Fungi associated with food and feed commodities from Ecuador

MycopathologÜ 156: 87-92, 2002. " @ 2003 Kluwer Academic Publishers. Printed in the Netherlands. 87 Fungi associated with food and feed commodities from Ecuador A.M. Pacin1.2, H.H.L. González3,4*, M. Etcheverry5, S.L. Resnik2,6, L. Vivas7 & S. Espin8 1Centro de Investigaciones en Micotoxinas Universidad Nacional de Luján, Argentina; 2Comisión de Investigaciones Científicas de la Provincia de Buenos Aires, Argentina; 3Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina; 4Facultad de Ingeniería, Universidad de Buenos Aires, Argentina; 5Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Universidad Nacional de Río Cuarto, Argentina; 6Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires, Argentina; 7Instituto Nacional Autónomo de Investigación Agropecuaria INIAP-Guayaquil, Ecuador; 8Instituto Nacional Autónomo de Investigación Agropecuaria INIAP-Quito, Ecuador Received 12 October 2000; accepted in final form 16 September 2002 Abstract Freshly harvested soybean, rice and corn from farms and corn-based pelleted feeds were collected from ranches from the coastal and mountain regions in Ecuador during 1998, and assessed for fungal contamination. The most prevalent fungi on pelleted feed were Aspe rgillus fiavus and Fusarium graminearum. The prevalent fungi recovered from soybean were F verticillioides, F semitectum, Aspergillus fiavus and A. ochraceus. In rice, F oxysporum was the most prevalent toxigenic fungal species recorded, followed by F verticillioides and A. fiavus. In corn, F verticillioides was the most prevalent fungus isolated in both the coastal and mountain regions, with high isolation frequencies of A. fiavus and A. parasiticus at the coast. Based on the toxigenic species recovered, ochratoxin A may pose a contamination risk for soybean. A higher probability of aflatoxin contamination of corn was found in the coastal samples compared to those of the mountain region, while a risk of fumonisin contamination of corn exists in both regions. Key words: Aspergillus, corn, Fusarium, maize, rice, soybean, toxigenic fungi Introduction Information about fungi associated with cereal grains and oil seeds is important in assessing the potential risk of mycotoxin contamination. A number of fungal species associated with these commodities belonging mainly to the genera Fusarium, Aspergillus and Penicillium, have been reported to produce mycotoxins that cause mycotoxicoses of domestic animal s and man [1, 2]. In some grain-producing countries, information regarding the mycoflora of freshly harvested grains is available. In Ecuador, corn, rice and soybean are produced and consumed, but very little information is available on the incidence of toxigenic fungal species associated with Ecuadorian grains used in food and feed [3]. Ecuador presents a unique variety of * PubJished in 2003. climates: inter-andean tropical; subtropical; moderate; cold and glacial; coast tropical humid; tropical monsoon; tropical dry and tropical orient [4, 5]. Rainfalls vary from 200 to 5000 mmjyear and relative humidities are seasonal, reaching up to 85% in the rainy, humid winter season in the mountain regions and up to 100% in the coastal regions and the east. Therefore, climatic conditions may be conducive for fungal growth and mycotoxin production. Rice is produced along the coast and in the east; hard endosperm corn in the coast, the east, and the tropical and inter- Andean regions; medium hard and soft endosperm corn are produced at the inter-andean subtropical to cold regions and soybean is produced throughout the country. In this paper, the incidence and significance of the mycoflora in Ecuador on freshly harvested grains prior to storage and corn-based pelleted feeds are

88 considered. To achieve this objective the following steps were carried out: (1) the fungi associated with soybean, rice, mountain and coasta1 grawn corn (co1-1ected fram farms), and pelleted feeds (collected fram ranches, in Ecuador in 1998) were identified; (2) the distribution of funga1 species of mycotoxico1ogica1 interest was estab1ished and (3) the corn mycoflora between coastal and mountain regions were compared. Materials and methods Samples A total of 52 samples (1 kg minimum) were collected in the pravinces of Pichincha, Cotopaxi, Chimborazo and Imbabura in the mountain region; and Los Rios and Guayas in the coastal region. Twenty four samples of corn fram farms at the time of harvest and seven random samples of pelleted feed were collected in the mountain region. The corn-based pellets were samp1ed at the ranches, and were made of corn grawn in the same region. Ten samples of corn, six samp1es of rice and five samples of soybean were collected fram farms at the time of harvest in the coasta1 region. The samples were later submitted to the Centra de Investigación en Micotoxinas, Universidad Nacional de Luján, and then to the Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, and to the Facultad de Ciencias Exactas, Físico-Químicas y Naturales, Universidad Nacional de Río Cuarto, Argentina, for isolation and identification of contaminant mycoflora. Isolation offungi Subsamples (appraximately 200 g) of each of the soybean, rice or corn samples, were surface-disinfected using a commercia1 water solution of sodium hypochlorite (1 %) for 1 min, rinsed twice with sterile distilled water, and dried in a laminar flow cabinet. Pelleted feed was not surface-disinfected. One hundred seeds, kerne1s or pellets per subsamp1e were plated, 20 per p1ate, on se1ective, differential and general media to recover the species of mycologica1 interest. Aspergillus jlavus-aspergillus parasiticus agar (AFPA), dich1oran- rase benga1-ch1oramphenico1 agar (DRBC), Nash and Snyder medium and yeast extractglucose-chloramphenico1 agar (YGCA) were used [6-9]. The p1ates were incubated in the dark at 30 C for 2-4 days for AFPA, at 28 C for 4-7 days for DRBC and YGCA, and 7 days at 25 C with 12 hour cycles of white light and 12 hour cycles of darkness, for a Nash and Snyder medium. The resulting funga1 colonies were subcultured onto potato-dextrase agar (PDA) prior to identification. Whenever several fungi were iso1ated fram a single grain it was recorded. Identification offungi Isolates of fungi were identified according to the leading authorities. Fusarium spp. according to Ne1son et al. [10]; Penicillium spp. According to Pitt & Hocking [11] and Ramírez [12]; Aspergillus spp. according to Pitt & Hocking [11] and other fungi according to EIlis [13], Von Arx [14] and Pitt & Hocking [11]. The methodologies recommended by the Commonwealth Mycologica1 Institute were used for the preservation of identified isolates [15]. The iso1ation frequency (Fr) and relative density (RD) of species were calculated according to González et al. [16] as follows: Fr (%) = Number of samples with a specles or genus/total number of samples x 100 RD (%) = Number of isolates of a species or genus/tota1number of fungi isolated x 100 Statistical analysis Asymptotic tests for equality of praportions were used to compare relative densities of species fram the mountain and coastal regions [17]. Fischer exact test was used to analyze possib1e differences in the iso1ation frequencies of species isolated fram corn kernels collected at the two geographic regions. The analysis was performed using the Statistix 4.1 package [18]. Results and discussion Soybean The fungal species isolated on different agar media fram soybean and rice samples harvested in the Ecuadorian coasta1 region in 1998 are shown in Table l. Among the Aspergillus species recovered fram Ecuadorian soybean seeds, A. jlavus Link and A. ochraceus Wi1he1m were the most preva1ent, while, A. parasiticus Speare and A. niger van Tieghem were isolated at a lower Fr. The most frequent Penicillium species iso1ated fram soybean seeds in Ecuador

89 Table J. Fungal species isolated from soybean seeds and paddy rice collected in the Ecuadorian coastal region in 1998. were P. janthinellum Biourge and P. simplicissimum (Oudem.) Thom. The most prevalent Fusarium species recovered were F. verticillioides (Sacc.) Nirenberg (syn. F. moniliforme Sheldon) belonging to Section Liseola and F. semitectum Berk. & Ravenel (Section Arthrosporiella). The high F. semitectum isolation frequency found in the Ecuadorian soybean samples is similar to that reported for soybean from Thailand [19]. In comparison to the main soybean production area in Argentina, with dry and cold weather conditions, it was found that Fusarium species were present in only 5% of the samples surveyed, with the most common species being F. equiseti, F. semitectum and F. verticillioides [20]. Other additional fungal species identified in the soybean seeds harvested in Ecuador inc1uded: Nigrospora oryzae (Berk. & Broome) Petch, Cladosporium cladosporioides (Fresen.) G.A. de Vries, and Arthrinium phaeospermum (Corda) M.B. Ellis. Rice Isolation frequency (%) Fungal species Soybean Rice Aspergillusfiavus 60.0 50.0 Aspergillus niger 10.0 Aspergillus ochraceus 60.0 16.7 Aspergillus parasiticus 20.0 Fusarium verticillioides 100.0 66.7 Fusarium oxysporum - 83.3 Fusarium proliferatum 16.7 Fusarium semitectum 100.0 33.3 Fusarium solani 16.7 Penicilliumjanthinellum 60.0 50.0 Penicillium simplicissimum 20.0 - Other fungi 20.0 66.7 Paddy rice harvested in the Ecuadorian coastal region had fewer fungal contaminants than soybean (Table 1). Among the Aspergillus species isolated from rice, A. fiavus was the most prevalent, and the only Penicil/ium species isolated from the Ecuadorian paddy rice was P. janthinellum. Among the potential1y toxigenic Fusarium species [21] recovered from paddy rice, F. oxysporum Schlechl. (Section Elegans) was the most frequently Table 2. Fungal species present in corn-based pellets collected from ranches in the Ecuadorian mountain region in 1998 (YGCA medium, 25 cc, 7 days). isolated (83.3% of samples). Other Fusarium species recovered were F. verticillioides, F.semitectum, F.proliferatum (Mats.) Nirenberg (Section Liseola) and F. solani (Mart.) Appl. & Wollenw. (Section Martiella and Ventricosum). The F. proliferatum isolates were differentiated from F. verticillioides by the presence of polyphialides on microscopy of cultures [10] and these observations were confirmed on carnation leaf agar containing 4 gil KCI [22]. Additional species identified in the paddy rice harvested in the Ecuadorian coast inc1uded: Epicoccum nigrum Link, Curvularia lunata (Wakker) Boedjin, Cladosporium cladosporioides (Pers.) Link, Bipolaris oryzae (Breda de Haan) Shoemaker, Rhizopus stolonifer (Ehrenb.) Lind. and Nigrospora oryzae (Berk. & Broome) Petch. Corn-based pellets The fungi associated with corn-based pellets destined for animal feed (poultry, pigs and cows) in the Ecuadorian mountain region are shown in Table 2. Based on the isolation frequency (Fr) and relative density (RD), A. fiavus was the most prevalent fungus present followed by F. graminearum Schwabe (Section Discolor), F. verticillioides and A. parasiticus. Mucor and Rhizopus spp. were also present in the samples. Corn Number Species Fr* of isolates RD** Aspergillusfiavus 71.4 313 57.3 Aspergillus parasiticus 14.3 39 7.1 Fusarium graminearum 28.6 124 22.7 Fusarium verticillioides 14.3 57 10.7 Mucar racemosus 14.3 1 0.2 Rhizopus stolonifer 71.4 12 2.2 * Fr =Isolation frequency (%). ** RD =Isolation relative density (%). The number of isolates, Fr and RD of the fungi recovered from corn kernels of the coastal and mountain regions are given in Table 3. Based on the total number of kernels plated (3400), members of the genus Fusarium were isolated most frequently. The

90 second mgst prevalent genus present was Penicillium, followed by Aspergillus. Other genera isolated in the Ecuadorian corn inc1ude Acremonium, Alternaria, Arthrinium, Cladosporium, Curvularia, Diplodia, Mucor and Rhizopus. The predominant Aspergillus spp., based on the RD, was A. parasiticus in the coastal region and A. fiavus in the mountain region. A. niger was isolated in corn from both regions. A highly significant difference (P < 0.01) in the RD of A. fiavus and A. parasiticus was observed between the two regions, but there was no significant difference for A. niger (P = 0.7940). Similar findings were observed in Costa Rica [23] where approximately 80% of corn samples collected were contaminated with A. fiavus, and the presence of aftatoxins was confirmed. Due to the high Fr and DR of A. fiavus in corn kernels grown in Ecuador, aftatoxins may also be of concern, especially for corn when it is grown in coastal regions. Among the Penicillium spp. isolated, P. funiculosum Thom was the most prevalent species (Table 3). P. citrinum Thom was found only in the mountain region at low RD and Fr, and other Penicillium spp. present at low levels were P. simplicissimum in the mountain region, and P. Janthinellum in the coastal region. A highly significant difference was observed between the RD of the coastal and mountain regions (P < 0.01) for P. citrinum and P. simplicissimum, but there was no significant difference detected for P. funiculosum (P = 0.4801). In both regions, F. verticillioides was the most prevalent fungus of the corn mycoftora with respect to Fr and RD. F. subglutinans (Wollenw. & Reinking) Nelson et al. of the Liseola Section was also isolated, but at a lower frequency, in each Ecuadorian region. The F subglutinans isolates were differentiated from F verticillioides by the presence of false heads and polyphialides on microscopic cultures [10] and these observations were confirmed according to Fischer et al. [22]. F. verticillioides, F. proliferatum and F subglutinans were the most frequent Fusarium species isolated from corn in Ecuador in 1996 [3]. Other Fusarium spp that were present at low incidence levels were F graminearum in both regions and F. heterosporum Nees in the mountain region. F oxysporum was isolated only in the coastal region. A significant difference was observed between the RD ofboth regions for F. graminearum (P = 0.0239), while for F heterosporum and F. subglutinans highly significant differences were detected between the RD ofthe coastal and mountain regions (P < 0.01). Additional species isolated from corn kernels harvested in Ecuador inc1ude: Acremonium strictum W. Gams, Alternaria alternata (Fr.) Keissler, Arthrinnium phaeospermum, Cladosporium cladosporioides, Curvularia lunata, Mucor racemosus Fres. and Rhizopus stolon~fer (Ehrenb.) Lind. Diplodia zeae (Schw.) Lév. was isolated at low Fr and RD in the mountain region. The present study has provided information on the contaminant mycoftora of grains and corn-based pellets in Ecuador. Fungi associated with these substrates in Ecuador that should be of concern due to their toxigenic potential, inc1ude A. fiavus, A. parasiticus, A. ochraceus, F verticillioides, F oxysporum, F proliferatum, F. graminearum, F subglutinans, P. citrinum and P.funiculosum. The significant differences observed in the Fr and/or RD of contaminant mycoftora between the coastal and mountain regions could be due to the different environmental conditions. A. ochraceus, was only recovered from soybean and is known to produce the ochratoxin A (OA) mycotoxin. The presence of OA in rice, corn, and beans has previously been reported in Ecuador [24]; thereby, it is important to evaluate if the A. ochraceus strains recovered from soybean in Ecuador are capable of mycotoxin production, and whether the substrate and environmental conditions are presumably conducive to the production of OA by this fungus. The recovery of Aspergillus and Fusarium species from rice suggests possible contamination by several mycotoxins. Aftatoxins [4] and deoxynivalenol [24] have previously been reported in rice grown in Ecuador. Based on the fungal species isolated in this study, fumonisins and fusaric acid are additional contaminants that may be present. Rice grown in Ecuador should be assayed for the presence of these mycotoxins in order to evaluate the potential risk they may pose for the safety of both food and livestock feed. Aftatoxin BI, B2 and G1, T-2 Toxin, deoxynivalenol and fumonisins were previously reported in corn produced in Ecuador [4, 24]. Based on the Fr and RD of A. parasiticus and A. fiavus, it is probable that higher aftatoxin concentrations may occur in the coastal region compared to the mountain region, and that fumonisins may occur in both regions. Due to F. oxysporum occurrence, fusaric acid contamination may also be possible; thus, the presence of this mycotoxin in corn from the coast should be evaluated.

91 Table 3. Fungal species present in corn harvested in the Ecuadorian coastal and mountain regions in 1998 (YGCA medium, 25 C, 7 days). Species Aspergillus fiavus Aspergillus niger Aspergillus parasiticus Fusariurn grarninearurn Fusariurn heterosporurn F usariurn verticillioides Fusariurn oxy.'porurn Fusariurn suhglutinans Peniciliurn citrinwn Penicilliurn funiculosurn Penicilliurn janthinellwn Penicilliurn sirnplicissirnurn Acrernoniurn stricturn Alternaria alternata Arthriniurn phaeosperrnwn Cladosporiurn cladosporioides Curvularia lunata Diplodia zeae Mucor racernosus Rhizopus stolonifer Coast (n = 897) Fr* No. isolates RD** 60.0 28 3.1 20.0 6 0.7 10.0 76 8.5 40.0 29 3.2 100.0 375 41.8 40.0 123 13.7 10.0 2 0.2 50.0 200 22.3 10.0 55 6.1 10.0 20.0 2 0.1 0.2 Mountain (n = 2000) Fr No. isolates RD 25.0 192 8.3 12.5 16 0.7 4.2 I1 0.5 ]2.5 56 2.4 4.2 94 4.1 79.2 932 40.5 20.8 96 4.2 4.2 76 3.3 37.5 641 27.9 8.3 40 1.7 4.2 89 3.9 4.2 3 O.] 4.2 4 0.2 ]2.5 5 0.2 8.3 8 0.3 8.3 22 0.9 4.2 ]1 0.5 12.5 9 0.4 * Fr =Isolation frequency ('lo). ** RD =Isolation relative density ('lo). Acknowledgements This work was possible due to the support of the Fondo Argentino de Cooperación Horizontal, Ministerio de Relaciones Exteriores, República Argentina. The authors acknowledge the financial support from Consejo Nacional de Investigaciones Científícas y Técnicas, Universidad de Buenos Aires, Universidad Nacional de Río Cuarto, Comisión de Investigaciones Científicas de la Provincia de Buenos Aires and Agencia Nacional de Promoción Científica y Tecnológica. The authors are also indebted to Mrs. M.E. Módena for her technical assistance. References 1. Marasas WFO, Nelson PE. Mycotoxicology. Introduction to the mycology, plant pathology, chemistry, toxicology and pathology of naturally occurring mycotoxicoses in animals and mano University Park, Pennsylvania: The Pennsylvania State University Press, ]987. 2. FAO/WHO. Joint FAO/WHO Expert Committee on Food Additives. Summary and Conclusions. Fifty-sixth meeting, Geneva, 6-15 February, 2001; 19 pp. 3. Farnochi MC, Ramírez ML, Reynoso MM, Chulze S. Mating type of Fusariurn strains (Section Liseola) isolated from Ecuadorian maize. Proceedings of II Congreso Latinoamericano de Micotoxicología, Maracay, Venezuela, ]997; pp. 4. 128-129. Mühlemann M, Lüthy J, Hübner P. Mycotoxin contamination of Food in Ecuador. A: Aftatoxins. Mitt Gebiete Lebensm Hyg 1997a; 88:474-496. 5. Hurtado O, Cueva C, Lara J, Carrión A, Espinoza R, Ceva]]os G, Cordero L, Borrero A. Manual de informacién cultural del Ecuador. Quito: Científica Latina Editores CIA, Ltd, ] 986. 6. Heineken H, González HHL, Resnik SL. Selección de medios de cultivo agarizados. CALIBA 1999; 6: 12-14. 7. Pitt n, Hocking AD, Glenn DR. An improved medium for the detection of Aspergillus fiavus and A. parasiticus. J Appl Bacteriol1983; 54: 109-114. 8. King AD, Hocking AD, Pitt n. Dichloran-rose bengal medium for enumeration and isolation of molds from foods. App] Environ Microbiol1983; 37: 959-964. 9. Nash SM, Snyder Wc. Quantitative estimation by plate counts of propagules of the bean root rot Fusariurn in field soils. Phytopatho]ogy ]962; 52: 567-572. 10. Nelson PE, Toussoun TA, Marasas WFO. Fusariurn species: An illustrated manual for identification. University Park, Pennsylvania: The Pennsylvania State University Press, ] 983. 11. Pitt n and Hocking AD. Fungi and Food Spoilage. London- New York: Blackie Academic & Professional, ]997. 12. Ramírez C. Manual and Atlas of the Penicillia. EIsevier Biomedical Press, 1982.

92 13. Ellis M. Dematiaceous Hyphomycetes. Kew, Surrey, England: Commonwealth MycologicaJ Institute, 1971. 14. Von Arx JA. The genera of Fungi SporuJating in Pure Culture. J. Cnlmer Press, 1981. 15. Smith D, Onions HS. The Preservation and Maintenance of Living Fungi. Kew, Surrey, England: Commonwealth Mycological Institute, 1983. 16. González HHL, Resnik SL,Boca RT, Marasas WFO. Mycoflora of Argentinian corn harvested in the main production area 17. in 1990. Mycopathologia 1995; 130: 29-36. Devore JL. Probability and Statistics for Engineering and the Sciences. Monterrey, California: Brooks-Cole Publishing Company, 1987; pp. 352-354. 18. Statistix Version 4.1. User's Manual. Analytical Software, Borland International Inc. and Fleming Software, 1994. 19. Pitt JI, Hocking AD, Bhudhasamai K, Miscamble BF, Wheeler KA, Tamboon-Ek P. The normal mycoflora of commodities from Thailand 2. Beans, rice, small grains and other commodities. Int J Food Microbiol1994; 20: 211-226. 20. Vaamonde G, Scarmato G, Bonera N. Zearalenone production by Fusarium species isolated from soybean. Int J Food Microbiol1987; 4: 129-133. 21. Marasas WFO, Nelson PE, Toussoun TA. Toxigenic Fusarium species. Identity and Mycotoxicology. University Park and London: The Pennsylvania State University Press, 1984. 22. Fischer NL, Marasas WFO, Toussoun TA. Taxonomic importance of microconidial chains in Fusarium Section Liseola and effects of water potential on their formation. Mycologia 1983; 75: 693-698. 23. Mora M, Lacey J. Handling and aflatoxins contamination of 24. white maize in Costa Rica. Mycopathologia 1999; 138: 77-89. Mühlemann M, Lüthy J, Hübner P. Mycotoxin contamination of food in Ecuador. B: Ochratoxin A, deoxynivalenol, T-2 toxin and fumonisin. Mitt Gebiete Lebensm Hyg 1997b; 88: 593-612. Address for correspondence: H.H.L. González, Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Ciudad Universitaria, C1428EGA, Núñez, Buenos Aires, Argentina Phone and Fax: (54-011) 4631-1148 E-mails: lucas@di.fcen.uba.ar and glucas@speedy.com.ar