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

Abstract

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.

Fusarium graminearum: pathogen or endophyte of North American grasses?

“…In environmentally collected plants from which FGSC cultures were isolated, 30% of the investigated grass species contained low levels of mycotoxins characteristic of the FGSC (DON, 15ADON, 3ADON, fusarenone‐X and zearalenone). The trichothecene chemotype frequencies of strains were similar to those found in strains isolated from wheat in the upper Midwestern USA (Kelly et al ., , ; Liang et al ., ), except for a slightly higher occurrence of NX‐2 strains and the presence of NIV isolates. We also found that strains in the FGSC derived from non‐symptomatic native grasses are fully capable of causing FHB disease on wheat.…”

TRI Genotyping and Chemotyping: A Balance of Power

“…A genotype is not a chemotype or vice versa and should not be used interchangeably. As such, on the basis of the proportion of potentially 'toxic' genotypes, it should be possible to predict whether there is a risk of toxin production and subsequent exposure [40]. A number of studies have utilized genotyping as a proxy to determining chemotype [41][42][43][44][45][46][47][48][49].…”

“…TRI1 specific PCR assays are based on amplification of TRI1 gene sequence using a reverse primer (TRI1-R; 5'-TTCCTGCAGGGGCTTGATG-3') and one of two forward primers for the detection of 3-ADON (5'-AATGCTCGCGAACTAATCAC-3'), and for the detection of 3-ANX (5'AATGCTAGCGAAATGATCAA-3') genotypes. Polymorphisms in the amplified sequence allows cleavage by ApoI restriction enzyme into a specific banding pattern (PCR fingerprinting) that is characteristic of NX-2-producing F. graminearum strains, and hence, the NX-2 genotype can be distinguished from Type B genotypes (i.e., 15-ADON, 3-ADON and NIV) of F. graminearum [40]. Polymorphisms in the TRI13 gene sequence is used to distinguish between the DON and the NIV genotypes based on differential size of the amplicon produced:~227 bp is produced for a DON genotype when primers Tri13F and Tri13DONR are used, whereas,~312 bp is produced for a NIV genotype by primers Tri13NIVF and Tri13R [99-101].…”

Comparative Sequence Analysis of TRI1 of Fusarium

“…Fu and Li's D* statistic was also positive and significant (2.05433; p < 0.02), and thus, it is inferred that TRI1 gene sequences are under positive selection and under selective functional constraints. However, Kelly et al [49] reported that tests of positive selection were not significant and the TRI1 gene sequences of the FGNX-2 were identical except for one nucleotide. In a subsequent F. graminearum genome study by Kelly and Ward [50], it was reported that among several genomic regions, TRI genes exhibit the strongest signals of selection.…”