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Presence of Fusarium Species and Other Toxigenic Fungi in Malting Barley and Multi-Mycotoxin Analysis by Liquid Chromatography− High-Resolution Mass Spectrometry Giovanni Beccari,†,§ Leonardo Caproni,†,‡,§ Francesco Tini,† Silvio Uhlig,‡ and Lorenzo Covarelli*,† †

Department of Agricultural, Food and Environmental Sciences, University of Perugia, Borgo XX Giugno, 74, 06121 Perugia, Italy Norwegian Veterinary Institute, Ullevålsveien, 68, P.O. Box 750, Sentrum, N-0106 Oslo, Norway

‡

S Supporting Information *

ABSTRACT: A study was carried out on 43 malting barley samples collected in 2013 across the Umbria region (central Italy) to determine the incidence of the principal mycotoxigenic fungal genera, to identify the Fusarium species isolated from the grains, and to detect the presence of 34 fungal secondary metabolites by liquid chromatography−high-resolution mass spectrometry. The multimycotoxin-method development involved the evaluation of both a two-step solvent and QuEChERS protocol for metabolite extraction. The former protocol was selected because of better accuracy, which was evaluated on the basis of spikerecovery experiments. The most frequently isolated fungal species belonged to the genera Alternaria and Fusarium. The predominant Fusarium species was F. avenaceum, followed by F. graminearum. HT-2 toxin was the most frequently detected mycotoxin, followed by enniatin B, enniatin B1, T-2 toxin, and nivalenol. As a consequence of the observed mixed fungal infections, mycotoxin co-occurrence was also detected. A combination of mycological and mycotoxin analyses allowed the ability to obtain comprehensive information about the presence of mycotoxigenic fungi and their contaminants in malting barley cultivated in a specific geographic area. KEYWORDS: Fusarium spp., barley, Hordeum vulgare L., mycotoxins, LC−HRMS, food contamination, beer

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INTRODUCTION Barley (Hordeum vulgare L.) is one of the most cultivated cereals worldwide. In Italy, barley cultivation is of great economic importance, with a production of 873 213 tons in 2013 from a land area of 213 300 ha. Italian barley is mainly used for animal feeding, but an important part is also destined to the beer industry.1 Considering the importance of this crop and of its derived products, such as beer, it is essential to achieve a high quality of the product, giving special attention to crop-cultivation phases. In fact, a number of microorganisms may have a harmful impact on barley production. In particular, barley heads may be subject to Fusarium head blight (FHB), a complex disease caused by several Fusarium species, resulting in yield losses and mycotoxin accumulation in the grain. Moreover, other toxigenic fungal species (e.g., Alternaria spp., Aspergillus spp., Penicillium spp., etc.) may colonize barley kernels. Mycotoxins are secondary fungal metabolites causing immunosuppressive, carcinogenic, mutagenic, and hepatotoxic effects in humans. For this reason, some of these molecules have been classified as carcinogenic by the International Agency for Research on Cancer. The European Union (EU) Regulation 1881/2006 and its amendments set maximum levels for 11 mycotoxins in food: aflatoxins (AF) (the sum of AFB1, AFB2, AFG1, and AFG2 as well as AFB1 alone and AFM1), the sum of fumonisins B1 and B2 (FB1 and FB2), ochratoxin A (OTA), patulin, deoxynivalenol (DON), and zearalenone (ZEN). Furthermore, the EU Recommendation 2013/165 of March 27, 2013 on the presence of T-2 toxin (T-2) and HT-2 toxin (HT-2) established a tolerable contamination level for the sum © XXXX American Chemical Society

of these toxins. The indicated level for unprocessed barley is 200 μg kg−1. However, there are other mycotoxins, such as enniatins (ENs) and beauvericin (BEA), Alternaria toxins, and ergot alkaloids (EAs), that have not been legislated yet. Thus, mycotoxin monitoring should ideally not be performed only for those with legislations. For this purpose, recent studies have focused on the presence of other fungal metabolites in different cereals such as soft and durum wheat and cereal-based food.2−4 Fusarium head blight represents one of the most important sources of mycotoxin contamination in malting barley.5 In Europe, Fusarium graminearum, Fusarium culmorum, Fusarium avenaceum, and Fusarium poae are considered the main FHB causal agents, even if changes in the relative composition of the different species may occur throughout the years as a function of the cultivation area and of weather conditions.6−8 In Italy, FHB has been permanently present since 1995, with variable incidence in relation to time and cultivation areas.4,9−12 The most important mycotoxins associated with FHB infections belong to type A and type B trichothecenes. The main type A trichothecenes are represented by T-2, HT-2, and diacetoxyscirpenol (DAS). Fusarium sporotrichiodies, Fusarium langsethiae, and F. poae are some of the principal type A trichothecene producers.13,14 The most important type B trichothecene is DON that during the last decades has been considered the main mycotoxin associated with FHB.15 F. Received: February 11, 2016 Revised: April 22, 2016 Accepted: April 29, 2016

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DOI: 10.1021/acs.jafc.6b00702 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry graminearum and F. culmorum mainly produce DON and its derivatives 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyldeoxynivalenol (15-ADON) and nivalenol (NIV). F. poae is also able to biosynthesize NIV.14 The spectrum of mycotoxins biosynthesized by the Fusarium spp. associated with the FHB complex includes also other less-studied but highly bioactive secondary metabolites such as the hexadepsipetides ENs and BEA. F. avenaceum and F. poae are reported as two of the main EN and BEA producers, respectively.16,4 In addition, Fusarium equiseti and F. sporotrichioides have the ability to produce ENs and BEA.17,14,16 Fumonisin contamination can also occur on wheat and barley kernels, even if their producing species (for example, Fusarium proliferatum) are not considered typical FHB causal agents. Moreover, other fungal genera potentially infecting barley kernels are able to produce mycotoxins. For example, Alternaria spp. may play an important role in terms of mycotoxin contamination because they are able to biosynthesize more than 70 phytotoxins, even if only a few of them have been reported to act as mycotoxins.18,19 For example, alternariol (AOH), alternariol−monomethyl ether (AME), tenuazonic acid, and altertoxins are described to induce both fetotoxic and teratogenic effects.20 There are no previous risk assessments on Alternaria toxins in food and feed carried out at international level, and to date, there are no regulations on Alternaria toxins in food and feed in Europe or in other world regions.19 Therefore, it is very important to investigate the presence of Alternaria toxins because this genus is often associated with malting barley kernels.21−23 Claviceps spp. are also able to produce mycotoxins belonging to the EAs group.24 The physiological effects of this class of compounds have been known since ancient times.25 To date, more than 50 different EAs have been identified.26 In Europe, Claviceps purpurea is the most common Claviceps species, and it is known to infect barley as well.24 On the basis of the EAs identified in the sclerotia of C. purpurea and of literature data, chemical analyses should focus on the main EAs, namely ergotamine, ergocristine, α- and β-ergocryptine, ergocornine, ergosine, ergometrine, and their corresponding -inine epimers.27 Even if the -inine forms are described to be biologically inactive, interconversion may occur under various conditions and, consequently, the risk assessment is based on both forms (-ine and -inine).28,26 Mycotoxins produced by the abovementioned fungal species during barley head infection and colonization and, later on, in the grain during storage could be transferred into malt and also into beer, thus reducing the malting and brewing quality.29−36 The transfer of mycotoxins from grains to beer could have a harmful effect on consumer health because, even if population exposure through beer consumption did not show high toxicological concerns, the contribution of this beverage to total mycotoxin daily intake is not negligible.37 Therefore, it is important to investigate the presence of mycotoxigenic fungal species infecting barley kernels and to detect grain mycotoxin contaminations. With regard to this last aspect, the validation and application of an analytical method for the simultaneous detection of a large number of mycotoxins (multimycotoxin analysis) is an important step to realize the wide monitoring of unprocessed products, such as malting barley,38 and processed ones, such as malt39 and beer.40 This is particularly important because in many instances, more than one mycotoxin may occur at the same time in a single sample, determining the co-occurrence of fungal secondary metabolites.41 For these reasons, the aim of

this work was to (1) evaluate the presence of fungal microorganisms, with particular attention to mycotoxigenic species, on 43 malting barley samples collected in the Umbria region (central Italy) in 2013; (2) identify the Fusarium strains isolated from malting barley kernels to determine the FHB complex composition in the surveyed area; and (3) evaluate the simultaneous presence of 34 fungal secondary metabolites by liquid chromatography−high-resolution mass spectrometry (LC−HRMS).

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MATERIALS AND METHODS Malting Barley Sampling and Mycological Analysis. The survey was conducted by collecting a total of 43 malting barley samples cultivated during the season 2012−2013 in several fields across the Umbria region in central Italy. The sampling strategy aimed at covering as many cultivation areas as possible to have a representative overview of the examined region. Samples (500 g) were collected immediately after barley harvest and stored at 4 °C until analysis. For each sample, information about cultivation area, variety, and previous crop are summarized in Table S1. Each sample was divided into two portions; one (250 g) was used for mycological seed analysis, and the other one (250 g) for mycotoxin analysis. Mycological analyses were realized following the method indicated by Covarelli and co-workers12 with slight modifications. In brief, a subsample of 30 g was surface-sterilized for 2 min using a water−95% ethanol−7% sodium hypochlorite solution (82:10:8, v/v) and rinsed for 1 min with sterile water. A total of 100 surface-sterilized kernels per each sample were placed on potato dextrose agar (PDA, Biolife Italiana, Milan, Italy), supplemented with streptomycin sulfate (0.16 g l−1, SigmaAldrich, Saint Louis, MO) and 2,6 dichloro-4-nitroaniline (0.006 g l−1, Sigma-Aldrich), into 10 Petri dishes (100 mm diameter) containing 10 kernels each. After 5 days of incubation in the dark at 22 °C, a combination of visual and stereomicroscope (SZX9, Olympus, Tokyo, Japan) observations were carried out on each kernel to determine the possible development of fungal species. The incidence (%) of the fungal genera recovered during the whole survey was expressed as the average (±standard error, SE) of the 43 analyzed barley samples, while the incidence (%) of the fungal genera recovered on each sample was expressed as the average (±SE) of 10 replicates. Data were subject to the analysis of variance using the program DSAASTAT.42 To verify the affiliation of a fungal specimen to the genus Fusarium, we transferred a subset of about two colonies per samples into new plates containing PDA for their identification following the obtainment of monosporic cultures. Identification of Fusarium spp. Isolates. DNA was obtained from Fusarium spp. colonies grown on PDA for 2 weeks at 22 °C in the dark and extracted with extraction solution and dilution solution (Sigma-Aldrich). In brief, 50 μL of the extraction solution was placed inside 200 μL PCR plastic tubes (Eppendorf, Hamburg, Germany), and then a small portion of mycelium was scraped from the surface of the colonies of each isolate. After being heated to 99 °C for 10 min, tubes were centrifuged for 3 min (12000g) on a 1−14 Sigma centrifuge. The supernatant was transferred into new 200 μL plastic tubes, and 50 μL of the dilution solution was added. The extracted DNA was diluted to obtain a working solution of ∼30 ng μL−1 and stored at −20 °C until further processing. Speciesspecific primers (Table S2) were used for the identification of seven Fusarium species that were suspected to be present in the B


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Journal of Agricultural and Food Chemistry

B1 (ENB1) (homologue composition 3%, 20%, 19%, and 54%, respectively; ca. 97% purity) were purchased from SigmaAldrich. Culmorin, 15-OH-culmorin, and 5-OH-culmorin had previously been isolated and purified from cultures of F. graminearum and F. culmorum at the Norwegian Veterinary Institute (Oslo, Norway). Ergonovine, ergocornine, ergotamine, ergocristine, and α-ergocryptine were gifts from the Center for Analytical Chemistry, IFA-Tulln, Austria. The following U-[13C]-labeled compounds were obtained from Romer Laboratories: [13C]DON, [13 C]HT-2, [13C]T-2, [ 13 C]NIV, [ 13 C]ZEN, [ 13 C]FB 1 , [ 13 C]3-ADON, and [13C]OTA. ACN and deionized H2O, both LC−MS grade, were supplied by Thermo Fisher Scientific (Bremen, Germany). Formic acid and ammonium acetate (both proanalysis grade) were purchased from Merck KGaA (Darmstadt, Germany). Two-Step Solvent Extraction. This method was adapted from Varga and colleagues.45 Briefly, ground and homogenized barley samples (2.5 ± 0.01g) were weighted into 50 mL polypropylene tubes (VWR International, Milan, Italy). The first extraction was performed with 10 mL of extraction solvent 1 (ACN/H2O/formic acid; 80:19.9:0.1; v/v/v) on an Edmund Bühler SM 25 rotary shaker (Hechingen, Germany) for 60 min at room temperature. After the extraction, tubes were centrifuged for 10 min at 20 °C (2000g) on a Beckman Coulter Allegra X-30R centrifuge (Brea, CA), and the raw extract was decanted into a new 50 mL polypropylene tube. The residue was extracted a second time. The second extraction was performed with 10 mL of extraction solvent 2 (ACN/H2O/formic acid; 20:79.9:0.1; v/v/v) on the same rotary shaker for 30 min. Afterward, samples were centrifuged for 10 min using the same parameters as above. Then, an aliquot (400 μL) was filtered through a 0.22 μm nylon filter (Corning, New York). QuEChERS Extraction. The QuEChERS roQ kit from Phenomenex (Torrance, CA) was employed to extract mycotoxins from the examined matrix. In detail, a homogenized and representative portion of 2 g (±0.01 g) of barley kernels were weighted into 50 mL polypropylene tubes (VWR International). The first step of the QuEChERS extraction was performed with 10 mL of 0.1% formic acid in deionized H2O. The mixture was shaken for 3 min on a rotary shaker (Edmund Bühler SM 25) and incubated for 10 min at room temperature until the next step. Then, the second step consisted of adding 10 mL of ACN and subsequently shaking the mixture for another 3 min using the same rotary shaker. Subsequently, 4 g of magnesium sulfate, 1 g of sodium chloride, 1 g of sodium citrate tribasic dihydrate, and 0.5 g of citrate dibasic sesquihydrate were added, and then the mixture was shaken for 3 min. Once the extraction was completed, the samples were centrifuged for 5 min at 20 °C (2000g) on a Beckman Coulter Allegra X-30R. Next, an aliquot (400 μL) was filtered through a 0.22 μm nylon filter (Corning). Sample Preparation and LC−HRMS. Aliquots (160 μL) of the centrifuged raw extracts (using the two-step solvent extraction process described above) were transferred into chromatography vials with a microinsert (VWR International), and 40 μL of the working solution containing a mixture of [13C]-labeled mycotoxins (Table S3) was added to each vial. Vials were shaken, and 2 μL was injected into the mobile phase. Thus, using the two-step solvent extraction, for instance, a concentration of 10 ng mL−1 corresponded to 80 μg kg−1 (conversion factor of 8).

samples, as these had been identified in previous studies conducted in the same geographic area.12,22 Specific PCR assays for F. graminearum were performed using the primer pair Fg16 F/R, which produces polymorphic products using DNA from F. graminearum lineage 7.43 A single PCR protocol was optimized using a total reaction volume of 20 μL. Each reaction contained 9.2 μL of sterile water for molecular biology use (5prime, Hilden, Germany), 1.5 μL of cresol red (Sigma-Aldrich), 2 μL of 10× PCR Buffer (Microtech, Pozzuoli, Naples, Italy), 1.2 μL of magnesium chloride (Microtech), 2 μL of 10 mM DNTP mix (Microtech), 1 μL of 10 μM forward and reverse primers, 0.1 μL of 5 U μL−1 Taq polymerase (Microtech), and 2 μL of template DNA. The PCR cycle consisted of an initial denaturation step at 94 °C for 2 min followed by 35 cycles of denaturation (95 °C for 35 s), annealing (30 s) at the specific annealing temperatures (Table S2), extension (72 °C for 30 s), and a final extension at 72 °C for 5 min.12 Each PCR assay contained a positive control (template DNA of the target species), and a negative control with no DNA added. The Fusarium isolates that were not identified by species-specific PCR were subject to translation elongation factor 1-α (TEF1α) gene amplification, purification, and sequencing. A single PCR protocol was optimized by using a total reaction volume of 50 μL. Each reaction contained 26 μL of sterile water for molecular biology use (5Prime), 3.75 μL of cresol red (Sigma-Aldrich), 5 μL of 10X PCR Buffer (Microtech), 3 μL of magnesium chloride (Microtech), 5 μL of 10 mM DNTP mix (Microtech), 2.5 μL of 10 μM forward and reverse universal primers (Table S2), 0.25 μL of 5 U μL−1 Taq polymerase (Microtech), and 2 μL of template DNA. The PCR cycle consisted of an initial denaturation step at 94 °C for 5 min, followed by 30 cycles of denaturation (94 °C for 1 min), annealing (1 min) at the specific annealing temperatures (Table S2), extension (72 °C for 1 min), and a final extension at 72 °C for 10 min. The PCR fragments of the TEF1α gene were purified and sequenced by Macrogen Europe (Amsterdam, The Netherlands) and sequences analyzed by the Basic Local Alignment Search Tool (BLAST).44 All PCR analyses were performed on a T-100 thermal cycler (Bio Rad, Hercules, CA). All PCR fragments were separated by electrophoresis by applying a tension of 110 V for ∼45 min. Electrophoretic runs were visualized using a Euroclone UV Image analyzer (Pero, Milan, Italy). The composition of Fusarium spp. involved in the FHB complex in the surveyed area was expressed as the incidence (%) of the identified Fusarium species on the total isolates subject to identification. To consider the simultaneous occurrence of different Fusarium spp. in a single malting barley sample, we calculated the percentage of barley samples infected by one, two, and three Fusarium spp. on the total analyzed samples. To assess the association between any two specific different Fusarium spp., we also calculated the percentage of the samples infected by each couple of the isolated Fusarium spp. on the total analyzed samples. Mycotoxin Analysis. Chemicals and Reagents. The following mycotoxin calibrant solutions, either in acetonitrile (ACN) or in acetonitrile−water (ACN/H2O, 1:1),were from Romer Laboratories GmbH (Tulln, Austria): 3-ADON, DON, deoxynivalenol-3-glucoside, NIV, HT-2, T-2, ZEN, DAS, 15ADON, T-2 triol, T-2 tetraol, neosolaniol (NEO), OTA, FB1, FB2, sterigmatocystin, BEA, moniliformin (MON), AOH, AME, and mycophenolic acid. A mixture of enniatin A (ENA), enniatin A1 (ENA1), enniatin B (ENB), and enniatin C


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Table 1. Recoveries of Individual Mycotoxins and Fungal Metabolites after Spiking Barley at Three Concentration Levels (n = 3 for Each Concentration Level) Using the Two-Step Solvent-Extraction Protocol compound

SCa level 1 (mg kg‑1)

SC level 2 (mg kg‑1)

SC level 3 (mg kg‑1)

ERb level 1 (%)

RSDc (%)

ER level 2 (%)

RSD (%)

ER level 3 (%)

RSD (%)

CMd

15-OH-culmorin 5-OH-culmorin acetyl-deoxynivalenolf alternariol alternariol-methyl ether beauvericin culmorin deoxynivalenol deoxynivalenol-3-glucoside diacetoxyscirpenol enniatin A enniatin A1 enniatin B enniatin B1h ergocornineh ergocryptineh ergocrystineh ergonovineh ergotamineh fumonisin B1 fumonisin B2 fusarenon-X HT-2 toxin moniliformin mycophenolic acid neosolaniol nivalenol ochratoxin A sterigmatocystin T-2 tetraol T-2 toxin T-2-triol zearalenone

50 255 76 5 5 24 275 50 31 50 2 15 14 41 25 29 25 25 25 126 127 50 50 50 51 50 50 3 1 50 51 50 51

399 2041 606 40 40 192 2198 400 251 400 18 120 114 324 200 234 200 201 201 1006 1014 400 400 403 407 400 400 20 10 401 404 399 404

798 4081 1212 81 80 384 4396 800 502 800 36 240 228 648 400 468 400 403 401 2012 2028 800 800 806 814 800 800 40 20 802 808 798 808

103 31 69 49 119 27 90 87 88 107 64 52 75 62 76 84 68 109 71 95 85 65 92 106 107 112 88 NDi 91 102 105 97 87

4 4 2 4 2 1 4 2 0 4 0 3 12 5 2 6 3 3 3 5 5 2 8 10 6 3 5 2 1 5 7 3

106 33 59 35 109 31 84 91 88 105 56 55 55 57 77 93 72 109 69 93 84 78 88 80 96 87 94 94 107 103 106 93 87

4 2 6 3 6 1 2 5 3 2 1 2 3 1 2 1 2 2 4 3 2 3 3 4 2 1 8 23 6 5 1 0 3

107 32 57 37 109 32 82 92 87 104 59 54 61 58 75 90 69 107 68 94 86 80 92 96 94 87 87 79 103 96 105 91 92

6 3 6 2 9 2 1 4 10 2 1 4 6 3 3 2 1 2 1 4 2 5 1 7 11 4 4 6 6 4 6 7 4

MMe MM MM + IS MM MM MM MM MA + ISg MM MM MM MM MM MM MM MM MM MM MM MM + IS MM MM MM + IS MM MM MM MM + IS MM + IS MM MM MM + IS MM MM + IS

a

Spiked concentration. bExtraction recoveries are reported as mean values of each spike level. cRelative standard deviation. dCalibration method. Matrix-matched calibration. f3- and 15-acetyl-deoxynivalenol coeluted in the liquid-chromatography method. gInternal-standard calibration. hOnly the 8-R epimer was included in the analyses. iNot detected.

e

Scientific), equipped with a heated electrospray interface HESIII (Thermo Fisher Scientific). Important interface settings included a capillary temperature of 250 °C, a heater temperature of 300 °C, an S-lens voltage of +60/-50 V, and a spray voltage of +4/-3.1 kV for positive and negative ESI, respectively. The system was operated in the full-scan and ddMS2 mode. The instrument continuously scanned all ions in a defined mass range, and once it detected a target ion (identified as such in the inclusion list), it performed a single MS2 scan on the target ion. The rationale of the ddMS2 scan was the verification of analyte identity. Each batch of samples was run twice, one analysis was done using full-scan and ddMS2 after positive ESI and another following negative ESI. The mass spectrometer was operated in the m/z range of 200−1000 during positive ionization and m/z 90−1000 during negative ionization mode at a mass resolution of 70 000 fwhm at m/z 200 (Table S4). While mass spectrometric parameters for the full-scan analyses were globally set, the optimum normalized collision energies for each target ion were obtained during flow injection of single analyte solutions separately for each compound included in the method.

The instrumentation for liquid chromatography consisted of an Acquity sample manager with a 10 uL sample loop, an Acquity ultrahigh-performance−liquid chromatography (UHPLC) binary solvent manager, and an Acquity 30 cm column heater and cooler, all from Waters (Milford, MA). Separation was performed using an Atlantis T3 reverse-phase analytical column (100 mm × 2.1 mm i.d.; 3 μm; Waters) at 30 °C. Mobile phase A was ACN/H2O/acetic acid (AcOH) (95:4.9:0.1; v/v), and mobile phase B was H2O/AcOH (99.9:0.1; v/v), both containing 5 mM ammonium acetate. The linear gradient was as follows: start with 5% A for 1 min and then linearly increase to 15% A in 14 min, linearly increase for the next 15 min mobile phase A from 15% to 100% and then 100% A for 3 min, switching in 0.10 min to 5% A and column equilibration for 4.40 min before starting the next injection. The total run time was thus 37.5 min. The flow rate was 300 μL min−1, and the injection volume was 2 μL using the partial loop mode. The UHPLC was interfaced to a Q-Exactive Fourier transform High-Resolution Mass Spectrometer (Thermo Fisher D


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Journal of Agricultural and Food Chemistry For the quantification of the individual fungal metabolites, 1/ x weighted calibration curves were obtained for each analyte by plotting the peak area (or peak area ratios for internal standard calibration) versus the analyte concentration using Xcalibur 2.2 (Thermo Fisher Scientific). Either matrix-matched calibration or internal standard calibration using U-[13C]-labeled compounds was performed to minimize matrix effects. A U-[13C]labeled mycotoxin working solution was prepared containing: [13C]DON, [13C]HT-2, [13C]T-2, [13C]NIV, [13C]ZEN, [13C]FB1, [13C]3-ADON, and [13C]OTA (Table S3). Matrixmatched calibration standards combined with internal calibration standards were prepared at seven concentration levels using the following procedure: 160 μL of standard working solution (unlabeled mycotoxin mixture) was dried using nitrogen in a HPLC vial with microinsert; after the complete evaporation of the solvent, 160 μL of blank barley raw extract was added, and then 40 μL of [13C]-labeled mycotoxin working solution was added. The vials were placed on a Branson 3200 ultrasonic bath for 5 min to ensure dissolution. Furthermore, 40 μL of the [13C]-labeled mycotoxin working solution was added to the aliquots (160 μL) of the spiked ”blank” extracts as well as to the malting barley samples, and 2 μL of the obtained solutions was injected into the highperformance liquid chromatography−high-resolution mass spectrometry (HPLC−HRMS) instrument. Method Evaluation. We aimed not to fully validate the multimycotoxin method within the present study as it was beyond its scope. However, we intended to evaluate certain basic method-performance characteristics to assess the quality of the analytical data to some extent. The method performance parameters taken into account included extraction recovery for estimation of trueness and precision, working range, and limits of detection (LOD) and quantification (LOQ). “Blank” barley samples (2.5 ± 0.01 g) were spiked with the appropriate amount of spiking solution at three spiking levels before extraction (Table 1). A pair of separate spiking solutions were prepared to keep the maximum volume for spiking below 100 μL. Fortified sample aliquots were kept uncapped at room temperature overnight to allow solvent evaporation. The QuEChERS extraction protocol was evaluated at only one concentration level (Table S5). The percent spike recovery was used as an estimate of trueness, while the relative standard deviation of repeated spike-recovery experiments gave information about the method’s precision (Table 1). The LOD and LOQ were inferred from signal-to-noise (S/N) measurements where a S/N ratio of 3:1 and 10:1 in spiked barley samples was equivalent to the LOD and LOQ, respectively.

Figure 1. Incidence (%) of the fungal genera infecting malting barley kernels as detected by visual examination. Columns represent the mean (±SE) of 43 analyzed samples.

Fusarium spp. (Figure S2) were found to infect up to 56% of kernels (sample 22). A statistically different Fusarium spp. presence (P ≤ 0.05) was recorded by ANOVA among the analyzed samples. A total of 84 Fusarium isolates were subject to species identification. Species-specific PCR assays identified 86% of selected Fusarium strains. Unidentified strains (14%) were subject to TEF1α gene sequencing followed by BLAST analysis. The FHB complex composition, indicating the incidence of each isolated Fusarium species, is reported in Figure 2 and

Figure 2. Incidence (%) of the Fusarium spp. associated with the FHB complex in the surveyed area as identified by PCR and translation elongation factor 1α sequencing on the total Fusarium strains subject to identification.

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shows the prevalence of F. avenaceum strains (63%). F. graminearum (19%) was the second-most-important species of the complex, while the remaining species were identified as F. poae (5%), F. brachygibbosum (5%), F. sporotrichioides (4%), F. incarnatum−equiseti complex (3%), and F. langsethiae (1%). In the majority of samples (60%, n = 24) a single Fusarium spp. was found to infect the kernels, while two Fusarium spp. cooccurred in 37% (n = 15) of the samples. The co-occurrence of three species was detected in only one sample (3%) (Figure S3A). The two most co-occurring species were F. avenaceum and F. graminearum and accounted for 47% of the samples that were infected by two Fusarium spp. at the same time (Figure S3B). Mycotoxin Extraction and Analysis. The trueness of the method was assessed from spike-recovery experiments. The

RESULTS Incidence of Fungal Microorganisms in Malting Barley Kernels. The examination of barley kernels after 5 days of incubation on growth medium allowed the ability to determine the incidence (%) of the fungal genera infecting the kernels of each sample. As previously mentioned, we focused on the genera Fusarium, Alternaria, Epicoccum, Penicillium, and Aspergillus. Fungi not belonging to any of these genera were classified as “other”. Figure 1 shows the fungal community isolated from the analyzed malting barley samples. Alternaria spp. were the most detected species, followed by Fusarium spp. Both Alternaria and Fusarium species were isolated from every sample. The maximum number of kernels infected with Alternaria spp. (Figure S1) was 93% (sample 12), while E


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Journal of Agricultural and Food Chemistry two-step solvent extraction resulted generally in good spike recoveries and reasonable precision (Table 1). Although the QuEChERS protocol was merely assessed to a limited extent, the obtained data indicated that the two-step solvent extraction protocol was superior and would give better accuracy compared to the former (Table S5). For this reason, the two-step extraction method was chosen to analyze the barley samples of this study. For some of the metabolites (e.g., AOH and 5-OHculmorin), the spike recoveries were rather low with both extraction methods (e.g., the recovery for 5-OH-culmorin was 32% for the two-step solvent extraction and 29% for QuEChERS; Tables 1 and S5). However, the determination of both AOH and 5-OH-culmorin was highly reproducible, as can be seen by the reasonably low relative standard deviations for the spike recoveries (Table 1). The two-step extraction method and LC−HRMS analysis of 34 fungal metabolites showed that all analyzed samples were contaminated by at least one mycotoxin (Table 2). Mycotoxins biosynthesized by Fusarium spp. were detected, alone or in combination with other fungal metabolites, in 95%

(n = 41) of the analyzed samples. Type A trichothecenes (DAS, NEO, HT-2, T-2, T-2 triol, and T-2 tetraol) were detected in 72% (n = 31) of samples, followed by hexadepsipetides (BEA and ENs), detected in 70% (n = 30) of samples, and by type B trichothecenes (DON, fusarenon-X, and NIV), detected in 37% (n = 16) of the analyzed samples. The other Fusarium secondary metabolites such as hydroxy-culmorins, ZEN, MON, and FB1+FB2 showed incidences of 14%, 12%, 12%, and 4%, respectively. In detail, HT-2 (65% of positive samples) was the most frequently detected Fusarium mycotoxin, followed by ENB (51%); ENB1 (47%); T-2 (36%); NIV (35%); DAS (32%); T-2 tetraol (28%); BEA (23%); ENA1 (19%); ENA, 15-OH-culmorin, and NEO (15%); T-2 triol, ZEN, and MON (12%); 5-OH-culmorin (9%); DON (7%); fusarenon-X, FB1, and FB2 (2%) (Table 2). Considering the mycotoxins subject to EU regulation (DON) or EU recommendation (sum of T-2 and HT-2), the analysis showed that the highest concentration of DON in our set of samples (108.7 μg kg−1) was well below the maximum level of 1250 μg kg−1 indicated for unprocessed barley by EU regulation (1881/2006). The sum of T-2 and HT-2 toxins was above the EU recommended limit of 200 μg kg−1 in one sample (2% of total samples) in which the maximum value for the sum of these compounds was 400.1 μg kg−1 (262.5 μg kg−1 of T-2 + 137.6 μg kg−1 of HT-2). The sum of T-2 + HT-2 was above 100 μg kg−1 (Table 2) in four samples (9% of total samples). With regard to nonlegislated metabolites, the mycotoxins detected in highest concentrations, ENs and NIV, were present with maximum values of 171.4 (ENB) and 434.5 μg kg−1, respectively. ENs were detected with the gradient ENB > ENB1 > ENA1 > ENA (Table 2). Alternaria toxins were present, always in combination with other metabolites, in 35% of the samples (n = 15). AOH was detected in 28% of samples, followed by AME (21%) (Table 2). Conversely, AME was detected with a maximum concentration of 28.3 μg kg−1, which was above the maximum value recorded for AOH (14.7 μg kg−1). EAs biosynthesized by Claviceps spp. were found, alone or in combination with other metabolites, in 21% (n = 9) of samples. Ergonovine and ergocornine were the most frequently detected EAs (15% of positive samples) followed by ergocristine (13%) and ergocryptine and ergotamine (9%) (Table 2). Ergocristine was the EA analogue that was found in highest concentrations with a maximum of 1084 μg kg−1. In one sample, the sum of the EAs included in the method (ergocornine, ergocryptine, ergocristine, ergotamine, and ergonovine) was 1557 μg kg−1. The EAs were detected with the following concentration gradient: ergocristine > ergotamine > ergonovine > ergocryptine > ergocornine (Table 2). With regard to the natural co-occurrence of the analyzed mycotoxins, merely 9% (n = 4) of the samples was contaminated by only one mycotoxin (Figure 3A). Consequently, all the other samples (91%) resulted to be cocontaminated by two or more mycotoxins. Most often, three to five mycotoxins co-occurred in the samples (14% of samples), while the co-occurrence of 10 mycotoxins was evident in 12% of the samples. A single sample (2%) was co-contaminated by 12 mycotoxins (Figure 3A). Mycotoxins related to Fusarium spp. and Alternaria spp. co-occurred in 30% of the samples, followed by the concurrent occurrence of Fusarium mycotoxins and EAs (14%). A total of 49% of samples (n = 21) were contaminated by Fusarium mycotoxins only (Figure 3B). With regard to the co-occurrence of mycotoxins biosynthesized by Fusarium spp., the most frequent combinations were

Table 2. Mycotoxin Occurrence in the Analyzed Barley Samples mycotoxin 15-OH-culmorin 5-OH-culmorin acetyldeoxynivalenol alternariol alternariol-methyl ether beauvericin culmorin diacetoxyscirpenol deoxynivalenol deoxynivalenol-3glucoside enniatin A enniatin A1 enniatin B enniatin B1 ergocornine ergocryptine ergocristine ergonovine ergotamine fumonisin B1 fumonisin B2 fusarenon-X HT-2 toxin moniliformin mycophenolic acid neosolaniol nivalenol ochratoxin A sterigmatocystin T-2 toxin T-2 tetraol T-2 triol zearalenone a

incidence (n = 43)

positive samples (%)

average of positive samples (μg kg‑1)

max value (μg kg‑1)

7 4 NDa

15 9 ND

24.3 103 ND

41.2 141.3 ND

12 9

28 21

7.5 6.2

14.7 28.3

11 ND 15 3 ND

23 ND 32 7 ND

54 ND 4.1 39.2 ND

316.3 ND 8.8 108.7 ND

7 9 24 22 7 4 6 7 4 1 1 1 28 5 ND 7 15 ND ND 17 12 5 5

15 19 51 47 15 9 13 15 9 2 2 2 65 12 ND 15 35 ND ND 36 28 12 12

17.5 28.4 56.6 39.9 36.7 46.1 482.4 116 239.7 156.3 65 153.7 36.1 317 ND 22.6 95.5 ND ND 27.5 102.7 20.4 1.8

17.5 51.2 171.4 101.4 64.3 91.9 1084.1 238.8 290.7 156.3 65 153.7 262.5 425.6 ND 35.8 434.5 ND ND 137.6 260 31.9 1.8

Not detected. F


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detected in the analyzed samples, both potentially being able to biosynthesize mycotoxins. In fact, as a consequence of fungal infection, the adopted multimycotoxin analysis showed the presence of both Alternaria and Fusarium toxins. However, the different infection levels of the samples, in particular for Fusarium spp., highlight the possibility for barley kernels to present different fungal infection levels at the malting process. This demonstrates that the production of high-quality malt requires the control of fungal infections already from the field as fungal seed infections strongly influence the malting process and decrease the malting quality.50 In our study, the recorded differences in Fusarium infections were not a result of different susceptibilities of barley varieties to FHB (all samples were represented by the variety “Quench”) but were mainly due to other factors such as local climatic conditions in the different cultivation areas and agronomic factors.51 The composition of the species associated with the FHB complex in the examined malting barley samples harvested throughout the Umbria region showed the predominance of F. avenaceum. This species, often classified as a “secondary” FHB causal agent for its weak pathogenic behavior. However, this species has recently gained more attention due to the dramatic increase of its presence and of its mycotoxins in both wheat4,12 and malting barley, also in the surveyed area.22,23 This is in accordance to what is observed in Northern European countries, where this species represents one of the most important FHB agent and its mycotoxins have been considered of concern for several years.52 F. graminearum, which is generally classified as the major FHB causal agent, was also present in the surveyed area, but, in accordance with earlier reports, it did not represent the main species.22,23 In fact, previous studies conducted in a single location of the same area showed that F. avenaceum increased its presence from an incidence of 2% in 2011 to 19% in 2012 and 39% in 2013.22,23,47 Furthermore, during an investigation conducted on durum wheat in the same year and in the same geographic area, F. avenaceum was also the most isolated species, followed by F. graminearum.53 This composition change of the FHB complex could be related to climatic conditions coupled to the use of particularly efficient fungicides against the main FHB species, such as F. graminearum and F. culmorum. Simpson and co-workers54 reported that fungicides caused a differential control of wheat FHB agents. In fact, a prolonged use of a restricted group of fungicides for the control of FHB in wheat may have caused a selective pressure toward the main FHB agents, such as F. graminearum and F. culmorum, with respect to the other species. The species associated with the FHB complex may also cooccur in the same field. In this work, we show that 40% of the investigated samples were infected by more than one Fusarium species, suggesting that different mycotoxins could co-occur in the same field. This was confirmed by mycotoxin analyses, which showed that the majority of samples was contaminated by more than one mycotoxin. The Fusarium spp. recovered from the barley samples were potentially able to biosynthesize a wide range of secondary metabolites. For example, F. avenaceum and F. poae potentially led to EN or BEA kernel contaminations, while F. graminearum and F. poae could be responsible for type B trichothecene contamination. In addition, the typical type A trichothecene producers such as F. sporotrichioides and F. langsethiae55 were also found. Even if the incidence of F. langsethiae in our study was low, this species was found for the first time in the

Figure 3. Incidence (%) of malting barley samples contaminated by 1 to 12 secondary fungal metabolites on the total analyzed samples (A). Presence (%) of malting barley samples contaminated by Fusarium toxins (Ftox), Alternaria toxins (Atox), and ergot alkaloids (EAs) on the total analyzed samples (B). Incidence (%) of malting barley samples contaminated by trichothecenes (Tri), hexadepsipeptides (Hexa), and other Fusarium toxins (Others) on the total analyzed samples (C).

represented by trichothecenes + hexadepsipetides (37%), followed by the combination of trichothecenes + hexadepsipetides + other Fusarium secondary metabolites (17%) (Figure 3C).

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DISCUSSION The present study reports the results of an investigation on the presence of toxigenic fungal microorganisms, with a particular emphasis on the presence of Fusarium species causing FHB and on the quantification of 34 fungal secondary metabolites in malting barley kernels cultivated in 2013 across the Umbria region in central Italy. To our knowledge, this is the first complete survey conducted on this matrix in this geographic area that takes into consideration both fungal infections and derived mycotoxins. Previous studies on malting barley in the examined area were limited to investigations conducted on different varieties cultivated in a single location,22,23 while data from other Italian areas are still scarce.46 Therefore, this work sheds light onto this important crop from mycological, toxicological, and analytical points of view. The fungal community recovered from the analyzed samples was similar to that observed in other countries.47,21,48,49 Alternaria and Fusarium were the two main fungal genera G


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characterized by the high presence of potential mycotoxigenic species, including those belonging to the genus Fusarium. Several Fusarium species that are associated with FHB were identified in the samples, and these were often co-occurring in the same field. Considering that one species biosynthesizes many different secondary metabolites, the co-occurrence of several Fusarium species may lead to contaminations caused by a wide range of different mycotoxins. Thus, the co-occurrence of different mycotoxins was evident in almost all of the analyzed samples. In addition to Fusarium mycotoxins, secondary metabolites biosynthesized by other fungal genera (e.g., Alternaria and Claviceps) were present on barley kernels, thus increasing contamination complexity. In this respect, the applied HPLC−HRMS-based method for mycotoxin detection and quantification following the two-step solvent extraction was especially useful for monitoring malting barley contaminations in relation to fungal infections present on barley kernels. Notably, running HRMS in the full-scan mode allows for the retrospective analyses of the data without compromising sensitivity, which is an important advantage of the technique.

examined area. However, the detected low incidence of this species may have been caused by its slower growth with respect to the other species on the medium used in this study. For this reason, the adoption of additional fungal isolation methods, such as the deep-freezing blotter test, could be useful to favor the development of less competitive Fusarium spp., such as F. langsethiae, from infected barley kernels.56 The varied chemical structures of the 34 fungal secondary metabolites analyzed in this work made the choice of the extraction method difficult. For this reason, two different extraction methods were compared, which have been both used before in extraction protocols for multicompound analysis. The two-step solvent extraction method has commonly been linked to analyses based on mass spectrometry, and with or without cleanup, it has been applied to many cereals and derivative products for mycotoxin extraction.57−59 However, the latest trends have demonstrated that QuEChERS-like extraction protocols are especially simple and fast to perform and can be applied for many types of organic molecules and different matrixes.60 In this study, we found that the two-step solvent extraction in combination with the HPLC−HRMS instrumental method performed well for the quantification of the majority of fungal metabolites. In particular, the two-step solventextraction method led to good extraction recoveries for most of the considered compounds, with the exception of BEA, AOH, OTA, and culmorin. However, even if the extraction recoveries for these compounds were rather low, individual measurements were highly repeatable. In general, the use of matrix-matched calibration as well as the use of isotopically labeled internal standards (where available) enables the ability to correct for matrix effects,45 thereby improving the accuracy of LC−MSbased analytical methods.58,61,62 Even though we did not specifically investigate the effect of the barley matrix on the ionization of target analytes, the generally reasonable spike recoveries indicated that the (expected) matrix effect had been well-corrected for by using matrix-matched or internal standard calibration. During the present study, a combination of mycological and mycotoxin analyses allowed the ability to obtain comprehensive information about the presence of mycotoxigenic fungi and their mycotoxins in malting barley cultivated in a specific geographic area. In fact, our data on mycotoxin contaminations reflect the high prevalence of F. avenaceum by a relatively high occurrence of ENs, which were detected in 51% of samples, with ENB being the analogue that was detected most frequently and at the highest concentrations. However, ENs do not seem to represent an important risk for beer consumers, as several studies recently demonstrated that no more than 0.2% of total ENs and BEA originally present in barley grains are found in the final product.63 Results concerning DON occurrence, detected only in 7% of samples, reflect the relatively low presence of F. graminearum and the absence of F. culmorum. At the same time, it came to our surprise that EAs, which are mainly produced by C. purpurea, which was not part of the mycological survey, were detected at relatively high concentrations. Thus, monitoring C. purpurea infections in the examined area could be important. Similarly, the relatively high presence of HT-2 and T-2 highlights the need to specifically investigate the presence of their biosynthesizing species. In conclusion, this study shows that the mycoflora associated with malting barley cultivated in the surveyed area of central Italy, in which this crop and brewing are currently increasing, is

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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.6b00702. Tables showing information about barley samples, primers used for PCR identification, and information about parameters used for mycotoxin extraction and determination. Figures showing the incidence of Alternaria spp. and Fusarium spp. and the co-occurrence of Fusarium in the analyzed barley samples. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*L. Covarelli tel: +39 075 585 6464; fax: +39 075 585 6482; email: [email protected]. Author Contributions

§ G. Beccari and L. Caproni contributed equally to this work and should both be considered as first authors.

Funding

This project was co-funded by Fondazione Cassa di Risparmio di Perugia, project no. 2013.0269.021. L. Caproni was supported by an Erasmus traineeship grant from the University of Perugia during his stay at the Norwegian Veterinary Institute (Oslo, Norway). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Bavicchi S.p.A. (Perugia, Italy) for providing barley samples and Dr. M. Orfei, L. Ceccarelli, and M.V. Consalvi for technical assistance.

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ABBREVIATIONS USED FHB, Fusarium head blight; EU, European Union; AF, aflatoxins; FB1 and FB2, fumonisins B1 and B2; OTA, ochratoxin A; DON, deoxynivalenol; ZEN, zearalenone; T-2, T-2 toxin; HT-2, HT-2 toxin; ENs, enniatins; BEA, beauvericin; EAs, ergot alkaloids; DAS, diacetoxyscirpenol; NIV, nivalenol; 3-ADON, 3-acetyl-deoxynivalenol; 15-ADON, 15-acetyl-deoxynivalenol; AOH, alternariol; AME, alternariol−monomethyl H


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ether; LC−HRMS, liquid chromatography−high-resolution mass spectrometry; PDA, potato dextrose agar; SE, standard error; TEF1α, translation elongation factor 1-α; BLAST, Basic Local Alignment Search Tool; ACN, acetonitrile; ACN/H2O, acetonitrile−water; NEO, neosolaniol; MON, moniliformin; ENA, enniatin A; ENA1, enniatin A1; ENB, enniatin B; ENB1, enniatin B1; UHPLC, ultrahigh-performance liquid chromatography; AcOH, acetic acid; NCE, normalized collision energies; HPLC-HRMS, high-performance liquid chromatography−highresolution mass spectrometry; LOD, limits of detection; LOQ, limits of quantification; RSD, relative standard deviation; S/N, signal-to-noise

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