internatwnal Journal of Food Microbwlogy. 12 (1991 ) 157 - 166 ~' 1991 Elsevier Science Pubhshers B.V. 0168-1605/91/$03.50
157
F O O D 00353
Production of zearalenone in vitro and in corn grains stored under modified atmospheres N a c h m a n Paster, Jill B l u m e n t h a l - Y o n a s s i , R i v k a B a r k a i - G o l a n and Mazal Menasherov Department of Stored Products, A.R.O., The Volcam Center, Bet Dagan. Israel (Received 24 November 1989; revision recmved 2 July 1990; accepted 31 July 1990)
The production of zearalenone by an isolate of Fusarmm equtseti was studied m chemically defined medium and in corn grains stored under modified atmospheres. An increase m the concentrations of sucrose or xylose in Czapek's m e d m m resulted in increased toxin production, while no toxin was produced when lactose was present in the m e d m m . Methionme ( 1 0 - : and 10-3 M) and cystme ( 1 0 - 3 M) added to Czapek's medium inhibited zearalenone production. When armno acids or mtrogen salts were added as the sole nitrogen source, only alanine, tryptophan and NH4CI totally inhibited zearalenone production. Zearalenone producuon was inhibited almost completely m hlgh-motsture corn grains (27%) kept under atmospheres enriched with hagh CO 2 levels (60%. 40% or 20%) with either 20% or 5% 02. However, a lower a m o u n t of C O 2 was needed to inh~bit fungal development and toxin formation when a reduced O 2 level was applied. Key words: Zearalenone; Fusaraum equtsett
Introduction
Fusarium equiseti is widely accepted as being toxigenic, producing a range of mycotoxins (Marasas et al., 1984). The fungus has been recorded frequently on cereals (Gordon, 1956; Marasas et al., 1984), including maize and wheat, in America, Europe and the U.S.S.R. (Joffe, 1960; Marasas et al., 1984). Its presence on barley and wheat was reported by lchinoe et al. (1984) in Japan, and by Bottalico et al. (1977, 1984) in Italy during several years of harvests. F. equiseti has also been isolated from wheat in India (Mall et al., 1979) and from barley in Alaska (Marasas et ai., 1984). Various isolates of the fungus have been reported as capable of producing zearalenone. In Italy, Bottalico et al. (1984) conducted surveys over a number of years to detect zearalenone production by Fusarmm isolates collected from wheat, barley, corn and rice. They found that some isolates of F. culmorum, F. equiseti and F. Correspondence address: N. Paster, Department of Stored Products. A.R.O.. The Volcani Center. P.O. Box 6, Bet Dagan 50250, Israel.
158
grammearum produce the mycotoxin at levels of 43-829 m g / k g medium (corn or rice substrate). In Argentina, Fusarium species were collected from soybeans. F eqmsett (5 out of 11) isolates and F semttectum (13 out of 24) isolates were found to produce zearalenone when cultivated on rice medium, while none of the six F. momhforme strains isolated produced the toxin (Vaamonde et al., 1987). EI-Kady and EI-Maraghy (1982) studied the ability of 296 isolates of Fusartum spp. isolated from cereal grains in Egypt to produce zearalenone and detected the mycotoxin in 45 of them (F. oxvsporum, 36: F montliforme, 8; and F. equtsett, one isolate). Hence, most of the information on zearalenone production by the fungus is based upon surveys and data on the effect of nutritional factors or controlled environmental conditions on toxin formation, seems to be lacking. The present study was aimed at supplying information on the ability of F. equisen to produce zearalenone on different nutritional sources in v~tro as well as on corn grain stored under atmospheric conditions or modified atmospheres.
Materials and Methods
Organism and growth conditions The fungus Fusarium equiseti N R R L 6470 was used throughout the studies. The culture medium was Czapek's medium (El-Kady and E1-Maraghy, 1982), composed of N a N O 3, 3.0 g / l ; K 2 H P O 4, 1.0 g / l ; MgSO 4 - 7 H 2 0 , 0.5 g / l ; KCL, 0.5 g / l : FeSO 4, 0.01 g / l ; Bacto-agar (Difco), 15.0 g / l ; sucrose, 30.0 g / l ; and distilled water, 1 1. The media were autoclaved for 15 rain at 121°C. All trials were carried out in three replicates. Additton of sugars, amino acMs and nitrogen salts The effect of carbon source (type and amount) on fungal growth and zearalenone production was studied by replacing the sucrose with lactose or xylose and by increasing the amount of the sucrose in Czapek's medium. The sugars were tested in concentrations of 0.1, 0.2 and 0.4 M. To study the effect of sulphur-containing amino acids, methionine or cystine (L-form) was added to Czapek's medium at 10-2 or 10 -3 M. The effect of nitrogen sources on the growth and zearalenone production was determined by adding nitrogen salts ( N a N O 2, NH4CI or N H a N O 3 ) or amino acids to the basic Czapek's medium (devoid of NaNO3). Tenfold concentrated solutions of these compounds were sterilized by passage through a 0.45 HA millipore filter and then added to autoclaved medium at a final concentration of 0.35 M. All the compounds were reagent grade. Cultural conditions and fungal growth assessment Uniform discs (0.3 cm diameter) including mycelia and conidia were transferred from the edge of a 7-day-old culture to petri dishes containing 15 ml of the above described medium. The dishes were held for 14 days at 25 ° C and the linear growth
159 of the colony was measured daily. In addition, mycelium dry weight was determined as follows. The entire contents of a petri dish were transferred to a flask and boiling water was added until the medium was completely dissolved. The mycelium was isolated by filtration and then washed with sterile water, dried over a filter paper, W h a t m a n No. 1, and weighed after drying overnight at 80 ° C.
Studies for determination of zearalenone production on corn stored under modified atmospheres Corn grains at 14% moisture content (MC) were sterilized by g a m m a irradiation applied at a level of 1000 krad. After irradiation, batches of 50 g each were transferred to 250-ml Erlenmeyer flasks and 5 ml of spore suspension was added to each flask. The spore suspension of the fungus was prepared in sterile water by collecting spores from the periphery of 7-day-old colonies grown on Czapek's medium. The suspension was adjusted to 10 s p o r e s / m l using a haemocytometer. Sterilized grains wetted with 5 ml sterile water served as control. The flasks which served as test chambers were then connected to the system described by Navarro and D o n a h a y e (1972) which enables the formation of modified atmospheres (MA). Six test chambers (three containing inoculated grain and three containing uninoculated grain) were connected to each system and flushed with the desired gas mixture which was passed (before entering the test chambers) through water to maintain a relative humidity of approximately 99%. All the experiments were conducted for 14 days at 26 + I ° C . After disconnection from the MA system, the contents of one test chamber were taken for zearalenone determination while grain MC and fungal level were assessed in samples taken from the remaining two flasks. Fungal infestation was determined by direct plating and propagule counts, using the dilution method (Clark, 1968); CO 2 measurements served as an additional indirect parameter for assessing fungal growth as described below. Direct plating was undertaken by plating 100 corn kernels after surface sterilization (2 min in a 2% solution of sodium hypochlorite, followed by two rinses in sterile water) on Czapek's medium in ten petri dishes. Dilution counts were done as follows: 10 g of corn was blended in a commercial blender, and 5 g was then transferred to 45 ml of 0.1% sterile agar solution. After shaking for 15 min, four additional decimal dilutions were prepared, and three samples (two of 0.25 ml and one of 0.5 ml) were taken from each dilution and seeded onto the surface of the medium. With both methods plates were incubated at 26 _+ I ° C and the number of infested kernels (in the direct plating method) or of fungal colonies (in the dilution count) was counted daily during 7 days of incubation. C O 2 measurement in a grain sample was determined by gas chromatography as described by Paster and Menasherov, 1988. Zearalenone analysis The method of Eppley (1968) was used for zearalenone determination both in the in vitro studies and in corn grains. In the first case, the entire contents (medium and mycelium) of six petri dishes from each of the treatments were taken for the assay.
160 In b o t h cases the tested m a t e r i a l s were b l e n d e d (twice, for 1 min) in a rmxture of 25 ml distilled water: 250 ml c h l o r o f o r m a n d filtered t h r o u g h W h a t m a n No. 1 filter p a p e r . T h e first 50 mi of the c h l o r o f o r m filtrate was collected a n d p l a c e d on a c o l u m n that c o n s i s t e d of three layers of 5 g N a 2 S O 4, 10 g of silica gel a n d 15 g o f N a 2 S O 4. T h e c o l u m n was w a s h e d with 150 ml h e x a n e a n d 150 ml benzene. Z e a r a l e n o n e was eluted with 250 ml of a c e t o n e : benzene. 5 : 95 v / v . T h e ehiate was e v a p o r a t e d to d r y n e s s a n d the residue was d i s s o l v e d in c h l o r o f o r m . Z e a r a l e n o n e was d e t e r m i n e d b y t h i n - l a y e r c h r o m a t o g r a p h y using plates of sihca gel (E. Merch, c a t a l o g No. 5553). T h e p l a t e s were d e v e l o p e d using t o l u e n e - e t h y l a c e t a t e - f o r m i c a c i d - w a t e r ( 5 0 : 4 0 : 4 . 5 : 0 . 5 ) as the solvent system a n d the toxin c o n c e n t r a t i o n was initially e v a l u a t e d b y c o m p a r i s o n of fluorescent i n t e n s i t y with various s t a n d a r d ( S i g m a C h e m i c a l Co., St. Louis, M O , U.S.A.) spots. T h e results were c o n f i r m e d by s p r a y i n g the T L C plates, when dry, with bts-diazotized b e n z i d i n e ( M a l a i y a n d i et al., 1976). which t u r n e d the s p o t s b r o w n i s h red.
Results
Effect of various sugars on colon)' growth and toxin accumulation C z a p e k ' s m e d i u m n o r m a l l y c o n t a i n s sucrose at 0.1 M. T h e effect o f r e p l a c i n g the s u g a r c o n t e n t of the m e d i u m with sugars at various c o n c e n t r a t i o n s was studied. In all cases, after 10 d a y s the fungal colonies filled the p e t r i dishes ( d i a m e t e r 90 mm). In a l m o s t every case, an increase in the sugar c o n c e n t r a t i o n resulted in a m a r k e d increase in g r o w t h ( T a b l e I), the e x c e p t i o n b e i n g that an increase in lactose from 0.2 to 0.4 M d i d not result in e n h a n c e d g r o w t h of the fungus. A n increase in the c o n c e n t r a t i o n of sucrose or xylose resulted in higher toxin p r o d u c t i o n . C z a p e k ' s
TABLE I The effect of various sugars on colony growth and toxin accumulation by Fusanum equlsett Compound
Concentration ( × 10- t M)
Dry weight (mg)
Zearalenone (mg/60 ml medium)
Sucrose
2 2
170b a 300d 600e
2.Oh 4.0c
4
190bc 300d 320d
tr b tr. tr.
l 2 4
110a 150b 230c
tr. 2.0b 6.0d
4
Lactose
Xylose
1 2
4.0c
a Within columns, means followed by the same letter do not differ significantly (P < 0.05). b tr. ~ t r a c e .
161
medium with 0.1 M sucrose supports 2 mg zearalenone/60 ml medium, while Czapek's medium with 0.2 or 0.4 M sucrose supported twice this amount of zearalenone production (4 m g / 6 0 ml medium). Replacing sucrose by xylose at 0.1 M reduced zearalenone production to trace amounts. However, at higher concentrations xylose supported a greater toxin production: 2 m g / 6 0 ml and 6 r a g / 6 0 ml medium at 0.2 M and 0.4 M, respectively. Only trace amounts of the toxin were produced when lactose was present in the medium (Table I).
Effect of amino acids and various nttrogen salts Methionine and cystine were added at 10 -2 and 10 -3 M to Czapek's medium, while in another series of trials the N a N O 3 nitrogen source in the medium was replaced by a variety of amino acids and nitrogen salts at an equivalent concentration (0.035 M). Data show that both S-containing amino acids inhibit zearalenone production at 10-2 M (Table II), a concentration at which methionine also reduced fungal growth, as recorded by dry weight. At the 10 -3 M concentration, neither amino acid affected fungal growth, but methionine restricted zearalenone synthesis to a trace amount. At the 10-3 concentration, cystine had no effect on toxin production. The other amino acids could be divided into four groups according to their effect on zearalenone production (Table III). Thus, in the presence of alanine, histidine or tryptophane, no toxin was produced. A slight reduction was noted when asparagine or aspartic acid was present, while tyrosine, valine and glycine enhanced zearalenone production. The amount of the toxin produced in the presence of glutamic acid, serine or threonine was the same as m the control. All of the inorganic nitrogen sources tested supported less zearalenone production by E. equiseti than the standard N a N O 3 (Table III). In tests with N H 4 C L , colony growth was reduced (from 90 m m diameter to 35 m m diameter) and no zearalenone was produced. N H 4 N O 3 also reduced growth (colony diameter = 75 m m ) and toxin production (0.1 m g / 6 0 ml medium). Although N a N O : supported
T A B L E !1 M y c e h u m d r y w e i g h t a n d z e a r a l e n o n e p r o d u c t i o n on m e d i a s u p p l e m e n t e d with m e t h l o n i n e a n d c y s t i n e Compound
Concentrauon ( x 1 0 - I M)
Dry weight (mg)
Zearalenone ( m g / 6 0 ml m e d i u m )
Methlomne
10- 3 10 - 2
260b a 200a
tr. b 0
Cystme
10- 3 10 -2
260b 290b
2.0 tr.
260b
2.0
Control
" M e a n s followed by the s a m e letter d o not differ sJgntflcantJy ( P < 0.05). b tr. ~ trace.
162 TABLE Ill The effect of some salts and different anuno acids added at a concentrauon of 0.035 M. on mycehal growth and zearalenone production Compound
Linear growth (mm)
Zearalcnone (rag/60 ml medmm )
NaNO 3 NaNO2 NH4CL NH4NO 3 Glycme Tyrosme Vahne Serine Threomne Glutarmc acid Aspargine Aspartic acid H~stldine Tryptophan Alamne
90 90 35 75 90 90 90 90 90 90 90 47 90 90 90
2.0c " 1.0b nd b tr.a c 5.0e 4.0d 4.0d 2.0c 2.0c 2.0c 1.0c 1.0b tr.a nd b nd b
Means followed by the same letter do not differ sxgnificantly ( P < 0.05). b Non-detectable. c tr. = trace. "
normal
fungal
reduced
t o h a l f o f t h e c o n t r o l (1 m g / 6 0
growth,
as
measured
by
colony
diameter,
toxin
production
was
ml medium).
Zearalenone production on corn stored under modified atmospheres The CO 2 accumulated by fungi which developed under low 02 pressure (5%) was almost half of that developed by fungi grown on the control grains (16.0 and 17.5% vs. 31.2%) (Table IV). At 20% 02 in combination with 20 or 60% CO 2, the amount of CO 2 produced was also less than that measured in corn stored under atmospheric
TABLE IV Growth of Fusartum equtsett and zearalenone production tn corn gram stored under modified atmospheres Gas composmon (%)
Corn moisture
Colony
Infested
Accumulated CO 2
Zearalenone
CO 2
02
N2
content (%)
counts ( c f u / g × l O 6)
grams (%)
(%/25 g / 2 4 h)
(/,tg/50 g)
20 60 20 40
Air 20 20 5 5
60 20 75 55
26.5 27.0 26.0 25.6 26.2
2.4 1.7 1.2 0.9 04
100 100 100 100 100
31.2 24.8 27.0 17.5 16.0
200 tr. * tr. tr. tr.
•
tr.
=
trace.
163 conditions. The number of cfu counted in grains stored under low 02 levels was also low as compared with that of grains stored under elevated CO 2 with 20% 02, or in air. In all cases, direct incubation of surface sterilized corn showed 100% infection by F. equisetz. Zearalenone was present in the corn grains inoculated with the fungus and stored under air. However, in grains stored under any of the gaseous combinations used (high CO 2 with either 20% or 5% 02), only trace amounts of zearalenone were found.
Discussion
The production of mycotoxins is reported to vary on different substrate~. While zearalenone may be accumulated on corn or rice, none was found on soybeans inoculated with toxin-producing fungus (Badewey et al., 1987). More accurate investigations of substrate effect on mycotoxin production are those employing synthetic media. In the present study, the effect of various additions to a defined culture medium on the production of zearalenone by F. equiseti was investigated. Of the sugars tested, lactose did not support toxin formation, while xylose and sucrose supported high concentrations of zearalenone. For both sugars maximum production occurred on the highest concentration tested. These findings emphasize the importance of testing a range of concentrations of substrates in order to understand their effect on toxin synthesis. The findings on the effect of carbon sources on zearalenone production contrast with those reported for F. oxysporum. In this fungus sucrose completely inhibited zearalenone production, with maltose being the only sugar to promote high toxin yields (EI-Kady and El-Maraghy, 1982). Working with strains of Gibberella zeae. Hidy et al. (1977) reported that xylose and sucrose were among the carbohydrates that supported zearalenone production when applied in combination with glucose. The glucose concentration was a very important factor in the process. The significance of different levels of zearalenone production supported by different sugars is not yet clear. However, the results point to the importance of testing fungi on a variety of media when seeking to determine their toxigenicity. The effect of substituting nitrogen sources in the culture medium on the production of zearalenone by F. equiseti, was also investigated. Sulphur-containing amino acids were found to inhibit zearalenone production. Methionine was the most effective, inhibiting toxin production at 10 -~ M concentration. A similar effect has been found on Aspergillus ochraceus, where methionine at 10 -2 M inhibited ochratoxin production (Paster, 1982). Of other amino acids tested, glycine, valine and tyrosine enhanced zearalenone production and alanine, tryptophan and histidine inhibited formation of the toxin. These results correspond generally with the effects of amino acids on zearalenone production by F. oxysporum (EI-Kady and EI-Maraghy, 1982). Glycine was found also to support good zearalenone production by G. zeae (Hidy et al., 1977).
164 Of the inorganic salts tested, N H 4 N O 3. and NH4CI inhibited both fungai growth and zearalenone production, while N a N O , reduced the amount of toxin formed without affecting myceliai growth in comparison with the control ( N a N O 3). These results do not match those obtained by El-Kady and EI-Maraghy (1982), who reported that ammonium nitrogen was suitable for zearalenone production by F oxysporum, while biosynthesis was completely inhibited in the presence of N a N O , . Hence, the ability of different fungi to utilize a certam nitrogen source for formation of the same toxin may vary. Following the in vitro studies we investigated the effect of MA on zearalenone production in vivo, since physical methods have attracted worldwide interest as alternative means for the chemicals used in storage. The results indicate the potential of F. equtsett to produce zearalenone on corn stored under atmospheric conditions, with the amount produced being larger than that produced on different nutritional sources in vitro. The occurrence of F. equiseti in stored corn may therefore be accompanied by the risk of zearalenone presence. The results of the MA studies showed that raised CO 2 levels, together with decreased 0 2 tension, reduced fungal development and zearalenone production, with a clear connection existing between these gases. Thus, lowering the 0 2 level enabled us to use also less CO 2 in order to inhibit the toxin production. Working with T-2, Paster and Menasherov (1988) reported that toxin production by Fusarium sporotrichioides also was inhibited under high CO2/low 02 levels (40%/5% respectively), but the fungal growth was not inhibited even under 60% CO 2. Such a phenomenon of blocking mycotoxin synthesis with CO 2 levels lower than those required to inhibit fungal growth was reported also inter alia as regards Aspergillus flaous and aflatoxin (Epstein et al., 1970) and A. ochraceus and ochratoxin (Paster et al., 1983). It is concluded that MA can be used successfully to inhibit a large range of mycotoxins. However, the marked difference existing among species in their tolerance to high CO 2 may raise difficulties in using MA for fungi inhibition. The results of the present work demonstrate the relationships between nutritional factors and zearalenone formation by F. equiseti in vivo and indicate that MA can be used to inhibit toxin production, albeit not fungus growth, on corn grains.
References Badawey. A., Sawmsky. J and Halasz. A. (1987) Influence of substrate on mycotoxm productton of Fusarm .7 spectes. Acta Ahment. 16, 105- l 10. BottalK,~ A., Frisullo. S. and Ptglomca. V. (1977) Surveyof freshly harvested maize from some European Iocalittes for Fusarmm spp. and zearalenone, m 1976. Phytopath. Medtt. 16, 142-144. Bottalico. A.. Lerano. P and Vtsconh. A. (1984) Production of zearalenone, trichothecenes and mondiformm by Fusanum specms from cereals m Italy. In: H. Kurata and Y Ueno (Eds.). Elsevier, Amsterdam, pp. 199-209. Clarke. J.H. (1968) Fungi m stored products. Trop. Stored Prod. Inf. 15, 3-14. EI-Kady, I.A. and EI-Maraghy, S.S. (1982) Screening of zearalenone-producing Fusar#um spectes in Egypt and chemicallydefined medmm for production of the toxin. Mycopathologia7. 25-29. Eppley. R.M. (1968) Screening method for zearalenone aflatoxin and ochratoxin. J. Assoc. Off. Anal. Chem. 51.74-78.
165 Epstein. E.. Steinberg, M.P., Nelson, A.I. and Wel, L.S. (1970) Aflato~dn production as affected by environmental condiuons. J. Food Sci. 35. 389-391. Gordon. W.L. (1956) The taxonomy and habitats of the Fmarmm species m Tnmdad, B.W.I. Can. J. Bot. 34. 847-864. Hidy, P.H., Baldwin, R.S., Greasham, R.L.. Keith, C.L. and McMullen. J.R. (1977) Zearalenone and some derivatives: Production and biological actiwties. Adv. Appl. Microbtol. 22, 59-82. Ichinoe, M., Uchiyama, S., Amano, R. and Kurata. H. (1984) Tnchothecene producing Fusarmm m barley and wheat in Japan. In: J. Lacey (Ed.), Tnchothecenes and Other Mycotoxins. J. Wtley& Sons. Chichester, U.K. pp. 21-33. Joffe, A.Z. (1960) The mycoflora of overwintered cereals and ~ts toxaclty. Bull. Res. Counc. Israel 9D, 101-126. Malaiyandi. M., Barrette, J.P. and Wavrock, P.L. (1976) Bis-dlazouzed benzidine as a spray reagent for detecting zearalenone on thin layer chromatoplates. J. Assoc. Off. Anal. Chem. 59. 956-962. Mall, O.P., Metha, P.. Agrawal, S.C. and Vora, V.C. (1979) A survey of fungal contaminations of f~eld crops with a v~ew to evahiate thetr abihty to produce mycotoxms. 1. Fungal contaminants of wheat and barley harvested during spring 1975. Indian J. Microbiol. 19, 118-122. Marasas, W.F.O., Nelson, P.E. and Toussoun, T.A. (1984) ToxJgemc Fusarmm species. The Pennsylvania State University Press, Umversity Park, PA. Navarro, S. and Donahaye. E. (1972) An apparatus for studying the effect of controlled low pressure and composition of atmospheric gases on insects. J. Stored Prod. Res. 8, 223-226. Paster, N. (1982) Studies of the growth and morphogenesis of Asperg~llus ochraceus Wilhelm. a fungus causing deterioration of stored gram. Ph.D. thesis, The Hebrew Umverslty of Jerusalem, Faculty of Agriculture, Rehovot, Israel. Paster, N.. Lisker, N. and Chet, I. (1983) Ochratoxin A production by Asperglllus ochraceus Wilhelm grown under controlled atmospheres. Appl. Environ. Microbiol. 45, 1136-1139. Paster, N. and Menasherov, M. (1988) Inhibition of "1"-2 toxin production on high-moisture corn kernels by modified atmospheres. Appi. Environ. Microbiol. 54, 540-543. Vaamonde, G., Scarmoto, G. and Borena, B. (1987) Zearalenone producuon by Fusarmm species isolated soybeans. Int. J. Food Microbiol. 4, 129-133.
157
F O O D 00353
Production of zearalenone in vitro and in corn grains stored under modified atmospheres N a c h m a n Paster, Jill B l u m e n t h a l - Y o n a s s i , R i v k a B a r k a i - G o l a n and Mazal Menasherov Department of Stored Products, A.R.O., The Volcam Center, Bet Dagan. Israel (Received 24 November 1989; revision recmved 2 July 1990; accepted 31 July 1990)
The production of zearalenone by an isolate of Fusarmm equtseti was studied m chemically defined medium and in corn grains stored under modified atmospheres. An increase m the concentrations of sucrose or xylose in Czapek's m e d m m resulted in increased toxin production, while no toxin was produced when lactose was present in the m e d m m . Methionme ( 1 0 - : and 10-3 M) and cystme ( 1 0 - 3 M) added to Czapek's medium inhibited zearalenone production. When armno acids or mtrogen salts were added as the sole nitrogen source, only alanine, tryptophan and NH4CI totally inhibited zearalenone production. Zearalenone producuon was inhibited almost completely m hlgh-motsture corn grains (27%) kept under atmospheres enriched with hagh CO 2 levels (60%. 40% or 20%) with either 20% or 5% 02. However, a lower a m o u n t of C O 2 was needed to inh~bit fungal development and toxin formation when a reduced O 2 level was applied. Key words: Zearalenone; Fusaraum equtsett
Introduction
Fusarium equiseti is widely accepted as being toxigenic, producing a range of mycotoxins (Marasas et al., 1984). The fungus has been recorded frequently on cereals (Gordon, 1956; Marasas et al., 1984), including maize and wheat, in America, Europe and the U.S.S.R. (Joffe, 1960; Marasas et al., 1984). Its presence on barley and wheat was reported by lchinoe et al. (1984) in Japan, and by Bottalico et al. (1977, 1984) in Italy during several years of harvests. F. equiseti has also been isolated from wheat in India (Mall et al., 1979) and from barley in Alaska (Marasas et ai., 1984). Various isolates of the fungus have been reported as capable of producing zearalenone. In Italy, Bottalico et al. (1984) conducted surveys over a number of years to detect zearalenone production by Fusarmm isolates collected from wheat, barley, corn and rice. They found that some isolates of F. culmorum, F. equiseti and F. Correspondence address: N. Paster, Department of Stored Products. A.R.O.. The Volcani Center. P.O. Box 6, Bet Dagan 50250, Israel.
158
grammearum produce the mycotoxin at levels of 43-829 m g / k g medium (corn or rice substrate). In Argentina, Fusarium species were collected from soybeans. F eqmsett (5 out of 11) isolates and F semttectum (13 out of 24) isolates were found to produce zearalenone when cultivated on rice medium, while none of the six F. momhforme strains isolated produced the toxin (Vaamonde et al., 1987). EI-Kady and EI-Maraghy (1982) studied the ability of 296 isolates of Fusartum spp. isolated from cereal grains in Egypt to produce zearalenone and detected the mycotoxin in 45 of them (F. oxvsporum, 36: F montliforme, 8; and F. equtsett, one isolate). Hence, most of the information on zearalenone production by the fungus is based upon surveys and data on the effect of nutritional factors or controlled environmental conditions on toxin formation, seems to be lacking. The present study was aimed at supplying information on the ability of F. equisen to produce zearalenone on different nutritional sources in v~tro as well as on corn grain stored under atmospheric conditions or modified atmospheres.
Materials and Methods
Organism and growth conditions The fungus Fusarium equiseti N R R L 6470 was used throughout the studies. The culture medium was Czapek's medium (El-Kady and E1-Maraghy, 1982), composed of N a N O 3, 3.0 g / l ; K 2 H P O 4, 1.0 g / l ; MgSO 4 - 7 H 2 0 , 0.5 g / l ; KCL, 0.5 g / l : FeSO 4, 0.01 g / l ; Bacto-agar (Difco), 15.0 g / l ; sucrose, 30.0 g / l ; and distilled water, 1 1. The media were autoclaved for 15 rain at 121°C. All trials were carried out in three replicates. Additton of sugars, amino acMs and nitrogen salts The effect of carbon source (type and amount) on fungal growth and zearalenone production was studied by replacing the sucrose with lactose or xylose and by increasing the amount of the sucrose in Czapek's medium. The sugars were tested in concentrations of 0.1, 0.2 and 0.4 M. To study the effect of sulphur-containing amino acids, methionine or cystine (L-form) was added to Czapek's medium at 10-2 or 10 -3 M. The effect of nitrogen sources on the growth and zearalenone production was determined by adding nitrogen salts ( N a N O 2, NH4CI or N H a N O 3 ) or amino acids to the basic Czapek's medium (devoid of NaNO3). Tenfold concentrated solutions of these compounds were sterilized by passage through a 0.45 HA millipore filter and then added to autoclaved medium at a final concentration of 0.35 M. All the compounds were reagent grade. Cultural conditions and fungal growth assessment Uniform discs (0.3 cm diameter) including mycelia and conidia were transferred from the edge of a 7-day-old culture to petri dishes containing 15 ml of the above described medium. The dishes were held for 14 days at 25 ° C and the linear growth
159 of the colony was measured daily. In addition, mycelium dry weight was determined as follows. The entire contents of a petri dish were transferred to a flask and boiling water was added until the medium was completely dissolved. The mycelium was isolated by filtration and then washed with sterile water, dried over a filter paper, W h a t m a n No. 1, and weighed after drying overnight at 80 ° C.
Studies for determination of zearalenone production on corn stored under modified atmospheres Corn grains at 14% moisture content (MC) were sterilized by g a m m a irradiation applied at a level of 1000 krad. After irradiation, batches of 50 g each were transferred to 250-ml Erlenmeyer flasks and 5 ml of spore suspension was added to each flask. The spore suspension of the fungus was prepared in sterile water by collecting spores from the periphery of 7-day-old colonies grown on Czapek's medium. The suspension was adjusted to 10 s p o r e s / m l using a haemocytometer. Sterilized grains wetted with 5 ml sterile water served as control. The flasks which served as test chambers were then connected to the system described by Navarro and D o n a h a y e (1972) which enables the formation of modified atmospheres (MA). Six test chambers (three containing inoculated grain and three containing uninoculated grain) were connected to each system and flushed with the desired gas mixture which was passed (before entering the test chambers) through water to maintain a relative humidity of approximately 99%. All the experiments were conducted for 14 days at 26 + I ° C . After disconnection from the MA system, the contents of one test chamber were taken for zearalenone determination while grain MC and fungal level were assessed in samples taken from the remaining two flasks. Fungal infestation was determined by direct plating and propagule counts, using the dilution method (Clark, 1968); CO 2 measurements served as an additional indirect parameter for assessing fungal growth as described below. Direct plating was undertaken by plating 100 corn kernels after surface sterilization (2 min in a 2% solution of sodium hypochlorite, followed by two rinses in sterile water) on Czapek's medium in ten petri dishes. Dilution counts were done as follows: 10 g of corn was blended in a commercial blender, and 5 g was then transferred to 45 ml of 0.1% sterile agar solution. After shaking for 15 min, four additional decimal dilutions were prepared, and three samples (two of 0.25 ml and one of 0.5 ml) were taken from each dilution and seeded onto the surface of the medium. With both methods plates were incubated at 26 _+ I ° C and the number of infested kernels (in the direct plating method) or of fungal colonies (in the dilution count) was counted daily during 7 days of incubation. C O 2 measurement in a grain sample was determined by gas chromatography as described by Paster and Menasherov, 1988. Zearalenone analysis The method of Eppley (1968) was used for zearalenone determination both in the in vitro studies and in corn grains. In the first case, the entire contents (medium and mycelium) of six petri dishes from each of the treatments were taken for the assay.
160 In b o t h cases the tested m a t e r i a l s were b l e n d e d (twice, for 1 min) in a rmxture of 25 ml distilled water: 250 ml c h l o r o f o r m a n d filtered t h r o u g h W h a t m a n No. 1 filter p a p e r . T h e first 50 mi of the c h l o r o f o r m filtrate was collected a n d p l a c e d on a c o l u m n that c o n s i s t e d of three layers of 5 g N a 2 S O 4, 10 g of silica gel a n d 15 g o f N a 2 S O 4. T h e c o l u m n was w a s h e d with 150 ml h e x a n e a n d 150 ml benzene. Z e a r a l e n o n e was eluted with 250 ml of a c e t o n e : benzene. 5 : 95 v / v . T h e ehiate was e v a p o r a t e d to d r y n e s s a n d the residue was d i s s o l v e d in c h l o r o f o r m . Z e a r a l e n o n e was d e t e r m i n e d b y t h i n - l a y e r c h r o m a t o g r a p h y using plates of sihca gel (E. Merch, c a t a l o g No. 5553). T h e p l a t e s were d e v e l o p e d using t o l u e n e - e t h y l a c e t a t e - f o r m i c a c i d - w a t e r ( 5 0 : 4 0 : 4 . 5 : 0 . 5 ) as the solvent system a n d the toxin c o n c e n t r a t i o n was initially e v a l u a t e d b y c o m p a r i s o n of fluorescent i n t e n s i t y with various s t a n d a r d ( S i g m a C h e m i c a l Co., St. Louis, M O , U.S.A.) spots. T h e results were c o n f i r m e d by s p r a y i n g the T L C plates, when dry, with bts-diazotized b e n z i d i n e ( M a l a i y a n d i et al., 1976). which t u r n e d the s p o t s b r o w n i s h red.
Results
Effect of various sugars on colon)' growth and toxin accumulation C z a p e k ' s m e d i u m n o r m a l l y c o n t a i n s sucrose at 0.1 M. T h e effect o f r e p l a c i n g the s u g a r c o n t e n t of the m e d i u m with sugars at various c o n c e n t r a t i o n s was studied. In all cases, after 10 d a y s the fungal colonies filled the p e t r i dishes ( d i a m e t e r 90 mm). In a l m o s t every case, an increase in the sugar c o n c e n t r a t i o n resulted in a m a r k e d increase in g r o w t h ( T a b l e I), the e x c e p t i o n b e i n g that an increase in lactose from 0.2 to 0.4 M d i d not result in e n h a n c e d g r o w t h of the fungus. A n increase in the c o n c e n t r a t i o n of sucrose or xylose resulted in higher toxin p r o d u c t i o n . C z a p e k ' s
TABLE I The effect of various sugars on colony growth and toxin accumulation by Fusanum equlsett Compound
Concentration ( × 10- t M)
Dry weight (mg)
Zearalenone (mg/60 ml medium)
Sucrose
2 2
170b a 300d 600e
2.Oh 4.0c
4
190bc 300d 320d
tr b tr. tr.
l 2 4
110a 150b 230c
tr. 2.0b 6.0d
4
Lactose
Xylose
1 2
4.0c
a Within columns, means followed by the same letter do not differ significantly (P < 0.05). b tr. ~ t r a c e .
161
medium with 0.1 M sucrose supports 2 mg zearalenone/60 ml medium, while Czapek's medium with 0.2 or 0.4 M sucrose supported twice this amount of zearalenone production (4 m g / 6 0 ml medium). Replacing sucrose by xylose at 0.1 M reduced zearalenone production to trace amounts. However, at higher concentrations xylose supported a greater toxin production: 2 m g / 6 0 ml and 6 r a g / 6 0 ml medium at 0.2 M and 0.4 M, respectively. Only trace amounts of the toxin were produced when lactose was present in the medium (Table I).
Effect of amino acids and various nttrogen salts Methionine and cystine were added at 10 -2 and 10 -3 M to Czapek's medium, while in another series of trials the N a N O 3 nitrogen source in the medium was replaced by a variety of amino acids and nitrogen salts at an equivalent concentration (0.035 M). Data show that both S-containing amino acids inhibit zearalenone production at 10-2 M (Table II), a concentration at which methionine also reduced fungal growth, as recorded by dry weight. At the 10 -3 M concentration, neither amino acid affected fungal growth, but methionine restricted zearalenone synthesis to a trace amount. At the 10-3 concentration, cystine had no effect on toxin production. The other amino acids could be divided into four groups according to their effect on zearalenone production (Table III). Thus, in the presence of alanine, histidine or tryptophane, no toxin was produced. A slight reduction was noted when asparagine or aspartic acid was present, while tyrosine, valine and glycine enhanced zearalenone production. The amount of the toxin produced in the presence of glutamic acid, serine or threonine was the same as m the control. All of the inorganic nitrogen sources tested supported less zearalenone production by E. equiseti than the standard N a N O 3 (Table III). In tests with N H 4 C L , colony growth was reduced (from 90 m m diameter to 35 m m diameter) and no zearalenone was produced. N H 4 N O 3 also reduced growth (colony diameter = 75 m m ) and toxin production (0.1 m g / 6 0 ml medium). Although N a N O : supported
T A B L E !1 M y c e h u m d r y w e i g h t a n d z e a r a l e n o n e p r o d u c t i o n on m e d i a s u p p l e m e n t e d with m e t h l o n i n e a n d c y s t i n e Compound
Concentrauon ( x 1 0 - I M)
Dry weight (mg)
Zearalenone ( m g / 6 0 ml m e d i u m )
Methlomne
10- 3 10 - 2
260b a 200a
tr. b 0
Cystme
10- 3 10 -2
260b 290b
2.0 tr.
260b
2.0
Control
" M e a n s followed by the s a m e letter d o not differ sJgntflcantJy ( P < 0.05). b tr. ~ trace.
162 TABLE Ill The effect of some salts and different anuno acids added at a concentrauon of 0.035 M. on mycehal growth and zearalenone production Compound
Linear growth (mm)
Zearalcnone (rag/60 ml medmm )
NaNO 3 NaNO2 NH4CL NH4NO 3 Glycme Tyrosme Vahne Serine Threomne Glutarmc acid Aspargine Aspartic acid H~stldine Tryptophan Alamne
90 90 35 75 90 90 90 90 90 90 90 47 90 90 90
2.0c " 1.0b nd b tr.a c 5.0e 4.0d 4.0d 2.0c 2.0c 2.0c 1.0c 1.0b tr.a nd b nd b
Means followed by the same letter do not differ sxgnificantly ( P < 0.05). b Non-detectable. c tr. = trace. "
normal
fungal
reduced
t o h a l f o f t h e c o n t r o l (1 m g / 6 0
growth,
as
measured
by
colony
diameter,
toxin
production
was
ml medium).
Zearalenone production on corn stored under modified atmospheres The CO 2 accumulated by fungi which developed under low 02 pressure (5%) was almost half of that developed by fungi grown on the control grains (16.0 and 17.5% vs. 31.2%) (Table IV). At 20% 02 in combination with 20 or 60% CO 2, the amount of CO 2 produced was also less than that measured in corn stored under atmospheric
TABLE IV Growth of Fusartum equtsett and zearalenone production tn corn gram stored under modified atmospheres Gas composmon (%)
Corn moisture
Colony
Infested
Accumulated CO 2
Zearalenone
CO 2
02
N2
content (%)
counts ( c f u / g × l O 6)
grams (%)
(%/25 g / 2 4 h)
(/,tg/50 g)
20 60 20 40
Air 20 20 5 5
60 20 75 55
26.5 27.0 26.0 25.6 26.2
2.4 1.7 1.2 0.9 04
100 100 100 100 100
31.2 24.8 27.0 17.5 16.0
200 tr. * tr. tr. tr.
•
tr.
=
trace.
163 conditions. The number of cfu counted in grains stored under low 02 levels was also low as compared with that of grains stored under elevated CO 2 with 20% 02, or in air. In all cases, direct incubation of surface sterilized corn showed 100% infection by F. equisetz. Zearalenone was present in the corn grains inoculated with the fungus and stored under air. However, in grains stored under any of the gaseous combinations used (high CO 2 with either 20% or 5% 02), only trace amounts of zearalenone were found.
Discussion
The production of mycotoxins is reported to vary on different substrate~. While zearalenone may be accumulated on corn or rice, none was found on soybeans inoculated with toxin-producing fungus (Badewey et al., 1987). More accurate investigations of substrate effect on mycotoxin production are those employing synthetic media. In the present study, the effect of various additions to a defined culture medium on the production of zearalenone by F. equiseti was investigated. Of the sugars tested, lactose did not support toxin formation, while xylose and sucrose supported high concentrations of zearalenone. For both sugars maximum production occurred on the highest concentration tested. These findings emphasize the importance of testing a range of concentrations of substrates in order to understand their effect on toxin synthesis. The findings on the effect of carbon sources on zearalenone production contrast with those reported for F. oxysporum. In this fungus sucrose completely inhibited zearalenone production, with maltose being the only sugar to promote high toxin yields (EI-Kady and El-Maraghy, 1982). Working with strains of Gibberella zeae. Hidy et al. (1977) reported that xylose and sucrose were among the carbohydrates that supported zearalenone production when applied in combination with glucose. The glucose concentration was a very important factor in the process. The significance of different levels of zearalenone production supported by different sugars is not yet clear. However, the results point to the importance of testing fungi on a variety of media when seeking to determine their toxigenicity. The effect of substituting nitrogen sources in the culture medium on the production of zearalenone by F. equiseti, was also investigated. Sulphur-containing amino acids were found to inhibit zearalenone production. Methionine was the most effective, inhibiting toxin production at 10 -~ M concentration. A similar effect has been found on Aspergillus ochraceus, where methionine at 10 -2 M inhibited ochratoxin production (Paster, 1982). Of other amino acids tested, glycine, valine and tyrosine enhanced zearalenone production and alanine, tryptophan and histidine inhibited formation of the toxin. These results correspond generally with the effects of amino acids on zearalenone production by F. oxysporum (EI-Kady and EI-Maraghy, 1982). Glycine was found also to support good zearalenone production by G. zeae (Hidy et al., 1977).
164 Of the inorganic salts tested, N H 4 N O 3. and NH4CI inhibited both fungai growth and zearalenone production, while N a N O , reduced the amount of toxin formed without affecting myceliai growth in comparison with the control ( N a N O 3). These results do not match those obtained by El-Kady and EI-Maraghy (1982), who reported that ammonium nitrogen was suitable for zearalenone production by F oxysporum, while biosynthesis was completely inhibited in the presence of N a N O , . Hence, the ability of different fungi to utilize a certam nitrogen source for formation of the same toxin may vary. Following the in vitro studies we investigated the effect of MA on zearalenone production in vivo, since physical methods have attracted worldwide interest as alternative means for the chemicals used in storage. The results indicate the potential of F. equtsett to produce zearalenone on corn stored under atmospheric conditions, with the amount produced being larger than that produced on different nutritional sources in vitro. The occurrence of F. equiseti in stored corn may therefore be accompanied by the risk of zearalenone presence. The results of the MA studies showed that raised CO 2 levels, together with decreased 0 2 tension, reduced fungal development and zearalenone production, with a clear connection existing between these gases. Thus, lowering the 0 2 level enabled us to use also less CO 2 in order to inhibit the toxin production. Working with T-2, Paster and Menasherov (1988) reported that toxin production by Fusarium sporotrichioides also was inhibited under high CO2/low 02 levels (40%/5% respectively), but the fungal growth was not inhibited even under 60% CO 2. Such a phenomenon of blocking mycotoxin synthesis with CO 2 levels lower than those required to inhibit fungal growth was reported also inter alia as regards Aspergillus flaous and aflatoxin (Epstein et al., 1970) and A. ochraceus and ochratoxin (Paster et al., 1983). It is concluded that MA can be used successfully to inhibit a large range of mycotoxins. However, the marked difference existing among species in their tolerance to high CO 2 may raise difficulties in using MA for fungi inhibition. The results of the present work demonstrate the relationships between nutritional factors and zearalenone formation by F. equiseti in vivo and indicate that MA can be used to inhibit toxin production, albeit not fungus growth, on corn grains.
References Badawey. A., Sawmsky. J and Halasz. A. (1987) Influence of substrate on mycotoxm productton of Fusarm .7 spectes. Acta Ahment. 16, 105- l 10. BottalK,~ A., Frisullo. S. and Ptglomca. V. (1977) Surveyof freshly harvested maize from some European Iocalittes for Fusarmm spp. and zearalenone, m 1976. Phytopath. Medtt. 16, 142-144. Bottalico. A.. Lerano. P and Vtsconh. A. (1984) Production of zearalenone, trichothecenes and mondiformm by Fusanum specms from cereals m Italy. In: H. Kurata and Y Ueno (Eds.). Elsevier, Amsterdam, pp. 199-209. Clarke. J.H. (1968) Fungi m stored products. Trop. Stored Prod. Inf. 15, 3-14. EI-Kady, I.A. and EI-Maraghy, S.S. (1982) Screening of zearalenone-producing Fusar#um spectes in Egypt and chemicallydefined medmm for production of the toxin. Mycopathologia7. 25-29. Eppley. R.M. (1968) Screening method for zearalenone aflatoxin and ochratoxin. J. Assoc. Off. Anal. Chem. 51.74-78.
165 Epstein. E.. Steinberg, M.P., Nelson, A.I. and Wel, L.S. (1970) Aflato~dn production as affected by environmental condiuons. J. Food Sci. 35. 389-391. Gordon. W.L. (1956) The taxonomy and habitats of the Fmarmm species m Tnmdad, B.W.I. Can. J. Bot. 34. 847-864. Hidy, P.H., Baldwin, R.S., Greasham, R.L.. Keith, C.L. and McMullen. J.R. (1977) Zearalenone and some derivatives: Production and biological actiwties. Adv. Appl. Microbtol. 22, 59-82. Ichinoe, M., Uchiyama, S., Amano, R. and Kurata. H. (1984) Tnchothecene producing Fusarmm m barley and wheat in Japan. In: J. Lacey (Ed.), Tnchothecenes and Other Mycotoxins. J. Wtley& Sons. Chichester, U.K. pp. 21-33. Joffe, A.Z. (1960) The mycoflora of overwintered cereals and ~ts toxaclty. Bull. Res. Counc. Israel 9D, 101-126. Malaiyandi. M., Barrette, J.P. and Wavrock, P.L. (1976) Bis-dlazouzed benzidine as a spray reagent for detecting zearalenone on thin layer chromatoplates. J. Assoc. Off. Anal. Chem. 59. 956-962. Mall, O.P., Metha, P.. Agrawal, S.C. and Vora, V.C. (1979) A survey of fungal contaminations of f~eld crops with a v~ew to evahiate thetr abihty to produce mycotoxms. 1. Fungal contaminants of wheat and barley harvested during spring 1975. Indian J. Microbiol. 19, 118-122. Marasas, W.F.O., Nelson, P.E. and Toussoun, T.A. (1984) ToxJgemc Fusarmm species. The Pennsylvania State University Press, Umversity Park, PA. Navarro, S. and Donahaye. E. (1972) An apparatus for studying the effect of controlled low pressure and composition of atmospheric gases on insects. J. Stored Prod. Res. 8, 223-226. Paster, N. (1982) Studies of the growth and morphogenesis of Asperg~llus ochraceus Wilhelm. a fungus causing deterioration of stored gram. Ph.D. thesis, The Hebrew Umverslty of Jerusalem, Faculty of Agriculture, Rehovot, Israel. Paster, N.. Lisker, N. and Chet, I. (1983) Ochratoxin A production by Asperglllus ochraceus Wilhelm grown under controlled atmospheres. Appl. Environ. Microbiol. 45, 1136-1139. Paster, N. and Menasherov, M. (1988) Inhibition of "1"-2 toxin production on high-moisture corn kernels by modified atmospheres. Appi. Environ. Microbiol. 54, 540-543. Vaamonde, G., Scarmoto, G. and Borena, B. (1987) Zearalenone producuon by Fusarmm species isolated soybeans. Int. J. Food Microbiol. 4, 129-133.
