versão impressa ISSN 1517-8382
Braz. J. Microbiol. v.35 n.3 São Paulo jul./set. 2004
Avaliação da toxigenidade das cepas de Aspergillus flavus e Fusarium spp. isoladas de amostras de sorgo
Josefa B. da SilvaI; Paulo DilkinII; Homero FonsecaIII; Benedito CorrêaII
IInstituto Butantan, São Paulo, SP, Brasil
IIDepartamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, São Paulo, SP, Brasil
IIIEscola Superior de Agricultura "Luís de Queiroz", Universidade de São Paulo, São Paulo, SP, Brasil
Fifty-nine Aspergillus flavus and 35 Fusarium verticillioides strains, isolated from freshly harvested (10) and stored (130) Brazilian sorghum samples, were tested regarding their ability to produce aflatoxins (coconut milk agar) and fumonisins (rice culture), respectively. Aflatoxins B1 and B2 were detected by TLC, and fumonisins B1 and B2 were analyzed by HPLC. Thirty-eight (64.4%) A. flavus strains produced detectable levels of aflatoxins at concentrations ranging from 12.00 to 3282.50 µg/kg (AFB1 + AFB2), while thirty two (91%) F. verticillioides strains produced FB1 at concentrations ranging from 0.12 to 5.38 µg/g. Two F. proliferatum strains produced low fumonisin levels. The toxigenic potential of A. flavus (64.4%) and F. verticillioides (91.5%) strains observed in sorghum samples indicates that rigorous control should be directed at the storage conditions of these products to minimize contamination with toxigenic deteriorating fungi, preventing further hazard to human and animal health.
Key words: toxigenicity, Aspergillus flavus, Fusarium verticillioides, mycotoxins, sorghum, aflatoxin, fumonisin
A produção de aflatoxinas por 59 cepas de Aspergillus flavus e fumonisinas por 35 cepas de Fusarium verticillioides isoladas de amostras de grãos de sorgo recém colhido (10 amostras) e armazenado (130 amostras), foram avaliadas. A detecção de aflatoxinas (AFB1 e AFB2) foi efetuada por Cromatografia em Camada Delgada (CCD) e fumonisinas (FB1 e FB2) foram analisadas por Cromatografia Líquida de Alta Eficiência (CLAE). Os resultados demonstram a produção de AFB1 e AFB2 em 38 cepas (64,4%) de A. flavus cujos níveis variaram de 12,00 a 3282,50 µg/kg. Referente às cepas de F. verticillioides, 32 (91%) produziram FB1, nas concentrações de 0,12 a 5,38 µg/g. Baixos níveis de fumonisinas foram detectados em 2 cepas de F. proliferatum. A constatação da potencialidade toxígena das cepas de A. flavus (64,4%) e de F. verticillioides (91,5%) nesta investigação, revelam a importância da pesquisa de aflatoxinas e fumonisinas nas amostras de sorgo. Diante disto, sugere-se o controle rigoroso das condições de armazenamento de sorgo, visando minimizar a contaminação por fungos deteriorantes toxígenos, evitando riscos à saúde humana e animal.
Palavras-chave: toxigenicidade, Aspergillus flavus, Fusarium verticillioides, micotoxinas, sorgo, aflatoxina, fumonisina
Sorghum (Sorghum bicolor L., Moench) is a worldwide grass originating from the African and Asian continents, which has spread to other temperate and tropical regions. Sorghum has been ranked as the seventh most cultivated grain in the world and the fourth in Africa (32).
Sorghum grains are used as raw material for poultry, swine and bovine feeds, but are also destined for human use (37), constituting the staple food in India, China, and some African and Asian countries.
The presence of deteriorative fungi with ability to produce mycotoxin in grains and food represents a great hazard for human and animal health, and it has been reported for sorghum in many countries with a high frequency of Aspergillus and Fusarium genera (1,7,11,10,25).
Aflatoxins are bifuranocumarin mycotoxins produced by A. flavus and A. parasiticus, with aflatoxin B1 (AFB1) being the most hepatotoxic, showing mutagenic and carcinogenic and, probably, teratogenic properties in animals (34,35). According to the International Agency for Research on Cancer, AFB1 is classified as a human carcinogen class 1.
Fumonisins are mycotoxins produced mainly by F. verticillioides Sacc Nirenberg (= F. moniliforme Sheldon), and F. proliferatum in several agricultural products worldwide, especially maize and sorghum (1,5,18). The toxic effects of fumonisins depend on the animal specie and the toxigenicity of Fusarium strains (26). This toxin causes leukoencephalomalacia in equines (18) and rabbits, pulmonary edema in swine (3,12), and it has been reported as a probable cause of esophageal cancer in humans (19,36).
Taking into account the lack of mycotoxigenicity studies of Aspergillus and Fusarium strains isolated from Brazilian sorghum, the objective of the present study was to determine the toxigenic potential of A. flavus, F. verticillioides and F. proliferatum strains isolated from both freshly harvested and stored sorghum in São Paulo State, Brazil.
MATERIALS AND METHODS
Aspergillus and Fusarium strains
Fifty-nine A. flavus, 35 F. verticillioides, and 3 F. proliferatum isolates, obtained from freshly harvested (10) and stored (130) sorghum grains cultivated in Nova Odessa, São Paulo State, were evaluated. Samples (10 g) were collected monthly, and the grains were ground and homogenized in 90 mL water. Decimal dilutions of up to 10-6 were accomplished and 1-mL aliquots of the dilutions were inoculated onto potato dextrose agar. After incubation (5 days at 25ºC), the colonies were counted, isolated, and identified. Aspergillus and Fusarium strains were identified according to Rapel and Fennell (27) and Nelson et al. (21,23) methods, and stored on Sabouraud dextrose agar (SDA) slants at 4-8ºC.
Production and determination of aflatoxins
Culture Preparation: A small fragment of A. flavus colony activated in SDA at 25ºC was inoculated onto the centre of a coconut milk agar plate (17), and incubated at 25ºC for 10 days.
Extraction and Analytical Method: Aflatoxins were extracted with methanol / 4% KCl (9:1), followed by clarification with ammonium sulphate and partition with chloroform. AFB1 and AFB2 were detected by thin layer chromatography as described by Soares and Rodriguez-Amaya (33), followed by confirmation using trifluoroacetic acid (31). The detection limit was 2 µg/kg for both AFB1 and AFB2.
Production and determination of fumonisins
Culture preparation: fumonisin production by 35 F. verticillioides and 3 F. proliferatum strains was carried out in 50 g polished rice grains humidified with 50 mL distilled water (121ºC for 15 min). The rice medium was inoculated with an aqueous suspension of conidia (2 mL), containing 107 spores obtained from potato dextrose agar, and incubated in the dark at 25ºC for 3 weeks. Then, rice cultures were dried, ground finely with a laboratory mill and stored at 4ºC until fumonisin analysis.
Extraction and analytical method: fumonisins were extracted and determined according to Ross et al. (29) with some modification. Ten grams of rice culture were added to 50 mL acetonitrile/water (1:1) and stirred for 30 min, and the extract was filtered through Whatman No. 1. Following, 2 mL of the filtrate were added to 5 mL water, and the mixture was applied onto a Sep-Pak C18 cartridge (Waters, Division Millipore Corp., Milford, MA), preconditioned with 2 mL methanol and washed with 2 mL Milli Q water (Millipore, Belford, MA, USA). The cartridge was washed with 2 mL acetonitrile/water (20:80), and the toxin was eluted with 2 mL of the same solvent, but at a ratio of 70:30. The final extract was collected in Eppendorf tubes and stored at -20ºC until use.
Two hundred microliters of the final extract were derivated with 50 µL o-phthaldialdehyde (OPA) solution prepared by dissolving 40 mg OPA in 1 mL methanol and diluted in 5 mL 0.1 M sodium tetraborate, with 50 µL mercaptoethanol. The derivated product was analysed by reverse-phase isocratic HPLC system (Shimadzu SCL-6B pump, RF55 fluorescent detector with excitation and wavelength emission of 355 and 400 nm, respectively), using a 150 x 4.6 mm C18 column (50 ODS-20, O-Phenomenex-ultracarb). The mobile phase consisted of methanol/sodium borate acetate buffer (77:23), pH 3.6.
Calibration was carried out with fumonisin standard solutions (Sigma) prepared with 0.0125, 0.025, and 0.05 µg FB1, and 0.005, 0.01, and 0.02 µg FB2 per mL. In the recovery experiment, four samples of polished rice grains (10 g each contaminated with 12.5 to 75.0 µg/g FB1, and 25.0 to 175.0 µg/g FB2) were analysed. The coefficients of variation were 4.8 (FB1) and 7.5 (FB2), and the recovery rate was 88% for FB1 and 94% for FB2. The detection limit was approximately 50 ng/g for both FB1 and FB2.
RESULTS AND DISCUSSION
Aflatoxin analysis showed that 38 (64.4%) of 59 tested A. flavus strains produced detectable levels of aflatoxins at concentrations ranging from 12.00 to 3282.50 µg/kg (AFB1 + AFB2). Fifteen strains produced only AFB1, while 23 produced both AFB1 and AFB2 (Table 1). Aflatoxin group B (AFB1 and AFB2), producing A. flavus strains, has also been described by Pier (24) and Pitt (25), who identified 10% AFB1 producer strains and 90% strains producing both AFB1 and AFB2. In addition, other researchers (13,15) have also been reported higher AFB1 levels compared to AFB2. Our results agree with those ones reported by Kichou et al. (14), who demonstrated that 23% of A. flavus strains isolated from sorghum in Morocco produced AFB1 and AFB2. In India, Sashidhar et al. (30), analysing 150 sorghum grain samples, found high rates of contamination by A. flavus (67%) and Fusarium (59%); however, only two strains produced AFB1 at concentrations of 16 and 40 µg/kg. Production of AFB1 and AFB2 in sorghum and wheat inoculated with A. flavus was also reported (39).
Fumonisin analysis showed that 32 (91.5%) of 35 tested F. verticillioides strains produced detectable levels of fumonisins at concentrations ranging from 0.12 to 5.38 µg/g (FB1 + FB2). Twenty-three strains produced only FB1 and 9 produced FB1 + FB2 (Table 2). The mean recovery rate for fumonisins was approximately 85%. Fumonisin production by almost every F. verticillioides strains (28,38) has been observed in 100% of F. verticillioides strains isolated from corn.
Fumonisin-producing F. verticillioides strains have also been analyzed by other investigators (6,16), who detected high fumonisin producer strains in corn, but low producers in sorghum. According to Nelson et al. (22), the low production of fumonisins by F. verticillioides strains from sorghum grains may be related to the substrate and/or to the geographical area.
The higher production of FB1, when compared to FB2, has also been reported (4,8,9), with FB1 accounting for 70% of all fumonisins both in culture and in naturally contaminated corn. FB2 and FB3 concentrations detected in foods or produced in culture by F. verticillioides strains are approximately 15 to 25% of the produced FB1. However, Apsimon (2) isolated F. verticillioides strains producing more FB2 than FB1.
Moretti et al. (20) concluded all strains isolated from sorghum belonged to the "F" mating population characterized by little or no FB1 and FB2 production. In contrast, majority of strains isolated from maize belonged to the "A" mating population, which produces moderate to high levels of FB1 and FB2.
Two strains of the 3 F. proliferatum isolates produced FB1 + FB2 at concentrations of 0.12 and 0.18 µg/g (Table 2). Fumonisin production by other Fusarium species, mainly F. proliferatum, has been reported (23,38); however, F. verticillioides continues to be the main producer of this toxin.
In the present study, a small number of A. flavus strains was isolated from freshly harvested sorghum samples (1 strain), although a larger number of toxigenic strains were isolated from stored sorghum (S7-S13). This result might be explained by the fact that Aspergillus is classified in the literature as a storage fungus, which has already been detected in the field. Concerning F. verticillioides, which is typically considered to be a field fungus, a larger number of strains was detected in freshly harvested samples. Nevertheless, this fungus was isolated until the seventh month of storage.
The occurrence of toxin production by strains isolated from foods and animal feed does not necessarily imply the presence of mycotoxins. However, it indicates a potential risk for a possible contamination with mycotoxins. Furthermore, if these foods represent a good substratum for mycotoxin production and if the abiotic factors (especially moisture and temperature) are appropriate, the contaminant hazard tends to increase.
We would like to thank the Instituto de Zootecnia, Nova Odessa, São Paulo State, for supplying sorghum grain samples and for help with this study, and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for financial support.
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Submitted: February 17, 2003; Returned to authors for corrections: July 11, 2003; Approved: June 04, 2004.