Mycopathologia (2006) 162: 5763 DOI 10.

1007/s11046-006-0027-8

Springer 2006

Natural occurrence of mycotoxins in staple cereals from Ethiopia

Amare Ayalew1, Hartmut Fehrmann2, Johann Lepschy3, Robert Beck3 & Dawit Abate4
1

Department of Plant Sciences, Alemaya University, P.O. Box 241Alemaya, Ethiopia; 2Institut fur Planzenpathologie und Panzenschutz, Universitat Gottingen, Grisebachstrae 6, 37077, Gottingen, Germany; 3 Bayerische Landesanstalt fur Landwirtschaft, Lange Point 6, 85354, Freising, Germany; 4Department of Biology, Addis Ababa University, P.O. Box 1176Addis Ababa, Ethiopia
Received 20 October 2005; accepted in revised form 22 March 2006

Abstract The occurrence of mycotoxins in barley, sorghum, te (Eragrostis tef) and wheat from Ethiopia has been studied. Samples were analyzed for aatoxin B1 (AFB1), ochratoxin A (OTA), deoxynivalenol (DON), nivalenol (NIV) and zearalenone (ZEN) using high performance liquid chromatography (HPLC) and for fumonisins (FUM) using enzyme linked immunosorbent assay (ELISA). AFB1 and OTA were detected in samples of all the four crops. AFB1 was detected in 8.8% of the 352 samples analyzed at concentrations ranging from trace to 26 lg kg)1. OTA occurred in 24.3% of 321 samples at a mean concentration of 54.1 lg kg)1 and a maximum of 2106 lg kg)1. DON occurred in barley, sorghum and wheat at 40 2340 lg kg)1 with an overall incidence of 48.8% among the 84 mainly suspect samples analyzed; NIV was co-analyzed with DON and was detected at 40 lg kg)1 in a wheat sample and at 50, 380, and 490 lg kg)1 in three sorghum samples. FUM and ZEN occurred only in sorghum samples with low frequencies at concentrations reaching 2117 and 32 lg kg)1, respectively. The analytical results indicate higher mycotoxin contamination in sorghum, which could be related to the widespread storage of sorghum grain in underground pits leading to elevated seed moisture contents. This is the rst report on the occurrence of OTA in te. Key words: barely, Eragrostis tef, mycotoxin, natural occurrence, sorghum, wheat Abbreviations: AFB1 Aatoxin B1 ; DON Deoxynivalenol; FUM Fumonisins; NIV Nivalenol; OTA Ochratoxin A; ZEN Zearalenone

Introduction Invasion of cereal grain by fungi is frequently associated with a substantial risk of contamination by mycotoxins. On the basis of adverse eects on human and animal health and widespread contamination, aatoxins, deoxynivalenol (replaced in some areas by nivalenol), fumonisins, ochratoxin A and zearalenone are considered as the most important mycotoxins on a worldwide scale [1]. Species of Aspergillus, Fusarium and Penicillium are known to produce these mycotoxins. Such fungi are commonly found in the mycobiota of

Ethiopian grains [24]. However, it is not possible to predict the presence of mycotoxins in the grain and it is essential to determine mycotoxin occurrence through survey and experimental studies. The occurrence of mycotoxins in food grains has been the subject of many investigations all over the world [5, 6]. In Ethiopia, information on the incidence and levels of aatoxins in cereals is very limited [7], whereas reports on OTA and Fusarium mycotoxins are so far not available. Comprehensive reviews on the global occurrence of mycotoxins [5, 6] cite few episodes from Africa. There is a need to further extend the data base on

58 the nature and extent of mycotoxin contamination in the region. This has been echoed by the conclusions of an international workshop [8]. The present study reports the natural occurrence of six mycotoxins in samples of the staple food crops barley, wheat, sorghum and te (Eragrostis tef (Zucc.) Trotter) from Ethiopia. Te alone provides two-thirds of the human nutrition in Ethiopia. Chromnet integrator. The HPLC system for DON, NIV, OTA, and ZEN included a Merck L-6200 HPLC pump and a Merck F-1050 uorescence detector. In addition, for DON and NIV, a Merck 655A-40 auto sampler, a post-column system of Besta E-100 (2) pumps and reaction loops of 0.5 mm 8 m and 0.5 mm 4 m (Omnit), and the chromatography data system Chromeleon Version 6.0 (Dionex) were used. For OTA and ZEN, the auto sampler was Gilson Abimed (Model 231) and the chromatography data system Gynkosoft Version 5.5 (Softron) was used for recording chromatograms. A reversed phase Lichrospher 100 RP-18 column, 125 4 mm, 5 lm particle size (Merck) was used for all HPLC analyses. Analysis of AFB1 Aatoxin B1 was analyzed by the method of Werner [10] using ve volumes of acetonewater (85:15) for extraction and immunoanity (RIDA aatoxin) columns (R-Biopharm, Darmstadt, Germany) for clean-up of extracts. Eluates from the columns were evaporated and the residue was dissolved in 1 ml of a 30% solution of the HPLC mobile phase (watermethanolacetonitrile, 130: 70:40) in water. Two hundred microliters was injected manually. Post-column derivatization was carried out in a reaction coil of 1 m 0.25 mm with 0.1% (wt/vol) iodine solution at 0.4 ml min)1 at 60 C. Detection was carried out at 444 nm (365 nm excitation). Analysis of DON and NIV A ground sub-sample (12.5 g) was extracted in 50 ml of acetonitrilewater (84:16) by stirring for 2 h. Initial clean-up of portions of extracts through a column of 0.75 g charcoalceliteneutral alumina, 25:15:5 (wt/wt) was followed by a further purication through a column of 0.5 g Florisil [11]. The nal eluate was evaporated and the residue was taken up in 1 ml of the HPLC mobile phase. One hundred microliters were injected. Acetonitrilewater (1:9) was used as eluent at a ow rate of 1 ml min)1. Post-column derivatization using 0.1 N NaOH at 0.4 ml min)1 was followed by reaction with a solution of acetyl acetone (5 ml) and ammonium acetate (62.5 g) in 1 l of water at a ow rate of 0.75 ml min)1 and 115 C in oil bath (MGW Lauda C6) and detection was at 503 nm (397 nm excitation).

Materials and methods Samples Grain samples of the 1999 harvest intended for human consumption were collected at harvest from threshing yards, and four to six months later from traditional storage structures, mainly, underground pits (sorghum), gota and gotera (barley, wheat), or from bags (te). Gota is a cylindrical chamber made of mud mixed with straw, whereas gotera is basketwork with thatched conical roong. Samples were collected from major-growing regions; sorghum samples from Wollo (north Ethiopia) and Hararghe (east), wheat and barley from West Shoa, Arsi, Bale (central and south), and te samples from East Shoa (central Ethiopia). Samples were 1 kg each, kept in portable cool-box during collection trips, and then in a refrigerator (ca. 4 C) before transport to the analytical laboratory in Freising, Germany, where they were maintained at )18 C until determination of mycotoxins. A representative sub-sample of 100 g was ground to pass a 1-mm sieve and used for mycotoxin analysis. The numbers of samples analyzed for each mycotoxin were as indicated in Tables 13. Samples analyzed for Fusarium mycotoxins were primarily (ca. 70%) suspect samples that had shown fusarial infection in mycological examination of the seed reported elsewhere [9]. High performance liquid chromatography analysis of AFB1, OTA, DON, NIV and ZEN HPLC conditions The HPLC equipment for AFB1 analysis consisted of a Milton Roy (model ConstaMetric I) pump, a Kratos PCRS 520 post-column pump, a Shimadzu RF535 uorescence detector, and a Spectra-Physics

59
Table 1. Aatoxin B1 content of samples of cereal grains of the 1999 harvest from Ethiopia Crop Number of samples analyzed AFB1 positive samples (%) Samples (%) with the indicated AFB1 content a (lg kg)1) a Barley Sorghum Te Wheat
a b

Median (lg kg)1)

Averageb (lg kg)1)

Maximum (lg kg)1)

b 3.5 0.0 11.4 0.0

c 1.7 0.0 0.0 1.7

d 1.7 0.0 5.7 1.7

e 0.0 2.4 0.0 0.0 1.8 0.9 2.5 9.5 3.8 10.0 5.1 8.7 11.7 25.9 15.6 12.3

115 82 35 120

11.3 6.1 22.9 4.2

4.4 3.7 5.7 0.8

a = trace, <1; b = 1 to <5; c = 5 to <10, d = 10 to < 20, e = >20. Average for the positive samples.

Table 2. Incidence and levels of ochratoxin A in cereal grains of the 1999 harvest in Ethiopia Crop Number of samples analyzed OTA positive samples (%) Samples (%) with the indicated OTA contenta a Barley Sorghum Te Wheat
a b

Median (lg kg)1)

Averageb (lg kg)1)

Maximum (lg kg)1)

b 13.6 5.1 15.2 20.6

c 1.0 7.7 6.1 1.9

d 1.0 0.0 0.0 0.0

e 0.0 2.6 0.0 0.0 6.1 42.8 31.9 14.9 17.2 174.8 32.7 19.6 164 2106 80 66

103 78 33 107

26.2 21.8 27.3 23.4

10.7 6.4 6.1 0.9

a = 1 to <5; b = 5 to <50, c = 50 to <100, d = 100 to <200, e = >200 lg kg)1. Average for the positive samples.

Table 3. Occurrence of Fusarium mycotoxins in cereal grains of the 1999 harvest from Ethiopia Mycotoxin Crop Number of samples analyzed 20 33 8 23 20 33 8 23 5 39 8 15 15 29 9 16 Number of positive samples 7 30 0 4 0 3 0 1 0 3 0 0 0 2 0 0 Mean (range) of concentration (lg kg)1) 70 (40110)a 360 (502340) b 50, 90, 90, and 110c 50, 380, and 490 40 1370, 1653, and 2117 19 and 32

DON

NIV

FUM

ZEN

Barley Sorghum Te Wheat Barley Sorghum Te Wheat Barley Sorghum Te Wheat Barley Sorghum Te Wheat

Values in parenthesis represent the concentration range in positive samples. = mycotoxin not detected. c Individual measurements are presented where the number of positive samples was few.
b

60 Analysis of OTA and ZEN A representative ground grain sample (12.5 g) was extracted with acidied chloroform (10 ml of 0.33 mol l)1 ortho-phosphoric acid and 125 ml chloroform). Clean-up was carried out using Extrelut columns (Merck) [12]. OTA was eluted by a chloroformformic acid (30 ml + 1 ml) mixture, followed by chloroform. For samples that were analyzed for both OTA and ZEN, chloroform/acetic acid (90:10) was employed for the simultaneous elution of the mycotoxins [13]. The eluate was evaporated, taken up in 1 ml of methanolwaterortho-phosphoric acid (685:400:4), and used for HPLC analysis. The mobile phase was acetonitrilewaterglacial acetic acid (50:50:2) with a ow rate of 1 ml min)1. Detection was at 470 nm (with excitation at 330 and 276 nm for OTA and ZEN, respectively). Enzyme linked immunosorbent assay of FUM ELISA Commercially available ELISA kits (RidascreenFast Fumonisin, R-Biopharm, Darmstadt, Germany) were used according to the procedures given by the manufacturer. After a competitive enzyme immunoassay, the A450 was measured using a microplate reader (Multiscan Plus, TITERTEK). FUM analysis Five grams ground grain sample was extracted with 25 ml of 70% methanol by stirring for 3 min on a magnetic stirrer. After ltration, 1 ml of the extract was mixed with 6 ml distilled water and assayed for FUM content in the ELISA. A calibration curve using A450 values was constructed from four standard concentrations between 0.1 and 6.4 mg kg)1. All the standard and sample values were previously adjusted as percentages of the zero standard; 0 mg kg)1 was equal to 100% absorbance. wheat from Ethiopia. AFB1 occurred in 31 (8.8%) of the samples of the four dierent crops at levels ranging from trace (less than 1 lg kg)1) to 25.9 lg kg)1 (Table 1). The mean concentrations ranged from 3.8 in barley to 10 lg kg)1 in sorghum. The overall mean AFB1 concentration in the positive samples was 5.9 lg kg)1. OTA occurred with an average concentration of 54.1 lg kg)1 in 24.3% of the samples (Table 2). OTA levels were between 2.7 and 2106 lg kg)1. Comparatively higher OTA concentrations were recorded in sorghum. Incidence and levels of Fusarium mycotoxins The extent of occurrence of DON, NIV, ZEN, and FUM was analyzed in the crop samples (Table 3). None of these mycotoxins was detected in te samples. DON occurred in the other three crops. It was the most common of the Fusarium mycotoxins occurring in 48.8% of the total samples most of which were suspect samples. The concentrations of DON ranged from 40 to 2340 lg kg)1. ZEN and NIV were detected in 4.8% and 2.9% of the samples, respectively, and their concentrations were low too (Table 3). FUM occurred in 4.5% of the samples but at concentrations that exceeded 2100 lg kg)1. NIV occurred in wheat and sorghum while FUM and ZEN were encountered only in sorghum samples.

Discussion The levels of aatoxins in barley, sorghum, te and wheat in the present study agree with previous reports that the incidence of aatoxins in grains other than maize is generally low [5]. However, the concentrations of AFB1 encountered in the present study were relatively low (maximum of 26 lg kg)1) compared to certain reports from tropical areas such as Alpert et al. [14] for sorghum (reaching up to 1000 lg kg)1) and Quitet et al. [15] for wheat (1388 lg kg)1). On the other hand, high levels of incidence and concentrations of OTA were encountered in the samples. The maximum level recorded (2106 lg kg)1) is much higher compared to reports even from central/east Europe where OTA contamination is known to be prevalent; OTA was detected at 0.539 lg kg)1 in a recent analysis of Bulgarian wheat samples [16].

Results Incidence and levels of aatoxin B1 and ochratoxin A Aatoxin B1 and ochratoxin A positive samples were detected in barley, sorghum, te as well as

61 OTA in wheat from Ethiopia ranged from 4.2 to 66 lg kg)1. In general, OTA has been found as a contaminant of grain primarily in north-temperate areas [17], although it occurs in many commodities throughout the world [18]. The deviation from this widespread tenet among mycotoxicologists observed in the present work might be due, at least in part, to the mild sub-tropical climate in most of the sampling areas. Ethiopia is a highland country, where tropical temperature conditions are limited to the lowlands in the peripheries [19]. Moreover, the highest levels of OTA were detected in sorghum samples drawn from underground storage pits which appeared to be favorable to OTA accumulation. Sorghum grain stored in pits showed steady increase in moisture content [20]. The highest AFB1 was also detected in sorghum that had been stored in underground pits. From the angle of mycotoxin contamination, the current practice of grain storage in underground pits in Ethiopia appears to be inadequate. The incidence of AFB1 in the crops varied from 4.2% in wheat to 22.9% in te, while the incidence of OTA was comparable in the four crops ranging from 21.8% in sorghum to 27.3% in te. These trends cannot be explained based on results of the present study. The high frequency of both the mycotoxins in te, however, is a cause for concern from the human health perspective. Te is a major staple in the Ethiopian diet. It is made into injera, a pancake like at fermented bread, which people eat once or twice (occasionally three times) every day. Because of the high te consumption per day per person, even low levels of mycotoxins in the grain could lead to signicant extended exposure of humans to the mycotoxins. It is well established that mycotoxins generally withstand the fermentation and baking process. Aatoxin B1 was reported earlier in te [7] but this is the rst report on the occurrence of OTA in te. In 2003, tolerance limits for OTA in cereals have been enforced by 37 countries, 29 of which have set the limit at 5 lg kg)1 [21]. OTA is generally produced by isolates of A. ochraceus and P. verrucosum [18]. It is worth noting that grain samples of the present study were subjected to mycological examination [9] which revealed that A. ochraceus occurred in 24.4% of the OTA-positive and 4.5% of the OTA-negative samples; the corresponding gures for Penicillium spp. were 18% and 16%, while both groups co-occurred in 6.4% and 1.5% of the OTA-positive and OTA-negative samples, respectively. Penicillia were not identied on the species level. Despite the strong association between the incidence of A. ochraceus and OTA contamination, the presence of ochratoxigenic species among the Penicillium isolates cannot be ruled out. Yet, the frequency of any potential OTA producers detected could not account for the widespread occurrence of the mycotoxin among the samples. Since OTA is generally stable, it might be detected long after the producing fungi have died out or have been outgrown by other species. In general Aspergilli occur more commonly in warmer regions while Penicillia are more frequent in colder climates [22]. In Ethiopia, the altitude-mediated ecological diversity and storage in underground pits might expose the grain to a range of fungi. The incidence and levels of Fusarial mycotoxins found in the present study were low when compared to reports from other parts of the world. About 60% of European wheat is found contaminated with DON at concentrations ranging from 2 to 3960 lg kg)1 [23]. DON concentrations reaching 40,000 lg kg)1 were further reported from Europe [24]. In wheat from Japan average values of DON for the years 19761980 were 1009180 lg kg)1 [25]. In the current study, the average DON level in positive samples of wheat was 85 lg kg)1 and the overall mean in the positive samples was 280 lg kg)1. NIV, which was even less frequent than DON in the Ethiopian samples, is widely reported in some parts of the world. Yoshizawa [26] found NIV contents of up to 4400 and 26,000 lg kg)1, respectively, in wheat and barley from Japan. ZEN levels of 20 8100 lg kg)1 were reported for sorghum from Australia [27]. About 25% of European wheat is contaminated with ZEN at concentrations reaching 2000 lg kg)1 [23]. In the present study, the two positive samples contained 19 and 32 lg kg)1 ZEN; both the incidence and mycotoxin levels were low. Regardless of geographical location, DON occurs predominantly in wheat, barley and maize and less often in sorghum [28]. ZEN commonly occurs as a co-contaminant with trichothecenes including DON [16]. The more frequent occurrence of DON in sorghum and the absence of detectable ZEN in wheat from Ethiopia thus call for further investigation. FUM has been reported

62 mainly as a common contaminant of maize worldwide [6]. Miller [1] considered strains of F. moniliforme isolated from sorghum to be poor producers of fumonisins. The incidence of FUM in the present study (in sorghum) was also low although its level reached 2117 lg kg)1. It is pertinent that most of the samples (57 out of 84) analyzed for DON and NIV had shown Fusarium infection (generally by F. graminearum, F. avenaceum or F. verticillioides) in mycological investigations reported elsewhere [9]. By and large the same suspect samples of the four crops were analyzed for FUM, with wider coverage of sorghum samples based on the higher frequency of F. moniliforme infection. Fusarium mycotoxins generally show low acute toxicity. The no observable eect level (NOEL) of DON ranges from 0.08 mg kg)1 of body weight in pigs to 0.1 mg kg)1 of body weight per day in mice [28]. The NOEL of FUM for renal toxicity in rats is 0.67 mg kg)1 body weight per day, while the corresponding NOEL value for liver cancer is 0.8 mg kg)1 [28]. Nearly 40 countries have enforced limits for DON in wheat and other cereals generally at 750 lg kg)1, reaching up to 2000 lg kg)1 in some countries; only 6 countries have set limits for fumonisins in maize at 1000 to 3000 lg kg)1 [21]. It can therefore be concluded that the levels of DON with mean 280 lg kg)1 and median of 90 lg kg)1 in cereal grains in Ethiopia are too low to be of major concern to farmers. The three FUM positive sorghum samples contained the mycotoxins at an average of 1713.2 lg kg)1. None of the Fusarium mycotoxins was detected in te samples, which agrees with the low level of infection of the grain by Fusarium spp. [9]. It is worth noting that FUM was found only in sorghum samples from northern Ethiopia but not in any of the samples from the east. Regional and seasonal dierences in the distribution of mycotoxin contamination are well established [29]. A clear picture would emerge by analyzing samples over several seasons. Time course studies at the dierent stages of grain production and utilization are also required to nd out the important period of accumulation of particular mycotoxins. In the present study, the data sets for freshly harvested and stored grain samples were merged for transport of the former to the analytical laboratory was delayed because of the vague plant material movement regulations in Ethiopia. The freshly harvested samples stayed for considerable time at the airport of origin, outside the cold chain, which made it dicult to explain the trends in the incidence of mycotoxins in the two batches of samples. In conclusion, OTA was the most widespread mycotoxin in cereals of the 1999 harvest in Ethiopia. AFB1 and Fusarium toxins appeared to play a minor role in the contamination of the crop samples, particularly in crops other than sorghum. The existing gap between supply and demand of food in Ethiopia forces many people to consume what they might have otherwise rejected, even when the food is visibly moldy or organoleptically unacceptable. This would lead to increased risk to the consumer health from mycotoxins and emphasizes the urgency for establishing regular monitoring programs for mycotoxins in staple grains in developing countries with chronic food decits. Further monitoring of mycotoxins in Ethiopia is justied. Moreover, the producing fungi and their frequency of occurrence as well as the conditions for production of particular mycotoxins in dierent agro-ecological regions need to be thoroughly investigated. Acknowledgements We thank Dr. Schuster, Mr. Zach, Ms. Anni John, Ms. Gitta Clasen, Ms. Sabine Topor and Ms. Edith Bock for cooperation and technical help. The German Academic Exchange Service (DAAD) sponsored Amare Ayalew during the study.

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Address for correspondence: Amare Ayalew, Department of Plant Sciences, Alemaya University, P.O. Box 241, Alemaya, Ethiopia Phone: +251-25-6610704; Fax: +251-25-6610719 E-mail: amareayalew@yahoo.com