Mike Roscoe

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.

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.

Considerations in the preparation of laboratory samples for the analysis of ochratoxin A in wheat

A process used to prepare the test portion of ground wheat from the whole grain laboratory sample for ochratoxin A (OTA) analysis using dry comminution with homogenisation and sub-sampling via a rotary sample divider was developed and evaluated. With respect to OTA content, the developed process produced a homogeneous sample of ground wheat from 10 kg of whole grain. Relative standard deviations of the mean OTA concentration for five naturally contaminated wheat samples processed using the developed method ranged from 9% to 19% over a relevant concentration range of 1.7 to 7.6 mg/kg. Additional studies demonstrated that OTA was stable in ground wheat with moisture content between 12 to 13% for at least a year when stored at ambient temperatures. Further examination of the developed comminution and dividing procedure demonstrated that higher concentrations were measured in smaller sized particles, indicating that the accuracy and precision of OTA analyses could be affected by the particle size of ground wheat.

Fate of moniliformin during milling of Canadian durum wheat, processing, and cooking of spaghetti

Tittlemier, S. A., Roscoe, M., Trelka, R., Patrick, S. K., Bamforth, J. M., Gräfenhan, T., Schlichting, L. and Fu, B. X. 2014. Fate of moniliformin during milling of Canadian durum wheat, processing, and cooking of spaghetti. Can. J. Plant Sci. 94: 555-563. Samples of clean Canadian durum were fortified with kernels damaged by Fusarium avenaceum at levels corresponding to the grading tolerances for Fusarium damaged kernels in Canadian durum wheat. Fusarium avenaceum produces the mycotoxin moniliformin (MON); the fortified durum samples contained MON ranging from 0.16 to 0.90 mg kg-1. The fate of MON was followed during milling of the fortified durum samples, as well as during the preparation and cooking of spaghetti made with the fortified durum. The majority of MON was associated with semolina, implying that removal of bran would not have a large impact on reducing MON concentrations. However, F. avenaceum DNA was more evenly distributed amongst the milling products, suggesting that MON underwent translocation from mycelium to endosperm in the damaged kernels. There was an apparent loss of MON when spaghetti was prepared from semolina. From an overall dietary exposure point of view, the cooking of MON-containing pasta in water will reduce dietary exposure to the water-soluble MON via leaching to the cooking water. However, the lack of quantifiable MON in samples prepared from higher grade Canada Western Amber Durum (which contained lower amounts of Fusarium damaged kernels) suggests that dietary exposure to MON from contaminated pasta is not likely to occur.

By-products of grain cleaning: an opportunity for rapid sampling and screening of wheat for mycotoxins

By-products of cereal grain cleaning were analysed for a number of mycotoxins. Deoxynivalenol (DON) was the most frequently detected in by-products from commercial-scale cleaning procedures (maximum 2.94 mg/kg), followed by zearalenone (ZEA; maximum 0.045 mg/kg) and ochratoxin A (OTA; maximum 0.019 mg/kg). These three mycotoxins were also the most frequently detected in four different fractions collected from wheat run through a dockage tester, a piece of equipment used in the Canadian inspection process to separate material other than grain from wheat. Concentrations of mycotoxins were highest in the ‘light dockage’ fraction that contained dust and roughage such as glumes, fragments of stem, or rachis. Mycotoxin concentrations in this fraction reached up to 32 mg/kg (DON), 0.532 mg/kg (ZEA), and 0.249 mg/kg (OTA). Concentrations of DON in light dockage were significantly correlated with concentrations in whole grain that was un-cleaned or had undergone basic cleaning, indicating that the light dockage fr...

Occurrence of ochratoxin A in Canadian wheat shipments, 2010–12

Randomly selected domestic and export shipments (n = 1907) of Canadian durum and other wheat that occurred between 1 January 2010 and 31 December 2012 were analysed for ochratoxin A (OTA). The majority of samples did not contain OTA above the LOQ of 1 μg kg–1. Only 37% of samples analysed contained quantifiable OTA; the median OTA of the positive results was 2.10 μg kg–1. Canada Western Amber Durum shipments contained OTA more frequently, and at slightly higher concentrations, than Canada Western Red Spring wheat. For both wheat classes the frequency of OTA occurrence and mean concentrations appeared to increase in the lower grades, but these increases were not statistically significant. A periodic trend of a late summer increase of mean monthly OTA concentrations in shipments appears tied to the cycle of producer deliveries of wheat to primary grain elevators.

Evaluation of a Commercially Available Enzyme-Linked Immunosorbent Assay and a Liquid Chromatography–Tandem Mass Spectrometric Method for the Analysis of Glyphosate in Wheat, Oats, Barley, Malt, and Lentils

An accurate and precise ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method was validated for the analysis of glyphosate and its main transformation product (aminomethylphosphonic acid) in barley, malt, wheat, oats, and lentils. The validation data demonstrated good performance of the method. This UHPLC-MS/MS method was also used to evaluate the performance of a commercially available enzyme-linked immunosorbent assay (ELISA) test kit. For all of the grain matrices examined, the ELISA showed poor accuracy and precision at its stated lower working limit of 0.075 mg/kg; however, performance was acceptable at 0.30 mg/kg, as well as higher concentrations relevant to established maximum residue limits. At these relevant concentrations, the ELISA also produced results higher than the UHPLC-MS/MS method. Although results from the two methods were linearly correlated, differences in the result values from the two methods differed among the grains studied and ranged from...

The effects of selected factors on measured ergot alkaloid content in Claviceps purpurea-infected hexaploid and durum wheat

Four wheat genotypes, including the ergot-susceptible durum ‘AC Avonlea’ and hard red spring wheat ‘AC Cadillac’, as well as the resistant durum wheat line 9260B-173A and the hard red spring wheat line ‘Kenya Farmer’ wereinoculated with different Claviceps purpurea isolates. Honeydew and sclerotia were collected and analysed for 10 ergot alkaloids. Total concentrations of the 10 ergot alkaloids ranged from 16 µg/kg in honeydew to 1,798 mg/kg insclerotia. Ergonovine and ergosine were the predominant alkaloids in honeydew obtained from plants inoculated with various isolates, whereas ergocristine and ergocryptine were the main alkaloids observed in sclerotia. Bothhost plant and C. purpurea isolate were significant factors affecting total ergot alkaloid concentrations in sclerotia. Irrespective of host plant line, all mean total ergot alkaloid concentrations were higher in sclerotia produced from the EI-2 isolate (695-1,010 mg/kg), as compared to EI-4 (255-594 mg/kg). The mass of total ergot alkaloids was al...

Analysis of quinclorac and quinclorac methyl ester in canola from the 2015 harvest using QuEChERS with liquid chromatography polarity-switching tandem mass spectrometry

ABSTRACT A method using QuEChERS sample preparation with liquid chromatography polarity-switching tandem mass spectrometry was developed and validated for the analysis of quinclorac and its degradation product quinclorac methyl ester in canola seed. The method was used to analyse canola treated with quinclorac, harvest sample composites and samples of canola shipments. Quinclorac residues were present in all samples of canola treated with a quinclorac-containing herbicide that were analysed. Quinclorac was found in 93% of samples, with an average of 0.018 mg kg–1. All samples contained quinclorac methyl ester, with an average of 0.061 mg kg–1. The average concentration of total residues (as quinclorac equivalents) on treated canola was 0.075 mg kg–1, with a range of 0.016–0.124 mg kg–1. The observed residues were all at least 10 times lower than the Canadian maximum residue limit of 1.5 mg kg–1. Quinclorac and quinclorac methyl ester were not found in any harvest and export composite samples, which represented the majority of canola grown in western Canada in 2015 and canola exported in late 2015. Even though usage of quinclorac-containing herbicide on canola can result in the presence of low concentrations of residues, the absence of quinclorac residues in harvest and shipment samples suggests that use of quinclorac-containing herbicide was not widespread, and that any residues present were diluted as canola was combined along the grain-handling chain into shipment lots, or segregated and prevented from entering shipment lots. GRAPHICAL ABSTRACT