Abstract
Fusarium fungal contaminants and related mycotoxins were investigated in eight maize feed samples submitted to the Iowa State University Veterinary Diagnostic Laboratory. Fusarium moniliforme, F. proliferatum, and F. subglutinans were isolated from seven, eight, and five samples, respectively. These strains belonged to mating populations A, D, and E of the teleomorph Gibberella fujikuroi. Fusaproliferin was detected at concentrations of 0.1 to 30 μg/g in four samples, and beauvericin was detected (0.1 to 3.0 μg/g) in five samples. Fumonisins were detected in all eight samples (1.1 to 14 μg/g). Ten of 11 strains of F. proliferatum and all 12 strains of F. subglutinans isolated from the samples produced fusaproliferin in culture on whole maize kernels (4 to 350 and 100 to 1,000 μg/g, respectively). Nine F. proliferatum strains also produced beauvericin in culture (85 to 350 μg/g), but none of the F. subglutinans strains produced beauvericin. Fumonisin B1 was produced by all nine F. moniliforme strains (50 to 2,000 μg/g) and by 10 of the F. proliferatum strains (1,000 to 2,000 μg/g). This is the first report of the natural occurrence of fusaproliferin outside Italy and of the natural occurrence of beauvericin in North America.
Fusarium moniliforme Sheldon, F. proliferatum (Matsushima) Nirenberg, and F. subglutinans (Wollenw. et Reinking) Nelson, Toussoun, et Marasas are members of Fusarium section Liseola, and all three are common in Iowa maize (20). These species can produce potent mycotoxins, including fumonisins (11), fusaric acid (16), and moniliformin (16). Contamination of livestock feed by these Fusarium species is associated with a variety of toxicity symptoms ranging from poor weight gain to mortality (5–7, 16, 21, 24). Symptoms occurring in animals that consume Fusarium-contaminated feeds cannot always be attributed to known toxins (10).
Fusaproliferin is a toxic metabolite originally isolated from cultures of F. proliferatum (15, 26, 29). It is a sesterterpene compound that has toxic activity against brine shrimp (Artemia salina L.), insect cells, and human B lymphocytes and has teratogenic effects on chicken embryos (13, 26–28). Logrieco et al. (13) showed that fusaproliferin also can be produced by F. subglutinans. Beauvericin is a cyclodepsipeptide compound that is known to have insecticidal properties (3). It also has been shown to be highly toxic to human cell lines, inducing apoptosis (14, 23). It can be produced by strains of F. proliferatum (17), F. semitectum, and F. subglutinans (4).
As described at present, Gibberella fujikuroi (Sawada) Ito apud Ito et Kimura is the teleomorph for F. moniliforme, F. proliferatum, and F. subglutinans (9). F. subglutinans from Midwestern maize usually corresponds to mating population E of G. fujikuroi, whereas F. proliferatum from Midwestern maize usually corresponds to mating population D (9). G. fujikuroi mating populations were tested for production of fusaproliferin in culture on maize kernels, and only strains from populations D and E produced the compound (19). Previous to this study, reports of the natural occurrence of fusaproliferin have been limited to Italy (28).
The objectives of this study were to determine whether fusaproliferin or beauvericin could be found in Fusarium-contaminated livestock feed from Iowa, whether Fusarium strains from Iowa are capable of producing fusaproliferin or beauvericin, and whether these two compounds occur in combination with other mycotoxins.
MATERIALS AND METHODS
Sample collection and fungal strain characterization.
The samples used in this study were maize or feed specimens submitted by field veterinarians to the Iowa State University Veterinary Diagnostic Laboratory (ISUVDL), Ames, for mycotoxin analysis. We included only samples that had no detectable deoxynivalenol. Fusarium strains were isolated from ground maize and feed samples by culturing 0.5-g subsamples on Nash-Snyder medium (22). A single spore of each putative Fusarium colony was transferred to carnation leaf agar for identification according to the criteria and synoptic keys of Nelson et al. (22). One or more strains from seven of the samples were chosen arbitrarily for further characterization. Chosen strains represented most of the Fusarium species isolated from each sample. The mycelium and conidia from each strain were frozen in sterile 18% glycerol-water and stored at −75°C in the Istituto Tossine e Micotossine da Parassiti Vegetali (ITEM) culture collection at Bari, Italy.
Fusarium strains identified as F. moniliforme, F. proliferatum, or F. subglutinans were characterized in terms of their G. fujikuroi mating populations. Tester strains for mating populations were obtained from J. F. Leslie, Kansas State University (9). Strains in the present study were crossed on carrot agar as male parents with tester strains of mating populations A through F as described by Klittich and Leslie (8). Mated cultures were considered interfertile if perithecia formed within 6 weeks. All strains were crossed twice with both testers from each mating population.
In vitro mycotoxin production.
Single-conidium isolates of F. moniliforme, F. proliferatum, and F. subglutinans were cultured on 100 g of autoclaved yellow maize kernels that were adjusted to about 45% moisture in 500-ml Erlenmeyer flasks and inoculated with 2 ml of an aqueous suspension containing approximately 107 conidia/ml. Cultures were incubated at 25°C for 4 weeks. The harvested culture material was dried in a forced draft oven at 60°C for 48 h, finely ground, and stored at 4°C until use. Controls were treated the same way, except that they were not inoculated.
Mycotoxin analyses.
Analyses for deoxynivalenol were performed at ISUVDL. Analyses for fumonisins, beauvericin, and fusaproliferin were performed at ITEM and at the Dipartimento di Scienze degli Alimenti, Portici, Italy.
For beauvericin and fusaproliferin extraction, 10 g of each sample was ground and homogenized in a Waring blender for 5 min with 15 ml of methanol (99.5%). Samples were filtered through Whatman no. 4 filter paper, and methanol was removed under reduced pressure. This extraction procedure yielded 150 mg of raw organic extract that was used to quantify beauvericin and fusaproliferin. An aliquot of 100 μl of methanol extracts, corresponding to 100 mg of raw material, was filtered through an Acrodisk filter (pore size, 0.22 μm) before the high-performance liquid chromatography (HPLC) injection (20 μl, corresponding to 20 mg of culture).
For fumonisin B1 extraction, the procedure of Shephard et al. (31), partially modified as follows, was adopted. Extracts were prepared by shaking 10 g of each sample in 100 ml of methanol-water (3:1 [vol/vol]) for 1 h, and the extracts were then filtered through rapid-flow Whatman no. 4 filter paper. A 10-ml aliquot of the extract was applied to a Bond Elut strong anion exchange cartridge, and fumonisin B1 was eluted from the column by a 99.5:0.5 (vol/vol) chloroform (99%)–acetic acid (100%) solution (2). The dried residue was dissolved in 100 μl of methanol and stored at 4°C.
Standards of fumonisins and beauvericin were purchased from Sigma Chemical Co., St. Louis, Mo. The fusaproliferin standard was isolated in the laboratory of the Dipartimento di Scienze degli Alimenti from maize kernels inoculated with an F. proliferatum strain (26). The contaminated natural samples were examined for fumonisins B1 and B2 by liquid chromatography (LC)-mass spectrometry (MS). A benchtop API 100 (Perkin-Elmer Sciex) single-quadrupole MS equipped with an atmospheric pressure ionization source and ionspray interface was used for the MS analyses. All quantitative results were performed in positive-ion mode with the orifice voltage set at 40 V. The acquired data were processed by using Multiview and MacQuan software (Perkin-Elmer Sciex). The resolution was set at 0.5 atomic mass unit (measured at half height), and the mass calibration and resolution adjustments on the resolving quadrupole were performed in ionspray by using a PPG 10-4 solution introduced via a model 22 Harvard infusion pump. Initial MS spectra were collected in continuous-flow mode by infusion directly to the ionspray probe. A standard mixture of fumonisin B1 and fumonisin B2 was infused at 10 μl/min. All other experiments were recorded by using a Perkin-Elmer LC-200 pump fully controlled from the API 100 data system.
The LC-MS analyses were performed by using a Brownlee C18 3- by 30-mm HPLC column (Perkin-Elmer). Column flow was 0.8 ml/min, and an aqueous eluent of 5 mM ammonium acetate acidified with 1% formic acid containing 80% methanol was used. Only 40 μl/min was delivered into the ionspray source. Standards of fumonisins were purchased from Sigma, and a stock solution was made in methanol (1 mg/ml). Successive dilutions were made by using a 50% aqueous solution of methanol containing 2 mM ammonium acetate and 0.1% formic acid. The quantification was performed by using the protonated signal at m/z 722 and 706 for fumonisins B1 and B2, respectively.
The detection limit determined for the fumonisin B1 standard was estimated to be equivalent to 0.5 ppb, and the limit of quantification was equivalent to 1.2 ppb. The detection limit determined for the fumonisin B2 standard at m/z 706 was estimated to be equivalent to 4.5 ppb, and the limit of quantification was equivalent to 9.0 ppb.
Analysis of fumonisin B1 in culture extracts was performed by comparing the extracts (spotted from 1 to 20 μl) with pure fumonisin B1 (0.1, 0.5, 1, and 2 μg of standard) by high-performance thin-layer chromatography (HPTLC) on precoated silica gel 60 F254 plates (10 by 20 cm; thickness, 0.25 mm; E. Merck, Darmstadt, Germany) with two different solvent systems: chloroform-methanol-water-acetic acid (55:36:8:1 [vol/vol/vol/vol]) and chloroform-methanol (60:40 [vol/vol]). A detection limit of 2.5 μg/g was obtained for fumonisin B1 by using the extraction procedure described above and the HPTLC analysis.
The amounts of beauvericin and fusaproliferin in feed samples and culture material were determined by HPLC. Samples (10 g) were extracted with 15 ml of methanol in a Waring blender. The raw extract obtained from each sample, after the removal of solvent, was redissolved to obtain a constant concentration of 1 g of raw material/ml of solvent. Then all the samples, feed and culture, were filtered through an Acrodisk filter and analyzed by HPLC. In this way each injection of 20 μl corresponded to 20 mg of contaminated starting sample.
For fusaproliferin, HPLC analysis was carried out with LC10AD pumps and an SPO-M10A diode array detector (both from Shimadzu) equipped with a Shiseido Capcell Pak C18 column (250 by 4.6 mm; 5 μm). The HPLC system was set up with a flow rate of 1.0 ml/min and with a CH3CN–H2O (65:35 [vol/vol]) eluent system. The retention time of the authentic standard of fusaproliferin was 4.11 min. Quantification by HPLC procedures was carried out by comparison of the peak areas of the investigated samples with the calibration curve of the authentic standard. There was a linear correlation between the sample concentration and the areas of the peaks in the HPLC profile from 0.005 to 14 μg of fusaproliferin.
The HPLC configuration for beauvericin included CH3CN–H2O (85:15 [vol/vol]) as the eluent system with a flow rate of 1.0 ml/min. The wavelengths used to quantify beauvericin and fusaproliferin were 205 and 261 nm, respectively. There was a linear correlation between the sample concentration and the areas of the peaks in the HPLC profile from 0.02 to 5 μg of beauvericin. The retention time of the authentic standard of beauvericin was 5.7 min. As with fusaproliferin, quantification of beauvericin by HPLC procedures was done by comparison of the peak areas of the investigated samples with the calibration curve of the authentic standard. To confirm mycotoxin occurrence, the standards were spiked with extracts and analyzed by HPLC. The detection limit was 0.1 μg/g for beauvericin and 0.025 μg/g for fusaproliferin. To confirm beauvericin occurrence, the positive natural samples were analyzed by 1H nuclear magnetic resonance spectra and by low-resolution electronic impact mass spectrometry (m/z 784) (17). All analyses were run in triplicate, and the mean values are reported. The calculated standard deviation was always less than 5%.
RESULTS
Two or more Fusarium species were isolated from each of the eight samples analyzed. F. moniliforme, F. proliferatum, and F. subglutinans were isolated from seven, eight, and five samples, respectively. F. graminearum Schwabe was isolated from one sample. Beauvericin was detected in five samples, and fusaproliferin was detected in four samples (Table 1). Beauvericin concentrations ranged from 0.1 to 3.0 μg/g, and fusaproliferin concentrations ranged from 0.1 to 30 μg/g. Fumonisins were detected in all eight samples (Table 1). Fumonisin B1 concentrations ranged from 0.3 to 9.5 μg/g, and fumonisin B2 concentrations ranged from 0 to 4.0 μg/g. Notably, fumonisin B2 concentrations were higher than fumonisin B1 concentrations in five samples. Four samples contained all four toxins: beauvericin, fusaproliferin, and fumonisins B1 and B2.
TABLE 1.
Fusarium species and concentrations of mycotoxins in maize and feed samples analyzed for fusaproliferin and beauvericina
| Sample | Type | Fusarium spp. | Toxin concn (μg/g)
|
|||
|---|---|---|---|---|---|---|
| Fumonisin
|
Fusaproliferin | Beauvericin | ||||
| B1 | B2 | |||||
| 41599 | Feed | F. moniliforme, F. proliferatum, F. subglutinans | 6.5 | 1.5 | NDb | 0.1 |
| 43232 | Feed | F. graminearum, F. moniliforme, F. proliferatum | 0.3 | 0.8 | 0.5 | 0.6 |
| 43438 | Maize | F. proliferatum, F. subglutinans | 0.3 | 0.8 | 0.3 | 0.5 |
| 43702-D1 | Maize | F. moniliforme, F. proliferatum | 0.4 | ND | ND | ND |
| 43702-1 | Maize | F. moniliforme, F. proliferatum, F. subglutinans | 0.3 | 1.5 | 0.1 | 0.5 |
| 43702-2 | Maize | F. moniliforme, F. proliferatum | 0.3 | 0.8 | ND | ND |
| 43702-3 | Maize | F. moniliforme, F. proliferatum, F. subglutinans | 0.3 | 0.8 | ND | ND |
| 40380 | Feed | F. moniliforme, F. proliferatum, F. subglutinans | 9.5 | 4.0 | 30 | 3 |
All samples had no detectable deoxynivalenol.
ND, none detected.
Of the 32 strains tested for mycotoxin production, 9 were F. moniliforme, 11 were F. proliferatum, and 12 were F. subglutinans. Ten of 11 strains of F. proliferatum and all 12 strains of F. subglutinans isolated from the samples produced fusaproliferin in culture on whole maize kernels (4 to 350 and 100 to 1,000 μg/g, respectively). Nine F. proliferatum strains also produced beauvericin in culture (85 to 350 μg/g), but none of the F. subglutinans strains produced beauvericin. Fumonisin B1 was produced by all 9 F. moniliforme strains (50 to 2,000 μg/g) and by 10 of the F. proliferatum strains (1,000 to 2,000 μg/g) (Table 2). Although other fumonisins were not measured, it is likely that they were present in the cultures containing fumonisin B1. Eight strains of F. proliferatum produced all three toxins.
TABLE 2.
Toxin production by strains of F. moniliforme, F. proliferatum, and F. subglutinans isolated from livestock feed samplesa
| Source | Fusarium sp. | ITEM strain | Mating populationb | Toxin concn (μg/g)
|
||
|---|---|---|---|---|---|---|
| Fumonisin B1 | Fusaproliferin | Beauvericin | ||||
| 43702-1 | F. subglutinans | 2281 | E+ | NDc | 300 | ND |
| F. subglutinans | 2283 | E− | ND | 200 | ND | |
| F. subglutinans | 2284 | E+ | ND | 140 | ND | |
| F. subglutinans | 2286 | E− | ND | 100 | ND | |
| F. moniliforme | 2282 | A− | 1,750 | ND | ND | |
| F. proliferatum | 2406 | D− | 1,000 | 350 | ND | |
| F. proliferatum | 2407 | D− | 780 | 4 | ND | |
| F. moniliforme | 2285 | A− | 1,500 | ND | ND | |
| 43702-D1 | F. proliferatum | 2287 | D− | 1,700 | 80 | 100 |
| F. proliferatum | 2291 | NF | 1,200 | 250 | 150 | |
| F. moniliforme | 2288 | A− | 1,400 | ND | ND | |
| F. moniliforme | 2289 | A− | 1,800 | ND | ND | |
| F. moniliforme | 2290 | A+ | 2,000 | ND | ND | |
| 43702-2 | F. proliferatum | 2292 | D− | 1,000 | 140 | 85 |
| 40380 | F. subglutinans | 2391 | NF | ND | 750 | ND |
| F. subglutinans | 2392 | E+ | ND | 440 | ND | |
| F. subglutinans | 2393 | E− | ND | 270 | ND | |
| F. subglutinans | 2394 | E− | ND | 390 | ND | |
| F. moniliforme | 2390 | A+ | 50 | ND | ND | |
| 41599 | F. subglutinans | 2398 | E+ | ND | 110 | ND |
| F. subglutinans | 2399 | E+ | ND | 230 | ND | |
| F. proliferatum | 2400 | D+ | ND | ND | 100 | |
| F. moniliforme | 2395 | A− | 750 | ND | ND | |
| F. moniliforme | 2396 | A− | 100 | ND | ND | |
| F. moniliforme | 2397 | A− | 75 | ND | ND | |
| 43438 | F. subglutinans | 2404 | E− | ND | 1,000 | ND |
| F. subglutinans | 2405 | E− | ND | 550 | ND | |
| F. proliferatum | 2401 | D+ | 2,000 | 180 | 150 | |
| F. proliferatum | 2402 | D+ | 1,500 | 150 | 200 | |
| F. proliferatum | 2403 | D+ | 1,100 | 120 | 180 | |
| 43232 | F. proliferatum | 2408 | D+ | 1,100 | 130 | 350 |
| F. proliferatum | 2409 | D+ | 1,000 | 80 | 100 | |
Strains were grown on maize kernels for 4 weeks at 25°C.
+ and −, mating types; NF, not fertile with any mating population testers after two crosses.
ND, none detected.
DISCUSSION
Our results indicate that fusaproliferin and beauvericin can occur in Fusarium-contaminated livestock feed in Iowa and probably in other parts of the United States. It also appears that the ability to produce fusaproliferin is common in Iowa strains of F. subglutinans and F. proliferatum and that the ability to produce beauvericin is common among Iowa strains of F. proliferatum. Overall, the capability of fumonisin B1, beauvericin, and fusaproliferin production by specific mating populations of G. fujikuroi agrees with previous reports (11, 13). Of particular interest is the lack of beauvericin production by F. subglutinans strains in this study. In our previous studies, most F. subglutinans strains isolated from Poland, Austria, Peru, and Canada produced beauvericin (12, 18, 19). On the other hand, none of the F. subglutinans strains from the United States, Argentina, and Italy produced beauvericin (19). Based on the limited number of strains tested in these studies, it appears that beauvericin production by strains of F. subglutinans is related to their geographic origins. In this respect, Schlacht et al. (30) showed by randomly amplified polymorphic DNA analysis that F. subglutinans could be subdivided into two different groups. Further research on a larger number of strains is needed to confirm this pattern of beauvericin production, and it would be interesting to assess a possible relation between the two different groups obtained by molecular data and beauvericin production.
Many toxicology studies using culture material of F. proliferatum strains that produce large amounts of fumonisins have been conducted. The high proportion of F. proliferatum strains that produced beauvericin and fusaproliferin in this study suggests that the culture material used in some previous studies may have contained one or both of these two toxins.
Little is known about the toxicity of fusaproliferin and beauvericin to mammals and other animals. Plattner and Nelson (25) showed that a strain of F. proliferatum isolated from maize associated with swine mycotoxicosis produced beauvericin, and they suggested a possible role of this toxin in the disease. Similarly, strains of F. subglutinans toxic to ducklings and rats (16) were able to produce both beauvericin and fusaproliferin (13, 18). In the present study, beauvericin production was limited to F. proliferatum strains, several of which also produced fumonisins and fusaproliferin.
The cooccurrence of these four mycotoxins in animal feed samples has not been reported previously. Because fusaproliferin can cause pathological and teratological effects on chicken embryos and because beauvericin is toxic to a broad range of mammalian cells, it could be supposed that the occurrence of these toxins with fumonisin B1 represents a risk for animal health. It has been proposed that fumonisin B1 can act synergistically with other Fusarium toxins (1). However, until feeding studies are conducted with pure beauvericin and fusaproliferin, it will not be possible to draw conclusions about their toxicity to livestock, either alone or in combination with other toxins.
ACKNOWLEDGMENTS
This work was supported by Hatch Act and State of Iowa funds. This research was supported, in part, by a grant from the Iowa State University Dean of Agriculture’s International Competitiveness and Sustainability Program.
We are grateful to Paula Imerman, ISUVDL, for performing analytical work.
Footnotes
Journal paper J-17768 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, project no. 3260.
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