APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1976, p. 423-427 Copyright 1976 American Society for Microbiology Vol. 32, No. 3 Printed in U.S.A. Screening of Toxic Isolates of Fusarium poae and Fusarium sporotrichioides Involved in Causing Alimentary Toxic Aleukia B. YAGEN* AND A. Z. JOFFE Department of Natural Products, School of Pharmacy,* and Laboratory of Mycology and Mycotoxicology, Department of Botany, The Hebrew University, Jerusalem, Israel Received for publication 19 April 1976 A total of 131 isolates offusarium poae and F. sporotrichioides from overwintered cereals, which were associated with the alimentary toxic aleukia toxicoses in the Soviet Union, were tested for their ability to produce T-2 toxin [4,f, 15 diacetoxy-8a-(3-methylbutyryloxy)-12,13-epoxytrichothec-9-en-3a-oll. The presence of T-2 toxin was determined by thin-layer chromatography, gas-liquid chromatography, spectroscopic analyses, and the rabbit skin test. A good correlation was demonstrated between T-2 toxin detection by thin-layer chromatography and inflammatory skin reactions of rabbits. The nature of the toxic principle produced by Fusarium poae and F. sporotrichioides, the causal agents of alimentary toxic aleukia (ATA) and various animal disorders, has been investigated with divergent results. Olifson (25-28) and Olifson et al. (29) had claimed that the toxins involved were steroids called poaefusarin and sporofusarin, but several American (1-7, 10, 11, 23, 24, 31-34) and Japanese (35-39) investigators have come to different conclusions. They isolated trichothecene compounds, mainly T-2 toxin, and could not confirm the presence of a steroid in the above Fusarium isolates. It has therefore been deemed of interest to study the toxins produced by isolates of the F. poae and F. sporotrichioides species collected at the time of ATA outbreaks in the Soviet Union and actually associated with overwintered cereal grains (millet, wheat, rye, oats, barley, and buckwheat) described by Joffe (12-17, 19), Joffe and Palti (20, 21), and Schoental and Joffe (30). MATERIALS AND METHODS Sources of isolates. The toxic fungi of Fusarium used in this investigation were isolated from various overwintered grains collected from various fields of the Orenburg district, Soviet Union, and from the homes of people who consumed such grain and subsequently died. A total of 131 isolates, 106 F. sporotrichioides and 25 F. poae, were examined. Identification and culturing of Fusarium. Abundant spores of the cultures were produced and were identified on standard potato-dextrose agar (PDA) medium, as based on the morphology of micro- and macroconidia obtained from pure, and chiefly monoconidial, cultures, according to the taxonomic system of Joffe (18) and Joffe and Palti (21). The toxic 423 Fusarium were subcultured on PDA for 15 days at 18 C, and pure cultures maintained as monoconidial isolates were stored on PDA slants in test tubes and on sterilized soil at 3 C. The isolates were grown on wheat in 150-ml Erlenmeyer flasks containing 10 g of grain and 15 ml of tap water. These media were twice autoclaved at 1 atm for 30 min, inoculated with each of the 131 isolates, and incubated at 12 C for 21 days. Preparation of crude extract. Wheat infected by different isolates was covered with 50 ml of ethyl alcohol (96%) and left at room temperature for 24 h. The contents of each flask were extracted for 1 to 2 min in a Waring blender. The wheat slurry was filtered under vacuum through filter paper, and the residue was washed twice with ethyl alcohol (50 ml). Evaporation of the solvents yielded a biologically active yellow oil. To purify the oil from water-soluble compounds, it was extracted five times with 5 ml of absolute ethyl alcohol. The combined alcoholic extracts were concentrated to about 1 ml, transferred to a small vial, and brought up to 2 ml with ethyl alcohol. Analyses at this point were made by the following methods. (i) TLC. For thin-layer chromatography (TLC), thin-layer plates of silica gel (E. Merck AG, Darmstadt, Germany) were prepared in the conventional manner and then activated at 120 C for 2 h. A 10-,ul amount of the crude extract from each isolate and a known amount of T-2 toxin solution were applied. The plate was developed for a distance of 15 cm in a solution of 30% acetone in hexane, and T-2 toxin was observed in visible light after spraying with a freshly prepared mixture of p-anisaldehyde reagent (40) and then heating at 130 C for 5 min. The spots of T-2 toxin were colored gray-brown. The presence of T-2 toxin in the sample of crude extract was estimated qualitatively by comparing the intensity of the gray-brown zone at the Rf of T-2 toxin (Fig. 1). Sensitivity in the T-2 toxin detection with this procedure was of the order of 1,.g of A sample
424 YAGEN AND JOFFE APPL. ENVIRON. MICROBIOL.,.;. * t )~~~~~~~~~~~~~~~~~~es ', +'4,,sS : ok X -illn X S *...w m;,0 1 2 3 4 5 6 7 8 9 lo 1 1 12 FIG. 1. Thin-layer chromatogram on Kiesel Gel-F of10 ethanolic extracts (2 to 11) from wheat, infected by different isolates, compared with standard T-2 toxin (1 and 12). Extracts 4 and 5 caused very strong skin reaction; extracts 2, 6, and 7, strong; extracts 3, 8, 9, and 10, moderately strong; extract 11, very slight. was judged to contain no T-2 toxin when 10 Al of crude extract did not give a visible spot of the Rf of (ii) GLC. The conditions for gas-liquid chromatography (GLC) were as follows: Packard model 845 with a hydrogen flame and a column of 6 feet, 0.13 inch (ca. 1.83 m) packed with 1.5% OV-17 on Gas Chrom Q, 80 to 100 mesh; column temperature, 240 C; injection block temperature, 250'C; nitrogen carrier gas flow rate, 20 ml/min. A 4-,ul amount of the crude extract from each isolate was used for the analyses by the gas chromatograph. The quantitative estimation of the amount of T-2 toxin present in the crude extract was carried out by GLC, using peak areas as a criterion. Peak areas (square centimeters) were calculated by multiplying the peak height by the peak width at one-half peak height. T- 2 toxin (3 mg/ml) solution was used as a standard; its retention time on the column was 32 min. (iii) Mass spectra. Low-resolution mass spectra were recorded on a Varian CH-5 spectrometer. To confirm the identity of T-2 toxin present in a thinlayer chromatoplate, the following procedure was used. A 1-ml amount of 2 ml of crude extract was jgim6iib P concentrated to about 0.2 ml and applied to a preparative Silica Gel P-254 plate (20 by 20 cm). A known T-2 toxin solution was applied to both edges of the plate and was developed in a solution of 40% acetone in hexane. The narrow section of the edges of this plate was sprayed with p-anisaldehyde reagent (40) and heated at 130 C. The chromatograph zone at the Rf value of T-2 toxin was scraped into a small flask and extracted several times with hot chloroformethyl acetate (1:1) solution. The combined organic solutions were evaporated. Mass spectra were run by using a direct inlet system. The following fragments were obtained: mwe 466 (M+), 382, 365, 364, 322, 321, 305, and 304. (iv) Toxicity tests on rabbit skin. The mycotoxic properties off. sporotrichioides and F. poae isolates were assessed by a skin test on male and female rabbits. Only rabbits with nonpigmented skin and weighing at least 1.5 kg were used because their skin is thinner and more sensitive to toxins. The extracts were applied on the back and sides of each rabbit (squares of skin measuring 3 by 3 were carefully cleared of hair). Each rabbit was kept in a separate cage and maintained on laboratory feed.
VOL. 32, 1976 The bioassay was performed by applying 10 1.l of Fusarium crude extract to the shaved skin with a Hamilton syringe. Two squares of each rabbit served for the control and were treated with an alcohol extract of autoclaved wheat or millet grains not infected by any fungus. Applications were made twice, at an interval of 24 h. The rabbits were kept under observation for at least 14 to 18 days after the first application. The intensity of the skin reaction produced by each isolate, grown at 12 C for 21 days, was assessed on the scale of grades reported elsewhere by Joffe (12, 19) and Joffe and Palti (20). This method was sensitive to the application of 0.1 jig of RESULTS The results of the rabbit skin screening tests are summarized in Table 1. They show that no less than 71 out of 106 isolates of F. sporotrichioides and 20 out of 25 isolates of F. poae produced very strong or strong dermatitic reactions. Pathological examination confirmed that toxic crude extracts applied to the skin of rabbits resulted in essentially the same injuries as those produced by pure T-2 toxin samples, i.e., edema, hemorrhage, and necrosis of the epidermis, dermis, and hair follicles. The TLC test that was performed clearly identified the toxic principle involved as T-2 toxin (see Fig. 1). This toxin is present in the wide range of overwintered grains investigated in this study. Mass spectroscopic identification of T-2 toxin was carried out on 35 random samples. The minimum dose inducing skin irritation was 0.1,ug; the lowest dose identifiable by TLC was approximately 1 gg. By 14 days after the application of only 0.1 ug of T-2 toxin to rabbit skin, severe degeneration of the epidermis and edematic-necrotic responses of the dermis were still observed. A comparison of amounts of T-2 toxin, determined by GLC, and corresponding rabbit skin reaction is presented in Table 2. DISCUSSION According to Keyl et al. (22), Ueno et al. (35-38), Chung et al. (8) and Epply et al. (9), the skin test of rabbits was widely applied for the screening and determination of isolates of Fusarium that produce trichothecene and, chiefly, This is the first screening report on the distribution of T-2 toxin-producing fungi in the Soviet Union associated with ATA disease. Reports of the natural occurrence of the isolates that produce T-2 toxin are rare. Only 13 out of the 136 isolates, mostly from the United States, that were examined by Burmeister et al. (7) produced F. POAE AND F. SPOROTRICHIOIDES ISOLATES 425 TABLE 1. Toxicity to the skin of rabbits of F. poae and F. sporotrichioides isolates from different sources Rabbit skin reactiona Species Source ++++ +++ ++ + + 0 F. sportori- Millet 22 24 9 6 5 2 chioides Wheat 2 5 2 6 2 Rye 8 4 1 Barley 2 4 2 F. poae Millet 8 5 1 1 Wheat 4 1 1 Barley 2 2 anumber of isolates producing various levels of T-2 toxin. Toxicity grades: ++++, very strong; +++, strong; + +, moderate; +, slight; ±, very slight; 0, none. TABLE 2. Comparison of amounts of T-2 toxin in crude extracts of F. sporotrichioides (106 isolates) and F. poae (25 isolates) and their effects on rabbit skin No. o T-2 toxin in Species No. of iso- crude extract (mg/ml) F. sporotrichioides +++ +,b very strongc 34 10-21 +++, strong 37 4-9 ++, medium 14 0.5-3 +, slight 12 0.1-0.5 ±, very slight 7 0.1 0, none 2 0 F. poae + + + +, very strong 14 16-21 +++, strong 6 9-15 ++, medium 1 0.2-4 +, slight 1 0.1-0.5 ±, very slight 3 0.1 0, none 0 0 a The weight of the crude extract, obtained from 10 g of infected wheat, ranged from 0.07 to 0.55 g. It is dependent upon the isolate used for the inoculation. bsee footnote to Table 1 for the toxicity grades of the rabbit skin reaction. c Intensity of the gray-brown color at Rf of T-2 toxin in TLC. In our investigation we found that more than 95% of the F. poae and F. sporotrichioides isolates from the Orenburg district produced T-2 toxin in various quantities (Table 1). This phenomenon is certainly connected with the meteorological and ecological conditions that existed in this district in 1943 and 1944 which promoted the development of the large number off. sporotrichioides and F. poae that are very active in the production of T-2 toxin, causing outbreaks of ATA. Among the isolates in our collection there were many that produced gram quantities of T-2 toxin when grown on 1 kg of wheat or millet at 12 or 5oC for a period of 21 and 45 days, respectively (Joffe and Yagen, personal communication). Those isolates were involved in
426 YAGEN AND JOFFE the more severe cases of ATA disease in the Soviet Union. In our investigations the skin irritation factor was in good agreement with the amount of T-2 toxin present in the sample, as established by GLC. A major disadvantage of the skin bioassay, in general, is its lack of specificity, since many naturally occurring trichothecenes and other fungal metabolites can cause skin irritation. Although T-2 toxin is, thus, not the only toxin that could have caused skin irritation, in 95% of our samples, it failed to cause such symptoms unless the gray-brown zone at the Rf corresponding to T-2 toxin was relatively intense. This confirms that in the isolates from the Orenburg district studied here T-2 toxin is the major principle responsible for rabbit skin irritation. These findings, are in contrast with the results published in the Soviet Union (25-29), which identified the toxins isolated from F. poae and F. sporotrichioides as steroids. Investigators in the United States (24) and Japan (37) have previously found that authentic ATAproducing isolates of F. sporotrichioides from the Soviet Union produced trichothecene compounds, chiefly, Our results fully confirm this. ACKNOWLEDGMENTS This work was supported by the Joint Research Fund of the Hewbrew University and Hadassah. We thank Miriam Goldstein and Rachel Levine for technical assistance. Purified crystalline T-2 toxin was kindly supplied by Y. Ueno, Microbial Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Ichigaya, Japan. LITERATURE CITED 1. Bamburg, J. R., W. F. 0. Marasas, N. V. Riggs, E. B. Smalley, and F. M. Strong. 1968. Toxic spiroepoxy compounds from Fusaria and other Hyphomycetes. Biotechnol. Bioeng. 10:445-455. 2. Bamburg, J. R., N. V. Riggs, and F. M. Strong. 1968. The structure of toxin from two strains of Fusarium tricinctum. Tetrahedron 24:3329-3336. 3. Bamburg, J. R., and F. M. Strong. 1969. 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