Vol. 56, No. 2
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1990, p. 520-525
0099-2240190/020520-06$02.00/0 Copyright © 1990, American Society for Microbiology
Absence of Trichothecenes in Toxigenic Isolates of Fusarium moniliformet C. J. MIROCHA,l* H. K. ABBAS,' AND R. F. VESONDER2 Department of Plant Pathology, University of Minnesota, St. Paul, Minnesota 55108,' and Agricultural Research Service, U. S. Department of Agriculture, Peoria, Illinois 616042 Received 11 August 1989/Accepted 18 November 1989
Thirty-four isolates of Fusarium moniliforme were obtained from cereal grains collected in various parts of the world. The isolates were grown on rice and tested as a diet for toxicity to rats. Of these isolates, 53% caused death, 12% caused congestion and hemorrhage of the stomach and intestine as well as hematuria, 21% caused diarrhea, 38% caused weight loss, and 9% were nontoxic. The cultures were tested for T-2, HT-2, neosolaniol, acetyl-T-2, T-2-tetraol, iso-T-2, diacetoxyscirpenol, monoacetoxyscirpenol, deoxynivalenol, nivalenol, fusarenone-X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, zearalenone, moniliformin, fusarochromanone, fusarin-C, and wortmannin; all were negative. In addition, F. moniliforme NRRL A25820 was grown on corn and banana fruit as solid substrates as well as on a defined liquid medium; none of the above toxins were found. When F. moniliforme NRRL A25820 was incorporated into a rat diet, no toxicity was noted. Twenty-eight additional isolates of F. moniliforme, isolated from feed associated with equine leukoencephalomalacia, were grown on cracked corn for 2 weeks. The cultures were negative when tested for deoxynivalenol, 15acetyldeoxynivalenol, diacetoxyscirpenol, monoacetoxyscirpenol, nivalenol, and fusarenone X. Seventy-five percent of the isolates were toxic to ducklings, indicating the presence of a toxin other than trichothecenes. Our results support the conclusion that F. moniliforme does not produce trichothecenes. Fusarium moniliforme Sheldon is frequently isolated from cereal grains; in corn (Zea mays L.) it is systemic and, although not readily visible, it can be isolated from almost all seeds (7, 13). Certain isolates of F. moniliforme produce toxic metabolites that affect experimental animals such as ducklings, mice, rabbits, and rats (2, 11, 12, 17, 20; R. F. Vesonder, Abstr. Phytopathology 1986, vol. 76, p. 1147). Some of the mycotoxins demonstrated and accepted as possible etiologic toxic agents are moniliformin, fusarin-C, fusaric acid, fumonisin, and the phytoestrogen zearalenone (5, 9, 11, 17). This organism has been associated with human esophageal cancer in Africa (15, 16) and China (14, 25). Isolates of F. moniliforme cause equine leukoencephalomalacia (4, 23); mycotoxins called fumonisins have been suggested as possible etiologic agents (5). Some but not all isolates of F. moniliforme, when grown in the laboratory in pure culture and fed to experimental animals as their sole diet, have been found to be toxic. However, some authors claim that F. moniliforme produces trichothecenes and that in some cases the concentrations may be sufficient to account for toxicity to animals (6, 10). However, there has been no verification that F. moniliforme produces trichothecenes. We believe that this species does not produce trichothecenes, and this thesis is the subject of this paper. Identification of F. moniliforme is as defined by Nelson et al. (19). To this end, we have gathered isolates of F. moniliforme from various sources and tested them for both toxicity and production of trichothecenes. Among these are 34 isolates obtained from feed samples associated with mycotoxicoses and 28 isolates from feed associated with leukoencephalomalacia.
Ghosal et al. (10) reported the occurrence of diacetoxyscirpenol and T-2 toxin from moldy corn found in the field and infected with F. moniliforme. However, they failed to demonstrate that F. moniliforme found in the corn produced the trichothecenes they found. A more likely candidate is Fusarium graminearum, which is almost always found in infected corn in the field and produces trichothecenes as well as zearalenone. Chakrabarthi and Ghosal (6) reported the presence of trichothecolone, diacetoxyscirpenol, and T-2 toxin from a culture of F. moniliforme grown in the laboratory on a synthetic medium as well as from a banana fruit infected with F. moniliforme. We attempted to obtain their isolate for confirmation purposes, but they refused to collaborate. Moreover, the Commonwealth Mycological Institute of Kew did not have a viable specimen of their isolate available for testing. Richardson (K. E. Richardson, Ph.D. dissertation, North Carolina State University, Raleigh, 1986) claimed the production of T-2, T-2-tetraacetate, acetyl-T-2, and iso-T-2 by F. moniliforme NRRL A-25820. The isolate used by Richardson was obtained from P. B. Hamilton (Poultry Science, North Carolina State University, Raleigh) and grown on various substrates in an effort to confirm his report. F. moniliforme is ubiquitous and widespread in corn growing in the field. The claim that this species produces trichothecenes is important because of its implication in public and animal health. For this reason, we thoroughly investigated the ability of this species to produce trichothecenes. We used at least three organic substrates and monitored 13 different trichothecenes to test our hypothesis. MATERIALS AND METHODS
*
Corresponding author.
Isolation and sources of F. moniliforme. The sources and code numbers of the isolates used in these studies are given in Table 1. Corn kernels or fragments of animal feed were
t Published as paper no. 16,960 of the contribution series of the Minnesota Agricultural Experiment Station based on research conducted under project 22-34. 520
ABSENCE OF TRICHOTHECENES IN F. MONILIFORME
VOL. 56, 1990
521
TABLE 1. Toxicity of F. tnonilifiotne isolates when grown on rice and fed to rats at 50% of an otherwise balanced diet Country oar or staite of state of
Source isolatte w'hich Avi sgs' lge (g) g no. of ~Code No. of died rats cAnge Country Toxic signs" Source origin"isltwhcdidcag g
Control 1 Control 2 China Haiti New Caledonia
Rice Corn Corn
South Africa
Pure cultures
South Korea
Corn
Taiwan
Pure cultures
United States Arizona Minnesota
Corn Animal feed
21 19
Maryland
Pure cultures
North Carolina District of Columbia
Pure culture Animal feed
R-1 651 7 68 MRC 1471 MRC 1920 K-3 K-4 PSE 6-1 PSA 5-3 PRD 3-3 PRC 3-3 PKH 5-3 PKD 6-4
+3
N.E. +L
+3 +3 +3
N.E. N.E.
+H.S,ln
+L
+3 +1 +3 +3 +3 +3
Rm 22.pl.12 83-19560-1 83-19560-2 83-19560-3 83-19560-4 83-19560-5 20153-A 20153-B 207965#1 #23 #33 A25820 FS#833-1 FS#833-2
FS#833-3 FS#833-4 FS#833-5 FS#833-6 FS#833-7
+3 +3 +2 +3
+L +L +L +L +L +L +L ND ND N.E. +H
+1
+L,D +L,D +2,D +L,D +D,H
+1 +1
+D +D
12 ND -6 ND ND ND 5 -12 ND ND ND ND ND ND -12 -17 -17 -7 -16 -18 -13.4 ND ND
ND ND 17 -10 -6 - 12 -7 ND ND ND
Two types of controls were used: rats fed a complete rat diet (Control 1) and rats fed an autoclaved rice-complete diet (1:1) (Control 2). Three rats were used for each treatment. Isolates of fungi were obtained from the following donors: China, the District of Columbia. and Haiti, C. J. Mirocha, 1981 to 1987: New Caledonia. D. Laurent, 1983: South Africa, W. F. 0. Marasas, 1985; Seoul. Korea, S. Ohh, Ginseng and Tobacco Institute; Nankang, Taipei, Taiwan. T. Tseng. Institute of Botany Academia Sinica; Maryland, G. Bean. University of Maryland. College Park; and North Carolina. P. Hamilton, North Carolina State University, Raleigh. -, No detectable toxic effect; +, definite detectable toxic effect: N.E., not evaluated because of autolysis; L, body weight lost; H, hematuria; S, stomach hemorrhage; In, intestinal hemorrhage; D, diarrhea; ND, not determined because death occurred.
selected, shaken in 2.5% NaOCI for 1 min, rinsed in sterile distilled water, and transferred to acidified potato dextrose agar and pentachloronitrobenzene agar supplemented with aureomycin, a medium selective for Fiusariiim species (18). Cultures were transferred to acidified potato dextrose agar and carnation leaf agar and examined for F. moniliforme 10 to 14 days later (19). Stock cultures of F. moniliforme isolated were maintained on moist autoclaved soil and stored at - 15°C. Preparation of F. moniliforme extracts. Crude fungus culture extracts were prepared as described elsewhere (3). Ground moldy rice (20 g) was used for trichothecene and zearalenone analyses. The moldy rice was extracted three times for 1 h with 75, 75, and 50 ml of 50% aqueous methanol on a shaker at high speed. The combined extracts (200 ml) were filtered through Whatman no. 4 filter paper and cleaned up by the methods described by Yoshizawa et al. (24). Separate samples of ground moldy rice (20 g) were extracted to determine moniliformin, fusarin-C, fusarochromanone (TDP-1), and hemorrhagic factor (wortmannin) by the methods of Scott and Lawrence (22), Gelderblom et al. (9), Pathre et al. (21), and Abbas and Mirocha (1), respectively. Toxins
were detected on precoated (0.25 mm thick) silica gel 60 plates without fluorescent indicator (E. Merek, Darmstadt, Federal Republic of Germany) (3). Toxins were confirmed by combined gas chromatography-mass spectroscopy (GCMS), using the full mass spectrum obtained in electron impact at 70 eV. Moniliformin was confirmed by its UV
spectrum.
Mycotoxin references. The sources of diacetoxyscirpenol, monacetoxyscirpenol, scirpenol, T-2 toxin, neosolaniol, HT2 toxin, acetyl-T-2 toxin, T-2 tetraol, iso-T-2, deoxynivalenol, nivalenol, fusarenone-X, 3-acetyldeoxynivalenol, 15acetyldeoxynivalenol, zearalenone (F-2), and moniliformin were those reported by Abbas et al. (3). Fusarochromanone (TDP-1) was produced and purified in our laboratory. Hemorrhagic factor (wortmannin) was produced and purified by Abbas and Mirocha (1). Fusarin-C was a gift of Leonard F. Bjeldanes, University California-Berkeley, Department of Nutritional Science, Berkeley, Calif. Rat feeding tests. The ground culture mixed 1:1 with a complete rat diet was fed to 20-day-old white virgin female Sprague-Dawley rats (Bio-Lab Corp., St. Paul, Minn.) housed in individual cages. The animals and feed were
522
MIROCHA ET AL.
weighed at the beginning and the end of the experiment. Three rats were used for each fungal isolate and the control in which rats were fed autoclaved rice and a complete rat diet (1:1 ratio). Rats were observed frequently for 5 days, and symptoms and days of death were recorded. Surviving rats were sacrificed by cervical dislocation and examined for gross pathological changes in tissues, including stomach, lungs, liver, heart, uterus, thymus, brain, bladder, kidney, and intestines. Duckling feeding test. One-day-old ducklings (five per treatment) were fed rations containing a corn medium on which F. moniliforme was grown. Cracked corn (300 g), contained in 3.8-liter flasks and with a moisture content of -60%, was autoclaved for 0.5 h, seeded with the appropriate Fusarium isolate, and grown for 2 weeks at 25°C. The cultures were harvested and air dried in a hood, mixed with Purina chick starter in a ratio of 1:1 by weight, and ground in a Wiley mill with 2-mm mesh. Two groups of five ducklings (controls) were fed Purina chick starter alone, and the other group was offered Purina chick starter with ground yellow corn (1:1 ratio). The test was conducted for 7 days, and then the number of deaths was recorded. Treatment of isolate F. moniliforme A-25820. (i) Source of fungus. A pure culture of F. moniliforme NRRL A-25820 (reported to produce trichothecenes) was received on 1 January 1987 from Pat Hamilton, North Carolina State University, Raleigh. The fungus was transferred to potato dextrose agar and carnation leaf agar for identification and to check for contamination. The fungus was also grown on various substrates such as banana, corn, rice, and liquid medium (Czapek Dox broth containing 1% peptone) and tested for toxicity in rat feeding and intubation tests. Extracts of these samples were tested for trichothecene production and other selected metabolites. (ii) Growth of fungus. All of the isolates were grown on a solid rice substrate as described by Abbas et al. (3). After 21 days of growth, the rice cultures were fragmented, transferred to a screen-bottomed tray, and allowed to air dry in a ventilated hood. The moldy rice was ground to the consistency of flour by using a coffee grinder in a fume hood, mixed 1:1 with a ground complete rat diet, and fed to rats. The same fungal isolates were grown on a corn kernel substrate as described above and checked for toxicity to rats.
The fungal isolates were grown on a banana fruit substrate in which five yellow bananas (with intact peel) were treated with absolute ethanol, flamed, and then inoculated with the Fusarium isolates. Other control banana fruits were treated as described above but not inoculated. The bananas were then incubated for 2 weeks at 25°C under moist conditions. The molded bananas were frozen for 24 h, broken up into small disks, transferred to a screen-bottomed tray, and allowed to air dry for 3 to 4 days in a ventilated hood. The molded as well as control bananas were each ground and then mixed separately 1:1 with a complete rat diet and fed to rats as described above. The same fungal isolates were grown on a liquid medium (Czapek Dox broth containing 1% peptone) in 500-ml flasks, each containing 200 ml of medium. The cultures were incubated at 27 to 29°C for 21 days. At harvest, the cultures were filtered through Whatman no. 4 filter paper, and a 1-ml portion was administered in a single oral dose to 20-day-old virgin Sprague-Dawley rats (Bio-Lab Corp.) by gastric intubation. Three rats were used for this experiment and observed frequently for 24 h after being dosed, and major signs of toxicity were recorded.
APPL. ENVIRON. MICROBIOL.
(iii) Qualitative detection of toxins in culture extracts. Molded rice or corn culture (20 g) was extracted with 50% ethyl acetate in acetonitrile (100, 75, and 75 ml) three times for 1 h each time in a high-speed automatic shaker. The extracts were filtered through Whatman no. 4 filter paper, and the combined filtrates (250 ml) were evaporated on a rotary evaporator at 45°C. The residue was dissolved in 50 ml of acetonitrile and defatted with 100 ml of petroleum ether (60 to 70°C) twice for 1 min each time. The acetonitrile layer was evaporated on a rotary evaporator and redissolved in 10 ml of chloroform-methanol (9:1, vol/vol). Half of this material was used for thin-layer chromatography (TLC) analysis, and the remainder was used for GC-MS analysis. A portion equivalent to 2 g of culture was saved for chemical identification of mycotoxins. The extracts were spotted on a TLC plate for the detection of trichothecenes (see Table 2) and zearalenone and wortmannin, using methods of detection described by Abbas et al. (3). All samples were extracted for moniliformin with acetonitrile-H20 (95:5, vol/vol) as described by Scott and Lawrence (22). The banana samples were homogenized with MeOH-H20 (55:45 1% N NaCl) for 5 min and filtered through Whatman filter paper no. 4. The filtrates were defatted twice with n-hexane and then reextracted three times with dichloromethane. The combined dichloromethane layers were evaporated I o dryness on a rotary evaporator, and the residue was dissolved in 10 ml of chloroform-methanol (9:1, vol/vol). Half of this material was used for TLC analysis, and the other half was used for analysis by GC-MS. The liquid culture media were extracted as described above for the banana samples, except that the extracts were filtered through ca. 5 cm (40 g) of anhydrous Na2SO4 and glass wool in a glass funnel. The anhydrous Na2SO4 WaS washed with 30 ml of dichloromethane. The combined dichloromethane extracts were evaporated to dryness on a rotary evaporator. The residue was dissolved in 10 ml of chloroform-MeOH (9:1, vol/vol) and used for both GC-MS and TLC analyses. The chemical identities of the trichothecenes, zearalenone, and wortmannin in culture extracts were confirmed by TLC analysis and by derivatizing with trifluoroacetic acid (Pierce Chemical Co., Rockford, Ill.) and subsequent analysis by GC-MS, using both selected ion monitoring and full electron impact mass spectra. The chemical identity of moniliformin was confirmed by TLC analysis, using visual comparison with standards, and by UV spectroscopy. Rice cultured with F. moniliforme NRRL A-25820 for 28 days at 25°C was dried overnight at ambient temperature in a forced-air oven and then finely ground in a Waring blender. The ground rice (100 g) was extracted twice with CH30H (5 ml/g), the solutions were combined, and the volume was reduced to about half on a rotary-vacuum apparatus at 400C. NaCl (5%) was added, and the solution was partitioned twice with ethyl acetate. The ethyl acetate extracts were pooled and evaporated to dryness. The remaining residue was analyzed by GC-MS for trichothecenes by conversion to their trimethylsilyl ethers. The residue from the ethyl acetate was dissolved in 95% ethanol, and the pH was adjusted to 12 with 0.2 N NaOH. The solution was heated at 60 to 80°C for 0.5 h and then cooled to room temperature, and the pH was adjusted to 7. The resulting solution was partitioned twice with ethyl acetate. The ethyl acetate extracts were pooled and taken to dryness, and the residue was acetylated with trifluoroacetic anhydride in pyridine at 50°C for 1.5 h. The reaction mixture was taken to dryness, and the remaining residue was ana-
TABLE 2. Mycotoxins produced by Fusarium species on various substrates grown in the laboratory Fusarium species
523
ABSENCE OF TRICHOTHECENES IN F. MONILIFORME
VOL. 56, 1990
Rice
Toxin concn (ppm Corn
F. graminearum Zea (685) #124 ND F. moniliforme A-25820b F. poae N.F. 5098 T-2 (600) HT-2 (50) T-2-TOL (10)
[ig/lg]) ona: Banana
Zea (570)
Zea (8.0)
ND
ND
T-2 (
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1990, p. 520-525
0099-2240190/020520-06$02.00/0 Copyright © 1990, American Society for Microbiology
Absence of Trichothecenes in Toxigenic Isolates of Fusarium moniliformet C. J. MIROCHA,l* H. K. ABBAS,' AND R. F. VESONDER2 Department of Plant Pathology, University of Minnesota, St. Paul, Minnesota 55108,' and Agricultural Research Service, U. S. Department of Agriculture, Peoria, Illinois 616042 Received 11 August 1989/Accepted 18 November 1989
Thirty-four isolates of Fusarium moniliforme were obtained from cereal grains collected in various parts of the world. The isolates were grown on rice and tested as a diet for toxicity to rats. Of these isolates, 53% caused death, 12% caused congestion and hemorrhage of the stomach and intestine as well as hematuria, 21% caused diarrhea, 38% caused weight loss, and 9% were nontoxic. The cultures were tested for T-2, HT-2, neosolaniol, acetyl-T-2, T-2-tetraol, iso-T-2, diacetoxyscirpenol, monoacetoxyscirpenol, deoxynivalenol, nivalenol, fusarenone-X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, zearalenone, moniliformin, fusarochromanone, fusarin-C, and wortmannin; all were negative. In addition, F. moniliforme NRRL A25820 was grown on corn and banana fruit as solid substrates as well as on a defined liquid medium; none of the above toxins were found. When F. moniliforme NRRL A25820 was incorporated into a rat diet, no toxicity was noted. Twenty-eight additional isolates of F. moniliforme, isolated from feed associated with equine leukoencephalomalacia, were grown on cracked corn for 2 weeks. The cultures were negative when tested for deoxynivalenol, 15acetyldeoxynivalenol, diacetoxyscirpenol, monoacetoxyscirpenol, nivalenol, and fusarenone X. Seventy-five percent of the isolates were toxic to ducklings, indicating the presence of a toxin other than trichothecenes. Our results support the conclusion that F. moniliforme does not produce trichothecenes. Fusarium moniliforme Sheldon is frequently isolated from cereal grains; in corn (Zea mays L.) it is systemic and, although not readily visible, it can be isolated from almost all seeds (7, 13). Certain isolates of F. moniliforme produce toxic metabolites that affect experimental animals such as ducklings, mice, rabbits, and rats (2, 11, 12, 17, 20; R. F. Vesonder, Abstr. Phytopathology 1986, vol. 76, p. 1147). Some of the mycotoxins demonstrated and accepted as possible etiologic toxic agents are moniliformin, fusarin-C, fusaric acid, fumonisin, and the phytoestrogen zearalenone (5, 9, 11, 17). This organism has been associated with human esophageal cancer in Africa (15, 16) and China (14, 25). Isolates of F. moniliforme cause equine leukoencephalomalacia (4, 23); mycotoxins called fumonisins have been suggested as possible etiologic agents (5). Some but not all isolates of F. moniliforme, when grown in the laboratory in pure culture and fed to experimental animals as their sole diet, have been found to be toxic. However, some authors claim that F. moniliforme produces trichothecenes and that in some cases the concentrations may be sufficient to account for toxicity to animals (6, 10). However, there has been no verification that F. moniliforme produces trichothecenes. We believe that this species does not produce trichothecenes, and this thesis is the subject of this paper. Identification of F. moniliforme is as defined by Nelson et al. (19). To this end, we have gathered isolates of F. moniliforme from various sources and tested them for both toxicity and production of trichothecenes. Among these are 34 isolates obtained from feed samples associated with mycotoxicoses and 28 isolates from feed associated with leukoencephalomalacia.
Ghosal et al. (10) reported the occurrence of diacetoxyscirpenol and T-2 toxin from moldy corn found in the field and infected with F. moniliforme. However, they failed to demonstrate that F. moniliforme found in the corn produced the trichothecenes they found. A more likely candidate is Fusarium graminearum, which is almost always found in infected corn in the field and produces trichothecenes as well as zearalenone. Chakrabarthi and Ghosal (6) reported the presence of trichothecolone, diacetoxyscirpenol, and T-2 toxin from a culture of F. moniliforme grown in the laboratory on a synthetic medium as well as from a banana fruit infected with F. moniliforme. We attempted to obtain their isolate for confirmation purposes, but they refused to collaborate. Moreover, the Commonwealth Mycological Institute of Kew did not have a viable specimen of their isolate available for testing. Richardson (K. E. Richardson, Ph.D. dissertation, North Carolina State University, Raleigh, 1986) claimed the production of T-2, T-2-tetraacetate, acetyl-T-2, and iso-T-2 by F. moniliforme NRRL A-25820. The isolate used by Richardson was obtained from P. B. Hamilton (Poultry Science, North Carolina State University, Raleigh) and grown on various substrates in an effort to confirm his report. F. moniliforme is ubiquitous and widespread in corn growing in the field. The claim that this species produces trichothecenes is important because of its implication in public and animal health. For this reason, we thoroughly investigated the ability of this species to produce trichothecenes. We used at least three organic substrates and monitored 13 different trichothecenes to test our hypothesis. MATERIALS AND METHODS
*
Corresponding author.
Isolation and sources of F. moniliforme. The sources and code numbers of the isolates used in these studies are given in Table 1. Corn kernels or fragments of animal feed were
t Published as paper no. 16,960 of the contribution series of the Minnesota Agricultural Experiment Station based on research conducted under project 22-34. 520
ABSENCE OF TRICHOTHECENES IN F. MONILIFORME
VOL. 56, 1990
521
TABLE 1. Toxicity of F. tnonilifiotne isolates when grown on rice and fed to rats at 50% of an otherwise balanced diet Country oar or staite of state of
Source isolatte w'hich Avi sgs' lge (g) g no. of ~Code No. of died rats cAnge Country Toxic signs" Source origin"isltwhcdidcag g
Control 1 Control 2 China Haiti New Caledonia
Rice Corn Corn
South Africa
Pure cultures
South Korea
Corn
Taiwan
Pure cultures
United States Arizona Minnesota
Corn Animal feed
21 19
Maryland
Pure cultures
North Carolina District of Columbia
Pure culture Animal feed
R-1 651 7 68 MRC 1471 MRC 1920 K-3 K-4 PSE 6-1 PSA 5-3 PRD 3-3 PRC 3-3 PKH 5-3 PKD 6-4
+3
N.E. +L
+3 +3 +3
N.E. N.E.
+H.S,ln
+L
+3 +1 +3 +3 +3 +3
Rm 22.pl.12 83-19560-1 83-19560-2 83-19560-3 83-19560-4 83-19560-5 20153-A 20153-B 207965#1 #23 #33 A25820 FS#833-1 FS#833-2
FS#833-3 FS#833-4 FS#833-5 FS#833-6 FS#833-7
+3 +3 +2 +3
+L +L +L +L +L +L +L ND ND N.E. +H
+1
+L,D +L,D +2,D +L,D +D,H
+1 +1
+D +D
12 ND -6 ND ND ND 5 -12 ND ND ND ND ND ND -12 -17 -17 -7 -16 -18 -13.4 ND ND
ND ND 17 -10 -6 - 12 -7 ND ND ND
Two types of controls were used: rats fed a complete rat diet (Control 1) and rats fed an autoclaved rice-complete diet (1:1) (Control 2). Three rats were used for each treatment. Isolates of fungi were obtained from the following donors: China, the District of Columbia. and Haiti, C. J. Mirocha, 1981 to 1987: New Caledonia. D. Laurent, 1983: South Africa, W. F. 0. Marasas, 1985; Seoul. Korea, S. Ohh, Ginseng and Tobacco Institute; Nankang, Taipei, Taiwan. T. Tseng. Institute of Botany Academia Sinica; Maryland, G. Bean. University of Maryland. College Park; and North Carolina. P. Hamilton, North Carolina State University, Raleigh. -, No detectable toxic effect; +, definite detectable toxic effect: N.E., not evaluated because of autolysis; L, body weight lost; H, hematuria; S, stomach hemorrhage; In, intestinal hemorrhage; D, diarrhea; ND, not determined because death occurred.
selected, shaken in 2.5% NaOCI for 1 min, rinsed in sterile distilled water, and transferred to acidified potato dextrose agar and pentachloronitrobenzene agar supplemented with aureomycin, a medium selective for Fiusariiim species (18). Cultures were transferred to acidified potato dextrose agar and carnation leaf agar and examined for F. moniliforme 10 to 14 days later (19). Stock cultures of F. moniliforme isolated were maintained on moist autoclaved soil and stored at - 15°C. Preparation of F. moniliforme extracts. Crude fungus culture extracts were prepared as described elsewhere (3). Ground moldy rice (20 g) was used for trichothecene and zearalenone analyses. The moldy rice was extracted three times for 1 h with 75, 75, and 50 ml of 50% aqueous methanol on a shaker at high speed. The combined extracts (200 ml) were filtered through Whatman no. 4 filter paper and cleaned up by the methods described by Yoshizawa et al. (24). Separate samples of ground moldy rice (20 g) were extracted to determine moniliformin, fusarin-C, fusarochromanone (TDP-1), and hemorrhagic factor (wortmannin) by the methods of Scott and Lawrence (22), Gelderblom et al. (9), Pathre et al. (21), and Abbas and Mirocha (1), respectively. Toxins
were detected on precoated (0.25 mm thick) silica gel 60 plates without fluorescent indicator (E. Merek, Darmstadt, Federal Republic of Germany) (3). Toxins were confirmed by combined gas chromatography-mass spectroscopy (GCMS), using the full mass spectrum obtained in electron impact at 70 eV. Moniliformin was confirmed by its UV
spectrum.
Mycotoxin references. The sources of diacetoxyscirpenol, monacetoxyscirpenol, scirpenol, T-2 toxin, neosolaniol, HT2 toxin, acetyl-T-2 toxin, T-2 tetraol, iso-T-2, deoxynivalenol, nivalenol, fusarenone-X, 3-acetyldeoxynivalenol, 15acetyldeoxynivalenol, zearalenone (F-2), and moniliformin were those reported by Abbas et al. (3). Fusarochromanone (TDP-1) was produced and purified in our laboratory. Hemorrhagic factor (wortmannin) was produced and purified by Abbas and Mirocha (1). Fusarin-C was a gift of Leonard F. Bjeldanes, University California-Berkeley, Department of Nutritional Science, Berkeley, Calif. Rat feeding tests. The ground culture mixed 1:1 with a complete rat diet was fed to 20-day-old white virgin female Sprague-Dawley rats (Bio-Lab Corp., St. Paul, Minn.) housed in individual cages. The animals and feed were
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MIROCHA ET AL.
weighed at the beginning and the end of the experiment. Three rats were used for each fungal isolate and the control in which rats were fed autoclaved rice and a complete rat diet (1:1 ratio). Rats were observed frequently for 5 days, and symptoms and days of death were recorded. Surviving rats were sacrificed by cervical dislocation and examined for gross pathological changes in tissues, including stomach, lungs, liver, heart, uterus, thymus, brain, bladder, kidney, and intestines. Duckling feeding test. One-day-old ducklings (five per treatment) were fed rations containing a corn medium on which F. moniliforme was grown. Cracked corn (300 g), contained in 3.8-liter flasks and with a moisture content of -60%, was autoclaved for 0.5 h, seeded with the appropriate Fusarium isolate, and grown for 2 weeks at 25°C. The cultures were harvested and air dried in a hood, mixed with Purina chick starter in a ratio of 1:1 by weight, and ground in a Wiley mill with 2-mm mesh. Two groups of five ducklings (controls) were fed Purina chick starter alone, and the other group was offered Purina chick starter with ground yellow corn (1:1 ratio). The test was conducted for 7 days, and then the number of deaths was recorded. Treatment of isolate F. moniliforme A-25820. (i) Source of fungus. A pure culture of F. moniliforme NRRL A-25820 (reported to produce trichothecenes) was received on 1 January 1987 from Pat Hamilton, North Carolina State University, Raleigh. The fungus was transferred to potato dextrose agar and carnation leaf agar for identification and to check for contamination. The fungus was also grown on various substrates such as banana, corn, rice, and liquid medium (Czapek Dox broth containing 1% peptone) and tested for toxicity in rat feeding and intubation tests. Extracts of these samples were tested for trichothecene production and other selected metabolites. (ii) Growth of fungus. All of the isolates were grown on a solid rice substrate as described by Abbas et al. (3). After 21 days of growth, the rice cultures were fragmented, transferred to a screen-bottomed tray, and allowed to air dry in a ventilated hood. The moldy rice was ground to the consistency of flour by using a coffee grinder in a fume hood, mixed 1:1 with a ground complete rat diet, and fed to rats. The same fungal isolates were grown on a corn kernel substrate as described above and checked for toxicity to rats.
The fungal isolates were grown on a banana fruit substrate in which five yellow bananas (with intact peel) were treated with absolute ethanol, flamed, and then inoculated with the Fusarium isolates. Other control banana fruits were treated as described above but not inoculated. The bananas were then incubated for 2 weeks at 25°C under moist conditions. The molded bananas were frozen for 24 h, broken up into small disks, transferred to a screen-bottomed tray, and allowed to air dry for 3 to 4 days in a ventilated hood. The molded as well as control bananas were each ground and then mixed separately 1:1 with a complete rat diet and fed to rats as described above. The same fungal isolates were grown on a liquid medium (Czapek Dox broth containing 1% peptone) in 500-ml flasks, each containing 200 ml of medium. The cultures were incubated at 27 to 29°C for 21 days. At harvest, the cultures were filtered through Whatman no. 4 filter paper, and a 1-ml portion was administered in a single oral dose to 20-day-old virgin Sprague-Dawley rats (Bio-Lab Corp.) by gastric intubation. Three rats were used for this experiment and observed frequently for 24 h after being dosed, and major signs of toxicity were recorded.
APPL. ENVIRON. MICROBIOL.
(iii) Qualitative detection of toxins in culture extracts. Molded rice or corn culture (20 g) was extracted with 50% ethyl acetate in acetonitrile (100, 75, and 75 ml) three times for 1 h each time in a high-speed automatic shaker. The extracts were filtered through Whatman no. 4 filter paper, and the combined filtrates (250 ml) were evaporated on a rotary evaporator at 45°C. The residue was dissolved in 50 ml of acetonitrile and defatted with 100 ml of petroleum ether (60 to 70°C) twice for 1 min each time. The acetonitrile layer was evaporated on a rotary evaporator and redissolved in 10 ml of chloroform-methanol (9:1, vol/vol). Half of this material was used for thin-layer chromatography (TLC) analysis, and the remainder was used for GC-MS analysis. A portion equivalent to 2 g of culture was saved for chemical identification of mycotoxins. The extracts were spotted on a TLC plate for the detection of trichothecenes (see Table 2) and zearalenone and wortmannin, using methods of detection described by Abbas et al. (3). All samples were extracted for moniliformin with acetonitrile-H20 (95:5, vol/vol) as described by Scott and Lawrence (22). The banana samples were homogenized with MeOH-H20 (55:45 1% N NaCl) for 5 min and filtered through Whatman filter paper no. 4. The filtrates were defatted twice with n-hexane and then reextracted three times with dichloromethane. The combined dichloromethane layers were evaporated I o dryness on a rotary evaporator, and the residue was dissolved in 10 ml of chloroform-methanol (9:1, vol/vol). Half of this material was used for TLC analysis, and the other half was used for analysis by GC-MS. The liquid culture media were extracted as described above for the banana samples, except that the extracts were filtered through ca. 5 cm (40 g) of anhydrous Na2SO4 and glass wool in a glass funnel. The anhydrous Na2SO4 WaS washed with 30 ml of dichloromethane. The combined dichloromethane extracts were evaporated to dryness on a rotary evaporator. The residue was dissolved in 10 ml of chloroform-MeOH (9:1, vol/vol) and used for both GC-MS and TLC analyses. The chemical identities of the trichothecenes, zearalenone, and wortmannin in culture extracts were confirmed by TLC analysis and by derivatizing with trifluoroacetic acid (Pierce Chemical Co., Rockford, Ill.) and subsequent analysis by GC-MS, using both selected ion monitoring and full electron impact mass spectra. The chemical identity of moniliformin was confirmed by TLC analysis, using visual comparison with standards, and by UV spectroscopy. Rice cultured with F. moniliforme NRRL A-25820 for 28 days at 25°C was dried overnight at ambient temperature in a forced-air oven and then finely ground in a Waring blender. The ground rice (100 g) was extracted twice with CH30H (5 ml/g), the solutions were combined, and the volume was reduced to about half on a rotary-vacuum apparatus at 400C. NaCl (5%) was added, and the solution was partitioned twice with ethyl acetate. The ethyl acetate extracts were pooled and evaporated to dryness. The remaining residue was analyzed by GC-MS for trichothecenes by conversion to their trimethylsilyl ethers. The residue from the ethyl acetate was dissolved in 95% ethanol, and the pH was adjusted to 12 with 0.2 N NaOH. The solution was heated at 60 to 80°C for 0.5 h and then cooled to room temperature, and the pH was adjusted to 7. The resulting solution was partitioned twice with ethyl acetate. The ethyl acetate extracts were pooled and taken to dryness, and the residue was acetylated with trifluoroacetic anhydride in pyridine at 50°C for 1.5 h. The reaction mixture was taken to dryness, and the remaining residue was ana-
TABLE 2. Mycotoxins produced by Fusarium species on various substrates grown in the laboratory Fusarium species
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Rice
Toxin concn (ppm Corn
F. graminearum Zea (685) #124 ND F. moniliforme A-25820b F. poae N.F. 5098 T-2 (600) HT-2 (50) T-2-TOL (10)
[ig/lg]) ona: Banana
Zea (570)
Zea (8.0)
ND
ND
T-2 (
