Toxigenic Fungi and Mycotoxins in Mature Corn Silage

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Food and Chemical Toxicology 45 (2007) 2420–2425 www.elsevier.com/locate/foodchemtox

Toxigenic fungi and mycotoxins in mature corn silage

E. Richard a, N. Heutte a, L. Sage b, D. Pottier a, V. Bouchart c, P. Lebailly a, D. Garon a,*

a Groupe Re´gional d’Etudes sur le Cancer, GRECAN-EA 1772, Universite´ de Caen Basse-Normandie, Centre François Baclesse, Avenue Geńe´ral Harris, BP 5026, 14076 Caen Cedex 05, France b Equipe Perturbations Environnementales et Xeńobiotiques, LECA UMR 5553, Universite´ J. Fourier, BP 53, 38041 Grenoble Cedex 09, France c De´partement Environnement-Alimentation, Laboratoire De´partemental Frank Duncombe, 14280 Saint Contest, France

Received 22 September 2006; accepted 13 June 2007

Abstract

To investigate the exposure of livestock and farm workers to mycotoxins during the last months of silage use, the mycoﬂora and the mycotoxins in a mature silage (11-months-old) were studied. A multimycotoxin method was developed to evaluate the toxigenic in vitro ability of fungal strains. The screening of potentially toxigenic fungi isolated from the mature silage showed that six Fusaria (Fusarium culmorum, Fusarium equiseti, Fusarium graminearum, Fusarium oxysporum, Fusarium solani and Fusarium verticillioides) and one Asper- gillus (Aspergillus fumigatus) were able to produce mycotoxins on nutrient agar. Seven major mycotoxins (aﬂatoxin B1, citrinin, deoxynivalenol, fumonisin B1, gliotoxin, ochratoxin A and zearalenone) were also searched in the corn silage by high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Among the three mycotoxins (citrinin, gliotoxin and deoxynivalenol) detected in the silage, gliotoxin, a strongly immunosuppressive mycotoxin, occured in the mature silage at level up to 877 ppb, which was associated with the presence of A. fumigatus in the silage. 2007 Elsevier Ltd. All rights reserved.

Keywords: Mature corn silage; Mycoﬂora; Multimycotoxin; HPLC-MS

1. Introduction post-harvest stage (during silage storage). Among these toxigenic species, Aspergillus flavus and Fusarium verticil- Corn silage (Zea mays L.) is one of the most important lioides were able to produce mycotoxins on corn (Cleveland cattle feeds in France (AGRESTE, 2000). Its production et al., 2003). was essentially based on the principle of preservation under The fungal growth reduced nutritional value and could anaerobic conditions together with the growth of lactic result in the production of mycotoxins (Frisvad et al., acid bacteria which promote a natural fermentation, lower- 2006) and allergenic spores (Adhikari et al., 2004) that ing the pH to a level at which clostridial growth is prohib- constitute a risk factor for human and animal health. Toxic ited. These conditions (low pH and anaerobiosis) were syndromes caused by mycotoxin ingestion were indicated considered as unfavourable for the growth of most molds. as mycotoxicosis. Surveillance for mycotoxins like aflatox- However, this matrix could be exposed to fungal develop- ins, ochratoxin A, zearalenone and fumonisins in cereals ment, particularly at the end of corn silage use. The pres- and animal feeds has shown that, where mycotoxins were ence of microfungi could be attributed to the infection of identified, mixtures of these toxins often occurred (Scuda- corn at the pre-harvest stage (in the field) and/or at the more et al., 1998). The daily handling of silage spoiled by fungal toxins could be a potential risk factor for human or animal safety. These observations showed the impor- * Corresponding author. Tel.: +33 231 45 52 21; fax: +33 231 45 51 72. tance of developing multimycotoxin analysis methods as E-mail address: [email protected] (D. Garon). tools for the exposure studies.

Few data were available on the fungal contamination 2.4. Multimycotoxin analysis by HPLC-MS and the ability of local strains to produce mycotoxins in corn silage, particularly in a mature silage predictably Liquid chromatography was performed using an Agilent Technologies series 1100 (Palo Alto, USA) quaternary pump coupled with an auto- exposed to factors promoting mold proliferation and sampler and a model SL mass spectrometric detector. The analytes were mycotoxin production such as oxygen, heat, rain or insect chromatographed at 40 C on a 150 · 2.1 mm i.d., 5 lm, Zorbax SB-C18 damage. column (Agilent Technologies, Palo Alto, USA) with a 1 mm Optiguard The aims of this multimycotoxin report were: (i) to C18 precolumn. Mycotoxins were separated using gradient elution with investigate the mycoflora of a mature corn silage (11- acetonitrile as mobile phase A and water acidified with 0.5% acetic acid (pH 3) as mobile phase B. The gradient program was: at time zero, 5% months-old), (ii) to develop a multimycotoxin method used solvent A; linear gradient to 50% solvent A within 15 min and to 80% at to assess the toxigenic in vitro ability of the fungal species, time 25 min. The flow rate was 300 ll/min. The injection volume was (iii) to compare the concentrations of seven mycotoxins at 25 ll. The retention times of mycotoxins were respectively 15.4, 16.7, 6.9, two levels in the corn silage (top and bottom). 15.1, 14.2, 20.3 and 20.4 min for aflatoxin B1, citrinin, deoxynivalenol, fumonisin B1, gliotoxin, ochratoxin A and zearalenone. Mass spectrometry was performed on a quadrupole analyser equipped 2. Materials and methods with electron spray ionization (ESI) source and operating in positive and negative modes. The parameters used for the mass spectrometer in all 2.1. Chemicals and reagents experiments were: capillary voltage 3.0 kV, solvent gas 720 l/h, evaporation temperature 350 C and pressure of nebulization 35 psi. The ions Seven mycotoxin standards (aflatoxin B1, citrinin, deoxynivalenol, monitored were 313, 335, 647 (aflatoxin B1); 251, 233, 273 (citrinin); 265, fumonisin B1, gliotoxin, ochratoxin A and zearalenone) were supplied by 295, 297, 355 (deoxynivalenol); 722, 723, 724 (fumonisin B1); 263 (glio- Sigma–Aldrich (St. Louis, MO, USA). The stock solutions of 250 lg/ml toxin); 404, 405, 426 (ochratoxin A); 317, 318, 319 (zearalenone). Full scan were prepared in LC grade methanol from Chromanorm VWR Prolabo mass spectra were recorded in order to select the most abundant m/z value (Fontenay-sous-Bois, France) and stored at À20 C in the dark. Diluted and then the selected ion monitoring mode (SIM) was used for the solutions were prepared immediately before use by diluting the stock quantification. solutions with mobile phase acetonitrile/water (10:90, v/v). The working solution was composed of the seven mycotoxins. It was prepared by combining suitable aliquots of each individual standard stock dilution in 2.5. Multimycotoxigenic in vitro ability of fungal strains order to obtain each mycotoxin at 1.25 lg/ml. Milli-Q quality water (Millipore, Bedford, USA) and all other chem- A multimycotoxin method was developed to simultaneously assess icals of LC grade were obtained from Prolabo. the ability of the fungal strains to produce seven major mycotoxins:

Purification assays used Oasis HLB (6 ml, 200 mg) cartridges pur- aflatoxin B1, citrinin, deoxynivalenol, fumonisin B1, gliotoxin, ochratoxin chased from Waters (Milford, MA, USA). A and zearalenone. For each dish, three agar plugs measuring 8 mm in diameter were removed from the central area of the colony (including 2.2. Sample collection conidia and mycelium), pooled, weighted and transferred to 5 ml glass vials. All mycotoxins were extracted by 2 ml of ethyl acetate acidified Corn silage was located in a dairy farm of Normandy (France) char- with 1% acetic acid except fumonisin B1 which required 2 ml of methanol acterized by 150 hectares of cultivated land with 21 hectares of corn and acidified with 1% acetic acid. After 15 min of centrifugation at 1500 rpm, 205 cattle daily fed corn silage. Samples of a mature corn silage were each extract was evaporated to dryness under a stream of nitrogen. The collected at the end of the silage use (11-months-old). final residue was dissolved in 0.5 ml of a mixture acetonitrile–water Samples were taken through 15 cm in the ‘‘trench-type’’ silo at two (10:90, v/v) and filtered through Millex HV 0.45 lm before the injection levels (0.75 m from top and bottom of the silage) and the temperature was into the HPLC-MS. Analytical recoveries, carried out by spiking exper- recorded. For each level, 800 g was mixed and ground in a blender in order iments, were 100% (aflatoxin B1 and gliotoxin), 96% (deoxynivalenol), to obtain an homogeneous sample. Hundred gram was then taken for the 86% (citrinin and ochratoxin A), 82% (zearalenone) and 65% analysis of mycoflora and eight aliquots of 5 g were stored at À20 C (fumonisin B1). before their multimycotoxin analysis. To evaluate the method, a screening of the potentially toxigenic fungal species isolated from the mature silage (Table 1) was conducted. These fungal strains were grown in triplicate on two different media, MEA and 2.3. Mycological analysis 1.5% corn steep medium (CS), a complex medium adapted from Cullen et al. (1982) during 2 weeks at 25 C before their multimycotoxin analysis. For each sample, 100 g of silage was suspended in 500 ml of sterile water containing sodium dodecyl sulfate (0.05%, w/v). After 1 h of mag- netic shaking, 1 ml of each suspension was sprayed in a Petri dish (90 mm 2.6. Determination of seven mycotoxins in corn silage diameter) containing malt extract (1.5%)/agar (1.5%) medium (MEA) complemented with chloramphenicol (0.05%, w/v) following the soil plates Mycotoxins were extracted and purified as previously described by method of Warcup (1950). To limit proliferous fungi like Trichoderma Garon et al. (2006). The method has been optimized with the extraction spp., MEA complemented with malachite green (0.0025%, w/v) was also and purification of fumonisin B1 from corn silage. The clean-up step on used (Davet and Rouxel, 1997). Oasis HLB cartridge consisted in a double elution with 5 ml of pure The plates were incubated at 24 and 37 C. The identity of each strain, methanol following by 5 ml of MTBE–methanol (90:10, v/v). After isolated and purified, was achieved through macro and microscopic evaporation, the final residue was dissolved in 1 ml of mobile phase, fil- examinations (Booth, 1966; Pitt, 1979; Domsch et al., 1980; Von Arx, tered through Millex HV 0.45 lm and injected into the HPLC-MS. The

1981; Klich, 2002; Samson et al., 2002; Samson and Frisvad, 2004). For analytical recoveries were found to be, respectively, 65% (aflatoxin B1, the species belonging to the Penicillium genus, growth was also observed deoxynivalenol, ochratoxin A and zearalenone), 70% (gliotoxin), and 80% on two selective media, Czapek yeast autolysate agar (CYA) and 25% (citrinin and fumonisin B1). The detection and quantification limits for glycerol nitrate agar (G25N), and incubated at 5, 22 and 37 C(Pitt, 1979). aflatoxin B1, citrinin, fumonisin B1 and ochratoxin A were, respectively, 1.5 Fusarium species were cultured on MEA and potato dextrose agar medium and 5 ppb, 6.5 and 20 ppb for deoxynivalenol, gliotoxin and zearalenone. (PDA). The flow chart of analytical procedure for the determination of All the purified strains were preserved on agar slants (MEA) at 4 C. mycotoxins in corn silage is summarized in Fig. 1. 2422 E. Richard et al. / Food and Chemical Toxicology 45 (2007) 2420–2425

Table 1 3. Results and discussion Mycoflora in a mature corn silage (11-months-old) Fungal species 3.1. Mycoflora associated with the 11-months-old Absidia corymbifera corn silage Aspergillus flavusa Aspergillus fumigatusa a Twenty-five fungal species were identified in the corn Aspergillus parasiticus silage mainly belonging to Aspergillus, Fusarium and Peni- Aspergillus terreusa Aspergillus versicolora cillium genera (Table 1). All species were isolated from the Byssochlamys niveaa,b two levels of the corn silage except Mucor hiemalis and Bys- Cladosporium cladosporioides sochlamys nivea only observed at the top. Three thermo- Fusarium culmoruma a philic species, Aspergillus fumigatus, Malbranchea Fusarium equiseti pulchella and Thermomyces lanuginosus, were identified at Fusarium graminearuma Fusarium oxysporuma the two levels of the mature silage where temperatures Fusarium solania around 30 C were observed. Fusarium verticillioidesa The fungal species, A. fumigatus, Monascus ruber, B. Geotrichum candidum nivea and Penicillium roqueforti were adapted to ensiling Malbranchea pulchella var. sulfurea conditions and were the most frequently determined con- Monascus rubera Mucor hiemalisb taminants of ensilaged feeds (Nout et al., 1993; Ohmomo Penicillium crustosum and Kitamoto, 1994; Boysen et al., 2000). In a survey of Penicillium loliense fungi infecting stored corn silage in France and Italy, Pel- Penicillium purpurogenum hate (1977) achieved a non-exhaustive account of fungal Penicillium roqueforti species among which Geotrichum candidum, M. ruber, Penicillium variabile Rhizomucor pusillus Mucor spp. and P. roqueforti were also identified in our Thermomyces lanuginosus study. P. roqueforti could secrete PR toxin in silage (Teu- a Potentially toxigenic species tested in vitro. ber and Engel, 1983) and was previously identified in an b Not detected at the bottom of the silage. analysis of spoiled corn silage carried out in the Nether- lands from 1986 to 1990 (Nout et al., 1993). The occurrence of A. fumigatus was also observed in spoiled forage crop (Cole et al., 1977) and presents a dual men- ace from the ingestion of pathogenic spores (associated with lung damage) and from potential mycotoxins production such as gliotoxin and several tremorgenic mycotoxins like fumigaclavins (Cole et al., 1977; Sutton et al., 1996). Some potentially toxigenic species were noticed in our study: A. flavus and Aspergillus parasiticus (aflatoxins), A. fumigatus (gliotoxin), B. nivea (patulin), Fusarium culmorum and Fusarium graminearum (trichot- hecenes and zearalenone), and F. verticillioides (fumonisins). Other species which are considered as uncommon toxigenic fungi were isolated in this study. Among Penicillium genus, Penicillium crustosum was the most important producer of penitrem A, a highly toxic tremorgenic indol- terpene (Mantle et al., 1983; Lewis et al., 2005)andPenicil- lium purpurogenum was likely to produce rubratoxin, an hepatotoxin (Unger and Hayes, 1978). Cladosporium clado- Fig. 1. Flow chart of analytical procedure for determination of seven sporioides was considered as a producer of cladosporic acid mycotoxins (aflatoxin B1, citrinin, deoxynivalenol, fumonisin B1, glio- and volatile organic compounds (Abbott, 2002; Pieckova´ toxin, ochratoxin A and zearalenone) in corn silage. and Wilkins, 2004).

3.2. Multimycotoxigenic in vitro ability of fungal strains 2.7. Statistical analysis Among the 13 potentially toxigenic fungal species, six Statistical analyses were performed for each detected mycotoxin. Fusaria (F. culmorum, Fusarium equiseti, F. graminearum, Student t-test was used to compare the level of each mycotoxin between the top and the bottom of the silage (normality on each sample were Fusarium oxysporum, Fusarium solani and F. verticillioides) assessed by Shapiro–Wilk test and equality of variances were assessed by a and one Aspergillus (A. fumigatus) produced mycotoxins two-tailed F-test). SAS 9.1 software was used to analyse the data. on solid medium (Table 2). E. Richard et al. / Food and Chemical Toxicology 45 (2007) 2420–2425 2423

Table 2 Screening for toxigenic in vitro ability of potentially mycotoxigenic fungi isolated from mature corn silage Fungal species Culture mediuma Production of mycotoxin (lg/g)b

Deoxynivalenol Fumonisin B1 Gliotoxin Zearalenone Aspergillus fumigatus MEA – – 4.039 ± 0.097 – CS – – 1.827 ± 0.101 – Fusarium culmorum MEA – – – 0.154 ± 0.053 CS – – – 0.013 ± 0.009 Fusarium equiseti MEA – – – – CS 0.023 ± 0.009 – – – Fusarium graminearum MEA – – – 2.954 ± 0.209 CS – – – 0.081 ± 0.048 Fusarium oxysporum MEA 0.021 ± 0.004 – – – CS 0.034 ± 0.007 – – – Fusarium solani MEA 0.032 ± 0.002 – – – CS – – – – Fusarium verticillioides MEA – 15.050 ± 0.730 – – CS – – – – – not detected mycotoxin. a MEA: malt extract agar and CS: corn steep agar. b Means and standard deviations (calculated on triplicate).

The production of gliotoxin on artificial media was observed at concentrations above 1.5 ppm. Although zearalenone was produced by F. culmorum and F. graminearum, no data could be given in the corn silage because of concentrations lower than the quantification limit. Deoxy- nivalenol was also produced on artificial media by F. equiseti, F. oxysporum and F. solani and has been quantified in the mature silage. F. verticillioides isolated from the silage produced fumonisin B1 at high amount (15 ppm) even though no fumonisin B1 has been detected in the corn silage. This difference could be explained by the composition of the growth matrices. The detection of citrinin in corn silage was probably linked to the presence of M. ruber, however, this species only produced red pigments on the 2 solid culture media tested. Production of citrinin by fungi belonging to the genus Monascus was previously Fig. 2. Detection of three mycotoxins (citrinin, deoxynivalenol and observed in batch-culture without relation with the pig- gliotoxin) at two levels (top and bottom) in mature corn silage. Each ments biosynthesis (Pisareva et al., 2005). symbol represents the concentration of one aliquot. the bottom of the silage which could be explained by the 3.3. Analysis of seven mycotoxins in corn silage difference in temperature and humidity during the silage maturation. The higher concentrations at the bottom Fig. 2 and Table 3 present the mycotoxin concentrations of the silage could be probably explained by the heating at two levels in the mature corn silage (top and bottom). of the corn silage near the concrete and the persistance of These results showed the quantification of three myco- humidity at this level. toxins: citrinin (26.9–36.6 ppb), deoxynivalenol (159.8– Moreover, data showed that the variability of the mea- 203.5 ppb) and gliotoxin (0–877.7 ppb). Among these three sures within each level of the silage was not significantly mycotoxins, citrinin and gliotoxin could be, respectively, different themselves which demonstrate the homogeneity produced by thermotolerant (M. ruber) and thermophilic in achievement and treatment of the silage aliquots. (A. fumigatus) species. In a previous monitoring of corn silage, gliotoxin was Citrinin and deoxynivalenol showed concentrations sig- not detected in corn silage until nine months after harvest- nificantly different (p < 0.01) between the two levels in the ing and citrinin concentration increased during storage silage. The concentration of gliotoxin was respectively before stabilized around 15 ppb in the following months below the detection limit in the eight aliquots of the top (Garon et al., 2006). This last concentration was lower than of the silage and significantly different from 0 (p < 0.01) the levels found in the studied silage (26.9–36.6 ppb) which at the bottom. This study revealed that highest amounts showed that citrinin could be really considered as a storage of citrinin, deoxynivalenol and gliotoxin were present at mycotoxin. Schneweis et al. (2001) also observed citrinin in 2424 E. Richard et al. / Food and Chemical Toxicology 45 (2007) 2420–2425

Table 3 Statistical analysis of mycotoxins concentrations in mature silage: comparison between the two levels in silage sampling Mycotoxin Level of samplinga Mycotoxin concentration p-value of Student t-testc p-value of Shapiro–Wilk testd p-value of F-teste (lg/kg silage dw)b Citrinin Top 26.9 ± 2.5 <0.01 0.41 0.86 Bottom 36.6 ± 2.3 0.60 Deoxynivalenol Top 159.8 ± 9.6 <0.01 0.99 0.13 Bottom 203.5 ± 17.7 0.74 Gliotoxin Top Not detected <0.01 Bottom 877.7 ± 58.3 0.06 a Eight aliquots were performed per level. b Mean and standard deviation. c Test of means comparison. d Test of normality sample. e Test of variance comparison. silage samples at similar concentrations. Another study Adhikari, A., Sen, M.M., Gupta-Bhattacharya, S., Chanda, S., 2004. reported that aflatoxin formed in corn prior to silaging Airborne viable, non-viable, and allergenic fungi in a rural agricultural has been shown to break down slowly in stored silage after area of India: a 2-year study at five outdoor sampling stations. Sci. Total Environ. 326, 123–141. two months (Kalac and Woodford, 1982). In the studied AGRESTE (Statistiques Agricoles, Ministe`re de l’Agriculture et de la mature silage, no aflatoxin could be detected after eleventh Peˆche), France, 2000. http://www.agreste.agriculture.gouv.fr/. month of storage. At last, zearalenone concentrations were Booth, C., 1966. The Genus Fusarium. Commonwealth Mycological below the quantification limit. This toxin was previously Institute, Kew, UK, p. 237. detected before harvesting up to 100 ppb in leaves of maize Boysen, M.E., Jacobsson, K.-G., Schnu¨rer, J., 2000. 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