Fusaria and fumonisins in maize from Ghana and their co-occurrence with aflatoxins

International Journal of Food Microbiology 61 (2000) 147–157 www.elsevier.nl / locate / ijfoodmicro

Fusaria and fumonisins in maize from Ghana and their co-occurrence with aflatoxins a, b c Kafui Kpodo *, Ulf Thrane , Benedicte Hald a

Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958, Frederiksberg C, Denmark b Department of Biotechnology, Technical University of Denmark, DK-2800 Lyngby, Denmark c Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, Stigbøjlen 4 DK-1870, Frederiksberg C, Denmark Received 1 November 1999; received in revised form 6 April 2000; accepted 29 May 2000

Abstract Fifteen maize samples from four markets and processing sites in Accra, Ghana were analysed for fumonisins B 1 , B 2 , and B 3 . All samples contained fumonisins. Total fumonisin levels for 14 samples ranged from 70 to 4222 mg kg 21 . One sample of visibly mouldy kernels contained 52 670 mg kg 21 total fumonisins. Mycological examination of the samples showed Aspergillus spp. as the most dominant fungi (76.4%) followed by Penicillium spp. (19.9%). Fusarium formed 2.6% with Fusarium verticillioides as the predominant Fusarium species. Thirty-two Fusarium strains representing five species isolated from the maize samples were tested for the production of fumonisins in maize substrates. From 95% (21 of 22) of the F. verticillioides strains tested, all three types of fumonisins were produced. Total fumonisin levels ranged from 127 to 11 052 mg g 21 . Additional studies on maize samples from 15 processing sites in Accra revealed a co-occurrence of both fumonisins and aflatoxins in 53% (8 of 15) of the samples.  2000 Elsevier Science B.V. All rights reserved. Keywords: Aflatoxin, Fumonisin; Fusarium; Ghana; Maize; Mycotoxins

1. Introduction The genus Fusarium is one of the most economically important genera of fungi and includes several pathogenic species which cause a wide range of plant diseases (Nelson et al., 1981). Fusaria have the ability to produce a diverse range of mycotoxins *Corresponding author. Present address: Food Research Institute, P.O. Box M.20, Accra, Ghana.

many of which are produced only under culture conditions. Marasas (1991) identified F. sporotrichioides, F. poae, F. equiseti, F. graminearum, and F. moniliforme ( 5 F. verticillioides) as the five most important toxigenic Fusarium species. Fusarium verticillioides commonly infects a wide range of crops throughout the world and is considered a major parasite of the Gramineae particularly in tropical and subtropical regions resulting in severe economic losses (Webster and Gunnel, 1992) de-

0168-1605 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00370-6

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pending on the crop. F. verticillioides has been reported as the most prevalent fungus in maize growing areas around the world (Nelson et al., 1981; Marasas et al., 1984) but much more widespread in tropical and subtropical regions. Maize kernel infection by F. verticillioides is of concern because of the loss of grain and seed quality and the potential occurrence of fumonisins and other mycotoxins. Several species of Fusarium are known to produce fumonisins with the principal producing strains being F. moniliforme, F. proliferatum, and F. nygami (Nelson et al., 1991,1992,1994). Fumonisins have been reported to induce leukoencephalomalacia (ELEM) in horses (Kellerman et al., 1990; Ross et al., 1990), pulmonary oedema in swine (Harrison et al., 1990) and to be hepatotoxic and carcinogenic to rats (Gelderblom et al., 1991). Furthermore, F. verticillioides has been shown to be statistically associated with human oesophageal cancer risk in the Transkei region of southern Africa (Marasas, 1982; Marasas et al., 1981,1988) and in China (Li et al., 1980; Yang, 1980). Other studies (Wang et al., 1991; Riley et al., 1994; Ramasamy et al., 1995) have demonstrated that fumonisin B 1 is a potent inhibitor of sphinganine N-acyltransferase, causing an elevation of the sphinganine:sphingosine ratio which results in a disruption of cell membrane function. The toxicity of fumonisins and their presence in maize and maize products has resulted in several surveys being conducted in many parts of the world to determine their levels (Murphy et al., 1993; Doko et al., 1995; Shephard et al., 1996). Maize (Zea mays) is a dietary staple in Ghana as well as in other West African countries. In Ghana it contributes a major part of the total calories of the diet of people in the coastal areas (National Food and Nutrition Board, 1962). With the growing awareness of the toxicity of fumonisins and other mycotoxins, it is of utmost importance that studies are conducted to give an indication of the extent of the problem in the country. Some work has been carried out on mycotoxins namely aflatoxins, citrinin, ochratoxin A, and zearalenone in maize and maize products in Ghana (Jespersen et al., 1994; Kpodo et al., 1996), however, no information is available regarding the occurrence of fumonisins in maize in the country or the potential for the production of these toxins and the possibility of the co-occurrence of fumonisins with aflatoxins. In this paper we

determine which Fusarium species are present on maize kernel samples from Accra, Ghana, their ability to produce fumonisins, the levels of fumonisins in maize from some markets and processing sites as well as the co-occurrence of fumonisins with aflatoxins in samples of maize kernels from Ghana.

2. Materials and methods

2.1. Maize samples Maize samples were obtained from two retail markets, A and B, and two processing sites, C and D, all in Accra, Ghana during the rainy season, August to September. Fourteen of the 15 samples collected were destined for human consumption and were in apparently good condition. One sample (MA-7) consisted of visibly mouldy kernels sorted out and was to be used for poultry feed. Additional samples were further purchased from 15 major maize processing sites all in Accra. Each of these sites has a capacity of several tonnes per week. These samples were used for studies on the co-occurrence of fumonisins and aflatoxins.

2.2. Isolation and identification of fusaria From each sample, 200 kernels were randomly selected and surface disinfected by soaking for 1 min in 1% NaOCl after which they were plated (5 per plate) onto Dichloran 18% Glycerol agar (DG18; Hocking and Pitt, 1980) and Czapek-Dox Iprodione Dichloran agar (CZID; Abildgren et al., 1987) under aseptic conditions. A total of 100 kernels per sample was plated on each medium. Plates were incubated at 258C for 5 days under alternating cycles of 12 h of near ultraviolet (NUV) light and darkness after which Fusarium colonies ¨ were isolated onto Spezieller Nahrstoffarmer Agar (SNA) defined by Nirenberg (1976). The SNA plates were incubated under alternating light for 6 days after which Fusarium species were then single-point inoculated onto fresh SNA plates, potato sucrose agar (PSA) (Booth, 1971), two-point inoculated on Tannin sucrose agar (TAN), (Thrane, 1986) and three-point inoculated on Yeast extract sucrose agar (YES) (Frisvad, 1981).

K. Kpodo et al. / International Journal of Food Microbiology 61 (2000) 147 – 157

Colony diameters on PSA were measured after 4 days in the dark at 258C and 7 days in the dark at 378C. Colony diameters on TAN were also measured after incubation in the dark at 258C for 7 days. SNA plates incubated at 258C in alternating light for 7 days were used for morphological characterisation and identification according to the descriptions by Burgess et al. (1994) and Samson et al. (1995). Representative Fusarium isolates from each maize kernel sample have been stored on soil tubes and deposited in the IBT culture collection of the Department of Biotechnology, Technical University of Denmark, Lyngby, Denmark.

2.3. Extraction and clean-up procedure of samples Fumonisin (B 1 , B 2 , and B 3 ) analyses were based on the method of Sydenham et al. (1992). Fumonisins were extracted from 50-g portions of finely ground maize kernels by blending with methanol–water (3:1, v / v). The slurry was centrifuged and the pH of the filtered supernatant adjusted to between 5.8 and 6.5 with 2 M NaOH solution. A 10-ml aliquot of the filtered extract was applied to a BondElut strong anion exchange (SAX) cartridge (Varian, Harbor City, CA, USA) fitted to a Supelco solid phase extraction (SPE) manifold (Supelco, Bellefonte, PA, USA), previously conditioned by the successive passage of 5 ml of methanol and 5 ml of methanol–water (3:1, v / v). The cartridge was then washed with 5 ml of methanol–water (3:1, v / v) followed by 3 ml of methanol. The fumonisins were eluted from the cartridge with 10 ml of a 1% solution of acetic acid in methanol, the eluent being collected and evaporated to dryness at 408C under a stream of nitrogen. The residue was stored at 48C until HPLC analysis which was carried out within 14days. The aflatoxin extraction procedure was based on that of Pons (1979). Finely ground maize kernels were extracted with methanol followed by precipitation of colour pigments using zinc acetate then extraction into dichloromethane with further clean-up by column chromatography using cellulose and silica gel. Aflatoxins were eluted with dichloromethane– acetone (80:20, v / v) which was evaporated off and the residue quantitatively transferred into 10 ml of HPLC-grade dichloromethane. Five millilitres were evaporated to dryness under a stream of nitrogen and

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the final residue dissolved in 0.1–1.0 ml of HPLC mobile phase and used for HPLC analysis.

2.4. Mycotoxin standards Fumonisin B 1 (FB 1 ), B 2 (FB 2 ), and B 3 (FB 3 ) standards were obtained from the Programme on Mycotoxins and Environmental Carcinogenesis (PROMEC), Medical Research Council, South Africa. The standard solutions were prepared by dissolving the pure fumonisins in acetonitrile–water (1:1) to give concentrations of 25 mg / ml each for FB 1 , FB 2 , and FB 3 . HPLC analysis of the o-phthaldialdehyde (OPA) derivatives of the individual toxin standards resulted in the observation of a single chromatographic peak for each. The solution was stored at 48C. Aflatoxin standards were obtained from Sigma Chemical Co. Ltd (St Louis, MO, USA). Standard stock and HPLC working solutions were prepared by evaporating and dissolving in mobile phase consisting of methanol–acetonitrile–water (10:30:60, v / v / v) to give concentrations of 0.1 mg / ml for aflatoxins B 1 and G 1 and 0.03 mg / ml for B 2 and G 2.

2.5. HPLC analyses Analysis for fumonisins was carried out by dissolving the residue in 200 ml of methanol. o-Phthaldialdehyde (OPA) reagent was prepared by dissolving 0.04 g of o-phthaldialdehyde in 1 ml of methanol followed by the addition of 5 ml of 0.1 M sodium tetraborate and 50 ml of 2-mercaptoethanol. OPA (225 ml) was added to a 25-ml sample solution. The fumonisin OPA derivatives (20 ml) were analysed between 1 and 2 min of derivatisation using a reversed-phase HPLC–fluorescence detection system. A Shimadzu HPLC system (SpectraChrom, Brondby, Denmark), consisting of an LC-6A solvent delivery system connected to a fluorescence detector Model RF 535 and an C-R3A integrator, was used. Chromatographic separations were performed on a Phenomenex Ultracarb 5 m ODS(20) column (150 3 4.6 mm, i.d.), connected to an Ultracarb 5 m ODS(20) guard column (30 3 4.6 mm) (Phenomenex, Torrance, CA, USA). Methanol–0.1 M sodium dihydogen phosphate (77:23) solution adjusted to a pH of 3.35 with orthophosphoric acid was used as mobile phase at a flow rate of 1.0 ml / min. Fluores-

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cence of the fumonisin OPA derivatives was recorded at excitation and emission wavelengths of 335 and 440 nm, respectively. Fumonisin quantification was performed by peak area measurements, by comparison with reference standard solutions at concentrations of 25 mg / ml for each fumonisin. Appropriate dilutions of sample extracts were made with methanol. The limit of detection of the analytical method was 50 mgkg 21 for each toxin. Average percentage recovery study values (six experiments) were 90.3, 87.4 and 90.1% for FB 1 , FB 2 , and FB 3 , respectively. HPLC analysis for aflatoxins was by reversedphase liquid chromatography with post-column iodine derivatization. Separation of aflatoxins was carried out on a Spherisorb S5 ODS-1 column of dimension 25 3 4.6 mm packed with 5-mm particles (Phase Separations Inc., Norwalk, CT, USA) maintained at 358C. The HPLC mobile phase flow-rate was 1.2 ml min 21 and post-column iodine derivatization of aflatoxins B 1 and G 1 was achieved using saturated iodine solution according to the procedure of Shepherd and Gilbert (1984). Iodine was pumped at a flow rate of 0.4 ml min 21 using an Eldex precision metering pump (Eldex Laboratories Inc., San Carlos, CA, USA). The derivatization tube consisted of stainless steel tubing (5 m 3 0.3 mm) maintained at 758C. The excitation and emission wavelengths used were 360 and 440 nm, respectively. The aflatoxins were identified by their retention times, and peaks areas were used to determine their concentrations in the samples by reference to standard curves obtained by chromatographing pure aflatoxin standard solutions under identical conditions.

2.6. Inoculation studies Fusarium cultures, isolated and identified as described above, were grown on potato sucrose agar (PSA) for 7 days on an alternating 12-h, 258C light– dark schedule after which the Petri dishes were washed with 5 ml of sterilized distilled water to produce conidial suspensions. Petri dishes (100 3 20 mm) containing 30 g of milled yellow maize with 30 ml of water added were then autoclaved for 1 h at 1218C on each of 2 consecutive days. After the second autoclaving, the maize was cooled overnight

and inoculated with 1 ml of inocula using a sterile syringe. Petri dishes of inoculated maize were incubated in the dark at 258C for 16 days after which they were dried in an oven at 458C for 48 h then milled to a uniform consistency and stored at 48C until assayed for fumonisins by HPLC.

2.7. Extraction and HPLC of maize cultures Each maize culture was assayed for fumonisin B 1 , B 2 and B 3 using the procedure of Sydenham et al. (1992) outlined earlier with slight modifications as follows: 5 g of culture material were extracted and 4 ml of filtrate were passed through the SAX cartridge instead of 10 ml for the maize kernels. HPLC was performed as described previously and the limits of detection for the procedure were 0.6, 1.3, and 0.7 mg g 21 for fumonisins B 1 , B 2 , and B 3 , respectively.

3. Results and discussion The occurrence (percentage of colonies of each genera for each sample) of genera in the 15 maize kernel samples obtained from the four retail markets and processing sites is summarised in Table 1. A total of 1912 colonies were counted on the DG18 plates prepared for the 15 samples. The predominant fungi were Aspergillus spp. (65%) and Penicillium spp. (28.1%). Fusarium spp. formed 5.1%. Other moulds not belonging to any of these genera constituted 1.8%. These included Acremonium, Chaetomium, Gliocladium and Zygomycetes. Traditionally, cereal fungi are grouped either as field or storage fungi. Field fungi are known to die out during drying and storage since they cannot survive under conditions of low moisture content. Storage fungi notably Aspergillus and Penicillium invade the grain after harvest, during drying and storage since they are capable of surviving at low moistures and water activities. The maize samples examined were dried kernels in which case most of the field fungi had died out at the time of collection. This explains the high incidence of Aspergillus and Penicillium species. This fact is further confirmed by sample KA-2 which consisted of maize from a previous crop season and had been in storage for 1 year and therefore did not contain any Fusarium species (Table 2). Similar observations have been

K. Kpodo et al. / International Journal of Food Microbiology 61 (2000) 147 – 157 Table 1 Percentage colonies of Aspergillus, Fusarium, and Penicillium species isolated from maize samples Sample no.

Aspergillus

Market A MA-1 MA-2 MA-3 MA-4 MA-5 MA-6 MA-7 b

56.1 64.4 98.9 86.0 48.5 51.7 50.0

Market B KA-1 KA-2 c KA-3 KA-4 KA-5 KA-6

72.5 20.9 57.1 60.0 85.7 90.9

Site C AJ-1

69.6

Site D AM-1

62.5

Fusarium 7.6 5.5 – – 16.7 10.3 21.4

1.4 – 4.8 5.5 1.6 –

1.8

–

Penicillium

Others

36.4 30.1 1.1 14.0 34.8 19.0 26.8

–a – – – – 19.0 1.8

26.1 72.1 38.1 34.5 12.7 9.1

–

28.6

–

37.5

–

7.0 – – – –

a

None isolated. Visibly mouldy kernels sorted out for use as poultry feed. c Maize from the previous year. b

made by other researchers (Akinrele, 1970; Jespersen et al., 1994). In this study, a total of 516 Fusarium colonies were counted from both DG18 and CZID plates for all the 15 samples plated. Of this number, 229 representative colonies were isolated and identified. Six different Fusarium species were identified (Table 2). These were: F. verticillioides (65.9%), F. semitectum (29.3%), F. equiseti (3.1%), F. oxysporum (0.9%), F. graminearum (0.4%), and F. chlamydosporum (0.4%). The dominant Fusarium species on maize kernels was F. verticillioides in South Africa (Marasas et al., 1979,1981; Sydenham et al., 1990; Rheeder et al., 1992) and in Nigeria (Gbodi et al., 1985). Jespersen et al. (1994) showed F. subglutinans as the dominant Fusarium species in Ghana in contrast to our results. Table 3 lists the results obtained for the fumonisin analyses of the 15 maize samples collected from the retail markets and processing sites. All the samples were found to contain fumonisins. Fumonisins B 1 , B 2 , and B 3 were detected in 100, 80, and 47% of the

151

samples, respectively. The 14 samples destined for human consumption had total fumonisin (FB 1 1 FB 2 1 FB 3 ) levels ranging from 70 to 4222 mg kg 21 . Market A samples were positive for FB 1 , FB 2 , and FB 3 at levels of 188–906 mg kg 21 , none detectable to 223 mg kg 21 and none detectable to 123 mg kg 21 , respectively (excluding sample MA-7). Samples from Market B were positive for fumonisins at the following ranges: 70–474 mg kg 21 below the detection level to 131 mg kg 21 and below the detection level to 72 mg kg 21 , for FB 1 , FB 2 , and FB 3 , respectively. The sample from Processing site C (AM-1) had the highest levels of all three types of fumonisins (2621, 1375, and 226 mg kg 21 ) for samples destined for human consumption. Earlier studies by Kpodo et al. (1996), showed that samples taken from this same processing site contained higher levels of aflatoxins and citrinin than those from Site D. These data on fumonisins in maize from Ghana indicate generally lower levels than those previously obtained in maize from Argentina and Costa Rica (Sydenham et al., 1993; Viquez et al., 1996). Similar studies by Doko et al. (1995) showed that maize hybrids from Benin and Zambia had fumonisin levels ranging from 0 to 3310 mg kg 21 for FB 1 and from 20 to 1710 mg kg 21 for FB 2 . Mean fumonisin contents of positive samples were 700 and 220 mg kg 21 with median values of 190 and 100 mg kg 21 for Benin and Zambia, respectively. In these studies FB 3 levels were not detected. In our studies, total mean fumonisin content (843 mg kg 21 ) and median value (579 mg kg 21 ) were higher than those obtained for Benin and Zambia. With the exception of three samples (MA-4; KA2; KA-5) which contained only FB 1 , the proportion of FB 1 and FB 2 in the samples (expressed as percentages of the total fumonisin concentrations) fell within the ranges 62.1–85.2% and 12.5–32.6%, respectively, (Table 3) confirming that FB 1 is the major fumonisin produced under natural conditions. Similar observations have been made by Thiel et al. (1992) and Sydenham et al. (1993). Fumonisin B 3 , when present, formed between 5.3 and 15.5% of the combined FB 1 , FB 2 , and FB 3 levels. Sample MA-7 which consisted of mouldy kernels destined to be used for poultry feed had very high levels of fumonisins FB 1 , FB 2 , and FB 3 : 33 103, 12 318, and 7249 mg kg 21 . Combined fumonisin

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Table 2 Fusarium species isolated from Ghanaian maize kernels Sample no.

No. of Fusarium

Individual Fusarium species (%)

and market

isolated

Fv a

Fs

Market A MA-1 MA-2 MA-3 MA-4 MA-5 MA-6 MA-7 b

22 23 10 14 25 36 20

77.3 78.3 90.0 85.7 58.3 86.1 60.0

18.2 13.0 10.0 14.3 29.2 13.9 35.0

4.5 0 0 0 8.3 0 0

Market B KA-1 KA-2 c KA-3 KA-4 KA-5 KA-6

9 0 17 13 8 7

33.3 0 35.3 53.8 25.0 71.4

66.7 0 52.9 46.2 75.2 28.6

Processing site C AM-1

14

64.3

Processing site D AJ-1

11

45.5

Fe

Fo

Fg

Fc

0 8.7 0 0 0 0 0

0 0 0 0 4.2 0 0

0 0 0 0 0 0 5.0

0 0 11.8 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

0 0 0 0 0 0

28.6

7.1

0

0

0

45.5

9.1

0

0

0

a

Fv 5 F. verticilloides; Fs 5 F. semitectum; Fe 5 F. equiseti; Fo 5 F. oxysporum; Fg 5 F. graminearum; Fc 5 F. chlamydosporum. b Visibly mouldy kernels sorted out for use as poultry feed. c Maize from the previous crop.

levels for this sample was 52 670 mg kg 21 which is well above levels previously shown to be associated with outbreaks of equine leukoencephalomalacia (ELEM) (Thiel et al., 1991a; Sydenham et al., 1992). According to Ross et al. (1992), a concentration of FB 1 greater than 10 000 mg kg 21 in horse feed could be related to ELEM. However, Thiel et al. (1991a) reported that lower concentrations of FB 1 averaging 7700 mg kg 21 may be associated with ELEM. Based on this, sample (MA-7) could be included in the intoxicant group. This could be used to emphasise the role hand-picking of mouldy kernels could play in the reduction of fumonisin levels in maize. The consequences of constant ingestion of naturally contaminated maize are not known and require further investigations. Studies by Chamberlain et al. (1993) and Kubena et al. (1995) showed that fumonisins acted additively in combination with aflatoxins in turkeys, the toxicological implications for humans of the co-occurrence of these two mycotoxins are unknown and need to be investigated. Table 4 shows details of fumonisin and

aflatoxin analyses on samples from 15 major processing sites in Accra. All the samples contained fumonisins. In addition, 8 of the 15 samples contained aflatoxins giving a 53% incidence of cooccurrence. The highest total aflatoxin level was 662 mg kg 21 recorded in a sample from site number 6.This sample, in addition, contained fumonisins at a level of 1043 mg kg 21 . The maize sample contaminated with the highest level of fumonisin (2534 mg kg 21 ) was co-contaminated with aflatoxins at a concentration of 89 mg kg 21 . The co-occurrence of fumonisins and aflatoxins has also been reported by some researchers (Wang et al., 1995; Yamashita et al., 1995; Yashizawa et al., 1996; Ali et al., 1998). A similar level of co-occurrence of 55% was obtained by Ali et al. (1998). This may be due to the fact that the same growing conditions favour both fumonisin and aflatoxin production and maize is always a prevalent host. F. verticillioides is also known to produce fusarins (Miller et al., 1995) of which fusarin C has been shown to be mutagenic and immunosuppressive (Bacon et al., 1990). Few strains

K. Kpodo et al. / International Journal of Food Microbiology 61 (2000) 147 – 157

153

Table 3 Fumonisin levels in maize kernel samples from markets and processing sites in Accra, Ghana 21

Sample no.

Fumonisin concentration (mg kg

and market

FB 1

FB 2

) FB 3

Total a

Fumonisin (%) FB 1

FB 2

FB 3

Market A MA-1 MA-2 MA-3 MA-4 MA-5 MA-6 MA-7 c

230 903 387 188 664 906 33 103

60 213 86 nd 122 223 12 318

nd b 68 nd nd nd 123 7249

290 1184 473 188 786 1252 52 670

79.3 76.3 81.8 100.0 84.5 72.4 62.8

20.7 18.0 18.2 0 15.5 17.8 23.4

0 5.7 0 0 0 9.8 13.8

Market B KA-1 KA-2 KA-3 KA-4 KA-5 KA-6

374 238 429 474 70 458

65 nd 123 131 nd 76

nd nd nd 70 nd 72

439 238 552 675 70 606

85.2 100.0 77.7 70.2 100.0 75.6

14.8 0 22.3 19.4 0 12.5

0 0 0 10.4 0 11.9

Processing site C AJ-1

581

116

128

825

70.4

14.1

15.5

Processing site D AM-1

2621

1375

226

4222

62.1

32.6

5.3

a

Combined FB 1 1 FB 2 1 FB 3 levels determined for each sample. b nd 5 None detected ( , 50 mg kg 21 ). c Visibly mouldy grain.

Table 4 Co-occurrence of fumonisins with aflatoxins in maize from processing sites in Accra a Site

Fumonisin concentration (mg kg 21 )

Aflatoxin concentration (mg kg 21 )

code

FB 1

FB 2

FB 3

Total b

AFB 1

AFB 2

AFG 1

AFG 2

Total c

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

191 71 11 41 1216 682 1655 64 20 136 69 11 1007 88 109

nd d 10 nd nd 772 289 695 51 nd 15 12 nd 641 20 24

nd nd nd nd 224 72 184 nd nd nd nd nd 127 nd nd

191 81 11 41 2212 1043 2534 115 20 151 81 11 1775 108 133

338 5 nd nd 89 204 36 nd 111 33 2 nd nd nd nd

54 1 nd nd 4 7 1 nd 13 6 nd nd nd nd nd

nd nd nd nd nd 434 48 nd nd nd nd nd nd nd nd

nd nd nd nd nd 17 4 nd nd nd nd nd nd nd nd

392 6 nd nd 93 662 89 nd 124 39 2 nd nd nd nd

Detection limits for FB 1 , FB 2 , FB 3 5 50 mg kg 21 ; AFB 1 and AFB 2 5 0.04 mg kg 21 , AFG 1 and AFG 2 5 0.06 mg kg 21 . Combined FB 1 1 FB 2 1 FB 3 levels. c Combined AFB 1 1 AFB 2 1 AFG 1 1 AFG 2 levels. d nd 5 None detected. a

b

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K. Kpodo et al. / International Journal of Food Microbiology 61 (2000) 147 – 157

of F. verticillioides have also been shown to produce another toxin, moniliformin (Marasas et al., 1984) which is toxic to chickens (Allen et al., 1981). The presence of fusarins or moniliformin in Ghanaian maize or the ability of F. verticillioides isolates from Ghanaian maize to produce these toxins have not been investigated. Table 5 shows results of the capacity of 22 isolates of F. verticillioides, four each of F. semitectum, and F. equiseti and one isolate each of F. oxysporum and F. graminearum to produce fumonisins. All the 22 isolates of F. verticillioides studied produced all three types of fumonisins (FB 1 , FB 2 , and FB 3 ) except for isolate IBT 9496 which produced only FB 1 . Fumonisin levels ranged from 2 to 8364, none detectable to 1928, and from none detectable to 1691 mg g 21 for fumonisins B 1 , B 2 , and B 3 , respectively. Similar studies conducted by several researchers (Bezuidenhout et al., 1988; Nelson et al., 1991; Thiel et al., 1991b) on the ability of F. verticillioides strains from corn-based feeds to produce fumonisin B 1 showed that 96% (52 of 54) produced the toxin. In the present study, 91% (20 of 22) of the F. verticillioides isolates can be described as high producers ( . 500 mg g 21 ) of fumonisins, whilst isolate IBT 9486 can be considered as an intermediate producer (50–500 mg g 21 ). In the light of the present studies and previous ones conducted by earlier researchers, this high percentage of fumonisin producing strains in F. verticillioides may be regarded as a general characteristic of this species. The proportion of FB 1 , FB 2 , and FB 3 produced in the inoculation studies expressed as percentages of the total fumonisin concentrations (Table 5) fell within the ranges 56.9–79.6% for FB 1 , 10.2–35.1% for FB 2 , and 1.9–20.2% for FB 3 . These data agree with those obtained for maize samples from Ghana (Table 3) as well as data for Argentinian maize samples reported by Sydenham et al. (1993). In these studies, extremely high levels of fumonisins were produced by most F. verticillioides isolates. The highest yet recorded FB 1 level is 8364 mg g 21 produced by isolate IBT 9488. Similarly, Sydenham et al. (1993) had earlier reported the highest level to be 8160 mg g 21 for FB 1 produced by a strain of F. verticillioides isolated from Argentinian maize. Other levels recorded from their study were found to be lower. Recent studies by Visconti

and Doko (1994) and Castella´ et al. (1996) showed lower fumonisin production for F. verticillioides isolates from maize and mixed poultry feeds. Results from the present study showed that none of the four tested isolates of F. semitectum produced fumonisins, likewise for one strain of F. graminearum tested. From the literature there is no report of these two species producing fumonisins which tallies with our findings although only a limited number was tested. F. graminearum is, however, associated with the production of other mycotoxins namely zearalenone and trichothecenes. Earlier studies (Kpodo et al., 1996) showed the absence of zearalenone in Ghanaian maize and maize products. No studies on tricothecenes in Ghanaian maize are available. One of four isolates of F. equiseti produced FB 1 in small or insignificant amounts (2 mg g 21 ) whilst one isolate of F. oxysporum produced all three types of fumonisins. Total fumonisins produced by this isolate was 14 162 mg g 21 (Table 5). Earlier studies (Thiel et al., 1991b) have shown that fumonisin production was restricted to the section Liseola which contains F. moniliforme, F. proliferatum, F. subglutinans, F. anthophilum, F. annulatum, and F. succisae (Nelson et al., 1983). Recent reports have, however, showed new Fusarium species with similar characteristics as those in the section Liseola except that they are able to form chlamydospores. These are F. dlamini, F. nygamai, F. napiforme, and F. beomiforme. Strains from these species except F. beomiforme have also been found to produce fumonisins (Nelson et al., 1992) likewise the production of fumonisin B 1 by Alternaria alternata (Chen et al., 1992). F. oxysporum belongs to the section Elegans. The fact that only one strain was tested suggests that this finding should be viewed with caution till further confirmatory tests are conducted.

Acknowledgements The authors express their appreciation to the Danish International Development Assistance (DANIDA) for funding this study and the Programme on Mycotoxins and Environmental Carcinogenesis, South African Medical Research Council for providing fumonisin standards used in this work.

K. Kpodo et al. / International Journal of Food Microbiology 61 (2000) 147 – 157

155

Table 5 Production of fumonisins by cultures of Fusarium species isolated from Ghanaian maize Isolate and Fusarium spp. Control

c

F. verticilloides IBT 9485 IBT 9486 IBT 9487 IBT 9488 IBT 9489 IBT 9490 IBT 9491 IBT 9492 IBT 9493 IBT 9494 IBT 9495 IBT 9496 IBT 9497 IBT 9498 IBT 9515 IBT 9499 IBT 9500 IBT 9501 IBT 9502 IBT 9503 IBT 9504 IBT 9505 F. semitectum IBT 9506 IBT 9507 IBT 9508 IBT 9509 F. equiseti IBT 9510 IBT 9511 IBT 9512 IBT 9513 F. oxysporum IBT 9514 F. graminearum IBT 9516 a

Fumonisin concentration (mg g FB 1

21 a

Fumonisin (%)a

)

FB 2

FB 3

Total

b

FB 1

FB 2

FB 3

58.4 70.1 74.2 75.7 66.9 73.4 56.9 70.0 68.6 66.5 75.9 100.0 71.4 70.7 68.2 73.3 79.6 69.4 70.3 71.3 68.6 70.4

35.1 10.2 17.8 15.9 12.9 24.7 24.0 16.8 14.1 14.0 12.7 0 18.1 19.0 12.9 18.5 11.4 16.4 19.1 19.2 21.0 11.8

6.5 19.7 8.0 8.4 20.2 1.9 19.1 13.2 17.3 19.5 11.4 0 10.5 10.3 18.9 8.2 9.0 14.2 10.6 9.5 10.4 17.8

nd nd nd nd

– – – –

– – – –

– – – –

nd nd nd nd

nd nd nd 2

– – – 100.0

– – – –

– – – –

4255

14 162

47.2

22.8

30.0

–

–

d

nd

nd

2751 89 7278 8364 3412 1625 1354 3978 3995 5765 1004 2 3301 1087 2740 1965 7380 2292 7086 1526 4705 533

1655 13 1744 1761 656 547 571 955 819 1213 168 nd 837 293 520 495 1062 541 1928 411 1446 89

306 25 783 927 1031 42 454 749 1009 1691 151 nd 487 158 760 219 835 469 1065 204 712 135

4712 127 9805 11 052 5099 2214 2379 5682 5823 8669 1323 2 4625 1538 4020 2679 9277 3302 10 079 2141 6863 757

nd e nd nd nd

nd nd nd nd

nd nd nd nd

nd nd nd 2

nd nd nd nd

6677

3230

nd

nd

nd 21

nd 21

nd 21

Detection limits: FB 1 5 0.6 mg g , FB 2 5 1.4 mg g , and FB 3 5 0.7 mg g . Total combined FB 1 1 FB 2 1 FB 3 levels determined for each isolate. c Control, maize used for the preparation of the cultures. d nd 5 None detected in control maize ( , 50 mg kg 21 ); none detected in culture material. e nd 5 None detected in culture material.

b

–

156

K. Kpodo et al. / International Journal of Food Microbiology 61 (2000) 147 – 157

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