Trichothecene Production in Liquid Stationary Cultures of Fusarium tricinctum NRRL 3299 (Synonym: F. sporotrichioides): Comparison of Quantitative ...
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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1985, p. 656-662 Vol. 50, No. 3
0099-2240/85/090656-07$02.00/0
Copyright X3 1985, American Society for Microbiology
Trichothecene Production in Liquid Stationary Cultures of Fusarium
tricinctum NRRL 3299 (Synonym: F. sporotrichioides): Comparison
of Quantitative Brine Shrimp Assay with Physicochemical Analysis
W. W. A. BERGERS,l* J. G. M. M. VAN DER STAP,1 AND C. E. KIENTZ2
Medical Biological Laboratory' and Prins Maurits Laboratory,2 TNO, 2280 AA Rijswijk, The Netherlands
Received 1 April 1985/Accepted 18 June 1985
Stationary liquid cultures of Fusarium tricinctum NRRL 3299 (synonym: F. sporotrichioides) produce T-2
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toxin, neosolaniol, diacetoxyscirpenol, and HT-2 toxin when cultured on peptone-enriched Czapek Dox
medium. At 15 and 27°C, maximum T-2 toxin yield (265 and 50 ,ug/ml) was found after 10 to 14 and 7 days,
respectively. The T-2 toxin in the culture medium was metabolized rapidly at 27°C and slowly at 15°C. Addition
of 0.025% (wt/vol) sorbic acid to the medium resulted in an increased production of trichothecenes at 15°C (400
,ug of T-2 per ml after 14 days). Trichothecenes in the culture liquid were determined by the brine shrimp
bioassay and physicochemical analysis. The brine shrimp assay was improved by using modern bioassay
equipment, including tissue culture trays and multipipettes, and by a standardized approach with positive and
negative controls. The physicochemical analysis was based on adsorption of the trichothecenes onto Amberlite
XAD-2 columns, derivatization with trifluoroacetic anhydride followed by capillary gas chromatography, and
identification by mass spectrometry (as many as 17 trichothecenes were detected in the culture medium). The
brine shrimp assay offers an interesting monitoring system for the quantitation of T-2 toxin and should be
useful for studies on production of this toxin in culture. Specific information on less toxic trichothecenes,
however, requires a more time-consuming chemical analysis.
Severe epidemics afflicting cattle and humans have been MATERIALS AND METHODS
ascribed to mouldy foods (17). Toxigenic Fusarium strains
are believed to play a major role as causative agents since Stationary cultures of F. tricinctum NRRL 3299. Spores of
they produce a specific range of toxic trichothecenes when F. tricinctum NRRL 3299 (originating from freeze-dried
grown on various natural substrates, e.g., rice, corn, and spores obtained from the Northern Regional Research
grains. In the laboratory, trichothecenes are usually pro- Center, Peoria, Ill.) were seeded on potato glucose agar plates
duced on solid substrates, such as autoclaved rice, by and grown for 7 days at 27°C. Spores were harvested from 10
long-term (3 to 4 weeks) incubation at either low (10°C) or plates by gently scraping the surface with cotton sticks which
relatively high (25 to 27°C) temperatures (1, 3, 10). However, were then suspended in 50 ml of sterile distilled water
Fusarium strains can also be cultured in liquid semisynthetic (concentration, 107 spores per ml) and stored at -80°C. Glass
media as stationary surface cultures or as shaken submerged tissue culture flasks (4.8 by 4.8 by 11 cm) containing 40 ml of
cultures (5, 13, 19, 21). Toxic trichothecenes can also be Czapek Dox medium (Oxoid Ltd., London) supplemented
produced under these conditions, although often in much with 1% casein peptone (Oxoid) were inoculated with 6 x 105
lower quantities. The advantages of liquid media over solid spores. Sorbic acid (0.025%, wt/vol) was sometimes added to
substrates are obvious: the composition of a culture liquid the medium. The liquid cultures were incubated at 15 or 27°C
can be easily varied, and analysis of trichothecenes requires for 3 weeks. Evaporation losses were less than 10%. At
fewer purification steps. Furthermore, the filtered culture regular intervals, samples of about 1 ml of the culture liquid
medium can be used as an inexpensive source of tri- were collected with sterile Pasteur pipettes with a bent end (to
chothecenes for toxicity studies. prevent disturbance of the mould layer). The samples were
Although several studies of trichothecene production by sterilized by filtration (Millipore filter; pore size, 0.22 ,um) and
Fusarium tricinctum NRRL 3299 (synonym: F. sporo- stored at 4°C until tested. Growth characteristics were
trichioides) (1, 4, 5, 11) have been made, information about followed visually during the culture period.
liquid cultures is still scanty. In addition, methods of chem- Brine shrimp test. Brine shrimp (Artemia sp.) eggs were
ical analysis of trichothecenes are complex, and therefore bought in a tropical fish shop (distributed by San Francisco
usually only a few culture parameters are studied. In this Bay, Far East, Ltd., Hong Kong) and stored at -20°C. A
study, liquid stationary cultures of F. tricinctum NRRL 3299 quantity of 25 mg of eggs was suspended in 25 ml of brine
were investigated for trichothecene production and metabo- shrimp medium in a 50-ml plastic tissue culture flask. Brine
lism at 15 and 27°C on standard Czapek Dox liquid medium
with and without sorbic acid (7). The performance of a rapid shrimp medium was composed of 3 g of dipotassium
bioassay was compared with a comprehensive chemical glycerophosphate, 30 g of NaCl, 0.3 g of CaC12 * 2H20, 0.5 g
analysis based on direct adsorption of the trichothecenes in of MgSO4 * 7H20, 1.5 g of MgCl2 * 6H20, 0.8 g of KCI, 0.1
the culture liquid onto XAD-2 columns, derivatization, gas g of MgBr2 * 6H20, 6 g of glycine, and distilled water to
chromatography, and identification by mass spectrometry. make 1,000 ml. The pH was adjusted to 6.5 with HCl before
the medium was autoclaved for 15 min at 121°C (8). Penicillin
G (10,000 U) and streptomycin (0.01 g; Gist Brocades, Delft,
The Netherlands) were sterilized by filtration and added
*
Corresponding author. after the medium was autoclaved. The flask was placed on a
656VOL. 50, 1985 BIOASSAY AND CHEMICAL ANALYSIS OF TRICHOTHECENES 657
shaker (18 times per min) in an incubator at 30°C with 1,000 (VG Lab Systems, Altrincham, England), based on peak
lx of illumination. area, with docosane as an internal standard. Pure
After the eggs were hatched for 24 h, empty shells floating trichothecenes were used as reference standards (recoveries
on the surface of the medium were sucked off. The nauplii on application of the total analytical procedure were esti-
clustering at the bottom of the flask were pipetted off in 5 ml, mated to be 100% for T-2 toxin, HT-2 toxin, DAS,
while the nonlarviparous eggs stayed on the bottom of the neosolaniol, and 50% for T-2 tetraol). Identification was
flask. The clustered nauplii were diluted with 15 ml of fresh carried out on a VG 7070 F mass spectrometer combined
brine shrimp medium to 200 to 300 nauplii per ml, and 0.1 ml with a Varian 1400 gas chromatograph. Samples were in-
was pipetted with a 12-channel pipette into each well of a jected by a solid injector onto a CP Sil 8 CB column (50 m by
96-well tissue culture tray (Costar, Cambridge, Mass.). 0.32 mm [inner diameter]). The temperatures of the injection
Before incubation with the toxic culture filtrates, dead port and ion source were 300 and 200°C, respectively. The
nauplii were counted under a stereomicroscope (magnifica- oven temperature was programmed from 180 to 300°C at 8°C
tion, x 18). At least three dilutions of culture filtrates in the per min. A VG 2050 data system was used for data acquisi-
range of 12.5 to 1,600 times, causing 5 to 100% mortality, tion.
were made with brine shrimp medium, and 25-,u volumes Chemicals. All chemicals used were of the highest purity
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were quadruply added to the wells. The nauplii were incu- available. The mycotoxin standards-T-2 toxin, HT-2 toxin,
bated for 24 h at 30°C in the dark, and dead nauplii were T-2 tetraol, neosolaniol, DAS, and deoxynivalenol-were
thereafter counted. Viable nauplii were subsequently killed supplied by Makor Chemicals (Jerusalem, Israel). Nivalenol
by adding 10 ,l of 37% Formalin per well, and the total and fusarenon-X were generous gifts from C. J. Mirocha,
number of dead nauplii was counted. Each series of tests University of Minnesota, St. Paul.
included negative controls (brine shrimp medium only) and
two positive controls, namely, potassium dichromate and RESULTS
pure T-2 toxin. The concentrations that killed 50% of the
nauplii (LC5os) were determined from the linear response by Toxicity of culture filtrates in the brine shrimp test. The
Finney log probit analysis (6) or estimated from log probit toxicities of liquid cultures of F. tricinctum NRRL 3299
paper by connecting points above and below the 50% mor- grown at 15 and 27°C, expressed as T-2 toxin equivalents,
tality level. The death rate of control nauplii was 0 to 5% in are shown in Fig. 1. At 27°C the toxicity of the culture
all experiments. The hatching rate of the eggs was unaffected medium increased steadily for 7 days, followed by a decline
during a year of storage (an average of 80% of the eggs during 3 days. Addition of sorbic acid caused a slower onset
hatched). of the production of toxic compounds, accompanied with
Physicochemical analysis. (i) Isolation and derivatization. growth inhibition; the maximum level reached after 7 days
Trichothecenes were isolated from liquid cultures by adsorp- was slightly higher. At 15°C the culture showed a later onset
tion onto purified (12) Amberlite XAD-2 columns (Rohm and of the production of toxic compounds, increasing rapidly
Haas Co., Philadelphia, Pa.). After dilution (one to four between days 4 and 14, whereas after reaching the maximum
times) of the culture liquid with distilled water, 1 ml was level almost no decline in toxicity was ~een. With sorbic acid
transferred to a glass column (250 by 15 mm) packed with 5 in the medium, a similar picture was obtained, but the onset
g of XAD-2. The resin was washed with 50 ml of distilled of production of toxic compounds was delayed and a higher
water and dried by a flow of air through the column for 15 maximum level was reached.
min. The trichothecenes were extracted with three 10-ml Chemical analysis of trichothecenes in culture tiftrates. The
portions of acetonitrile-water (9:1). The eluates were com- results of the gas chromatographic analysis are shown in
bined and concentrated to 4 ml under a flow of nitrogen in a Tables 1 and 2. During the first week of the stationary culture
laboratory-made Kuderna-Danish apparatus at 70°C; 1 ml of at 27°C (Table 1), the concentrations of the trichothecenes
the concentrated eluate was evaporated and derivatized with T-2 toxin, HT-2 toxin, DAS, and neosolaniol increased,
0.5 ml of trifluoroacetic anhydride (Pierce Chemical Co., whereas after day 7 T-2 toxin, DAS, and neosolaniol disap-
Rockford, Ill.) in the presence of 10 mg of sodium hydrogen peared rapidly from the culture medium and increasing
carbonate. After 30 min at 80°C, 100 RI of hexane containing quantities of T-2 tetraol were formed. During the first 10
3.9 ,ug of docosane as internal standard was added, and days, the stationary cultures at 15°C (Table 2) showed an
excess trifluoroacetic anhydride was evaporated at 50°C increase of T-2 toxin, neosolaniol, and DAS, whereas after
under a gentle flow of nitrogen. The residue was extracted day 10 the decrease of T-2 toxin was accompanied by an
for 1 min with 300 RI of toluene followed by 0.5 ml of water increase of HT-2 toxin, followed by an increase of T-2
to dissolve the residue of sodium hydrogen carbonate. After tetraol.
mixing, 100 RI of the toluene layer was transferred with a Figure 2 shows isometric plots of the gas chromatograms
syringe to a dry reaction vial (Pierce) containing anhydrous of the acetylated trichothecenes. In addition to T-2 tetraol,
sodium sulfate. A sample of 1 ,ul was injected onto the gas many intermediary metabolites, which were identified by
chromatographic column. mass spectrometry, were found (Table 3). These metabolites
(ii) Gas chromatographic analysis and identification. A were deacetylated or hydrolyzed forms of T-2 toxin and
Carlo Erba 5300 Mega gas chromatograph, equipped with a DAS.
flame ionization detector and an on-column injection system, Comparison of the brine shrimp assay with chemical anal-
was used. The separation was carried out on a wall-coated, ysis. LC50s for T-2 toxin, HT-2 toxin, DAS, neosolaniol, and
open, tubular, fused-silica column (50 m by 0.32 mm [inner T-2 tetraol can be obtained from the log probit data of the
diameter], CP Sil 8 CB; Chrompack, Middelburg, The Neth- pure compounds shown in Fig. 3. T-2 toxin showed the
erlands). A temperature program from 120 to 190°C at 15°C lowest LC50. The completely deacetylated compound T-2
per min and from 190 to 260°C at 3°C per min was used, with tetraol had the lowest toxicity. The toxicity of a 1:1 mixture
the detector temperature adjusted to 300°C. Quantitation of T-2 and HT-2 toxins was compared with that of the pure
was achieved by the internal standard quantitation program compounds. The mixture gave an LC50 of 72 ng/ml (95%
of the interfaced DEC PC 3501VG Minichrom data system fiducial limits, 67 and 78 ng/ml, respectively), whereas on the658 BERGERS, VAN DER STAP, AND KIENTZ APPL. ENVIRON. MICROBIOL.
E 300-
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200
100
0
0 5 10 15 20
d ays
FIG. 1. Brine shrimp toxicity of the culture liquid of stationary cultures of F. tricinctum NRRL 3299 grown at 15 and 27°C (expressed as
T-2 toxin equivalents in micrograms per milliliter). A, 15°C, Czapek Dox peptone medium; B, 15°C, Czapek Dox peptone medium plus 0.025%
(wt/vol) sorbic acid; A', 27°C, Czapek Dox peptone medium; B', 27°C, Czapek Dox peptone medium plus 0.025% (wt/vol) sorbic acid. Data
of the duplicate cultures A and B were evaluated statistically by the Welch test. The T-2 toxin equivalents at 7, 14, 18, and 21 days were
significantly different (P = 0.027, 0.013, 0.009, and 0.023, respectively). The estimated values at 10 days were not evaluated.
basis of the separate compounds an LC50 of 86 ng/ml (95% maximum toxicity, obtained after 7 days of culturing at 27°C,
fiducial limits, 68 and 120 ng/ml) was calculated. That these is equivalent to 63 ,ug of T-2 toxin per ml. This result can be
values are not significantly different indicates that the toxic- compared with the 50 p.g/ml found by chemical analysis
ity of culture liquids measured in the brine shrimp test is the (Table 1). The toxicity remaining after 18 to 21 days of
sum of the separate trichothecene toxicities; i.e., it is di- culturing is possibly due to T-2 tetraol, which has a lower
rectly comparable with the results of the chemical analysis. intrinsic toxicity than T-2 toxin. From the brine shrimp data
From the brine shrimp data in Fig. 1 it appears that the for the culture at 15°C, a maximum toxicity was observed
TABLE 1. Concentrations of several trichothecenes in the TABLE 2. Concentrations of several trichothecenes in the
medium of a F. tricinctum NRRL 3299 stationary surface culture medium of a F. tricinctum NRRL 3299 stationary surface culture
during 3 weeks at 27°C during 3 weeks at 15°C
Concn (,ug/ml) of following compound': Concn (pLg/ml) of following compound":
No. of T-2 HT-2 T-2 No. of T-2 HT-2 T-2
days toxin toxin Neosolaniol DAS tetraol days toxin toxin Neosolaniol DAS tetraol
4 35 b 25 4 _h
7 50 15 35 10 25 7 120 25 5 -
10 15 10 20 75 10 265 5 35 15
14 10 10 145 14 185 80 35 10 25
18 10 165 18 125 85 25 5 45
21 10 200 21 140 90 30 5 65
"Minor quantities of other trichothecenes are not given. a Complete chemical analyses are given in Fig. 2.
b _, Concentration of trichothecene below 5 jig/ml. b_, Concentration of trichothecene below 5 pg/ml.VOL. 50, 1985 BIOASSAY AND CHEMICAL ANALYSIS OF TRICHOTHECENES 659
70
£0
-. 0 _
50
E
40
30
20
10
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0
time (min )
120
-
100
E 80
60 IJ f
e
40 d
c
20 b
a
0
time (min)
FIG. 2. Isometric plots of gas chromatograms of the acetylated trichothecenes from the culture liquid of a stationary culture of F.
tricinctum NRRL 3299 at 15C during 21 days. a, b, c, d, e, and f, 4, 7, 10, 14, 18, and 21 days, respectively. The numbers of the peaks at
chromatogram f correspond with the numbers of the trichothecenes in Table 3; the major peak C-22 is the internal standard docosane. The
minor peak 6 belongs to chromatogram e.
after 10 days, which is equivalent to 255 p,g of T-2 toxin per method employed by us was suited to detect B-type
ml. By chemical analysis, 265 ,ug/ml was found (Table 2). trichothecenes, such as nivalenol, as well (17). However,
these were not present in the culture filtrates. In addition to
DISCUSSION the derivates which are thought to be derived from the T-2
toxin or DAS molecule, minor quantities were found with
Abbas et al. (1) recently demonstrated that T-2 toxin, slightly different side chains (Table 3). The detection of these
neosolaniol, HT-2 toxin, and T-2 tetraol, as well as five new trichothecenes was a result of the high resolution of the
related metabolites, are formed by the same strain on rice. gas chromatographic analysis.
With the exception of acetyl T-2 the same trichothecenes, Cullen et al. (5), who studied T-2 toxin production in liquid
together with DAS and several additional metabolites, were medium by the same strain and a related F. tricinctum strain,
found in our liquid cultures. The less water-soluble acetyl detected DAS and HT-2 toxin in addition to T-2 toxin. They
T-2 toxin, although intracellularly present, may not be reported that surface cultures of F. tricinctum NRRL 3299
excreted into the culture medium. Ichinoe and Kurata (9) produce 52.5 mg of T-2 toxin per liter in Czapek Dox
applied the Booth system of classification to toxigenic medium supplemented with peptone. Although our concen-
strains. The Fusarium strain NRRL 3299, originally identi- trations are much higher than those found by these authors,
fied as F. tricinctum and later characterized as F. poae, was their figures for liquid cultures with vermiculite (14 days at
identified by them as F. sporotrichioides, a major producer 19°C) are in the same range. Differences between these
of T-2 toxin. According to their data, these strains produce results may result from differences in temperature or time
only A-type trichothecenes such as T-2 toxin, HT-2 toxin, regime or both, surface-to-volume ratio of the liquid cul-
DAS, and neosolaniol (Fig. 4). The gas chromatographic tures, or the type of peptone used. The increased production660 BERGERS, VAN DER STAP, AND KIENTZ APPL. ENVIRON. MICROBIOL.
TABLE 3. Trichothecenes identified in the culture medium of stationary cultures of F. tricinctum NRRL 3299a
Compound Rl R2 R3
1. T-2 tetraol OH OH OH
2. 15-Acetyl-T-2 tetraol OH OCOCH3 OH
3. 15-Monoacetoxyscirpenol OH OCOCH3 H
4. 8-Acetyl-T-2 tetraol OH OH OCOCH3
5. 4-Acetyl-T-2 tetraol OCOCH3 OH OH
6. 15-Isovaleryl-T-2 tetraol OH OCOCH2CH(CH3)2 OH
7. 8-Isovaleryl-T-2 tetraol OH OH OCOCH2CH(CH3)2
8. Neosolaniol OCOCH3 OCOCH3 OH
9. DAS OCOCH3 OCOCH3 H
10. 4,8-Diacetyl-T-2 tetraol OCOCH3 OH OCOCH3
11. 8-Butyryl-15-acetyl-T-2 tetraol OH OCOCH3 OCO(CH2)2CH3
12. HT-2 toxin OH OCOCH3 OCOCH2CH(CH3)2
13. 8-Isovaleryl-4-acetyl-T-2 tetraol OCOCH3 OH OCOCH2CH(CH3)2
14. 8-Butenecarbonyl-15-acetyl-T-2 tetraolb OH OCOCH3 OCOC4H7
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15. 8-Butyryl-4,15-diacetyl-T-2 tetraol OCOCH3 OCOCH3 OCO(CH2)2CH3
16. T-2 toxin OCOCH3 OCOCH3 OCOCH2CH(CH3)2
17. 8-Butenecarboxyl-4,15-diacetyl-T-2 tetraolb OCOCH3 OCOCH3 OCOC4H7
a The position of the R groups is represented in Fig. 4. The trichothecenes 1, 8, 9, 12, and 16 were identified by comparison with the mass spectra of the
trifluoroacetylated reference compounds. The identification of the other trichothecenes was based on interpretation of their mass spectral fragments.
b
The 8-butenecarbonyl side chain is presumably a decomposition product of 3'-hydroxyisovaleryl caused by the acetylation method used (14).
of trichothecenes when sorbic acid is added to the culture duced 3-acetyldeoxynivalenol is transformed into deoxy-
seems to parallel the effect of sorbic acid on the production nivalenol. They demonstrated also that, of three Fusarium
of T-2 toxin by F. acuminatum, as found by Gareis et al. (7). species, the growing mycelium of F. solani (synonym: F.
Kotsonis and Ellison (13) studied the production of T-2 sporotrichioides) most efficiently converted added 3-
and HT-2 toxins in liquid cultures of F. poae (synonym: F. acetyldeoxynivalenol into deoxynivalenol. We described the
sporotrichioides) (9). They found that T-2 toxin was pro- course of production of several trichothecenes in liquid
duced before HT-2 toxin and that hexadeuterio-T-2 toxin cultures of F. tricinctum NRRL 3299 at 15 or 27°C; similarly,
was converted by the culture to trideuterio-HT-2 toxin. we found a transient increase of T-2 toxin, DAS, and
Further, Yoshizawa and Morooka (21) found that, in a neosolaniol which was accompanied by an increase of HT-2
stationary liquid culture of F. roseum, the previously pro- toxin, T-2 tetraol, and intermediates. Our data could be
90
0
0-
70
ai
10
50
3QF
10
5
/C / /e
1
I I I I I
25 50 100 200 400 800 1600 3200
trichothecenes ng/ml
FIG. 3. Log probit data of the mortality of brine shrimp (24 h exposition at 30°C) caused by several trichothecenes found in stationary
cultures of F. tricinctum NRRL 3299. a, T-2 toxin; b, HT-2 toxin; c, DAS; d, neosolaniol; e, T-2 tetraol. The LC50 of potassium dichromate
was 30 jig/ml.VOL. 50, 1985 BIOASSAY AND CHEMICAL ANALYSIS OF TRICHOTHECENES 661
H3C of the culture filtrates, as assayed with the brine shrimp test.
The brine shrimp assay therefore offers an interesting mon-
itoring system for the quantitation of toxic trichothecenes in
liquid cultures.
ACKNOWLEDGMENTS
R3
We thank E. R. J. Wils and A. G. Hulst for their help in
cH3 I trichothecene identification by mass spectral analysis. We further
Rl acknowledge the skillful technical assistance of J. van der Berg in
R2 analytical determinations. Finally, we thank J. L. F. Gerbrandy and
A. Verweij for helpful criticism.
FIG. 4. Structural diagram of A-type trichothecenes.
LITERATURE CITED
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explained by assuming that the previously produced T-2 1. Abbas, H. K., C. J. Mirocha, and W. T. Shier. 1984. Mycotoxins
produced from fungi isolated from foodstuffs and soil: compar-
toxin is converted into HT-2 toxin, T-2 tetraol, and interme- ison of toxicity in fibroblasts and rat feeding tests. Appl.
diates during prolonged culturing. These transformations are Environ. Microbiol. 48:654-661.
clearly dependent on culture aging, which proceeds faster at 2. BUI, J., D. Dive, and C. van Peteghem. 1981. Comparison of
27°C than at 15°C. It is somewhat surprising, furthermore, some bioassay methods for mycotoxin studies. Environ. Pollut.
that the HT-2 toxin in the culture fluid was hardly metabo- Ser. A 26:173-182.
lized and that the T-2 tetraol concentration in the cultures at 3. Bottalico, A., P. Lerario, and A. Visconti. 1983. Production of
27°C seemed to increase with time. Additional studies are mycotoxins (Zearalenone, Trichothecenes and Moniliformin) by
required to explain these phenomena. Siegfried (16) ana- Fusarium species in Italy. Microbiol. Aliments Nutr. 1:133-142.
lyzed the yields of T-2 toxin and DAS in liquid cultures of F. 4. Burmeister, H. R. 1971. T-2 toxin production by Fusarium
tricinctum on solid substrate. Appl. Microbiol. 21:739-742.
tricinctum at different temperatures and found that as much 5. Cullen, D., E. B. Smalley, and R. W. Caldwell. 1982. New
as 40% of the total T-2 toxin produced after 3 weeks of process for T-2 toxin production. Appl. Environ. Microbiol.
culturing at 8°C was contained in the mycelium. Therefore, 44:371-375.
incorporation of analyses of the trichothecenes in the 6. Finney, D. J. 1980. Probit analysis. Cambridge University Press,
mycelium would give additional information on liquid cul- Cambridge.
tures. 7. Gareis, M., J. Bauer, A. van Montgelas, and B. Gedek. 1984.
Several authors have used the brine shrimp test as a Stimulation of aflatoxin B, and T-2 toxin production by sorbic
qualitative screening test for the detection of toxigenic acid. Appl. Environ. Microbiol. 47:416-418.
Fusarium strains (8, 18). This test has also been compared 8. Harwig, J., and P. M. Scott. 1971. Brine shrimp (Artemia salina
L.) larvae as a screening system for fungal toxins. Appl.
with chemical analysis (thin-layer chromatography) by Scott Microbiol. 21:1011-1016.
et al. (15). They concluded that the wide range of toxicities 9. Ichinoe, M., and H. Kurata. 1983. Trichothecene-producing
of several trichothecenes limits the usefulness of this assay. fungi, p. 73-82. In Y. Ueno (ed.), Trichothecenes: chemical,
We likewise found a wide range of toxicities for A-type biological and toxicological aspects. Elsevier/North-Holland
trichothecenes (Fig. 3). The outstanding toxicity of T-2 Publishing Co., Amsterdam.
toxin, however, enables the use of the brine shrimp test as a 10. Ishii, K. 1983. Chemistry and bioproduction of non-macrocyclic
monitoring test for the production of T-2 toxin by Fusarium trichothecenes, p. 7-19. In Y. Ueno (ed.), Trichothecenes:
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adapted for use as a quantitative test with modem bioassay Holland Publishing Co., Amsterdam.
11. Joffe, A. Z., and B. Yagen. 1977. Comparative study of the yield
equipment, including tissue culture trays and multipipettes of T-2 toxin produced by Fusarium poae, F. sporotrichioides
and by a standardized approach with positive and negative and F. sporotrichioides var. tricinctum strains from different
controls (20). The results of the chemical analysis showed a sources. Mycopathologia 60:93-97.
good correlation with the brine shrimp toxicity data, espe- 12. Junk, G. A., J. J. Richard, M. D. Grieser, D. Witiak, J. U.
cially during the production period of T-2 toxin, reflecting Witiak, M. D. Arguello, R. Vick, H. J. Svec, J. S. Fritz, and
both the predominance of T-2 toxin as well as its high G. V. Calder. 1974. Use of macroreticular resins in the analysis
intrinsic toxicity. The quantitative brine shrimp bioassay can of water for trace organic contaminants. J. Chromatogr. 99:
be compared with other potent bioassays used to quantitate 745-762.
trichothecenes, such as those based on inhibition of cell 13. Kotsonis, F. M., and R. A. Ellison. 1975. Assay and relationship
of HT-2 toxin and T-2 toxin formation in liquid culture. Appl.
growth (1), reticulocytes assay (18), or immunoassays (18). Microbiol. 30:33-37.
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