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Pestka Date f” 979/ 0-7639 MSUis an Am'mnn'vv ‘ ’ '2, ' n, '_, Institution MSU RETURNING MATERIALS: Place in book drop to “angles remove this checkout from w. your record. FINES will be charged if book is returned after the date stamped below. 53%: 2 I? PRODUCTION OF DEOXYNIVALENOL (VOMITOXIN) IN LIQUID CULTURE by Abdalla Z. El-Bahrawy A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for MASTER OF SCIENCE Department of Food Science and Human Nutrition 198‘} DEDICATION To my great and wonderful country, EGYPT ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to his major advisor, Dr. James J. Pestka, for his patient counseling and guidance throughout the couse of these studies and during the preparation of the thesis. Grateful recognition is extended to Drs. Everett 5. Beneke and P. Hart for their valuable help. Appreciation is also extended to Dr. A. Pearson for his help. The author would also like to thank Ms. Barbara Reeves for her help during typing. Finally, a special thanks is extended to all of his labmates for their help and cooperation during the research work. TABLE OF CONTENTS Abstract Introduction Literature Review Historical Background Chemistry Toxicological Characteristics of DON Biosynthesis Microbial and Chemical Modification Natural Occurrence of DON Fate of Deoxynivalenol During Food Processing Regulation of DON Control DON Production in the Laboratory Extraction and Quantitation of DON Chapter 1 Abstract Introduction Materials and Methods Chemicals Culture Inoculum Preparation Media Analysis Results Discussion Chapter 11 Abstract Introduction Materials and Methods Chemicals Culture Inoculum Preparation Analysis Results Discussion Summary Appendix A Appendix B Appendix C Appendix D Appendix E Bibliography iii 49 50 51 52 53 54 10. LIST OF TABLES Emetic Doses of DON Natural Occurrence of DON Heat Stability of DON DON Levels Detected in Cereal Foods Production of DON and l5-A-DON in Liquid Culture by U-5373 Effect of Macroconidia Produced by CMC Shake Medium on DON Production Effect of Com Steep Liquor Concentration on DON Production Effect of Sucrose Concentration on DON Production Effect of Ammonium Tartrate Concentration on DON Production Comparison of DON Production Among Fusarium graminearum Strains iv 10 ll 22 23 25 25 26 39 10. ll. 12. LIST OF FIGURES Chemical structure of DON and its related compounds Trichothecene Biosynthesis Proposed pathways for the biological and chemical transformations of deoxynivalenols Single spore isolation technique Inoculum preparation technique Extraction and analysis technique Time course of DON and lS-A-DON production, mycelial growth, carbohydrate utilization, and pH changes of E. graminearum (U5373) in Fries medium at 28 C Time course of DON and lS-A-DON production, mycelial dry weight, carbohydrate utilization, and pH changes of E. graminearum (U5373) in Fries plus four percent corn steep liquor at 28 C Time course of DON and 15- A- DON production mycelial fl)? weight6 of four inoculum sizes (103, 02‘, and 10 6) of F. graminearum in FriesO plus four percent corn steep liquor at 28 C Time course of DON and l5-A-DON production, mycelial growth, carbohydrate utilization, and pH changes of _F_‘. graminearum (U5373) in GYEP medium at 28 C Time course of DON and 15-A-DON production, mycelial growth, carbohydrate utilization, and pH changes of E. graminearum (U5373) in GYEP plus four percent corn steep liquor at 28 C Time course of DON and lS-A-DON production, mycelial growth, carbohydrate utilization, and pH changes of E. graminearum (U5373) in Fries plus four percent corn steep liquor at 28 C l8 19 21 30 31 32 33 34 4L3 13. Time course of DON and lS-A-DON production, mycelial growth, carbohydrate utilization, and pH changes of E. graminearum (Van Wert A-l) in Fries plus four percent corn steep liquor at 28 C #4 114. Time course of DON and 15-A-DON production, mycelial growth, carbohydrate utilization, and pH changes of F. graminearum (Stuckey) in Fries plus four percent corn steep liquor at 28 C #5 15. Time course of DON and l5-A-DON production, mycelial growth, carbohydrate utilization, and pH changes of E. graminearum (NRRL-5883) in Fries plus four percent corn steep liquor at 28 C #6 vi ABSTRACT Growth and toxigenesis by Fusarium graminearum were studied in liquid media. Parameters monitored during fermentations included toxin production, fungal mass, carbohydrate utilization, and pH changes. Factors, which were varied, included basal medium composition, sucrose concentration, and ammonium tartrate concentration, inoculum size, and E. graminearum strain. Supplementation of both modified Fries medium and GYEP with four percent corn steep liquor greatly increased DON yields by E. graminearum strain U5373 compared to modified Fries medium or GYEP medium alone. Highest deoxynivalenol (DON) yield (16.5 mg/L) was in modified Fries medium supplemented with four percent corn steep liquor incubated for 20 days at 28°C. U5373 gave high mycelial dry weight when it was grown in modified Fries medium and four percent corn steep liquor (3.2 g/flask after five days) or in GYEP plus four percent corn steep liquor (2.8 g/flask after five days) when compared to growth in modified Fries or GYEP alone. In all of the media, pH rose from acidic levels and peaked to alkaline levels by the end of the fermentation. Lower yields of DON were obtained when sucrose concentration higher than three percent were used. Higher levels of ammonium tartrate ( 1.5%) reduced both the toxin yield and the final pH. Of a total of nine Fusarium graminearum strains tested, only four produced DON and l5-acetyl deoxynivalenol (lS-A-DON) in modified Fries plus corn steep liquor. Strains U5373, Van Wert A-l, and Stuckey produced mainly DON while strain NRRL 5883 produced lS-acetyl DON as a main trichothecene. Conditions for optimal production of DON in liquid culture is thus dependent on medium composition and strain of E. graminearum. INTRODUCTION Mycotoxins are toxic secondary metabolites produced by fungi in foods, feeds, and other agricultural products which can cause serious health problems and diseases in exposed humans and animals. Diseases caused by mycotoxins are known as mycotoxicoses (Bullerman, 1981). Due to the occurrence of mycotoxins in human foods and animal feeds, these compounds have been of concern to agricultural researchers during the past 25 years. Deoxynivalenol (DON), also known as vomitoxin, is a naturally occurring mycotoxin which has an LD50 of 70 mg/kg in male mice (Cole et al., 1981). In addition, it causes emesis and feed refusal in swine (Vesonder et al., 1973). DON is a secondary metabolite of E. gaminearum Schwabe, a fungus which is identified as a pathogen of corn, wheat, barley, oats, and rice in the USA, Canada, and Japan (Vesonder et al., 1979; Hart et al., 1983; Scott et al., 1984; Yoshizawa, 1983). DON is produced by the fungus and can remain in grains even after killing of the mold. Food processing procedures such as baking (El-Bana et al., 1983), milling (Hart et al., 1983), and cooking (Yoshizawa, 1983) have no effect on DON levels in the final product. Currently, it is impractical to inactivate DON once it is present as a contaminant in a grain (Christensen et al., 1978). DON has been reported as a contaminant of wheat breakfast cereals, wheat flour, bran, cookies, biscuits, crackers, and baby cereals in Canada (Scott, 1984). DON has also been reported to be a contaminant of cooked rice, bread, and noodles in Japan and Korea (Yoshizawa, 1983). Various laboratories have produced DON under laboratory conditions. Although most investigators have used autoclaved moist cereals such as wheat, corn, and rice for DON production, it is of more interest to study the production of DON in liquid cultures in order to achieve the following objectives. 1. Define the optimum environmental conditions for DON production. 2. 'Identify the key biosynthetic intermediates and key enzymes in these reactions. 3. Produce DON for 14¢ labeled DON to study the fate of DON during metabolism. 4. Mass produce the toxin for toxicity and metabolism studies in feeding trials. Production of DON in liquid culture has been described by Morooka et a1. (1972) in which Czapek-Dox medium supplemented with a 0.5% peptone at 25 C for 14 days was used. When E. graminearum was grown in glucose-yeast extract-peptone (GYEP) 0 medium at 28 C, DON and 15-acetyl DON were detected after 14 days by Miller et a1. (1983). These workers concluded that in liquid culture 15-acetyl DON was produced in higher levels by the North American isolates of E. graminearum compared to DON levels. This research was carried out using liquid culture to (a) define a suitable medium for high levels of DON production; (b) identify factors affecting DON production such as carbon source, nitrogen source, trace elements, time, and inoculum concentration; (c) determine the relationship of DON production, fungal growth kinetics, and carbohydrate utilization; and (d) screen various isolates of Fusarium graminearum for DON and l5-A-DON production in an optimal medium. LITERATURE REVIEW Historical Background DON was first isolated from Fusarium-damaged barley in the 1970 epidemic in Kagawa, Japan. At that time it was given the name Rd-toxin (Morooka et al., 1972). In 1972, several Fusarium outbreaks occurred in the United States' corn belt in which swine refused to eat infected corn which, in many cases, caused the swine to vomit. The refusal and emetic factors were identified as DON and given the trivial name vomitoxin (Vesonder et al., 1973). Chemistry Deoxynivalenol (Rd-toxin) (Vomitoxin) (30 7a l5-Trihydroxy-12,13- epoxytrichothec-9-en-8-one) (Cole et al., 1981) is a colorless, fine-needles compound obtained by crystalization from ethyl acetate-petroleum ether, with a melting point of 151-153 C and a molecular weight of 296.1296 (Yoshizawa, 1983). It is a highly polar molecule and thus soluble in polar organic solvents and water ( > lg/ml) (Poland, 1984). The chemical structure of DON and related metabolites are shown in Figure l. Toxicological Characteristics of DON Vomiting is one of the most significant symptoms occurring in animals when contaminated feed is consumed. DON has reported to have emetic activity in swine (Vesonder et a1, 1973), ducklings, cats, and dogs (Yoshizawa et al., 1977). Table 1 summarizes the data on the emetic effects of DON. 3 RT o 5 4 H2 ’ R3 I CH3 R2 Trichothecene 31 32 33 Deoxynivalenol OH OH OH 15- monoacetyldeoxynivalenol OH OH OAc 3- monoacetyldeoxynivalenol OAc OH OH 3,7- diacetyl deoxynivalenol OAc OAc OH 3,7,15- triacetyldeoxynivalenol OAc OAc OAc Figure 1. Chemical structure of DON and its related compounds. Table l Emetic Doses of DON Emetic Dose (mg /kg) Toxin Ducklinga _C_a_t_a Doga Swineb DON 10.0 0.1 0.1 0.05 aYoshizawa et al., 1977 bVesonder et al., 1973 DON has also been shown to be the etiological agent responsible for feed refusal exhibited by swine (Vesonder et al., 1976). DON makes corn unattractive to swine; they will starve rather than eat the com. If the ration contains more than about five percent of such corn, pigs refuse it (Christensen et al., 1978). DON lowers the feed efficiency, resulting in severe loss of body weight in farm animals (Yoshizawa, 1983). Biosynthesis DON is classified as a type B trichothecenes mycotoxins. It has been reported that trichothecene skeleton is formed from three molecules of mevalonate through the usual pathway of lipid biosynthesis involving isopentenyl, geranyl, and farnesyl pyrophosphate (Tamm, 1977). The farnesyl skeleton (an open chain) cyclizes to form trichodiene which is the parent hydrocarbon of the trichothecone series. According to Ciegler (1979), the biosynthesis of trichothecin and other trichothecene mycotoxins involves the sequence in Figure 2. 3 mevalonic acids farnesyl pyrophosphate trichodiene trichodiol 12,l3--epoxytrichothec--9-ene trichodermol 12,13-—epoxy, 4B, 8 dihyroxytrichothec-9-ene trichothecolone trichothecin Figure 2: Trichothecene Biosynthesis . Microbial and Chemical Modification According to Yoshizawa et al. (1975a, 1975b), DON and its acetylated derivatives can be converted to each other through several steps. 1:. w converted 3-acetyl DON to DON in the peptone supplemented Czapek-Dox medium. When 3-acetyl DON was added as a carbon source to sugar-free Czapek-Dox medium, E. ro_segrn_, E. n_iy11_e_ and 5. 2911711 were able to deacetylate 3-A-DON into DON. 3,7,15-tricetyl DON was converted by _F_. solani into 7,15- diacetyl DON which then deacetylated into 7-acetyl DON. It was noted that the ester at C-7 was not hydrolyzed by the fungal mycelium. Chemically, 3,7,15-triacetyl DON reacted with 1096 methanolic ammonium hydroxide (5 m1) at 5 C for 20 minutes; the analysis of the product indicated the presence of DON, 7,15-diacetyl DON, and 3,7-diacetyl DON (see Figure 3). Natural Occurrence of DON The trichothecene mycotoxins consist of about 40 kinds of derivatives; however, the mycotoxins detected in the natural environment are limited to a small number of toxins such as T-2 toxin, diacetoxyscirpenol, nivalenol, and DON. Among the natural occurring trichothecene, DON and nivalenol of 1:. graminearum are the major contaminants of cereal grains in Japan (Ueno, 1980). In 1973, 1978, 1979, and 1982, Vesonder et al. found detectable amounts of DON in corn which had been rejected by swine in the USA. DON had also been detected in Japanese barley (Morooka et al., 1972). In Canada, it has been reported by Scott et a1. (1984) that DON is a natural contaminant of Canadian wheat. Considerable evidence has indicated the natural occurrence of DON in grains grown in many parts of the world, as is summarized in Table 2. deoxynivalenol l5-acetyl deoxynivalenol 3,15-diacetyl deoxynivalenol 3,7,15-triacety1 deoxynivalenol 7,15-diacety1 deoxynivalenol 7-acetyl deoxynivalenol 3,7-diacetyl deoxynivalenol wwavewwr 3-acetyl deoxynivalenol Figure 3. Proposed pathways for the biological and chemical transformations of deoxynivalenols. Symbols: ------- , biological transformation; - - - -, chemical transformation (Yoshizawa, 1977). Table 2 Natural Occurrence of DON So_urc.e Corn Corn Corn Barley Corn Mixed feed Maize kernals Commercial pellets Corn Wheat Feed barley Malt barley Oats Wheat Wheat Rye Country USA USA USA Japan France USA USA USA USA Canada UK UK UK UK USA Canada Content (22m) 3 8 8 5 0.1-0.6 l 0.1 0.04-0.06 25 0.22-0.74 0.02—O.5 0.02-0.1 0.0 2—0.1 0.02-0.5 0.02-0.5 0.1 W Vesonder et a1. (1973) Ishii et a1. (1975) Yoshizawa et al. (1977) Yoshizawa et a1. (1977) Jemmali et a1. (1977) Mirocha et a1. (1976) Mirocha et a1. (1976) Mirocha et a1. (1976) Vesonder et a1. (1977) Scott et a1. (1984) Gilbert et a1. (1983) Gilbert et a1. (1983) Gilbert et a1. (1983) Gilbert et a1. (1983) Gilbert et al. (1983) Scott et a1. (1984) 10 Fate of Deoxynivalenol During Food Processing 0 Heat treatment has no effect on DON levels unless it is higher than 100 C for 30 minutes, as is illustrated in Table 3. Table 3 Heat Stability of DON 0 Time Recovery 9 _ 9.6. 100 30 100 150 30 80 180 30 4O Source: Kamimura et al., 1979. Trichothecenes are highly stable to heat and chemical treatments (Bamburg, 1976) which gives the indication that they might be stable during different food process applications. It is an area of interest to know the effect of food processing on the DON levels in contaminated grains. Baking Egyptian bread at high temperature (350°C for two minutes) did not reduce DON concentrations when compared to original spiked flour levels (El-Banna et al., 1983). Japanese bread baking and Chinese noodle preparation have a slight effect on DON levels in the final products (Kamimura et a1, 1979). Recently, it has been determined that treatment of corn which was naturally DON-contaminated with sodium bisulfite resulted in partial degradation of DON (Swanson et al., 1984). Although this might possibly be incorporated in feed and food processing, it is not yet a practical approach. 11 Since DON is heat stable and food processing procedures have no effect on DON levels which were present in the ingredients, Hart et a1. (1983) suggested that DON might be transferred to processed wheat foods. Scott (1984) reported that, in fact, there are detectable levels of DON in processed wheat cereals as illustrated in Table 4. Table 4 DON Levels Detected in Cereal Foods No. Samples DON, averaged Food Analyzed ma Wheat breakfast cereals 36 0.086 Wheat flour 43 0.40 Bran 14 0.17 Bread (including buns) 21 0.080 Cookies/biscuits 35 0.12 Crackers 20 0.27 Baby cereals (mixed) 30 0.043 aproduct basis Source: Scott, 1984. 12 Regulation of DON The United States' Food and Drug Administration has issued a level of concern of two ppm for wheat entering the milling process, one ppm for finished wheat products for human consumption, and four ppm for wheat and wheat- milling byproducts used in animal feeds (Poland, 1984). After determining that high levels of DON occurred in Ontario wheat, the Canadian government decided to limit human exposure to DON. Canadian Health Protection Branch established a DON guideline of 0.3 ppm for soft wheat and a zero level or no detectable level for wheat to be used in baby foods (Food Chemical News, 1983). Control Moisture content is the most important factor affecting fungal . . . ‘ 1 JP" . colonization of stored cereals. Mmsture contents below 1596 (wethleight ba51s) will prevent Fusarium graminearum colonization. The growth of toxigenic fungi and subsequently the contamination with DON can also be prevented by the use of antifungal agents. Antifungal agents such as sorbates, propionates, and benzoates have been used to prevent fungal growth. In addition, antifungal antibiotic, natamycin, and plant-derived products such as components of the essential oils of certain herbs and spices have inhibitory effects on both fungal growth and toxin production (Ray et al., 1982). Genetic resistance to Fusarium ear infection and/or mycotoxin synthesis in cereals are also desirable control possibilities. DON Production in the Laboratory A variety of cultural and environmental conditions support DON production. Most frequently, autoclaved moist cereals such as wheat, corn, and rice are used for DON production (Vesonder et al., 1982). Such natural substrates 13 sometimes yield high levels of DON but extraction and purification is complicated by many interfering substances making it a costly procedure. Furthermore, little can be learned about DON biosynthesis, nutrient uptake and fungal growth when solid substrate are used. Liquid culture fermentation for DON production has been described using different media systems. Morooka et a1. (1972) supplemented a semidefined medium containing Czapek-Dox (NaNO3, KHZPOq, MgSOg, KCl, FeSOq and sucrose) with 0.596 peptone to produce DON and named it Rd-toxin. GYEP, a medium containing glucose, yeast extract, and peptone, was used for DON production by E. graminaerum (Miller et al., 1983). The toxin production was mainly shunted towards 15-A-DON pathway production rather than DON. These results led the authors to conclude that the North American I_=_. graminaerum isolates produce mainly l5-A-DON in liquid culture. They also suggested that factors such as reduced oxygen levels, depletion of carbohydrate in the medium, pH, and nitrogen source may affect 15-A-DON and DON production. Extraction and Quantitation of DON For contaminated cereal samples and solid cultures, CH3OH-HZO (60:40 v/v) is typically used to extract toxin residues. The CH3OH was evaporated, then extracted with ethyl acetate (1:1) three times and evaporated to dryness under a vacuum (Vesonder et al., 1978). The most common organic solvents used for DON extraction from liquid culture are ethyl acetate (three times with equal volume) (Yoshizawa et al., 1975) and chloroform-methanol (4:1) containing 0.8% (v/v) HZO (Miller et al., 1983). Sodium sulfate is used to dry the extract and the extract evaporated (Yoshizawa et al., 1975a). For detection and quantitation of DON, gas liquid chromatography (GLC), gas chromatography/mass spectrometry (Scott et al., 1981), high pressure liquid chromatography (HPLC) (Bennett et al., 14 1981), and thin layer chromatography (TLC) have been used. TLC is simpler and less time- and money-consuming as compared with other methods. To carry out TLC, the silica gel plate is developed in chloroform-methanol (97:3 and 5:1), chloroform-acetone (3:1 and 3:2), ethylacetate toluene (3:1) (Yoshizawa, 1975a) or chloroform-acetone-isopropanol (8:1:1) (Trucksess et al., 1984). Different chromators have been used such as 20% aqueous sulfuric acid and heated at 1100 C for 10 minutes (Yoshizawa, 1975), three percent 4(p- nitrobenzyl), pryridine and heated at 150°C for 30 minutes (Takitani, 1978) and dipping plates in aluminum chloride (15%) and heated for six minutes at 120°C. In the latter method, DON is observed under longwave ultraviolet lights as blue spots and quantitated by comparing fluorescence intensity of a sample spot with the standard spots visually or densitometrically (Trucksess et al., 1984). CHAPTER I FACTORS EFFECTING DEOXYNIVALENOL PRODUCTION IN LIQUID CULTURE BY FUSARIUM GRAMINEARUM 15 ABSTRACT Growth and toxigenesis by Fusarium graminaerum U5373, an isolate of Michigan wheat, were studied in liquid media. Parameters monitored during fermentation included toxin production, fungal mass, carbohydrate utilization, and pH changes. Factors, which were varied, included basal medium composition, sucrose concentration, ammonium tartrate concentration, and inoculum concentration. Using modified Fries medium for deoxynivalenol (DON) production resulted low levels of DON (0.25 mg/L) and l5-acety1 deoxynivalenol (l5-A-DON) (0.25 mg/L) after 20 days. However, supplementation of modified Fries with four percent corn steep liquor increased DON level (16.5 mg/L) after 20 days. Mycelial dry weight was 2.8 g/flask after five days in modified Fries plus four percent corn steep liquor as compared to 1.0 g/flask in case of modified Fries medium alone. Moreover, the final pH and carbohydrate utilization in modified Fries medium plus four percent corn steep liquor were higher than in modified Fries medium. An inoculum size of 106 macroconidia/flask gave higher DON production (16.5 mg/L) after 20 days than inocula of 103, 10“, and 105 macroconidial/flask in modified Fries plus corn steep liquor. Increasing sucrose concentration increased the mycelial dry weight but decreased the DON level after 20 days in this medium. Increased concentrations of ammonium tartrate in modified Fries plus corn steep liquor reduced both the toxin level and the final pH after 20 days. Replacement of yeast extract with trace elements and/or vitamins resulted in decreased DON levels in 20 day cultures. Utilization of GYEP medium for toxin production resulted, after 20 days, in high levels of 15- A-DON (14.0 mg/L) compared to DON (5.5 mg/L). However, supplementation of GYEP with four percent corn steep liquor produced 4.5 mg/L of DON and no 16 detectable level of 15-A-DON after 20 days. Mycelial dry weight was 2.8 g/flask after five days in GYEP plus corn steep liquor compared to 0.4 g/flask in case of GYEP alone. 17 INTRODUCTION Ear rot is an important disease of corn in the north central region of the United States (Vesonder, 1978). The disease can reach epidemic proportions and result in severe economic loss not only in reduced grain quality, but also from intoxication of farm animals eating contaminated corn (Vesonder et a1, 1973). The common toxic responses, vomiting and feed refusal in animals, are caused by the secondary metabolite DON. DON is produced by Fusarium graminaerum Schwabe prior to and/or during storage of cereal grains (Hart, 1983). DON has been reported as a dominant natural contaminant of cereal grains (Ueno, 1980) and occurs in processed wheat foods (Scott, 1984). Since DON is heat stable (Kamimura et a1. , 1979) and food processing procedures have little effect on DON levels which are present in the contaminated grain (Hart et al., 1983; El-Banna et al., 1983), the mycotoxin is carried over into foods. Scott (1984) reported that there were detectable levels of DON in commercial wheat foods such as breakfast cereals, cookies, crackers, and baby cereals. The study presented herein was carried out to define suitable media for high DON production and identify possible factors affecting toxin production in liquid culture. 18 M ATERIALS AND METHODS Chemicals All inorganic chemicals and organic solvents were of reagent-grade quality or better. DON standard was purchased from Mycolabs Company (St. Louis, Missouri). Identity of 15-A-DON from our isolates was confirmed with GC-MS by C. Mirocha (University of Minnesota). 3-A-DON was supplied by Miller (Chemistry and Biology Research Institute, Agriculture Canada, Ottawa). Corn steep liquor was purchased from Corn Products Company (Cook County, Illinois). Culture Fusarium graminearum U5373 (Michigan wheat isolate) was obtained from L. P. Hart (Department of Botany and Plant Pathology, Michigan State University). Culture purity was assured by the single spore isolation method as described in Appendix D, and a summary is found in Figure 4. Fusarium species were preserved and stored in sterilized soil as described in Appendix E. Suspension of Conidia Prepared Using 10ml Water Pour the Suspension over Solidified Agar Plate (2%) Incubate in an Inclined Position for 16-24 Hours at Room Temperature 1 Examine the Germination Under Dissecting Microscope Y Single Germinating Conidia Are Cut Out with Dissecting Needle and Transferred to the Growth Medium Figure 4. Single spore isolation technique. 19 Inoculum Preparation Stock _Ii. graminearum strains were maintained in sterilized soil. For inoculum, strains from soil tubes were grown on potato dextrose agar (PDA) at 25°C for seven days under alternating fluorescent light and darkness (12 hours each). Agar plugs (from colony edge) were asceptically transferred to 250 ml Erlenmeyer flasks containing 40 m1 carboxymethyl-cellulose medium (CMC) (See Appendix B) and agitated on a rotary shaker at 250 rpm for three to five days at 25 C (Cappellini et al., 1965) (see Figure 5). Conidial concentrations were determined with a hemacytometer slide. Strains Grown on PDA (25 C) for Seven Days I Agar Plugs Transferred to CMC Medium 1 Agitation on a Rotary Shaker at 250 rpm V Filter the Culture Through Cheese Cloth Y Macrocondidia Counted by Hemacytometer Slide Figure 5. Inoculum preparation technique. Media Modified Fries (see Appendix A) and GYEP (see Appendix C) were prepared and added to Roux flasks in 200 m1 volumes. For comparative experiments, four percent (v/v) corn steep liquor was added to each and they were autoclaved (1210 C for 15 minutes). The standard experimental protocol involved inoculating each flask with 106 macroconidia and incubating it at 28°C in the dark. Each set (10 flasks) of media (modified Fries, modified Fries plus four percent corn steep liquor, GYEP, and GYEP plus four percent corn steep liquor) was incubated in 20 duplicate over a period of 25 days and flasks were analyzed after five-day intervals. For studying the inoculum effect on DON production, modified Fries medium plus four percent corn steep liquor was prepared, autoclaved (121 Cofor 15 minutes), inoculated with 103, 10“, 105, and 106 macroconidia/flask, and incubated at 28°C in the dark over a period of 25 days. Toxin production, carbohydrate, and pH were determined at five-day intervals from two individual flasks of each inoculum concentration. To study the effect of varying the concentration of corn steep liquor in modified Fries on DON production, modified Fries medium was prepared; then an amount of corn steep liquor, from one percent through six percent, was added. Two flasks containing 200 ml medium each (for each concentration) were autoclaved (121°C for 15 minutes), then inoculated with 106 macroconidia/flask and incubated at 28°C for 20 days. Similarly, sucrose concentrations (w/v) of one percent through five percent in modified Fries medium plus four percent corn steep liquor were prepared to study the effect of sucrose on DON production and concentrations of 0.5% - 2.096 of ammonium tartrate were added to modified Fries medium to study the effect of ammonium tartrate on DON production by 1:. graminearum after 20 days incubation in modified Fries plus four percent corn steep liquor. Trace elements formula (Difco Manual, 1953) were prepared, and 1 ml was added to each flask containing 200 m1 modified Fries medium without yeast extract and autoclaved (121°C for 15 minutes). The flasks were inoculated with 106 macroconidia each and incubated at 28°C in the dark. Duplicate flasks were used and studied over a 20-day period. Similarly, a vitamin formula (Difco Manual, 1953) was used to study the effect of vitamins on DON production in modified Fries medium without corn steep liquor added. 21 Analysis After incubation, the mycelial mat was separated from the broth by filtering through tared Whatman No. 4 filter paper. The mycelium was dried at 60°C for 12 hours and weighed to determine dry weight. The filtrate was assayed for pH and for total carbohydrate by the phenol method (Hansen 6t Philips, 1981). Aliquots (100 ml) of filtrate were then extracted three times with equal volumes fo ethyl acetate, the solvent dried by filtration through Na2504, and the extract evaporated to dryness under vacuum. Dried extracts were redissolved in one m1 ethyl acetate and 1-5 pl spotted onto ALCl3-dipped silica gel plates. These were developed in ethyl acetate-toluene (3 + l). Plates were heated at 110° C for five minutes, dipped in paraffin oil-hexane (3 + 7) and compared with DON, 15-A-DON, and 3-A—DON standard under longwave ultraviolet light (Trucksess et al., 1984). Standard DON fluorescence was used to quantitate DON and 15-A- DON. Extraction and quantitation methods are summarized in Figure 6. Filtration of the Culture Using Whatman No. 4 I The Aliquot of Filtrate Was Extracted (Three Times) with Ethyl Acetate (1 : 1) V Ethyl Acetate Dried with Sod Sulfate and Evaporated V Ethyl Acetate Dried with Sod Sulfate and Evaporated l TLC (Silica Gel)--Toulene:Ethyl Acetate (l:3)-(ALC13) V F j Rf 0.21 (DON) Rf 0.46 (l5-A-DON) Figure 6. Extraction and analysis technique. 22 RESULTS Macroconidia of E. graminearum were produced in shake cultures containing a simple salts-yeast extract-carboxymethylcellulose medium (CMC). The yield of macroconidia ranged from 106 to 107 macroconidia/ ml. The effect of basal medium composition on DON production was systematically examined and the results are summarized in Table 5. Table 5 Production of DON and 15-A-DON in Liquid Culture by U-5373a Toxin Concentration (mg/L) Medium DON l5-A-DON 1. Modified Fries 0.3 0.3 2. Modified Fries plus four percent corn steep liquor 16.5 n.d. 3. Four percent corn steep liquor n.d. n.d. 4. Modified Fries without ammoninum tartrate plus four percent corn steep liquor .15 .l 5. GYEP .55 14.00 6. GYEP plus four percent corn steep liquor 4.50 n.d. 7. Modified Fries without yeast extract 0.3 0.3 8. Modified Fries without yeast extract and trace element 0.3 n.d. 9. Modified Fries without yeast extract and vitamin n.d. n.d. O acultures were grown at 28 C for 20 days, then analyzed for toxin production n.d. = none detected 23 Utilizing modified Fries medium for toxin production yielded low levels of both DON (0.25 mg/L) and l5-A-DON (0.25 mg/L) after 20 days. In this medium, U5373 had a linear growth of 0.2 g/day (Figure 7). At day five, maximum levels (1 mg/L) of both DON and A-DON were found, but these decreased with time. Higher DON yields (16.5 mg/L) were obtained on modified Fries medium supplemented with four percent corn steep liquor (pH 4.0) at 28 C at day 20. Low levels (2 mg/L) of 15-A-DON were produced during the growth phase but were not found at day 20. In modified Fries plus corn steep liquor, DON production began and pH reached 5.5, then peaked at pH 8.0 (see Figure 8). The pH of the medium increased by the end of the fermentation. Using macroconidia produced in CMC medium for inoculum greatly increased DON production compared to using agar plugs from the colony edge. Representative results comparing the two procedures are shown in Table 6. Table 6 Effect of Macroconidia Produced in CMC Shake Medium on DON Productiona Type of Inoculum DON Production (mg/L) Macroconidia produced from CMC 16.5 Agar plug from the colony edge 2.0 O acultures were grown at 28 C for 20 days, then analyzed for toxin production. The effect of differing the inoculum concentrations was tested. The results shown in Figure 9 indicate that changing the inoculum concentrations from 103 to 106 macroconidia/200 m1 of modified Fries plus four percent corn 24 steep liquor gradually increased the DON production in modified Fries plus corn steep liquor but had no effect on the growth kinetics of the liquid culture (Figure 9). Interestingly, at all of the inoculum concentrations which were tried (103, 10", 105, and 106/f1ask), DON levels peaked at day 20. Levels of l5-A-DON were much lower and peaked at day five. Supplementation of modified Fries medium with four, five, and six percent corn steep liquor greatly increased DON yields to 15 mg/L (Table 7). Concentrations of less than four percent reduced the levels of DON. Using four percent corn steep liquor as the sole medium for DON production resulted in zero toxin being produced (Table 5). Generally, toxin production began to peak at the late stationary phase of growth (Figures 8 and 11) when the media were supplemented with corn steep liquor, but earlier when these media were not amended (Figures 7 and 10). The optimal sucrose concentration for DON production in modified Fries plus corn steep liquor was 2-3% (Table 8). Five percent sucrose increased the dry weight (3.9 g/flask), but decreased DON yield (10 mg/L). The optimum ammonium tartrate concentration for DON production was 0.5 - 1.0%. Higher concentrations decreased the toxin yield (1 mg/L) and the final pH, 8.6 (Table 9). Addition of vitamins and trace elements had no effect on DON production (Table 5). The utilization of GYEP medium for DON production resulted in a large increase in the production of l5-A-DON (14 mg/L) and a reduction in DON (5.5 mg/L) after 15 days (Figure 10). The growth rate in GYEP was 0.1 g/day and the pH first decreased then began to increase after 10 days. However, supplementation of GYEP with four percent corn steep liquor resulted in DON concentrations of 4.5 mg/ml, with no detectable levels of 15-A-DON after 20 days. The growth rate in GYEP plus corn steep liquor was 0.56 g/day 25 and the pH of the medium did not decrease at any time, but did increase to pH 8.6. Table 7 Effect of Corn Steep Liquor Concentration on DON Productiona Corn Steep Mycelial Liquor Final Dry Weight Carbohydrate DON (16) L”. g/flask mg/ml mg/L 1 8.0 4.1 1.80 2.5 2 8.2 4.0 1.85 5.0 3. 8.2 3.5 1.90 7.0 4 8.4 3.6 2.10 15.0 5 8.3 4.0 2.20 15.0 6 8.3 4.6 2.20 15.0 O aThe culture was grown on modified Fries plus corn steep liquor at 28 C for 20 days. Table 8 Effect of Sucrose Concentration on DON Productiona Sucrose Mycelium Final Concentration Dry Weight Carbohydrate DON (1) (gram/flask) mg/mL mg/L 1 2.6 0.3 20.0 2 2.9 1.2 18.5 3 3.0 1.9 18.5 4 3.8 2.2 12.5 5 3.9 2.2 10.0 0 8culture was grown on modified Fries plus four percent corn steep liquor at 28 C for 20 days. 26 Table 9 Effect of Ammonium Tartrate Concentration on DON Productiona Ammonium Tartrate Mycelium Final Concentration Final Dry Weight Carbohydrate DON (31) pH g/flask mg/ml mg/L 0.0 7.3 4.0 1.25 0.2 0.5 8.2 3.7 2.10 12 1.0 8.8 2.7 2.0 5 13 1.5 8.8 2.8 2.00 3 2.0 8.6 2.8 2.0 5 1 aculture was grown on modified Fries plus four percent corn steep liquor at 28 C for 20 days. DISCUSSION Secondary metabolites of fungal origin have been characterized as follows: 1. their production is extremely specific, often being confined to one specie or one strain of a species; 2. in general, they have no obvious function in the life of the producer organism; and 3. they are produced by cells in which growth is restricted. That is, they occur in the early stationary phase of a batch culture. In general DON production by 1:. graminearum U5373 followed the general pattern of other secondary metabolites. _F_. graminearum U5373 was isolated from infected Michigan wheat by Dr. Hart (Department of Botany and Plant Pathology, Michigan State University). It was used for this study because it produced high levels of DON in studies with inoculated wheat (Hart, unpublished) and corn (Hart, 1983). Fusarium 27 gaminearum strains produce perithecia and macroconidia on carnation leaf water agar. Growing E. graminearum in CMC medium in shake cultures induced the production of large quantities of macroconidia. The mechanism of CMC enhancement is not known. In general, utilization of macroconidia rather than agar plugs greatly improved the reproductibility of DON production in liquid media. Modified Fries medium and GYEP medium supplemented with four percent corn steep liquor produced the highest levels of DON (see Figures 8 and 11); decrease or removal of corn steep liquor greatly reduced toxin yields in both modified Fries and GYEP (see Figures 7 and 10). Corn steep liquor contains several precursors and stimulators (phenyl acetic acid, organic acids) of fungal secondary metabolites such as penicillins (Perlman, 1967), aflatoxins (Jarvis, 1971), and T-2 toxin (Cullen et a1, 1981). This is of further significance because corn steep liquor might contain similar components to those present in the corn kernels which also apparently stimulate DON production in the field. Modified Fries medium is a complex substrate used for fungi and has previously been used for the production of Periconia circinata toxin which inhibits the growth of sorghum roots (Pringle et al., 1963). GYEP medium has been used by Miller et a1. (1983) for the production of DON and 15-A-DON. The supplementation of modified Fries and GYEP with four percent corn steep liquor increased the mycelium dry weight because corn steep liquor might include nutrients which enhance the growth and give intact mycelium mat. Our results showed that high sucrose concentrations increased the mycelial dry weight and decreased the toxin yield; this is similar to the results of a study on the effect of glucose concentrations on DON production by E. graminearum by Miller et a1. (1983). An explanation of the effect of sucrose can be understood by examining the time course study (Figure 8). It is important to note that the 28 fungus reached the stationary phase while the carbohydrate concentration was high. Only during the stationary phase when the carbohydrates began to decline did the levels of DON increase. These results suggest that when the medium contains high levels of carbohydrates, the fungus uses it for growth, but when the carbohydrates are reduced to low levels, the fungus begins to produce the most DON (a secondary metabolite). The inhibitory effect of ammonium tartrate on toxin production might be due to the limiting the activity of secondary metabolite enzymes by high concentrations of ammonium ions in the medium (Morton et al., 1960). The results obtained concerning the ammonium tartrate effect are in agreement with secondary metabolite characteristics since the secondary metabolite does not begin as long as an adequate nitrogen supply for growth is available (Griffin, 1982). Increase in pH to an alkaline level might be responsible for the mycelial autolysis (Lahoz et al., 1968). Another possible explanation for mycelium autolysis is that the toxin itself might have antifungal activity since the mycelium dry weight begins to decrease after the toxin is produced. This explanation is in agreement with the biological activity of trichothecenes since trichothecenes are antibacterial, antifungal, antiviral, and cytostatic (Cole et al., 1981). The shunting of toxin production pathway towards l5-A-DON production in GYEP fermentation is consistent with a similar study by Miller et a1. (1983). However, these results of this latter study contrast with the results obtained when GYEP was optimized for DON production by the supplementation of four percent corn steep liquor. DON production began to peak when the pH reached 7.5 or higher which might indicate that the enzymes for DON biosynthesis require high pH for 29 maximal activity. The toxin production at high pH is in agreement with similar fermentation for the production of penicillin by Penicillium chrysogenium at pH 7.4 (Perlman, 1967). Further research is needed on the enzymes involved in the biosynthesis of DON and deacetylation of l5-A-DON to DON. 30 r—\ _l E; E "5 A V _‘l \ Z or O E D V 1 < Z 1 O m D H o-—o H 9 f; f‘ I ._ 30 U) E (U \’ a E \ \— CO V L #- +3 E 20 ‘ 6 go ”3 ”-4 >~ <1) .8 3 .2: ..._ >~ 5- -'~ I“ c': C— Q L} TOP 0M . 1 f . o O 5 IO '5 20 25 O——O L U D—J H Figure 7. Time course of DON and lS-A-DON production, mycelial growth, carbohydrate utilization, andopH changes of _F_'. graminearum (U5373) in modified Fries medium at 28 C. 31 3 \ on E :‘2 D00 Ea VI < 21 Om CLr—t A ‘Or ‘9 I: —4 U) E ('3 \ . v—t or: (1.. E \ 30+ 39 <1) ‘53 «o 2 L4 . ~a .3? E» a) g 20" . 3 S I i‘ G- D U .3 I 101- . I I o a . 1 a o 0 5 IO 15 2O 25 Figure 8. Time course of DON and 15- A- DON production, mycelial, dry weight, carbohydrate utilization, and pH changes of F. graminearum (U50373) in modified Fries medium plus four percent corn steep liquor at 28 C. 32 Z ,— ‘J. #16 A 2 v. : ~— - 5: \ _‘. g at \ t ,— L 13 6 V S u. EL 2 10 ..c: ._l 4 u 5 Cl‘. .... at (3 "EL < ,2 A; E 3 .._ Z 1 ‘ V ' 0 < 8 2 a“ g ' 12 ‘ 3 z: c: 2 if c: G 1 l 2 1 l I ‘ ’ l ,5 o .x 0 m A o s to 15 20 25 n: 0 0 f: .—-4 O 5 10 15 20 25 a r-: ‘41-- r-\ —" a .. E : ._‘ ‘4 "T V E g “EL : z I: 5 a '8 E“ .9: .E.‘ 2: 3 V g: a» Z ' >~ Z 1 3 c m l C: ‘- O m ._. :3 o .—. fi‘ 0 0- 5 10 15 20 25 Figure 9. Time course of DON and l5-A-DON production and mycelial dry weight of four inoculum sizes (103, 10“, 105, and 106) E. graminearum (U5373) in modified Fries medium plus four percent corn steep liquor at 28°C. o——-o DON (mg/ L) Figure 10. 33 IS—A—DON (mg/L) H .. '5 r ~ 9 2: ._. U) E to \ —4 or. £1— E \ x—x CI. 0 6 2: '° ‘ .2 $— 06 "O ”-1 >~ G) _c: 3 O >. e = e co 1:. c: U 5 p. q 3 I I O 4 J L 1 J O O 5 IO I5 20 15 Time course of DON and l5-A-DON production, mycelial growth, carbohydrate utilization, and pH changes of E. graminearum (U5373) in GYEP medium at 28 C. 34 DON (mg/L) lS—A-DON (mg/L) O——O H TO- A .x 2’8 '5? 19 .-4 . 1+: \ CC V 1.) .C: 00 46 '8 3 >\ $4 C3 Carbohydrate (mg/ml) pH o——-c1 U I ..._ H O 1 ' ' O 0 5 ‘0 ‘5 10 15 Figure 11. Time course of DON and 15-A-DON production, mycelial growth, carbohydrate utilization, and pH changes of _F_. gaminearum (U5373) in GYEP plus four percent corn steep liquor at 28°C. CHAPTER II PRODUCTION OF DEOXYNIVALENOL IN LIQUID CULTURE BY NINE STRAINS OF FUSARIUM GRAMINEARUM 35 ABSTRACT Four out of nine isolates of Fusarium gaminearum were found to produce deoxynivalenol (DON) and l5-monoacetyl deoxynivalenol (l5-A-DON) in stationary liquid culture consisting of modified Fries medium supplemented with four percent corn steep liquor. Strains U5373, Van Wert A-1, and Stuckey produced mainly DON (16.5, 5.5, and 2.5 mg/L, respectively) and low levels of 15- A-DON (2, 2.5, and 0.5 mg/L, respectively) after 20 days incubation at 28°C. Appearance of DON and disappearance of 15-A-DON coincided with both a rapid rise in pH and with the onset of the stationary phase. DON peaked after the exhaustion of carbohydrate and then began to decline. These three strains had high final mycelial dry weights of 3.3 g/flask. In contrast, strain NRRL 5883 had a lower mycelial dry weight (2 g/flask) and produced primarily l5-A-DON during an extended growth rate phase. Only small amounts of DON (0.5 mg/L) appeared during the late stationary phase. NRRL 5883 exhibited a slow rise in pH relative to the other three strains and utilized only 7596 of the available carbohydrate during the 25 day period. Qualitative and quantitative appearance of DON or 15- A-DON in liquid culture is thus dependent on strain of 1:. graminearum. 36 INTRODUCTION Deoxynivalenol (DON) is a trichothecene mycotoxin which is produced by Fusarium gaminearum Schwabe and occurs naturally in cereal grains grown in the United States (Vesonder et al., 1973), Canada (Scott, 1984), Japan (Ichinoe et al., 1983), the United Kingdom (Gilbert et al., 1983), and France (Jemmali et al., 1978). DON has been reported as a contaminant of wheat breakfast cereals, wheat flour, bran, cookies, biscuits, crackers, and baby cereals in Canada (Scott, 1984). The Canadian Health Protection Branch guidelines for DON are 0.3 ppm for soft wheat and zero level for wheat to be used in baby foods (Food Chemical News, 1982). Although DON is typically produced in the laboratory on grains such as rice, corn, and wheat, these methods are not suitable for biosynthetic studies or production of radiolabeled toxin. Liquid culture fermentation was first described by Morooka et al. (1972) who found that DON can be produced using Czapek-Dox medium supplemented with 0.596 peptone at 25°C within 14 days. Miller et a1. (1983) found that l5-A-DON was the main toxin produced by North American 5. gaminearum isolates on GYEP medium (glucose, yeast extract, and peptone). In a previous report, we found that E. graminearum U5373 fermentation of modified Fries medium supplemented with four percent corn steep liquor resulted in higher yields of DON when compared to three other media. We also optimized GYEP for DON production by supplementing it with four percent corn steep liquor. In the present study, DON production by eight North American E. graminearum strains were compared to strain U5373 for production of DON, growth kinetics, carbohydrate utilization, and pH change over a 25 day period fermentation. 37 MATERIALS AND METHODS Chemicals All inorganic chemicals and organic solvents were of reagent-grade quality or better. DON standard was purchased from Mycolabs Company (St. Louis, Missouri). Identity of l5-A-DON from our isolates was confirmed with GC-MS by C. Mirocha (University of Minnesota). 3-A-DON was donated by Miller (Chemistry and Biology Research Institute, Agriculture Canada, Ottawa). Corn steep liquor was purchased from Corn Products Company (Cook County, Illinois). Culture Fusarium graminearum strains U5373, Van Wert A-l, Stuckey, Sandusky A- 2, M-3, B507-U5371, and B601-U5372 were isolated from Michgian wheat and obtained from L. P. Hart (Department of Botany and Plant Pathology, Michigan State University), and strains NRRL5883 and Crawford-5 were donated by Dr. Smalley (University of Wisconsin). Culture purity was assured by the single spore isolation method as described in Appendix D, and a summary is found in Figure 4 (Chapter I). E. graminearum isolates were preserved and stored in sterilized soil as described in Appendix E. Inoculum Preparation Stock 5. graminearum strains were maintained in sterilized soil. For inoculum, strains from soil tubes were grown on potato dextrose agar (PDA) at O 25 C for seven days under alternating fluorescent light and darkness (12 hours each). Agar plugs (from colony edge) were asceptically transferred to 250 m1 Erlenmeyer flasks containing 40 ml carboxymethylcellulose medium (CMC) (see 38 Appendix B) and the flasks were agitated on a rotary shaker at 250 rpm for three to five days at 25°C (Capellini et al., 1965). Conidial concentrations were determined with a hemacytometer slide. Roux flasks containing 200 m1 modified Fries medium plus four percent corn steep liquor (see Appendix A) were inoculated with 106 macroconidia/flask and O incubated at 28 C in the dark. Each isolate was grown in duplicate flasks. Analysis After incubation, the mycelial mat was separated from the broth by filtering through tared Whatman No. 4 filter paper. The mycelium was dried at 60°C for 12 hours and weighed to determine dry weight. The filtrate was assayed for pH and total carbohydrate by the phenol method (Hansen & Philips, 1981). Aliquots (100 m1) of filtrate were then extracted three times with equal volumes of ethyl acetate, the solvent dried with Na2504, and the extract evaporated to dryness under vacuum conditions (Yoshizawa et al., 19753). Dried extracts were redissolved in 1 m1 ethyl acetate and 1-5111 spotted onto ALC13- dipped silica gel plates. These were developed in ethyl acetate-toluene (3 + 1). Plates were heated at 110°C for five minutes, dipped in paraffin oil-hexane (3 + 7), and compared with DON, l5-A-DON, and 3-A-DON standard under longwave ultraviolet light (Trucksess et a1. , 1984). Standard DON fluorescence was used to quantitate DON and l5-A-DON. Extraction and quantitation method were summarized previously in Figure 6 (Chapter I). 39 RESULTS When E. graminearum isolates were compared for DON and l5-A-DON production in modified Fries plus four percent corn steep liquor at 28°C, only four of the nine isolates were found to produce detectable levels of DON and 15- A-DON (see Figures 12-15). The relative 20 days toxin yields are summarized in Table 10. Levels ranged from 2.5 to 16.5 mg/L for DON and from 0.5 to 12.5 mg/L for l5-A-DON. The highest DON yields were produced by U5373 within 20 days (16.5 mg/L) at 28°C (Figure 12) and the lowest by the Stuckey isolate (Table 14). Table 10 Comparison of DON Production Among Fusarium graminearum Strains Toxin Production (mg/L)a m E l5-A-DON U5373 16.5 Nob NRRL 5883 ND 10.0 Crawford-5 ND ND Wert A-l 5.4 ND Stuckey 2.5 ND B507 ND ND B601 ND ND Sandusky A-2 ND ND M—3 ND ND aToxin product at 20 days bND = none detected 40 Strains U5373, Van Wert A-1, and Stuckey produced low amounts of l5-A- DON (0.5 to 2.5 mg/L) during the early growth phase. The appearance of DON and disappearance of 15-A-DON in these three strains coincided both with a rapid rise in pH and the onset of the stationary phase. In these fermentations, DON peaked after the exhaustion of carbohydrate and then declined rapidly (see Figures 12, 13, and 14). In contrast, Figure 15 indicates that strain NRRL 5883 produced primarily l5-A-DON during an extended growth phase while the mycelium dry weight after five days was only 1.0 g/flask as compared to 3.1 g(U5373), 3.0 g(Van Wert A-l), and 3.0 g(Stuckey)/flask for the other three strains respectively. NRRL 5883 strain also exhibited a slower rise in pH and utilized carbohydrate at a much lower rate than the other three strains (see Figures 12, 13, 14, and 15). Only small amounts of DON (0.5 mg/L) appeared during the late stationary phase of NRRL 5883. 41 DISCUSSION We have previously shown that modified Fries plus four percent corn steep liquor medium is optimal for the production of DON and l5-A-DON by E. graminearum U5373 (Michigan wheat isolate). Change in basal medium composition will determine whether l5-A-DON or DON is the primary trichothecene is produced by this culture. From the results presented here, it appears that 1:. graminearum strains can vary in ability to produce either DON or 15-A-DON in modified Fries plus corn steep liquor, which is in agreement with the general characteristics of secondary metabolites (Deacon, 1980) which can be listed as follows: 1. their production is extremely specific, often being confined to one specie or one strain of a species; 2. in general, they have no obvious function in the life of the producer organism; and 3. they are produced by cells whose growth is restricted. That is, they occur in the early stationary phase of a batch culture which is in agreement with DON production (Figures 12, l3, l4, and 15). For strains U5373, Van Wert A-1, and Stuckey, the disappearance of 15-A- DON was coincident with the accumulation of DON. These results might indicate that deacetylation of l5-A-DON is a step in the biosynthesis of DON (Yoshizawa et al., 1977) as was evidenced by the relative appearance of these toxins (see Figures 12-14). Yoshizawa et al. (1975a) found that disappearance of 3-A-DON was concurrent with DON production in the fermentation by _F_. w. Yoshizawa et a1. (1977) suggested that 3-A-DON synthesis is a step in the biosynthesis of DON. Our results also indicated that these three North American 1:. graminearum isolates (U5373, Van Wert A-1, and Stuckey) produce primarily DON under optimum culture conditions which is in contrast with the 42 results of Miller et a1. (1983) which suggested that North American isolates produce mainly l5-A-DON in liquid culture. While we have taken as large a sampling of strains as the Miller et a1. study, the difference between the two studies might be related to the use of corn steep liquor in the growth medium (see Chapter I). In contrast to the three above-mentioned isolates, strain NRRL 5883 produced mainly 15-A-DON which is consistent with the Miller et al. (1983) study. These results indicate that NRRL 5883 has limited ability to convert l5- A-DON to DON which might be due to an alteration of the deacetylation enzyme activity or its being blocked. Further research on the existence of such a deacetylation enzyme is, therefore, needed. 43 DON (mg/L) IS-A-DON (mg/L) Ch—o H E ‘Or I9 f2 IE m E E V \ 8 00 E 30» g .3 16 f? >~ o -g 3 3320 I r: U r. a ‘3 I no» ' I I o 1 . I L o 0 5 IO IS 10 25 Figure 12. Time course of DON and l5-A-DON production, mycelial growth, carbohydrate utilization, and pH changes of E. graminearum (U50373) in modified Fries medium plus four percent corn steep liquor at 28 C. 44 ,7 E. E 6 r-xv <2 CLO EC) V1 <1 21 Cm Cir—1 o—-o H '0 40r 1} I. .——-. Dry weight (g/flask) D——-O Carbohydrate (mg/m1) 3 .3 Figure 13. Time course of DON and 15- A- DON production, mycelial growth, carbohydrate utilization, and pH changes of F. graminearum (Van Werto A- 1) in modified Fries medium plus four percent corn steep liquor at 28° C. 45 A .J t. E3 (“V :2 2‘8 VIZ}. < 21 Om 0—4 H H d T o 1 S? 40r '19 :3 A . C v-< \ E 05 \ V 0.13 E +4 .16 'Ec Q) u-t :7: <1) .22 t 3 >.’° :t‘ .C 0.0 O E w ‘31I U o 1 1_ 1 1 L O O 5 IO 15 2O 25 Figure 14. Time course of DON and 15- A- DON production, mycelial growth, carbohydrate utilization, and pH changes of F. gaminearum (Stuckey) in modified Fries medium plus four percent corn steep liquor at 28 C. 46 18 IS—A—DON (mg/L) CF—O DON (mg/L) H - a E 40,-- -9 r: \ H 00 {a 5 En CD / V +4 «H 2 "En '0 ‘6 "-4 2‘ “3’ -§ 201 r ?: Cd C... C21 U i ‘3 I I o l 1 1 l l o 0 IO 15 20 25 Figure 15. Time course of DON and IS-A-DON production, mycelial growth, carbohydrate utilization, and pH changes of _E. graminearum (NRRL- 58§3) in modified Fries medium plus four percent corn steep liquor at 28(3. 47 SUMMARY This study was carried out to Optimize the liquid culture fermentation for DON production. Major achievements of this investigation can be summarized as follows. 1. Modified Fries medium plus four percent corn steep liquor was optimal for DON production when compared to modified Fries, GYEP, and GYEP plus four percent corn steep liquor. 2. Lower yields of DON were obtained when a sucrose concentration higher than three percent was used on the basic modified Fries plus corn steep liquor medium. 3. Trace elements and vitamins did not induce DON production in modified Fries medium deplete of yeast extract. 4. Levels of ammonium tartrate greater than one percent in modified Fries plus corn steep liquor reduce both the toxin yield and the final pH. 5. Using CMC media for inoculum preparation yielded a high concentration of macroconidia as compared to PDA plates. Utilizing macroconidia as inoculum increased reproducibility of DON production compared to using agar plugs from cultures grown on PDA. 6. Strain U537°3 (W- 8) produced the highest yields of DON after 20 days at 28° C compared to NRRL 5883, Van Wert A-,l and Stuckey. 7. Three strains tested in modified Fries plus corn steep liquor (U5373, Van Wert A-l, Stuckey) produced primarily DON while NRRL 5883 produced primarily l5-A-DON. Thus, Fusarium graminearum strains vary in ability to produce primarily lS-A- DON or DON as has been previously suggested by Miller et a1. (1983, 1984). 8. Deacetylation of l5-A-DON could be a step in the biosynthesis of DON as was evidenced by the relative appearance of these toxins in U5373, Van Wert A-1, and Stuckey cultures. #8 DON is now recognized as a major contaminant of cereal grains and, subsequently, the foods to be manufactured from these ingredients. Therefore, a number of problems need to be solved. These include the following. 1. Investigation of sensitive and easy methods for DON detection needs to be done. - 2. Establishment of detoxification procedures for DON such as chemical and thermal methods must be undertaken. 3. Study must be done of the metabolism and fate of DON in farm animals. 4. Classification and identification of trichothecene producing Fusarium. Of relevance to this study, further research is needed on the role of deacetylation enzymes in the biosynthesis of DON. Furthermore, identification of those components of corn steep liquor which stimulated DON production is of particular interest in understanding the possible DON precursors. The submerged culture conditions in shaker flasks and fermentors need to be optimized for DON production since they have been used for other trichothecene production. APPENDICES APPENDIX A MODIFIED FRIES MEDIUM Component gZL Ammonium tartrate 5.00 Ammonium nitrate 1.00 Magnesium sulphate 0.50 Potassium phosphate 1.00 Sodium chloride 0.10 Calcium chloride 0.13 Sucrose 30.00 Ferrous sulphate .02 Yeast extract 1.0 The components were added to one liter of distilled water, then autoclaved O at 121 C for 15 minutes (pH, 5.5) (Pringle, 1963). 49 APPENDIX B CARBOXYMETHYLCELLULOSE (CMC) MEDIUM Component g_/_I_. Ammonium nitrate 1.0 Potassium phosphate 1.0 Magnesium sulphate 0.5 Yeast extract 1.0 Carboxymethylcelluose 15.0 Dissolve 15 grams of CMC in a blender in warm water, followed by the 0 other components, autoclave at 121 C for 15 minutes (Tuite, 1969). 50 APPENDIX C GLUCOSE YEAST EXTRACT-PEPTONE (GYEP) MEDIUM Component gig Glucose 10 Yeast extract 1 Peptone l The components were dissolved in one liter of distilled water, then 0 autoclaved at 121 C for 15 minutes (pH, 6.1) (Miller, 1983). 51 APPENDIX D SINGLE SPORE ISOLATION METHOD The single spore technique devised by H. N. Hansen (1907) and modified by Toussoun and Nelson (1976). It consists of pouring three ml of two percent water agar into petri dishes and letting it solidify. A suspension of conidia was prepared in 10 m1 of sterile water so that it contained 1-10 conidia under low- power (10 x) microscope field when a drop from a three mm diameter loop was examined on a slide. The conidia suspension was poured over the solidified agar to cover the entire surface, and the excess was drained off. The dishes incubated in an inclined position at room temperature for 16 to 24 hours. Then the dishes were opened and examined under a dissecting microscope. Small squares of the agar containing single germinating conidia were cut out with a dissecting needle and transferred to optimum growth medium. Identification Medium The single germinating conidia were transferred to carnation-leaf agar (CLA) medium. After three or four days at room temperature under alternating fluorescent light and darkness (12 hours each), the growth was identified to make sure that there was no mutation (Nelson et al., 1983). 52 APPENDIX E PRESERVATION AND STORAGE OF FUSARIUM SPECIES Soil, peat moss, and perlite mixture were used as media for E. graminearum preservation. A few grams of soil mixture were placed in a test tube, and the test tube was autoclaved for one hour on each of two successive days. Two plugs from the colony edge of a culture grown on carnation leaf agar were transferred to sterile soil tubes. The tubes were held at room temperature 0 for one week, then stored at 5 C. 53 BIBLIOGRAPHY BIBLIOGRAPHY Bamburg, J. R. (1976). Chemical and biological studies of the trichothecene mycotoxins. In J. V. Rodricks (Ed.), Mycotoxins and other fungal related food problems. Washington, DC: American Chemistry Society. Bennett, G. A., Peterson, R. E., Plattner, R. D., & Shotwell, O. L. (1981). Isolation and purification of deoxynivalenol and a new trichothecene by high pressure liquid chromatography. Journal of the American Oil Chemistry Society. 5_8_, 1002A-1005A. Bullerman, L. 8., 6c Buchanan, R. L. (1981). Mycotoxins other than aflatoxins-- their relationship to food safety. Journal of Food Protection, 43, 701-702. Cappellini, R. 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Studies on the toxic barley infected with Fusarium spp. Journal of the Food Hygiene Society (Japan), l_3, 368-375. Nelson, P. E., Toussoun, T. A., 6c Marasas, W. F. O. (1983). Fusarium species--an illustrated manual for identification. College Station: Pennsylvania State University Press. Perlman, D. (1967). Microbial production of therapeutic compounds. In H. J. Pepper (Ed.), Microbial technology. New York: Reinhold. Pohland, A. (1984). The analytical chemistry of deoxynivalenol. Toxigenic fungi--their toxins and health hazard. Proceedings of the Mycotoxin Symposia held at the Third International Mycological Congress, Tokyo, August 30-September 3, 1983. New York: Elseiver. Pringle, R. B., 6: Scheffer, R. P. (1963). Purification of the selective toxin of Periconia circinata. Phytopathology, 23(7), 785-787. Ray, L. L., 6: Bullerman, L. (1982). Preventing growth of potentially toxic molds using antifungial agents. Journal of Food Protection, 42, 953-963. Scott, P. M. (1984). 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The biosynthesis of trichothecene mycotoxins. In P. S. Steyn (Ed.), The biosynthesis of mycotoxinsJ a study in secondary metabolism. New York: Academic Press. Toussoun, T. A., 6: Nelson, P. E. (1976). A pictorial guide to the identification of Fusarium species according to the taxonomic system of Snyder and Hansen, 2nd ed. College Station: Pennsylvania State University Press. Trucksess, M. W., Nesheim, 5., 6c Eppley, R. (1984). Thin layer chromatographic detrmination of deoxynivalenol in wheat and corn. Association of Official Analytical Chemists, 6_l, 40-43. 57 Tuite, J. (1969). Plant pathological methods--fungi and bacteria. Minneapolis: Burgess. Ueno, Y. (1980). Toxicological evaluation of trichothecene mycotoxins. In D. Baker or T. Wadstrom (Eds.), Natural toxins. New York: Pergamon Press. Vesonder, R. F., Ciegler, A., or Jensen, A. H. (1973). Isolatin of the emetic principle from Fusarium-infected corn. Applied Microbiology, 26, 1008- 1010. Vesonder, R. F., Ciegler, A., or Jensen, A. H. (1977). Production of refusal factors by Fusarium strains on grains. Applied and Environmental Microbiology, 34, 105-106. Vesonder, R. F., Ciegler, A., Jensen, H., Rohwedder, W. K., 6: Weisleder, D. (1976). Co-identity of the refusal and emetic principle from Fusarium- infected corn. Applied and Environmental Microbiology, 3_1, 280-285. Vesonder, R. F., Ciegler, A., Rogers, R. F., Burbridge, K. A., Bothast, R. J., 6c Jensen, A. H. (1978). Survey of 1977 crop year preharvest corn for vomitoxin. Applied and Environmental Microbiology, _36, 885-888. Vesonder, R. F., Ciegler, A., Rohwedder, W. K., or Eppley, R. (1979). Re- examination of 1972 Midwest corn for vomitoxin. Toxicon, 11, 658-660. Vesonder, R. F., Ellis, J. J., Kwolek, W. F., 6: Demarini, D. J. (1982). Production of vomitoxin on corn by Fusarium graminearum NRRL-5883 and E. roseum NRRL-6101. Applied and Environmental Microbiology, 4_3_, 967-970. Yoshizawa, T. (Ed.). (1983). Red-mold diseases and natural occurrence in Japan. In Y. Ueno (Ed.), Trichothecenes. New York: Elsevier. Yoshizawa, T., 6( Morooka, N. (1975a). Biological modification of trichothecene mycotoxins: Acetylation and deacetylation of deoxynivalenol by Fusarium spp. Applied Microbiolgy, Q, 54-58. Yoshizawa, T., 6: Morooka, N. (1975b). Comparative studies on microbial and chemical modifications of trichothecene mycotoxins. Applied and Environmental Microbiology, 30, 38-43. Yoshizawa, T., 6: Morooka, N. (1977). Trichothecenes from mold-infested cereals in Japan. In J. V. Rodricks, C. W. Hesseltine, 6c M. A. Mehlman (Eds.), Mycotoxins in humans and animal health. Park Forest South, IL: Pathotox. ABSTRACT PRODUCTION OF DEOXYNIVALENOL (VOMITOXIN) IN LIQUID CULTURE by Abdalla Z. El-Bahrawy Growth and toxigenesis by Fusarium graminearum were studied in liquid media. Parameters monitored during fermentations included toxin production, fungal mass, carbohydrate utilization, and pH changes. Factors, which were varied, included basal medium composition, sucrose concentration, and ammonium tartrate concentration, inoculum size, and E. graminearum strain. Supplementation of both modified Fries medium and GYEP with four percent corn steep liquor greatly increased DON yields by E. graminearum strain U5373 compared to modified Fries medium or GYEP medium alone. Highest deoxynivalenol (DON) yield (16.5 mg/L) was in modified Fries medium supplemented with four percent corn steep liquor incubated for 20 days at 280 C. U5373 gave high mycelial dry weight when it was grown in modified Fries medium and four percent corn steep liquor (3.2 g/flask after five days) or in GYEP plus four percent corn steep liquor (2.8 g/flask after five days) when compared to growth in modified Fries or GYEP alone. In all of the media, pH rose from acidic levels and peaked to alkaline levels by the end of the fermentation. Lower yields of DON were obtained when sucrose concentration higher than three percent were used. Higher levels of ammonium tartrate ( 1.596) reduced both the toxin yield and the final pH. Of a total of nine Fusarium graminearum strains tested, only four produced DON and l5-acetyl deoxynivalenol (l5-A-DON) in modified Fries plus corn steep liquor. Strains Abdalla Z. El-Bahrawy U5373, Van Wert A-1, and Stuckey produced mainly DON while strain NRRL 5883 produced 15-acetyl DON as a main trichothecene. Conditions for optimal production of DON in liquid culture is thus dependent on medium composition and strain of E. graminearum. "IIIIIIIIIIIIIIII