John P. Rheeder

Production of fumonisin analogs by Fusarium species.

The fumonisins, a family of food-borne carcinogenic mycotoxins, were first isolated in 1988 (21) from cultures of Fusarium verticillioides (Sacc.) Nirenberg (previously known as Fusarium moniliforme Sheldon). During the same year, the structures of the fumonisins were elucidated (6) and fumonisin B1 was shown to cause equine leukoencephalomalacia (34). There have been numerous publications dealing with this group of novel, carcinogenic mycotoxins, and comprehensive reviews of different aspects of the fumonisins are available (20, 22, 23, 24, 35, 36, 37, 41, 43, 46, 52, 55, 60, 61, 66). Due to the widespread occurrence of the fumonisins in maize, a dietary staple in many countries, the carcinogenic risk of fumonisins to humans was evaluated by the International Agency for Research on Cancer in 1993, and the toxins produced by F. moniliforme were evaluated as “Group 2B carcinogens,” i.e., probably carcinogenic to humans (24). This review focuses on the Fusarium species that produce fumonisins and the fumonisin analogs produced by each of these species.

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.

Exposure assessment for fumonisins in the former Transkei region of South Africa

The fumonisins are mycotoxins produced mainly by Fusarium verticillioides and F. proliferatum in maize, the predominant cereal staple for subsistence farming communities in southern Africa. In order to assess exposure to these mycotoxins in the Bizana (now known as Mbizana) and Centane magisterial areas of the former Transkei region of the Eastern Cape Province of South Africa, the actual maize consumption by different age groups in these communities was measured. In the groups 1–9 years (n = 215) and 10–17 (n = 240) years, mean consumption (±standard error) was 246 ± 10.8 and 368 ± 10.3 g per person day−1, respectively, with no significant difference (p > 0.05) between the magisterial areas. For adults (18–65 years) mean maize consumption in Bizana (n = 229) and Centane (n = 178) were significantly different (p < 0.05) at 379 ± 10.5 and 456 ± 11.9 g per person day−1, respectively. An exposure assessment was performed by combining the maize consumption distribution with previously determined levels of total fumonisin (fumonisins B1 and B2 combined) contamination in home-grown maize in these two areas. Assuming an individual adult body weight of 60 kg, fumonisin exposure in Bizana, an area of relatively low oesophageal cancer incidence, was 3.43 ± 0.15 µg kg−1 body weight day−1, which was significantly lower (p < 0.05) than that in Centane (8.67 ± 0.18 µg kg−1 body weight day−1), an area of high oesophageal cancer incidence. Mean fumonisin exposures in all age groups in both Bizana and Centane were above the provisional maximum tolerable daily intake (PMTDI) of 2 µg kg−1 body weight day−1 set by the Joint FAO/WHO Expert Committee on Food Additives.

Fusarium andiyazi sp. nov., a new species from sorghum

We describe a new Fusarium species in sec- tion Liseola from sorghum in Africa and the United States. This species is distinguished morphologically by the production of unique pseudochlamydospores in carnation leaf pieces on carnation leaf agar, and appears to be most closely related to F thapsinum. The new species also can be distinguished molecu- larly by means of Amplified Fragment Length Poly- morphisms, AFLPs. Strains with both MAT-1 and MAT-2 mating types were identified, but no sexual stage was generated in crosses made under labora- tory conditions.

Reduction of patulin in apple juice samples — influence of initial processing

Abstract Patulin is a secondary metabolite produced by a wide range of fungi including Penicillium expansum, a common contaminant of apples. Patulin is reputed to be a mutagen and recommendations have been made that levels should not exceed 50 ng g in apple juice intended for human consumption. The production of apple juice requires the use of ripe fruit, which may be pre-disposed to fungal contamination with P. expansum and concurrent contamination with patulin. During a study using selected over-ripe fruit, triplicate samples of apples were withdrawn at several points prior to maceration. The mean patulin level in the non-processed fruit was found to be 920 ng g , but this level dropped to 190 ng g following an initial water treatment step. Analyses of the wash water showed that appreciable levels had been transferred from the solid to the aqueous phase. Additional removal, by hand, of rotten and damaged fruit prior to further processing, significantly reduced the mean patulin level in the juice to 55 ng g . High patulin levels were recorded in the rotten fractions ( mean = 2335 ng g ). Mycological analyses tended to support the chemical data, in that removal of the rotten fruit significantly reduced the total fungal counts in the juice samples.

Individual fumonisin exposure and sphingoid base levels in rural populations consuming maize in South Africa

Low and high oesophageal cancer incidence areas of the former Transkei region of South Africa have been associated with corresponding low and high levels of fumonisin contaminated home-grown maize. This is the first study in South Africa assessing fumonisin B (FB) mycotoxin exposure by quantifying individual maize consumption with weighed food records and FB levels from maize in each participants household and concurrently evaluating sphinganine (Sa), sphingosine (So) and Sa/So ratios in plasma and urine of these participants as possible biomarkers of FB exposure. The high consumption of maize in Bizana (n=36) and Centane (n=30) of 0.41+/-0.21 and 0.39+/-0.19 kg/day, respectively, confirms the reliance on maize as the dietary staple. Mean total FB (FB(1)+FB(2)+FB(3)) levels in home-grown maize were 0.495+0.880 and 0.665+0.660 mg/kg in Bizana and Centane, respectively. Mean fumonisin exposure based on individual consumption was 3.9+/-7.3 and 4.1+/-7.6 microg/kg body weight/day, respectively, for Bizana and Centane. The mean combined sphinganine/sphingosine ratios in Bizana and Centane were similar and ranged from 0.10-0.55 in plasma (n=41) and urine (n=62). There was no association between sphingoid base levels and/or Sa/So ratios in the plasma and urine and individual fumonisin exposure, negating the sphingoid bases as potential biomarkers of fumonisin exposure in humans.

Fungal infection and mycotoxin contamination of maize in the Humid forest and the western highlands of Cameroon

Fungal incidence and mycotoxin contamination of farm-stored maize were assessed and compared in grain samples from three villages each in two agroecological zones over time. Maize samples were collected at 2 and 4 months after stocking from 72 farmers’ stores in 1996 and 1997 in the Humid Forest (HF) and Western Highlands (WHL) of Cameroon. Mycological assays of these samples revealed several fungal species.Nigrospora spp. were the most prevalent fungi in HF (32%) and WHL (30%) in 1996,Fusarium verticillioides (22%) andF. graminearum (27%) were also isolated from these samples. In the WHL in 1996, no significant difference in fungal incidence was found among villages for samples collected 2 months after harvest, but at 4 months incidence was significantly higherP<0.05). In 1997 the levels of fungal contamination were lower than in 1996. The incidence ofAspergillus spp. was low in general, ranging from 0.0 to 5.9% infected kernels. Analysis with thin layer chromatography detected low levels of aflatoxins in a few samples.F. verticillioides mycotoxin fumonisin Bi (300-26,000 ng/g) andF. graminearum metabolites deoxynivalenol (<100–l,300 ng/g) and zearalenone (<50–110 ng/g) were determined by means of polyclonal antibody competitive direct enzyme-linked immunosorbent assay. A significant correlation (r=0.72; P=0.0001) was found between the incidence ofF. graminearum and the contamination with deoxynivalenol. Storage time (2vs 4 months after stocking) had a significant positive effect (r=0.39; P=0.013) on the level of fumonisin B1. This is the first report of the natural occurrence of these mycotoxins in maize in Cameroon.

Sphingoid base levels in humans consuming fumonisin-contaminated maize in rural areas of the former Transkei, South Africa: a cross-sectional study

High incidences of oesophageal cancer are associated with the consumption of subsistence-grown maize by rural populations in the former Transkei region of Eastern Cape Province, South Africa. This cross-sectional study was conducted in the north-eastern magisterial area of Bizana (a previously low oesophageal cancer incidence area) and the south-eastern area of Centane (a previously high incidence area). Plasma and urine samples of male and female participants were analysed for the sphingoid bases, sphinganine and sphingosine. Good home-grown and visibly mouldy maize samples, collected from the households of the participants, were analysed for fumonisin B1, B2 and B3. Plasma sphinganine/sphingosine ratios in males and females were significantly lower (p < 0.05) due to lower sphinganine levels in Bizana compared to Centane. In contrast, the urinary female and combined (males + females) sphinganine/sphingosine ratios were significantly higher (p < 0.05) in Bizana due to the significantly lower (p < 0.05) urinary sphingosine levels. Interestingly, urinary sphingoid base levels were significantly lower (p < 0.05) in males than females within each area. Based on the mean total fumonisin levels in good maize, the estimated mean probable daily intake (PDI) was 5.8 µg kg−1 body weight day−1 in Bizana during 2000 and 4.4 and 6.7 5.8 µg kg−1 body weight day−1 in Centane during 1997 and 2000, respectively, exceeding the maximum tolerable daily intake proposed by JECFA. However, there was no significant difference in the mean total fumonisin levels in the maize between the magisterial areas. The observed differences in plasma and urinary sphingoid base levels could not be ascribed as a biomarker of fumonisin exposure and further studies at an individual level are required.

Fusarium globosum, a new species from corn in southern Africa

Fusarium globosum, a new species in Fu? sarium Section Liseola, is described and illustrated. This fungus is characterized by abundant globose mi? croconidia produced singly or in botryose clusters on monophialides and polyphialides in the aerial myce? lium. Clavate to ellipsoidal microconidia, with a trun? cate base, are produced in false heads and in chains in the aerial mycelium. Some pyriform to napiform microconidia are also found in culture. Macroconidia

Simple intervention method to reduce fumonisin exposure in a subsistence maize-farming community in South Africa.

In the Centane magisterial area of South Africa, high rates of oesophageal cancer have been associated with home-grown maize contaminated with fumonisins. The aim of this study was to implement a simple intervention method to reduce fumonisin exposure in a subsistence-farming community. The hand-sorting and washing procedures, based on traditional maize-based food preparation practices, were previously customised under laboratory-controlled conditions. Home-grown maize and maize-based porridge collected at baseline were analysed for fumonisin B1, B2 and B3. The geometric mean (95% confidence interval) of fumonisin contamination in the home-grown maize at baseline was 1.67 (1.21–2.32) mg kg−1 and 1.24 (0.75–2.04) mg kg −1 (dry weight) in the porridge. Fumonisin exposure was based on individual stiff porridge consumption and the specific fumonisin levels in the porridge (dry weight) consumed. Porridge (dry weight) consumption at baseline was 0.34 kg day−1 and fumonisin exposure was 6.73 (3.90–11.6) µg kg−1 body weight day−1. Female participants (n = 22) were trained to recognise and remove visibly infected/damaged kernels and to wash the remaining maize kernels. The discarded kernels represented 3.9% by weight and the fumonisins varied from 17.1 to 76.9 mg kg−1. The customised hand-sorting and washing procedures reduced fumonisin contamination in the maize and porridge by 84 and 65%, respectively. The intervention reduced fumonisin exposure by 62% to 2.55 (1.94–3.35) µg kg−1 body weight day−1. This simple intervention method has the potential to improve food safety and health in subsistence-farming communities consuming fumonisin-contaminated maize as their staple diet.