Table 1.
Summary of the F. pseudograminearum genome sequence assembly in comparison to selected fungal genome sequences obtained by short-read sequencing and the reference genome for F. graminearum.
Figure 1.
Alignment between F. graminearum and F. pseudograminearum.
F. graminearum chromosomes are ordered in decreasing size. (A) Dot-plot representing a whole genome alignment between F. graminearum isolate Ph1 and F. pseudograminearum isolate CS3096. The alignment was generated with NUCmer, part of the MUMmer 3 comparative sequence analysis package. Sequences were pre-masked for known repetitive elements and simple repeats using RepeatMasker. The dot plot represents the best 1∶1 alignment between the two genomes. Dots closest to the diagonal represent co-linearity between the two genomes. Red represents matches in the forward direction and blue is indicative of inversions in part of the mapped contig relative to the F. graminearum genome. (B) Distribution of gaps in the alignment between F. pseudograminearum and F. graminearum, relative to the F. graminearum genome. F. graminearum chromosomes were divided into 100 Kbp non-overlapping windows and the unaligned nucleotides in each window summed and expressed as a percentage.
Table 2.
Fusarium pseudograminearum genes that may be of bacterial origin and are candidates for roles in pathogenicity towards plants.
Table 3.
Examples of Fusarium pseudograminearum genes that may be of fungal origin and are candidates for roles in cereal pathogenicity.
Figure 2.
Phylogram of fungal amidohydrolases found in F. pseudograminearum that appear to be of bacterial origin and a number of bacterial amidohydrolases.
Fungal sequences are highlighted in grey boxes. Numbers on branches indicate approximate likelihood ratio test branch support values.
Table 4.
Summary of BLASTp analysis of FpAH1 versus the non-redundant protein database (nr) at NCBI.
Figure 3.
Nucleotide alignment of the end of F. graminearum chromosome 1 (top) and the F. pseudograminearum contig containing FpAH1 (bottom).
Clear orthologous relationships were evident between regions joined by grey shaded boxes. FpAH1 is indicated in red. The genes surrounding FpAH1 in the F. pseudograminearum genome that do not have clear orthologous relationships in F. graminearum are blown up and the putative function and species of the closest match in GenBank is indicated.
Figure 4.
Statistical parsimony haplotype network for FpAH1/PnAH1 in Fusarium pseudograminearum and Phaeosphaeria spp. Haplotype bubbles are proportional to sample size shown in parentheses.
Dots on line segments indicate single point mutations.
Figure 5.
Virulence assay of the Fusarium pseudograminearum amidohydrolase 1 mutants (ΔFpAH1) compared to the parental strain towards barley cultivar Golden Promise at 21 days post inoculation (dpi) (A–B).
(A) Survival of plants in the assay 21 dpi. N = 3 with each replicate consisting of 13–15 plants divided between two paper towel rolls and maintained in separate vessels. The difference between wild type and each mutant was highly statistically significant (P-value <0.01). (B) Representative rolls of plants from the assay. (C–D). Complementation of ΔFpAH1 (strains 96T681 and 96T682) restores virulence towards barley cultivar Golden Promise. (C) Proportion of plants surviving at 13 dpi. The total number of plants in each case was 30. (D) Photograph of assays at 13 dpi.
Figure 6.
Fusarium root-rot virulence assay of the Fusarium pseudograminearum amidohydrolase 1 mutant (ΔFpAH1) towards wheat cultivar Kennedy (A and B) and barley cultivar Golden Promise (C and D).
N = 16 individual plants. CS3096 is the parental strain for the FpAH1 mutant (96T492), which was complemented with FpAH1 driven by the TrpC promoter (96T681). Error bars represent the standard error of the mean. Different letters on A and C indicate statistically significant differences (P<0.05) in pair-wise t-tests. B and D are the plants used to score the shoot length shown in A and C.
Figure 7.
The FpDLH1-FpAMD1 locus in Fusarium pseudograminearum and the arrangement of the genes in this region in F. graminearum, F. verticillioides and Colletotrichum graminicola.
The DLH1-AMD1 locus is boxed in grey in each of the species. Genes of the same color in different species are reciprocal best blast hits and likely to be orthologous. Sequence contigs for each species are truncated to the relevant regions only.
Figure 8.
Virulence assay of the Fusarium pseudograminearum dienelactone hydrolase 1 mutant (ΔFpDLH1) mutant towards wheat cultivar Kennedy in crown rot (A and B) and root rot (C and D) assays.
For A and B N = 3 with each biological replicate consisting of 4 paper towel rolls consisting of 7–8 plants per roll. For C and D N = 15–16 plants. Error bars represent the standard error of the mean. Letters indicate statistically significant differences at P<0.05. CS3096 and CS3427 are the parental strains for the FpDLH1 mutants, 96T926 and 27T892 respectively. Mock treatments are inoculations performed with agar plugs that have not been colonized by Fusarium.