Biosynthesis of zearalenone: a simple and efficient method to incorporate [13C]acetate label by using solid cultures.

A suitable method was developed to efficiently incorporate 13C-labeled acetate into zearalenone by using solid cultures. Periodic feeding of the label during the zearalenone production phase significantly increased the label incorporation for the singly labeled acetate.

[The occurrence of Fusarium varieties and their mycotoxins in silo corn. 2. The formation of zearalenone in the field by artificial infection of silo corn with Fusarium culmorum (W. G. Smith) Sacc]. / Vorkommen von Fusarium-Arten und ihren Mykotoxinen auf Silomais. 2. Mitteilung. Bildung von Zearalenon auf dem Feld durch künstliche Infektion von Silomais mit Fusarium culmorum (W. G. Smith) Sacc.

The formation of zearalenone in a maize plot artificially infected with Fusarium culmorum was studied. The zearalenone concentration steeply increased only in the 8th week after inoculation and reached a maximum value of ca. 7 ppm, whereas zearalenone could not be detected in the control variants. The crude nutrient and dry matter content was not significantly influenced by the fungal infection. The infected crop showed average ear dry weights distinctly lower than that of the control variants (P less than or equal to 0.001). Apart from zearalenone, the mycotoxin deoxynivalenol was qualitatively detected in the infected maize. The toxicological relevance of the ascertained zearalenone content with regard to the health of dairy cattle and pigs was discussed.

Production of deoxynivalenol and zearalenone by isolates of Fusarium graminearum Schw.

The production of deoxynivalenol (DONI) on rice, corn, wheat, and barley grains by Fusarium graminearum Schw. NRRL 5883 was investigated. Highest yields (91.9-202 ppm) were obtained on rice; yields on the other substrates were: corn (34.1-84.5 ppm), wheat (3.6-24.4 ppm), and barley (0-6.6 ppm). Fifty isolates of Fusarium from corn inoculated in the field in 1979 with a mixture of strains of F. graminearum, originally collected from corn plants infected with stalk rot, were tested for DONI production on corn. Twenty of these were also tested for zearalenone production. One isolate produced more than 200 ppm DONI, 13 produced 20-50 ppm, 17 produced 10-20 ppm, and the rest produced less than 10 ppm. The only isolate that did not produce DONI was not identified as F. graminearum. All 20 isolates tested produced zearalenone; 18 produced higher levels of zearalenone (15.4-369 ppm) than of DONI. The other 2 isolates formed essentially the same levels of zearalenone and DONI-37 and 30 ppm, and 15 and 16 ppm, respectively.

Production of zearalenone, T-2 toxin, and deoxynivalenol by Fusarium spp. isolated from plant materials grown in North Carolina.

Fusarium spp. isolated from plant materials grown in the hot, humid climate of North Carolina were tested for production of mycotoxins. Isolates of F. acuminatum, F. graminearum, F. moniliforme, F. oxysporum, and F. solani produced zearalenone while isolates of F. equiseti and F. graminearum produced T-2 toxin and deoxynivalenol, respectively. This is the first report of zearalenone production by F. solani. The toxins were identified by capillary gas chromatography-mass spectrometry. These findings suggest that there are toxigenic strains of Fusarium indigenous to the warmer regions of the USA and that fasariotoxicoses of animals in this region are not necessarily the result of importing toxic grains from the cooler, upper midwestern USA.

Method for detecting production of zearalenone, zearalenol, T-2 toxin, and deoxynivalenol by Fusarium isolates.

Three methods for detecting toxigenic fusaria in culture were compared by using known producers of zearalenone, zearalenol, T-2 toxin, and deoxynivalenol. Moist, autoclaved rice cultures of known toxigenic isolates grown in 20-ml tubes yielded oily extracts containing compounds which interfered with qualitative and quantitative analysis for the mycotoxins. Vermiculite moistened with nutrient broth in 20-ml tubes yielded a much cleaner extract. Growing the fungi on a liquid medium required a shorter incubation period, but yields of T-2 toxin and deoxynivalenol were low and variable, and the method required greater space in the incubator. Screening of the extracts by thin-layer chromatography with colorimetric spray reagents to detect the presence of these toxins permitted reduction in the number of extracts quantified by the more lengthy gas-liquid chromatographic method. Culturing in nutrient broth on vermiculite in tubes coupled to a qualitative screen before quantitation proved to be a convenient, inexpensive, and relatively rapid method that enabled reliable screening of a large number of Fusarium isolates for toxin production as compared with prior methods.

Production of trichothecenes and zearalenone by Fusarium species isolated from wheat.

Fusaria isolates from wheat were tested for ability to produce trichothecenes and zearalenone. Four isolates of F. culmorum out of 13 produced vomitoxin (DON) and 3 Ac-DON, one produced diacetoxysirpenol and 12 zearalenone. Particularly high yield of zearalenone was observed in cultures of sever pathogenic isolates. Higher temperature (20 degrees C) during first week of incubation favoured yield of zearalenone. About 50% of zearalenone was produced by surface mycelium.

Chemotaxonomy of Gibberella zeae with special reference to production of trichothecenes and zearalenone.

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.

Deoxynivalenol, acetyl deoxynivalenol, and zearalenone formation by Canadian isolates of Fusarium graminearum on solid substrates.

Three isolates of Fusarium graminearum (DAOM 180377, 180378, and 180379) were screened for their ability to produce mycotoxins on the solid substrates corn and rice. They all produced deoxynivalenol and zearalenone on corn. On rice, only DAOM 180378 and 180379 produced significant amounts of these mycotoxins, with levels of deoxynivalenol being much higher than those of zearalenone. The effects of the initial moisture content before autoclaving, incubation temperature, and time were studied with isolate DAOM 180378. At 19.5 degrees C the main product was zearalenone, whereas at 25 degrees C both deoxynivalenol and zearalenone were formed. Higher incubation temperatures (28 degrees C) favored deoxynivalenol formation, the maximum amount being 515 ppm (515 micrograms/g) formed after 24 days at an initial moisture content of 40%. The maximum level of zearalenone produced at the same temperature was 399 ppm, but at an initial moisture content of 35%. Other factors, such as pH, oxygen and carbon dioxide concentrations, and size of the culture flask also appeared to affect the production of mycotoxins.

Screening of zearalenone-producing Fusarium species in Egypt and chemically defined medium for production of the toxin.

296 isolates of Fusarium spp. from 100 samples of ccereal grains were examined for their ability to produce zearalenone on liquid culture medium. Thin layer chromatographic analysis revealed that the mycotoxin was detected from 45 isolates, (F. oxysporum), 36; F. moniliforme, 8; and F. equiseti, one isolate). A suitable liquid medium and some optimal conditions for the biosynthesis of zearalenone were reported. Glutamine and riboflavin stimulated the production of the toxin. The maximum amount of zearalenone appeared at pH 7, after 12 days of incubation at 20 degrees C.

Elaboration of vomitoxin and zearalenone by Fusarium isolates and the biological activity of Fusarium-produced toxins.

Sixteen Fusarium isolates belonging to F. graminearium Schw. and F. culmorum (W.G. Smith) Sacc. produced vomitoxin and zearalenone on cracked corn at 28 degrees C. Quantitation for vomitoxin was by gas-liquid chromatography. This toxin was produced in quantities of 5 to 236 microgram/g of fermented corn. Vomitoxin showed weak antibiotic activity against Penicillium digitatum Sacc., Mucor ramannianus Möller, and Saccharomyces bayanus Sacc., but did not inhibit gram-positive, gram-negative, or acid fast bacteria. The two molds and the yeast were inhibited by T-2 toxin at 5 micrograms, and diacetoxyscirpenol inhibited the molds at 5 micrograms and the yeast at 50 micrograms.

Laboratory screening for zearalenone formation in corn hybrids and inbreds.

Grains from 14 corn inbreds and 4 single cross hybrids were inoculated with 3 isolates of Gibberella zeae to determine their inhibition of zearalenone production. The corn hybrids: Pa762 x A632 (50 mg/kg zearalenone production), A619 x A632 (17 mg/kg zearalenone production), H95 x Mo17 (132 mg/kg zearalenone production), and B73 x MO17 (33 mg/kg zearalenone production) appear to have less resistance than the inbreds to toxin formation. Inbred H95 (64 mg/kg zearalenone production) supported the highest toxin production of all inbreds. The remaining 13 inbreds did not exceed 15 mg/kg zearalenone production. The inbreds A632 (4 mg/kg zearalenone production) and Pa762 (2 mg/kg zearalenone production) demonstrated some resistance; the resulting cross, hybrid Pa762 x A632 (50 mg/kg zearalenone production), does have greater resistance than hybrid H95 x Mo17 (132 mg/kg zearalenone production). Analysis of variance indicated highly significant variation between corn varieties and fungal isolates. The coefficient of variation for 29 fermentations run in duplicate on inoculated control corn to produce zearalenone (212 mg/kg) was 37%, which would include variation in both the fermentation and analysis. Isolate and variety interaction is not significant.

Biosynthesis of [14C]zearalenone from [1-14C]acetate by Fusarium roseum 'Gibbosum'.

Addition of [1-14C]acetate or [1,2-14C]acetate to actively growing cultures of Fusarium roseum 'Gibbosum' on rice yielded zearalenone with a specific activity ranging between 1.63 and 46.5 microCi/mmol.

Identification of the naturally occurring isomer of zearalenol produced by Fusarium roseum 'Gibbosum' in rice culture.

One diastereomer of trans-zearalenol [2,4-dihydroxy-6-(6,10-dihydroxy-trans-1-undecenyl)-benzoic acid-mu-lactone] was isolated from cultures of Fusarium roseum 'Gibbosum.' This strongly estrogenic metabolite was identified by analysis of its mass spectrum and its behavior in thin-layer, high-pressure liquid and gas-liquid chromatographic systems. The concentration of zearalenol in cultures was 563 mu g/g, or 7% of the 8,000-mu g/g zearalenone content, while the two diastereomers of 8'-hydroxyzearalenone each occurred at 3% of the zearalenone level. Of the two possible diastereomers of zearalenol, the one occurring in cultures was identical to the low-melting-point (171 degrees C) isomer (alpha) obtained by synthesis. In the rat uterus bioassay, the alpha zearalenol isomer was three times more estrogenic than zearalenone while the beta isomer was equal in activity in zearalenone. The two diastereomers of zearalenol can be distinguished from each other by the intensity of the m/e+ 302 fragment of the mass spectrum of the pure underivatized compound.

[Fusarium graminearum in silages. Zearalenone production (author's transl)]. / Fusarium graminearumdans les ensilages. Production de zéaralénone.

Zearalenone natural contamination of transverse sections of recently opened silages has been observed. In our in vitro experiments, there is no zearalenone production on corn by Fusarium graminearum in anaerobic jar during 3 months. However, on confined atmosphere, zearalenone production is 1.5 to 3 ppm. Contamination of silage is not possible if there are anaerobic conditions in silages.

Media for identification of Gibberella zeae and production of F-2-(Zearalenone).

Media are described for the isolaton of Fusarium graminearum in the perithecial state, Gibberella zeae, and for the production of F-2 (zearalenone) by Fusarium species. On soil extract-corn meal agar isolated medium, G. Zeae produced perithecia in 9 to 14 days under a 12-h photoperiod. Species of Fusarium were screened for F-2 production on a liquid medium. From strains that produced F-2, the yields, from stationary cultures of G. zeae and F. culmorum after 12 days of incubation, ranged from 22 to 86 mg/liter. Three strains produced no F-2. Glumatic acid, starch, yeast extract,and the proper ratio of medium volume-to-flask volume were necessary for F-2 synthesis.