Marc E. Savard

Interaction of Fusarium graminearum and F. moniliforme in Maize Ears: Disease Progress, Fungal Biomass, and Mycotoxin Accumulation

ABSTRACT To investigate the interaction between two major ear-rotting pathogens, maize ears were inoculated with either Fusarium graminearum, F. moniliforme, or an equal mixture of the two. Silk and kernel tissues were periodically harvested throughout the growing season so that a time course of the experimental variables (disease severity, ergosterol content, fungal DNA content, and mycotoxin concentration) could be recorded. Over the 3 years tested (1992 to 1994), the highest levels of disease and ergosterol were found in the F. graminearum treatment, followed by the mixture treatment (F. graminearum plus F. moniliforme) and, finally, the F. moniliforme treatment. Kernel ergosterol content and disease rating were correlated for both pathogens, but the highest correlation coefficients were obtained in the F. graminearum treatment. The DNA analysis revealed that, in the mixed inoculum, F. moniliforme had a greater growth rate than did F. graminearum. In 1994, appreciable F. moniliforme from natural inoculum was found in the F. graminearum treatment. Fumonisin B(1) levels did not differ between the F. moniliforme treatment and the mixed inoculum treatment. The effect of temperature on the growth rate of the two species explained some of the field results, with temperatures in the silks being more favorable to F. moniliforme. Data on the growth rate on silks obtained by the incorporation of radiolabeled precursor to ergosterol demonstrated that F. graminearum was able to grow well at 26 to 28 degrees C, whereas F. moniliforme grew well over a broader range, including at higher temperatures.

Spoilage fungi and their mycotoxins in commercially marketed chestnuts

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.

Response of growing swine to dietary exposure to pure fumonisin B1 during an eight-week period: Growth and clinical parameters

Consumption of corn or corn-based products contaminated with Fusarium moniliforme/fumonisins has been associated with a variety of animal and human diseases and is a major food/feed safety issue. This study focused on the clinical toxicity and performance parameters in growing swing exposed to low to moderate levels of pure fumonisin B1 (FB.) for 8 weeks. Male (castrated) and female pigs were fed diets containing 0,0.1,1.0, and 10 mg FB1/kg diet (ppm). Weight gains and feed consumption were measured weekly. Blood samples were collected throughout the study, and various clinical and hematological parameters were measured. Because fumonisins are potent inhibitors of sphingolipid biosynthesis, sphinganine and sphingosine concentrations were determined in the liver, lung, and kidney. Organ weights and carcass quality were measured at the end of the trial. In general, male pigs were more adversely affected by FB1 in the diet than females. The average daily gain for males decreased by 8% for pigs fed 1.0 ppm and by 11% at 10.0 ppm, when compared to the control (0 ppm). Males fed 0.1 ppm showed an erratic growth pattern during the first 5 weeks of the experiment. Feed consumption for the same animals was somewhat higher than that of the controls during each of the first 4 weeks but thereafter was 6-7% lower each week as compared to controls. Female pigs fed FB1-diets showed a general enhancement of feed consumption until week 4. Among clinical chemistry parameters, cholesterol increased in males for the 1.0 and 10.0 ppm diets as compared to controls after 2 weeks, while the levels in both sexes were elevated for the 1.0 ppm diet only by the end of the experiment. Serum liver enzyme concentrations were altered during week 2 only. Changes were observed in the weight of the pancreas and adrenals for male pigs fed FB1 diets as compared to controls. The free sphinganine to free sphingosine ratio (biomarker of exposure in FB1-consuming animals) increased in all three organs for the 10 ppm diet, regardless of sex. The study indicated that FB1 can cause different effects at each dose level, at concentrations as low as 0.1 ppm (showing erratic growth) followed by a reduced growth and biochemical abnormalities in blood (1.0 ppm) and sphingolipid alterations in tissues (10.0 ppm). Some of these effects occurred below the exposure level that caused alteration in sphingolipid metabolism.

Cloning, expression, and characterization of a single-domain antibody fragment with affinity for 15-acetyl-deoxynivalenol.

A single-domain variable heavy chain (V(H)H) antibody fragment specific to the mycotoxin 15-acetyldeoxynivalenol (15-AcDON) was obtained after immunization of a llama (Llama glama) with the protein conjugate 15-DON-BSA plus TiterMax Classic adjuvant. After confirmation of a polyclonal response to DON toxin in both conventional (cIgG) and heavy chain antibody (HCAb) fractions, a V(H)H library was constructed from amplified cDNA by nested PCR. V(H)H fragments with binding affinity for the mycotoxin were selected by panning of the phagemid library against microtiter plates coated with 15-DON-OVA. The dominant clone (NAT-267) was expressed in E. coli and was purified as a V(H)H monomer (mNAT-267) at a final concentration of 1.3 mg mL(-1). Isolated NAT-267 V(H)H DNA was fused to the homopentamerization domain of the B subunit of verotoxin to generate the pentabody format of single-domain antibody (sAb). The V(H)H pentamer (pNAT-267) was expressed in E. coli and was purified at a final concentration of 1.0 mg mL(-1). Surface plasmon resonance (SPR) analysis of soluble mNAT-267 binding kinetics to immobilized 15-DON-Horse Radish Peroxidase (HRP) indicated a dissociation constant (K(D)) of 5microM. Competitive direct enzyme-linked immunosorbent assay (CD-ELISA) and fluorescence polarization assay (FPA) inhibition experiments with monomer and pentamer confirmed binding to 15-AcDON. Competitive inhibition FPAs with mNAT-267 and pNAT-267 determined IC(50) values of 1.24 and 0.50 microM, respectively, for 15-AcDON hapten. These values were similar to the IC(50) value of 1.42 microM for 15-AcDON given by polyclonal llama serum sampled 56 days after immunization. Competition formats for structurally related trichothecenes resulted in no cross-reactivity to: DON; 3-acetyldeoxynivalenol (3-AcDON); neosolaniol (NEO); diacetoxyscirpenol (DAS); and T-2 toxin. Our study confirmed that recombinant V(H)H fragments capable of binding low molecular weight haptens can be produced through the creation and panning of hyper-immunized single-domain (sdAb) libraries.

Analysis of Fusarium toxins in maize and wheat using thin layer chromatography.

Thin layer chromatography (TLC) methods for identifying and quantifying deoxynivalenol (DON), fumonisin B1 (FB1) and zearalenone in grain samples were compared to immunoassay (ELISA) and high performance liquid chromatography (HPLC) methods to determine the reliability of the less expensive TLC. There was a very good agreement between levels of DON measured by TLC and competitive-direct ELISA, and between levels of fumonisin B1 measured by TLC and HPLC, over a wide range of concentrations. Correlation coefficients (Pearsons) were 0.978, 0.914 and 0.953 for DON in maize, DON in wheat and FB1 in maize respectively. A lower correlation coefficient (r = 0.672) was obtained when zearalenone was quantified by TLC and HPLC. Possible reasons for this are discussed. A cost comparison of the various methods revealed that TLC was the least expensive for sample analysis. It is recommended that researchers choose which analytical method to use based upon individual considerations of cost and precision.

Biological Fate of Fumonisin B1 in Food-Producing Animals

The presence of mycotoxins in grains and feedstuffs causes not only animal health problems, but also a valid concern about the transmission of potentially toxic residues into animal-derived products intended for human consumption. In a series of studies at Agriculture and Agri-Food Canada, we investigated the biological fate of fumonisin B1 (FB1) in several food-producing animals (grower pigs, laying hens, dairy cattle), as well as monitored various parameters for evidence of toxicity in these species. In several experiments involving either single-dose protocols (iv, po) or longer-term feeding trials, the pharmacokinetic profiles of FB1 (purity > 95%) in these species were determined, including tissue accumulation and transmission of residues. Toxicological (and economical) implications such as performance (feed consumption, growth), productivity, and carcass quality were also measured when appropriate.

Oxidative deamination of hydrolyzed fumonisin B1 (AP1) by cultures of Exophiala spinifera

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).

Sequential distribution of the mycotoxin deoxynivalenol in wheat spikes after inoculation with Fusarium graminearum

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.

Biological control of fusarium head blight of wheat with Clonostachys rosea strain ACM941

Fusarium head blight (FHB), caused by Gibberella zeae, is a devastating disease of wheat. A strain of Clonostachys rosea, ACM941 (American Type Culture Collection ATCC 74447), was evaluated for antibiosis against G. zeae in vitro and for control of FHB under greenhouse and field conditions in comparison to the registered fungicide Folicur (tebuconazole). Strain ACM941 reduced mycelial growth of the pathogen by 52.6% in dual-culture after 6 days and completely suppressed spore germination for 6 h when cocultured with a macroconidial suspension of G. zeae. Strain ACM941 reduced G. zeae perithecial production by more than 99% in a leaf disk assay, 60%–77% on infected corn kernels, and 32%–57% on spikelet debris in the field. These effects were significant (P < 0.05) and not statistically different from those produced by tebuconazole. When strain ACM941 was sprayed onto wheat heads 2 days prior to inoculation with G. zeae, it significantly reduced infected spikelets (IS) by 64% and Fusarium -damaged kernels (FDK) by 65% in greenhouse experiments. Under simulated disease epidemic conditions during 2005–2007, strain ACM941 reduced the FHB index by 58%, IS by 46%, FDK by 49%, and deoxynivalenol (DON) in kernels by 21%. These effects were significant but lesser in magnitude than those achieved by tebuconazole, which reduced FHB index by 97%, IS by 82%, FDK by 73%, and DON by 62%. Results from this research suggest that strain ACM941 of C. rosea is a promising biocontrol agent against G. zeae and may be used as a control measure in an integrated FHB management program.

Analysis of Fusarium graminearum mycotoxins in different biological matrices by LC/MS.

The purpose of this study was to develop an LC/MS assay to accuratelydetect three mycotoxins produced by Fusarium graminearum in various matrices. Using different LC conditions, deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), and zearalenone (ZEN) were detected in four different matrices (fungalliquid cultures, maize grain, insect larvae and pig serum). The sensitivity of MS detection allowed us to detect concentrations as low as 8 ppb of DON and 12 ppb of ZEN. A very small quantity of matrix was therefore necessary for successful analysis of these toxinsand a variety of experimental situations were successfully investigated using this technique. Production of 15-ADON and butenolide was monitored in a liquid culture of F. graminearum under controlled conditions. Using simple extraction procedures,the differential accumulation of DON and 15-ADON was followed in inoculated maize genotypes varying in susceptibility to F. graminearum. Toxicokinetic studies were carried outwith maize insect pests reared continually on artificial diets containing ZEN and suggested that larvae may possess the ability to degrade ZEN. Finally, persistence of DON was assessed in pigs fed diet supplemented with DON, results indicated that DON accumulates quickly in pig blood and then levels decline progressively for 12 hours thereafter. TheLC/MS study reported here is very useful and flexible for the detection of these mycotoxins in different media and at very low concentrations.