Mycotoxins and Fusarium spp. associated with infected ears of corn in Minnesota

Five Fusarium species were isolated from the grain of dent corn (Zea mays) selected from 20 of 32 damaged fields in 10 counties in Minnesota on the basis of hyphal growth visible on kernels in the field. Three mycotoxins were identified in the infected ears: zearalenone, deoxynivalenol, and 15-acetyl-deoxynivalenol. This is the first report of the presence of 15-acetyl-deoxynivalenol on corn ears in the field prior to harvest and in combination with deoxynivalenol and zearalenone. Ninety-nine cultures were selected from colonies growing from kernels on an agar medium; 30% of the cultures were F. graminearum, 23% were F. subglutinans, 20% were F. moniliforme, 14% were F. oxysporum, and 12% were F. proliferatum.     

Natural occurrence of deoxynivalenol, 15-acetyl-deoxynivalenol, and zearalenone in refusal factor corn stored since 1972.

Two samples of "refusal factor" corn, one stored frozen in Minnesota and one stored dry in Indiana since 1972 or 1973, were analyzed for the presence of Fusarium spp. and Fusarium toxins. Both samples were from corn refused by swine in Indiana from 1972 to 1973. Sample FS 808 (stored in Indiana) contained 20 ppm of deoxynivalenol (20 micrograms/g), 16 ppm of 15-acetyl-deoxynivalenol, 5 ppm of zearalenone, and 0.2 ppm of alpha-zearalenol. Sample FS 362 (stored in Minnesota) contained 3 ppm of deoxynivalenol, 1 ppm of 15-acetyldeoxynivalenol, and 0.3 ppm of zearalenone. The presence of 15-acetyl-deoxynivalenol is significant because it is the first report of it occurring naturally in refusal factor corn, and it may account in part for the refusal that could not be solely attributed to deoxynivalenol.     

Natural occurrence of deoxynivalenol, 15-acetyl-deoxynivalenol, and zearalenone in refusal factor corn stored since 1972

Two samples of "refusal factor" corn, one stored frozen in Minnesota and one stored dry in Indiana since 1972 or 1973, were analyzed for the presence of Fusarium spp. and Fusarium toxins. Both samples were from corn refused by swine in Indiana from 1972 to 1973. Sample FS 808 (stored in Indiana) contained 20 ppm of deoxynivalenol (20 micrograms/g), 16 ppm of 15-acetyl-deoxynivalenol, 5 ppm of zearalenone, and 0.2 ppm of alpha-zearalenol. Sample FS 362 (stored in Minnesota) contained 3 ppm of deoxynivalenol, 1 ppm of 15-acetyldeoxynivalenol, and 0.3 ppm of zearalenone. The presence of 15-acetyl-deoxynivalenol is significant because it is the first report of it occurring naturally in refusal factor corn, and it may account in part for the refusal that could not be solely attributed to deoxynivalenol.     

Moniliformin and the European Corn Borer (<Emphasis Type="Italic">Ostrinia nubilalis</Emphasis>)

A representative survey was made of maize ears of the 1988 and 1989 crop in Austria to establish the influence of corn borer injuries onFusarium species involved in ear fusariosis andFusarium toxin production.TheFusarium species most frequently isolated from rot-damaged ears wereFsacchari var. subglutinans (about 50 %) andF. graminearum (about 30 %). There was a striking difference between theFusarium species of the Liseola and the Discolor section concerning their occurrence on corn borer-damaged ears. More than 80 % of the ears infected withF. sacchari var. subglutinans andF. verticillioides, but less than 15 % of the ears infected withF. graminearum, F. crookwellense andF. culmorum showed corn borer injuries.Toxin analyses of the infected ears corresponded to the known toxigenicity of the respectiveFusarium species. Ears infected withF. sacchari var. subglutinans contained moniliformin (up to 20 mg/kg), those infected withF. verticillioides fumonisin B₁ and B₂ (up to 15 mg/kg). In ears infected withF. graminearum, F. culmorum andF. crookwellense zearalenone (up to 40 mg/kg) and deoxynivalenol (up to 500 mg/kg) or nivalenol (up to 10 mg/kg), respectively, could be detected. Hence measures to combat the European corn borer will mainly reduce moniliformin and fumonisin contamination, but will affect zearalenone, deoxynivalenol and nivalenol contents of the ears to a much lesser extent.     

Cumulation of mycotoxins in maize cobs infected with<Emphasis Type="Italic">Fusarium gramihearum</Emphasis>

Maize cobs withFusarium ear rot were collected at 1986 season and five infected byFusarium graminearum were analyzed for presence of triohothecenes and zearalenone. Collected material was subsampled forFusarium damaged kernels and corresponding axial stems and healthy looking kernels. All investigated cobs contained deoxynivalenol (DON) (range 18.0–131.5 mg/kg) and zearalenone (ZEA) (range 0.38–2.17 mg/kg), in four cobs 15-acetyl-deoxynivalenol (15-AcDON) (range 5.2–6.2 mg/kg) was present and two cobs besides three all metabolites contained 3-acetyl-deoxynivalenol (3-AcD0N) (range 0.5–0.8 mg/kg).The average of individual toxins amount in axial stems: in mg/kg was equal to: DON — 110.36, ZEA — 4.57, 15-AcD0N — 16.66, and 3-AcD0N — 1.32.Fusarium damaged kernels contained in average the following amount (mg/kg) of: DON 77.00, ZEA 0.98, 15-AcD0N 3.78 and 3-AcD0N 0.06. Healthy looking kernels contained DON 1.96 mg/kg and ZEA 0.07 mg/kg only. Cooccurrence of 3-AcDON and 15-AcDON in two samples was an interesting finding. The amount of DON in total cob was highly correlated (r = 0.94) with percentage ofFusarium damaged kernels in given ear.     

Pathogenicity of Fusarium Isolates from Wheat, Rye and Triticale Towards Seedlings and their Ability to Produce Trichothecenes and Zearalenone

37 Fusarium isolates (F. culmorum 20, F. graminearum 4, F. avenaceum 3, F. solani 4, and F. equiseti 6 from wheat, rye and triticale) were examined for pathogenicity and tested for zearalenone (F-2) and trichothecenes production in vitro. Strong pathogens (F. culmorum and F. graminearum) produced deoxynivalenol and 3-acetyl-deoxynivalenol and zearalenone in considerable quantities.     

Occurrence of moniliformin,deoxynivalenol, and zearalenone in durum wheat (Triticum durum Desf.)

Forty-eight durum wheat samples from 5 locations in Austria were examined forFusarium infection andFusarium toxin content.F.gramlnearum andF.avenaceum were by far the prevailingFusarium species In durum wheat kernels, followed byEpoae, F.culmorum, andF.equlsetl. Ion-paired HPLC analyses of the samples showed moniliformin contents of kernels up to 0.88 mg/kg. All moniliformin contaminated samples also contained high levels of deoxynivalenol (up to 8.2 mg/kg) and lower levels of zearalenone (<0.33 mg/kg). The levels of zearalenone in naturally contaminated durum wheat samples did not correspond to the high yields of zearalenone found in cultures of the fusaria isolated from the durum wheat kernels. These conflicting results as well as some toxicologlcal aspects of the carry over ofFusarium toxins from durum wheat kernels into pasta are discussed.     

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.     

Fusarium species associated with banana fruit rot and their potential toxigenicity

Banana fruits exhibiting signs of decay were collected from markets in the United States and Italy. Fungi isolated from the lesions on the banana fruits wereFusarium moniliforme, F subglutinans, andF. semitectum var.majus. When the fungal strains were cultivated on maize kernels, the cultures did not produce zearalenone (ZON), zearalenols (á-, â-ZOH), and trichothecenes [deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), T-2 toxin (T-2), diacetoxyscirpenol (DAS)]. Fumonisins and fusarin C (FUS-C) were not detected naturally nor in bananas purchased in the U.S. and artificially infected withFusarium. Moniliformin (M) (up to 267 mg/kg) was detected in maize kernel cultures ofF. subglutinans from bananas. No mycotoxins were detected in naturally infected fruits. Although no mycotoxins were detected in the extracts from corn cultures ofF. semitectum var.majus, the extracts were toxic to brine shrimp and mice.     

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.     

Aflatoxin B1, zearalenone and deoxynivalenol production on irradiated corn kernels: Influence of inoculum size

The influence of inoculum size on aflatoxin B₁ (AFB₁), zearalenone (ZEN) and deoxynivalenol (DON) production was examined on irradiated corn kernels. Spore concentrations were determined in serial dilutions and adjusted to 10,10²,10³,10⁵ and 10⁶ spores/ml. Aflatoxin B₁ production was dependent on the inoculum size. The high levels of aflatoxin B₁ produced byA. parasiticus (21 and 30 mg/kg) were obtained with 10² and 10³ spores/ml after 35 and 20 days incubation. There was no spore concentration influence on zearalenone and deoxynivalenol production after 10, 20 and 35 days incubation. At 28°C and 0.97 water activity (aw), the mean levels of zearalenone production were 382, 267 and 520 μg/kg and the mean levels on deoxynivalenol production were 697,465 and 782 μg/kg.     

Bestimmung von DON-3-Glukosid in künstlich und natürlich kontaminiertem Weizen mittels LC-MS/MS

Naturally contaminated and artificiallyFusarium spp. inoculated wheat was analyzed for deoxynivalenol, deoxynivalenol-3-glucoside, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol. After extraction and clean-up with MycoSep columns, the trichothecenes were determined using a LC-ESI-MS/MS method. Deoxynivalenol-3-glucoside was detectable in 4 out of 4 artificially inoculated and in 22 out of 25 naturally contaminated wheat samples. For the latter, the average relative deoxynivalenol-3-glucoside concentration was about 6% of the deoxynivalenol concentration. The maximum relative deoxynivalenol-3-glucoside concentration was 12% as compared to the concentration of deoxynivalenol. In all samples, the concentration of deoxynivalenol-3-glucoside was higher than the concentrations of 3-acetyl-deoxynivalenol or 15-acetyl-deoxynivalenol.

Financial support: Christian Doppler Society, Austrian Genome Research Intiative (GEN-AU)     

Trichothecene chemotypes ofFusarium graminearum isolated from corn in Hungary

Twenty three strains ofFusarium graminearum isolated from corn were screened for their ability to produce type A and B trichothecenes and zearalenone (ZEA) on the solid substrate rice grains in the dark at 28 °C for 21 days. Toxin analyses were made with HPLC technique. Of 23 total isolates, 10 produced deoxynivalenol (DON), 4 produced DON and nivalenol (NIV), 1 produced DON and 15 acetyl-DON (15-ADON), 1 produced NIV and 4-acetyl-NIV (4-ANIV) and 1 produced NIV. Of 23 totalF. graminearum isolates, 20 produced ZEA. These results suggest that strains ofF. graminearum, prevailing in Hungarian corn growing regions, might belong to DON-and NIV-chemotypes. This is the first report demonstrating that DON-, DON-NIV-, DON-15-ADON-, NIV-4-ANIV and NIV-producingF. graminearum isolates are distributed in Hungary.     

Aflatoxin B1, zearalenone and deoxynivalenol production by Aspergillus parasiticus and Fusarium graminearum in interactive cultures on irradiated corn kernels

The influence of inoculum size in the production of aflatoxin B1 (AFB1), zearalenone (ZEN) and deoxynivalenol (DON) was determined when Aspergillus parasiticus NRRL 3000 and Fusarium graminearum ITEM 124 were cultured alone and in pairs on irradiated corn kernels at 28 °C and 0.97 water activity (aw). The highest levels of AFB1 produced by A. parasiticus were produced at the lowest levels of the inoculum (103 spores/ml). No significant differences were observed in ZEN and DON production at any inoculum level during the experimental period. When A. parasiticus was co-inoculated with F. graminearum both to the same inocula (106 spores/ml), AFB1 inhibition percentage were 60, 72 and 56% at 10, 20 and 35 days of incubation respectively, while at 106 spores/ml the percentages of inhibition were 34, 84 and 93% at 10, 20 and 35 days. In the mixture cultures A. parasiticus 103 × F. graminearum 106 spores/ml the percentage of inhibition of AFB1 oscillated in 99% during all the incubation. In the interaction A. parasiticus 106 spores/ml × F. graminearum 103 spores/ml the accumulation of AFB1 decreased in 80, 94 and 86% at 10, 20 and 35 days of incubation respectively. In single culture F. graminearum was inoculated with 10³ or 106 spores/ml and the highest levels of ZEN and DON were detected at 35 days of incubation. The levels oscillated in 538–622 μg/kg for ZEN and 870–834 μg/kg for DON respectively. In paired cultures there were no significant differences in the levels regardless of the spore concentrations during the incubation time. This revised version was published online in June 2006 with corrections to the Cover Date.     

Mycotoxin production and cytotoxicity of Fusarium strains isolated from Norwegian cereals

Thirty-four isolates of the eight most common Fusarium species isolated from Norwegian cereals; F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. poae, F. sporotrichioides, F. torulosum and F. tricinctum were studied for their cytotoxicity and ability to produce mycotoxins. The strains were cultivated on rice, and analysed for trichothecenes (all species), zearalenone (all species), fusarochromanone (F. equiseti), wortmannin (F. torulosum), moniliformin and enniatins (F. avenaceum, F. tricinctum and F. torulosum). The cytotoxicity of the extracts were examined with an (in vitro) MTT-cell culture assay. All F. graminearum and five of seven F. culmorum isolates belonged to chemotype IA, producing deoxynivalenol and 3-acetyl-deoxynivalenol, while the two other F. culmorum strains were nivalenol producers (chemotype II). The F. equiseti isolates and one of the F. poae isolates produced both type A and B trichothecenes, and relatively large quantities of fusarochromanone were detected in the F. equiseti cultures. All Fusarium species studied showed significant cytotoxicity, but with a large variation between species, and also within each species. F. sporotrichioides and F. equiseti showed the highest average cytotoxicity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Trichothecene mycotoxins in kernels and head fusariosis susceptibility in winter triticale

A single isolates ofFusarium graminearum Schwabe KF 366 andFusarium culmorum (W.G.Sm.) Sacc. KF 365 were used to infect 10 genotypes (9 lines and one cultivar) of winter triticale, 1 rye cultivar and 1 wheat cultivar, and amounts of mycotoxins in kernels were analysed at the same stage of development. One genotype of triticale CHD 353/79 and rye “Chodan” were found to be most resistant towards both species infection with lowest amount of mycotoxins (deoxynivalenol) content in kernels and also the lowest yield reduction. The most susceptible line CZR 142 cumulated in kernels about ten times higher amount of mycotoxins (up 53 mg DON/kg and 16 mg 3AcDON/kg, and 5 mg zearalenone/kg). GenerallyF, culmorum formed higher level of mycotoxins in kernels of infected heads thanF. graminearum. In kernels of more susceptible genotypes except deoxynivalenol, 3 acetyldeoxynivalenol and zearalenone also were present.     

Fusarium species and their mycotoxins in infected corn in Italy

Surveys of corn (infected plants and commercial kernels) forFusarium species and their mycotoxins were carried out on samples collected all over Italy and from some European and mediterranean countries.Investigations on samples of corn stalk and ear rot standing in the field, mainly collected in southern Italy, proved to be contaminated with zearalenone (ZON), zearalenols (ZOL), and deoxynivalenol (DON). TheFusarium species most frequently isolated, and their recorded toxigenic capability (in parentheses), were:F. moniliforme;F. culmorum (ZON, ZOL, DON, 3AcDON);F. equiseti (ZON, ZOL); andF. proliferatum (MF). Along with these species,F. graminearum group 2 (ZON, DON and/or 3AcDON or 15AcDON);F. chlamydosporum;F. acuminatum (type-A trichothecene derivatives); andF. semitectum were often found to be associated.F. heterosporum (ZON, ZOL);F. solani;F. crookwellense (ZON, ZOL, FUS, NIV);F. oxysporum (MF);F. avenaceum (MF);F. sporotrichioides (T-2 toxin and derivatives); andF. poae (DAS, MAS) were occasionally isolated.     

Multiplex PCR assay for the identification of nivalenol, 3- and 15-acetyl-deoxynivalenol chemotypes in Fusarium

The ability to rapidly distinguish trichothecene chemotypes in a given species/population of the genus Fusarium is important due to significant differences in the toxicity of these secondary metabolites. A multiplex PCR assay, based on primer pairs derived from the Tri3, Tri5 and Tri7 genes of the trichothecene gene cluster was established for the identification of the different chemotypes among Fusarium graminearum, F. culmorum and F. cerealis. Using the selected primers, specific amplification products of 625, 354 and 708 bp were obtained from Fusarium isolates producing nivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol, respectively. Moreover, the multiplex PCR was successfully used to identify the chemotype of the Fusarium species contaminating wheat kernels. Four picograms of fungal DNA were found to be necessary to obtain a visible amplification product.     

Impact of aggressiveness of <Emphasis Type="Italic">Fusarium graminearum</Emphasis> and <Emphasis Type="Italic">F. culmorum</Emphasis> isolates on yield parameters and mycotoxin production in wheat

Plant-associated isolates from Fusarium graminearum and F. culmorum were inoculated on wheat in field experiments in 2007 and 2008 to ascertain their influence on fungal colonization of the ears, as well as mycotoxin contamination (deoxynivalenol, DON; nivalenol, NIV; zearalenone, ZEA) and yield parameters in the mature crop after inoculation with or without irrigation. The isolates were assigned to four different groups of aggressiveness on the basis of pathogenic symptom development and mycotoxin production in vitro. Increased levels of trichothecene-producing Fusarium DNA in the ears indicated a successful inoculation of the plants, which resulted in increased DON content in the wheat kernels in 2007. Dry conditions at anthesis markedly suppressed fungal colonization as well as mycotoxin accumulation. However, due to precipitation during the ripening period, yield and thousand-kernel weight were similar whether or not irrigation was applied at the time of inoculation. The level of aggressiveness among the isolates as determined in vitro was not reflected in the field experiment. The activity of the extracellular invertase in developing ears increased as a plant response to pathogen infection, especially when the plants were irrigated at the time of inoculation. In 2008, the Fusarium inoculation of wheat heads did not cause fungal growth and mycotoxin contamination in the grain, because of the dry weather conditions that occurred over the entire period of anthesis and ripening. The risk of future mycotoxin contamination in grains was discussed based on climate change prognosis.     

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。