Randall M. Clear

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.

Trichothecene and Moniliformin Production by Fusarium Species from Western Canadian Wheat

Fusarium graminearum, Fusarium culmorum, and Fusarium avenaceum, isolated from Fusarium-damaged wheat harvested in western Canada, were cultured and evaluated for mycotoxin production. Extracts of the culture media were assayed for trichothecenes by gas chromatography-mass spectrometry and for moniliformin by liquid chromatography. Deoxynivalenol (DON) was found in 28 of 42 isolates of F. graminearum and 42 of 42 isolates of F. culmorum at levels ranging from 0.5 to 25.0 microg/g. 15-AcetylDON was found in 28 of 42 isolates of F. graminearum at levels ranging from 1.0 to 7.1 microg/g. 3-AcetylDON was found in 41 of 42 isolates of F. culmorum at levels ranging from 0.8 to 13.0 microg/g. Several other trichothecenes were assayed but not detected in the culture medium. Moniliformin was present in 40 of 42 isolates of F. avenaceum at levels ranging from 1.3 to 138.1 microg/g, but was not present in any of the isolates of F. graminearum or F. culmorum.

Microbiological and aflatoxin evaluation of Brazil nut pods and the effects of unit processing operations.

Harvesting of Brazil nuts not only helps to preserve the Amazon rainforest but also provides income to individuals who would otherwise have little means of making a livelihood. Recently, the European Community has tightened the quality requirements for Brazil nuts, particularly with regard to aflatoxin levels and microbiological contamination. The objectives of this research were to gain a better understanding of the origin of aflatoxins on Brazil nuts and to microbiologically evaluate some of the operations involved in processing. In this regard, five Brazil nut pods were aseptically picked from trees located in each of three concessions of the Peruvian Amazon rainforest (Madre de Dios province). The exteriors of the pods and the nuts were examined for yeast and molds, including Aspergillus flavus and Aspergillus parasiticus, and for bacteria, including Salmonella and Escherichia coli. Brazil nuts obtained from various commercial process operations located in Peru were similarly evaluated. Exteriors of all Brazil nut pods did not contain A. parasiticus, and only pods from one concession yielded A. flavus isolates. All isolates tested were aflatoxigenic (630 to 915 ppb total aflatoxin). Coliforms, E. coli, and salmonellae were not recovered from any of the pods. Whole, in-shell nuts obtained after opening the pods yielded no A. flavus or A. parasiticus. Aflatoxins were not detected (detection limit 1.75 ppb) in any of the nuts. Whole, in-shell and shelled nuts from various process operations were all positive for A. flavus but negative for E. coli and salmonellae. Soaking of whole, in-shell nuts before cracking or shelling increased coliform numbers, whereas levels of A. flavus decreased. In order to gain a better understanding of the sanitary performance of the unit process operations, additional evaluations should be conducted on product lots processed on different days. Also, the microbiology of product processed from common lots should be followed through the various unit operations and compared.

Diversity of Fusarium head blight populations and trichothecene toxin types reveals regional differences in pathogen composition and temporal dynamics

Analyses of genetic diversity, trichothecene genotype composition, and population structure were conducted using 4086 Fusarium graminearum isolates collected from wheat in eight Canadian provinces over a three year period between 2005 and 2007. The results revealed substantial regional differences in Fusarium head blight pathogen composition and temporal population dynamics. The 3ADON trichothecene type consistently predominated in Maritime provinces (91%) over the sampled years, and increased significantly (P<0.05) between 2005 and 2007 in western Canada, accounting for 66% of the isolates in Manitoba by the end of the sampling period. In contrast, 3ADON frequency was lower (22%, P<0.001) in the eastern Canadian provinces of Ontario and Québec and did not change significantly between 2005 and 2007, resulting in two distinct longitudinal clines in 3ADON frequency across Canada. Overall, genetic structure was correlated with toxin type, as the endemic population (NA1) was dominated by 15ADON isolates (86%), whereas a second population (NA2) consisted largely of 3ADON isolates (88%). However, the percentage of isolates with trichothecene genotypes that were not predictive of their genetic population assignment (recombinant genotypes) increased from 10% in 2005 to 17% in 2007, indicating that trichothecene type became an increasingly unreliable marker of population identity over time. In addition, there were substantial regional differences in the composition of recombinant genotypes. In western and maritime provinces, NA2 isolates with 15ADON genotypes were significantly more common than NA1 isolates with 3ADON genotypes (P<0.001), and the reverse was true in the eastern provinces of Québec and Ontario. Temporal trends in recombinant genotype composition also varied regionally, as the percentage of 15ADON isolates with NA2 genetic backgrounds increased approximately three fold in western and Maritime provinces, while the opposite trends were observed in Québec and Ontario. The results indicate that F. graminearum population dynamics in Canada have been influenced by a complex adaptive landscape comprising different regional selective pressures, and do not reflect a simple model of dispersal and integration following the introduction of a novel pathogen population. In addition, we identified F. graminearum strains that produce the recently discovered A-trichothecene mycotoxin (NX-2) for the first time in Canada, representing a significant expansion of the known range of NX-2 producing strains in North America.

The geographic distribution and complex evolutionary history of the NX-2 trichothecene chemotype from Fusarium graminearum

Fusarium graminearum and 21 related species comprising the F. sambucinum species complex lineage 1 (FSAMSC-1) are the most important Fusarium Head Blight pathogens of cereal crops world-wide. FSAMSC-1 species typically produce type B trichothecenes. However, some F. graminearum strains were recently found to produce a novel type A trichothecene (NX-2) resulting from functional variation in the trichothecene biosynthetic enzyme Tri1. We used a PCR-RFLP assay targeting the TRI1 gene to identify the NX-2 allele among a global collection of 2515 F. graminearum. NX-2 isolates were only found in southern Canada and the northern U.S., where they were observed at low frequency (1.8%), but over a broader geographic range and set of cereal hosts than previously recognized. Phylogenetic analyses of TRI1 and adjacent genes produced gene trees that were incongruent with the history of species divergence within FSAMSC-1, indicating trans-species evolution of ancestral polymorphism. In addition, placement of NX-2 strains in the TRI1 gene tree was influenced by the accumulation of nonsynonymous substitutions associated with the evolution of the NX-2 chemotype, and a significant (P<0.001) change in selection pressure was observed along the NX-2 branch (ω=1.16) in comparison to other branches (ω=0.17) in the TRI1 phylogeny. Parameter estimates were consistent with positive selection for specific amino-acid changes during the evolution of NX-2, but direct tests of positive selection were not significant. Phylogenetic analyses of fourfold degenerate sites and intron sequences in TRI1 indicated the NX-2 chemotype had a single evolutionary origin and evolved recently from a type B ancestor. Our results indicate the NX-2 chemotype may be indigenous, and possibly endemic, to southern Canada and the northern U.S. In addition, we demonstrate that the evolution of TRI1 within FSAMSC-1 has been complex, with evidence of trans-species evolution and chemotype-specific shifts in selective constraint.

A simple medium to aid the identification of Fusarium moniliforme, F. proliferatum, and F. subglutinans

Growth of Fusarium moniliforme , F. proliferatum , and F. subglutinans on Czapek solution agar containing 20% saccharose resulted in both cultural differences and enhanced micromorphological features. F. moniliforme could be reliably distinguished from the other two species based on differences in colony color and texture. These differences were intensified by lowering the pH of the media from 7.7 to 4.4 without adversely affecting micromorphology.

Genetic evidence for a recent geographic expansion of 15-acetyldeoxynivalenol chemotypes of Fusarium graminearum in Canada.

The genetic structure of 15-acetyldeoxynivalenol (15-ADON) chemotypes of Fusarium graminearum populations collected from Canada was examined using restriction length fragment polymorphism and inter simple sequence repeat molecular markers. The genetic analysis revealed that 15-ADON producing F. graminearum populations from different provinces of Canada are genetically homogeneous. Mismatch distribution analysis was used to examine the evolutionary factors leading to genetic homogeneity among populations. The results of mismatch distributions showed evidence of a recent burst in population growth and demographic expansion across Canada. These results suggested that vigilant monitoring of trends of population growth of F. graminearum in Canada may be useful for management of this fungal pathogen. Key words: Gibberella zeae, mycotoxin, genetic variation, population expansion, geographic distribution, fungi. La structure génétique de chimiotypes 15-ADON de populations de Fusarium graminearum collectés au Canada a été étudiée à l’aide de marqueurs moléculaires RFLP (restriction fragment length polymorphism) et ISSR (inter simple sequence repeat). L’analyse génétique a révélé que les populations de F. graminearum productrices de 15-ADON, provenant de différentes provinces canadiennes, sont génétiquement homogènes. L’analyse de la distribution des différences génétiques a permis d’examiner les facteurs évolutifs menant à l’homogénéité génétique chez les populations. Les résultats de cette analyse ont confirmé les observations relatives à une récente explosion des populations et à l’expansion démographique, et ce, d’un bout à l’autre du Canada. Ces résultats suggèrent qu’un suivi attentif de cette tendance de la croissance des populations de F. graminearum pourrait s’avérer utile en ce qui a trait à la gestion de cet agent pathogène mycosique. Mots-clés : Gibberella zeae, mycotoxine, variation génétique, expansion des populations, distribution géographique, champignons.