Historical genomics reveals the evolutionary mechanisms behind multiple outbreaks of the host-specific coffee wilt pathogen Fusarium xylarioides

Abstract

Background: Nearly 50% of crop yields are lost to pests and disease, with plants and pathogens locked in an amplified co-evolutionary process of disease outbreaks. Coffee wilt disease, caused by Fusarium xylarioides, decimated coffee production in west and central Africa following its initial outbreak in the 1920s. After successful management, it later re-emerged and by the 2000s comprised two separate epidemics on arabica coffee in Ethiopia and robusta coffee in east and central Africa.

Results: Here, we use genome sequencing of six historical culture collection strains spanning 52 years to identify the evolutionary processes behind these repeated outbreaks. Phylogenomic reconstruction using 13,782 single copy orthologs shows that the robusta population arose from the initial outbreak, whilst the arabica population is a divergent sister clade to the other strains. A screen for putative effector genes involved in pathogenesis shows that the populations have diverged in gene content and sequence mainly by vertical processes within lineages. However, 15 putative effector genes show evidence of horizontal acquisition, with close homology to genes from F. oxysporum. Most occupy small regions of homology within wider scaffolds, whereas a cluster of four genes occupy a 20Kb scaffold with strong homology to a region on a mobile pathogenicity chromosome in F. oxysporum that houses known effector genes. Lacking a match to the whole mobile chromosome, we nonetheless found close associations with DNA transposons, especially the miniature impala type previously proposed to facilitate horizontal transfer of pathogenicity genes in F. oxysporum. These findings support a working hypothesis that the arabica and robusta populations partly acquired distinct effector genes via transposition-mediated horizontal transfer from F. oxysporum, which shares coffee as a host and lives on other plants intercropped with coffee.

Conclusion: Our results show how historical genomics can help reveal mechanisms that allow fungal pathogens to keep pace with our efforts to resist them. Our list of putative effector genes identifies possible future targets for fungal control. In turn, knowledge of horizontal transfer mechanisms and putative donor taxa might help to design future intercropping strategies that minimize the risk of transfer of effector genes between closely-related Fusarium taxa.

Keywords: Comparative genomics; Effector; Fungi; Fusarium oxysporum; Host adaptation; Proteome.

Fig. 1

The emergence and spread of F. xylarioides. A map of Africa detailing the year collected, country of origin and coffee plant host for the 62 F. xylarioides strains in the CABI-IMI culture collection. These strains illustrate the spread west of CWD from the pre-1970s strains to the post-1970s strains, and the emergence of the host-specific arabica and robusta populations. The six strains sequenced in this study are labelled on the map as: Coffea674, from Cote D’Ivoire; Coffea659 from the Central African Republic; robusta254, from Uganda; robusta277, from Tanzania; arabica563 and arabica908, from Ethiopia. Map created in Rstudio 1.2.1335 using the Standard Features package [18] and drawn in ggplot2 [19]

Fig. 2

Representative whole-genome alignments of F. xylarioides and F. oxysporum f. sp. lycopersici. Representative whole-genome alignments of F. xylarioides strain robusta277 against the 15 F. oxysporum f. sp. lycopersici chromosomes, including the 4 mobile chromosomes, annotated in red, and the 11 core chromosomes shared with sister Fusarium species [9]. Each dot represents chromosomal correspondence, with absences representing the absent Fol chromosomes. Alignments for each F. xylarioides genome are in Additional file 2: Figure S2. Genomes were aligned using Mummer 4.0.0 (http://mummer.sourceforge.net/) with outputs processed using Dotprep.py before visualizing using Dot in DNA Nexus (https://dnanexus.github.io/dot/). Blue indicates forward alignments, green indicates reverse alignments, orange indicates repetitive alignments

Fig. 3

Global view of genome metrics plotted across 20 Kb windows of each F. xylarioides genome. Global view of genome metrics plotted across 20 Kb windows of each F. xylarioides genome. Light blue = fraction of nucleotides annotated as a gene. Orange = fraction of nucleotides annotated as transposable element. Dark green = %GC. Vertical solid lines demark the 11 inferred chromosomes matching to the F. verticillioides genome. Dashed lines demark: FV, scaffolds that are shared with F. verticillioides but not assembled into chromosomes; FXU, scaffolds that are absent from F. verticillioides but are shared with Coffea659 and F. udum; LS, scaffolds that are not shared with F. verticillioides, Coffea659 nor F. udum and are therefore lineage specific. Coloured points indicate the location of putative effector genes: square = pre-defined in the literature, circle = CAZyme, diamond = small cysteine-rich proteins. Colours allocated across spectrum arbitrarily but same colour indicates belongs to same ortholog. The lower strip on each plot shows Large RIP Affected Regions in black

Fig. 4

Phylogenetic relationships between Fusarium species. Phylogenetic relationships between Fusarium species reconstructed from 13 782 orthogroups support monophyly of the F. xylarioides clade, with little consistent support for alternate topologies. Strain accession numbers are in Additional file 2: Table S3. The brackets denote the FFC, with the Asian (blue) and African (red) clades. Pie chart colours: pink = proportion of genes (orthogroups) recovering the depicted node; dark green = the proportion of genes recovering the second most common topology; light blue = the proportion of genes recovering all other topologies

Fig. 5

Gene sharing across the F. xylarioides strains. a) Orthogroups shared between F. xylarioides arabica, robusta and the two Coffea strains. Drawn using 13 782 orthogroups (excluding 449 F. xylarioides robusta orthogroups and 175 F. xylarioides arabica orthogroups that differed in presence/absence between the two strains of each population. The two Coffea strains are more divergent (with 1123 orthogroups that differ in presence/absence between the two strains) and so are drawn separately. The two arabica strains (563 and 908) share 13 062 genes and the two robusta strains (254 and 277) share 13 104 genes in total. b) Putative effectors shared between F. xylarioides arabica, robusta and the two Coffea strains. Drawn using 64 putative effector proteins that differed in presence/ absence across the host-specific populations

Fig. 6

Putative effectors’ characteristics and presence or absence across F. xylarioides strains and F. udum. The four effector classes are shown in: yellow for predefined effectors; purple for small and cysteine-rich effectors; blue for carbohydrate-active enzymes; and red for transposon-adjacent effectors. The presence of transposons is represented by names in bold with its distance from the genes promoter described if less than 1500bp (if not, the transposon is over 1500bp away), genes under positive selection by an asterisk, secreted proteins with a signal peptide by a caret, genes in an AT-rich block by a tilde, genes with evidence of horizontal transfer from F. oxysporum are a darker shade and function is represented by the Pfam domain, where a hit was returned. Genes which are absent from the FFC with F. oxysporum the closest match with a percent identity (%) >=90 are represented by a quotation mark

Fig. 7

Horizontal gene transfer between F. xylarioides and F. oxysporum. Decision tree showing the numbers of putative effector genes in F. xylarioides (Fxyl) displaying different patterns in their phylogenetic relationships, focusing on possible horizontal acquisition from F. oxysporum (Foxy). FFC = F. fujikuroi complex that F. xylarioides belongs to. Interpretations consistent with each pattern are shown on the right

Fig. 8

Four putative effector genes on the robusta254 scaffold and their phylogenetic trees. a Phylogenetic tree for og14741, b Phylogenetic tree for og9441, c Phylogenetic tree for og14743, d Phylogenetic tree for og13478. For each gene, the FFC is absent, F. xylarioides is nested within F. oxysporum and F. oxysporum f. sp. raphani is the closest match (also shown in Additional file 2: Figure S7). All branch support values = 100%, all trees drawn in Geneious 9.1. e The four effector genes are surrounded by mimps and DNA transposons on a robusta254 scaffold which shares a high (>96%) nucleotide sequence identity with the mobile and pathogenic Fol chromosome 14. Similar scaffolds with the same four putative effector genes are present in robusta277, Coffea659 and Coffea674. Alignments were made with nucmer (MUMmer3). The blue annotations indicate the effector genes, the yellow annotations indicate mimps and the purple annotations indicate DNA transposons

Fig. 9

mimp families found in F. xylarioides. Phylogeny of three mimp families in F. oxysporum, F. xylarioides and F. culmorum (tree rooted with F. culmorum in a). a, mimp family 1. b, mimp family 4. c, mimp family mn4. No other species matches were returned compared to the species in Additional file 2: Table S3 and the nr database (BLAST, 1e-50). Nodes which are coloured in black share a branch support value >90%, F. xylarioides mimps are annotated in red. Phylogeny was inferred using Chi2 support values. Drawn in Geneious 9.1