Animal Feed Science and Technology

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1 Animal Feed Science and Technology 152 (2009) Contents lists available at ScienceDirect Animal Feed Science and Technology journal homepage: Short communication Fungal contamination and mycotoxin natural occurrence in oats for race horses feeding in Argentina C. Sacchi a, H.H.L. González b,c,, L.E. Broggi a, A. Pacin d,e, S.L. Resnik d,f, G. Cano d,e, D. Taglieri d,e a Facultad de Bromatología, Universidad Nacional de Entre Ríos, Entre Ríos, Argentina b Facultad de Ingeniería, Universidad de Buenos Aires, Argentina c Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina d Comisión de Investigaciones Científicas de la Provincia de Buenos Aires, Argentina e Fundación de Investigaciones Científicas Teresa Benedicta de la Cruz. Luján, Provincia de Buenos Aires, Argentina f Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina article info abstract Article history: Received 26 May 2008 Received in revised form 18 February 2009 Accepted 9 April 2009 Available online 12 May 2009 Keywords: Oats Alternaria Fusarium Mycotoxins A mycological survey was carried out for the first time, on oat samples with hull collected in the Argentinean province of Entre Ríos in Twenty-three oat samples were evaluated. Aflatoxins, zearalenone, deoxynivalenol and fumonisins were also analysed. Species of the genus Alternaria were the most prevalent component of the oats mycoflora. Species belonging to Aspergillus, Fusarium, Arthrinium, Acremonium and Curvularia genera were also recorded. The predominant species of these isolated genera were Alternaria alternata (0.59 of specific relative density) and Aspergillus flavus (0.34). Aflatoxins, zearalenone and deoxynivalenol were not detected in all samples and only fumonisin B 1 was detected in two samples from Entre Ríos Province at low concentration levels (108.0 and g/kg). Based on the observed prevalence of Alternaria alternata, the potential presence of toxins such as tenuazonic acid, alternariol and alternariol monomethyl ether may pose a contamination risk for oats produced in Entre Ríos Province. On the other hand, according to the incidence of Aspergillus flavus in the samples coming from Abbreviations: RD, relative density; Fr, isolation frequency; AF, aflatoxins; AFB 1, aflatoxin B 1; AFB 2, aflatoxin B 2; AFG 1, aflatoxin G 1; AFG 2, aflatoxin G 1; ZEN, zearalenone; DON, deoxynivalenol; FB 1, fumonisin B 1;FB 2, fumonisin B 2;FB 3, fumonisin B 3; OPA, O-pthaldialdehyde. Corresponding author at: Departamento de Industrias, F.C.E. y N., Universidad de Buenos Aires, Ciudad Universitaria, (C1428EHA), Buenos Aires, Argentina. Tel.: ; fax: address: glucas@speedy.com.ar (H.H.L. González) /$ see front matter 2009 Elsevier B.V. All rights reserved. doi: /j.anifeedsci

2 C. Sacchi et al. / Animal Feed Science and Technology 152 (2009) Buenos Aires, under inappropriate storage conditions aflatoxins and cyclopiazonic acid might be produced. The fumonisins concentration found in the Argentinean oats did not represent a threat to animal health for high consumers of oats. The present study has provided, for the first time, information on the internal mycoflora of oats and natural occurrence of fumonisins in Argentina Elsevier B.V. All rights reserved. 1. Introduction Oats an important cereal for human and animal nutrition is used as horse feed and as fodder for animal grazing (Suttie and Reynolds, 2004). Molds are the predominant spoilage flora of cereal grains, and their presence can result in the production of mycotoxins. In oats-producing countries, especially in Northern and Eastern Europe there is information regarding the mycoflora of oats and the occurrence of NIV, T-2, HT-2 and especially of DON and ZEN (Lõiveke et al., 2003; Hietaniemi et al., 2004; Kosiak et al., 2004; Mankevičienè et al., 2007; Schollenberger et al., 2006; Buckley et al., 2007; Krysińska-Traczyk et al., 2007; Scudamore et al., 2007). In Argentina no information has been reported on potentially toxigenic fungi or the natural occurrence of mycotoxins. In Entre Ríos the main destination of oats is for race horses feeding and for fodder rolls to feed cattle and small amounts are used as seed for the next crop season. The aims of this study were to identify the fungi associated with oats destined to race horses feeding in Entre Ríos Province, Argentina; focusing on the fungal species of mycotoxicological interest and to analyse the natural occurrence of AF, ZEN, DON, and FB. 2. Materials and methods 2.1. Oat grain samples Race horses breeding is an important activity in two Departments of the Entre Ríos Province. Samples of oat grains (Avena byzantina C. Koch: yellow oats ) were collected during 2006 (17 samples) from Urdinarraín (lat South; long West), Gualeguaychú (lat S; long W) and Larroque (lat S; long W) in the Gualeguaychú Department and from Basavilbaso (lat S; long W) in the Concepción del Uruguay Department. At harvest time, incremental samples of oats with hulls were taken in the storage plants from the trucks coming from different crop fields. Aggregate samples were reduced with the quarter to 10 kg. The oats production in 2006 in Entre Ríos was 12,600 ton, 396 ton being produced in Gualeguaychú and Concepción del Uruguay Departments, this implied that each sample represented 23.3 ton. In Entre Ríos race horses feeding is supplemented with oats grain coming from Buenos Aires Province. For this reason samples of Avena sativa L. (white oats) with hulls coming from Tres Lomas (lat S; long W), were collected from the six commercial stores for animal feeding in Gualeguaychú Department. Bags of 40 kg were sampled according to the EC 401/2006 and one aggregate sample was collected in each store. Mycological analysis was immediately performed on oat samples of 300 g and for mycotoxin analysis the remnant of the laboratory samples were grounded in a laboratory mill (Romer Labs Inc., USA). Only one analysis of mycotoxins and fungal contamination was performed on each laboratory sample Isolation of fungi Oat kernels were surface-disinfected in a commercial 5% (v/v) aqueous solution of sodium hypochlorite for 1 min, rinsed twice, and dried in a laminar flow cabinet. This disinfection is to determine the endogenous mycoflora colonizing during cereal development. Then 100 oat kernels per sample were

3 332 C. Sacchi et al. / Animal Feed Science and Technology 152 (2009) placed in Petri dishes, 20 kernels per plate, on Yeast Extract Glucose Chloramphenicol Agar, Biokar BK007 (González et al., 2008). The plates were incubated at 25 C for 72 h and the resulting fungal colonies subcultured onto Potato Dextrose Agar, Biokar BK095, and then identified. Where several fungi were isolated from a single seed, all were recorded Identification of fungi The fungi were identified according to Nelson et al. (1983), Pitt and Hocking (1997) and Samson et al. (2004). The Fr and RD of each genera and species were calculated (González et al., 2008). Some of the cultures were stored in the BAFC Culture Collection from the Facultad de Ciencias Exactas y Naturales of the University of Buenos Aires in the Biodiversity and Experimental Biology Department Mycotoxins analysis Aflatoxins, deoxynivalenol and zearalenone analysis Briefly, 25 g were extracted with 100 ml acetonitrile:water (84:16, v/v) by blending it for 3 min at high speed with an Osterrizer blender. The supernatant (8 ml) were placed in a 15 mm 85 mm culture tube and 4 ml of the filtrate were passed through a Mycosep 226 column (Romer Labs. Inc., USA). The extract was evaporated to dryness. The residue was dissolved in toluene:acetonitrile (95:5, v/v) and a 50 L aliquot was applied on Merck silica gel TLC plates (# ) and developed in toluene:acetone (1:1, v/v). Standards of AFB 1, AFB 2, AFG 1 and AFG 2, DON and ZEN were purchased from Sigma Chemical Company (St Louis, MO, USA). AF and ZEN were visualised by fluorescence (AF: 366 nm, ZEN: 254 nm). Plates were dried, sprayed with 20% AlCl 3 ethanol:water (1:1, v/v) solution and heated at 150 Cfor 5 min and DON was visualised under long wave UV light. The detection limit was 1 g/kg for each AF and 25 g/kg for ZEN and DON. Average recoveries for total AF were 95.2% at a level of 4 g/kg. The average recoveries for DON and ZEN were 90.3% and 93.4% at a level of 100 g/kg, respectively Fumonisins analysis Briefly, 50 g were extracted with 100 ml methanol + water (3:1, v/v). The ph was adjusted in the filtrates before they were applied to strong anion exchange solid-phase extraction cartridges (J.T. Baker cat. # , USA). The cartridges were washed and the FB were selectively eluted with acetic acid/methanol solution and dried. OPA derivatives were prepared and analysed by reversedphase HPLC using fluorescence detection ( ex and em : 335 and 440 nm) with LiChrosper C (25 cm 4 mm i.d., 5 m, Merck Co., Germany). Column temperature: 23 C; flow rate: 1 ml/min; injection volume: 54 L; mobile phase: methanol:nah 2 PO M (77:23, v/v) adjusted to ph 3.3 with H 3 PO 4. Derivative reagent: 80 mg OPA in 1 ml methanol plus 5 ml Na 2 B 2 O M, catalyst: 2- mercaptoethanol. Limits of detection were: FB 1 : 10 ng/g; FB 2 : 6 ng/g and FB 3 : 10 ng/g (signal-to-noise ratio = 3:1). Limits of quantification were: FB 1 : 18 ng/g; FB 2 : 30 ng/g and FB 3 : 18 ng/g (signal-to-noise ratio = 5:1). Average recoveries at 100, 200, 400 and 600 g/kg of spiked sample were: for FB 1 and FB 3 greater than 99% and at the same levels for FB 2 was 103%. Precision of methods was calculated in terms of relative standard deviation in percent and ranged from 7.5 to 17.0 for all fumonisins Statistical analysis The asymptotic normal test for the equality of proportions was used to compare relative densities (RD) of fungal genera and species between the samples coming harvest and those coming from stores (Devore, 1987). The Fisher s exact test (Conover, 1980) was used to analyse possible differences in the isolation frequencies (Fr). This test calculates exact P-values when the samples are small. We declare a difference as statistically significant when the corresponding P-value is less than 0.05 and highly significant when it is less than The statistical analysis was done using software Statistix version 7.0 (2000).

4 C. Sacchi et al. / Animal Feed Science and Technology 152 (2009) Table 1 Isolation frequency (Fr) and relative density (RD) of fungi isolated from oats collected in Entre Ríos Province, Argentina in From harvest From stores Fr RD Fr RD Acremonium strictum Alternaria alternata Arthrinium phaeospermum Aspergillus flavus Aspergillus niger Aspergillus ochraceus Nd Nd Aspergillus parasiticus Nd Nd Aspergillus penicillioides Nd Nd Aspergillus ustus Nd Nd Cladosporium cladosporioides Nd Nd Curvularia lunata Chaetomium globosum Epicoccum nigrum Eurotium chevalieri Nd Nd Fusarium graminearum Nd Nd Fusarium poae Nd Nd Fusarium verticillioides Mucor racemosus Nd Nd Nigrospora oryzae Penicillium citrinum Nd Nd Penicillium funiculosum Nd Nd Penicillium rugulosum Nd Nd Phoma glomerata Nd Nd Rhizopus stolonifer Fr: number of samples where a genus or species of fungi occurred over total number of samples. RD: number of isolates of a genus or species over the total number of fungal isolates. Nd: not detected. 3. Results 3.1. Fungi associated with oats harvested at Entre Ríos Province The internal mycoflora associated with oats is shown in Table 1, with the number of fungal genera and species isolated being 15 and 22, respectively. Based on Fr, the genus Alternaria was the most common component of the internal seed-borne mycoflora, followed by Fusarium, Aspergillus, Curvularia, Acremonium, Arthrinium, Eurotium, Cladosporium and Epicoccum. Other genera recovered at low incidence levels were Mucor, Nigrospora, Penicillium, Chaetomium, Phoma and Rhizopus. Alternaria alternata was the most prevalent isolated species followed by Arthrinium phaeospermum, Curvularia lunata, Fusarium graminearum and Aspergillus flavus. The potentially toxigenic fungal species Alternaria alternata showed the highest RD (0.59). F. graminearum was the most prevalent Fusaria and F. verticillioides and F. poae were found with low incidence levels Fungi associated with oats coming from Tres Lomas and collected in commercial stores The number of fungal genera and species isolated were 11 and 13, respectively. Based on Fr, the genus Rhizopus was the most prevalent, followed by Acremonium, Alternaria and Aspergillus. Other genera recovered at low incidence levels were Arthrinium, Curvularia, Chaetomium, Epicoccum, Fusarium, Nigrospora and Penicillium. Aspergillus flavus was the most prevalent fungus, followed by Alternaria alternata, Acremonium strictum and Rhizopus stolonifer. Other Aspergillus species were found at low incidence levels (Aspergillus niger and Aspergillus penicillioides). The potentially toxigenic fungal species were Aspergillus flavus (RD: 0.34) and Alternaria alternata (0.32).

5 334 C. Sacchi et al. / Animal Feed Science and Technology 152 (2009) Statistical comparisons Statistical differences were detected for Fr of Alternaria alternata (P=0.011) and for Fr of R. stolonifer (P<0.01) in the comparison with the samples coming from harvest and those coming from stores. RD differences were observed for Acremonium strictum, Alternaria alternata, Aspergillus flavus, C. lunata, Ch. globosum and R. stolonifer (P<0.01) Mycotoxins analysis AF, ZEN and DON were not found in all samples and only FB 1 was detected in two oat samples from Basavilbaso, at low concentration levels (108.0 and g/kg). 4. Discussion The endogenous mycoflora recovered from oats in Entre Ríos was qualitative and quantitatively different from those observed in the samples coming from Buenos Aires. Considering Fr and RD, Alternaria alternata (640 isolates) was the most prevalent component of the endogenous mycoflora in Entre Ríos. Similar results were found on other cereals in Entre Ríos (Broggi et al., 2007). The high incidence level of Alternaria alternata observed in this work (Fr: 1.0) was different from those observed in the Norwegian and Polish oats, and 0.037, respectively (Kosiak et al., 2004; Krysińska-Traczyk et al., 2007). In the samples coming from Tres Lomas Aspergillus flavus prevailed. The differences observed in the oat mycoflora from both regions can be explained by the environmental factors in the production areas. Entre Ríos Province is surrounded by two big rivers, the Paraná on the West and the Uruguay on the East. The average temperature in the Southern departments (Gualeguaychú and Concepción del Uruguay) reaches 31 C in summer and 10 C in winter and the presence of lagoons and swampy lands raises the humidity. The average annual rainfall is 1100 mm. In Tres Lomas the climate is temperate intermediate between continental and oceanic with average temperatures of 29 C in summer, severe winters with frequent freezing weather and the average annual rainfall is 800 mm. The absence of AF is in accordance with results observed by Buckley et al. (2007). FB has been verified in maize and has occasionally been detected in wheat (Shephard et al., 2005). Despite the low incidence level of F. verticillioides (Fr: 0.12 and RD: 0.02) on the oats from Entre Ríos, the presence of FB 1 is remarkable in two samples. Buckley et al. (2007) did not detected FB on Irish oats destined to equine feeding. DON, T-2 toxin and HT-2 toxin have been frequently reported in oats destined to animal and human consumption (Mankevičienè et al., 2007; Schollenberger et al., 2006; Buckley et al., 2007; Krysińska-Traczyk et al., 2007). The low incidence of F. graminearum and F. poae observed in the oat samples from Entre Ríos could explain that no DON and ZEN were detected. 5. Conclusions The present study has, for the first time, provided information on the endogenous mycoflora of oats grain in Argentina. Based on the Fr and RD of Alternaria alternata observed in Entre Ríos, Alternaria toxins such as tenuazonic acid, alternariol and alternariol monomethyl ether might be produced. On the other hand, according to the incidence of Aspergillus flavus in the samples coming from Buenos Aires, despite the absence of aflatoxins, this fungus should be taken into account, because under inappropriate storage conditions it could grow and produce aflatoxins and cyclopiazonic acid. In the present study the presence of fumonisin B 1 in two samples is remarkable at concentrations that might not be a threat to animal health. Further studies on mycoflora and screenings of trichothecenes and ZEN should be carried out in oats for race horse feeding in Argentina under other weather conditions, in order to determine the risk of this contamination with mycotoxins.

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