An adaptive evolutionary shift in Fusarium head blight pathogen populations is driving the rapid spread of more toxigenic Fusarium graminearum in North America.

Analysis of Fusarium head blight (FHB) pathogen diversity revealed that 3ADON producing Fusarium graminearum are prevalent in North America and identified significant population structure associated with trichothecene chemotype differences (F(ST)>0.285; P<0.001). In addition, we identified a trichothecene chemotype cline in Canada and documented a recent and significant shift in FHB pathogen composition by demonstrating that the 3ADON chemotype frequency in western Canada increased more than 14-fold between 1998 and 2004. On average, isolates from 3ADON populations produced significantly (P<0.05) more trichothecene and had significantly (P<0.005) higher fecundity and growth rates than isolates from the 15ADON population. These results indicate that selection is driving the rapid spread of an introduced pathogen population that is more toxigenic and potentially more vigorous. The discovery of this previously unrecognized pathogen diversity has significant implications for food safety and cereal production in North America.

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

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.

Species-specific PCR-based assays for the detection of Fusarium species and a comparison with the whole seed agar plate method and trichothecene analysis.

Species-specific PCR was used for the identification of nine Fusarium species in pure mycelial culture. A PCR-based method was compared with the whole seed agar plate method and trichothecene analysis for three toxin-producing Fusarium species using 85 grain samples of wheat, barley, oat, corn and rye. A simple SDS-based DNA extraction system followed by potassium acetate precipitation resulted in consistent PCR amplification of DNA fragments from cultures and grain samples. The species-specific PCR assays correctly identified pure cultures of Fusarium avenaceum ssp. avenaceum (9 isolates), Fusarium acuminatum ssp. acuminatum (12 isolates), Fusarium crookwellense (7 isolates), Fusarium culmorum (12 isolates), Fusarium equiseti (11 isolates), Fusarium graminearum (77 isolates), Fusarium poae (10 isolates), Fusarium pseudograminearum (23 isolates), and Fusarium sporotrichioides (10 isolates). Multiplex PCR was developed for the simultaneous detection of F. culmorum, F. graminearum and F. sporotrichioides, the three most important trichothecene producing species in Canada. In grain samples, results of PCR assays for these same three species related well with whole seed agar plate method results and determination of Fusarium trichothecenes. The PCR assay described in this study can be used for routine detection and identification of Fusarium spp. in Canada.

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.

Single laboratory evaluation of a planar waveguide-based system for a simple simultaneous analysis of four mycotoxins 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.

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.

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.