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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.

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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.