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA-glc, purity ≥84%), DIMBOA-glc (≥89%), double hexose derivative of 2-hydroxy-1,4-benzoxazin-3-one (HBOAglc-hex), double hexose derivative of 2,4-dihydroxy-1,4-benzoxazin-3one (DIBOA-glc-hex), 2-β-glucopyranoside-2-hydroxy-1,4-benzoxazin3-one (HBOA-glc, > 99%), 2-β-glucopyranoside-2-hydroxy-7-methoxy-1,4-benzoxazin-3-one (HMBOA-glc, 70%), and 4-acetyl-benzoxazolin-2-one (4-ABOA, purity 99.2%) were obtained as part of an ongoing patenting process. 20 2-Hydroxy-1,4-benzoxazin-3-one (HBOA) and 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one (HMBOA) were synthesized according to Krogh et al.21 Homoorientin, orientin, luteolin-4′-O-glc, luteolin, and apigenin were obtained from ExtraSynthese (Genay Cedex, France). Kaempferol-3′-O-glucoside was purchased from Carl Roth GmbH (Karlsruhe, Germany). Glacial acetic acid was obtained from Fischer Scientific (Roskilde, Denmark). DON (99.4%) and 13C-DON (≥99.5%) were obtained from Biopure (Tulln, Austria). All other chemicals were obtained from Sigma (Brøndby, Denmark). Omnifix-F syringes (B. Braun, Melsungen, Germany) and Q-Max RR syringe filters (0.45 μm pore size, Frisenette ApS, Knebel, Denmark) were used to filter all extracts prior to analysis. Field Design, Fusarium Inoculation, and Harvesting of Wheat Heads. Nine cultivars of winter wheat (WW) were sown in Autumn 2012 and nine cultivars of spring wheat (SW) were sown in Spring 2013. Spring and winter cultivars with different susceptibility to FHB were selected for the project based on knowledge from previous years of Fusarium disease assessment. The cultivars were arranged in a randomized block design with 2 m rows and four replicates for each cultivar. Autoclaved wheat kernels infected with F. culmorum were C

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Journal of Agricultural and Food Chemistry 450 nm (HP 1100 DAD detector), with retention times 21.1, 22.6, and 36.6 min for lutein, zeaxanthin and β-carotene, respectively. Tocopherols were ionized by electrospray ionization (fragmentor voltage 140 V, nebulizer pressure 40 psig, drying gas temperature 300 °C, drying gas flow 9 L/min, capillary voltage 4500 V) and detected by single ion monitoring mass spectrometry in negative polarity on Agilent 1100 LC/MSD. α-Tocopherol and β-/γ-tocopherol eluted at 26.0 and 28.6 min, respectively. β- and γ-Tocopherol were quantitated together due to separation challenges, as also experienced by Hejtmánková et al.23 Carotenoids and tocopherols were quantitated from external calibration curves (6.25−400 ng/mL) using Agilent ChemStation, ver. B.04.03. Quantitated carotenoid and tocopherol concentrations were corrected for the recovery values and corrected concentrations were calculated relative to sample weight. A representative chromatogram for carotenoid and tocopherol standards is shown in Figure 2.

and tocopherols in CHCl3 was added to 200 mg of wheat material and allowed to dry down at room temperature. Samples were extracted as described above, performed in six replicates. Limit of detection (LOD) was calculated from 3s and limit of quantitation (LOQ) from 10s; s is standard deviation for each analyte, calculated from eight paired measurements (from samples with concentrations close to visual assessment of detection limits) according to Engineering Statistics Handbook:24

s=

1 N−1

8

∑ (xi1 − xi2)2 i=1

where N is the number data pairs from which the standard deviation is calculated, and xi1 and xi2 is the pairwise measurement for the ith pair. Polar Compounds. Polar compounds are defined here as benzoxazinoids, phenolic acids, and flavonoids. Extraction and Analysis of Polar Compounds. One hundred milligrams of wheat material was extracted with 5 mL of 70% acetonitrile + 1% acetic acid by shaking for 2 h at 60 °C and then centrifuged at room temperature for 10 min at 3800g. The supernatant was filtered and diluted 1:1 with water before analysis (20 μL) by liquid chromatography interfaced with time-of-flight MS. Chromatography was performed at 30 °C on a Synergi Polar column, 250 mm × 2 mm i.d., 4 μm, from Phenomenex (Værløse, Denmark) equipped with a 4.0 mm × 2.0 mm i.d. guard column of the same material. Flow rate was 0.2 mL/min with gradient elution: 0−3 min at 5% B, 3−30 min at 5−31% B, 30−38 min at 31−100% B, 38−42 min at 100% B, 42−43 min at 100−5% B, and 43−50 min at 5% B. Eluents were (A) 7% acetonitrile + 20 mM acetic acid and (B) 78% acetonitrile + 20 mM acetic acid. Compounds were ionized by electrospray ionization (125 V fragmentor voltage, nebulizer pressure 40 psig, drying gas temperature 300 °C, drying gas flow 12 L/min, capillary voltage 3500 V) and detected by time-of-flight mass spectrometry (Agilent 6224) in scan mode (m/z 100−1700) in negative polarity. Ion chromatograms for polar compounds were extracted using Agilent MassHunter Quantitative Software v. B.04.00, from m/z values and retention times for individual analytes and quantitated from external calibration curves (6.25−200 ng/mL). Concentrations relative to sample amount were calculated as for carotenoids and tocopherols. A

Figure 2. Chromatographic separation of carotenoids and tocopherols quantitated in this study. Carotenoid absorbance (450 nm) and tocopherol MS intensity is shown on the primary and secondary y-axis, respectively. Peaks are numbered according to Table 1. Method Validation for Carotenoids and Tocopherols. For recovery studies 100 μL of a 2 μg/mL stock solution of carotenoids

Figure 3. Chromatographic separation of polar compounds quantitated in this study. Peaks have been normalized for easy comparison. Peaks are numbered according to Table 1. D

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representative chromatogram for standards of polar compounds is shown in Figure 3. Method Validation for Polar Compounds. For recovery studies, 5 μL of a 10 μg/mL stock solution of a mixture of benzoxazinoids or phenolic acids or flavonoids in acetonitrile was added to 200 mg of wheat material; the samples were allowed to dry at room temperature, extracted, and quantitated as described above. Recovery samples were performed in six replicates. LOD and LOQ were determined as described for carotenoids and tocopherols. Deoxynivalenol (DON). Extraction and Analysis of DON. One hundred milligrams of wheat sample was extracted with 800 μL of 84% acetonitrile by 10 min sonication followed by shaking for 2 h and centrifuged at room temperature for 10 min at 3800g. One hundred microliters of the supernatant was mixed with 350 μL water, and 13Clabeled DON internal standard was added to a final concentration of 25 ng/mL. Samples were then filtered through syringe microfilters before analysis by liquid chromatography interfaced with MS/MS (AB Sciex 3200, Nærum, Denmark). Ten microliters of filtered extract was chromatographed on a BDS Hypersil C18 column, 250 mm × 2.1 mm i.d., 5 μm (Fischer Scientific, Roskilde, Denmark) equipped with a 10 mm × 2.1 mm guard column of the same material. Flow rate was 0.2 mL/min using eluents (A) 1% MeOH and (B) 90% MeOH by gradient elution with 0.2 mL/min flow rate: 0−1 min at 0% B, 1−10 min at 0−100% B, 10−14 min at 100% B, 14−14.5 min at 100−0% B, and 14.5−23 min at 0% B. Analytes were ionized by ESI in negative polarity (capillary voltage 4500 V, curtain gas 20 psi, ion source gas-1 50 psi, ion source gas-2 50 psi, drying gas temperature 475 °C) and analyzed by MSMS (fragmentation parameters: declustering potential 30 V, entrance potential 11 V, collisional cell entrance potential 15 V, collisional energy 15 V and collisional cell exit potential 15 V). Deoxynivalenol and 13C labeled deoxynivalenol (retention time 12.2 min) were analyzed for using m/z pairs 295.0/265.0 and 310.0/279.0, respectively, with a dwell time of 200 ms. Analyst 1.6.2 software was used for quantitation from an external calibration curve of DON in the range (0.78−200 ng/mL in 25% acetonitrile). Concentrations relative to sample amount were calculated as described for carotenoids and tocopherols. Method Validation for DON. For recovery experiments, 100 mg of wheat material was spiked with 50 μL of a 10 μg/mL solution of DON and allowed to dry at room temperature. Samples were then extracted and analyzed by LC-MS/MS as described above. Standards and samples were spiked with 25 ng/mL 13C-labeled DON to correct for matrix effects. LOD and LOQ were determined as described in Nicolaisen et al.25 Data Analysis. Principal component analysis (PCA)26 was performed to investigate the relationship between the concentration of secondary metabolites and DON (in μg/g of wheat dry matter) in nine different cultivars of SW and WW. PCA analysis was performed separately for WW and SW cultivars, and since DON was quantitated only at harvest times 3−5, the PCA analysis did not include harvest times 1 and 2. Data were standardized using autoscaling where each variable has its mean removed and the result is scaled by the standard deviation. Root mean square error of cross-validation (RMSECV) plotted against the number of PCA components was used to select the optimum number of components in the PCA model. The optimum number of PCA components was chosen as the first minimum in the smooth declining RMSECV curve. Venetian blinds cross-validation with 10 data splits was used for validation of the PCA model. The PCA analysis was performed using MATLAB version 7.9 (R2009b) (The Mathworks, Inc., Natick, MA) along with the PLS_Toolbox ver. 7.9.3 (eigenvector Research, Inc., Manson, WA). Correlation coefficient matrices were made for secondary metabolites and DON at harvest times 3−5 for SW and WW individually. The matrices were made using the procedure PROC CORR of the Statistical Analysis System version 9.3, software package.27 In the matrices, correlation coefficients are marked in green for positive and in yellow for negative correlations, and color intensity indicates the degree of correlation.

RESULTS AND DISCUSSION Method Validation. Recovery, LOD, and LOQ for each analyte in this study are given in Table 1. Recoveries were Table 1. Method Validation Parameter Values for Analytes Quantitated in This Studya no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

analyte

recovery (RSD) %

Carotenoids 87 (15) 64 (32) 82 (11) 86 (8) 101 (10) Benzoxazinoids DIBOA-glc 92 (9) DIMBOA-glc 113 (9) HBOA-glc-hex 53 (13) Phenolic Acids gallic acid 81 (4) protocatechuic acid 100 (11) chlorogenic acid 56 (6) p-hydroxybenzoic acid 75 (7) caffeic acid 97 (6) vanillic acid 75 (5) syringic acid 90 (6) salicylic acid 90 (5) p-coumaric acid 81 (8) sinapic acid 85 (4) ferulic acid 80 (8) cinnamic acid 101 (3) Flavonoids homoorientin 96 (9) orientin 113 (10) luteolin-4′-O-glc 112 (5) kaempferol 112 (6) luteolin 88 (3) pratensein 91 (4) Mycotoxin DON 80 (2) lutein zeaxanthin β-carotene α-tocopherol β-/γ-tocopherol

LOD [μg/g]

LOQ [μg/g]

0.19 0.39 0.06 0.19 0.15

0.62 1.30 0.21 0.62 0.50

0.68 0.48 4.1

2.25 1.60 14

0.19 1.3 0.75 1.01 0.73 0.70 0.41 0.17 0.13 0.37 2.7 0.16

0.63 4.2 2.5 3.4 2.4 2.3 1.4 0.56 0.43 1.2 8.9 0.54

0.53 0.12 0.15 0.05 0.08 0.35

1.8 0.39 0.49 0.15 0.28 1.2

0.01

0.03

a

Recovery is given in percent. RSD denotes relative standard deviation for recoveries in percent (for n = 6). LOD denotes limit of detection and LOQ denotes limit of quantitation both given in μg/g of dry weight.

found to be 75−113%, except for zeaxanthin, HBOA-glc-hex, and chlorogenic acid with recoveries between 52% and 64%. LOD was relatively high for the secondary metabolites HBOAglc-hex, protocatechuic acid, p-hydroxybenzoic acid, and ferulic acid (4.08, 1.27, 1.01, and 2.66 μg/g, respectively). A similar LOD for HBOA-glc-hex was also calculated by Adhikari et al.28 HBOA, HMBOA, HBOA-glc, HMBOA-glc, BOA, MBOA, 4ABOA, β-cryptoxanthin, δ-tocopherol, isorhamnetin, kaempferol-3′-O-glc, and apigenin were also analyzed but could not be detected in any samples. Data Analysis. The PCA score and loading plots, also known as PCA biplots (Figures 4A−C), illustrate the relationships between samples and variables for PCA components 1 and 2 for WWs and PCA components 1, 2, and 3 for SWs. The first principal component (PC1) is the best fit to all data points, and PC2 is orthogonal to PC1 in the direction of the second largest variance. For WWs, PC1 and E

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components were used to describe the variance for the SWs, with PC1, PC2, and PC3 accounting for 32%, 19%, and 13% of the variance, respectively (Figure 4B,C). The relationship between the scores and loadings are that cultivars and harvest times with an elevated score of principal components had a high content of variables with high loadings from the same PC. Three main clusters could be deduced in each biplot, marked in yellow, blue, and red ovals with each cluster comprising one harvest time. This illustrates that wheat harvest time had the strongest impact on variance (Figure 4A−C). Some similarities were observed for secondary metabolite cluster patterns for both WWs and SWs for the same harvest times, that is, such secondary metabolites accumulate at highest concentration at a particular harvest time independent of wheat type. DON, lutein, β-carotene, HBOA-glc-hex, chlorogenic acid, and cinnamic acid clustered with harvest time 3, protocatechuic acid clustered with harvest time 4, whereas α-tocopherol and pratensein clustered with harvest time 5 (Figure 4A−C). DIMBOA-glc and sinapic acid were located between clusters of harvest time 3 and 4 but less apparent for SWs than for WWs. Accumulation of other secondary metabolites clustered differently depending on wheat type, that is, maximum accumulation for those secondary metabolites occurred at different time points for WWs and SWs. p-Hydroxybenzoic acid, caffeic acid, vanillic acid, syringic acid, and salicylic acid clustered with harvest time 5 for WWs but with harvest time 3 for SWs. Ferulic acid clustered with WWs at harvest time 3 but with SWs at harvest time 4. DON. DON quantitated in WWs (for mature grain) were in agreement with the Danish national crop protection report for 2013.29 Concentrations of DON in the wheat cultivars were in good agreement with the disease severity index (FHB index). Very susceptible cultivars SW1, SW2, SW4, and SW6 generally accumulated high concentrations of DON, while the toxin was virtually absent in the FHB resistant Sumai-3 cultivar (SW9). WW cultivars had a lower disease index, which corresponded well with their lower accumulation of DON compared with SWs. The cultivars could mainly be split in two groups, the susceptible cultivars (index 2−3) and the cultivars with highest resistance (index 1). The four cultivars with high resistance all accumulated low levels of DON. However, the correlation between susceptibility and accumulation of DON was not direct for all cultivars. Some cultivars with a high or moderate disease index accumulated similar concentrations of DON, which has been observed in other studies as well.30 Relatively large standard deviations were associated with DON quantitation, most likely due to “natural infection” with Fusarium infected wheat kernels and maize stalks, resulting in a heterogeneous infection differing for each random plot. For some cultivars there was a decrease in DON concentration from harvest time 3 to 4 followed by an increase to maturity (time 5). In wheat, several DON derivatives have been identified including acetylated DON and DON-3-glucoside31 as well as DON-glutathione.32 Possibly, an interconversion between DON and such derivatives could explain this type of dynamic behavior. For PC1 vs PC2, DON was clustered (red cluster, Figures 4A and 4C) with WW and SW cultivars at harvest time 3, when first FHB symptoms were observed, while DON also clustered with harvest time 4 for SWs when looking at PC1 vs PC3 (Figure 4C). This indicates that the dynamic pattern for DON in SWs might be more variable than that in WWs, supported by the dynamic plots for DON.

Figure 4. Principal component analysis biplots for secondary metabolites and deoxynivalenol to different harvest times. Biplot A shows PC1 vs PC2 for winter wheat, biplot B shows PC1 vs PC2 for spring wheat, and biplot C shows PC1 vs PC3 for spring wheat. Harvest time three is shown as circles, four as triangles, and five as squares. The numbers in the plots indicate secondary metabolites (see Table 1 for numbers).

PC2 described 42% and 17% of the variance, respectively (Figure 4A) and no further information was available from additional principal components. Three orthogonal principal F

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Figure 5. Correlation coefficient matrix for wheat secondary metabolites and deoxynivalenol in spring wheat for harvest times 3−5. Positive and negative correlations are marked in green and yellow color, respectively, with probabilities given in italic numbers below. Numbers 1−26 are wheat secondary metabolites as given in Table 1.

agreement with their clustering in the biplot analysis for WW cultivars (Figure 4A). In mature wheat heads only low concentrations of lutein (LOQ to 1.2 μg/g) and β-carotene (below LOQ) were found, comparable to other studies.35,36 Delgado et al.18 showed that DON and DON-3-glucoside was stimulated by lutein. Conversely, β-carotene and lutein inhibit accumulation of fumonisins in maize grains;36 thus toxin inhibition appears to depend on type of Fusarium fungus or toxin analyzed. In our studies, both carotenoids clustered with DON and SW cultivars at harvest time 3 in the biplot analyses (Figure 4A,B). The more resistant SW cultivars 3, 7, 8, and 9 exhibited a more significant drop in lutein concentration from harvest time 2 to 3 compared with other SW cultivars. Altogether, DON accumulation appears to be stimulated by lutein and β-carotene especially for SW cultivars. Carotenoids lutein, zeaxanthin and β-carotene were positively correlated for both WWs and SWs (corr. coef. of +0.61 to +0.84) (Figures 5 and 6), most likely a consequence of related biosynthesis pathways for these carotenoids. Carotenoids were also correlated to several phenolic acids (with correlation coefficients up to +0.86 for salicylic acid) for SWs, which will be discussed in the section of Phenolic Acids. This correlation is reflected in the PCA plots for SWs where lutein clusters with the five phenolics (Figure 4B,C). A significant positive correlation was observed between βcarotene and sinapic acid (corr. coef. +0.73). The reason for this is not clear but might stem from a simultaneous

The correlation coefficient matrices for SWs (Figure 5) and WWs (Figure 6) illustrate overall correlations between all individual analytes for harvest times 3−5. Only modest correlations could be observed between DON and wheat secondary metabolites. In SWs salicylic acid and p-coumaric acid exhibited the highest correlation with DON with correlation coefficients of +0.34 and +0.40, respectively. For WWs gallic acid and caffeic acid exhibited the highest positive correlations with DON with coefficients of +0.34 and +0.42, respectively. Carotenoids and Tocopherols. α-Tocopherol has been shown to inhibit fumonisin accumulation in liquid cultures of Fusarium verticillioides,33 and Boba et al. correlated αtocopherol to Fusarium resistance in transgenic flax.34 In our studies α-tocopherol clustered distinctly with harvest time 5 (mature wheat heads) opposite of DON for both SWs and WWs (Figure 4A,B), indicating that α-tocopherol inhibits DON accumulation independent of wheat type. Production of α-tocopherol could thus be a common response to infection by DON producing Fusarium species. Most cultivars exhibiting a steep increase in α-tocopherol content to maturity (harvest time 5) generally accumulated low levels of DON, supporting a suppressive role of α-tocopherol against DON accumulation in wheat heads. Large positive correlation coefficients were observed between α-tocopherol and the phenolics gallic acid, p-hydroxybenzoic acid and caffeic acid in WWs with correlation coefficients of +0.84, +0.83, and +0.77, respectively (Figure 6). This is in G

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Figure 6. Correlation coefficient matrix for wheat secondary metabolites and deoxynivalenol in winter wheat for harvest times 3−5. Positive and negative correlations are marked in green and yellow color, respectively, with probabilities given in italic numbers below. Number 1−26 are wheat secondary metabolites as given in Table 1.

acid, vanillic acid, and cinnamic acid with corr. coef. +0.78 to +0.86 and to a lesser degree to p-hydroxybenzoic acid and syringic acid (corr. coef. +0.62 and +0.65). The explanation for the observed correlation could be that salicylic acid accumulates as a response to Fusarium infection and subsequently induces the production of lutein and the four phenolic acids. SW cultivars 1 and 2 exhibited a large boost in the content of salicylic acid from harvest time 2 to 3, which also correlates with the FHB susceptibility for the cultivars. The more resistant WWs generally contained lower levels of salicylic acid with maximum concentrations observed at harvest time 2 compared with SW cultivars. Thus, we propose that salicylic acid plays a role in the cultivar susceptibility to FHB, both as a hormonal regulator and a secondary metabolite itself. Chlorogenic acid clustered with DON for both SWs and WWs (Figure 4A,B); that is, this phenolic acid was positively correlated with the trichothecene. Inverse correlations were observed when comparing dynamic plots for DON and chlorogenic acid, and in general chlorogenic acid at harvest time 2 (where susceptibility to Fusarium infection is highest) was inversely correlated to the FHB index. Overall, this points to a stimulative effect for chlorogenic acid on DON accumulation. Significant differences were observed for concentrations of caffeic acid in SWs and WWs peaking at harvest time 3 for SWs and at time 5 for WWs. Caffeic acid clustered with DON in the biplot analysis (Figure 4A,B) indicating that caffeic acid provokes accumulation of DON in SWs. The correlation

accumulation of the two secondary metabolites as a common response to the Fusarium infection or accumulation of DON. Phenolic Acids (Benzoic Acids and Cinnamic Acids). Phenolic acids are ubiquitous constituents of the plant kingdom, and their role as defense compounds is well established.37,38 Several in vitro studies have shown that some phenolic acids stimulate accumulation of Fusarium toxins.9,13,39,40 Conversely, ferulic acid is also known to inhibit Fusarium toxin accumulation in some in vitro studies.14 Gauthier et al. showed that effects of chlorogenic acid and caffeic acid on trichothecene accumulation depended on Fusarium strain.41 This illustrates that experimental conditions have important impact on phenolic acid activity against trichothecene accumulation. Chlorogenic acid inhibited fumonisin accumulation in vitro42 and was correlated with Fusarium resistance in maize,7 and protocatechuic acid exhibited resistance to F. graminearum penetration in seeds treated with chitosan.43 In the biplot analyses, benzoic acids were located opposite of DON for WW cultivars, whereas for SW cultivars benzoic acids were clustered with DON, that is, appear to stimulate DON accumulation in SWs. Thus, benzoic acids affect DON accumulation in a wheat type dependent manner. Salicylic acid is a plant hormone known to regulate the biosynthesis of other plant secondary metabolites44 and is also important in signaling processes in wheat infected with Fusarium.45 For SWs, correlation coefficient matrices (Figure 5) revealed a strong positive correlation between lutein, salicylic H

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harvest time 5. For WWs, no DIMBOA-glc was detected at harvest time 1, but it increased toward harvest time 4 with the most significant increase observed from harvest time 3 to 4. This could reflect that DIMBOA-glc plays a role against DON accumulation after harvest time 3. HBOA-glc-hex was quantitated in all samples with the highest content (concentrations up to 116 ± 5 μg/g) observed at harvest time 2 declining to below 6 μg/g in mature wheat heads. Tanwir et al.46 found 468 ± 8 ng/g HBOA-glc-hex in cleaned grains of wheat, a concentration approximately ten times lower than that quantitated from mature wheat heads in our studies. This could be explained by a heterogeneous distribution of benzoxazinoids in the cereal tissue, that is, HBOA-glc-hex could also be present in other parts of the wheat heads than wheat grains. HBOA-glc-hex was clustered with DON (Figure 4A,B), that is, positively correlated with the trichothecene. This might indicate that wheat cultivars with high levels of HBOA-glc-hex accumulate more DON or vice versa. Thus, the higher concentration of HBOA-glc-hex compared with results by Tanwir et al.46 might also be due to induction by Fusarium infection and DON accumulation. Flavonoids. Flavonoids in general are known for their antioxidant properties, and orientin, for example, is also known to possess antifungal activity.48 Studies of flavonoid effects on FHB or accumulation of Fusarium toxins are sparse, though. This is the first study quantitating homoorientin and orientin in wheat heads and correlating it with accumulation of DON. Homoorientin and orientin correlated strongly for SW cultivars (corr. coef. +0.84; Figure 5) probably reflecting their connected biosynthesis, supported by the dynamic plots for the two flavonoids. Homoorientin and orientin correlated less significantly in WW cultivars (corr. coef. +0.47; Figure 6). In general, the content of both homoorientin and orientin were higher in SWs than in WWs. In the biplot analyses for SWs, homoorientin and orientin are located opposite of DON along both PC2 and PC3 (Figure 4B,C) suggesting that the flavonoids might be inversely correlated to DON. Resistant SW cultivars 7, 8, and 9 contained very high concentrations of homoorientin at harvest time 1 and 2 with up to 1008 ± 46 μg/ g Comparable concentrations of some flavonoids were observed by Buśko et al.49 in grains from wheat cultivars infected with Fusarium. At harvest time 2, relatively higher levels of orientin were quantitated in especially SW cultivars 7 and 9 where wheat plants are most susceptible to infection by Fusarium. Furthermore, the susceptible SW cultivars 1 and 2, which accumulated high amounts of DON, contained low levels of orientin at all harvest time points. Altogether, this indicates that homoorientin and orientin play important roles against accumulation of DON, particularly for SW cultivars. WW and SW cultivars exhibited significant differences in their dynamic patterns of secondary metabolites from wheat in our field experiments with heads infected “naturally” with Fusarium. Using PCA and correlation coefficient analysis, several secondary metabolites were shown to affect the accumulation of DON in heads of both wheat types. The effect on DON accumulation differed for several of the secondary metabolites with inhibition of DON in SW cultivars but stimulation of DON in WWs and vice versa. Such differences for SW and WW cultivars have not been identified in previous studies but are important to consider when selecting wheat cultivars with an optimized composition of secondary metabolites for increased resistance against FHB and accumulation of trichothecenes. Different media or Fusarium

matrix for WWs suggests a slight positive correlation to DON (corr. coef. +0.42). In vitro studies by Ponts et al.13 identified caffeic acid as a stimulator of trichothecene production. Accordingly, we suggest caffeic acid to be a potential stimulator of DON accumulation in the wheat heads. Vanillic acid clustered with DON for SW cultivars but not for WW cultivars (Figure 4A,B). Susceptible SW1, SW2, and SW6 maintained high levels of vanillic acid until after harvest time 3, while vanillic acid content in the resistant Sumai-3 (SW cultivar 9) decreased significantly from harvest time 1 to harvest time 2. Furthermore, the more resistant WWs generally contained low levels of vanillic acid at harvest time 3. This suggests that vanillic acid plays a role in the cultivar propensity to accumulate DON in a wheat type dependent manner. Protocatechuic acid clustered with both WWs and SWs at harvest time 4 in our biplot analyses (Figure 4A,B), suggesting that it is important for both types of wheat for this time point. Protocatechuic acid is located opposite of DON and could be a potential inhibitor of DON accumulation in the wheat heads. The biplot analyses correlate cinnamic acid with DON (Figure 4A,B), which is supported by the dynamic plots for cinnamic acid in SW and WW. A “burst” in cinnamic acid production was observed from time 2 to 3, which correlates well with a higher accumulation of DON in the highly susceptible SW cultivars 1 and 2. Cinnamic acid was thus suggested to stimulate accumulation of DON in the wheat heads. p-Coumaric acid and ferulic acid clustered with DON for WWs (Figure 4A) and for SWs for PC1 vs PC3 (Figure 4C), suggesting that these two cinnamic acids stimulate DON accumulation in our field experiments. Studies by Etzerodt et al.9 and Ponts et al.13 showed that p-coumaric acid and ferulic acid provoked accumulation of trichothecenes in vitro in agreement with our observations in wheat heads. Benzoxazinoids. Benzoxazinoids were previously believed to be absent from mature cereal grain but were recently identified from mature wheat grain46 and grain products.47 In those studies, however, most of the benzoxazinoids were found in concentrations lower than what could be detected by our timeof-flight mass detector, which might explain why only DIBOAglc, DIMBOA-glc, and HBOA-glc-hex could be detected in our studies. In the biplot analyses for both WWs and SWs, DIBOA-glc and DIMBOA-glc were located opposite of DON whereas HBOA-glc-hex clustered with DON (Figure 4A,B). Thus, the benzoxazinoid monoglucosides appears to inhibit DON accumulation, whereas clustering of HBOA-glc-hex with DON suggests the diglucoside as a stimulator of DON accumulation. In wheat, the major benzoxazinoid is DIMBOA-glc,10 which is converted to the biologically active DIMBOA aglucone when cell integrity is compromised. DIMBOA inhibits different pests,10 inhibits trichothecene accumulation in liquid cultures of F. graminearum,9 and has been correlated with FHB resistance in wheat.12 DIMBOA-glc was located opposite of DON in our biplot analyses (Figure 4A,B), which indicates that DIMBOA-glc inhibits DON accumulation. Thus, DIMBOA-glc might play an important role in the inhibition of DON accumulation in planta for the cultivars of this study. In our studies, DIMBOA-glc concentration generally decreased toward grain maturity in SW cultivars, while the reverse was observed for the more resistant WW cultivars. In SWs, the highest levels of DIMBOAglc were observed at harvest time 1 followed by a decrease until I

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(7) Atanasova-Penichon, V.; Pons, S.; Pinson-Gadais, L.; Picot, A.; Marchegay, G.; Bonnin-Verdal, M. N.; Ducos, C.; Barreau, C.; Roucolle, J.; Sehabiague, P.; Carolo, P.; Richard-Forget, F. Chlorogenic acid and maize ear rot resistance: A dynamic study investigating Fusarium graminearum development, Deoxynivalenol production, and phenolic acid accumulation. Mol. Plant-Microbe Interact. 2012, 25, 1605. (8) Boutigny, A.-L.; Atanasova-Penichon, V.; Benet, M.; Barreau, C.; Richard-Forget, F. Natural phenolic acids from wheat bran inhibit Fusarium culmorum trichothecene biosynthesis in vitro by repressing Tri gene expression. Eur. J. Plant Pathol. 2010, 127, 275. (9) Etzerodt, T.; Maeda, K.; Nakajima, Y.; Laursen, B.; Fomsgaard, I. S.; Kimura, M. 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)one (DIMBOA) inhibits trichothecene production by Fusarium graminearum through suppression of Tri6 expression. Int. J. Food Microbiol. 2015, 214, 123. (10) Niemeyer, H. M. Hydroxamic acids derived from 2-hydroxy-2H1,4-benzoxazin-3(4H)-one: Key defense chemicals of cereals. J. Agric. Food Chem. 2009, 57, 1677. (11) Etzerodt, T.; Mortensen, A. G.; Fomsgaard, I. S. Transformation kinetics of 6-methoxybenzoxazolin-2-one in soil. J. Environ. Sci. Health, Part B 2008, 43, 1. (12) Søltoft, M.; Jorgensen, L. N.; Svensmark, B.; Fomsgaard, I. S. Benzoxazinoid concentrations show correlation with Fusarium head blight resistance in Danish wheat varieties. Biochem. Syst. Ecol. 2008, 36, 245. (13) Ponts, N.; Pinson-Gadais, L.; Boutigny, A.-L.; Barreau, C.; Richard-Forget, F. Cinnamic-derived acids significantly affect Fusarium graminearum growth and in vitro synthesis of type B trichothecenes. Phytopathology 2011, 101, 929. (14) Boutigny, A.-L.; Barreau, C.; Atanasova-Penichon, V.; VerdalBonnin, M.-N.; Pinson-Gadais, L.; Richard-Forget, F. Ferulic acid, an efficient inhibitor of type B trichothecene biosynthesis and Tri gene expression in Fusarium liquid cultures. Mycol. Res. 2009, 113, 746. (15) Desjardins, A. E.; Plattner, R. D.; Spencer, G. F. Inhibition of trichothecene toxin biosynthesis by naturally-occurring shikimate aromatics. Phytochemistry 1988, 27, 767. (16) Pani, G.; Scherm, B.; Azara, E.; Balmas, V.; Jahanshiri, Z.; Carta, P.; Fabbri, D.; Dettori, M. A.; Fadda, A.; Dessi, A.; Dallocchio, R.; Migheli, Q.; Delogu, G. Natural and natural-like phenolic inhibitors of type B trichothecene in vitro production by the wheat (Triticum sp.) pathogen Fusarium culmorum. J. Agric. Food Chem. 2014, 62, 4969. (17) Lorenc-Kukula, K.; Zuk, M.; Kulma, A.; Czemplik, M.; Kostyn, K.; Skala, J.; Starzycki, M.; Szopa, J. Engineering flax with the GT family 1 Solanum sogarandinum glycosyltransferase SsGT1 confers increased resistance to Fusarium infection. J. Agric. Food Chem. 2009, 57, 6698. (18) Delgado, R. M.; Sulyok, M.; Jirsa, O.; Spitzer, T.; Krska, R.; Polisenska, I. Relationship between lutein and mycotoxin content in durum wheat. Food Addit. Contam., Part A 2014, 31, 1274. (19) Norton, R. A. Effect of carotenoids on Aflatoxin B1 synthesis by Aspergillus f lavus. Phytopathology 1997, 87, 814. (20) Fomsgaard, I. S.; Mortensen, A. G.; Holm, P. B.; Gregersen, P. L. European Patent EP 2 265 133 A1, 2010. (21) Krogh, S. S.; Mensz, S. J. M.; Nielsen, S. T.; Mortensen, A. G.; Christophersen, C.; Fomsgaard, I. S. Fate of benzoxazinone allelochemicals in soil after incorporation of wheat and rye sprouts. J. Agric. Food Chem. 2006, 54, 1064. (22) Meier, U. Growth stages of mono- and dicotyledonous plants: BBCH-monograph; Blackwell Wissenschafts-Verlag: Berlin, 1997. (23) Hejtmankova, K.; Lachman, J.; Hejtmankova, A.; Pivec, V.; Janovska, D. Tocols of selected spring wheat (Triticum aestivum L.), einkorn wheat (Triticum monococcum L.) and wild emmer (Triticum dicoccum Schuebl Schrank) varieties. Food Chem. 2010, 123, 1267. (24) Engineering Statistics Handbook, http://www.itl.nist.gov/ div898/handbook/prc/section3/prc311.htm, last accessed May 27, 2016. (25) Nicolaisen, M.; Supronienė, S.; Nielsen, L. K.; Lazzaro, I.; Spliid, N. H.; Justesen, A. F. Real-time PCR for quantitation of eleven

strains influence the activity of phenolic acids, for example. This could explain why some secondary metabolites stimulate DON accumulation in our studies but inhibit trichothecene accumulation in other studies. Other defense compounds, such as alkylresorcinols, lignans, and pathogenesis-related proteins are also known to possess antifungal effects and inhibit mycotoxin accumulation. Future studies should include such metabolites for an extended profiling of Fusarium-related defense compounds. It would also be highly interesting to compare two or more consecutive years to assess environmental influence on the wheat-Fusarium interaction based on secondary metabolite profiles.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.6b01162. Table of wheat cultivars used in this study, table of parameters used for mass spectrometric analysis of polar compounds analyzed in this study, and dynamic plots for deoxynivalenol and selected analytes for all nine winter and spring wheat cultivars (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +45-87158213. E-mail: [email protected]. Funding

All work related to this research project was funded by Teknologi og Produktion, Det Frie Forskningsråd (Grant Number 12-132600). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Field technicians Helene Saltoft and Sidsel M. Kirkegaard are gratefully acknowledged for all work related to wheat cultivars prior to harvesting including infection with Fusarium via infected maize stalks.

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ABBREVIATIONS DIBOA-glc, 2-β-glucopyranoside-2,4-dihydroxy-1,4-benzoxazin3-one; DIMBOA-glc, 2-β-glucopyranoside-2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one; DON, deoxynivalenol; HBOA-glchex, double hexose derivative of 2-hydroxy-1,4-benzoxazin-3one; SW, spring wheat; WW, winter wheat

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REFERENCES

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K

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