1. 1. Mycotoxins 63 (1), 27-38 (2013) www.jstage.jst.go.jp/browse/myco Occurrence, mycotoxins and toxicity of Fusarium species from Abelmoschus esculentus and Sesamum indicum seeds Chibundu N. EZEKIEL 1,* , Cyril C. NWANGBURUKA 2 , Gibson O. CHIOMA 2 , Michael SULYOK 3 , Benedikt WARTH 3 , Clement G. AFOLABI 4 , Adenike A. OSIBERU 1 , Michael O. OLADIMEJI 1 , Olarenwaju A. DENTON 2 , Grace O. TAYO 2 , Rudolf KRSKA 3 1 Mycology/Mycotoxicology Research Unit, Department of Biosciences and Biotechnology, Babcock University, Ilishan Remo, Ogun State. Nigeria 2 Department of Agriculture and Industrial Technology, Babcock University, Ilishan Remo, Ogun State. Nigeria 3 Center for Analytical Chemistry, Department of Agrobiotechnology (IFA-Tulln), University of Natural Resources and Life Sciences Vienna, Konrad Lorenzstr. 20, A-3430 Tulln, Austria 4 Department of Crop Protection, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria Keywords: equisetin; fumonisins; Fusarium; moniliformin; mycotoxins; okra; sesame; zearalenone (Received September 30, 2012; Accepted February 8, 2013) Abstract Seeds of two okra accessions and 17 samples of sesame seeds were examined for contamination by Fusarium species. Altogether, 37 Fusarium isolates were obtained from the two crops: 6 isolates from two okra accessions and 31 isolates from 10 sesame samples. The isolates belonged to three species: F. oxysporum, F. semitectum and F. verti- cillioides. All isolates from okra were identified as F. semitectum while the three species of Fusarium occurred in sesame. Six randomly selected isolates from the two crops were screened for their ability to produce mycotoxins in ofada rice culture and the crude extracts of the mycotoxins were tested on week-old Clarias gariepinus fingerlings. Six metabolites were produced by the isolates in ofada rice: equisetin (EQUS), fumonisin B1 (FB1), FB2, methyl-equisetin (M-EQUS), moniliformin (MON) and zearalenone (ZEA). All isolates produced EQUS at concentrations ranging 454-29,983 g/kg. All isolates except F. semitectum BUFC 041 and F. oxysporum BUFC 024 produced MON and ZEA, while three F. semitectum isolates produced M-EQUS. Only F. verticillioides isolates produced fumonisins. There was evidence of variation within species obtained from both crops based on toxin profile and level of toxin produced. The culture extracts of all isolates induced 100% lethality to C. gariepinus fingerlings except for extracts of F. semitectum BUFC 041 which killed only 62.2% of the fingerlings, possibly due to the absence of MON. Our data suggest that okra and sesame may be potential sources of toxigenic Fusarium. Introduction Okra (Abelmoschus esculentus), a major tropical vegetable, is directly sown and exported outside its cultivation region after harvest. The annual global production of okra fruit is estimated at 6,000,000 tonnes, Research Paper 27 Corresponding Author Mycology/Mycotoxicology Research Unit, Department of Biosciences and Biotechnology, Babcock University, Ilishan Remo, Ogun State. Nigeria. Tel.: 234-7038167130. E-mail: chaugez@gmail.com A full color PDF reprint of this article is available at the journal WEB site.
2. 2. and countries in West and Central Africa and the Middle East are noted to contribute a high quota to okra production and consumption1, 2) . In particular, Nigeria ranks second to India as the highest producers of okra globally2) . Okra is edible in different forms which include the leaves, fruit and seeds. The fruit serves as soup thickeners while the seeds could be dried as a nutritious material for vegetable curds, or roasted and ground for use as coffee additive or substitute3, 4) . Okra contains carbohydrate, proteins and vitamin C in large quantities5) . The amino acids in okra fruits and oil (60-70% unsaturated fatty acids) contained in the seeds are comparable to amino acids and oils in soybean. The oil has potential to reduce cholesterol in the human body while the seed flour could be used to fortify cereal flour1) . It is assumed that the numerous benefits derived from okra may be threatened due to Fusarium attack on this crop which has been observed in agricultural fields in Nigeria; this has recently led researchers to investigate this observation. Consequently, there are few data on fungal contamination of okra and among the few available reports in Nigeria6, 7) ; none considered the toxigenicity of Fusarium species though Fusarium species were found to be the most predominant species in okra seeds. Sesame (Sesame indicum L.) is an oil rich seed grown in many parts of the world especially within the dry tropics between latitudes of 40 N and S. In 2007, Nigeria ranked among the five largest producers of sesame; producing seeds for oil which is used in local and international delicacies as well as livestock feed2, 8) . Sesame is exported outside Nigeria due to its oil-rich quality. Nutritionally, sesame is also rich in amino acids, vitamins and other essential nutrients; this makes the crop susceptible to colonization by microorganisms especially fungi. During colonization, the fungi utilize the nutrients in the seeds and this leads to seed deterio- ration. In the process, toxic substances known as mycotoxins may be produced9) . For example, up to 28 mycotoxins and other less important fungal metabolites have been detected in sesame seeds in Nigeria10) . The examples of fungal species that have been reported to attack sesame especially on field causing huge yield losses include Cercospora sesame, Fusarium spp., Pythium spp. and Sclerotium spp9, 11, 12, 13) . However, no report is available on the toxigenicity of Fusarium spp. in sesame in Nigeria. Mycotoxins produced by Fusarium species include: butenolide, equisetin (EQUS), enniatins, fusarins, beauvericin, fumonisins, fusaproliferin, moniliformin (MON), trichothecenes and zearalenone derivatives14,15) . These toxins occur as important contaminants, both alone and in mixture, in food and feed. Several studies have been carried out on Fusarium toxins to assess their toxicity using brine shrimps or catfish finger- lings16, 17, 18, 19) . However, there is limited information on the occurrence and toxigenicity of Fusarium spp. and Fusarium toxins that invade and colonize okra globally and to a lesser extent, sesame in Nigeria. Since both crops are widely consumed in Nigeria and there is the constant need to assess the mycotoxicological risk associated with such regularly utilized crops by determining the diversity and toxigenicity profile of isolates obtained from these crops, we embarked on this study. Knowledge of the toxigenic fungal profile in these crops may be useful in searching for better avenues towards the reduction of pre-harvest and post-harvest spoilage which tend to threaten food safety especially in tropical countries. Therefore, this research aimed at determining the presence of Fusarium in okra and sesame seeds, evaluating the isolates for their potential to produce mycotoxins in rice culture and testing the culture extracts of the isolates for toxicity to Clarias garie- pinus fingerlings. 28EZEKIEL et al. Mycotoxins
3. 3. Materials and Methods SamplesSeeds (500 g each) of two okra accessions (BAB 002 and BAB 003) were obtained from the germplasm of the Department of Agriculture and Industrial Technology, Babcock University, Nigeria, while 17 bulk samples (1.8-2 kg each) of sesame seeds were collected from four markets in Plateau State, Nigeria as described by Ezekiel et al10 ) . Both crops were collected between April and May 2011. The markets are situated in B/Ladi, Bokkos, Mekera and Vwang. About 125 g representative of each sesame sample was randomly obtained by quartering. The okra and sesame samples were stored at 4 C to prevent further accumulation of microbes until analysis. Isolation and characterization of Fusarium species in the seedsSeed belonging to the two crops (A. esculentus and S. indicum) were surface sterilized for 30 s in separate vials containing 2 % NaOCl. The sterilized seeds were rinsed twice with distilled water and blotted dry in between two folds of sterile Whatman #1 filter paper. Five seeds of each crop were plated per Petri dish of peptone-pentachloronitrobenzene agar (PPA), a semi selective medium for Fusarium20) . Triplicates of each PPA plate per crop were set up. Inocu- lated plates were incubated for 5 days under fluorescent lights on a 12 h day/ night schedule at 22-24 C. Fusarium species were transferred to a fresh PPA and incubated for 7 days under the same condition. A single spore of each isolate was manipulated, and incubated overnight on a water agar plate at 22-24 C. After germination, the fungus was maintained on a modified Czapek Dox complete medium (CM) and stored at 4 C until identification. Identification of Fusarium isolates was performed based on morphological characteristics using carnation leaf agar (CLA for examination of sporodochia and uniform macro conidia under the Olympus BX51 Digital Microscopy, Olympus Optical Co., LTD, Japan) and potato dextrose agar (PDA, for evaluation of pigmentation and colony morphology). Fusarium species were identified according to the taxonomic criterion of Leslie and Summerell21) . Fusarium verticillioides ATCC MYA 836 strain22) was used as reference for the identification. Fusarium isolates identified were cultured on CM slants in a 4 ml vial, and stored at 4 C. In vitro toxin production by Fusarium species on ofada riceAbout 5 kg of uncooked ofada rice (locally produced rice) was purchased from a fast-food company in Ibadan, Nigeria. Six randomly selected Fusarium isolates were grown on 100 g of autoclaved ofada rice in 500 ml Erlenmeyer flask to determine the toxigenic potentials of each isolate. The rice in each flask was inoculated with 2 ml of 1106 conidia per ml of corre- sponding isolate after moisture equilibration to 45 %19) . Duplicate flasks per isolate were inoculated. A control flask of autoclaved ofada rice without inoculation was set up. The flasks were incubated for 4 weeks at 25 C in the dark. Then, the rice was dried in a hot air oven at 55 C until a constant dry weight was achieved. The dried rice in each flask was ground using a Waring blender. A 20 g portion of the rice powder was transferred to a 50 ml polypropylene tube and sent to the University of Natural Resources and Life Sciences, Austria for multi-mycotoxin analysis. The remaining rice powder samples were extracted with acetonitrile/water/acetic acid (79 : 20 : 1, v/v/v)23, 24) . The extracts obtained were used for the bioassay at Babcock University, Nigeria. 29Vol. 63, No. 1, 27-38 (2013)
4. 4. Analysis of Fusarium toxins on ofada riceAll analytical samples were examined for Fusarium metabo- lites by LC-MS/MS according to Sulyok et al 24 ) . and Vishwanath et al25) . The analytical method has been extended to cover 320 metabolites, transferred to a more sensitive mass spectrometer and pre-validated for four matrices (Malachova et al., unpublished data). In brief, 5 g of sample was weighed into a 50 ml polypro- pylene tube (Sarstedt, Germany) and extracted with 20 ml acetonitrile/water/acetic acid (79 : 20 : 1, v/v/v) for 90 min on a GFL 3017 rotary shaker (GFL, Burgwedel, Germany). Extracts were diluted in a ratio of 11 in dilution solvent (acetonitrile/water/acetic acid 20 : 79 : 1 , v/v/v) and directly injected into the LC-MS/MS instrument. Chromatographic separation was performed at 25 C on a Gemini C18-column, 1504.6 mm i.d., 5 m particle size, equipped with a C18 43 mm i.d. security guard cartridge (all from Phenomenex, CA, US) and coupled to an 1290 Series HPLC System (Agilent, Waldbronn, Germany). After an initial time of 2 mins at 100 % eluent A, the proportion of eluent B was increased linearly to 50 % within 2-5 mins and 100 % within 5-14 mins, followed by a holding-time of 4 mins at 100% eluent B and 2.5 mins column re-equilibration at 100 % eluent A pumped at a flow rate of 1 ml/min. A QTrap 5500 LC-MS/MS System (Applied Biosystems, CA, US) equipped with a TurboIonSpray electrospray ionization (ESI) source was used to detect and quantify the fungal metabolites. ESI-MS/MS was performed in the scheduled multiple reaction monitoring (sMRM) mode both in positive and negative polarities in two separate chromatographic runs per sample by scanning two fragmentation reactions per analyte with the following settings: source temperature 550 C; curtain gas 30 psi; ion source gas 1 (sheath gas) 80 psi, ion source gas 2 (drying gas) 80 psi, ion spray voltage 4500 V and5500 V respectively, collision gas (nitrogen) medium. The MRM detection windows were 54 and 96 secs in the positive and negative ionization mode, respectively and the cycle time was set to one sec. Mycotoxins were quantified by external calibration (1/x weighted) using a multi-component standard prepared from authentic standards and later adjusted for apparent recoveries. For the determination of the apparent recovery, sub-samples (0.25g each) of the control (autoclaved but uninoculated rice) were spiked prior to extraction at one concentration level and analyzed as described above. Limits of detection (LOD) were estimated from calibration standards at low concentration levels based on the signal-to-noise ratio (S/ N=3 : 1). Toxicity assay of Fusarium culture materialsThe lethal effects of the Fusarium culture materials were determined using week-old larvae of freshwater African catfish, C. gariepinus, otherwise called fingerlings as previously illustrated by Ezekiel et al26) . for aflatoxins and ochratoxin A. Clarias gariepinus was used instead of brine shrimp (Artemia salina L.) larvae16) because it is the commonly used species for bioassay in sub-Saharan Africa. In addition, Gbore et al18) . determined the effect of fumonisin B1 (FB1) on hematological parameters of C. gariepinus. Briefly, 1000 actively motile fingerlings were purchased from the Fishery Section of May-Flower School, Ikenne, Ogun State, Nigeria and maintained in purified oxygen bags filled with freshwater. Prior to exposure and before acclimatization, the fingerlings were sorted randomly into groups of 45, which consisted of triplicate sub-groups of 15 fingerlings. The fingerlings in each group were exposed to the fungal culture extracts after being acclimatized for 12h. The extract concentrations tested on the fingerlings were 0.25g/ml, 0.5g/ml and 1 g/ml. The number of dead fingerlings per treatment was recorded after 24 h and this was deter- mined by loss of locomotion. Two controls were set up in same manner only that no crude extract was added. 30EZEKIEL et al. Mycotoxins
5. 5. Instead, the extraction solvent was added to a set of control to determine if the lethality data obtained was due to the toxins or extraction solvent. The other set received no treatment. Data from this experiment were reported as means of triplicate observations/concentrations. Data analysisAnalysis of all data was performed using SPSS version 15.0. Mean and standard error of the lethality data was calculated. Results and Discussion Fusarium species are important plant pathogens that colonize crops on field including grains, seeds and vegetables. This represents a high risk of mycotoxicoses in pre-harvested or freshly harvested crops14 ) . The seeds of the two okra accessions and 58.8 % (10 out of 17) of sesame were contaminated with Fusarium species. A total of 37 Fusarium isolates were obtained from the okra (6 isolates) and sesame samples (31 isolates), and they belonged to three species: F. oxysporum, F. semitectum and F. verticillioides. All isolates obtained in this study were characterized based on morphological features and some distinguishing character- istics are given below. Isolates of the three Fusarium species produced macroconidia of different sizes. F. semitectum did not produce microconidia but produced macroconidia with slightly curved and tapered apical cell and foot shaped basal cell in contrast to macroconidia with curved apical cell and notch or foot shaped basal cell that were produced by Fusarium oxysporum and F. verticillioides. Fusarium oxysporum and F. semitectum isolates produced smooth and rough chlamydospores respectively, while F. verticillioides produced chains of microconidia but no chlamydospores (Fig1). The characteristic rabbit-ear macroconidia was also observed in all isolates of F. semitectum. The pigmentation on PDA plates was brown and violet for F. semitectum, and F. oxysporum and F. verticillioides. The isolates obtained from okra were identified as F. semitectum while the three species of Fusarium were obtained from sesame samples (Table 1). F. semitectum had the highest occurrence (38.7 %) in the sesame samples while F. verticillioides had the least occurrence (25.8%). On the overall, F. semitectum repre- sented 48.6% of all fusaria obtained from the two crops while F. oxysporum and F. verticillioides represented 29.7% and 21.6 respectively. A wide range of vegetables including okra, tomato, pepper and cucurbits have been reported to harbor Fusarium species6, 27, 28, 29) . Therefore, the recovery of isolates belonging to only F. semitectum from okra seeds in this study is in line with those reports. However, there may be a limitation in the use of only morphological parameters for the characterization of the Fusarium isolates which we obtained in this study; the possible reason previous studies6, 29) that detected Fusarium in okra considered the isolates as Fusarium spp. Fusarium semitectum has been associated with bovine pulmonary emphysema30) . On the other hand, not much has been documented for Fusarium contamination in sesame. In this study, three species were identified from nearly 60% of the sesame seeds; this indicates the relative susceptibility of sesame seeds to Fusarium contamination9, 11) . Data obtained from the LC-MS/MS analysis of extracts of the six cultured Fusarium isolates showed that six metabolites were produced on ofada rice grains (Fig. 2 and Table 2). The six metabolites and their respective LODs and recoveries (%) are given: EQUS (0.09, 108.1%), FB1 (6.01, 83.2%), FB2 (6.09, 82.2%), methyl-equisetin (M-EQUS, 0.08, 100.5%), MON (0.69, 115.3%) and zearalenone (ZEA, 0.19, 103.1%). Fusarium semitectum isolates obtained from okra produced four of the metabolites in the rice cultures (EQUS, 31Vol. 63, No. 1, 27-38 (2013)
6. 6. Table 1.Incidence of Fusarium species in A. esculentus and S. indicum seeds from Nigeria Seed Na Nb Nc Occurrence of Fusarium species (%) F. semitectum F. oxysporum F. verticillioides Okra 2 2 6 6/6 (100.0) Sesame 17 10 31 12/31 ( 38.7) 11/31 (35.5) 8/31 (25.8) Total 19 12 37 18/37 ( 48.6) 11/37 (29.7) 8/37 (21.6) a Number of samples analyzed. b Number of samples positive for the presence of Fusarium species. b Number of isolates obtained. Fig. 1.Chlamydospore and conidial characters of F. semitectum (a and b), F. oxysporum (c and d) and F. verticillioides (e and f) isolated from A. esculentus and S. indicum seeds. a, b, d and e = macroconidia; b = characteristic Rabbit-ear macroco- nidia of F. semitectum; c = chlamydospore; f = characteristic chains of microconidia of F. verticillioides. a b c d e f 32EZEKIEL et al. Mycotoxins
7. 7. Table 2.Toxin profile and toxicity of the culture extracts of Fusarium species isolated from seeds of A. esculentus and S. indicum in Nigeria Fusarium species and strains Source Toxin profilea and concentration (g/kg) Mean SE Mortality (%)EQUS FB1 FB2 M-EQUS MON ZEA F. oxysporum BUFC 024 Sesame 911 0.5 100.00.00 F. semitectum BUFC 067 Okra 452 0.5 0.3 100.00.00 BUFC 059 Okra 29,983 92 0.7 0.5 100.00.00 BUFC 031 Sesame 25,245 34 4.0 0.3 100.00.00 BUFC 041 Sesame 28,280 77 0.1 62.21.73 F. verticillioides BUFC 021 Sesame 2,147 20 2 1.7 0.3 100.00.00 Blankb 3 LOD LOD LOD LOD LOD a EQUS = equisetin, FB1= fumonisin B1, FB2= fumonisin B2, M-EQUS= methyl-equisetin, MON= moniliformin, ZEA = zeara- lenone. b Un-inoculated rice incubated for 4 weeks. Fig. 2.Chromatograms of six metabolites produced in ofada rice culture of Fusarium species. AD, metabolites from F. semitectum BUFC 059; E and F, metabolites from F. verticillioides BUFC 021. A = equisetin, B = methylequisetin, C = moniliformin, D = zearalenone, E = fumonisin B1, F = fumonisin B2. For better visibility, only the quantifier ions are displayed. 33Vol. 63, No. 1, 27-38 (2013)
8. 8. M-EQUS, MON and ZEA) while the six metabolites were produced by the three Fusarium species isolated from sesame: F. oxysporum, F. semitectum and F. verticillioides (Table 2 ). The ranges of the metabolites produced by all Fusarium isolates were 452-29, Please dose up the space in the figure/number (29,983 is correct and not 29,983) 983 g/kg (EQUS), 20 g/kg (FB1), 2 g/kg (FB2), 34 - 92 g/kg (M-EQUS), 0.5-4 g/kg (MON) and 0.1-0.5 g/kg (ZEA). F. oxysporum is the most widely dispersed of the Fusarium species and its strains are generally viewed as non-toxigenic although they often can synthesize various polyketide secondary metabolites with unknown functions. Some strains of F. oxysporum have been reported to produce fumonisins, MON, ZEA and other mycotoxins21) . The F. oxysporum isolate (BUFC 024) obtained from sesame seeds in Plateau State, produced only EQUS and MON. The absence of fumonisin production by F. oxysporum (BUFC 024) affirms variation in strains of F. oxysporum. F. semitectum is commonly found in the soil and diverse aerial plant parts in tropical and sub-tropical areas. All the F. semitectum isolates obtained from the two crops exhibited similar morpho-characters on standardized media21 ) . However, the two cultured isolates from each crop produced varying toxins and concentration levels of the toxins on rice. F. semitectum is known to produce apicidin, beauvericin, EQUS, fusapyrone, MON, sambutoxin, trichothecenes and ZEA21, 31) . Only EQUS, M-EQUS, MON and ZEA were produced by the four isolates of F. semitectum as confirmed by the LC-MS/MS protocol used in this study. However, one of the isolates from okra (BUFC 067) and sesame (BUFC 041) did not produce M-EQUS and MON respectively. In addition, F. semitectum BUFC 067 produced 98.5 % less EQUS than F. semitectum BUFC 059, while F. semitectum BUFC 031 from sesame produced 10.7 % and 55.8 % less EQUS and M-EQUS respectively, than F. semitectum BUFC 041. These variations in type and concentration of metab- olite produced by each set of the two tested F. semitectum isolates from the two crops lead us to suggest that this study identified four F. semitectum strains in okra and sesame. This corroborates previous reports of Vogelgsang et al32) . that variation in Fusarium strains as well as type of cereal substrate on which the strains are grown may influence the type and levels of mycotoxin production by the isolates. In the present report, each set of the two tested F. semitectum isolates were obtained from the same crop/host and location, and were grown on ofada rice from the same source and incubated under similar conditions. Therefore, molecular studies on the four isolates may be helpful in resolving the assumption since the sole use of morphological features in characterizing Fusarium species is a limitation of this study. This paper reports for the first time M-EQUS production by F. semitectum in culture. Fusarium verticillioides, previously referred to as F. moniliforme, produces fumonisins21, 33) . Some strains are capable of producing very high concentrations of this toxic metabolite while other strains especially when grown on rice produce MON at trace level21) . The findings from the present study showed that F. verticilli- oides BUFC 021 obtained from sesame and grown on rice produced low concentrations of fumonisins and traces of MON. This is in line with the above reports. However, contrary to this report and that of Nelson et al34 ) . is the fact that the F. verticillioides BUFC 021 isolate we obtained from sesame in Nigeria produced additional mycotoxins (EQUS and ZEA) in rice as confirmed by LC-MS/MS. In fact, Nelson et al. 34) discounted ZEA production by F. verticillioides. This may then imply that some F. verticillioides strains are capable of producing additional mycotoxins including EQUS which was produced at a much higher concen- tration than fumonisins. We recommend further investigation into this observation, especially using strains obtained from diverse crops and geographical locations including sub-tropical Africa. 34EZEKIEL et al. Mycotoxins
9. 9. The mean temperature (24.5-27.0 C) and pH (6.0-6.5) values of the water used in raising the fingerlings were within the recommended ranges for catfish production35) . At 24 hours, the three concentrations of crude extracts from all the six isolates that were tested showed 100% mortality towards the fingerlings except for F. semitectum BUFC 041. The three concentrations (0.25 g/ml, 0.5 g/ml and 1 g/ml) of the culture extracts of F. semitectum BUFC 041 killed 62.2%, 60.0% and 64.4% of the fingerlings respectively (data not shown), with an overall mean percentage mortality of 62.2% (28/45) (Table 2). The decrease in mortality of fingerlings shown by extracts from F. semitectum BUFC 041 as compared to extracts from other isolates may be due to the absence of MON in the extract of F. semitectum BUFC 041. MON has been previously reported to exert relatively high toxic effects in brine shrimp larvae and day old quails in single administration17, 36) . In addition, co-occurrence of mycotoxins is are known to influence toxicity of the mycotoxins involved37) . Pedrosa and Borutova38) also reported that the administration of two or more mycotoxins could result in synergistic effects. The presence of MON in the extracts of other isolates which showed 100% mortality may therefore be justified for synergism. Furthermore, Sharma et al36) . reported synergistic effects for FB1 and MON, stating that FB1 and MON had less toxicity when administered alone while their combination was more toxic. This may be the reason for the 100% lethality observed in extracts from F. verticillioides BUFC 021. Gbore et al18) . also reported the toxic effects of FB1 to adult C. gariepinus. Conclusion This paper has revealed the possible contamination of okra and sesame by toxigenic Fusarium. This may pose a threat to animals including birds that pick up the okra seeds and humans who consume sesame and its products. The toxigenic Fusarium in the okra seeds may also be transferred to the edible fruits thus creating a higher risk situation for a wider range of consumers. Good agricultural practices such as crop rotation, weed control and post-harvest handling practices (e.g. sorting, cleaning, proper drying and storage) should therefore be encouraged to prevent the crops from contamination prior to harvesting and thereafter39, 40) . Acknowledgements The permission to use laboratory facilities and technical support received from Pathology/Mycotoxin laboratory of the International Institute of Tropical Agriculture, Nigeria is hereby acknowledged. Conict of Interest Authors hereby declare no conflict of interest. References Kumar, S., Dagnoko, S., Haougui, A., Ratnadass, A., Pasternak, D., Kouame, C.: Okra (Abelmoschus spp.) in West and Central Africa: Potential and progress on its improvement, Review. Afr J Agric Res, 5, 3590-3598 (2010) FAOSTAT.: Food and Agriculture Organization of the United Nations for 2011, (2011) 35Vol. 63, No. 1, 27-38 (2013)
10. 10. Schippers, R.R.: African indigenous vegetable an overview of the cultivated species, (2000), National Resources Institute (NRI), University of Greenwich, London Rao, P.S., Rao, P.U., Serikeran, B.: Serum cholesterol, triglycerides and total total fatty acid of rates in response to okra (Hibiscus esculentus) seed oil. J Am Oil Chem Ass, 68, 433 (2001) Adeboye, O.C., Oputa, C.O.: Effects of galex on growth and fruit nutrient composition of okra (Abelmoschus esculentus). Ife J Agric, 18, 1-9 (1996) Nwangburuka, C.C., Oyekale, K., Ezekiel, C.N., Anokwuru, P.C., Badaru, O.: Effect of Moringa oleifera leaf extract and sodium hypochlorite on seedling growth rate and fungal abundance in two accessions of Abelmoschus esculentus (L) Monech. Arch Appl Sci Res, 4, 875-881 (2012) Nwangburuka, C.C., Ezekiel, C.N., Oyekale, K., Anokwuru, C.P., Evbuomwan, I.K.: Cytomorphological and antifungal analysis of Acalypha wilkesiana and Moringa oleifera extracts, and sodium hypochlorite on Abelmoschus esculentus L. Moench. treated seeds. Nature Sci, 11, 31-39 (2013) Alegbejo, M.D., Iwo, G.A., Abo, M.E., Idowu, A.A.: Sesame: A potential industrial and export oilseed crop in Nigeria. J Sust Agric, 23, 59-76 (2003) Li, D-H., Wang, L-H., Zhang, Y-X., LV, H-X., QI, X-G., Wei, W-L., Zhang, X-R.: Pathogenic variation and molecular characterization of Fusarium species isolated from wilted sesame in China. Afr J Microbiol Res, 6, 149-154 (2012) 10Ezekiel, C.N., Sulyok, M., Warth, B., Krska, R.: Multi-microbial metabolites in fonio millet (acha) and sesame seeds in Plateau State, Nigeria. Eur Food Res Technol, 235, 285-293 (2012) 11Jonsyn, F.E.: Seedborne mould fungi of sesame (Sesamum indicum) in Sierra Leone and their potential aflatoxin/mycotoxin production. Mycopathol, 104, 123-127 (1988) 12Enikuomehin, O.A.: Cercospora leaf spot disease management in sesame (Sesamum indicum L.) with plant extracts. J Trop Agric, 43, 19-23 (2005) 13Afolagboye, F.M.: Seed-borne fungi of sesame (Sesamum indicum L.) and their control with plant extracts, (2011), Abstract of M.Sc. Dissertation. Federal University of Agriculture, Abeokuta,http://www. unaab.edu.ng/journal/index.php/theses/thesis/view/765 (Date accessed: 24/7/2012) 14Logrieco, A., Bottalico, A., Mul, G., Moretti, A., Perrone, G.: Epidemiology of toxigenic fungi and their associated mycotoxins for some Mediterranean crops. Euro. J Plant Pathol, 109, 645-667 (2003) 15Desjardins, A.E., Proctor, R.H.: Molecular biology of Fusarium mycotoxins. Int J Food Microbiol, 119, 47-50 (2007) 16Logrieco, A., Moretti, A., Fornelli, F., Fogliano, F., Ritieni, A., Caiaffa, M.F., Randazzo, G., Bottalico, A., Macchia, L.: Fusaproliferin production by Fusarium subglutinans and its toxicity to Artemia salina, SF-9 insect cells, and IARC/LCL 171 human B lymphocytes. Appl Environ Microbiol, 62, 3378-3384 (1996) 17Jimenez, M., Huerta, T., Mateo, R.: Mycotoxin production by Fusarium species isolated from bananas. Appl Environ Microbiol, 63, 364-369 (1997) 18Gbore, F.A., Adewole, A.M., Oginni, O., Oguntolu, M.F., Bada, A.M., Akele, O.: Growth performance, haematology and serum biochemistry of African catfish (Clarias gariepinus) fingerlings fed graded levels of dietary fumonisin B1. Mycotox Res, 26, 221-227 (2010) 19Moretti, A., Ferracane, L., Somma, S., Ricci, V., Mul, G., Susca, A., Ritieni, A., Logrieco, A.F.: Identi- fication, mycotoxin risk and pathogenicity of Fusarium species associated with fig endosepsis in Apulia, 36EZEKIEL et al. Mycotoxins
11. 11. Italy. Food Addit Contam A., 27, 718-728 (2010) 20Nash, S.M., Snyder, W.C.: Quantitative and estimations by plate counts of propagules of the bean rot Fusarium in field soils. Phytopathology, 73, 458-462 (1962) 21Leslie, J.F., Summerell, B.A.: The Fusarium Laboratory Manual, (2006), Iowa State University Press, Iowa 22Afolabi, C.G., Ojiambo, P.S., Ekpo, E.J.A., Menkir, A., Bandyopadhyay, R.: Evaluation of maize inbred lines for resistance to Fusarium ear rot and fumonisin accumulation in grain in Tropical Africa. Plant Dis, 91, 279-286 (2007) 23Sulyok, M., Berthiller, F., Krska, R., Schuhmacher, R.: Development and validation of a liquid chroma- tography/tandem mass spectrometric method for the determination of 39 mycotoxins in wheat and maize. Rapid Commun Mass Spectrom, 20, 2649-2659 (2006) 24Sulyok, M., Krska, R., Schuhmacher, R.: A liquid chromatography/tandem mass spectrometric multi- mycotoxin method for quantification of 87 analytes and its application to semi-quantitative screening of mouldy food samples. Anal Bioanal Chem, 389, 1505-1523 (2007) 25Vishwanath, V., Sulyok, M., Labuda, R., Bicker, W., Krska R.: Simultaneous determination of 186 fungal and bacterial metabolites in indoor matrices by liquid chromatography/tandem mass spectrometry. Anal Bioanal Chem, 395, 1355-1372 (2009) 26Ezekiel, C.N., Odebode, A.C., Fapohunda, S.O., Tayo, G.O., Olawuyi, O.J., Olaoye, O.B., Olarinmoye, A.O., Adeyemi, O.O.: Toxigenic potential of co-occurring aflatoxin and ochratoxin A detected in poultry feed on Clarias gariepinus larvae. Nature Sci, 9, 186-192 (2011) 27Chehri, K.: Occurrence of Fusarium species associated with economically important agricultural crops in Iran. Afr J Microbiol Res, 5, 4043-4048 (2011) 28Chehri, K., Salleh, B., Yli-Mattila, T., Reddy, K.R.N., Abbasi, S.: Molecular characterization of patho- genic Fusarium species in cucurbit plant from Kermanshah province, Iran Saudi J Biol Sci, 18, 341-351 (2011) 29Kobina, M.J., Ebenezer, O.: Fruit borne mycoflora of Capsicum annum L (pepper), Abelmoschus escul- entus L. Moench (okra), and Lycopersicon esculentum Mill. (tomato) from Accra metropolis. Afr J Food Sci, 6, 1-7 (2012) 30Linnabary, R.D., Tarrier, M.P.: Acute bovine pulmonary emphysema caused by the fungus Fusarium semitectum. Vet. Human Toxicol, 30, 255-256 (1988) 31Rabie, C.J., Marasas, W.F.O., Thiel, P.G., Lbben, A., Vleggaar, R.: Moniliformin production and toxicity of different Fusarium species from Southern Africa. Appl Environ Microbiol, 43, 517-521 (1982) 32Vogelgsang, S., Sulyok, M., Bnziger, I., Krska, R., Schuhmacher, R., Hans-Rudolf, F.: Effect of fungal strain and cereal substrate on in vitro mycotoxin production by Fusarium poae and Fusarium avenaceum. Food Addit Contam A, 25, 745-757 (2008) 33Neish, G.A., Farnworth, E.R., Greenhalgh, R., Young, J.C.: Observations on the occurrence of Fusarium species and their toxins in corn in eastern Ontario. Can J Plant Pathol, 5, 11 (1983) 34Nelson, P.E., Toussoun, T.A., Marasas, W.F.O.: Fusarium species. An illustrated Manual for Identifi- cation, (1983), The Pennsylvania State Press, University Park, PA 35Viveen, W.J.A.R., Richter, C.J.J., Van Oordt, P.G.W.J., Janssen, J.A.L., Huisman, E.A..: Practical manual for the culture of the African Catfish (Clarias gariepinus), (1986), Directorate General for International 37Vol. 63, No. 1, 27-38 (2013)
12. 12. Cooperation, Ministry of Foreign Affairs, The Hague 36Sharma, D., Asrani, R.K., Ledoux, D.R., Jindal, N., Rottinghaus, G.E., Gupta, V.K.: Individual and combined effects of fumonisin B1 and moniliformin on clinicopathological and cell-mediated immune response in Japanese quail. Poult Sci, 87, 1039-1051 (2008) 37Stoev, S., Denev, S., Dutton, M., Nkosi, B.: Cytotoxic effect of some mycotoxins and their combinations on human peripheral blood mononuclear cells as measured by the MTT assay. The Open Toxinol J, 2, 1-8 (2009) 38Pedrosa, K., Borutova, R.: Synergistic effects of mycotoxins discussed. FeedStuffs, 83, 1-3 (2011) 39Fandohan, P., Hell, K., Marasas, W.F.O., Wingfield, M.J.: Infection of maize by Fusarium species and contamination with fumonisin in Africa: A review. Afr J Biotech, 2, 570-579 (2003) 40Edwards, S.G.: Influence of agricultural practices on Fusarium infection of cereals and subsequent contamination of grain by trichothecenes mycotoxins. Toxicol Lett, 153, 29-35 (2004) 38EZEKIEL et al. Mycotoxins