Don Gaba

Retention of the fusarium mycotoxin deoxynivalenol in wheat during processing and cooking of spaghetti and noodles

Samples of Canadian Western Red Spring (CWRS) wheat of the variety Sinton and Canadian Western Amber Durum (CWAD) wheat of the variety Coulter containing high levels of naturally incurred Fusarium graminearum Schwabe and deoxynivalenol (DON) were conditioned, milled and processed to noodles and spaghetti respectively. Mycological examination revealed greater penetration of F. graminearum into Coulter wheat kernels than Sinton wheat kernels. The highest mill fraction mould counts were found in the bran of the Sinton wheat and the shorts of the Coulter wheat. Scouring of Sinton wheat preceding and following tempering reduced the level of DON in the wheat. Conditioning of Coulter wheat without scouring did not affect DON levels. The amount of DON retained in Sinton flour and Coulter semolina averaged 29% and 52% respectively of the amounts found in the respective conditioned wheats. DON concentrations were highest in the bran for both wheats. The weight of DON retained in cooked Japanese and Chinese noodles prepared from Sinton flour averaged 52 % and 42 % respectively of the weight of DON in the flour. Retention of DON in optimally cooked Coulter spaghetti averaged 43 % to 53 % of the amount present before cooking for both high temperature and low temperaturedried spaghetti. Overcooking resulted in a slight further decrease in retention of DON in cooked spaghetti.

Prevalence of fungi and fusariotoxins on barley seed from western Canada, 1995 to 1997

The mycoflora and levels of deoxynivalenol (DON) on oat seed (Avena sativa) grown in western Canada was determined by analyzing a total of 511 grain samples collected from 39 crop districts in Alberta, Saskatchewan, and Manitoba during 1995-1997. Fungi representing a minimum of 85 species were recovered. Alternaria alternata was the most frequently isolated species from each province. Levels of A. alternata, Bipolaris sorokiniana, and Fusarium graminearum were highest in Manitoba and eastern Saskatchewan, whereas Cladosporium species and Drechslera avenacea were highest in samples from Alberta and western Saskatchewan. DON levels ≥ 0.10 ppm were found in seeds from two Manitoba crop districts in 1996 and three in 1997, with a maximum level of 0.34 ppm. In 1997, DON was also detected in composite samples of seed from three Saskatchewan and two Alberta crop district composites.

Relative aggressiveness and production of 3- or 15-acetyl deoxynivalenol and deoxynivalenol by Fusarium graminearum in spring wheat

Abstract Fusarium graminearum is the principal cause of fusarium head blight in North America, a disease that has caused severe losses in yield and quality of cereals. In North America, the vast majority of F. graminearum isolates produce 3- or 15-acetyl deoxynivalenol (ADON) in addition to DON. Until recently, 15-ADON isolates predominated, but a rapid shift from 15-ADON to 3-ADON producers in Canada and north central USA has been documented. In order to better understand the effect of this population shift on relative aggressiveness of isolates and mycotoxin accumulation, we tested a total of 58 isolates for 3- and 15-ADON production on two Canadian spring wheat cultivars, ‘Roblin’ (susceptible) and ‘5602 HR’ (moderately resistant). In Experiment 1, three isolates from the Canadian provinces of Manitoba, Saskatchewan and Alberta, each of which produced either 15-ADON or 3-ADON, were tested using spray inoculation. In Experiment 2, 20 isolates which produced 15-ADON and 20 which produced 3-ADON from Manitoba, were tested using point inoculation. There were no significant differences in aggressiveness among isolates based either on geographic origin or mycotoxin type. Analysis of seeds from inoculated heads by gas chromatography/mass spectrometry indicated that the 3-ADON producing isolates had significantly higher DON levels than the 15-ADON isolates in ‘Roblin’ after both spray and point inoculation and in ‘5602HR’ after point inoculation. DON levels following point inoculation by 15-ADON isolates were similar in the two cultivars. The 15-ADON isolates from Alberta produced less DON than 15-ADON isolates from Manitoba and Saskatchewan. Consistently, more ADON was produced by 15-ADON isolates than by 3-ADON isolates. The results of the study suggest that if the percentage of 3-ADON isolates in Canada increases, DON levels in cereals are likely to increase in epidemic years.

Development of a specific TaqMan real-time PCR assay for quantification of Fusarium graminearum clade 7 and comparison of fungal biomass determined by PCR with deoxynivalenol content in wheat and barley.

A Fusarium graminearum clade 7 specific real-time quantitative PCR (qPCR) assay was developed in this study based on unique polymorphisms in sequences of the mating type protein (MAT) gene. PCR amplification was not observed in eight phylogenetic lineages of the F. graminearum complex and four other closely related Fusarium species. Accuracy of the quantification of the real-time PCR assay was verified with wheat DNA spiked with F. graminearum clade 7 DNA. Wheat samples representing two Canadian wheat classes, CWRS (Canadian Western Red Spring) and CWRW (Canadian Western Red Winter) were used to determine the relationships among F. graminearum DNA, deoxynivalenol (DON) and Fusarium damaged kernel (FDK). The amount of DON and F. graminearum DNA remaining after removal of FDK varied among samples, but was sometimes substantial. Positive correlations were observed between F. graminearum clade 7 DNA (in picograms) and DON as well as FDK. There was also a strong correlation between FDK and DON in CWRS and CWRW wheat composite samples, but the inherent variability in individual producer samples precluded a definitive correlation. For barley, a positive correlation was observed between Fusarium DNA and DON values. Real-time PCR assays can be a valuable tool for barley as there are no reliable symptoms to visually assess the level of Fusarium head blight in this crop.

Prevalence of fungi and fusariotoxins on hard red spring and amber durum wheat seed from western Canada, 2000 to 2002

Producer samples of Canada western red spring (CWRS) wheat (Triticum aestivum) and Canada western amber durum (CWAD) wheat (Triticum turgidum var. durum) grown in Manitoba, Saskatchewan, and Alberta between 2000 and 2002 were composited by year and crop district, then analyzed for fungal infection and contamination by trichothecenes and moniliformin. One hundred and fourteen CWRS and 79 CWAD samples were composited from 1136 CWRS and 643 CWAD producer samples. Alternaria alternata was the most frequently isolated species from each province. Levels of Alternaria alternata, Bipolaris sorokiniana, and Fusarium graminearum were highest in Manitoba and eastern Saskatchewan, whereas Cladosporium spp., Pyrenophora tritici-repentis, and Stagonospora nodorum were highest in samples from further west. The frequency of the toxigenic species F. graminearum greatly exceeded that reported 20 or more years earlier. Samples from all 3 years were analyzed for the presence of eight trichothecenes, and from 2002, for moniliformin. Deoxynivalenol (DON) was found almost exclusively in wheat from the eastern Prairies, at levels up to 7.9 ppm, where it sometimes cooccurred with 15-acetyl deoxynivalenol. HT-2 was detected at low levels in 7 of 79 CWAD composites but not in the CWRS composites. Low levels of moniliformin were found in one CWRS and nine CWAD composite samples, the first time that this compound has been reported from naturally infected wheat in North America. Not detected were nivalenol, fusarenon X, trichothecin, 3-acetyl deoxynivalenol, neosolaniol, diacetoxyscirpenol, and T-2 toxin.

Fusarium Damage in Small Cereal Grains from Western Canada. 2. Occurrence of Fusarium Toxins and Their Source Organisms in Durum Wheat Harvested in 2010

Samples of Canadian western amber durum harvested in 2010 were obtained as part of the Canadian Grain Commission Harvest Sample Program, inspected, and graded according to Canadian guidelines. A subset of Fusarium -damaged samples were analyzed for Fusarium species as well as mycotoxins associated with these species, including deoxynivalenol and other trichothecenes, moniliformin, enniatins, and beauvericin. Overall, Fusarium avenaceum and F. graminearum were the top two most frequently recovered species. Phaeosphaeria nodorum (a.k.a. Septoria nodorum ), F. culmorum , F. poae , F. acuminatum , and F. sporotrichioides were observed in samples as well. All samples analyzed for mycotoxins contained quantifiable concentrations of enniatins, whereas beauvericin, deoxynivalenol, and moniliformin were measured in approximately 75% of the samples. Concentrations in Fusarium -damaged samples ranged from 0.011 to 34.2 mg/kg of enniatins plus beauvericin, up to 4.7 mg/kg of deoxynivalenol, and up to 6.36 mg/kg of moniliformin. Comparisons of enniatins, beauvericin, and moniliformin concentrations to the occurrence of various Fusarium species suggest the existence of an infection threshold above which these emerging mycotoxins are present at higher concentrations. The current grading factor of Fusarium -damaged kernels manages concentrations of these emerging mycotoxins in grain; lower provisional grades were assigned to samples that contained the highest concentrations of enniatins, beauvericin, and moniliformin.

Monitoring of Fusarium Trichothecenes in Canadian Cereal Grain Shipments from 2010 to 2012

A method involving dry grinding, rotary sample dividing, and gas chromatography-mass spectrometry was evaluated for the analysis of eight Fusarium trichothecenes in cereal grains. Processing of whole cereal grains by the method produced representative test portions for the analysis of deoxynivalenol (DON). Method validation data, as well as the successful participation in various international proficiency tests, demonstrated the analytical method produced accurate and precise results. The evaluated method was used to monitor DON, 3- and 15-acetyldeoxynivalenol, nivalenol (NIV), T-2 toxin, HT-2 toxin, diacetoxyscirpenol, and fusarenon-X in shipments of Canadian wheat, durum, barley, corn, rye, and oats transported between August 1, 2010, and July 31, 2012. DON was the most frequently measured trichothecene, found in 231 of the 303 samples at concentrations up to 2.34 mg/kg; NIV was the next most frequently observed trichothecene, but its occurrence was limited to barley. Concentrations of DON were significantly associated with wheat class and grade. The median DON concentration in durum (0.09 mg/kg) was lower than that for hard red spring (0.21 mg/kg). Lower grades of wheat also contained higher median concentrations of DON than higher grades, supporting the current use of Fusarium damaged kernels as a grading factor to manage DON.

Fusarium Damage in Cereal Grains from Western Canada. 1. Phylogenetic Analysis of Moniliformin-Producing Fusarium Species and Their Natural Occurrence in Mycotoxin-Contaminated Wheat, Oats, and Rye

Harvest samples of common wheat (Triticum aestivum), oats (Avena sativa), and rye (Secale cereale) from producers in western Canada were analyzed for fungal infection by toxigenic Fusarium species and contamination by trichothecenes and moniliformin (MON). Fusarium graminearum and F. avenaceum were the two most frequently isolated species from samples of rye and wheat collected in 2010. F. poae and F. sporotrichioides were more commonly detected in randomly selected oat seeds. Other toxigenic Fusarium species including F. acuminatum, F. culmorum, and F. pseudograminearum as well as Phaeosphaeria nodorum (a.k.a. Septoria nodorum) were recovered primarily from fusarium-damaged kernels of wheat. Pure cultures of F. avenaceum, F. acuminatum, and other related species known to produce moniliformin were isolated from incubated seeds based on micro- and macromorphological criteria. The phylogenetic analysis inferred from partial DNA sequences of the acl1 and tef-1α genes revealed two major clades representing F. avenaceum and F. acuminatum, respectively. These clades comprised all Canadian isolates of the two species and a number of reference cultures studied earlier for their propensity to form moniliformin in vitro and in planta. However, some reference cultures previously reported to produce significant amounts of moniliformin formed minor phylogenetic lineages that represent rather distinct but closely related species. Concomitantly, cereal samples were analyzed for the presence of deoxynivalenol and moniliformin. These two Fusarium toxins were observed most frequently in common wheat, at concentrations up to 1.1 and 4.0 mg/kg, respectively. There was no apparent relationship between moniliformin concentrations and detection of F. avenaceum and F. acuminatum in rye and oat samples. Geographical analysis of the distribution of moniliformin and F. avenaceum and F. acuminatum across the Canadian Prairies also did not indicate a strong relationship.

Effects of electron beam irradiation on deoxynivalenol levels in distillers dried grain and solubles and in production intermediates.

Wheat contaminated with deoxynivalenol (DON), and distillers dried grain and solubles (DDGS) obtained after ethanol production from the contaminated wheat, were irradiated to doses ranging from 2.0 to 55.8 kGy using an electron accelerator. Samples of wet distillers grain, distillers solubles and stillage obtained during production of DDGS were also irradiated. All samples were analysed for Fusarium trichothecene mycotoxins by a method involving use of gas chromatography-mass spectrometry (GC-MS). The three production intermediates showed dose-dependent reductions in their DON contents ranging from 47.5 to 75.5% at the highest doses. Electron beam treatment produced a 17.6% reduction in the DON level of wheat at the highest dose used, but had no effect on DON in DDGS. These results indicate that electron beam treatment may provide a method for reducing DON levels in DDGS on an industrial scale.

Trichothecene and zearalenone production, in culture, by isolates of Fusarium pseudograminearum from western Canada

One hundred and twenty-seven isolates of Fusarium pseudograminearum were obtained from seeds and vegetative parts of cereals and other gramineae grown in western Canada. Culture of the isolates on agar selective for Fusarium graminearum allowed a successful differentiation from F. graminearum. Sterilized rice (40% moisture content) was inoculated with a single germinated spore of isolates identified as F. pseudograminearum and was incubated at 23 °C. After 14 days of incubation, mycelium was taken from each culture, and the DNA was extracted to detect the tri5 gene by polymerase chain reaction analysis and confirm the species identity. After 21 days of incubation, mycotoxins were quantified in dried rice. All isolates contained the tri5 gene. Deoxynivalenol (DON) was produced in 125 of the 127 isolates, 3-acetyldeoxynivalenol (3-ADON) in 122 isolates, 15-acetyldeoxynivalenol (15-ADON) in 2 isolates, diacetoxyscirpenol (DAS) in 17 isolates, and zearalenone in 100 isolates; both nivalenol (NIV) and fusarenon X (FX) were detected in 1 isolate. There appears to be three chemotypes: DON–3-ADON, DON–15-ADON, and NIV. Neither of the two 15-ADON producers or the NIV producer formed DAS. This is the first time that the production of DAS and FX is associated with F. pseudograminearum. None of the isolates produced HT-2 toxin or T-2 toxin at detectable levels.