The screening for genetic resistance to fusarium head blight (FHB) in wheat has been challenged by the lack of efficacy of various artificial inoculation techniques in disease nurseries. In 2001-2003, we examined the influence of inoculum sources (conidial suspension, infested barley and corn kernels, or infested wheat debris) and inoculation frequency on the development of FHB and the deoxynivalenol (DON) content. The study was conducted on three spring wheat genotypes in a disease nursery of Ottawa, Ontario. The development of FHB was monitored by visually estimating disease severity on a scale of 0 to 9, six times during each growing season. Severity of FHB over time was summarized as the area under the disease-progress curve (AUDPC). Symptoms of FHB were also rated, at a critical developmental stage (soft dough), as critical-time disease severity (DS), percentage of infected spikelets (IS), and FHB index. The percentage of Fusarium-damaged kernels (FDK) and DON content in the wheat kernels were assessed after harvesting. All inoculation treatments had significantly greater AUDPC and DS in 2002, FHB index and IS in 2001 and 2002, and FDK in all three years than the noninoculated control. Regardless of inoculum source, two inoculations produced as much FHB, FDK, and DON as three inoculations. Among the inoculum sources, the conidial suspension or infested kernels yielded significantly greater AUDPC, DS, FHB index, IS, and DON content than the infested debris. Inoculation with conidial suspension resulted in greater FDK than inoculation with infested kernels or infested debris, between which there were no differences. The results of the present study confirm that conidial suspension and infested kernels are equally effective inoculants for FHB nurseries. Infested debris, though a natural source of inoculum, is less effective.

The Canadian grain industry uses visual estimation of Fusarium-damaged kernels (FDK) for grading, but this method does not always accurately predict deoxynivalenol (DON) levels in specific lots. The imminent introduction of lower limits for DON in wheat and the increasing trend of producers and grain companies to market wheat directly, with DON limits specified in contracts, are challenging the methods now applied to measure or estimate the DON levels in wheat. Among the detection methods reviewed for the grain trade, image analysis and near infrared spectoscopy are the most attractive because they are nondestructive, simple, and have multiple applications. However, the adoption of these methods may be restricted by capital cost and their limit of detection. Both methods estimate DON indirectly, from other Fusarium parameters, and neither is commercially proven. The problem with near-infrared spectoscopy is that it measures factors associated with the presence of DON, such as kernel hardness and color, which are not exclusive to the presence of DON. linage analysis has the advantage of measuring FDK objectively, but also suffers from the shortcomings of estimating DON indirectly, by FDK. If the new DON limits proposed are adopted, these will be near the limit of detection of the immunomethods commonly employed for measuring DON. Although immunomethods require laboratory facilities, more time, and technical skills and may not account for other Fusarium factors that affect grain quality, they require only a low capital investment, are reasonably priced, and have wide acceptance in the trade. Immunomethods that target the fungus to genus or species can be designed as either qualitative or quantitative, and some DNA-based kits are now commercially available. Quantitative methods provide a better estimate of toxin levels resulting from the growth of Fusarium spp., and of their effects on grain quality, but the measure remains an approximation that relies on the strength of the relationship between the fungal content and DON level. Ultimately, the methods chosen will be determined by guidelines for DON, here and abroad, the purpose of the testing, and the needs of each sector of the grain industry; more than one method may be appropriate.

A nationwide survey was carried out to assess mould spoilage of Castanea sativa nuts sold in Canadian grocery stores in 1998-99. Morphological and cultural characters, along with secondary metabolite profiles derived from thin-layer chromatography, were used to sort and identify fungi cultured from nut tissue. Three mycotoxigenic fungi dominated (Penicillium crustosum, Penicillium glabrum/spinulosum and Penicillium discolor) and were isolated at frequencies of 67.1%, 18.6% and 17.7%, respectively, from a total sample size of 350 nuts. Another mycotoxin producer, Aspergillus ochraceus was also isolated, but at a much lower frequency. HPLC and diode array detection were used to confirm the suspected presence of the mycotoxins penitrem A, chaetoglobosin A and C, emodin and ochratoxin A in extracts prepared from naturally infected nut tissue. To the best of our knowledge, this is the first time emodin has been found in a naturally contaminated food source.

A new 1-hydroxy-2,6-pyrazinedione, sclerominol (1), was isolated from cultures of hypovirulent isolates of Sclerotinia minor, a fungal plant pathogen associated with lettuce drop and other plant diseases. This compound was characterized by NMR, mass spectrometry, and X-ray crystallography. One other 1-hydroxy-2,6-pyrazinedione, flutimide, has been reported. Flutimide has activity as an inhibitor of influenza virus endonuclease, and therefore, sclerominol was evaluated for related biological activity. Sclerominol (1) displayed some activity against cancer cell lines but little activity against three influenza virus strains. The role of 1 in the physiology of hypovirulent isolates of S. minor has not been determined, but 1 has also been recovered from debilitated isolates of S. sclerotiorum.

There has been only one report of fumonisin antibodies produced from a fumonisin-bovine serum albumin (BSA) immunogen in the past. Other, more exoteric, carriers, such as cholera toxin and keyhole limpet hemocyanin, have also been used. The resulting antibodies have a wide range of sensitivity and selectivity for the most common fumonisins B1, B2 and B3. We report on fumonisin monoclonal antibodies produced in typical fashion from an immunogen consisting of FB1 conjugated to BSA through its terminal amino group. These antibodies have excellent cross-reactivity with the three main fumonisins: FB1, 100%, FB2, 94%, and FB3, 72%, adequate sensitivity with an IC50 of 430 ng/mL for FB1, and show excellent correlation between ELISA and total fumonisins as determined by HPLC, with a slope of 0.985 and an r2 of 0.987.

Three types of commercial sweet corn hybrids [surgary (su1), shrunken or 'supersweet' (sh2) and surgary enhancer (se1)] were silk channel inoculated in 1996 and 1997 with a macroconidial suspension of Fusarium graminearum to determine how early the mycotoxin deoxynivalenol accumulates in kernels. Disease symptoms rapidly developed on all hybrids and were apparent 4 days after inoculation. Symptoms stabilized by 28 days after inoculation. Toxin levels were greater than 1 microgram/g in kernels as early as 2 weeks after silk emergence and rapidly increased to extremely high levels. Susceptibility in all hybrids decreased as the silk dried out. Deoxynivalenol concentrations were correlated to disease severity. There was some indication that the sh2 genotype was more susceptible than the su1 or se1 genotypes. These results suggest that improvement needs to be made in sweet corn with respect to resistance to gibberella ear rot.

One central spikelet of spring wheat (Triticum aestivum) cv. Roblin spikes was inoculated with macroconidia of Fusarium graminearum and the entire spikes were harvested at 2- to 4-day intervals from 2 to 25 days after inoculation. The spikes were dissected and the amount of deoxynivalenol (DON) in each spikelet and in each internode of the rachis was measured by enzyme-linked immunosorbent assay (ELISA) with monoclonal antibodies. High concentrations of DON were first detected in the inoculated spikelets, 4 days after inoculation. DON concentrations in the spikelets below the inoculation point eventually reached 500-600 ppm while the corresponding internodes of the rachis contained 1000-1200 ppm. Much lower amounts of DON were found in spikelets and rachis above the inoculation point.

Fumonisins B1 and B2 (FB1 and FB2) are fungal secondary metabolites produced by members of the genus Fusarium. Although FB1 is usually detected in greater quantities, FB2 frequently co-occurs in contaminated feeds and foods and contributes to the total toxin load. In the present study, the comparative toxicity of FB1 and FB2 was examined in male Sprague-Dawley rats administered toxin (0.75 mg/kg body weight) or vehicle control intraperitoneally (ip) for 2, 4 or 6 consecutive days. Clinical changes, including elevated serum cholesterol, alanine aminotransferase (ALT), creatinine and protein, were slightly more pronounced in FB1-treated rats. The most consistent hematological change was an increase in vacuolated bone marrow cells, which was more pronounced in FB1-treated rats. Histopathological changes were similar in FB1- and FB2-treated rats and included single cell necrosis in kidneys and liver, cytoplasmic vacuolation in adrenal cortex and lymphocytolysis in thymus. In the liver mRNA expression for the cyclin kinase inhibitor p21 gene was significantly increased in FB1- and FB2-treated rats, compared to controls. Expression of mRNA for the cyclin D1 gene was significantly depressed in FB2-treated rats. Hepatic cyclin E mRNA was elevated in response to FB1 and FB2 compared to controls. In FB2-treated animals this corresponded with decreased liver p27 mRNA expression. Hepatic proliferating cell nuclear antigen (PCNA) transcription was elevated in FB1- but not FB2- treated rats. Changes in liver microsomal protein levels of p27, cyclin E and PCNA were similar to changes in gene expression. In contrast, cyclin D1 protein levels were elevated in rats treated with FB1 and, to a lesser extent, FB2. The data indicate that FB1 and FB2 can alter the expression of genes associated with the cell cycle, and indicate a need for a further understanding of the mechanistic basis of FB1 and FB2 toxicity.

Fumonisins are mycotoxins of world-wide distribution in maize infected by the fungus Fusarium verticillioides. They are highly toxic to certain livestock and are potential carcinogens. Exophiala spinifera, a black yeast fungus found on moldy maize kernels, was identified previously as capable of growing on fumonisin B1 as a sole carbon source and thus is a potential source for fumonisin detoxifying enzymes. Pure cultures of E. spinifera transform fumonisin B(1) to the amino polyol AP(1) plus free tricarballylic acid through the activity of a soluble extracellular esterase, and further transformation is evidenced by accumulation in culture supernatant of a less polar compound(s) lacking a fluorescamine-reactive amino group. A free amine is thought to be critical for biological activity of FB(1) or AP(1). As a first step towards characterizing this amine-modifying activity, we investigated the biotransformation of AP(1) by E. spinifera liquid cultures that had been previously grown in liquid medium containing AP(1) as a sole carbon source. Accumulation of AP(1)-derived metabolites was monitored by thin-layer chromatography of culture supernatants, and product metabolites were purified and evaluated by mass spectrometry and nuclear magnetic resonance. Two products of treatment of purified AP(1) with cultures of E. spinifera are shown to be N-acetyl AP(1) and a new compound, 2-oxo-12,16-dimethyl-3,5,10, 14,15-icosanepentol hemiketal (or 2-OP(1) hemiketal).

Toxicokinetic studies were conducted using single doses of fumonisin B-1 (FB1) and B-2 (FB2) in rats and vervet monkeys. After ip injection in rats (7.5 mg fumonisin/kg body weight), both toxins were rapidly absorbed and their levels in plasma reached a maximum within 20 min. The subsequent elimination phases were characterised by mono-exponential declines in plasma levels with half-lives of 18 and 26 min for each toxin, respectively. After a similar gavage dose, no toxin was detected in plasma (<50 ng/ml). The toxins were recovered unmetabolised from excreta After ip injection, up to 32% of FB1 (as determined by radioactivity) and 1.2% of FB2 were recovered in urine, compared to 0.1% and 0.2% in urine after dosing by gavage. The remainder of the dose was recovered in faeces. Both toxin analogues were rapidly eliminated from plasma of vervet monkeys following iv injection (1.6 - 2 mg/kg body weight). After a short distributional phase, the elimination phase was characterised by a half life of 40 min for FB1 and 18 min for FB1. Following a gavage dose of 6.4 and 7.5 mg/kg body weight for FB1 and FB2 respectively, only limited amounts were absorbed. For FB1, plasma levels peaked after 1 - 2 hr with maximum levels below 210 ng/ml, while FB2 showed a maximum level of 40 ng/ml after 5 hr. Urinary excretion was significant after iv dosing (mean 13.1% for FB1 and 4.1% for FB2 dose), but considerably less after the gavage dose (mean 1.3% and 0.2%, respectively). Analysis of faeces for up to 7 days after dosing failed to recover the full dose and also indicated that, for both analogues, significant removal by hydrolysis of one of the tricarballylic acid moieties of fumonisin had occurred, yielding the corresponding partially hydrolysed fumonisin. Although the fully hydrolysed backbones of both analogues were detected, they accounted for less than 2% of the dose.

A new fumonisin, iso-fumonisin B1 (iso-FB1, 1), has been isolated from liquid cultures of the fungus Fusarium moniliforme (Sheldon) NRRL 13616. On the basis of its spectroscopic data, its structure has been determined to differ from that of fumonisin B1 only in the presence of a hydroxyl function at C-4 instead of C-5.

The fungal toxin fumonisin B1 (FB1) is a contaminant of corn-based foods and feeds produced by members of the genus Fusarium. Fumonisin B1 toxicity was examined using gavage administration of purified toxin to female Sprague-Dawley rats. For 11 consecutive days each rat received a single dose of FB1 at the following concentrations: control (saline), 1, 5, 15, 35, or 75 mg FB1/kg body weight/d. Significantly depressed body weight and food consumption occurred at 35 and 75 mg FB1/kg/d. By the end of the dosing period there were no significant changes in food consumption. Kidneys and bone marrow were most sensitive to FB1 exposure. Changes in renal morphology were observed from 5 to 75 mg FB1/kg/d, accompanied by transient changes in urine osmolality and urine enzyme levels. Increased cellular vacuolation was the primary change associated with bone-marrow toxicity, starting at doses of 5 mg FB1/kg/d. Hepatotoxicity was indicated by reduced liver weight, elevated serum alanine amonitransferase (ALT), and mild histopathological changes occurring at doses of 15 mg FB1/kg/d and higher. Increased cytoplasmic vacuolation of adrenal cortex cells occurred in rats treated with 15 mg FB1/kg/d and higher, indicating that the adrenals are also potential targets of FB1. Elevated serum cholesterol, which is a consistent response to FB1 was observed at 5 mg FB1/kg/d and higher. Based on responses in this study, gavage is an appropriate substitute for longer feeding studies. Compared to previous work with male rats, gender-related difference in FB1 responses lacked consistency but indicated that males may be marginally more sensitive than female Sprague-Dawley rats.

The production of fusaric acid and other toxins by a strain of Fusarium oxysporum used for control of the weed Striga hermonthica (Del.) Benth. was investigated. Culturing of the strain under optimal conditions for toxin production produced small amounts of fusaric acid and dehydrofusaric acid but no other toxins reported to be produced by some strains of F. oxysporum. Culturing of the fungus under conditions similar to those that would be used in the field produced no detectable toxins of concern for human health.

Fumonisin B1 (FB1) is a fungal toxin produced by members of the genus Fusarium. Ingestion of FB1 causes species-specific neurotoxic, nephrotoxic, hepatotoxic and pulmonary effects. The clinical, haematological and pathological responses of adult male and female B6C3F1 mice to FB1 were assessed following 14 daily gavage doses ranging from 1 to 75 mg FB1/kg body weight/day. There were no consistent sex-related changes. Although all responses were modest, the most notable effects of FB1 were on the liver, bone marrow, adrenals and kidneys. In the liver, hepatocellular single cell necrosis, mitosis and anisokaryosis were observed, accompanied by elevated serum ALT. In the kidneys, minor histopathological changes were confined to female mice, while mild decreases in ion transport and increases in blood urea nitrogen were seen only in males. Small changes in glutathione levels were observed in the kidneys and livers of male mice. Adrenal cortical cell vacuolation was observed at 15 mg FB1/kg and higher in females and from 35 mg FB1/kg in males. Serum cholesterol was elevated in both male and female mice, possibly due to FB1-induced changes in lipid metabolism in the liver and adrenals. Although bone marrow cell numbers were unchanged, increases in vacuolated myeloid cells and lymphocytes were observed in female mice. In general, the degree of changes observed indicate that mice are not as sensitive a model of FB1 toxicity as rats.

Mycotoxin fumonisin B1 (FB1) is a common contaminant of corn and a causative agent of different animal and human diseases. An experiment was conducted to assess the impact of pure FB1 on carcass quality of growing-finishing swine. Pigs were fed diets containing 0, 0.11, 0.33 and 1.0 mg FB1 kg−1 (ppm) until market weight. Although performance characteristics were not different among the respective treatments, an increase in feed consumption variability was observed between weeks 3 and 9 for the 1 ppm FB1 fed pigs as compared with controls. The same animals showed an increased variability in carcass characteristics, in particular in the fat content of loin and ham. The estimated lean yield tended to decrease with increasing dose, but a high standard deviation abolished treatment differences. An elevated cholesterol value at the end of the experiment for the 1 ppm FB1 fed pigs suggested a disruption of lipid metabolism. No other significant (P > 0.06) changes were observed. We conclude that a diet containing 1 ppm FB1 could have a detrimental effect on the carcass quality of a market pig and be a source of a monetary loss to the producer. Key words: Mycotoxin fumonisin B1, swine, carcass

An improved method of extraction of deoxynivalenol (DON) from small samples of plant tissues is described. Using a ball mill, the extraction time was reduced from 60 minutes to 1 minute and the amount of monoclonal antibodies required for a reliable competitive direct (CD)-ELISA was reduced from 100 mu g/mL to 5 mu g/mL. Using this method, we analysed the kernels, chaff. rachis and peduncle of individual heads of wheat plants that had been field-inoculated with Fusarium graminearum. The DON concentration varied considerably among the same tissues from different plants. The median concentration of DON was highest in the rachis (93 ppm). followed by chaff (50 ppm), kernels (25 ppm), and peduncles (16 ppm). We also determined the DON concentration in single kernels obtained from a naturally-affected commercial wheat field. One hundred each of visibly unaffected (''normal-looking'') kernels, shrivelled kernels, and fusarium damaged (''white tombstone'' and ''pink tombstone'') kernels were analysed. DON was detected in about 50% of the normal-looking and shrivelled kernels, but typically at concentrations of 5 ppm or less. DON levels in tombstone kernels varied from less than 1 ppm to 600 ppm, with a mean of 275 ppm and 118 ppm in pink tombstone and white tombstone kernels, respectively.

Polyclonal antibodies against fumonisin B-1 (FB1) were produced in rabbits after immunization with FB1 conjugates. Protection of the amino group prior to coupling the carboxylic acid of FB1 to human serum albumin, and then deprotecting the amine before immunization, generated high-affinity antibodies. The antibodies produced were specific to fumonisins and their hydrolyzed products (HFB1, HFB2) but not to any other mycotoxins commonly found in grains. The relative crossreactivities of FB1, fumonisin B-2 (FB2), fumonisin B-3 (FB3), HFB1, and HFB2 were 100%, 55%, 13%, 11%, and 3%, respectively. Using these antibodies, a sensitive indirect competitive ELISA was developed. The detection limit and IC50 for FB1 in water were 0.05 and 0.66 ng/mL, respectively. The linear range was determined to be 0.05-0.75 ng/mL. The method was applied directly to 75% methanol extracts of corn. Recoveries of FB1 were better than 87% from 10 to 10 000 ng/g at five levels of fortification. The detection limit in corn was 5 ng/g.

The deoxynivalenol (DON) concentration in grain samples of wheat and corn, inoculated with Fusarium graminearum in the field, was determined by gas chromatography and competitive direct (CD)-ELISA using monoclonal antibodies. The two methods gave similar results with no false positives or negatives. Based on analyses of 140 wheat and 83 corn samples, the correlation coefficient (r) between the two methods was 0.984 +/- 0.015 for wheat and 0.986 +/- 0.018 for corn (P = 0.0001 for both). The slopes for the regression analysis of the corn and wheat data were 1.012 +/- 0.019 and 1.099 +/- 0.017, respectively. If only lower concentrations were considered, the 28 corn samples with DON concentrations below 5 ppm yielded a slope of 1.251 +/- 0.109 (r = 0.914 +/- 0.063), and the 63 wheat samples with DON concentrations below 2 ppm produced a slope of 1.220 +/- 0.132 (r = 0.763 +/- 0.083).

Monoclonal antibodies (MABs) against the mycotoxin deoxynivalenol (DON) were produced using an immunogen consisting of DON conjugated to bovine serum albumin (BSA) through its 15-hydroxyl group (15-DON-BSA). From 80 hybridomas, the four best stabilized hybridoma cell lines secreting anti-DON MABs of subclass IgG(1) were used to prepare ascites in mice. The MABs purified from ascites were used to detect DON by direct competitive ELISA. The MABs were very specific to 15-acetyl-DON and DON but had negligible binding to 3-acetyl-DON, T-2 toxin, sambucinol, and neosolaniol. The effective range of detection was 0.05-20 mu g/mL of DON or 15-acetyl-DON. These antibodies also showed a high stability toward grain extracts containing up to 40% methanol.

Strains of Fusarium moniliforme and Fusarium proliferatum were collected in a survey of corn disease in Ontario. These were tested for the production of fumonisins (B-1, B-2) and fusarins (A,C,F) and, additionally, for moniliformin in the case of the latter species. Almost all strains tested were capable of producing fumonisins and fusarins and most strains of F. proliferatum could also produce moniliformin. A survey of fumonisin in the 1993 Ontario corn crop was undertaken. Fumonisins were detected in 9 of 98 samples at concentrations ranging from 0.4 to 2.5 mu g/g fumonisin B-1 plus B-2. All Of the locations where fumonisins were found experienced temperatures slightly above normal and drought during ear development. This is the first report of fumonisins in corn from Ontario and of the production of fusarins and fumonisins from strains of F. moniliforme and F. proliferatum from Ontario.