Analysis of deoxynivalenol from cultures of Fusarium species.

Eight isolates of Fusarium roseum and three of Fusarium colmorum were found to produce deoxynivalenol in rice cultures. Deoxynivalenol was extracted with aqueous methanol (40%) and purified by partitioning with ethyl acetate and acetonitrile-petroleum ether (boiling point, 60--70 degrees C). The toxin was identified by gas chromatography/mass spectrometry and quantified by gas-liquid chromatography. High recoveries (80%) of deoxynivalenol were obtained from rice cultures, and as low as 0.250 microgram of the toxin per g was detected.     

Toxin production by Fusarium culmorum IMI 309344 and F. graminearum NRRL 5883 on grain substrates

The production of deoxynivalenol, acetyl deoxynivalenol and zearalenone by Fusarium culmorum and F. graminearum on autoclave-sterilized grain (maize, rice, wheat and barley) was investigated. Fusarium culmorum produced significantly greater levels of toxins than F. graminearum. The four substrates examined differed in their ability to support toxin production. Toxin production on maize and rice was significantly greater than toxin production on barley or wheat.     

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.     

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 high quantities of 3-acetyldeoxynivalenol and deoxynivalenol

The time course of 3-acetyldeoxynivalenol (AcDON) formation was analysed over a 11 day culture period ofFusarium graminearum (DSMO 4258) on rice. The maximum of 2840 mg/kg AcDON was detected after 9 days of culture. To overcome the complicated clean up of the solid substrate extract, 10% methanol in water was applied as extract solvent. After liquid-liquid partition with ethyl acetate, more than 75% of the toxin could be detected. After one simple clean up step (column chromatography over florisil) AcDON could be crystallised. After ion exchange chromatography deoxynivalenol (DON) could be crystallised and an overall yield of 1,44 g DON per kg of culture was obtained.     

Exogenous H(2)O(2) and catalase treatments interfere with Tri genes expression in liquid cultures of Fusarium graminearum

Effect of exogenous H(2)O(2) and catalase was tested in liquid cultures of the deoxynivalenol and 15-acetyldeoxynivalenol-producing fungus Fusarium graminearum. Accordingly to previous results, H(2)O(2) supplementation of the culture medium leads to increased toxin production. This study indicates that this event seems to be linked to a general up regulation of genes involved in the deoxynivalenol and 15-acetyldeoxynivalenol biosynthesis pathway, commonly named Tri genes. In catalase-treated cultures, toxin accumulation is reduced, and Tri genes expression is significantly down regulated. Furthermore, kinetics of expression of several Tri genes is proposed in relation to toxin accumulation. Biological meanings of these findings are discussed.     

中国小麦赤霉病菌优势种——禾谷镰刀菌产毒素能力的研究

试验测定了分离自中国小麦赤霉病常发生地区病麦穗上的47个禾谷镰刀菌(Fusariumgraminearum)菌株的产毒紊能力。结果表明,它们可以产生25种包括单端孢霉烯族化合物(Trichothecenes)、倍半萜类化合物(sesquiterpenes)、赤霉烯酮(Zearalenone)和丁烯羟酸内酯(Butenolide)等类的已知次生代谢物。这些菌株属于化学型 I,其中,来自我国温暖麦区的菌株都属化学型IA (deoxynivalenol,3-acetyl),并在气候冷凉地区发现化学型IB(de-oxynivalenol,15-acetyl)菌株。     

Formation of trichothecenes by Fusarium solani var. coeruleum and Fusarium sambucinum in potatoes.

Fusarium solani var. coeruleum can form deoxynivalenol in potato tubers and in liquid medium, although concentrations observed in the rot were highly variable; acetyldeoxynivalenol and HT-2 toxin were detected in 1 to 3 tubers only (of 57). Trichothecenes were also detected in a very few (3 of 20) cultures of Fusarium sambucinum in potato tubers.     

Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates.

The fungal species isolated from Korean cereals (barley, polished barley, wheat, rye, and malt) were Alternaria spp., Aspergillus spp., Chaetomium spp., Drechslera spp., Epicoccum sp., Fusarium spp., and Penicillium spp., etc. The number of Fusarium strains isolated was 36, and their ability to produce Fusarium mycotoxins on rice was tested. Nivalenol (NIV) was produced by Fusarium graminearum (7 of 9 isolates), Fusarium oxysporum (3 of 10 isolates), and Fusarium spp. (7 of 15 isolates). Of 15 isolates of Fusarium spp., 6 formed deoxynivalenol (DON). Fusarenon-X and 3-acetyl-DON were produced by most NIV- and DON-forming isolates, respectively. Zearalenone was produced by 3 isolates of F. graminearum, 1 isolate of Fusarium equiseti, and 11 isolates of Fusarium spp. T-2 toxin was not produced by any Fusarium isolates. The highest concentrations of mycotoxins produced by Fusarium isolates were 77.4 (NIV), 5.3 (DON), 138.3 (fusarenon-X), 40.6 (3-acetyl-DON), and 23.2 (zearalenone) micrograms/g.     

Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates

The fungal species isolated from Korean cereals (barley, polished barley, wheat, rye, and malt) were Alternaria spp., Aspergillus spp., Chaetomium spp., Drechslera spp., Epicoccum sp., Fusarium spp., and Penicillium spp., etc. The number of Fusarium strains isolated was 36, and their ability to produce Fusarium mycotoxins on rice was tested. Nivalenol (NIV) was produced by Fusarium graminearum (7 of 9 isolates), Fusarium oxysporum (3 of 10 isolates), and Fusarium spp. (7 of 15 isolates). Of 15 isolates of Fusarium spp., 6 formed deoxynivalenol (DON). Fusarenon-X and 3-acetyl-DON were produced by most NIV- and DON-forming isolates, respectively. Zearalenone was produced by 3 isolates of F. graminearum, 1 isolate of Fusarium equiseti, and 11 isolates of Fusarium spp. T-2 toxin was not produced by any Fusarium isolates. The highest concentrations of mycotoxins produced by Fusarium isolates were 77.4 (NIV), 5.3 (DON), 138.3 (fusarenon-X), 40.6 (3-acetyl-DON), and 23.2 (zearalenone) micrograms/g.     

Absence of trichothecenes in toxigenic isolates of Fusarium moniliforme

Thirty-four isolates of Fusarium moniliforme were obtained from cereal grains collected in various parts of the world. The isolates were grown on rice and tested as a diet for toxicity to rats. Of these isolates, 53% caused death, 12% caused congestion and hemorrhage of the stomach and intestine as well as hematuria, 21% caused diarrhea, 38% caused weight loss, and 9% were nontoxic. The cultures were tested to T-2, HT-2, neosolaniol, acetyl-T-2, T-2-tetraol, iso-T-2, diacetoxyscirpenol, monoacetoxyscirpenol, deoxynivalenol, nivalenol, fusarenone-X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, zearalenone, moniliformin, fusarochromanone, fusarin-C, and wortmannin; all were negative. In addition, F. moniliforme NRRL A25820 was grown on corn and banana fruit as solid substrates as well as on a defined liquid medium; none of the above toxins were found. When F. moniliforme NRRL A25820 was incorporated into a rat diet, no toxicity was noted. Twenty-eight additional isolates of F. moniliforme, isolated from feed associated with equine leukoencephalomalacia, were grown on cracked corn for 2 weeks. The cultures were negative when tested for deoxynivalenol, 15-acetyldeoxynivalenol, diacetoxyscirpenol, monoacetoxyscirpenol, nivalenol, and fusarenone X. Seventy-five percent of the isolates were toxic to ducklings, indicating the presence of a toxin other than trichothecenes. Our results support the conclusion that F. moniliforme does not produce trichothecenes.     

A simple method with liquid chromatography/tandem mass spectrometry for the determination of the six trichothecene mycotoxins in rice medium

A selective and speedy LC-MS/MS method was developed to determine six trichothecene mycotoxins (nivalenol, deoxynivalenol, fusarenon X, 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, and T-2 toxin) in rice medium where Fusarium graminearum were cultivated for in vitro tests. The analytes were extracted from the rice medium with acetonitrile/water (85/15, v/v), and diluted with acetonitrile/water (5/95, v/v) in order to minimize the effects of matrices. Diluted solutions were analyzed by LC-MS/MS with electrospray ionization (ESI) interface in negative or positive ion mode and the multiple reaction monitoring mode. Recovery rates were 76-106% with a spiked level at 1-6 microg/kg of mycotoxins that corresponded to the limit of quantitation. The method was applied to study the time courses of trichothecene production and the biomass of fungi by three Fusarium graminearum strains. Three strains have different mycotoxin biosynthesis pathways, wFg14 and 03E-1 were DON producer, and 03N-1 was NIV producer.     

Comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides.

The range and comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides, i.e., NRRL 3299, NRRL 3510, M-1-1, HPB 071178-13, and F-38, were determined. Lyophilized cultures of the five strains maintained in the International Toxic Fusarium Reference Collection were used to inoculate autoclaved corn kernels. Corn cultures were incubated at 15 degrees C for 21 days and analyzed for trichothecenes by thin-layer chromatography and capillary gas chromatography. All five strains produced T-2 toxin, HT-2 toxin, T-2 triol, and neosolaniol. Two strains also produced T-2 tetraol, and two others produced diacetoxyscirpenol. The highest producer of T-2 toxin (1,300 mg/kg), HT-2 toxin (200 mg/kg), T-2 triol (1.9 mg/kg), and neosolaniol (170 mg/kg) was NRRL 3510, which was originally isolated from millet associated with outbreaks of alimentary toxic aleukia in the USSR. The second highest producer of T-2 toxin (930 mg/kg) was NRRL 3299. The other three strains produced T-2 toxin at levels ranging from 130 to 660 mg/kg. Thus, the five strains differed considerably in the amounts of T-2 toxin and other trichothecenes produced under identical laboratory conditions. These strains are being maintained under optimal conditions for the preservation of Fusarium cultures and are available from the Fusarium Research Center, The Pennsylvania State University, University Park.     

Comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides

The range and comparative yields of T-2 toxin and related trichothecenes from five toxicologically important strains of Fusarium sporotrichioides, i.e., NRRL 3299, NRRL 3510, M-1-1, HPB 071178-13, and F-38, were determined. Lyophilized cultures of the five strains maintained in the International Toxic Fusarium Reference Collection were used to inoculate autoclaved corn kernels. Corn cultures were incubated at 15 degrees C for 21 days and analyzed for trichothecenes by thin-layer chromatography and capillary gas chromatography. All five strains produced T-2 toxin, HT-2 toxin, T-2 triol, and neosolaniol. Two strains also produced T-2 tetraol, and two others produced diacetoxyscirpenol. The highest producer of T-2 toxin (1,300 mg/kg), HT-2 toxin (200 mg/kg), T-2 triol (1.9 mg/kg), and neosolaniol (170 mg/kg) was NRRL 3510, which was originally isolated from millet associated with outbreaks of alimentary toxic aleukia in the USSR. The second highest producer of T-2 toxin (930 mg/kg) was NRRL 3299. The other three strains produced T-2 toxin at levels ranging from 130 to 660 mg/kg. Thus, the five strains differed considerably in the amounts of T-2 toxin and other trichothecenes produced under identical laboratory conditions. These strains are being maintained under optimal conditions for the preservation of Fusarium cultures and are available from the Fusarium Research Center, The Pennsylvania State University, University Park.     

Toxigenicity of some fusaria associated with plant and human diseases in the Malaysian peninsula

In the course of a plant disease survey of the Malaysian Peninsula (Malaysia comprises the Malaysian Peninsula, Sabah and Sarawak) during the period 1981-1986, more than 1000 isolates of Fusarium were obtained from diseased plants and seeds. Two further isolates were obtained from patients admitted to hospitals in the same area. The occurrences of F. proliferatum, F. nygamai and F. longipes are new records for the Malaysian Peninsula and the association of F. solani and F. oxysporum var. redolens with human diseases does not seem to have been reported previously. Ten representative species which could be classified into seven sections of the genus were selected for studies of their toxigenicity in liquid cultures and/or on rice. Crude toxin preparations from culture filtrates or extracts of the inoculated rice were tested for toxicity to brine shrimp larvae and tobacco mesophyll protoplasts. The protoplasts were more sensitive than the brine shrimp larvae to the toxin preparations, except those from the isolates of F. solani and F. oxysporum var. redolens obtained from either humans or tobacco. The toxicity of the preparations from rice cultures per g rice was always greater than the toxicity per ml of culture filtrates from cultures grown on Czapek-Dox broth, Czapek-Dox supplemented with 1% (w/v) peptone or Czapek-Dox supplemented with 5% (w/v) tobacco extract. The activity of all toxin preparations was stable to heat. It is concluded that the occurrence of toxigenic species of Fusarium in the Malaysian Peninsula is widespread and that they may pose a serious threat to the health of human, animal and plant populations.     

A Simple Method with Liquid Chromatography/Tandem Mass Spectrometry for the Determination of the Six Trichothecene Mycotoxins in Rice Medium

A selective and speedy LC–MS/MS method was developed to determine six trichothecene mycotoxins (nivalenol, deoxynivalenol, fusarenon X, 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, and T-2 toxin) in rice medium where Fusarium graminearum were cultivated for in vitro tests. The analytes were extracted from the rice medium with acetonitrile/water (85/15, v/v), and diluted with acetonitrile/water (5/95, v/v) in order to minimize the effects of matrices. Diluted solutions were analyzed by LC–MS/MS with electrospray ionization (ESI) interface in negative or positive ion mode and the multiple reaction monitoring mode. Recovery rates were 76–106% with a spiked level at 1–6 μg/kg of mycotoxins that corresponded to the limit of quantitation. The method was applied to study the time courses of trichothecene production and the biomass of fungi by three Fusarium graminearum strains. Three strains have different mycotoxin biosynthesis pathways, wFg14 and 03E-1 were DON producer, and 03N-1 was NIV producer.     

Natural multi-occurrence of mycotoxins in rice from Niger State,Nigeria

Twenty-one rice samples from field (ten), store (six) and market (five) from the traditional rice-growing areas of Niger State, Nigeria were analysed for aflatoxins (AFs), ochratoxin A (OTA), zearalenone (ZEA), deoxynivalenol (DON), fumonisin B₁ (FB₁) and B₂ (FB₂), and patulin (PAT) by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) respectively. T-2 toxin was determined using TLC only. AFs were detected in all samples, at total AF concentrations of 28–372 μg/kg. OTA was found in 66.7% of the samples, also at high concentrations (134–341 μg/kg) that have to be considered as critical levels in aspects of nephrotoxicity. ZEA (53.4%), DON (23.8), FB₁ (14.3%) and FB₂ (4.8%) were also found in rice, although at relatively low levels. T-2 toxin was qualitatively detected by TLC in only one sample. Co-contamination with AFs, OTA, and ZEA was very common, and up to five mycotoxins were detected in a single sample. The high AF and OTA levels as found in rice in this study are regarded as unsafe, and multi-occurrences of mycotoxins in the rice samples with possible additive or synergistic toxic effects in consumers raise concern with respect to public health.     

Identification of mycotoxins produced by species of Fusarium and Stachybotrys obtained from Eastern Europe.

Isolates of Fusarium and Stachybotrys spp. and crude extracts from these fungi were obtained from Hungary and the U.S.S.R. and used for the evaluation of the mycotoxins they produced. The cultures were grown on millet and oats and extracted in Budapest, Hungary (Veterinary Medical Research Institute) and chemically analyzed at the University of Minnesota using thin-layer chromatography (TLC), gas-liquid chromatography (GLC), gas chromatograph-mass spectrometry (GC-MS), and the rat skin bioassay. Zearalenone was found in most of the Fusarium cultures, T-2 toxin, neosolaniol, T-2 tetraol, and HT-2 toxin were found in extracts of Fusarium poae and F. sporotrichioies. A special effort was made to isolate the steroid-like toxins reported in the early Russian literature as sporofusarin and poaefusarin. None of the extracts from the Fusarium species yielded poaefusarin or sporofusarin when analyzed by our chemical methods or by those of L.E. Olifson, S.M. Kenina, and V.L. Kartashova, 1972. We therefore accounted for the toxicity of the Fusarium extracts as due to the 12,13,epoxytrichothecenes. One culture of Stachybotrys alternans yielded a macrocyclic ester of 12,13-epoxytrichothecene which, upon hydrolysis, yielded verrucarol; a steroid-like molecule (SB-3) was also isolated. The former had skin-irritant activity but SB-3 did not; the latter exhibited cardiac activity on the heart of the cockroach.     

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.     

Assay and Relationship of HT-2 Toxin and T-2 Toxin Formation in Liquid Culture

Both T-2 toxin and HT-2 toxin can be conveniently quantitated in crude extracts by using a combination of thin-layer chromatography and fluorodensitometry. This technique was used to follow the production of these toxins by liquid cultures of Fusarium poae (NRRL 3287). T-2 toxin was produced prior to HT-2 toxin and hexadeuterio-T-2 toxin was converted by the culture to trideuterio-HT-2 toxin.