Beauvericin Production by Fusarium Species

Abstract

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.

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.

TABLE 1.

Production of beauvericin by species of Fusarium on autoclaved maize kernels

Fusarium species	Strain		Original host	Sourceb	Geographic origin	Beauvericin production (μg/g)c
	Original no.	Other designation(s)
Discolor
F. culmorum	KF-833		Triticum aestivum	JC	Poland	ND
	KF-838		Triticum aestivum	JC	Poland	ND
	KF-839		Triticum aestivum	JC	Poland	ND
	KF-1144		Triticum aestivum	JC	Poland	ND
	KF-1147		Triticum aestivum	JC	Poland	ND
	KF-1158		Triticum aestivum	JC	Poland	ND
	ITEM-478		Zea mays	AL	Italy	ND
	ITEM-627		Triticum aestivum	AL	Yugoslavia	ND
F. cerealis	KF-501		Zea mays	JC	Poland	ND
	KF-582		Zea mays	JC	Poland	ND
	KF-1154		Triticum aestivum	JC	Poland	ND
	ITEM-619		Triticum aestivum	AL	Yugoslavia	ND
	ITEM-667		Solanum tuberosum	AL	Italy	ND
F. graminearum	ITEM-644		Panicus crusgalli	AL	Italy	ND
	ITEM-646		Triticum durum	AL	Italy	ND
	KF-1413		Zea mays	JC	Poland	ND
	ITEM-645		Triticum durum	AL	Italy	ND
	ITEM-635		Zea mays	AL	Italy	ND
F. sambucinum	ITEM-847	BBA 64995	Brassica oleracea	HN	The Netherlands	2
	ITEM-934	BBA 64678	Triticum aestivum	HN	Switzerland	21
	ITEM-952	BBA 62433	Beta vulgaris	HN	Spain	53
	ITEM-954	BBA 64960	Soil	HN	The Netherlands	38
	ITEM-955	BBA 64737	Solanum tuberosum	HN	Germany	20
	ITEM-848	BBA 65009	Solanum tuberosum	HN	Italy	76
	ITEM-956	BBA 62434	Solanum tuberosum	HN	Iran	130
	ITEM-957	BBA 64226	Solanum tuberosum	HN	England	190
	ITEM-958	BBA 64998	Solanum tuberosum	HN	France	38
	ITEM-933	BBA 64996	Solanum tuberosum	HN	France	17
	ITEM-846	BBA 62397	Solanum tuberosum	HN	Germany	230
	ITEM-961	BBA 64480	Solanum tuberosum	HN	Finland	3
	ITEM-960	BBA 64262	Glycine max	HN	Brazil	ND
	ITEM-959	BBA 64484	Solanum tuberosum	HN	Finland	ND
F. venenatum	ITEM-831	BBA 64935	Solanum tuberosum	HN	Poland	ND
	ITEM-835	BBA 65030	Zea mays	HN	Germany	ND
	ITEM-836	BBA 64478	Solanum tuberosum	HN	Finland	ND
	ITEM-834	BBA 64757	Humulus lupulus	HN	Germany	ND
F. torulosum	ITEM-838	BBA 64479	Solanum tuberosum	HN	Finland	ND
	ITEM-840	BBA 62398	Betula verrucosa	HN	Germany	ND
	ITEM-841	BBA 64990	Buxus sp.	HN	The Netherlands	ND
	ITEM-843	BBA 64988	Hordeum vulgare	HN	Hungary	ND
	ITEM-844	BBA 64465	Triticum sp.	HN	Germany	TR
	ITEM-953	BBA 64993	Unknown	HN	The Netherlands	ND
	ITEM-839	BBA 63933	Triticum aestivum	HN	Australia	ND
F. flocciferum	KF-2108		Soil	JC	England	ND
	KF-2109		Soil	JC	England	ND
Gibbosum
F. acuminatum var. acuminatum	KF-332	ITEM-995	Potato	JC	Poland	8
	ITEM 728		Zea mays kernels	AL	Peru	ND
	ITEM-993	NRRL-13909	Aspergillus sclerotia	AL	United States	ND
	ITEM-1042	BBA 64641	Soil	HN	Denmark	ND
F. acuminatum var. armeniacum	ITEM-992	NRRL-6227	Fescue hay	SWP	United States	ND
	ITEM-797	MRC-3826	Oats	WFOM	South Africa	ND
	KF-359	NRRL-13334, ITEM-998		JC	Poland	2
F. compactum	ITEM-488		Zea mays	AL	Italy	ND
	ITEM-616		Cicer arietinum	AL	Italy	ND
	ITEM-1289		Musa sp.	AL	Cretan island	ND
F. scirpi	ITEM-1166	NRRL-13156, FRC-R6252	Soil	SWP	Australia	ND
F. equiseti	KF 403	R-7617	Corn feed	PEN	United States	ND
	KF-1011	ITEM 2892	Lycopersicon esculentum fruit	JC	Poland	12
	KF-1017	ITEM-2889	Lycopersicon esculentum fruit	JC	Poland	3
F. longipes	KF-475	R-7459, ITEM-3202	String bean	PEN	Philippines	200
	ITEM-870	NRRL-13368	Soil	SWP	Australia	ND
Liseola
F. subglutinans	ITEM-805		Musa fruit	AL	Panama	10
	ITEM-807		Musa fruit	AL	Panama	300
	ITEM-817		Musa fruit	AL	Ecuador	300
F. anthophilum	KF-391	NRRL 13286	Sugarcane	JC	India	1,300
		ITEM-3197
	KF-461	M-1134	Plantago lanceolata	PEN	United States	ND
Elegans (F. oxysporum)	KF-75	ITEM-2890	Triticum aestivum	JC	Poland	13
	KF-93	ITEM-2469	Zea mays	JC	Poland	83
	KF-1230	ITEM-2470	Zea mays stalk	JC	Poland	3,200
	ITEM-1508		Zea mays	AL	Italy	TR
	ITEM-1461		Asparagus sp.	AL	Italy	ND
	ITEM-1463		Asparagus sp.	AL	Italy	ND
	ITEM-1443		Triticum durum	AL	Italy	ND
Sporotrichiella
F. chlamydosporum	KF-333	BBA 62169	Triticum aestivum	HN	Canada	ND
F. poae	KF-1404	ITEM-2891	Zea mays	JC	Poland	36
	KF-1409	ITEM-2893	Zea mays	JC	Poland	63
	ITEM-1446		Triticum durum	AL	Italy	TR
	ITEM-1523		Zea mays	AL	Poland	20
F. sporotrichioides	ITEM-550	KF-96, ATTC 62360	Triticum aestivum	JC	Poland	ND
	ITEM-710	NRRL-3510, FRC-T345, MRC 1704	Panicum milaceum	SWP	USSR	ND
F. tricinctum	KF-248	ITEM-706	Triticum aestivum	JC	Poland	ND
	KF-260	ITEM-649	Triticum aestivum	JC	Poland	ND
Roseum (F. avenaceum)	KF-203		Triticum aestivum	JC	Poland	ND
	KF-831		Triticum aestivum	JC	Poland	ND
	KF-1215	ITEM-3187	Zea mays	JC	Poland	7
	KF-1337	DAOM 170472	Pea pod	JC	Canada	ND
	ITEM-620		Triticum aestivum	AL	Yugoslavia	ND
	ITEM-859		Triticum durum		Italy	ND
Recently described species
F. polyphialidicum	KF-464	M-2405, MRC-3389*	Citrus debris in soil	PEN	South Africa	ND
F. beomiforme	KF-1906	ITEM-3188	Soil	LWB	Australia	5
F. dlamini	KF-463	M-1637, MRC-3032*, ITEM3198	Plant debris in soil	PEN	South Africa	19
	KF-338	BBA 64596, ITEM-3199	Vitis vinifera	JC	Germany	94
F. nygamai	KF-434	M-1540, ITEM-3200	Soil debris	PEN	Australia	19
	KF-437	BBA-64375, ITEM-3201	Cajanus indicus	HN	India	3

^aFrom other collections. *, ex-holotype culture. 

^bLWB, L. W. Burgess, Fusarium Research Laboratory, University of Sydney, Sydney, Australia; JC, J. Chelkowski; AL, A. Logrieco; WFOM, W. F. O. Marasas, Programme on Mycotoxins and Experimental Carcinogenesis, Medical Research Council, Tygerberg, South Africa; PEN, P. E. Nelson, Fusarium Research Center, Department of Plant Pathology, Pennsylvania State University, University Park, Pa.; HN, H. Nirenberg, Institut für Mikrobiologie, Biologische Bundesanstalt für Land und Forstwirtschaft, Berlin, Germany; SWP, S. W. Peterson, National Center for Agricultural Utilization Research, Peoria, Ill. 

^cND, not detected. TR, trace. 

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 (λ = 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 λ of 204 nm and 0.8 μg/g at a λ 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.

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.