InternationalJournal of Food Microbiology, 13 (1991) 329-334
© 1991 Elsevier Science Publishers B.V. 0168-1605/91/$03.50 FOOD 00422
Short c o m m u n i c a t i o n
Influence of temperature on zearalenone production by regional strains of Fusarium graminearum and Fusarium oxysporum in culture G . D . M i l a n o 1 and T.A. L o p e z 2 I Departamento de Fisiopatolog[a, Facultad de Ciencias Veterinarias, UNCPBA, Tandil, and 2 Laboratorio de Toxicolog[a Veterinaria, Departamento de Produccidn Animal, INTA, Balcarce, Argentina
(Received 7 September 1990;revision received 10 April 1991; accepted 18 May 1991)
Zearalenone production by Fusarium graminearum and Fusarium oxysporum was studied under two temperature conditions. Incubation at 25 °C for 4 weeks enhanced zearalenone synthesis, improving detection of zearalenone-producing strains of Fusarium oxysporum. Zearalenone production was either totally or partially inhibited when temperature was lowered to 12-14°C during the last 2 weeks of incubation. Key words: Zearalenone; Fusarium graminearum; Fusarium oxysporum
Zearalenone is an estrogenic secondary metabolite synthesized by certain species of the genus F u s a r i u m . Its estrogenic activity in mammals, extensively described for swine, causes morphological changes and functional disorders of the reproductive tract, leading to retention of corpora lutea (Long and Diekman, 1986), hyperplasia of endometrium, ovary atrophy, nymphomania (Chang et al., 1979), pseudopregnancy (Etienne and Jemmali, 1982), and embryonic loss (Long and Diekman, 1986). Zearalenone occurs naturally in corn and other grains infected by zearalenoneproducing F u s a r i u m spp. before or after harvest. Mirocha et al. (1967), Eugenio et al. (1970), and Sherwood and Peberdy (1974) have stated that high moisture content and temperature stress (exposure to 1 2 - 1 4 ° C ) are necessary for the fungus to produce high levels of zearalenone. However, later studies by Naik et al. (1978) and Montani et al. (1988) have indicated that response to temperature stress depends greatly on the strain tested, exposure to low temperature not always being required.
Correspondence address." G.D. Milano, Departamento de Fisiopatologia, Facultad de Ciencias Veteri-
narias, UNCPBA, Pinto 399, 7000, Tandil, Argentina.
330 Corn samples from the southeast of Buenos Aires province have been shown to be contaminated with zearalenone, particulary those stored in open cribs (L6pez and Tapia, 1980; Odriozola et al., 1985). Fusarium graminearum, Fusarium oxysporum, and Fusarium moniliforme have been frequently isolated from corn samples of the same area (Winter et al., 1974; Lori, 1985), but no suggestions have been made regarding the species involved in zearalenone contamination. Similary, zearalenone production by regional strains of Fusarium spp. under different t e m p e r a t u r e conditions has scarcely been studied in Argentina (Montani et al., 1988).
Experimental procedures Four regional strains of Fusarium graminearum Schwabe and three regional strains of Fusarium oxysporum Schlecht (Booth, 1971) were used in this study to investigate the effect of t e m p e r a t u r e stress on zearalenone production. The strains were isolated from infected corn and wheat kernels collected in the southeast of Buenos Aires province. Kernels were surface disinfected in 2% sodium hypochlorite solution for 2 rain, rinsed twice in sterile distilled water, and placed in moist chambers for 3 days at 24-25 °C. Isolates were grown on potato-dextrose agar (PDA, 18 g of agar, 260 g of potato, 20 g of dextrose in 1 1 of distilled water; p H 6.8) for 7 days at 24-25 ° C. Sporulation was induced by placing 7-day-old cultures under cold white fluorescent light for 72 h at 20 °C. Isolates from P D A were identified according to Booth (1971). A conidial suspension of each strain (about 1.0 × 105 conidia per ml) was p r e p a r e d in 10 ml sterile distilled water from cultures on PDA, and inoculated onto 100 g of moist autoclaved cracked corn (moisture content adjusted to 35-38%, w / w ) , in a 250-ml Erlenmeyer flask. One set of flasks of each strain was incubated for 4 weeks at 24-25 ° C. The other set was incubated for 2 weeks at 24-25 o C, followed by 2 weeks at 12-14 ° C. Cultures at both t e m p e r a t u r e conditions were done in duplicate. Z e a r a l e n o n e was extracted and analysed by a multimycotoxin test, as previously described by Tapia (1985). The presence of zearalenone was confirmed by spraying the chromatographic plate with 5% A1CI 3 in ethanol (w/v), followed by heating the plate at 110 ° C for 5 rain. Monodimensional thin-layer chromatography was used to quantify zearalenone under shortwave ultraviolet light (250 nm), comparing fluorescence intensity of 10 /xl of sample extract with those of aliquots of a zearalenone standard in methanol (minimum detectable zearalenone concentration achieved by the method is 100 ng g - I ) (Tapia, 1985).
Results and Discussion Results are shown in Table I. All strains of Fusariurn grarninearurn and Fusarium oxysporurn produced zearalenone when incubated at a constant temperature of 25 °C. At this t e m p e r a t u r e both species produced the highest levels of
331 TABLE I In vitro zearalenone production on corn by strains of Fusarium graminearum and Fusarium oxysporum under two t e m p e r a t u r e conditions Species
F. graminearum F. oxysporum
Z e a r a l e n o n e production (mg k g - l) 25oc b
25 o C / 1 2 _ 1 4 o C c
1 095 + 398 7+ 3
270_+ 161 nd
Numb er of isolates. b 4 weeks at 24-25 ° C. c 2 weeks at 2 4 - 2 5 ° C followed by 2 weeks at 1 2 - 1 4 ° C .
toxin. Three F. graminearum strains produced larger amounts of zearalenone when incubated at 2 5 ° C during 4 weeks, while one strain had a 71% increase in its production when temperature was lowered to 12-14 ° C during the last 2 weeks of incubation (results not shown). Exposure to low temperatures completely inhibited synthesis of zearalenone by F. oxysporum strains. Several workers have established that incubation at 1 2 - 1 4 ° C after the fungus has invaded the substrate, is necessary to obtain high levels of zearalenone (Christensen et al., 1965; Mirocha et al., 1967; Eugenio et al., 1970; Sherwood and Peberdy, 1974). Temperatures of 12-14°C, although not appropriate for fungal growth, are supposed to activate the pathways to zearalenone synthesis (Mirocha and Christensen, 1974). Our results suggest that strains of F. graminearum and F. oxysporum from the southeast of Buenos Aires province do not always require low temperatures to yield maximum levels of zearalenone. Moreover, such temperatures can either reduce or arrest zearalenone biosynthesis. This inhibitory effect together with the favourable response observed at 25 °C suggest that zearalenone production may occur in seasons in which minimum temperatures are not lower than 20 ° C. It is interesting to remark that production of zearalenone by F. oxysporum, although not unknown (Mirocha and Christensen, 1974; Willie and Morehouse, 1977), is not frequently observed. Fifty strains of F. oxysporum tested in previous reports failed to produce detectable amounts of zearalenone (Caldwell and Tuite, 1968; Caidwell et al., 1970; Eugenio et al., 1970; Ishii et al., 1974; Bottalico, 1977; Ichinoe et al., 1977). Exposure to low temperatures ( 1 2 - 1 6 ° C ) for a variable number of weeks was a common step of the culture procedures described in these reports. In the present study, low temperatures inhibited zearalenone production by F. oxysporum strains able to produce zearalenone at 25 °C. Incubation at a constant temperature of 25 °C seems to be a more adequate procedure to detect zearalenone-producing strains of F. oxysporum. Further studies to ascertain which culture procedures allow for a better expression of zearalenone-production of F. oxysporum and other Fusarium species would improve laboratory detection of strains responsible for natural contamination of grains, and would clarify our current knowledge of field conditions for natural synthesis of zearalenone.
Acknowledgements G.D. Milano has a fellowship from the Consejo Nacional de Investigaciones Cientificas y Tecnicas (CONICET). The authors thank Grisela Botta and Celia Garcia for technical assistance and advice, and Dr. Julio Rodriguez for critical review of the article. They also thank Marina Dosantos and Maria Salom6n for their assistance.
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