Chemotaxonomy of Gibberella zeae with Special Reference to

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1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1983, p Vol. 46, No /83/ $02.00/0 Copyright 1983, American Society for Microbiology Chemotaxonomy of Gibberella zeae with Special Reference to Production of Trichothecenes and Zearalenone M. ICHINOE,1 H. KURATA,1 Y. SUGIURA,2 AND Y. UENO2* Department of Microbiology, National Institute of Hygienic Sciences, Kami-yoga, Setagaya-ku, Tokyo 158,1 and Department of Toxicology and Microbial Chemistry, Faculty of Pharmaceutical Sciences, Tokyo University of Sciences, Ichigaya, Tokyo 162, Japan2 Received 19 April 1983/Accepted 26 September 1983 By adopting a single-spore isolation technique, 113 isolates of Gibberella zeae, the perfect stage of Fusarium graminearum, were isolated from rice stubbles in barley and wheat fields and tested for production of trichothecenes and zearalenone on rice grains. Of the isolates, 93% produced the trichothecenes, and they could be subdivided into two chemotaxonomic groups: nivalenol and fusarenon-x producers and deoxynivalenol and 3-acetyldeoxynivalenol producers. No cross production of these two types of trichothecenes was observed in these isolates. Zearalenone was detected in 68% of the isolates, but no clear relationship could be observed regarding its position with respect to the two chemotaxonomic groups. Gibberella zeae, the perfect stage of Fusarium graminearum, has been reported to occur on barley, wheat, corn, and other cereals as a reddish mold. This mold is known to produce mycotoxins such as trichothecenes and zearalenone (ZEN) (10, 19, 20). Head blight disease (Akakabi-byo in Japanese) of barley and wheat was prevalent in regions facing the Pacific Ocean, especially in southern Japan (1, 7). Major outbreaks of Fusarium invasion in this region occurred in 1890, 1901, 1934, 1946, 1958, 1963, and 1970 when low temperatures and high humidity prevailed during harvest time (29). Nivalenol (NIV) and 4-acetylnivalenol (fusarenon-x, FX) (15, 22) were isolated from one of the causative fungi, Fusarium nivale Fn-2B. Deoxynivalenol (DON) had been detected in Fusarium-infested barley in 1970 (28). In the United States, large-scale G. zeae infection occurred in the corn belt in 1966, 1972, and 1975 (18), and the northwest region of Ohio appears to be more susceptible than others to G. zeae ear rot. DON (vomitoxin; swine feed refusal factor) was detected in the 1972 corn contaminated with F. graminearum (24). Recent studies have revealed that NIV and DON heavily contaminate foodstuffs and animal feed in different parts of the world (2, 3, 9, 23, 25), and the natural occurrence of these trichothecenes is one of the great concerns of health authorities. However, there are some regional differences in the occurrence of these trichothecenes. For example, in the United States, Canada, Austria, and South Africa, DON is the major trichothecene in corn and other cereals and feed of animals (5, 6, 9, 16, 23-26), whereas in Japan and France, both NIV and DON occur in Fusarium-infested crops (2-4). In the present paper, we aim to disclose the regional differences in G. zeae strains regarding the production of NIV and DON. MATERIALS AND METHODS Sampling of G. zeae. In May and June 1981, G. zeae was collected from rice stubbles in barley, malting barley, and wheat fields located in two geographically different regions: (i) Miki-cho, Kagawa Prefecture, in southern Japan, where head blight disease occurred frequently, and (ii) Gyoda, Kohnosu, Higashi-matsuyama, and Kawagoe in Saitama Prefecture and Tsuchiura, Sakura, Akeno, Oho, and Tsukuba in Ibaraki Prefecture, all in central Japan, where head blight disease is very rare. In all of these regions, rice is common as a second crop after the harvest of barley and wheat. In addition to rice stubbles, F. graminearum was also collected from barley and wheat grains in the Saitama and Ibaraki Prefectures. Single-spore isolation of G. zeae. Small pieces of wet peat moss and cellulose filters were placed on petri dishes (9 cm in diameter) and autoclaved. Pieces of rice stubbles with perithecia were placed on the filters in the petri dishes and incubated at 25 C under 12-h light-dark cycles. After several days of culture, singlespore isolation of ascospores was made under a dissecting microscope (x 30). From each perithecium, seven strains were isolated. Mycotoxin production was studied in five of seven strains. Isolation of F. graminearum from grains. Seventy grains were added to a 300-ml Erlenmeyer flask, immersed in 1% sodium hypochlorite solution for 1 min, and washed three times with sterilized water. Five grains each were placed on potato-dextrose agar plates, and after 7 days at 25 C, the fungi were isolated. Analysis of trichothecene mycotoxins. Thirty grams 1364

2 VOL. 46, 1983 of polished rice grains, which were previously immersed in tap water for 1 h, were transferred to a 300- ml Erlenmeyer flask and autoclaved at 120 C for 15 min. After 3 ml of 10% peptone solution was added, the isolated fungus was inoculated and cultured at 25 C for 7 days. The method of Suzuki et al. (13) was adopted with slight modifications for the extraction of the trichothecenes from rice cultures, as summarized below. The rice culture was mixed with 5 g of Hyflo Super-Cel (Johns-Manville Sales Corp., Wako Pure Chemicals, Tokyo) and 200 ml of n-hexane-saturated acetonitrile and extracted in a 500-ml Waring blender for 5 min. After filtration, the residue was again extracted with 100 ml of the same solvent. The combined acetonitrile layer was extracted with 100 ml of n-hexane for 10 min in an automatic shaker, and 20 g of anhydrous Na2SO4 was added to the acetonitrile layer. After removal of the solvent under reduced pressure, 0.5 ml of methanol was added to the residue, and then 1.5 ml of chloroform was added to dissolve the crude extract. This sample was subjected to an analysis of the trichothecenes. For thin-layer chromatography (TLC) analysis, 10 Fl each of the crude extract and standard trichothecene solutions was spotted on silica gel 60 precoated plates (E. Merck AG, Darmstadt, Federal Republic of Germany), and the plates were developed with two solvent systems: chloroform-methanol (7:3) and chloroform-acetone (3:2). The trichothecenes were visualized either by spraying with a 20%o aluminum chlorideethanol solution and heating at 110 C for 10 min (3), or by spraying with 4-(p-nitrobenzyl)pyridine (NBP)-tetraethylenpentamine (TEPA) (14). With the former method, the 8-ketotrichothecenes exhibit blue fluorescence under a UV lamp (265 nm) with a detection limit of 0.05 F±g, whereas with the latter method, all of the trichothecenes give blue spots on a white background, and the detection limit ranges from 0.02 to 0.2 F±g, depending on the trichothecenes examined. For semiquantitative determination of the trichothecenes, TLC plates were spotted with 10-,ul solutions of the standard trichothecenes NIV, FX, DON, and 3- acetyldeoxynivalenol (3-AcDON). For each solution of standard mycotoxin, 0.5, 1, 2, 5, 10, and 20,ug were applied on plates, in comparison with 10,ul of test extract. The plates were developed with chloroformacetone (3:2) and sprayed with aluminum chloride solution, and the relative amounts of the trichothecenes in the crude extracts were estimated by visual comparison. This simplified semiquantitative method was very useful for estimation of the trichotheceneproducing ability of many isolates. Confirmation of the trichothecenes was done by gasliquid chromatography (GLC) and gas chromatography-mass spectrometry analyses. For this purpose, cleanup of the crude extracts of rice culture was done as follows. The crude extract was applied on a silica gel (Wakogel S-1; Wako Pure Chemicals) column (1.7 by 28 cm), and after passage of 300 ml of chloroform, the trichothecenes were eluted with 50 ml of chloroform-methanol (9:1). The residue, dissolved in a small amount of chloroform, was applied to a column of Florisil (2.5 g). The column was eluted with 100 ml of chloroform, followed by elution with chloroformmethanol (9:1). The latter elute was evaporated, and the residue was subjected to GLC analysis after silyla- CHEMOTAXONOMY OF G. ZEAE 1365 tion with reagent B as previously described (8). Gas chromatography-mass spectrometry analysis was carried out with a gas chromatograph-mass spectrometer (model M-80; Hitachi, Ltd., Tokyo). The GLC conditions were the same as for the GLC analysis described above, and the spectra were obtained in the electron impact mode at an ionizing voltage of 70 ev. Analysis of ZEN production. The culture of G. zeae on rice grains was essentially the same for analysis of ZEN production as for trichothecenes, except that the fungi were cultured at 25 C for 7 days, followed by culture at 15 C for 14 days. The rice cultures thus obtained were homogenized in a 500-ml Waring blender for 5 min with 200 ml of a methanol-1% NaCl solution (55:45). After filtration over filter paper, 100 ml of the filtrate was extracted with 50 ml of chloroform in an automatic shaker for 10 min. After an additional extraction, the combined chloroform extracts were dehydrated with 20 g of Na2SO4 and evaporated to dryness, and the residue, dissolved in 1 ml of benzene, was spotted on a precoated silica gel 60 TLC plate. After being developed with chloroformethanol (95:5) or benzene-acetone (9:1), ZEN was monitored under a UV lamp (365 and 254 nm). For confirmation of the presence of ZEN, a multilayer column composed of silica gel (5 g), Florisil (5 g), and Na2SO4 (3 g) was charged with the extract dissolved in 1 ml of benzene, and after passage of 75 ml of benzene, ZEN was eluted with 75 ml of benzeneacetone (9:1) and confirmed by GLC analysis (12). Chemicals. NIV and FX were isolated from the culture filtrate of F. nivale Fn-2B (21), and DON and 3-AcDON were kindly donated by T. Yoshizawa (Kagawa University, Kagawa, Japan). ZEN was purchased from Makor Chemicals, Jerusalem, Israel. All of the organic solvents used were of analytical reagent grade. Precoated silica gel 60 TLC plates (layer thickness, 0.25 mm) were purchased from E. Merck. Wakogel S-1 and Florisil ( mesh; Wako Pure Chemicals) were activated at 130 C for 16 h and 105 C for 2 h, respectively, before use. NBP (Tokyo Kasei Co., Tokyo) was recrystallized from n-hexane and used as a 1% solution in chloroform-carbon tetrachloride (2:3), and TEPA (Tokyo Kasei) was used as a 10o solution in the same solvent mixture. RESULTS TLC analysis of trichothecenes. The standard trichothecenes and the crude extracts of rice grains artificially infected with four isolates of G. zeae were spotted on the TLC plates, developed in two solvent systems, and sprayed either with NBP-TEPA (blue spot test) or aluminum chloride solution. The standard trichothecenes, namely, NIV, FX, DON, and 3-AcDON, were clearly separated from each other (Fig. 1). The crude extracts of rice cultures exhibited blue spots on the plates after being sprayed with NBP-TEPA, and some false, violet-colored spots very close to FX and 3-AcDON also appeared. However, on being sprayed with the aluminum chloride solution, only the trichothecenes exhibited fluorescent spots under a UV

3 1366 ICHINOE ET AL. Rf V V (A) NBP-TEPA DON NIV a b ST c d (B) AIC13 a 3-AcDON SS DON f * NIV a b ST c d CHCI3:MeOH (7:1) CHCQ3:(CH3)20 (3:2) FIG. 1. Thin-layer chromatograms of standard trichothecenes and crude extracts of G. zeae-infested rice grains. Silica gel TLC plates spotted with the standards (NIV, DON, FX, and 3-AcDON), and the crude extracts of four strains (a, MB-II-2; b, MB-II-5; c, B-VI-1; and d, B-VI-2) of G. zeae listed in Table 2 were (A) developed in chloroform-methanol (7:1) and sprayed with NBP-TEPA (V, violet spots; B, blue spot) or (B) developed in chloroform-acetone (3:2) and sprayed with a 20% aluminum chloride-ethanol solution. ST, standard. lamp. Employing two solvent systems and spraying reagents, we identified and quantified the four 8-ketotrichothecenes in the crude extracts of the rice cultures. Trichothecene-producing ability of G. zeae. Twenty-five strains of G. zeae were isolated from the perithecia collected from five cereal fields (two barley and three wheat) in Miki-cho, Kagawa; 45 strains were isolated from nine fields (three barley, two malting barley, and four wheat) in Saitama; and 25 strains were isolated from five fields (three barley and two wheat) in Ibaraki (Table 1). All 95 isolates were tested for trichothecene production. Among the 25 isolates obtained from Kagawa, 10 isolates produced both NIV and FX, 3 isolates produced NIV only, 1 isolate produced FX only, 9 isolates produced both DON and 3-AcDON, and 1 isolate produced only DON. Thus, all of the strains isolated from Kagawa could be grouped into two types: (i) DON-3-AcDON producers and (ii) NIV-FX producers. Furthermore, the strains isolated from barley fields produced DON-type trichothecenes, and those from wheat fields produced NIV-type trichothecenes. From Saitama, 40 of 45 isolates produced NIV and FX, and only 5, isolated from malting barley fields, produced DON and 3-AcDON. Of 25 isolates from Ibar- APPL. ENVIRON. MICROBIOL. aki, only 5, which were isolated from barley fields, produced DON and 3-AcDON, and all the remaining strains produced NIV and FX. These data revealed that 87 (92%) of the 95 isolates produced the 8-ketotrichothecenes on rice cultures, and these 87 toxigenic isolates could be divided into two chemotaxonomic types: NIV (70%) and DON (30%). No trichothecenes were detected in eight isolates. No strain of G. zeae examined produced both types of trichothecenes (Table 1). To confirm the above results and to see whether the trichothecene-producing ability of G. zeae differs with different areas of the same field, we isolated strains of G. zeae from different sites in five fields in Saitama and one field in Ibaraki; three strains were collected from each field, and their trichothecene-producing ability was examined under the same conditions as described above. Among the 18 isolates, 15 were the NIV type, whereas 3 were the DON type. The data support the results of Table 1 regarding studies of isolates from Saitama and Ibaraki. Survey of the trichothecene-producing ability of F. graminearum isolated from barley kernels. Barley kernel samples were picked in seven fields located in two different areas where G. TABLE 1. Production of trichothecenes and ZEN by G. zeae Location Fielda Trichothecenesb ZENc Miki-cho, W-1 5/5 (NIV, FX) 5/5 Kagawa W-II 5/5 (NIV, FX) 4/5 W-III 3/5 (NIV, FX) NT B-I 5/5 (DON, 3-AcDON) 5/5 B-II 5/5 (DON, 3-AcDON) 4/5 Saitama Gyoda W-IV 5/5 (NIV, FX) 1/3 B-Ill 5/5 (NIV, FX) 3/3 Kohnosu W-V 5/5 (NIV, FX) 2/3 B-IV 5/5 (NIV, FX) 1/3 Higashi- W-VI 5/5 (NIV, FX) NT matsu- MB-I 4/5 (NIV, FX) NT yama Kawagoe W-VII 2/5 (NIV, FX) NT B-V 4/5 (NIV, FX) 1/3 MB-lI 5/5 (DON, 3-AcDON) 1/3 Ibaraki Tsuchiura B-VI 4/5 (NIV, FX) 3/3 Sakura B-VII 5/5 (DON, 3-AcDON) 0/3 Akeno W-VIII 5/5 (NIV, FX) 2/3 B-VIII 5/5 (NIV, FX) 2/3 Oho W-IX 4/5 (NIV, FX) NT a W, Wheat; B, barley; MB, malting barley. b Number of strains in which trichothecenes were detected/number of strains tested. c Number of strains in which ZEN was detected/ number of strains tested. NT, Not tested.

4 VOL. 46,1983 CHEMOTAXONOMY OF G. ZEAE 1367 zeae-infected rice stubbles remained. The barley kernels were collected from three barley fields and one malting barley field in Saitama and three barley fields in Ibaraki. Nineteen isolates of F. graminearum were obtained and examined for production of mycotoxins. All of the 19 isolates produced only NIV-type trichothecenes; no DON-type producer was detected. Semiquantitative data on the mycotoxin-producing ability of G. zeae isolated from stubbles and of F. graminearum isolated from kernels in the same fields are listed in Table 2. Although isolates of G. zeae from two fields, MB-Il and B- VII, produced DON and 3-AcDON, F. graminearum isolated from barley kemels in the same fields produced NIV and FX. ZEN-producing ability of G. zeae and F. graminearum. To examine the relationship between the trichothecenes and ZEN production, 50 randomly selected strains of G. zeae isolated from TABLE 2. Location 14 fields in Kagawa, Saitama, and Ibaraki were cultured on rice grains, and the production of ZEN was examined by TLC analysis. In Table 1, 34 (68%) of 50 isolates produced this mycotoxin. In the case of F. graminearum, 10 (53%) of 19 isolates were able to produce ZEN on rice culture (Table 2). DISCUSSION Presence of NIV and DON types in G. zeae. As reported earlier (8, 21), rice, barley, wheat, and corn supported the production of NIV and FX by F. nivale Fn-2B, and the highest production of the trichothecenes was observed with rice grains as a substrate. Fusarium roseum (F. graminearum) was also reported to produce DON and an acetate derivative of DON on rice grains (30). Hence, rice grains were selected as a substrate for the production of trichothecenes. Trichothecenes and ZEN production by G. zeae from rice stubbles and by F. graminearum from barley grain in the same fieldsa G. zeae F. graminearum and fieldb Strain Trichothecenes Strain Tnchothecenes nlo. no. ZEN no. ZEN NIV DON FX AcDON NIV FX Saitama B-Ill , B-IV , B-V ,5 +2,+1 MB-II ,5 +4, +5 +4, +5 Ibaraki B-VI , B-VII , 5 +5, +3 +4, +2 B-VIII , a Semiquantitative amounts of trichothecenes: -, not detected; +1, 1.0 to 3.0 ±glg; +2,3.0 to 7.0.g/g; +3,7.0 to 34 Pg/g; +4, 34 to 67 F&g/g; +5, 67 to 134 jg/g. ZEN: -, not detected; +, detected. b W, Wheat; B, barley; MB, malting barley.

5 1368 ICHINOE ET AL. The trichothecenes were detected in 86 of 95 (90.5%) isolates of G. zeae (Table 1). Furthermore, all of the trichothecene producers could be subdivided into NIV type (66 strains, 76.7%) and DON type (20 strains, 23.3%), and no strains produced both types of 8-ketotrichothecenes. There were no detectable differences in the physiological and morphological characteristics between the two types of trichothecene producers. Chemically, FX and 3-AcDON are primarily produced in the fungal mycelia, but some intraor extracellular deacetylases catalyze the removal of acetyl groups from these parent products. Therefore, the resulting NIV and DON are considered to be end products of these trichothecene-producing fungi (22, 30). This is why, in the proposed chemotaxonomic classification, the terms "NIV chemotypes" and "DON chemotypes" are introduced instead of "FX chemotypes" and "3-AcDON chemotypes." Geographic difference in the distribution of NIV and DON chemotypes. For collecting the perithecia of G. zeae, we selected two regions where there is a prevalence of head blight disease (Akakabi-byo in Japanese) in crops: Kagawa, where head blight disease occurs frequently, located in southern Japan, and Saitama and Ibaraki, where head blight disease is not so frequent, located in central Japan. Among the isolates obtained from the former region, 52 were the NIV type and 44 were the DON type, whereas most of the isolates obtained from Saitama and Ibaraki were the NIV type. These data suggest a regional difference in the distribution of the two chemotypes of G. zeae. Yoshizawa et al. (27) have demonstrated that the toxigenic strains of F. graminearum collected from barley and wheat in southern Japan could be classified into NIV type (46.5%) and DON type (44.8%). Suzuki et al. (13) reported on the central region, where the percentages were 93% NIV type and 7% DON type. As for the crop difference, our present data indicate that both NIV and DON types of G. zeae were detected in barley and malting barley fields and that no DON type was isolated from wheat fields. Relationship between the trichothecene- and ZEN-producing ability of G. zeae. ZEN was first isolated from the metabolites of G. zeae as an estrogenic mycotoxin (11). Recent data indicated that this mycotoxin coexists with the trichothecenes in natural conditions (5, 6, 28). In an attempt to clarify whether this estrogenic mycotoxin is coproduced by NIV-type gr DON-type G. zeae, we selected 50 strains of the trichothecene producer and analyzed their ZEN production. Of these strains, 34 (68%) produced this mycotoxin (Table 1). Among the 32 NIV and 16 APPL. ENVIRON. MICROBIOL. DON types that constituted the 50 strains examined, 24 (71%) and 10 (62.5%) isolates, respectively, were positive for ZEN-producing ability. Therefore, no exclusive relationship could be observed between ZEN production and the NIV or the DON types. Experiments with F. graminearum isolated from barley kernels also indicated that 10 (53%) of 19 isolates were capable of producing ZEN (Table 2). Because all of the isolates of F. graminearum tested here were the NIV type and no analysis was done with the DON type, we could not ascertain whether or not the NIV type is the predominant producer of ZEN. Significance of the life cycle of G. zeae in the natural occurrence of trichothecene mycotoxins. In Japan, it is well known that the Gibberella stage of F. graminearum is formed on rice stubbles remaining in the rice-wheat or ricebarley rotation fields. The perithecia of G. zeae mature from late March to May, and their ascospores, liberated in the air, infect barley and wheat in their heading stage, mostly between April and May. When high humidity and low temperatures continue during the preharvest time, F. graminearum starts to grow and produces the trichothecenes and ZEN on the parent crops. The present data, indicating that both NIV and DON types of G. zeae are almost equally distributed in southern Japan, explain why NIV and DON were natural contaminants of barley and wheat harvested in this area (3, 4). It is interesting to note that the high distribution of NIV-type G. zeae was observed in the central region, where severe head blight disease had not been prevalent in the past. Our present results lead to the hypothesis that NIV is the major trichothecene polluting central Japan when climatic conditions are favorable for the growth of G. zeae and for the bioproduction of trichothecenes during preharvest time. These basic approaches on the chemotaxonomy of G. zeae could explain the geographic differences of trichothecene contamination in the world. ACKNOWLEDGMENTS This experiment was partly supported by a grant-in-aid from the Ministry of Health and Welfare, Japan. We thank T. Yoshizawa of Kagawa University, M. Kakishima of The University of Tsukuba, and K. Fujita of the Saitama Agriculture Experiment Station for their help collecting the field samples. we also thank R. V. Bhat of the National Institute of Nutrition, Hyderabad, India, for his helpful discussion and Isha Bhattacharyya for assistance in preparing the manuscript. LITERATURE CITED 1. Ishii, H Studies on the mechanism of prevalence of the wheat scab and barley blight by Gibberella zeae (Schw.) Petch in Japan. Shokubutsu Boeki 8: (In Japanese.)

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