Production of Volatile Sesquiterpenes by Fusarium sambucinum ...

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1995, p. 3815–3820 0099-2240/95/$04.0010 Copyright q 1995, American Society for Microbiology

Vol. 61, No. 11

Production of Volatile Sesquiterpenes by Fusarium sambucinum Strains with Different Abilities To Synthesize Trichothecenes ´ ,1* CHESTER J. MIROCHA,1 ERWIN WA HENRYK H. JELEN ¸ SOWICZ,2

AND

´ SKI2 EDWARD KAMIN

Department of Plant Pathology, University of Minnesota, St. Paul, Minnesota 55108,1 and Institute of Food Technology, Agricultural University of Poznan ´, Poznan ´, Poland2 Received 22 June 1995/Accepted 29 August 1995

Twenty-five strains of Fusarium sambucinum grown on wheat kernels were examined for trichothecene production and the synthesis of volatile sesquiterpenes. The volatiles were purged with air and collected on Tenax traps. Adsorbed compounds were eluted from the traps and injected into a gas chromatograph coupled with a mass spectrometer. Ten strains isolated from potato tubers produced high amounts of diacetoxyscirpenol and its derivatives. These strains were characterized by the production of high amounts of diverse sesquiterpenes. In 10 cultures, 19 compounds were detected, of which 6 were predominant and composed as much as 82% of the volatile sesquiterpene fraction (e.g., b-farnesene, b-chamigrene, b-bisabolene, a-farnesene, trichodiene, and an unidentified compound). Fifteen strains isolated from various sources that did not produce trichothecenes produced much less volatile sesquiterpenes, with less chemical diversity. No more than six compounds were present in cultures. Two of these compounds were present in the toxigenic strains isolated from potatoes (b-farnesene and acoradiene), but four were unique to the strains not producing trichothecenes (longifolene, isocaryophyllene, d-elemene, and an unidentified one). The pattern of volatile sesquiterpenes was characteristic and distinctive for both toxic and nontoxic strains. Fusarium sambucinum (Fuckel), a field fungus occurring in moderate climatic zones, is distributed widely on vegetables, fruits, conifer trees, and soil (14). As a plant pathogen, along with Fusarium coeruleum (Libert) ex Sacc., it is the main species that causes dry rot of potato tubers in North America and Europe (6, 18). F. sambucinum is known to produce toxic metabolites which can be injurious to human and animal health (10). Among the toxins produced by this fungus, trichothecenes form a major group; diacetoxyscirpenol (DAS) is the major toxin, whereas monoacetoxyscirpenol (MAS), T-2 toxin, and neosolaniol are produced in lesser amounts (4, 8, 17, 19). Trichothecenes are biosynthesized from 3-R mevalonic acid in the isoprenoid pathway. The biosynthesis of trichothecenes has been studied extensively, and it is assumed that, the 12,13epoxytrichothec-9-ene skeleton, which is basic for all trichothecenes, is formed from farnesyl PPi with trichodiene and trichodiol being the key intermediates in this synthesis (7, 12, 20). Apart from trichothecenes, strains of F. sambucinum causing hematuria and death in rats were reported to produce wortmannin (1) and H-8 toxin (3). Kim and Lee (11) recently reported the presence of a novel toxin (sambutoxin) which was isolated from rotted potato tubers in Korea. Our main objective was to establish whether the isoprenoid pathway of sesquiterpene production in strains of toxigenic F. sambucinum results only in trichothecene production or, if it is less specific, whether other volatile sesquiterpenoid compounds are produced as well.

isolates (pure culture) were transferred to plates containing water agar and were grown for 14 days. The surfaces of the plates were washed with 10 ml of sterile water, the concentration of spores was adjusted to 4 3 105 spores per ml, and 3 ml of this conidial suspension was used for inoculation of the medium. Hard red spring wheat (‘‘Stoa’’ variety) was used as a solid medium. Portions of 200 g were placed in 2,000-ml Erlenmeyer flasks, and 100 ml of distilled water was added to each flask. The flasks were autoclaved for 1 h at 1218C on 2 subsequent days and then cooled and inoculated. All strains were incubated in triplicate. As a control sample, uninoculated, autoclaved wheat was used. The cultures were incubated at 258C for 5 days and were shaken daily to ensure even distribution of the inoculum. Sampling procedure. On the 5th day of incubation, the cotton stoppers in the flasks were exchanged for stoppers with a gas inlet and a gas outlet, equipped with a Tenax trap. The flasks were purged with compressed air for 1 h at a flow of 90 ml/min. To eliminate impurities, the air was passed through the Tenax trap before purging the culture. All traps were made of glass tubes (6-mm outer diameter and 4-mm inner diameter) and were filled with 120 mg of Tenax GR (60/80 mesh; Alltech Associates, Carnforth, United Kingdom), which was placed between two silanized glass wool plugs. The traps had been conditioned prior to analyses at 2308C with a nitrogen flow rate of 50 ml/min for 3 h and then cooled to ambient temperature. The connections were made with Swagelock fittings with Teflon ferules; the tubing used was Teflon. After sampling of the volatile compounds, 10 g of culture material was taken from each flask and analyzed for DAS and its derivatives. Volatile compound analysis. Volatiles adsorbed on Tenax traps were eluted with 1 ml of an isopentane–ethyl ether mixture (1/1 [vol/vol]), with tetradecane added as a standard at a concentration of 1 mg/ml. The eluate was concentrated under a gentle stream of nitrogen to approximately 20 ml, and 2 ml was injected in a splitless mode into a gas chromatograph-mass spectrometer. Data were obtained by using a Shimadzu GC-17A gas chromatograph interfaced with a Shimadzu QP-5000 mass spectrometer, operating in electron impact mode at 70 eV, in a scan range of 40 to 440 atomic mass units (amu). The compounds were separated with a DB-5 column (30 m by 0.25 mm by 0.25 mm; J&W Scientific, Folsom, Calif.). The injection port and transfer line temperatures were maintained at 240 and 2808C, respectively; the temperature program was 358C for 1 min and then increased at the rate of 58C/min to 2308C and 208C/min to 2808C. The flow rate was kept constant at 1 ml/min through pressure programming. The concentrations of volatile compounds were calculated from their peak areas found in total ion chromatograms, with tetradecane as a reference and with no correction factors. The amounts of volatiles are given in nanograms per liter of air used for purging per 200 g of culture. The spectra of identified compounds were matched with those in the computerized U.S. National Institute of Standards and Technology library and with those in the Wiley library (13). For major sesquiterpenes, high-resolution mass spectrometry was performed to confirm the empirical formula of the compounds. The chosen samples were analyzed on a Carlo Erba gas chromatograph coupled with a Kratos M-25 sector mass spectrometer. The gas chromatography parameters were carrier gas helium

MATERIALS AND METHODS Fungal cultures, inoculation, and incubation. Twenty-five strains of F. sambucinum were obtained from the Plant Pathology Department of the University of Minnesota and Institute of Food Technology, Agricultural University of Poznan ´. The strains were isolated between 1972 and 1989 in various countries and were stored on sterile soil and/or silica gel prior to the experiment. The

* Corresponding author. Present address: Institute of Food Technology, Agricultural University of Poznan ´, Wojska Polskiego 31, 60624 Poznan ´, Poland. 3815

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APPL. ENVIRON. MICROBIOL.

TABLE 1. Total volatile sesquiterpenes and trichothecenes produced by F. sambucinum strains of different originsa Trichothecene amt (mg/g) Strain

UM 8 UM 15 KF 710 UM 9 UM 14 KF 735 UM 7 UM 18 UM 17 KF 736 UM N64 UM N32B UM N35B UM 24 UM 29 UM 27 UM N37B UM N60B UM 28 UM N32A UM 38 UM N58 UM N72A UM 26 UM N71B

Commodity

Potato tuber Potato tuber Potato tuber Potato tuber Potato tuber Potato tuber Potato tuber Potato tuber Potato tuber Potato tuber Barley Barley Barley Haybarn Soil Soil Timothy Barley Soil Barley Hay Soil Grass Soil Grass

Origin

United States United States Poland United States United States Poland United States United States United States Poland Norway Norway Norway New Zealand Alaska Alaska Norway Norway Alaska Norway New Zealand Norway Norway Alaska Norway

Sesquiterpene concn (ng/liter)

4,15-DAS

15-MAS

4-MAS

Total trichothecenes

442.9b 394.1b 394.8b 318.2c 379.3b 422.9b 411.5b 284.1b 129.9b 430.0b 19.7b 18.4b 20.4b 27.6c 22.2b 23.8b 9.3b 45.6b 6.3b 10.8b 24.5b 31.8b 23.4b 18.4c 11.8b

266.9b 153.8c 237.4b 133.9b 286.6c 279.8c 218.8b 327.7b 107.9c 395.8b ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

6.2c 5.2b 5.3c 5.6b 14.1c 2.9c 4.2b 14.3b 3.9c 4.8c ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

1.3b 1.0b 1.4c 1.2c 0.9c 1.3d 1.1b 1.6b 1.1c 1.6b ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

274.4b 160.0c 244.1b 140.7b 301.6c 284.0c 224.1b 343.6b 112.9c 402.2b ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

a The contents of trichothecenes and volatile sesquiterpenes were evaluated after 5 days of incubation. Values are means of three replicates. The amounts of volatile sesquiterpenes are expressed in nanograms per liter of air used for purging. ND, trichothecenes not detected. b r, #25%. c 25% , r # 50%. d 50% , r # 75%.

FIG. 1. Total ion chromatograms (TIC) of volatile sesquiterpene fractions that are typical for F. sambucinum isolates with different abilities to produce trichothecenes. (A) Strain UM 18; (B) strain UM N60B. IS, tetradecane peak (internal standard); 1, unidentified sesquiterpene (see the fourth compound listed in Table 2); 2, b-farnesene; 3, b-chamigrene; 4, b-bisabolene; 5, trichodiene; 6, a-farnesene; 7, longifolene; 8, unidentified sesquiterpene (see the fourth compound listed in Table 4); C, compound originating from the sampling device, also present in control samples.

F. SAMBUCINUM PRODUCTION OF SESQUITERPENES

VOL. 61, 1995 TABLE 2. Sesquiterpene hydrocarbons produced by F. sambucinum UM 18 after 5 days of incubation on wheat kernels Compound (no.)

Retention time (min)

RIa

Contents (%)b

Unidentified (1) a-Bergamotene (2) Unidentified (3) Unidentified (4) Unidentified (5) Unidentified (6) Elixene (7) b-Farnesene (8) b-Santalene (9) Acoradiene (10) b-Chamigrene (11) Diepi-a-Cedrene (12) Ar-Curcumene (13) b-Himachalene (14) b-Bisabolene (15) Unidentified (16) Trichodiene (17) a-Farnesene (18) b-Selinene (19)

22.12 22.33 22.73 22.92 23.02 23.46 23.58 24.01 24.14 24.35 24.47 24.62 24.70 25.20 25.34 25.54 25.72 25.92 26.15

1387 1394 1409 1419 1422 1440 1445 1462 1468 1476 1481 1487 1490 1510 1516 1526 1533 1538 1550

0.6 0.7 0.9 3.8 3.6 0.7 0.8 7.9 1.9 2.3 5.5 1.6 2.5 1.2 20.3 3.2 17.8 22.6 2.1

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pestle, transferred to an Erlenmeyer flask, and extracted with a mixture of acetonitrile and water (40 ml; 84/16 [vol/vol]) for 1 h in a shaker. The extract was filtered through Whatman no. 4 filter paper, and 4 ml was used for the cleanup procedure. The extracts were purified with columns filled with 1.5 g of an aluminum oxide neutral (EM Science, Gibstown, N.J.) and C18 (Bakerbond octadecyl; 40-mm particle size; J. T. Baker, Inc., Philipsburg, N.J.) mixture (1/1 [wt/wt]). One milliliter of eluate was evaporated to dryness with nitrogen. To the dried sample, 100 ml of trifluoroacetic acid anhydride was added, and the sample was heated in a water bath at 608C for 20 min. After that time, the reagent was evaporated, the residue was dissolved in 1,000 ml of isooctane, and 1 ml was injected onto a gas chromatograph. The gas chromatograph was the same Shimadzu instrument that had been used for volatile compound analysis. The column used was the same DB-5 column. The injection port temperature was 2808C, the transfer line temperature was 2808C, and the analysis was performed with the programmed temperature (from 808C [1 min] to 2808C at 258C/min), the final temperature being kept for 10 min. The helium flow rate was constant at 1 ml/min. DAS and its derivatives were quantified in selected ion monitoring mode, in which the following ions were recorded: m/z 402, 374, 329 for DAS; 456 and 473 for 15-MAS; 402, 501 374 for 4-MAS; and 456 and 555 for scirpentriol. Ion m/z 402 was used for quantitation of DAS and 4-MAS, and ion 456 was used for quantitation of 15-MAS, with standard curves for standards added to a wheat matrix. To confirm the identities of the toxins, full-scan analysis in the range of 100 to 650 amu was performed, and the spectra of analyzed toxins were compared with the spectra of toxin standards.

RESULTS

a

RI, Kovacs indices calculated for sesquiterpenes analyzed on a DB-5 column at the programmed temperature (see Materials and Methods). b Values are percentages of the total sesquiterpene fraction produced.

The 25 strains of F. sambucinum that were examined differed in both trichothecene and volatile sesquiterpene production. On the basis of their abilities to synthesize trichothecenes, all strains could be placed into groups either of efficient producers or of nonproducing strains. Both groups were characterized by qualitative and quantitative differences in the amounts of volatile sesquiterpenes produced. Volatile sesquiterpenes of F. sambucinum strains producing trichothecenes. Our preliminary trials showed that the maximum sesquiterpene concentration was observed in 5- to 7-day-

at 1 ml/min, splitless injection (2 ml), an injector temperature of 2408C, and an ionization chamber temperature of 2658C. The column used was a DB-5 column (15 m by 0.25 mm by 0.3 mm; J&W Scientific). Perfluorokerosene was used for instrument calibration, and spectra were acquired at 3 s/decade in a mass range of 169 to 255 amu. Trichothecene extraction and analysis. While still moist, 10 g of inoculated culture, obtained after sampling of the volatiles, was ground with a mortar and

TABLE 3. Predominant, characteristic volatile sesquiterpenes produced by toxigenic and nontoxigenic F. sambucinum strains grown on wheat kernels Amt (%)a Strain

UM 8 UM 15 KF 710 UM 9 UM 14 KF 735 UM 7 UM 18 UM 17 KF 736 UM N64 UM N32B UM N35B UM 24 UM 29 UM 27 UM N37B UM N60B UM 28 UM N32A UM 38 UM N58 UM N72A UM 26 UM N71B a

Unknownb

b-Farnesene

b-Chamigren

b-Bisabolene

Trichodiene

a-Farnesene

Unknownc

Longifolene

4.5 4.9 4.5 3.9 4.3 4.4 4.3 3.8 4.4 4.6 ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND

10.6 8.9 8.0 9.8 7.9 9.3 10.5 7.8 6.7 10.8 9.6 7.3 16.6 17.4 22.9 10.2 TR 3.5 TR 12.0 TR 8.2 TR 10.2 ND





ND ND ND ND ND ND ND ND ND ND 57.9 40.2 36.8 39.1 34.2 44.9 24.7 43.9 59.6 20.4 22.4 30.9 35.9 38.6 59.3

ND ND ND ND ND ND ND ND ND ND 12.7 12.5 7.4 8.7 7.7 5.4 22.5 20.6 4.2 22.2 12.2 10.3 15.8 12.5 7.6

Values are percentages of the total fraction of volatile sesquiterpenes. ND, not detected. See compound 4 in Table 2. c See compound 4 in Table 4. b

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FIG. 2. Mass spectra (electron impact at 70 eV) of two of C15H24 compounds, which are characteristic for F. sambucinum strains producing high levels of trichothecenes. (A) Unidentified sesquiterpene (see fourth compound listed in Table 2; measured molecular weight of the most abundant C15H24 isotope, 204.1819 6 0.0062); (B) trichodiene (measured molecular weight of the most abundant C15H24 isotope, 204.1870 6 0.0045).

old cultures, depending on the strain. We chose 5 days for sampling both volatiles and toxins, even though a longer period of incubation usually increases the yield of trichothecenes. Ten strains produced high levels of trichothecenes, ranging from 112.9 to 402.2 ppm, in the 5-day time (Table 1). All of these strains were isolated from potato tubers with dry rot symptoms in either the United States (in 1974 to 1981) or Poland (1986 to 1987). Of the trichothecenes examined, 4,15-DAS was the major toxin, whereas 15-MAS and 4-MAS were detected in relatively small amounts. Scirpentriol was present in trace amounts in two of the samples. The volatile sesquiterpene fraction eluted between 22 and 27 min (Fig. 1) and consisted of 19 compounds. They were present in every culture, although the concentrations of some of them were very low. The percent abundance and retention indices of typical sesquiterpene fraction are given for culture UM 18 in Table 2. There were no volatile sesquiterpenes observed in a control sample (autoclaved, uninoculated wheat). The compounds exhibited mass spectra characteristic for C15H24 hydrocarbons, i.e., the presence of molecular ion at m/z 204, product of its demethylation (m/z 189), or other ions characteristic for sesquiterpene fragmentation ions (m/z 175, 161, 119, 105, 93, 67, 55). All but one compound were identified as a C15H24 hydrocarbons; the only exception was Ar-curcumene, which had the formula C15H22. The mass spectra of all of the compounds were compared with those in libraries; however, identification of some of them was not possible on the basis only of mass spectral matches. Six compounds were predominant, forming 71 to 82% of the total sesquiterpenes detected in

FIG. 3. Profiles of volatile sesquiterpenes produced by 10 toxigenic strains of F. sambucinum isolated from potato tubers. Compound numbers correspond to those listed in Table 2. Concentrations are in nanograms per liter of air used for purging volatiles from the culture.

VOL. 61, 1995

FIG. 4. Profiles of volatile sesquiterpenes produced by nontoxigenic strains of F. sambucinum. Compound numbers correspond to those listed in Table 4. Concentrations are given in nanograms per liter of air used for purging volatiles from the culture. Compounds 5 and 6 are identical to compounds 8 and 10, respectively, found in Fig. 3.

the strains examined (Table 3). Five of these were identified as b-farnesene, b-chamigrene, b-bisabolene, trichodiene, and a-farnesene. The unidentified compound (the fourth compound in Table 2) had a spectrum with a relatively intensive ion at m/z 204 and a characteristic, abundant m/z 108 (Fig. 2). The compound identified tentatively as trichodiene showed a characteristic spectrum with a base peak at m/z 109 (Fig. 2), originating from the cleavage between the two rings forming this molecule. The intensity of higher mass ions up to 204 amu (the highest mass observed) was very weak. The trichodiene was not present in searched spectral libraries; however, the mass spectral data agreed with data cited elsewhere (9). To confirm the elemental composition of the predominant sesquiterpenes, high-resolution mass spectrometry was performed. For the unidentified compound, the exact mass measured for the most abundant ion at m/z 204 was 204.1819 6 0.0062. The masses for b-farnesene, b-chamigrene, b-bisabolene, trichodiene, and a-farnesene were 204.1802 6 0.0019,

F. SAMBUCINUM PRODUCTION OF SESQUITERPENES

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204.1827 6 0.0049, 204.1867 6 0.0064, 204.1870 6 0.0045, and 204.1851 6 0.0038, respectively. The theoretical mass for the most abundant ion for C15H24 is 204.1878; therefore, the empirical formula for analyzed compounds was assumed to be correct. The profiles of volatile sesquiterpenes were similar for all examined toxigenic isolates (Fig. 3), despite the different yields of sesquiterpenes from different strains. When the values for relative abundance (as percentages) of sesquiterpenes produced among strains were compared, the numbers varied within a relatively narrow range, i.e., b-farnesene formed 6.7 to 10.8% of the sesquiterpene fraction, b-chamigrene formed 5.3 to 6.0%, b-bisabolene formed 17.1 to 22.7%, trichodiene formed 15.4 to 19.1%, and a-farnesene formed 21.1 to 25.9% (Table 3). Volatile sesquiterpenes of F. sambucinum strains not producing trichothecenes. Fifteen of the F. sambucinum strains, which were isolated from various commodities in Alaska, Norway, and New Zealand, did not produce trichothecenes (2), and their efficiency in volatile sesquiterpene production was several times lower than that found in toxigenic strains (Table 1). The profiles of volatile sesquiterpenes were also quite different from those of the toxigenic isolates (Fig. 4), i.e., only as many as six sesquiterpenes were present in cultures: isocaryophyllene, longifolene, d-elemene, b-farnesene, acoradiene, and an unidentified one (Table 3). Two of the sesquiterpenes (bfarnesene and acoradiene) were detected also in cultures of toxigenic F. sambucinum isolated from potatoes. The remaining sesquiterpenes were not detected in toxigenic strains. Isocaryophyllene, b-farnesene, and d-elemene were not detected in some cultures; however, this may be associated with the low yield of the sesquiterpene fraction produced by these isolates. The identities of isocaryophyllene and longifolene were confirmed with standards of these compounds. The unidentified compound (fourth compound listed in Table 4) was the most abundant constituent in all but two cultures, reaching 22.4 to 59.6% of the sesquiterpene fraction. Its mass spectrum (Fig. 5) resembles that of trans-caryophyllene, with only minute differences in m/z 120, 106, and 93 ion intensities. However, the retention time of this compound did not match that of the trans-caryophyllene standard. Striking similarities in mass spectra suggest that it may be a different isomer of caryophyllene. DISCUSSION All strains isolated from potatoes were able to synthesize large amounts of DAS and its derivatives. The high amounts of volatile sesquiterpenes produced by these strains show that

FIG. 5. Mass spectrum (electron impact at 70 eV) of the predominant volatile C15H24 compound (see the fourth compound listed in Table 4) that is characteristic for F. sambucinum strains not producing trichothecenes.

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TABLE 4. Sesquiterpene hydrocarbons produced by F. sambucinum UM N60B after 5 days of incubation on wheat kernels Compound (no.)

Retention time (min)

RIa

Content (%)b

Isocaryophyllene (1) Longifolene (2) d-Elemene (3) Unidentified (4) b-Farnesene (5) Acoradiene (6)

22.64 22.80 23.00 23.63 24.01 24.35

1406 1413 1421 1447 1462 1474

11.2 20.6 9.6 43.9 3.5 11.2

a

to be a stable characteristic of F. sambucinum on the basis of its metabolites. The detection of trichodiene in vapors over the toxigenic strain cultures shows that this compound can be isolated along with other sesquiterpenes by a purge and trap method. Its association with trichothecene biosynthesis makes trichodiene an important volatile marker in monitoring fungal contamination. ACKNOWLEDGMENT

RI, Kovacs indices calculated for sesquiterpenes analyzed on a DB-5 column at the programmed temperature (see Materials and Methods). b Percentages of the total sesquiterpene fraction produced.

We thank Tom Krick of the Biological Sciences Department, University of Minnesota, for assistance in obtaining high-resolution mass spectra of analyzed compounds.

other compounds of isoprenoid origin are formed along with trichothecenes. It is assumed (20) that the biosynthesis of trichothecenes commences with the formation of farnesyl PPi from three molecules of mevalonate. The first unique sesquiterpene intermediate in the formation of the trichothecene skeleton is the trichodiene (7, 15, 16). Trichodiene is considered a precursor of trichodiol and 12,13-epoxytrichothec-9-ene (12, 20). The presence of b-farnesene was noted both for toxigenic and for nontoxigenic strains, since farnesyl PP1 is a common substrate for the synthesis of all sesquiterpenes. Its concentration was higher in toxigenic strains, which is associated with high efficiency in sesquiterpene production by these strains. The production of trichodiene was characteristic only of the toxigenic strains, and this compound formed a significant portion of the sesquiterpene fraction. In all strains examined, trichodiene and b-farnesene were accompanied by other characteristic sesquiterpenes. The nontoxigenic strains were isolated in different countries, and all exhibited similar patterns of volatile sesquiterpene production, (Fig. 4). The ranges in which the sesquiterpenes were present were wider than those of compounds isolated from potatoes (e.g., longifolene formed 4.2 to 22% of the sesquiterpene fraction and b-farnesene formed 0 to 22.9%) (Table 3). The nontoxigenic strains in our experiment were those that did not produce trichothecenes, although nontrichothecene-producing strains were shown to be toxic to rats (2) and to be capable of producing toxic metabolites of a nonsesquiterpenoid character (1, 3). The sesquiterpene fraction of aflatoxigenic Aspergillus strains was examined by Zeringue et al. (21), who observed a correlation between the initiation of aflatoxin biosynthesis and sesquiterpene production; however, the pattern of sesquiterpenes produced was unique to each strain, not to the entire set of toxigenic isolates. The presence of a sesquiterpene fraction in volatiles of Fusarium culmorum was reported by Bo ¨rjesson et al. (5), but the compounds were not identified. The volatile sesquiterpenes produced by F. sambucinum (and other Fusarium spp.) have not been characterized in the literature. Our experiment revealed that F. sambucinum isolates are a source of many volatile sesquiterpenes, which form their chemical ‘‘fingerprints.’’ This term can be found in many papers on volatile compounds of microbial origin. In most cases, the volatile metabolites produced vary among species and can also vary among the isolates within the same species. For the isolates analyzed in our study, we found that within one species there were only two possible patterns of sesquiterpenes produced, which were associated with strain toxigenicity; this seems

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