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Applied and Environmental Microbiology, August 1998, p. 3084-3088, Vol. 64, No. 8
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Beauvericin Production by Fusarium Species

A. Logrieco,^1,* A. Moretti,¹ G. Castella,^2, M. Kostecki,² P. Golinski,² A. Ritieni,³ and J. Chelkowski⁴

Istituto Tossine e Micotossine da Parassiti Vegetali, Consiglio Nazionale delle Ricerche, 70125 Bari,¹ and Dipartimento di Scienze degli Alimenti dell'Università degli studi di Napoli Federico II, 80055 Portici, Naples,³ Italy, and Department of Chemistry, University of Agriculture, 60-625 Poznan,² and Institute of Plant Genetics, Polish Academy of Sciences, 60-479 Poznan,⁴ Poland

Received 14 November 1997/Accepted 1 June 1998

	ABSTRACT

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Beauvericin is a cyclohexadepsipeptide mycotoxin which has insecticidal properties and which can induce apoptosis in mammalian cells. Beauvericin is produced by some entomo- and phytopathogenic Fusarium species (Fusarium proliferatum, F. semitectum, and F. subglutinans) and occurs naturally on corn and corn-based foods and feeds infected by Fusarium spp. We tested 94 Fusarium isolates belonging to 25 taxa, 21 in 6 of the 12 sections of the Fusarium genus and 4 that have been described recently, for the ability to produce beauvericin. Beauvericin was produced by the following species (with the number of toxigenic strains compared with the number of tested strains given in parentheses): Fusarium acuminatum var. acuminatum (1 of 4), Fusarium acuminatum var. armeniacum (1 of 3), F. anthophilum (1 of 2), F. avenaceum (1 of 6), F. beomiforme (1 of 1), F. dlamini (2 of 2), F. equiseti (2 of 3), F. longipes (1 of 2), F. nygamai (2 of 2), F. oxysporum (4 of 7), F. poae (4 of 4), F. sambucinum (12 of 14), and F. subglutinans (3 of 3). These results indicate that beauvericin is produced by many species in the genus Fusarium and that it may be a contaminant of cereals other than maize.

	TEXT

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Beauvericin is a toxic cyclic hexadepsipeptide first studied for its insecticidal properties (5, 7, 8). Beauvericin is a specific cholesterol acyltransferase inhibitor (20) and is toxic to several human cell lines (12). In particular, beauvericin induces programmed cell death similar to apoptosis and causes cytolysis accompanied by internucleosomal DNA fragmentation into multiples of 200 bp (12, 17).

In spite of the toxicological importance of beauvericin, the extent of human, animal, and plant exposure to this toxin has not been established. One approach is to screen fungal isolates for their abilities to produce beauvericin. Beauvericin was first reported to be produced by entomopathogenic fungi such as Beauveria bassiana (Balsamo) Vuill. and Paecilomyces fumosoroseus (Wize) Brown et Smith (8). In 1991, Gupta et al. (7) detected beauvericin in cultures of entomopathogenic strains of Fusarium moniliforme Sheldon var. subglutinans Wollenw. et Reinking and F. semitectum Berk et Rav. Beauvericin also is produced by F. subglutinans (Wollenw. et Reinking) Nelson, Toussoun, et Marasas isolated from maize ears from Austria, Canada, Italy, Poland, Peru, and South Africa, including some strains reported to be toxigenic to experimental animals (9, 10, 14). Beauvericin also is produced by F. proliferatum (Matsushima) Nirenberg isolated from maize and asparagus (13, 15, 18). In Gibberella fujikuroi (Sawada) Ito in Ito et K. Kimura, beauvericin was produced in large amounts by isolates belonging to mating populations B (F. subglutinans), C (F. proliferatum), D (F. proliferatum), and E (F. subglutinans), whereas isolates of mating populations A (F. moniliforme Sheldon) and F (F. thapsinum Klittich, Leslie, Nelson, et Marasas, sp. nov.) produce little, if any, of this toxin (15).

In the present study we measured the beauvericin production capabilities of Fusarium isolates representing 25 taxa, 21 in 6 of the 12 sections of Fusarium (16) and 4 that have been described recently.

Materials and methods. The strains we used (Table 1) are deposited in culture collections at the Institute of Plant Genetics (KF), Polish Academy of Sciences, Poznan, Poland (on autoclaved wheat kernels), and the Istituto Tossine e Micotossine da Parassiti Vegetali (ITEM), Bari, Italy, in sterile 18% glycerol at 75°C.

The abilities of different isolates to produce beauvericin were determined by analyzing maize kernel fungal cultures grown in duplicate as previously reported (11). Control maize meal was produced in the same way, except that it was not inoculated.

Samples of Fusarium cultures (15 g) were dried, ground (powdered), extracted overnight with 75 ml of solvent (acetonitrile-methanol-water, 16:3:1 [vol/vol/vol]), and filtered (Whatman no. 4 filter paper). The filtrate was defatted twice with 25 ml of heptane, and the bottom layer was evaporated to dryness. The residue was dissolved in 50 ml of solvent (methanol:water, 1:1 [vol/vol]) and extracted twice with 25 ml of methylene chloride. The methylene chloride phase (containing beauvericin) was collected and evaporated to dryness.

A beauvericin standard was purchased from Sigma Chemical Co. (St. Louis, Mo.). Beauvericin was analyzed by high-performance thin-layer chromatography (13) and high-pressure liquid chromatography. Evaporated extract containing BEA was dissolved in 1 ml of methanol, and 0.5 ml was applied to the top of a column containing 2 g of silica gel 60 (200/400 mesh; Aldrich), activated for 2 h at 110°C. The column was preconditioned with 5 ml of chloroform-2-propanol (95:5 [vol/vol]). The extract on the column was washed with the same solvent (3 ml), and then beauvericin was eluted with another 5 ml of the same solvent. Beauvericin was quantified by using a Waters 501 apparatus with a C₁₈ Nova Pack column (3.9 by 300 mm) and a Waters 486 UV detector ( = 204 nm; Y = 225) at a flow rate of 0.6 ml/min; the retention time was 10.5 min and the beauvericin detection limit was 0.07 µg/g at a  of 204 nm and 0.8 µg/g at a  of 225 nm. The production of beauvericin by F. oxysporum (ITEM-2470), F. poae (ITEM-1523), and F. sambucinum (ITEM-846) was confirmed by ¹H nuclear magnetic resonance (NMR) spectra and by low-resolution electronic-impact mass spectrometry (m/z 784) performed separately on the toxin purified from the fungal culture. In particular, the molecular peak at m/z 783 and the fragments at m/z 261 and 522 obtained by low-resolution electronic-impact mass spectrometry confirmed the trimeric structure of beauvericin. Proton and carbon NMR spectra were run in CDCl₃ (2 mg/ml) on a Bruker AMX600 spectrometer operating at 600.13 and 150.92 MHz, respectively. The ¹H and ¹³C data were consistent with previous results (13).

Results. Results of beauvericin production by 94 Fusarium isolates on maize cultures are summarized in Table 1. In the Discolor section, 12 of 14 isolates of F. sambucinum Fuckel sensu stricto produced 2 to 230 µg of beauvericin/g.

View this table: [in this window] [in a new window]   TABLE 1.   Production of beauvericin by species of Fusarium on autoclaved maize kernels

In the Gibbosum section, beauvericin production was mostly at low levels. The highest beauvericin producer was one of the two tested strains of F. longipes Wollenw. et Reinking (ITEM-3202) (200 µg/g). Other beauvericin-producing species of this section were Fusarium acuminatum Ell. et Ev. var. acuminatum (one of four isolates), Fusarium acuminatum var. armeniacum Forbes et al. (one of three isolates), and F. equiseti (Corda) Sacc. (two of three isolates).

In the Liseola section, all three isolates of F. subglutinans from bananas and one of two isolates of F. anthophilum (A. Braun) Wollenw. produced beauvericin (from 10 to 300 µg/g and 1,300 µg/g, respectively). Four of seven tested strains of F. oxysporum Schlecht. emend. Snyd. et Hans (Elegans section) produced beauvericin, including ITEM-2470, the highest-producing strain of this study, which was isolated from Polish maize and produced 3,200 µg/g.

In the Sporotrichiella section, all four tested strains of F. poae (Peck) Wollenw. produced the toxin, ranging from traces (ITEM-1446 from wheat) to 63 µg/g (ITEM-2893 from maize). One of the F. avenaceum (Fz.) Sacc. isolates (Roseum section) produced beauvericin at a very low level (7 µg/g).

Finally, of four recently described species, three produced beauvericin. In particular, one isolate of F. beomiforme Nelson, Toussoun, et Burgess, two isolates of F. dlamini Marasas et al., and two isolates of F. nygamai Burgess et Trimboli all produced low levels of beauvericin.

Discussion. Fourteen Fusarium species now are known to produce beauvericin. To our knowledge, this report is the first of beauvericin production by strains of F. sambucinum, F. acuminatum var. acuminatum, F. acuminatum var. armeniacum, F. equiseti, F. longipes, F. anthophilum, F. oxysporum, F. poae, F. avenaceum, F. beomiforme, F. dlamini, and F. nygamai.

The species that produce beauvericin occur worldwide and can grow in various ecological niches as well as on various host plants (3). Previous studies reported the natural occurrence of beauvericin only in maize (19) and identified F. subglutinans and F. proliferatum as the main beauvericin producers and the species responsible for its accumulation (9, 14, 15, 18). Our findings suggest that other species occurring on maize can contribute to beauvericin contamination, especially F. poae. We suspect that beauvericin could be a common wheat contaminant because F. poae is a common wheat pathogen (3).

Further study of beauvericin production by some species not commonly isolated from maize is needed. In this study, F. oxysporum ITEM-2470 was the highest beauvericin producer, even though some other strains in this species did not produce any detectable beauvericin. These differences suggest that beauvericin might play a role in the plant diseases induced by these fungi and that beauvericin might be specific for some formae speciales.

Most of the strains of F. sambucinum analyzed in this study were beauvericin producers. The highest producers (up to 230 µg/g) were isolated from European potatoes. The strains of F. sambucinum we used were previously studied in a European F. sambucinum project, and they produced trichothecenes (specifically diacetoxyscirpenol and/or neosolaniol and T-2 toxin) and enniatin B (1). The ability of these strains to synthesize beauvericin suggests that further studies should be made on the occurrence of beauvericin together with other toxins in infected potatoes.

The abilities of several species of the Liseola and Elegans sections and of three recently described species to produce beauvericin agree with their proposed taxonomic and molecular affinities (6). F. beomiforme, F. nygamai, and F. dlamini are often isolated from tropical and subtropical niches and plants (e.g., Striga hermontica, Sudan [21]; Cajanus indicus, India; soil debris, Australia). Thus, we suspect that beauvericin could be a potential contaminant of plants and commodities in those areas. This hypothesis is supported by the production of beauvericin by all three strains of F. subglutinans isolated from banana fruits in Ecuador and Panama. If the toxigenic ability of a fungal population from a specific plant host were known, it could indicate the possible toxin contaminants on the plant products as well as possible synergistic effects of the toxins on the plant.

Many strains analyzed in our study produced little, if any, beauvericin. Many of these have been maintained in culture collections for extended periods of time and may have lost their ability to produce toxins. As an example, F. dlamini ITEM-3198, which produced 19 µg of beauvericin/g (Table 1), was also received from another source, but that specimen failed to produce any detectable toxin. Studies of freshly isolated field strains may be necessary to accurately determine the abilities of some species to produce beauvericin.

In conclusion, beauvericin appears to be one of the toxins most widely produced by species of Fusarium. Additional data on its possible interactions with other toxins produced by these fungi, e.g., trichothecenes, enniatins, fumonisins, fusaric acid, moniliformin, and fusaproliferin (1, 2, 4, 9, 15), are needed to evaluate the potential toxicity and synergistic effects of beauvericin.

	ACKNOWLEDGMENTS

This work was supported by a grant from the Italian Ministry of Agriculture and Forestry, D.M. 131/7240/94 del 14/02/1992, prog. 451, and promoted by a scientific collaboration project sponsored by CNR (National Council of Research of Italy) and PAN (Polish Academy of Sciences).

We thank L. W. Burgess (Fusarium Research Laboratory, University of Sydney, Sydney, Australia), W. F. O. Marasas (Programme on Mycotoxins and Experimental Carcinogenesis, Medical Research Council, Tygerberg, South Africa), H. Nirenberg (Biologische Bundesanstalt für Land und Forstwirtschaft, Berlin, Germany) and S. W. Peterson (National Center for Agricultural Utilization Research, Peoria, Ill.) for providing isolates.

	FOOTNOTES

^* Corresponding author. Mailing address: Istituto Tossine e Micotossine da parassiti vegetali, CNR, Viale Einaudi 51, 70125 Bari, Italy. Phone: 39-80-5491570. Fax: 39-80-5486063. E-mail: logrieco{at}area.ba.cnr.it.

This work is dedicated to the memory of Professor P. E. Nelson.

Present address: Departamento Patologia Animal, Facultad de Veterinaria, Universidad Autonoma, 08193 Bellaterra, Barcelona, Spain.

	REFERENCES

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Applied and Environmental Microbiology, August 1998, p. 3084-3088, Vol. 64, No. 8
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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