The Fusarium graminearum Transporters Abc1 and Abc6 Are Important for Xenobiotic Resistance, Trichothecene Accumulation, and Virulence to Wheat.

The plant pathogenic fungus Fusarium graminearum is the causal agent of Fusarium head blight (FHB) disease on small-grain cereals. F. graminearum produces trichothecene mycotoxins such as deoxynivalenol (DON) that are required for full virulence. DON must be exported outside the cell to cause FHB disease, a process that may require the involvement of membrane-bound transporters. In this study, we show that the deletion of membrane-bound transporters results in reduced DON accumulation as well as reduced FHB symptoms on wheat. Deletion of the ATP-binding cassette (ABC) transporter gene Abc1 results in the greatest reduction in DON accumulation and virulence. Deletion of another ABC transporter gene, Abc6, also reduces FHB symptoms to a lesser degree. Combining deletions fails to reduce DON accumulation or virulence in an additive fashion, even when a ∆abc1 deletion is included. Heterologous expression of F. graminearum transporters in a DON-sensitive strain of yeast confirms Abc1 as a major DON resistance mechanism; furthermore, it suggests that Abc1 is directly participating in DON transport rather than facilitating DON transport though other means. Yeast expression further indicates that multiple transporters, including Abc1, play an important role in resistance to the wheat phytoalexin 2-benzoxazolinone (BOA) and other xenobiotics. Thus, Abc1 may contribute to virulence on wheat both by facilitating export of DON and by providing resistance to the wheat phytoalexin BOA. This research provides useful information that may aid in designing novel management techniques of FHB or other destructive plant diseases.

Fusarium graminearum Species Complex: A Bibliographic Analysis and Web-Accessible Database for Global Mapping of Species and Trichothecene Toxin Chemotypes.

Fusarium graminearum is ranked among the five most destructive fungal pathogens that affect agroecosystems. It causes floral diseases in small grain cereals including wheat, barley, and oats, as well as maize and rice. We conducted a systematic review of peer-reviewed studies reporting species within the F. graminearum species complex (FGSC) and created two main data tables. The first contained summarized data from the articles including bibliographic, geographic, methodological (ID methods), host of origin and species, while the second data table contains information about the described strains such as publication, isolate code(s), host/substrate, year of isolation, geographical coordinates, species and trichothecene genotype. Analyses of the bibliographic data obtained from 123 publications from 2000 to 2021 by 498 unique authors and published in 40 journals are summarized. We describe the frequency of species and chemotypes for 16,274 strains for which geographical information was available, either provided as raw data or extracted from the publications, and sampled across six continents and 32 countries. The database and interactive interface are publicly available, allowing for searches, summarization, and mapping of strains according to several criteria including article, country, host, species and trichothecene genotype. The database will be updated as new articles are published and should be useful for guiding future surveys and exploring factors associated with species distribution such as climate and land use. Authors are encouraged to submit data at the strain level to the database, which is accessible at https://fgsc.netlify.app.

Fusarium BP1 is a reader of H3K27 methylation.

Histone H3 lysine 27 methylation catalyzed by polycomb repressive complex 2 (PRC2) is conserved from fungi to humans and represses gene transcription. However, the mechanism for recognition of methylated H3K27 remains unclear, especially in fungi. Here, we found that the bromo-adjacent homology (BAH)-plant homeodomain (PHD) domain containing protein BAH-PHD protein 1 (BP1) is a reader of H3K27 methylation in the cereal fungal pathogen Fusarium graminearum. BP1 interacts with the core PRC2 component Suz12 and directly binds methylated H3K27. BP1 is distributed in a subset of genomic regions marked by H3K27me3 and co-represses gene transcription. The BP1 deletion mutant shows identical phenotypes on mycelial growth and virulence, as well as similar expression profiles of secondary metabolite genes to the strain lacking the H3K27 methyltransferase Kmt6. More importantly, BP1 can directly bind DNA through its PHD finger, which might increase nucleosome residence and subsequently reinforce transcriptional repression in H3K27me3-marked target regions. A phylogenetic analysis showed that BP1 orthologs are mainly conserved in fungi. Overall, our findings provide novel insights into the mechanism by which PRC2 mediates gene repression in fungi, which is distinct from the PRC1-PRC2 system in plants and mammals.

Metatranscriptomic Comparison of Endophytic and Pathogenic Fusarium-Arabidopsis Interactions Reveals Plant Transcriptional Plasticity.

Plants are continuously exposed to beneficial and pathogenic microbes, but how plants recognize and respond to friends versus foes remains poorly understood. Here, we compared the molecular response of Arabidopsis thaliana independently challenged with a Fusarium oxysporum endophyte Fo47 versus a pathogen Fo5176. These two F. oxysporum strains share a core genome of about 46 Mb, in addition to 1,229 and 5,415 unique accessory genes. Metatranscriptomic data reveal a shared pattern of expression for most plant genes (about 80%) in responding to both fungal inoculums at all timepoints from 12 to 96 h postinoculation (HPI). However, the distinct responding genes depict transcriptional plasticity, as the pathogenic interaction activates plant stress responses and suppresses functions related to plant growth and development, while the endophytic interaction attenuates host immunity but activates plant nitrogen assimilation. The differences in reprogramming of the plant transcriptome are most obvious in 12 HPI, the earliest timepoint sampled, and are linked to accessory genes in both fungal genomes. Collectively, our results indicate that the A. thaliana and F. oxysporum interaction displays both transcriptome conservation and plasticity in the early stages of infection, providing insights into the fine-tuning of gene regulation underlying plant differential responses to fungal endophytes and pathogens.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

No to Neocosmospora: Phylogenomic and Practical Reasons for Continued Inclusion of the Fusarium solani Species Complex in the Genus Fusarium.

This article is to alert medical mycologists and infectious disease specialists of recent name changes of medically important species of the filamentous mold FusariumFusarium species can cause localized and life-threating infections in humans. Of the 70 Fusarium species that have been reported to cause infections, close to one-third are members of the Fusarium solani species complex (FSSC), and they collectively account for approximately two-thirds of all reported Fusarium infections. Many of these species were recently given scientific names for the first time by a research group in the Netherlands, but they were misplaced in the genus Neocosmospora In this paper, we present genetic arguments that strongly support inclusion of the FSSC in Fusarium There are potentially serious consequences associated with using the name Neocosmospora for Fusarium species because clinicians need to be aware that fusaria are broadly resistant to the spectrum of antifungals that are currently available.

Chromosome-Scale Genome Assembly of Fusarium oxysporum Strain Fo47, a Fungal Endophyte and Biocontrol Agent.

Here, we report a chromosome-level genome assembly of Fusarium oxysporum Fo47 (12 pseudomolecules; contig N50: 4.52 Mb), generated using a combination of PacBio long-read, Illumina paired end, and high-throughput chromosome conformation capture sequencing data. Although F. oxysporum causes vascular wilt to over 100 plant species, the strain Fo47 is classified as an endophyte and is widely used as a biocontrol agent for plant disease control. The Fo47 genome carries a single accessory chromosome of 4.23 Mb, compared with the reference genome of F. oxysporum f. sp. lycopersici Fol4287. The high-quality assembly and annotation of the Fo47 genome will be a valuable resource for studying the mechanisms underlying the endophytic interactions between F. oxysporum and plants as well as for deciphering the genome evolution of the F. oxysporum species complex.

The Fusarium graminearum t-SNARE Sso2 Is Involved in Growth, Defense, and DON Accumulation and Virulence.

The plant-pathogenic fungus Fusarium graminearum, causal agent of Fusarium head blight (FHB) disease on small grain cereals, produces toxic trichothecenes that require facilitated export for full virulence. Two potential modes of mycotoxin transport are membrane-bound transporters, which move toxins across cellular membranes, and N-ethylmaleimide-sensitive factor attachment receptor (SNARE)-mediated vesicular transport, by which toxins may be packaged as cargo in vesicles bound for organelles or the plasma membrane. In this study, we show that deletion of a gene (Sso2) for a subapically localized t-SNARE protein results in growth alteration, increased sensitivity to xenobiotics, altered gene expression profiles, and reduced deoxynivalenol (DON) accumulation in vitro and in planta as well as reduced FHB symptoms on wheat. A double deletion mutant generated by crossing the ∆sso2 deletion mutant with an ATP-binding cassette transporter deletion mutant (∆abc1) resulted in an additive reduction in DON accumulation and almost complete loss of FHB symptoms in planta. These results suggest an important role of Sso2-mediated subapical exocytosis in FHB progression and xenobiotic defense and are the first report of an additive reduction in F. graminearum DON accumulation upon deletion of two distinct modes of cellular export. This research provides useful information which may aid in formulating novel management plans of FHB or other destructive plant diseases.

The genome of opportunistic fungal pathogen Fusarium oxysporum carries a unique set of lineage-specific chromosomes.

Fusarium oxysporum is a cross-kingdom fungal pathogen that infects plants and humans. Horizontally transferred lineage-specific (LS) chromosomes were reported to determine host-specific pathogenicity among phytopathogenic F. oxysporum. However, the existence and functional importance of LS chromosomes among human pathogenic isolates are unknown. Here we report four unique LS chromosomes in a human pathogenic strain NRRL 32931, isolated from a leukemia patient. These LS chromosomes were devoid of housekeeping genes, but were significantly enriched in genes encoding metal ion transporters and cation transporters. Homologs of NRRL 32931 LS genes, including a homolog of ceruloplasmin and the genes that contribute to the expansion of the alkaline pH-responsive transcription factor PacC/Rim1p, were also present in the genome of NRRL 47514, a strain associated with Fusarium keratitis outbreak. This study provides the first evidence, to our knowledge, for genomic compartmentalization in two human pathogenic fungal genomes and suggests an important role of LS chromosomes in niche adaptation.

A fungal ABC transporter FgAtm1 regulates iron homeostasis via the transcription factor cascade FgAreA-HapX.

Iron homeostasis is important for growth, reproduction and other metabolic processes in all eukaryotes. However, the functions of ATP-binding cassette (ABC) transporters in iron homeostasis are largely unknown. Here, we found that one ABC transporter (named FgAtm1) is involved in regulating iron homeostasis, by screening sensitivity to iron stress for 60 ABC transporter mutants of Fusarium graminearum, a devastating fungal pathogen of small grain cereal crops worldwide. The lack of FgAtm1 reduces the activity of cytosolic Fe-S proteins nitrite reductase and xanthine dehydrogenase, which causes high expression of FgHapX via activating transcription factor FgAreA. FgHapX represses transcription of genes for iron-consuming proteins directly but activates genes for iron acquisition proteins by suppressing another iron regulator FgSreA. In addition, the transcriptional activity of FgHapX is regulated by the monothiol glutaredoxin FgGrx4. Furthermore, the phosphorylation of FgHapX, mediated by the Ser/Thr kinase FgYak1, is required for its functions in iron homeostasis. Taken together, this study uncovers a novel regulatory mechanism of iron homeostasis mediated by an ABC transporter in an important pathogenic fungus.

A phosphorylated transcription factor regulates sterol biosynthesis in Fusarium graminearum.

Sterol biosynthesis is controlled by transcription factor SREBP in many eukaryotes. Here, we show that SREBP orthologs are not involved in the regulation of sterol biosynthesis in Fusarium graminearum, a fungal pathogen of cereal crops worldwide. Instead, sterol production is controlled in this organism by a different transcription factor, FgSR, that forms a homodimer and binds to a 16-bp cis-element of its target gene promoters containing two conserved CGAA repeat sequences. FgSR is phosphorylated by the MAP kinase FgHog1, and the phosphorylated FgSR interacts with the chromatin remodeling complex SWI/SNF at the target genes, leading to enhanced transcription. Interestingly, FgSR orthologs exist only in Sordariomycetes and Leotiomycetes fungi. Additionally, FgSR controls virulence mainly via modulating deoxynivalenol biosynthesis and responses to phytoalexin.

Fusarium graminearum Trichothecene Mycotoxins: Biosynthesis, Regulation, and Management.

Fusarium head blight (FHB) of small grain cereals caused by Fusarium graminearum and other Fusarium species is an economically important plant disease worldwide. Fusarium infections not only result in severe yield losses but also contaminate grain with various mycotoxins, especially deoxynivalenol (DON). With the complete genome sequencing of F. graminearum, tremendous progress has been made during the past two decades toward understanding the basis for DON biosynthesis and its regulation. Here, we summarize the current understanding of DON biosynthesis and the effect of regulators, signal transduction pathways, and epigenetic modifications on DON production and the expression of biosynthetic TRI genes. In addition, strategies for controlling FHB and DON contamination are reviewed. Further studies on these biosynthetic and regulatory systems will provide useful knowledge for developing novel management strategies to prevent FHB incidence and mycotoxin accumulation in cereals.

Expression of the Fusarium graminearum terpenome and involvement of the endoplasmic reticulum-derived toxisome.

The sesquiterpenoid deoxynivalenol (DON) is an important trichothecene mycotoxin produced by the cereal pathogen Fusarium graminearum. DON is synthesized in specialized subcellular structures called toxisomes. The first step in DON synthesis is catalyzed by the sesquiterpene synthase (STS), Tri5 (trichodiene synthase), resulting in the cyclization of farnesyl diphosphate (FPP) to produce the sesquiterpene trichodiene. Tri5 is one of eight putative STSs in the F. graminearum genome. To better understand the F. graminearum terpenome, the volatile and soluble fractions of fungal cultures were sampled. Stringent regulation of sesquiterpene accumulation was observed. When grown in trichothecene induction medium, the fungus produces trichothecenes as well as several volatile non-trichothecene related sesquiterpenes, whereas no volatile terpenes were detected when grown in non-inducing medium. Surprisingly, a Δtri5 deletion strain grown in inducing conditions not only ceased accumulation of trichothecenes, but also failed to produce the non-trichothecene related sesquiterpenes. To test whether Tri5 from F. graminearum may be a promiscuous STS directly producing all observed sesquiterpenes, Tri5 was cloned and expressed in E. coli and shown to produce primarily trichodiene in addition to minor, related cyclization products. Therefore, while Tri5 expression in F. graminearum is necessary for non-trichothecene sesquiterpene biosynthesis, direct catalysis by Tri5 does not explain the sesquiterpene deficient phenotype observed in the Δtri5 strain. To test whether Tri5 protein, separate from its enzymatic activity, may be required for non-trichothecene synthesis, the Tri5 locus was replaced with an enzymatically inactive, but structurally unaffected tri5N225D S229T allele. This allele restores non-trichothecene synthesis but not trichothecene synthesis. The tri5N225D S229T allele also restores toxisome structure which is lacking in the Δtri5 deletion strain. Our results indicate that the Tri5 protein, but not its enzymatic activity, is also required for the synthesis of non-trichothecene related sesquiterpenes and the formation of toxisomes. Toxisomes thus not only may be important for DON synthesis, but also for the synthesis of other sesquiterpene mycotoxins such as culmorin by F. graminearum.

Nanoscale enrichment of the cytosolic enzyme trichodiene synthase near reorganized endoplasmic reticulum in Fusarium graminearum.

Trichothecene mycotoxin synthesis in the phytopathogen Fusarium graminearum involves primarily endoplasmic reticulum (ER)-localized enzymes of the mevalonate- and trichothecene biosynthetic pathways. Two exceptions are 3-hydroxy-3-methylglutaryl CoA synthase (Hms1) and trichodiene synthase (Tri5), which are known cytosolic enzymes. Using 3D structured illumination microscopy (3D SIM), GFP-tagged Tri5 and Hms1 were tested for preferential localization in the cytosol proximal to the ER. Tri5 protein was significantly enriched in cytosolic regions within 500 nm of the ER, but Hms1 was not. Spatial organization of enzymes in the cytosol has potential relevance for pathway efficiency and metabolic engineering in fungi and other organisms.

Kinome Expansion in the Fusarium oxysporum Species Complex Driven by Accessory Chromosomes.

The Fusarium oxysporum species complex (FOSC) is a group of soilborne pathogens causing severe disease in more than 100 plant hosts, while individual strains exhibit strong host specificity. Both chromosome transfer and comparative genomics experiments have demonstrated that lineage-specific (LS) chromosomes contribute to the host-specific pathogenicity. However, little is known about the functional importance of genes encoded in these LS chromosomes. Focusing on signaling transduction, this study compared the kinomes of 12 F. oxysporum isolates, including both plant and human pathogens and 1 nonpathogenic biocontrol strain, with 7 additional publicly available ascomycete genomes. Overall, F. oxysporum kinomes are the largest, facilitated in part by the acquisitions of the LS chromosomes. The comparative study identified 99 kinases that are present in almost all examined fungal genomes, forming the core signaling network of ascomycete fungi. Compared to the conserved ascomycete kinome, the expansion of the F. oxysporum kinome occurs in several kinase families such as histidine kinases that are involved in environmental signal sensing and target of rapamycin (TOR) kinase that mediates cellular responses. Comparative kinome analysis suggests a convergent evolution that shapes individual F. oxysporum isolates with an enhanced and unique capacity for environmental perception and associated downstream responses.IMPORTANCE Isolates of Fusarium oxysporum are adapted to survive a wide range of host and nonhost conditions. In addition, F. oxysporum was recently recognized as the top emerging opportunistic fungal pathogen infecting immunocompromised humans. The sensory and response networks of these fungi undoubtedly play a fundamental role in establishing the adaptability of this group. We have examined the kinomes of 12 F. oxysporum isolates and highlighted kinase families that distinguish F. oxysporum from other fungi, as well as different isolates from one another. The amplification of kinases involved in environmental signal relay and regulating downstream cellular responses clearly sets Fusarium apart from other Ascomycetes Although the functions of many of these kinases are still unclear, their specific proliferation highlights them as a result of the evolutionary forces that have shaped this species complex and clearly marks them as targets for exploitation in order to combat disease.

The fungal myosin I is essential for Fusarium toxisome formation.

Myosin-I molecular motors are proposed to function as linkers between membranes and the actin cytoskeleton in several cellular processes, but their role in the biosynthesis of fungal secondary metabolites remain elusive. Here, we found that the myosin I of Fusarium graminearum (FgMyo1), the causal agent of Fusarium head blight, plays critical roles in mycotoxin biosynthesis. Inhibition of myosin I by the small molecule phenamacril leads to marked reduction in deoxynivalenol (DON) biosynthesis. FgMyo1 also governs translation of the DON biosynthetic enzyme Tri1 by interacting with the ribosome-associated protein FgAsc1. Disruption of the ATPase activity of FgMyo1 either by the mutation E420K, down-regulation of FgMyo1 expression or deletion of FgAsc1 results in reduced Tri1 translation. The DON biosynthetic enzymes Tri1 and Tri4 are mainly localized to subcellular structures known as toxisomes in response to mycotoxin induction and the FgMyo1-interacting protein, actin, participates in toxisome formation. The actin polymerization disruptor latrunculin A inhibits toxisome assembly. Consistent with this observation, deletion of the actin-associated proteins FgPrk1 and FgEnd3 also results in reduced toxisome formation. Unexpectedly, the FgMyo1-actin cytoskeleton is not involved in biosynthesis of another secondary metabolite tested. Taken together, this study uncovers a novel function of myosin I in regulating mycotoxin biosynthesis in filamentous fungi.

Fusarium graminearum: pathogen or endophyte of North American grasses?

Mycotoxin-producing Fusarium graminearum and related species cause Fusarium head blight on cultivated grasses, such as wheat and barley. However, these Fusarium species may have had a longer evolutionary history with North American grasses than with cultivated crops and may interact with the ancestral hosts in ways which are biochemically distinct. We assayed 25 species of asymptomatic native grasses for the presence of Fusarium species and confirmed infected grasses as hosts using re-inoculation tests. We examined seed from native grasses for the presence of mycotoxin-producing Fusarium species and evaluated the ability of these fungi to produce mycotoxins in both native grass and wheat hosts using biochemical analysis. Mycotoxin-producing Fusarium species were shown to be prevalent in phylogenetically diverse native grasses, colonizing multiple tissue types, including seeds, leaves and inflorescence structures. Artificially inoculated grasses accumulated trichothecenes to a much lesser extent than wheat, and naturally infected grasses showed little to no accumulation. Native North American grasses are commonly inhabited by Fusarium species, but appear to accommodate these toxigenic fungi differently from cultivated crops. This finding highlights how host identity and evolutionary history may influence the outcome of plant-fungal interactions and may inform future efforts in crop improvement.

Plant diversity and plant identity influence Fusarium communities in soil.

Fusarium communities play important functional roles in soil and in plants as pathogens, endophytes, and saprotrophs. This study tests how rhizosphere Fusarium communities may vary with plant species, changes in the diversity of the surrounding plant community, and soil physiochemical characteristics. Fusarium communities in soil associated with the roots of two perennial prairie plant species maintained as monocultures or growing within polyculture plant communities were characterized using targeted metagenomics. Amplicon libraries targeting the RPB2 locus were generated from rhizosphere soil DNAs and sequenced using pyrosequencing. Sequences were clustered into operational taxonomic units (OTUs) and assigned a taxonomy using the Evolutionary Placement Algorithm. Fusarium community composition was differentiated between monoculture and polyculture plant communities, and by plant species in monoculture, but not in polyculture. Taxonomic classification of the Fusarium OTUs showed a predominance of F. tricinctum and F. oxysporum as well of the presence of a clade previously only found in the Southern Hemisphere. Total Fusarium richness was not affected by changes in plant community richness or correlated with soil physiochemical characteristics. However, OTU richness within two predominant phylogenetic lineages within the genus was positively or negatively correlated with soil physiochemical characteristics among samples within each lineage. This work shows that plant species, plant community richness, and soil physiochemical characteristics may all influence the composition and richness of Fusarium communities in soil.

Effector Gene Suites in Some Soil Isolates of Fusarium oxysporum Are Not Sufficient Predictors of Vascular Wilt in Tomato.

Seventy-four Fusarium oxysporum soil isolates were assayed for known effector genes present in an F. oxysporum f. sp. lycopersici race 3 tomato wilt strain (FOL MN-25) obtained from the same fields in Manatee County, Florida. Based on the presence or absence of these genes, four haplotypes were defined, two of which represented 96% of the surveyed isolates. These two most common effector haplotypes contained either all or none of the assayed race 3 effector genes. We hypothesized that soil isolates with all surveyed effector genes, similar to FOL MN-25, would be pathogenic toward tomato, whereas isolates lacking all effectors would be nonpathogenic. However, inoculation experiments revealed that presence of the effector genes alone was not sufficient to ensure pathogenicity on tomato. Interestingly, a nonpathogenic isolate containing the full suite of unmutated effector genes (FOS 4-4) appears to have undergone a chromosomal rearrangement yet remains vegetatively compatible with FOL MN-25. These observations confirm the highly dynamic nature of the F. oxysporum genome and support the conclusion that pathogenesis among free-living populations of F. oxysporum is a complex process. Therefore, the presence of effector genes alone may not be an accurate predictor of pathogenicity among soil isolates of F. oxysporum.

Examining the Transcriptional Response in Wheat Near-Isogenic Lines to Infection and Deoxynivalenol Treatment.

head blight (FHB) is a disease caused predominantly by the fungal pathogen that affects wheat and other small-grain cereals and can lead to severe yield loss and reduction in grain quality. Trichothecene mycotoxins, such as deoxynivalenol (DON), accumulate during infection and increase pathogen virulence and decrease grain quality. The locus on wheat chromosome 3BS confers Type II resistance to FHB and resistance to the spread of infection on the spike and has been associated with resistance to DON accumulation. To gain a better genetic understanding of the functional role of and resistance or susceptibility to FHB, we examined DON and ergosterol accumulation, FHB resistance, and the whole-genome transcriptomic response using RNA-seq in a near-isogenic line (NIL) pair carrying the resistant and susceptible alleles for during infection and DON treatment. Our results provide a gene expression atlas for the resistant and susceptible wheat- interaction. The DON concentration and transcriptomic results show that the rachis is a key location for conferring Type II resistance. In addition, the wheat transcriptome analysis revealed a set of -responsive genes that may play a role in resistance and a set of DON-responsive genes that may play a role in trichothecene resistance. Transcriptomic results from the pathogen show that the genome responds differently to the host level of resistance. The results of this study extend our understanding of host and pathogen responses in the wheat- interaction.

Effector profiles distinguish formae speciales of Fusarium oxysporum.

Formae speciales (ff.spp.) of the fungus Fusarium oxysporum are often polyphyletic within the species complex, making it impossible to identify them on the basis of conserved genes. However, sequences that determine host-specific pathogenicity may be expected to be similar between strains within the same forma specialis. Whole genome sequencing was performed on strains from five different ff.spp. (cucumerinum, niveum, melonis, radicis-cucumerinum and lycopersici). In each genome, genes for putative effectors were identified based on small size, secretion signal, and vicinity to a "miniature impala" transposable element. The candidate effector genes of all genomes were collected and the presence/absence patterns in each individual genome were clustered. Members of the same forma specialis turned out to group together, with cucurbit-infecting strains forming a supercluster separate from other ff.spp. Moreover, strains from different clonal lineages within the same forma specialis harbour identical effector gene sequences, supporting horizontal transfer of genetic material. These data offer new insight into the genetic basis of host specificity in the F. oxysporum species complex and show that (putative) effectors can be used to predict host specificity in F. oxysporum.