Fusarium graminearum is responsible for production of the mycotoxin deoxynivalenol (DON) on maize and wheat in Ontario, Canada. It has been understood since the early 1980s that in most parts of Canada, the predominant chemotype of F . graminearum is 15ADON, and not the 3ADON chemotype mainly found in Europe and Asia. The discovery of F . graminearum strains that did not produce DON but the structurally related 7-α hydroxy, 15-deacetylcalonectrin (3ANX) and its hydrolysis product 7-α hydroxy, 3,15-dideacetylcalonectrin to (NX) demonstrated that we still have a lot to learn about this well studied but complicated fungus. We conducted a survey of maize and wheat samples from Ontario farms. In the 2015 crop year, we isolated 86 strains and tested a representative subset of 20 using the published genetic probes for assessing genotype. We also developed a targeted LC-MS/MS method for the identification and quantitation of known toxins from this species to determine chemotype. The results showed that 80% of our strains produced some 3ANX in addition to 15ADON and one strain produced 3ANX and no 15ADON. Comparison of chemical data with genotyping revealed that in more than 50% of the cases there was no clear agreement. These data demonstrate the importance of chemical analysis for understanding the toxigenic potential of strains, especially using a LC-MS method that is capable of differentiating 3ADON and 15ADON. For this collection, genotyping of isolates did not produce reliable information on the chemotype. This is the first report of 3ANX toxin production concurrently with 15ADON and suggests that the 3ANX producers in North America likely originated from the 15ADON background.

Ontario has suffered widespread epidemics of Fusarium Head Blight or Gibberella Ear Rot roughly every five years since the late 1970s. We undertook a study of the chemotype and genotype of Fusarium graminearum isolated from 1,800 samples of wheat and maize collected across the cereal growing areas over three years. 468 isolates obtained were genotyped and 60 were selected for chemotyping. The dominant genotype has remained the native 15-acetyldeoxynivalenol (15-ADON) population. Approximately 20% of the strains tested were of the native chemotype producing only 15-ADON and one strain producing solely 7α-hydroxy,15-deacetylcalonectrin (3ANX) was observed. The majority of the 15-ADON strains were also capable of producing 3ANX. There was consistent mismatch between chemotype and genotype. This reflects the considerable plasticity in the genes associated with trichothecene biosynthesis documented in several Fusarium species. Although there is a large gradient in climate from southern to eastern Ontario, we did not detect differences in the distribution of the chemotypes. Grain from which strains were isolated for chemotyping were analysed. Approximately half of the 53 samples had >2 mg/kg deoxynivalenol with a maximum of 400 mg/kg and median of 14 mg/kg. 7α-hydroxy,3,15-dideacetylcalonectrin (NX toxin) was detected in three of these samples at an average of 4.5 mg/kg. The stability of the F. graminearum genotype in Ontario can be explained by several factors. Since 1980, the area planted to maize has remained stable, however, the area given to wheat has about doubled. Minimum tillage was rare in 1980 but it is now the norm. Increased crop residue on the soil has greatly increased the biomass of ascocarps that overwinter. Overall, these data demonstrate the need to monitor the mycotoxins in Fusarium populations and for the need to consider the potential toxicity of NX in the feed supply.

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Supported lipid bilayer systems were evaluated following various experimental procedures in an effort to determine their appropriateness for visualization using total internal reflection fluorescence (TIRF) microscopy. The incorporation and distribution of Texas Red® 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE) was studied when incorporated into bilayers of variable lipid composition using different forms of mechanical shearing. Results showed that 0.8 mol% TR-DHPE provides the most optimum TIRF images. At this concentration, a sufficient level of photostability can be achieved without an undesirable increase in TR-DHPE aggregates caused by excess probe molecules. Solutions composed of a 3:1 molar ratio of DOPC:DPPC with 0.8 mol% TR-DHPE produce bilayers that consistently display clear, distinct, rounded domains, whereas other lipid compositions did not. This optimum phase separation appears to be influenced by an increase in mechanical shearing during the vesicle formation process, when the lipid solutions were exposed to sonication and extrusion processes. The combination of a sonication and extrusion process also helped with eliminating the presence of TR-DHPE aggregates within the model membranes. It was also shown that bilayers formed on conditioned glass, placed on a slide, produced more highly detailed bilayers in which distinct lipid phase separation could be optimally visualized using TIRF microscopy.