Mycotoxins produced by Discolor section Fusarium species

An improved TLC method ofFusaria metabolites detection and quantitation has been elaborated. A total 92 isolates of Discolor sectionFusaria from cereals and potato have been examined from the point of view of cultures morphology and ability to produce characteristic mycotoxins. Low nutrient media (CLA, SNA) were found as suitable for production of uniform and typical macroconidia in studied cultures. All 26 isolates ofF. sambucinum Fuckel (=F.sulphureum, Schlecht) formed diacetoxyscirpenol in amount 20-1000 mg/kg and all 17.F. crookwellense Burgess N. & T. produced zearalenone (16-602 mg/kg).F. graminearum Schwabe produced: zearalenone 14/14 isolates, deoxynivalenol 11/14 isolates, both up to 77 mg/kg. Out of 26F. culmorum cultures originating from Poland 22 produced zearalenone up to 675 mg/kg, 17/26 3 acetyldeoxynivalenol up to 280 mg/kg and 16/26 deoxynivalenol up to 220 mg/kg. The difference in metabolism agrees with the difference in morphology of those species.     

Fusarium Head Blight Reactions and Accumulation of Deoxynivalenol (DON) and Some of its Derivatives in Kernels of Wheat, Triticale and Rye

Fifty-three commercially grown cultivars and germplasm lines of winter triticale (n = 18), wheat (n = 13), and rye (n = 5) and spring triticale (n = 8), wheat (n = 7) and rye (n = 2) were inoculated at mid anthesis with a spore suspension consisting of a mixture of Fusarium culmorum, Fusarium avenaceum and Fusarium graminearum isolates of known toxinogenic activity. Reactions to Fusarium head blight were measured as disease severity, reductions of kernel number/head, kernel weight/head and 1000 kernel weight, number of Fusarium-damaged kernels and kernel content of deoxynivalenol (DON) and its acetyl-derivatives 3-AcDON, 15-AcDON, and moniliformin. None of the cereal genotypes was completely resistant to Fusarium head blight. Wheat suffered from the largest kernel weight reductions, and accumulated the largest amounts of deoxynivalenol (up to 39.5 mg/kg) and 3AcDON (up to 6.0 mg/kg) in kernels. Deoxynivalenol was not detected in grain samples of winter rye cv. Dańkowskie Z?ote, and spring rye cv. Ludowe. 15-AcDON was only detected in genotypes of triticale, and 3AcDON only in a few genotypes of winter wheat and rye. Moniliformin was detected at low concentrations (up to 0.092 mg/kg) in kernels of some genotypes selected for the mycotoxin analysis. A moderately strong Pearson correlation was found between head blight severity parameters and the accumulation of deoxynivalenol and its derivatives in grain of the cereal genotypes studied. Fusarium head blight severity parameters were correlated with the percentage of Fusarium-damaged kernels and reductions of yield components. However, some head blight-susceptible genotypes realized their potential yields, but accumulated high levels of mycotoxins in kernels. Both Fusarium head blight resistant and susceptible genotypes of the three cereal species accumulated deoxynivalenol in kernels. This finding suggests that the system regulating deoxynivalenol accumulation may be independent of Fusarium head blight reaction.     

A study of the correlation between the amount of deoxynivalenol in grain of wheat and triticale and percentage of<Emphasis Type="Italic">Fusarium</Emphasis> damaged kernels

The correlation between the amount of deoxynivalenol (DON) and the percentage ofFusarium damaged kernels (FDK) in samples of wheat and triticale was studied.Samples of naturally infected wheat grain, collected in 1986, 1987 and 1988 and of triticale collected in 1986 were used.Additionally, artificial inoculated wheat samples (10 genotypes inoculated with 3F. Culmorum strains of weak, medium and severe pathogenicity and samples of 10 triticale genotypes inoculated withF. culmorum. andF. graminearun) were studied. Using statistical methods (the variance analysis, method of least significant difference (LSD), orthogonal contrast (OC) and minimum within groups sum of squares criterion (MSSC)), the samples were divided into two groups with respect to the attribute DON/FDK.To the first group belong samples of wheat and triticale, of which the heads were artificially inoculated with severely pathogenic strainsF. culmorum. In the samples of this group the amount of DON in kernels damaged withFusarium increased by 0,46 mg/kg per 1% of FDK.In the second group, consisting of naturally infected samples and samples from artificially inoculated heads the amount of DON increased 0,30 mg DON/kg per 1% of FDK.The equation for the calculation of approximated amount of DON in farm and commercial lots of wheat and triticale after examination of percentage of FDK is given.     

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.     

Zearalenone formation by<Emphasis Type="Underline">Fusarium crookwellense</Emphasis> /Burgess,Nelson and Toussoun/ isolates from Poland and their pathogenicity towards cereals

Fusarium crookwellense /B.N. and T./ isolated from affected cereals in Poland formed zearalenone on wheat grain up to 602 mg/kg. Tested isolates have been found strong to severe pathogens of wheat, rye, triticale and barley seedlings and corn ears with pathogenicity similar to that ofF. culmorum andF. graminearum.     

A mini-survey of moulds and mycotoxins in locally grown and imported wheat grains in Nigeria

A preliminary survey involving limited sample size was conducted to determine the spectrum of moulds and mycotoxins in wheat grains from flour mills and local markets in Nigeria. Fourteen wheat samples were analyzed for moulds using standard mycological methods and for toxic fungal metabolites using a liquid chromatography-tandem mass spectrometric method. Fusarium (range of incidence 12.5–61.7%) dominated in the wheat grains though species of Aspergillus (range of incidence 2.24–3.86%) were also recovered from the samples. The identified fungal species were Aspergillus flavus (7.7%), Aspergillus niger clade (2.6%), Fusarium avenaceum (10.9%), Fusarium culmorum (22.4%) and Fusarium graminearum (56.4%). A total of 54 microbial metabolites were detected in the samples at concentration ranging between 0.01 μg/kg for macrosporin and 2560 μg/kg for deoxynivalenol. Among the four mycotoxins addressed by regulations in the European Union (EU) found in the samples, deoxynivalenol (incidence 100%) dominated in the samples and its levels exceeded the maximum acceptable EU limit (750 μg/kg) in 36% of the samples. This report underscores the need for more robust surveys with larger sample sizes and across several agro-ecologies in the country.     

Pathogenicity of Fusarium graminearum Schwabe isolates from Poland towards seedlings of cereal species

Ten isolates ofFusarium graminearum Schwabe originating from diseased cereal plants and kernels were tested for pathogenicity to various cultivars of wheat, rye, triticale and oats. The isolates varied greatly in their pathogenicity to the seedlings of the species, and were most pathogenic to rye and triticale, less pathogenic to barley and wheat and least pathogenic to oats.     

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.     

Characterisation of<Emphasis Type="Italic">fusarium graminearum</Emphasis> and<Emphasis Type="Italic">F. culmorum</Emphasis> isolates by mycotoxin production and aggressiveness to wheat

A total of 27Fusarium culmorum isolates from Germany and 41F. graminearum isolates from Kenya were investigated for aggressiveness and mycotoxin production on wheat ears. In addition, ergosterol content of the kernels from ears inoculated withF. graminearum was determined and theF. culmorum isolates were tested for mycotoxin productionin vitro. For both pathogens, isolates markedly differed in aggressiveness. 59% and 37% of theF. culmorum isolates produced NIV and DON, respectively,in vivo andin vitro. The DON-producing isolates also produced 3-acDONin vitro. The more aggressive isolates produced mainly DON while the less aggressive isolates produced mainly NIV. 12% and 85% of theF. graminearum isolates produced NIV and DON, respectively. The highly aggressive isolates produced higher amounts of DON, aggressiveness being highly correlated to DON content in the kernels. NIV-producing isolates were less aggressive. Ergosterol content of kernels was moderately correlated to aggressiveness but highly correlated to DON content. Disease severity was associated with kernel weight reduction.     

Toxicity,of extracts of barley kernels inoculated withFusarium spp. toArtemia salina

Toxicity toA. salina, of the Fusarium metabolites: deoxynivalenol (DON), its acetylated derivatives (3- and 15-AcDON), zearalenone (ZON), neosolaniol (NEO), nivalenol (NIV), T-2, HT-2 toxins, has been examined and compared with toxicity of extracts of barley kernels (8 cultivars and 4 lines) inoculated withFusarium culmorum, F. graminearum andF. sporotrichioides respectively. Estimated LC₅₀ values were expressed as relative toxicity (RT) in mg DON/kg for samples inoculated withF. culmorum, F. graminearum or in mg T-2/kg forF. sporotrichioides inoculations. Toxicity of extracts of the same genotype/line kernels was compared among different pathogens used for inoculation and differences in Fusarium head blight susceptibility of different genotypes/lines inoculated with the sameFusarium strain were found. Significant correlation between toxicity of extracts (LC₅₀, RT) and toxic metabolites concentration was found ( $\bar r = 0.82$ ; P = 0.01). Bioassays withA. Salina offer a fast, easy and inexpensive method to examine cereal genotypes susceptibility to Fusarium head blight and mycotoxins accumulation in kernels.     

Toxinogenicity of<Emphasis Type="Italic">Microdochium nivale (Fusarium nivale)</Emphasis> isolates from cereals in Poland

Microdochium nivale (Fusarium nivale) was found to be frequently occuring in Poland pathogen of small grain cereals heads, causing symptoms similar to those observed after infection ofFusarium species. In consecutive years since 1985 till 1989 the following percentage of wheat and rye ears infected withM. Nivale and withFusarium head blight symptoms was found: 34%, 21%, 42%, 9%, 46% (wheat) and 57%, 43%, 65%, 4%, 47% (rye) heads.However, in naturally infected rye and wheat samples (kernels and chaff), we did not detect toxins usually present in samples infected with fungi of genusFusarium — such as deoxynivalenol and derivatives. TypicalFusarium trichothecene metabolites were also not present in cultures of 11M. nivale strains, growing 3–5 weeks on rice (45% water content) at 20°C. Cultures of two typical isolates on wheat grain (strain KF 1124) and on rice (KF 245) were found to be non toxic to broiler chickens when present in amount 20–40% in their diet. It can be concluded thatM. nivale (F. nivale) representatives in Poland did not produce toxic metabolites neither under laboratory condition nor after cereal ears infection under field conditions.     

Deoxynivalenol and 3-acetyldeoxynivalenol and Fusarium species in winter triticale

Fusarium species infecting heads of Triticale and mycotoxins presence in infected kernels and chaff were studied during two seasons. The most important species observed on infected heads were in 1986F. avenaceum (39%),F. nivale (21%),F. culmorum (20%),F. graminearum (14%), and others (6%). In 1987 after long and snowy winterF. nivale dominated (64%), followed byF. avenaceum (24%),F. culmorum (6%), andF. graminearum (5%). The mycotoxins deoxynivalenol (DON) and 3-acetyl DON were present in all 11 subsamples of kernels from heads infected byF. culmorum and/orF. Graminearum (1.6–16.4 mg and 0.7–2.4mg/kg, respectively). Chaff from the same subsamples contained 9.9–33.2mg/kg of DON and 5.2–16.0mg/kg of 3-AcDON. Kernels with visibleFusarium-damage contained 2.4–31.2 mg/kg of DON and 1.2–6.0 mg/kg of 3-AcDON. Remaining part of kernels without symptoms of visibleFusarium-damage contained only DON in an amount of 0.9–5.9 mg/kg.     

<Emphasis Type="Italic">Fusarium</Emphasis> mycotoxins in conventionally and organically grown grain from Thuringia/Germany

Thefusarium mycotoxins deoxynivalenol (DON) and zearalenone (ZON) were determined in conventionally and organically grown grain harvested 1998 in Thuringia/Germany. A total of 196 wheat samples and 69 rye samples was analysed.In this year with heavy rainfalls during the summer months, high concentrations offusarium mycotoxins were typical in grain grown in Germany, as the DON concentrations found here. DON concentrations in conventionally grown wheat were found to be significantly higher than in organically grown wheat. 69% of the conventionally grown wheat were tested positive, containing a mean concentration of 1540 µg/kg DM. In 54% of the organically grown wheat samples DON was detected with a mean value of 760 µg/kg DM. DON concentration in rye and ZON concentration in wheat showed similar tendencies.The different cultivars of conventionally grown wheat showed large differences in DON contamination.     

Trichothecene resistance in wheat: Development of molecular markers for PDR-type ABC transporter genes

Infection withFusarium graminearum andF. culmorum not only causes severe yield and quality losses, the most relevant concern is the contamination of cereal foods and feeds with trichothecenes (e.g. deoxynivalenol, DON). The ability to synthesize trichothecenes has been shown to be a virulence factor ofF. graminearum on wheat and, on the other hand, toxin resistance is most likely an important component of field resistance. Our hypothesis is that pleiotropic drug resistance mediated by PDR-type ABC transporter proteins (acting as membrane located drug efflux pumps) is a relevant mechanism of DON resistance not only in yeast but also in wheat. Goal of this project is the development of molecular markers for this gene family for use in marker-assisted plant breeding programs. The technical difficulties caused by the large size of the PDR-family are discussed.     

Feasibility of 3D UV-C treatment to reduce fungal growth and mycotoxin loads on maize and wheat kernels

Fungal disease of grain crops is a concern for the agricultural industry, resulting in economic losses. Aside from severe yield losses, mycotoxigenic fungi such as Penicillium and Fusarium can produce harmful mycotoxins, including deoxynivalenol (DON), zearalenone (ZEN), and ochratoxin A (OTA). This proof-of-concept study explored the feasibility and effects of ultraviolet (UV) C light at 253.7 nm to reduce fungal and mycotoxin loads on model surfaces as well as on maize and wheat kernels using benchtop 2D and 3D illumination strategies. Reduction of Penicillium verrucosum (98.6%) and Fusarium graminearum (88.8%) on agar was achieved using a UV-C dose of 100 mJ cm^?2. Naturally occurring fungal growth resembling P. verrucosum on maize was reduced by 79% after exposure to 5000 mJ cm^?2. Similarly, fungal growth resembling F. graminearum on maize was reduced by 60% with 1000 mJ cm^?2. On wheat, significant reduction of fungal growth was not observed. Maximal reduction of DON (97.3%), ZEN (75.4%), and OTA (91.2%) on filter paper was obtained using 15,000 mJ cm^?2. The overall reduction of DON (30%; 14%), ZEN (52%; 42%), and OTA (17%; 6%) on maize and wheat, respectively, was lower than on filter paper. Moisture and crude protein content as well as percent germination of maize kernels were not affected by UV-C treatment up to 5000 mJ cm^?2. This study has shown that 3D UV-C treatment is a feasible option for reducing Fusarium and Penicillium growth on maize kernels and, at higher doses, decreasing ZEN by ~?50%.     

Deoxynivalenol and 3-acetyldeoxynivalenol — mycotoxins associated with wheat head fusariosis in Poland

Samples of wheat naturally infected in the field byFusarium culmorum (W.G.Sm.) Sacc. andFusarium graminearum Schwabe were analyzed for deoxynivalenol, 3-acetyldeoxynivalenol, and zearalenone. No zearalenone was detected at levels higher than 0.5mg/kg. Deoxynivalenol was present in 100% and 3-acetyldeoxynivalenol in 80% of the examined samples at levels of 0.21 to 30.4 mg/kg and 0.54 to 29.54 mg/kg, respectively. The mycotoxin levels in the chaff were 5 to 50 times higher than in the kernels. This is the first report on natural occurrence of 3-acetyldeoxynivalenol in wheat. This toxin, in addition to deoxynivalenol, was highly correlated with wheat head fusariosis. These findings suggest that more attention should be given to the occurrence of 3-acetyldeoxynivalenol in cereal grains during the growth as well as during storage.     

Occurrence of<Emphasis Type="Italic">Fusarium</Emphasis> toxins in the 1999’s harvest

Continuing the monitoring ofFusarium toxins in cereals, we investigated 245 samples of wheat, barley, triticale and oats in 1999. 84 samples out of 100 analysed forFusarium could be found to be infected. The most prominentFusarium species detected whereF. avenaceum, F. poae, F. detected whereF. avenaceum, F. poae, F. graminearum, andF. sporotrichoides. The level of mycotoxin contamination of the samples varied depending on their origins and was in general very low in comparison with the result obtained in samples of the previous year. There where only some wheat samples with deoxynivalenol (DON) concentrations beyond existing advisory levels. The average DON concentration of all samples was 0.35 mg/kg with a median of 0.007 mg/kg. 3-Acetyldeoxynivalenol and zearalenone (ZEA) could only be detected at minor concentrations (below 0.1 and 0.05 mg/kg, respectively) in less than 10% of the samples. The analysis of commercial cereal flour reflects this situation. Flour bought in the first quarter of 1999, which was suspected to contain a high portion of the 1998 harvest, was contaminated by DON to a higher extent than those purchased in 2000. The average DON concentration in the flour samples of 1999 and 2000 was 0.35 mg/kg and 0.23 mg/kg, respectively. Although the general mycotoxin level in the 1999 harvest was lower as in 1998 there were some highly contaminated samples that had mainly been grown in fields with either maize or other cereals as previous crop and reduced tillage. The combination of maize as previous crop and non-tillage could be stated as most unsuitable, which promotes enhanced mycotoxin contamination, and should therefore be avoided.     

Biosynthesis,isolation, purification and seperation of Zearalenone,deoxynivalenol and 15-Acetyldeoxynivalenol

Fusarium graminearum KF-376 isolate was found to be able to form simultaneously three toxic metabolites: zearalenone (FF-2), deoxynivalenol (DON) and 15-acetyldeoxynivalenol (15-AcDON). Toxins were extracted with methanol — water 3:1 (v/v) and purified by liquid chromatography on charcoal — Kieselgel 60 column (preliminary) and Aluminiumoxid 90 column. Final separation of the metabolites was achived on Kieselgel 60 — Aluminiumoxid 90 column.     

The use of yeast for microbial degradation of some selected mycotoxins

Several biodegradation experiments were carried out using 10 different yeast strains.Saccharomyces spp., Kluyveromyces spp. andRhodotorula spp. were tested for biodegradation of selected mycotoxins (ochratoxin A, nivalenol, deoxynivalenol and fumonisin B₁) standardsin vitro. There was confirmed that some yeast strains are able to degrade some mycotoxins. However, great differences between individual strains were observed. Moreover, 12Saccharomyces cerevisiae strains were tested for their potential capability to degrade zearalenone and fumonisins in Sabouraud broth. Two strains were capable to degrade zearalenone totally, one strain decreased the mycotoxin concentration up to 25%, and one strain up to 75% of original amount. Two strains were capable to degrade fumonisins partially.     

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.