Toxin Production by Fusarium Species from Sugar Beets and Natural Occurrence of Zearalenone in Beets and Beet Fiberst

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1992, p /92/ $02.00/0 Copyright 1992, American Society for Microbiology Vol. 58, No. 10 Toxin Production by Fusarium Species from Sugar Beets and Natural Occurrence of Zearalenone in Beets and Beet Fiberst RSLA BOSCH AND C. J. MIROCHA* Department of Plant Pathology, niversity of Minnesota, St. Paul, Minnesota Received 30 March 1992/Accepted 22 July 1992 Fifty-five Fusarium isolates belonging to nine species were collected from fungus-invaded tissue of stored sugar beets and identified as F. acuminatum (11 isolates), F. avenaceum (1 isolate), F. culmorum (1 isolate), F. equiseti (23 isolates), F. graminearum (4 isolates), F. oxysporum (1 isolate), F. solani (4 isolates), F. sporotrichioides (7 isolates), and F. subghtfinans (2 isolates). All isolates were cultured on autoclaved rice grains and assayed for toxicity by feeding weanling female rats the ground-rice cultures of the isolates in a 50%o mixture with a regular diet for 5 days. Fifty-eight percent of the isolates were acutely toxic to rats, 26% caused hematuria, 18% caused hemorrhages, and 29% caused uterine enlargement. In most cases, toxicity could not be accounted for by the known toxins found. The following mycotoxins were found in extracts of the rice cultures: zearalenone (22 to 6,282,ug/g), chlamydosporol (HM-8) (68 to 4,708 ig/g), moniliformin (45 to 400,ug/g), deoxynivalenol (10 to 34 pg/g), 15-acetyldeoxynivalenol (5 to 10 pg/g), diacetoxyscirpenol (22 to 63,ug/g), monoacetoxyscirpenol (21 to 26,ug/g), scirpenetriol (24,g/g), T-2 toxin (4 to 425,ug/g), HT-2 toxin (2 to 284,Ig/g), neosolaniol (2 to 250,ug/g), and T-2 tetraol (4 to 12,g/g). F. equiseti was the predominant species found on visibly molded beets in the field. Six of 25 moldy sugar beet root samples collected in the field contained zearalenone in concentrations ranging between 12 and 391 ng/g, whereas 10 samples from commercial stockpiles were negative for zearalenone. Zearalenone was detected in 31 of 75 sugar beet fiber samples (13 to 4,650 ng/g). Three of 31 samples contained cis-zearalenone (1 to 8 ng/g), and two contained trace amounts of trans-a- and trans-1-zearalenol. This is the first report of zearalenone in sugar beet roots and fiber. Species of the genus Fusarium occur widely in nature as saprophytes and plant parasites; they are found in a great variety of plants and agricultural products. In addition to the losses caused by infection of plants by Fusarium before or during harvest, some species are capable of producing mycotoxins in affected products. Many surveys revealed the presence of trans-zearalenone (ZEA) and several trichothecene toxins (deoxynivalenol [DON], acetyldeoxynivalenol [ADON], nivalenol [NIV], diacetoxyscirpenol, and T-2 toxin) as the most abundant natural contaminants of foodstuff and animal feed, especially in diets containing cereals and other grains (3, 6, 43, 44). Ingestion of food and feed contaminated with Fusarium toxins was suspected to be implicated in numerous diseases of man and animal (28). The wide range and frequent presence of Fusarium toxins found naturally occurring on cereals reveal an increasing need for research on the toxigenic potential of Fusarium spp. grown on plants other than cereals to assess the extent of mycotoxin hazard to man and animals. In this respect, sugar beets (Beta vulgaris L.) are important because they are subject to attack by various soilborne fungi, including Fusarium spp. Species of Fusarium have been reported worldwide to be pathogens of beet seedlings (9, 26, 47) and mature plants (7, 21, 24, 26) that are implicated in diseases such as damping-off, root rot, and stalk blight. Moreover, beets are predisposed to storage rot by fungi as a result of mechanical damage during harvest and then storage for several months in unprotected, large, outdoor stockpiles (10, 49). In Minnesota, Bugbee (10) studied * Corresponding author. t Paper no. 19,723 of the contribution series of the Minnesota Agricultural Experiment Station the decay-causing fungi on 2,246 rotted sugar beet samples and found that 31% were invaded by Fusarium spp. However, the mycotoxins produced by the Fusarium isolates were not studied, although their presence was suspected because of infrequent reports that sugar beet pulp caused problems of infertility when fed to dairy cattle. Thus, in an attempt to gain more information on the kind of toxigenic Fusarium spp. causing sugar beet storage rot, surveys on stored beets and beet fibers were made at a Minnesota sugar beet factory during the 1987 and 1988 processing seasons. In September 1988, sugar beets grown in fields of northwestern Minnesota were observed to be diseased with symptoms of chlorosis, wilting, and death of foliage as well as rot of taproots infected by Fusarium spp. Symptoms on foliage and roots of sugar beets similar to those observed in this study were recently reported by Martyn et al. (26) in Texas, who identified Fusarium oxysporum as the causal agent. Moreover, similar symptoms have been found with the disease known as Fusarium yellows, which has been characterized by wilt and chlorosis of foliage but without external root symptoms. The disease was first described by Stewart (41) as early as 1931 and since then has been reported by others (26, 27, 49) from all sugar-beet-growing regions of the world. Fusarium yellows is caused by F. oxysporum f. sp. betae. The purpose of this study was to (i) determine the Fusarium species present on decayed beet roots before harvest and during outdoor storage, (ii) test Fusarium spp. obtained from stored sugar beets for toxicity to animals, (iii) analyze the toxins they produce in culture, and (iv) determine the concentration of ZEA and its derivatives as well as DON in moldy beets and beet fibers.

2 3234 BOSCH AND MIROCHA MATERIALS AND METHODS Sampling. Sugar beets from crops of 1987 and 1988 stored in outdoor stockpiles of a sugar-processing company in northwestern Minnesota were surveyed monthly for visible Fusarium mold. Samples were collected, placed in plastic bags, and transferred to the laboratory at the niversity of Minnesota, where they were stored at + 10 C for up to 2 days before being assayed for fungi. Simultaneously, 75 sugar beet fiber samples were collected (6% moisture content) and stored at room temperature until analyzed for Fusanum toxins. In addition, in September 1988, sugar beets growing in two fields randomly selected in northwestern Minnesota (i.e., Crookston) showed symptoms of chlorosis, wilting, and death of foliage. Diseased plants were solitary and scattered throughout the fields. An investigation of the roots revealed heavy infestation by Fusarium spp.: mycelium was visible, covering the roots to various degrees. Twenty-five sugar beets heavily infested with Fusaium spp. were selected and handled the same as samples from the stockpiles. All beets were then cut in half and examined inside and out for visible Fusarium mycelium. Rotted tissue, visibly covered with mycelium, as well as surrounding healthy tissue was excised and assayed for fungi. The 25 moldy samples from fields as well as 10 selected at random from stockpiles on the same day were analyzed for ZEA and DON. Isolation and identification of Fusarium species. One piece (1 cm3) of each rotted sugar beet tissue sample was cut, surface disinfected in a 2.5% aqueous solution of NaOCI for 1 min, rinsed twice in sterile distilled water, and placed on acidified potato dextrose agar and aureomycin supplemental pentachloronitrobenzene agar medium (32). The latter medium is selective for Fusanum species. The plates were incubated in the light (from a northern exposure) at 22 C and examined at regular intervals. The dominant fungi were isolated in pure culture. To reduce the number of cultures, 55 cultures from sugar beets stored in stockpiles and 12 cultures from beets grown in the field were selected at random as representatives of the different Fusanium species encountered and were single spored by using the techniques of Nelson et al. (33). Single-spored cultures were then placed on potato dextrose agar medium and carnation leaf agar (13). The fungal colonies were incubated at approximately 24 C under fluorescent and V lamps (53,000 lx) for 12 h per day to foster growth and sporulation of fungi. Species identifications were determined on the basis of the manual of Nelson et al. (33). Stock cultures were maintained on moist autoclaved soil and stored at -15 C. Reference standards. The following mycotoxins were produced and purified in our laboratory: diacetoxyscirpenol, monoacetoxyscirpenol, scirpenetriol, T-2 toxin, acetyl-t-2 toxin, T-2 triol, T-2 tetraol, HT-2 toxin, neosolaniol, ZEA, zearalenol (ZOL) in the trans-a and trans-13 configurations, DON, 3-ADON, fusarenon-x (FX), NIV, chlamydosporol (also termed HM-8), fusarochromanone (also termed TDP- 1), and wortmannin. Moniliformin (MON) was purchased from Sigma Chemical Co., St. Louis, Mo., and 15-ADON was a gift from J. J. Pestka, Michigan State niversity, East Lansing. Chlamydosporol was identified in our laboratory as HM-8 (cytotoxic factor), but its structure was determined previously by Grove and Hitchcock (17). Preparation of Fusarium cultures. The 55 Fusarium isolates obtained from stored sugar beets were tested for their toxigenic potential by being grown on autoclaved moist rice seeds. The rice cultures were prepared by the method APPL. ENvIRON. MICROBIOL. reported by Eugenio et al. (12) and modified by Abbas et al. (4) Ṙat feeding test. The ground-rice cultures of the 55 Fusarium isolates were mixed separately with normal rat diet (50% [wt/wt]) and fed for 5 days to laboratory rats (20-day-old virgin female Sprague-Dawley rats; Bio-Lab Corp., St. Paul, Minn.) as described by Abbas et al. (4). Three rats were used for each treatment. During the experiment, mortality was recorded daily. After 5 days, weight changes of surviving rats and the food consumption of all rats were recorded. Surviving rats were sacrificed by cervical dislocation and examined for gross pathological changes in organs and tissues. Mycotoxin analyses. The 55 fungal rice cultures were extracted and analyzed for the presence of type-a trichothecene toxins such as diacetoxyscirpenol, monoacetoxyscirpenol, scirpenetriol, T-2 toxin, HT-2 toxin, acetyl-t-2 toxin, T-2 triol, T-2 tetraol, and neosolaniol and type B trichothecene toxins such as DON, 3-ADON, 15-ADON, FX, and NIV as well as ZEA, MON, chlamydosporol, wortmannin, and TDP-1 as reported in detail by Abbas et al. (1, 2, 4) and Lee (22). Briefly, 20 g of fungal rice culture was moistened with 5 ml of distilled water and extracted with different solvents depending on the toxin: methanol-water (55:45 [vol/vol]) was used for trichothecenes, ZEA, and chlamydosporol; the same solvent was used for TDP-1, with only 2% ammonium hydroxide added. The cultures were extracted, defatted with petroleum ether (bp, 60 to 70 C), and partitioned with dichloromethane. The solvent was evaporated to dryness, the residue was dissolved in 2 ml of methanol, and an aliquot of each sample was resolved by thin-layer chromatography (TLC) by using visual comparison with standards. Extraction and cleanup of MON was done as described by Scott and Lawrence (40). TLC was carried out on silica gel plates (E. Merck, Darmstadt, West Germany) in the following systems: dichloromethane-methanol, 9:1 or 4:1 (vol/vol), and petroleum ether-acetone, 1:1 (vol/vol). Detection and confirmation of mycotoxins were accomplished by spraying the developed plates with the following colorimetric reagents: methanolic sulfuric acid, p-anisaldehyde, 2,4- dinitrophenylhydrazine, and 4-(p-nitrobenzyl)pyridine-tetraethylenepentamine, with appropriate heating and examination under V light (254 and 364 nm) before and after spraying and heating. The 75 fiber samples and 25 sugar beet samples from the field as well as the 10 samples from stockpiles were extracted and analyzed for the presence of cis-zearalenone, ZEA, and trans-at,,-zol as well as DON. Extraction and purification of cis-zearalenone, ZEA, and trans-ot,1-zol were performed by using a slight modification of the method of Bennett et al. (8). The fiber samples were ground to the consistency of flour in a laboratory mill. Fifty grams was moistened with water (10% [vol/wt]) and extracted with 400 ml of chloroform. The fiber samples and the beet tissue were cut in small pieces (2 by 2 cm), which were uniformly mixed and then divided into two equal portions. One portion of each sample was kept frozen (-15 C) until analysis for DON, while the other was extracted for analysis for ZEA by blending for 4 min with chloroform (four separate extractions). All samples were filtered, and an aliquot (10-g equivalent) of each extract was cleaned up by sequential base-acid partition. The solvent was evaporated to dryness, and the residue was dissolved in 4 ml of methanol and transferred to a 2-dram (1 dram = 3.7 ml) vial. The solution was evaporated to dryness under a stream of nitrogen with gentle heating. The residue was

3 VOL. 58, 1992 TOXIN PRODCTION BY FSARIM SPECIES FROM SGAR BEETS 3235 dissolved in 2 ml of methanol before analysis by TLC, high-performance liquid chromatography (HPLC), and capillary gas chromatography-mass spectrometry. Portions of the same samples were extracted and purified for DON by a slight modification of the method reported by Chang et al. (11). Fifty grams of ground-fiber sample was weighed, and 10 g of basic copper carbonate was added and extracted with 400 ml of acetonitrile-water (84:16 [vol/vol]). All extracts were filtered, and 10-ml portions of the filtrate were loaded onto an activated charcoal column (Myco Lab Co., Chesterfield, Mo.) and eluted with 10 ml of extracting solvent. Two charcoal columns were used for each fiber sample, and eluates were combined. The eluates were evaporated to dryness on a rotary evaporator. The residue was dissolved in 4 ml of methanol-acetone (1:1 [vol/vol]) and transferred to a 2-dram vial. The solution was evaporated to dryness under a gentle flow of nitrogen gas, with gentle heating. The residue was dissolved in 2 ml of methanol prior to analysis by TLC and HPLC. Quantitation and confirmation of mycotoxins. Quantitation and additional confirmation of trichothecene toxins were performed by gas-liquid chromatography by comparison with standards. Calculations of toxin concentrations in cultures were based on external standards in concentrations of 50 ng/g and on dry weight. The gas chromatograph, model 5890 (Hewlett-Packard Co., Avondale, Pa.), was equipped with a flame ionization detector. Analysis was performed on a DB-5 fused silica capillary column (30 m by 0.25 mm) (J & W Scientific, Inc., Rancho Cordova, Calif.) by utilizing a splitless injection method. The temperature of the injector port and the detector was 280 C; the column temperature was programmed to increase from 80 to 230 C at 30 C/min and then from 230 to 280 C at 8 C/min. The carrier gas was He, with a flow rate of 2 m/min at the exit. The type A trichothecene toxins were quantified by derivatizing an aliquot of each extract with trifluoroacetic acid anhydride (Pierce Chemical Co., Rockford, Ill.), and the type B trichothecene toxins were quantified by derivatizing an aliquot of each extract with Tri-Sil-TBT (Pierce) as reported previously by Pawlosky et al. (35). The chemical identity of the toxins was confirmed by using TLC and by visually comparing them with standards and after resolution by capillary gas chromatography-mass spectrometry (using a Hewlett-Packard 5890 gas chromatograph interfaced with a VG 7070EQ mass spectrometer) and electron impact mass spectrometry. Selected-ion monitoring was used for detection of cis-zearalenone or ZEA in extracts of field samples by selecting ions at m/z 462 (M+), mlz 447 (or m/z 333), and m/z 305 and for detection of trans-a- and trans-1-zol by selecting mlz 536 (M+), mlz 447 (or mlz 333), and mlz 305. Analyses were conducted on a 10-m, narrowbore (inside diameter, 0.25 mm) DB5 fused silica capillary column inserted directly into the source of the mass spectrometer. The samples were introduced by splitless injection with a delay of 0.5 min. The flow rates were 60 ml/min at the inlet and 1.5 ml/min at the exit. The gas chromatography oven was programmed from 80 to 300 C at 25 C/min. The mass spectrometer was tuned with perfluorotributylamine with methane as the reagent gas. The source temperature was set at 150 C. The electron energy was set at 70 ev. RESLTS Fifty-five isolates belonging to nine Fusanium species were obtained from postharvest moldy sugar beets and identified as Fusarium acuminatum (11 isolates), Fusarium avenaceum (1 isolate), Fusarium culmorum (1 isolate), Fusarium equiseti (24 isolates), Fusarium graminearum (4 isolates), F. oxysporum (1 isolate), Fusarium solani (4 isolates), Fusarium sporotrichioides (7 isolates), and Fusarium subglutinans (2 isolates). In addition, 12 isolates were obtained from diseased sugar beets before harvest and identified as F. equiseti (nine isolates) and single isolates of F. oxysporum, Fusarium moniliforme, and F. solani were identified. F. culmorum, F. moniliforme, F. oxysporum, and F. solani have been frequently isolated from rotted tissue of diseased sugar beet plants by others (9, 19, 21). The finding of these Fusarium species is significant since all of them belong to the 20 recognized toxigenic Fusarium species as compiled by Marasas et al. (25) and have the potential to produce one or more toxins on various substrates. Of the 55 isolates that were grown on rice and fed to rats, 32 (58%), representing eight Fusarium species (F. acuminatum, F. avenaceum, F. culmorum, F. graminearum, F. oxysporum, F. solani, F. sporotrichioides, and F. subglutinans), caused death in rats, while 40 (73%) showed toxic signs in the gross pathological examination, i.e., 14 isolates caused hematuria (blood in the bladder), 9 caused intestinal hemorrhages, 1 caused hemorrhage in the duodenum, and 16 caused uterine enlargement (an indication of ZEA production). Thirteen isolates (23%) did not cause death or any visible pathological signs in the rats. The majority of the isolates caused reduced food consumption and decreased weight gains. Results of the analyses for mycotoxins in the rice culture extracts of the 55 isolates are presented in Table 1. One or more of 12 toxins were detected in the extracts. ZEA was produced by F. acuminatum, F. culmorum, F. equiseti, F. graminearum, F. oxysporum, and F. sporotrichioides in concentrations ranging between 22 and 6,282,ug/g. All isolates caused uterine enlargement in rats, an indication of the presence of ZEA. SB3 and SB15, two ZEA-producing isolates of F. equiseti, were lethal to rats, although in culture extracts the only toxin found was ZEA, in concentrations of 567 and 232,ug/g, respectively. Thirty-two isolates caused death when fed to rats, but only 12 could be accounted for by known toxins. HM-8 was found in extracts from F. acuminatum, F. avenaceum, F. equiseti, and F. subglutinans in amounts ranging between 68 and 4,708,ug/g. In culture extracts of five isolates, the only mycotoxin detected was HM-8. Of these, two isolates (SB14 and SB16) of F. acuminatum and one (SB20) of F. subglutinans caused death. Isolates SB14 and SB20 caused hemorrhages of the intestine and hematuria, whereas a highly toxic isolate of F. acuminatum (SB32) caused death but no other gross pathological signs. Studies with the pure compound chlamydosporol in levels as high as 2 mg/g of body weight indicated that this toxin could be responsible for food refusal but not for hemorrhaging and death in rats (1). MON was produced by F. acuminatum, F. avenaceum, and F. subglutinans in amounts ranging from 45 to 400,ug/g. All MON-producing isolates were found to be chlamydosporol producers as well (750 to 4,708,ug/g). Although there seems to be a correlation between the two Fusarium toxins, previous studies (1) on the toxigenic potential of Fusarium species showed that many but not all MON-producing isolates are also chlamydosporol producers. In our study, four of seven isolates were lethal to rats, while one isolate (SB7) of F. acuminatum caused hematuria as well as death. Studies by Abbas et al. (5) revealed that crystals of MON incorporated in diets of rats at concentrations of 2 mg/g and

4 3236 BOSCH AND MIROCHA APPL. ENvIRON. MICROBIOL. TABLE 1. Mycotoxins produced by species of Fusarium grown in a solid rice medium" Fusarium species and Toxic" Mycotoxin produced (p,g/g [dry wt])c code no. of isolate signs ZEA HM-8 MON DON 15-ADON DAS MAS T-2 NEO HT-2 T-2-tet F. acuminatum SB6 SB7 SB13 SB14 SB16 SB22 SB25 SB29 SB32 SB33 SB53 F. avenaceum SB5 F. culmorum SB26 F. equiseti SB3 SB8 SB9 SB15 SB17 SB18 SB19 SB23 SB24 SB31 SB35 SB36 SB37 SB39 SB40 SB41 SB42 SB45 SB46 SB48 SB50 SB51 SB52d F. graminearum SB1 SB2 SB4 SB55 F. oxysporum SB54 F. solani SB10 SB21 SB38 SB44 F. sporotrichioides SB11 SB12 SB27 SB28 SB34 SB47 SB49 F. subglutinans SB20 SB30, N.E., H 1D, In, H, In 1D,, 1D, In 3D, In 3D, In, Ind, H, In, 3D, N.E. 3D, 3D, N.E. 1D 1D, 3D,, H, In 3D,H, In, H, D* 364 6, , , ,503 1,616 4, , , , a The cultures were fed to rats to measure toxicity. 1, 2, or 3 dead rats, respectively;, uterine enlargement; H, hematuria; In, intestinal hemorrhage; D*, only duodenum hemorrhage; N.E., not evaluated owing to death and/or autolysis. C DAS, diacetoxyscirpenol; MAS, monoacetoxyscirpenol; NEO, neosolaniol; T-2-tet, T-2 tetraol; -, no detectable toxin. Results are means of two replications. The isolates were also analyzed for fusarochromanone and wortmannin as well as acetyl-t-2 toxin, T-2 triol, NIV, and FX and found to be negative. d The isolate SB52 of F. equiseti produced scirpenetriol in a concentration of 24,g/g.

5 VOL. 58, 1992 higher caused intestinal hemorrhages and death; concentrations of 1 mg/g and higher caused death in rats without accompanying symptoms, whereas levels lower than 0.75 mg/g did not show any toxic effect. Thus, the concentration of MON produced by the isolates in this study (45 to 400,g/g) could not account for the toxicity observed. DON and 15-ADON were produced by F. graminearum and F. sporotrichioides, respectively. F. sporotrichioides is well known as a type A trichothecene producer but uncommonly found as a type B producer (25). In the present study, only one isolate of F. equiseti produced DON in a concentration of 10 p,g/g but no 15-ADON. No FX or NIV was found in the culture extracts. Although DON and 15-ADON proved to be toxic in higher concentrations (oral 50% lethal doses for mice, 78 and 34 mg/kg, respectively [14]), the low concentrations found in our culture extracts were not sufficient to be lethal to rats but could account for feed refusal and reduced weight gains. Forsell et al. (15) reported that 2,ug of DON per g in feed was required to cause reduced weight gains in female mice, whereas feed refusal consistently occurred when 25 p,g of DON per g was added to the diet of the mice. A comparative study by Pestka et al. (36) with 15-ADON revealed that a dietary exposure of 5 p,g of 15-ADON per g caused reduced body weight gains after 16 days and marked feed refusal after 44 days in weanling female mice. Because of the high toxigenic potential of Fusanium species isolated from stored sugar beets, it was thought that sugar beet root tissue invaded by some Fusarium species might already contain one or more toxins before or after harvest and that these toxins might pass the processing procedure. The mycotoxicological investigation for the natural occurrence of DON, cis-zearalenone ZEA, and transa3,-zol (diastereoisomeric mixture) in rotted sugar beet root tissue revealed the presence of ZEA in sugar beet samples collected before harvest. Six of 25 samples contained ZEA in concentrations ranging between 12 and 391 ng/g. The actual amounts were as follows: RSB6, 15 ppm; RSB10, 205 ppm; RSBII, 12 ppm; RSB15, 221 ppm; RSB20, 79 ppm; and RSB21, 391 ppm. The total-ion chromatogram and mass spectrum of the ZEA-trimethylsilyl derivative are shown in Fig. 1. The sugar beet samples were collected in September 1988 from two fields randomly selected in northwestern Minnesota. The most prevalent Fusarium toxin in sugar beet fibers was ZEA (Table 2). It was detected in 31 of 75 samples in concentrations ranging from 13 to 4,650 ng/g. Three of the 31 fiber samples contained cis-zearalenone in concentrations of 1.2 (FIB 24), 8.3 (FIB 28), and 14 (FIB 63) ng/g in addition to ZEA, whereas two samples (FIB 41, FIB 69) contained trans-a4,-zol in trace amounts. This is the first report of the presence of ZEA in sugar beets and sugar beet fibers. The natural occurrence of ZEA in these samples might be due to the high incidence of isolates of F. equiseti obtained from sugar beets before harvest and from stored sugar beets; F. equiseti is an important ZEA producer (22). DISCSSION It is apparent from Table 1 that in most cases death in rat toxicity tests was due to unknown toxins. This supports the view that only a few of the Fusanium toxins have been characterized and many more exist that could be of economic importance in animal health. Only the type A trichothecene (45) toxins found in 12 of 32 isolates could account for acute toxicity. z 0 l - * w -~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ TOXIN PRODCTION BY FSARIM SPECIES FROM SGAR BEETS 3237 lee10 l TM+ 125 l-i -. - M-.8B ' 0. TM+ 4T7 I1L a.- 33Ti AL-M.... I IL~~~~~~ 1E-L-.. X1 ~.L-6 LAM I6 1i M G88 FIG. 1. (A) Total ion chromatogram of an extract from sugar beet sample RSB10 after reaction with silylating reagent and analysis by gas chromatography-mass spectrometry. Peak 328 is trimethylsilyl-zea. (B) Electron impact mass spectrum of peak 328. m/z 149 is due to phthalate plasticizer in the sample partially obscuring m/z 151. (C) Mass spectrum of the trimethylsilyl ether derivative of the ZEA standard. Although wortmannin has also been reported as an important hemorrhagic toxin (2) produced by Fusarium spp., it was not detected in any of the culture extracts analyzed. Likewise, fusarochromanone (34) was not detected in any of the cultures. Only F. equiseti has been described as a producer of fusarochromanone (23, 50). We found 15-ADON, as opposed to 3-ADON, to be the major derivative of DON produced by three isolates of F. graminearum. The absence of FX and NIV producers among the F. graminearum isolates obtained from sugar beets from Minnesota agrees with the findings of Ichinoe et al. (20) and others (16, 20, 44), who demonstrated that isolates of F. graminearum were chemotaxonomically subdivided into DON-ADON and NIV-FX producers with differences in their geographical distribution. While the DON-

6 3238 BOSCH AND MIROCHA TABLE 2. Occurrence of ZEA in sugar beet fibers during the and processing seasons Sample no. ZEA concn (ng/g [dry wt]) FIB FIB FIB FIB FIB FIB 24a FIB FIB 28a FIB FIB FIB 41b ,650 FIB FIB FIB FIB FIB FIB FIB FIB FIB FIB FIB FIB FIB FIB FIB FIB FIB 69b FIB FIB a cis-zearalenone was detected in three fiber samples in concentrations of 1.2 ng/g (dry weight) (FIB 24), 8.3 ng/g (dry weight) (FIB 28), and 14 ng/g (dry weight) (FIB 63). b Two fiber samples contained the diastereoisomeric mixture of trans-a,3- ZOL in detectable concentrations. ADON chemotype was most frequently isolated from cereals from Canada, North America, Argentina, China, Poland, and Germany, the NIV-FX chemotype occurred in France, Japan, and Korea in higher abundance. However, Sugiura et al. (42) found isolates of Gibberella zeae that produced NIV and trace amounts of DON. The presence of ZEA in samples of rotted tissue of sugar beets in the field before harvest as well as cis-zearalenone, ZEA, and trans-ot,1-zol in sugar beet fibers is important in that all possess estrogenic activity. The trans-ot-zol isomer has about three to four times more estrogenic activity than ZEA, whereas the trans-o-zol isomer has about the same activity as ZEA (18, 31). Mirocha et al. (31) demonstrated that cis-zearalenone has a significantly higher estrogenic activity to rats in the feeding and skin application test. In contrast, Peters (37) reported that both isomers have about the same uterotrophic activity when administered orally to mice. The findings of mycotoxins with estrogenic activity in moldy sugar beets and beet products might explain the infertility problems observed in cows when sugar beet pulp was fed to dairy cattle. This conclusion is supported by various reports of ZEA being associated with infertility in cattle. For example, Mirocha et al. (30) implicated the presence of ZEA in hay in a case of infertility in cattle. Vanyi et al. (46) demonstrated a degeneration of bull sperm when bulls were fed corn naturally contaminated with 20 ppm of APPL. ENvIRON. MICROBIOL. ZEA for 21 days. In Finland, dairy cows showed signs of vaginitis, a prolonged estrous cycle, and infertility after consuming mixed feed containing 25 ppm of ZEA (38). Moreover, Schuh and Baumgartner (39) reported a case of unspecific infertility in dairy cows in Austria suspected of being caused by ZEA-contaminated feed in that ZEA was detected in concentrations of 100 ppb in grass silage and of 50 ppb in maize silage. In contrast, Weaver et al. (48) found that pure ZEA in concentrations up to 500 mg/day given orally to nonlactating cows did not affect the health of the animals. The data of our study revealed that Fusanium species were able to produce toxins on sugar beets in the field and that isolates obtained from sugar beets produced potent toxins in culture in the laboratory. Moreover, ZEA was also found in sugar beet fiber, which suggests that it somehow survived commercial processing. The prevalence on sugar beets of Fusarium species from the sections Gibbosum and Sporotrichiella that are known for their capability to produce ZEA and type A trichothecene toxins suggests that suspect food and feed samples should be analyzed for ZEA as well as for T-2 toxin, HT-2 toxin, neosolaniol, and other type A trichothecenes. ACKNOWLEDGMENT This material was based on research conducted under project 22-34H of the Minnesota Agricultural Experiment Station and a grant from Environ Corp. REFERENCES 1. Abbas, H. K Characterization and biological activity of two new mycotoxins produced by species of Fusanum. Ph.D. thesis. niversity of Minnesota, St. Paul. 2. Abbas, H. 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