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.
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.
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.
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 fraction could be used as a readily available matrix for the rapid screening of DON in wheat. This would eliminate the time required for additional sampling and preparation of whole grain, and move towards a truly rapid method for the screening of DON in wheat.
The accuracy and precision of a commercially available system based on an indirect competitive immunoassay and planar waveguide technology was evaluated for the analysis of deoxynivalenol (DON), ochratoxin A (OTA), zearalenone (ZEAR), and T-2 toxin in wheat. The system generally performed well at the tested concentrations that were close to the regulatory limits of DON and OTA in wheat. The mean percent recovery of OTA from certified and in-house reference materials ranged from 90 to 111 %, with a relative standard deviation of 8-16 % (at 4.2, 4.9, and 7.0 μg/kg). Mean percent recoveries of DON ranged from 75 to 103 %, with a relative standard deviation of 14-20 % (at 610, 940, and 1300 μg/kg). As analyte concentrations approached the lower limits of the working range of 3 μg/kg OTA and 400 μg/kg DON, the mean percent recoveries and relative standard deviation increased for both DON and OTA. A lack of reference materials precluded a thorough evaluation of the method for the analysis of ZEAR and T-2. The particular strength of the technology was that multiple mycotoxins were analyzed simultaneously.
Fifteen lots of wheat were sampled to characterise the total variance and distribution among sample test results associated with measuring deoxynivalenol (DON) in bulk wheat lots. An unbalanced nested experimental design based on past research was used to determine contributions to the total variance from sampling, sample preparation, and analysis. The wheat lots used in the study contained average DON concentrations that ranged from 0.17 to 24.5 mg/kg. Sampling was determined to be the largest contributor to the total variance of measuring DON at low mg/kg concentrations, which are relevant to existing maximum levels. With the experimental design parameters of 1 kg laboratory samples, sub-division of whole and ground grain using rotary sample division, sample comminution using a commercial-grade coffee grinder, extraction of 100 g test portions, and making one measurement of DON in the test portion by gas chromatography-mass spectrometry, the total variance of DON measurement at 2 mg/kg was 0.046 mg2/kg2 (coefficient of variation=10.7%). At this concentration, sampling contributed 67% to the total variance, followed by sample preparation (18%) and analysis (15%). The DON distribution among sample test results was accurately described by the normal distribution. The mathematical model of variance was used with the normal distribution of DON measurement results to construct operating characteristics curves to model the likelihood of mischaracterising a wheat lot as (non) compliant with a certain decision limit. With realistic laboratory sample and test portion sizes, as well as a practicable decision limit of 1.5 mg/kg, the estimated probability of mischaracterising a wheat lot containing 2 mg/kg DON as less than this concentration was reduced to 1%.
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