Mycological and Multiple Mycotoxin Surveillance of Sorghum and Pearl Millet Produced by Smallholder Farmers in Namibia.

Mycological (mycotoxigenic Fusarium and aflatoxigenic Aspergillus spp.) and multiple mycotoxins [aflatoxin B1 (AFB1), fumonisin B (FB), deoxynivalenol and zearalenone] surveillance was conducted on raw whole grain sorghum (Sorghum bicolor) and pearl millet (Pennisetum glaucum) produced on smallholder farms, and processed products sold at open markets in northern Namibia. Fungal contamination was determined with morphological methods as well as with quantitative Real-Time PCR (qPCR). The concentrations of multiple mycotoxins in samples were determined with liquid chromatography tandem mass spectrometry. The incidence of mycotoxigenic Fusarium spp., Aspergillus flavus and A. parasiticus, as well as the concentrations of AFB1 and FB were significantly (P < 0.001) higher in the malts as compared to the raw whole grains, with Aspergillus spp. and AFB1 exhibiting the highest contamination (P < 0.001). None of the analysed mycotoxins were detected in the raw whole grains. Aflatoxin B1 above the regulatory maximum level set by the European Commission was detected in sorghum (2 of 10 samples; 20%; 3-11 µg/kg) and pearl millet (6 of 11 samples; 55%; 4-14 µg/kg) malts. Low levels of FB1 (6 of 10 samples; 60%; 15-245 µg/kg) were detected in sorghum malts and no FB was detected in pearl millet malts. Contamination possibly occurred postharvest, during storage, and/or transportation and processing. By critically monitoring the complete production process, the sources of contamination and critical control points could be identified and managed. Mycotoxin awareness and sustainable education will contribute to reducing mycotoxin contamination. This could ultimately contribute to food safety and security in northern Namibia where communities are exposed to carcinogenic mycotoxins in their staple diet.

Marasas et al. 1984 "Toxigenic Fusarium Species: Identity and Mycotoxicology" revisited.

This study was conducted to determine the species identity and mycotoxin potential of 158 Fusarium strains originally archived in the South African Medical Research Council's Mycotoxigenic Fungal Collection (MRC) that were reported to comprise 17 morphologically distinct species in the classic 1984 compilation by Marasas et al., Toxigenic Fusarium Species: Identity and Mycotoxicology. Maximum likelihood and maximum parsimony molecular phylogenetic analyses of single and multilocus DNA sequence data indicated that the strains represented 46 genealogically exclusive phylogenetically distinct species distributed among eight species complexes. Moreover, the phylogenetic data revealed that 80/158 strains were received under a name that is not accepted today (ex F. moniliforme) or classified under a different species name. In addition, gas chromatography-mass spectrometry (GC-MS) and/or high-performance liquid chromatography-mass spectrometry (HPLC-MS)-based mycotoxin analyses were conducted to determine which toxins the strains could produce in liquid and/or solid cultures. All of the trichothecene toxin-producing fusaria were nested within the F. sambucinum (FSAMSC) or F. incarnatum-equiseti (FIESC) species complexes. Consistent with this finding, GC-MS analyses detected trichothecenes in agmatine-containing broth or rice culture extracts of all 13 FSAMSC and 10/12 FIESC species tested. Species in six and seven of the eight species complexes were able to produce moniliformin and beauvericin, respectively, whereas B-type fumonisins were only detected in extracts of cracked maize kernel cultures of three species in the F. fujikuroi (FFSC) species complex.

Relative severity of fumonisin contamination of cereal crops in West Africa.

Traditional and improved varieties of maize, pearl millet and sorghum were planted by small-scale farmers under the direction of the International Institute for Tropical Agriculture in two Nigerian agro-ecological zones: the Sudan Savanna and the Northern Guinea Savanna. Samples were collected for the determination of Fusarium infection and fumonisin (B1, B2 and B3) contamination. A previous paper reported Aspergillus infection and aflatoxin contamination of these samples. Fusarium infection levels, measured by per cent kernels infected, were modest with mean levels for the above cereals of 16% ± 11% (SD), 12% ± 7% and 13% ± 16%, respectively. However, the Fusarium species recovered from maize were predominantly the fumonisin producers F. verticillioides and F. proliferatum, together making an infection rate of 15% ± 10%, whereas these species were present to a limited extent only in the other two cereals, 1% ± 1% for pearl millet and 2% ± 6% for sorghum. Fumonisin contamination was variable but reflected the diversity of Fusarium producers in these three cereals. Mean levels were 228 ± 579 µg kg(-1) (range < 5-2860 µg kg(-1)) for maize, 18 ± 7 µg kg(-1) (range = 6-29 µg kg(-1)) for pearl millet and 131 ± 270 µg kg(-1) (range < 5-1340 µg kg(-1)) for sorghum. Together with previous results on aflatoxin, this study confirmed the co-occurrence of aflatoxins and fumonisins in maize as well as in the traditional African cereals, millet and sorghum (89% co-occurrence across all three cereals). The low fumonisin levels may be ascribed to the use of good agricultural practices. Of the Fusarium species present, those in maize consisted mainly of fumonisin producers, the opposite of what was observed in pearl millet and sorghum. It is concluded that replacement of maize by pearl millet and sorghum could improve food safety with regards to aflatoxin B and fumonisin B exposure.

Mycological and aflatoxin contamination of peanuts sold at markets in Kinshasa, Democratic Republic of Congo, and Pretoria, South Africa.

Peanut (Arachis hypogaea L.) is an important food crop in sub-Saharan Africa. In this survey, the mycological and aflatoxin contamination of peanuts collected from Kinshasa, Democratic Republic of Congo, and Pretoria, South Africa, was assessed. Twenty peanut samples were purchased randomly at informal markets in the two cities and analysed for mycoflora and aflatoxins (AFB1, AFB2, AFG1 and AFG2) using standard methods. The results indicated that 95% of the Kinshasa samples and 100% of the Pretoria samples were contaminated with aflatoxigenic fungi in the ranges 20-49,000 and 40-21,000 CFU/g, respectively. Seventy-five per cent of the Kinshasa samples and 35% of the Pretoria samples exceeded the maximum limits of AFB1 as set by The Joint FAO/WHO Expert Committee on Food Additives. Residents of both cities are at a high risk of aflatoxin exposure despite their apparent cultural, socio-economic, geographic and climatic differences. Further work needs to be done to understand the supply chains of peanut trade in informal markets of the two countries so that interventions are well targeted on a regional rather than a national level.

Mycological analysis and multimycotoxins in maize from rural subsistence farmers in the former Transkei, South Africa.

Maize harvested in the Centane region of the former Transkei, Eastern Cape Province, South Africa, by subsistence farmers has been shown over many seasons to be contaminated with fumonisin mycotoxins. However, there are limited data on the presence of other mycotoxins. Two multimycotoxin LC-MS/MS methods were applied to good and moldy maize samples, as separated by the farmers themselves from the 2011 harvest. One method involved extract cleanup on multitoxin immunoaffinity columns before LC-MS/MS analysis for aflatoxins, fumonisins, deoxynivalenol (DON), zearalenone (ZEN), and T-2 and HT-2 toxins. The other method was based on a "dilute-and-shoot" approach for the above mycotoxins and a wide range of other fungal secondary metabolites. Both methods showed high incidences of fumonisins B1 and B2 (FB1 and FB2) in good maize (100% for both by the first method, means were 2083 and 927 µg/kg for the two analogues; 93% for both by the second method, positive means of 2764 and 1050 µg/kg, respectively). All samples of moldy maize were contaminated (mean FB1 of 27.64 and 35.98 mg/kg, respectively; mean FB2 of 10.58 and 14.14 mg/kg, respectively). Comparison of the two methods for FB1 and FB2 over the entire range of samples gave R(2) values 0.9144 and 0.8859, respectively. Low levels of DON were found by both methods (positive means of 12 and 4.7 µg/kg in good maize, respectively, and of 14 and 5.8 µg/kg in moldy maize, respectively). ZEN was determined with positive means of 108 and 25 µg/kg in good maize, respectively, and of 111 and 135 µg/kg in moldy maize, respectively. No aflatoxins, OTA, or T-2 or HT-2 toxins were detected. A wide range of other Fusarium , Aspergillus , Alternaria , and Penicillium mycotoxins and secondary metabolites were determined.

Multiple mycotoxin exposure determined by urinary biomarkers in rural subsistence farmers in the former Transkei, South Africa.

Subsistence farmers are exposed to a range of mycotoxins. This study applied novel urinary multi-mycotoxin LC-MS/MS methods to determine multiple exposure biomarkers in the high oesophageal cancer region, Transkei, South Africa. Fifty-three female participants donated part of their maize-based evening meal and first void morning urine, which was analysed both with sample clean-up (single and multi-biomarker) and by a 'dilute-and-shoot' multi-biomarker method. Results were corrected for recovery with LOD for not detected. A single biomarker method detected fumonisin B1 (FB1) (87% incidence; mean±standard deviation 0.342±0.466 ng/mg creatinine) and deoxynivalenol (100%; mean 20.4±49.4 ng/mg creatinine) after hydrolysis with ß-glucuronidase. The multi-biomarker 'dilute-and-shoot' method indicated deoxynivalenol-15-glucuronide was predominantly present. A multi-biomarker method with ß-glucuronidase and immunoaffinity clean-up determined zearalenone (100%; 0.529±1.60 ng/mg creatinine), FB1 (96%; 1.52±2.17 ng/mg creatinine), α-zearalenol (92%; 0.614±1.91 ng/mg creatinine), deoxynivalenol (87%; 11.3±27.1 ng/mg creatinine), ß-zearalenol (75%; 0.702±2.95 ng/mg creatinine) and ochratoxin A (98%; 0.041±0.086 ng/mg creatinine). These demonstrate the value of multi-biomarker methods in measuring exposures in populations exposed to multiple mycotoxins. This is the first finding of urinary deoxynivalenol, zearalenone, their conjugates, ochratoxin A and zearalenols in Transkei.

Fumonisin B1 as a urinary biomarker of exposure in a maize intervention study among South African subsistence farmers.

BACKGROUND: The consumption of maize highly contaminated with carcinogenic fumonisins has been linked to high oesophageal cancer rates. The aim of this study was to validate a urinary fumonisin B1 (UFB1) biomarker as a measure of fumonisin exposure and to investigate the reduction in exposure following a simple and culturally acceptable intervention. METHODS: At baseline home-grown maize, maize-based porridge, and first-void urine samples were collected from female participants (n=22), following their traditional food practices in Centane, South Africa. During intervention the participants were trained to recognize and remove visibly infected kernels, and to wash the remaining kernels. Participants consumed the porridge prepared from the sorted and washed maize on each day of the two-day intervention. Porridge, maize, and urine samples were collected for FB1 analyses. RESULTS: The geometric mean (95% confidence interval) for FB1 exposure based on porridge (dry weight) consumption at baseline and following intervention was 4.84 (2.87-8.14) and 1.87 (1.40-2.51) µg FB1/kg body weight/day, respectively, (62% reduction, P<0.05). UFB1C, UFB1 normalized for creatinine, was reduced from 470 (295-750) at baseline to 279 (202-386) pg/mg creatinine following intervention (41% reduction, P=0.06). The UFB1C biomarker was positively correlated with FB1 intake at the individual level (r=0.4972, P<0.01). Urinary excretion of FB1 was estimated to be 0.075% (0.054%-0.104%) of the FB1 intake. CONCLUSION: UFB1 reflects individual FB1 exposure and thus represents a valuable biomarker for future fumonisin risk assessment. IMPACT: The simple intervention method, hand sorting and washing, could positively impact on food safety and health in communities exposed to fumonisins.

Molecular characterization of Fusarium globosum strains from South African maize and Japanese wheat.

The fungus Fusarium globosum was first isolated from maize in South Africa and subsequently from wheat in Japan. Here, multiple analyses revealed that, despite morphological similarities, South African maize and Japanese wheat isolates of the fungus exhibit multiple differences. An amplified fragment length polymorphism-based similarity index for the two groups of isolates was only 45%. Most maize isolates produced relatively high levels of fumonisins, whereas wheat isolates produced little or no fumonisins. The fumonisin biosynthetic gene FUM1 was detected in maize isolates by Southern blot analysis but not in the wheat isolates. In addition, most of the maize isolates produced sclerotia, and all of them produced large orange to dark purple sporodochia in carrot agar culture, whereas wheat isolates did not produce either structure. In contrast, individual isolates from both maize and wheat carried markers for both mating type idiomorphs, which indicates that the fungus may be homothallic. However, a sexual stage of F. globosum was not formed under standard self-fertilization conditions developed for other homothallic species of Fusarium. The inability to produce the sexual stage is consistent with the high similarity of 87-100% and G (ST) index of 1.72 for the maize isolates, which suggests that these isolates are undergoing asexual but not sexual reproduction. Together, the results suggest that the South African maize and Japanese wheat isolates of F. globosum are distinct populations and could be different species.

Incidence of Fusarium verticillioides and levels of fumonisins in corn from main production areas in Iran.

A total of 52 corn samples collected in 2000 from four main corn production provinces of Iran (Fars, Kermanshah, Khuzestan, and Mazandaran) were analyzed for contamination with Fusarium verticillioides and fumonisins (FB(1), FB(2), FB(3), and 3-epi-FB(3)). The mean incidence of F. verticillioides (percent of kernels infected) for these four areas was 26.7, 21.4, 24.9, and 59.0%, respectively. The incidence in Mazandaran was significantly (p < 0.05) above that of the other areas. All samples from Mazandaran were contaminated with fumonisins with a mean level of total fumonisins of 10674 microg/kg. In contrast, the incidence of fumonisin contamination above 10 microg/kg was 53 (8/15), 42 (5/12), and 57% (8/14) in the samples from Fars, Kermanshah, and Khuzestan, respectively, and the corresponding mean total fumonisin levels were 215, 71, and 174 microg/kg, respectively. No statistical differences (p > 0.05) were observed in the fumonisin levels of the corn samples from these three provinces, which were significantly (p < 0.05) lower than the fumonisin contamination in samples from Mazandaran.

Production of fumonisin B and C analogues by several fusarium species.

Six strains of Fusarium verticillioides, two of F. oxysporum, one strain of F. proliferatum, and a strain of an unidentified species were cultured on maize patties and rice and evaluated for their ability to simultaneously produce fumonisin B (FB) and C (FC) series analogues. Fumonisins were quantified by LC-MS-MS using positive ion electrospray ionization. FC1 provided characteristic fragment ions at m/z 690, 672, 654, 532, 514, and 338 corresponding to sequential loss of H2O and tricarboxylic acid moieties from the alkyl backbone, while FC3 and FC4 provided equivalent product ions 16 and 32 amu lower than the corresponding FC1 fragments, respectively. All isolates cultured on maize produced FC4. All isolates except for that of F. proliferatum also produced FC1, and three of the six strains of F. verticillioides produced FC3. All isolates except those of F. oxysporum produced detectable amounts of FB1, FB2, and FB3. Isolates that produced fumonisin B analogues produced at least 10 fold more of the B series analogues than they did of the C series analogues. The results confirm that at least some strains of F. oxysporum produce FC, but not FB, fumonisin analogues and also suggest that the genetics and physiological regulation of fumonisin production may be more complicated than previously envisaged since some strains of F. verticillioides and F. proliferatum as well as the strain of the unidentified species can simultaneously produce both FB and FC analogues.

Toxicity, pathogenicity, and genetic differentiation of five species of fusarium from sorghum and millet.

ABSTRACT Fusarium isolates recovered from sorghum and millet are commonly identified as F. moniliforme, but with the recognition of new species in this group, the strains given this name are being re-evaluated. We analyzed five strains each from five Fusarium species (F. andiyazi, F. nygamai, F. pseudonygamai, F. thapsinum, and F. verticillioides) often associated with sorghum and millet for their ability to produce fumonisin and moniliformin, their toxicity to ducklings, and their ability to cause disease on sorghum seedlings in vitro. These species can be distinguished with isozymes (fumarase, NADP-dependent isocitrate dehydrogenase, and malate dehydrogenase) and with banding patterns resulting from amplified fragment length polymorphisms. Two species, F. pseudonygamai and F. thapsinum, produced high levels of moniliformin, but little or no fumonisins, and were consistently highly toxigenic in the duckling tests. Two species, F. verticillioides and F. nygamai, produced high levels of fumonisins, but little or no moniliformin, and also were toxigenic in the duckling tests. F. andiyazi produced little or no toxin and was the least toxigenic in the duckling tests. In sorghum seedling pathogenicity tests, F. thapsinum was the most virulent followed by F. andiyazi, then F. verticillioides, and finally F. nygamai and F. pseudonygamai, which were similar to each other. Thus, these five species, which would once have all been called F. moniliforme, differ sufficiently in terms of plant pathogenicity and toxin production profile, that their previous misidentification could explain inconsistencies in the literature and differences observed by researchers who thought they were all working with the same fungal species.

Fumonisin contamination and fusarium incidence in corn from Santa Catarina, Brazil.

In Brazil, the southern region has the highest incidence of esophageal cancer and also the highest production and consumption of corn (Zea mays) products. Corn samples intended for human consumption from the western, northern, and southern regions of the state of Santa Catarina, southern Brazil, had mean total fumonisin B (B(1), B(2), and B(3)) levels of 3.2, 3.4, and 1.7 mg/kg, respectively. Fusarium verticillioides, the predominant fungus in the corn samples, had mean incidences (percent of kernels infected) of 14, 11, and 18% for the three regions, respectively. Additional corn samples intended for animal feed from the southern region had a mean total fumonisin level of 1.5 mg/kg and a mean F. verticillioides incidence of 10%. The fumonisin levels in corn from the state of Santa Catarina, Brazil, were similar to the high levels determined in other high esophageal cancer incidence regions of the world.

Mycoflora and fumonisin mycotoxins associated with cowpea [Vigna unguiculata (L.) Walp] seeds.

Cowpea seed samples from South Africa and Benin were analyzed for seed mycoflora. Fusariumspecies detected were F. equiseti, F. chlamydosporum, F. graminearum, F. proliferatum, F. sambucinum, F. semitectum, and F. subglutinans. Cowpea seed from South Africa and Benin and F. proliferatum isolates from Benin, inoculated onto maize patty medium, were analyzed for fumonisin production. Samples were extracted with methanol/water and cleaned up on strong anion exchange solid phase extraction cartridges. HPLC with precolumn derivatization using o-phthaldialdehyde was used for the detection and quantification of fumonisins. Cowpea cultivars from South Africa showed the presence of fumonisin B(1) at concentrations ranging between 0.12 and 0.61 microg/g, whereas those from Benin showed no fumonisins. This is believed to be the first report of the natural occurrence of FB(1) on cowpea seed. Fumonisin B(1), B(2), and B(3) were produced by all F. proliferatum isolates. Total fumonisin concentrations were between 0.8 and 25.30 microg/g, and the highest level of FB(1) detected was 16.86 microg/g.

Polyclonal Antibody-Based ELISA and HPLC Methods for the Determination of Fumonisins in Corn: A Comparative Study.

The performance of an experimental polyclonal antibody (PAb)-based competitive direct enzyme-linked immunosorbent assay (CD-ELISA) developed for the analysis of fumonisins in corn was assessed by comparison with an established high-performance liquid chromatography (HPLC) method. The comparative study was conducted using a series of 20 corn samples naturally contaminated with combined fumonisin levels ranging from <0.05 to >5 µg/g (ppm). Linear regression analysis between the results generated by HPLC and CD-ELISA provided correlation coefficients (r) and regression slopes (b) of r = 0.960, b = 1.493 (P < 0.001); r = 0.865, b = 3.903 (P < 0.001); and r = 0.832, b = 0.107 (P < 0.001) for the individual fumonisins B1 (FB1), B2 (FB2) and B3 (FB3), respectively, while corresponding values of r = 0.967, b = 1.059 (P < 0.001) were obtained for the combined FB1, FB2, and FB3 concentrations. In 3 of 18 fumonisin-positive corn samples, combined fumonisin levels determined by CD-ELISA were between 85 and 100% higher than those determined in the same extracts by HPLC, while in 13 other samples, CD-ELISA results were between 1.8 and 53% higher than those determined by HPLC. Conversely, in 2 of 18 samples, CD-ELISA results were lower than those determined by HPLC. The differences recorded between HPLC and the experimental PAb-based CD-ELISA were far less than those previously recorded for other mono- and polyclonal antibody-based CD-ELISA systems. The results indicate that the experimental PAb-based CD-ELISA may be effectively applied for the initial screening for fumonisins in corn.