Characterization of the Fusarium circinatum biofilm environmental response role.

The capacity to form biofilms is a common trait among many microorganisms present on Earth. In this study, we demonstrate for the first time that the fatal pine pitch canker agent, Fusarium circinatum, can lead a biofilm-like lifestyle with aggregated hyphal bundles wrapped in extracellular matrix (ECM). Our research shows F. circinatum's ability to adapt to environmental changes by assuming a biofilm-like lifestyle. This was demonstrated by varying metabolic activities exhibited by the biofilms in response to factors like temperature and pH. Further analysis revealed that while planktonic cells produced small amounts of ECM per unit of the biomass, heat- and azole-exposed biofilms produced significantly more ECM than nonexposed biofilms, further demonstrating the adaptability of F. circinatum to changing environments. The increased synthesis of ECM triggered by these abiotic factors highlights the link between ECM production in biofilm and resistance to abiotic stress. This suggests that ECM-mediated response may be one of the key survival strategies of F. circinatum biofilms in response to changing environments. Interestingly, azole exposure also led to biofilms that were resistant to DNase, which typically uncouples biofilms by penetrating the biofilm and degrading its extracellular DNA; we propose that DNases were likely hindered from reaching target cells by the ECM barricade. The interplay between antifungal treatment and DNase enzyme suggests a complex relationship between eDNA, ECM, and antifungal agents in F. circinatum biofilms. Therefore, our results show how a phytopathogen's sessile (biofilm) lifestyle could influence its response to the surrounding environment.

Chromosome-Level Assemblies for the Pine Pitch Canker Pathogen Fusarium circinatum.

The pine pitch canker pathogen, Fusarium circinatum, is globally regarded as one of the most important threats to commercial pine-based forestry. Although genome sequences of this fungus are available, these remain highly fragmented or structurally ill-defined. Our overall goal was to provide high-quality assemblies for two notable strains of F. circinatum, and to characterize these in terms of coding content, repetitiveness and the position of telomeres and centromeres. For this purpose, we used Oxford Nanopore Technologies MinION long-read sequences, as well as Illumina short sequence reads. By leveraging the genomic synteny inherent to F. circinatum and its close relatives, these sequence reads were assembled to chromosome level, where contiguous sequences mostly spanned from telomere to telomere. Comparative analyses unveiled remarkable variability in the twelfth and smallest chromosome, which is known to be dispensable. It presented a striking length polymorphism, with one strain lacking substantial portions from the chromosome's distal and proximal regions. These regions, characterized by a lower gene density, G+C content and an increased prevalence of repetitive elements, contrast starkly with the syntenic segments of the chromosome, as well as with the core chromosomes. We propose that these unusual regions might have arisen or expanded due to the presence of transposable elements. A comparison of the overall chromosome structure revealed that centromeric elements often underpin intrachromosomal differences between F. circinatum strains, especially at chromosomal breakpoints. This suggests a potential role for centromeres in shaping the chromosomal architecture of F. circinatum and its relatives. The publicly available genome data generated here, together with the detailed metadata provided, represent essential resources for future studies of this important plant pathogen.

Identification and Characterization of a QTL for Growth of Fusarium circinatum on Pine-Based Medium.

Fusarium circinatum is an economically important pathogen of pine and resides in the Fusarium fujikuroi species complex. Here we investigated the molecular processes underlying growth in F. circinatum by exploring the association between growth and the nutritional environment provided by the pine host. For this purpose, we subjected a mapping population consisting of F. circinatum X F. temperatum hybrid progeny to an analysis of growth rate on a pine-tissue derived medium. These data, together with the available genetic linkage map for F. circinatum, were then used to identify Quantitative Trait Loci (QTLs) associated with growth. The single significant QTL identified was then characterized using the available genome sequences for the hybrid progeny's parental isolates. This revealed that the QTL localized to two non-homologous regions in the F. circinatum and F. temperatum genomes. For one of these, the F. circinatum parent contained a two-gene deletion relative to the F. temperatum parent. For the other region, the two parental isolates encoded different protein products. Analysis of repeats, G+C content, and repeat-induced point (RIP) mutations further suggested a retrotransposon origin for the two-gene deletion in F. circinatum. Nevertheless, subsequent genome and PCR-based analyses showed that both regions were similarly polymorphic within a collection of diverse F. circinatum. However, we observed no clear correlation between the respective polymorphism patterns and growth rate in culture. These findings support the notion that growth is a complex multilocus trait and raise the possibility that the identified QTL contains multiple small-effect QTLs, of which some might be dependent on the genetic backgrounds. This study improved our current knowledge of the genetic determinants of vegetative growth in F. circinatum and provided an important foundation for determining the genes and processes underpinning its ability to colonize its host environment.

Characterization of Host-Specific Genes from Pine- and Grass-Associated Species of the Fusarium fujikuroi Species Complex.

The Fusarium fujikuroi species complex (FFSC) includes socioeconomically important pathogens that cause disease for numerous crops and synthesize a variety of secondary metabolites that can contaminate feedstocks and food. Here, we used comparative genomics to elucidate processes underlying the ability of pine-associated and grass-associated FFSC species to colonize tissues of their respective plant hosts. We characterized the identity, possible functions, evolutionary origins, and chromosomal positions of the host-range-associated genes encoded by the two groups of fungi. The 72 and 47 genes identified as unique to the respective genome groups were potentially involved in diverse processes, ranging from transcription, regulation, and substrate transport through to virulence/pathogenicity. Most genes arose early during the evolution of Fusarium/FFSC and were only subsequently retained in some lineages, while some had origins outside Fusarium. Although differences in the densities of these genes were especially noticeable on the conditionally dispensable chromosome of F. temperatum (representing the grass-associates) and F. circinatum (representing the pine-associates), the host-range-associated genes tended to be located towards the subtelomeric regions of chromosomes. Taken together, these results demonstrate that multiple mechanisms drive the emergence of genes in the grass- and pine-associated FFSC taxa examined. It also highlighted the diversity of the molecular processes potentially underlying niche-specificity in these and other Fusarium species.

Intra-Species Genomic Variation in the Pine Pathogen Fusarium circinatum.

Fusarium circinatum is an important global pathogen of pine trees. Genome plasticity has been observed in different isolates of the fungus, but no genome comparisons are available. To address this gap, we sequenced and assembled to chromosome level five isolates of F. circinatum. These genomes were analysed together with previously published genomes of F. circinatum isolates, FSP34 and KS17. Multi-sample variant calling identified a total of 461,683 micro variants (SNPs and small indels) and a total of 1828 macro structural variants of which 1717 were copy number variants and 111 were inversions. The variant density was higher on the sub-telomeric regions of chromosomes. Variant annotation revealed that genes involved in transcription, transport, metabolism and transmembrane proteins were overrepresented in gene sets that were affected by high impact variants. A core genome representing genomic elements that were conserved in all the isolates and a non-redundant pangenome representing all genomic elements is presented. Whole genome alignments showed that an average of 93% of the genomic elements were present in all isolates. The results of this study reveal that some genomic elements are not conserved within the isolates and some variants are high impact. The described genome-scale variations will help to inform novel disease management strategies against the pathogen.

IMA genome - F14 : Draft genome sequences of Penicillium roqueforti, Fusarium sororula, Chrysoporthe puriensis, and Chalaropsis populi.

Draft genomes of Penicillium roqueforti, Fusarium sororula, Chalaropsis populi, and Chrysoporthe puriensis are presented. Penicillium roqueforti is a model fungus for genetics, physiological and metabolic studies, as well as for biotechnological applications. Fusarium sororula and Chrysoporthe puriensis are important tree pathogens, and Chalaropsis populi is a soil-borne root-pathogen. The genome sequences presented here thus contribute towards a better understanding of both the pathogenicity and biotechnological potential of these species.

Repeat-Induced Point Mutations Drive Divergence between Fusarium circinatum and Its Close Relatives.

The Repeat-Induced Point (RIP) mutation pathway is a fungal-specific genome defense mechanism that counteracts the deleterious effects of transposable elements. This pathway permanently mutates its target sequences by introducing cytosine to thymine transitions. We investigated the genome-wide occurrence of RIP in the pitch canker pathogen, Fusarium circinatum, and its close relatives in the Fusarium fujikuroi species complex (FFSC). Our results showed that the examined fungi all exhibited hallmarks of RIP, but that they differed in terms of the extent to which their genomes were affected by this pathway. RIP mutations constituted a large proportion of all the FFSC genomes, including both core and dispensable chromosomes, although the latter were generally more extensively affected by RIP. Large RIP-affected genomic regions were also much more gene sparse than the rest of the genome. Our data further showed that RIP-directed sequence diversification increased the variability between homologous regions of related species, and that RIP-affected regions can interfere with homologous recombination during meiosis, thereby contributing to post-mating segregation distortion. Taken together, these findings suggest that RIP can drive the independent divergence of chromosomes, alter chromosome architecture, and contribute to the divergence among F. circinatum and other members of this economically important group of fungi.

Genomic analysis of the aggressive tree pathogen Ceratocystis albifundus.

The overall goal of this study was to determine whether the genome of an important plant pathogen in Africa, Ceratocystis albifundus, is structured into subgenomic compartments, and if so, to establish how these compartments are distributed across the genome. For this purpose, the publicly available genome of C. albifundus was complemented with the genome sequences for four additional isolates using the Illumina HiSeq platform. In addition, a reference genome for one of the individuals was assembled using both PacBio and Illumina HiSeq technologies. Our results showed a high degree of synteny between the five genomes, although several regions lacked detectable long-range synteny. These regions were associated with the presence of accessory genes, lower genetic similarity, variation in read-map depth, as well as transposable elements and genes associated with host-pathogen interactions (e.g. effectors and CAZymes). Such patterns are regarded as hallmarks of accelerated evolution, particularly of accessory subgenomic compartments in fungal pathogens. Our findings thus showed that the genome of C. albifundus is made-up of core and accessory subgenomic compartments, which is an important step towards characterizing its pangenome. This study also highlights the value of comparative genomics for understanding mechanisms that may underly and influence the biology and evolution of pathogens.

The synergistic effect of concatenation in phylogenomics: the case in Pantoea.

With the increased availability of genome sequences for bacteria, it has become routine practice to construct genome-based phylogenies. These phylogenies have formed the basis for various taxonomic decisions, especially for resolving problematic relationships between taxa. Despite the popularity of concatenating shared genes to obtain well-supported phylogenies, various issues regarding this combined-evidence approach have been raised. These include the introduction of phylogenetic error into datasets, as well as incongruence due to organism-level evolutionary processes, particularly horizontal gene transfer and incomplete lineage sorting. Because of the huge effect that this could have on phylogenies, we evaluated the impact of phylogenetic conflict caused by organism-level evolutionary processes on the established species phylogeny for Pantoea, a member of the Enterobacterales. We explored the presence and distribution of phylogenetic conflict at the gene partition and nucleotide levels, by identifying putative inter-lineage recombination events that might have contributed to such conflict. Furthermore, we determined whether smaller, randomly constructed datasets had sufficient signal to reconstruct the current species tree hypothesis or if they would be overshadowed by phylogenetic incongruence. We found that no individual gene tree was fully congruent with the species phylogeny of Pantoea, although many of the expected nodes were supported by various individual genes across the genome. Evidence of recombination was found across all lineages within Pantoea, and provides support for organism-level evolutionary processes as a potential source of phylogenetic conflict. The phylogenetic signal from at least 70 random genes recovered robust, well-supported phylogenies for the backbone and most species relationships of Pantoea, and was unaffected by phylogenetic conflict within the dataset. Furthermore, despite providing limited resolution among taxa at the level of single gene trees, concatenated analyses of genes that were identified as having no signal resulted in a phylogeny that resembled the species phylogeny of Pantoea. This distribution of signal and noise across the genome presents the ideal situation for phylogenetic inference, as the topology from a ≥70-gene concatenated species phylogeny is not driven by single genes, and our data suggests that this finding may also hold true for smaller datasets. We thus argue that, by using a concatenation-based approach in phylogenomics, one can obtain robust phylogenies due to the synergistic effect of the combined signal obtained from multiple genes.

Multiple independent origins for a subtelomeric locus associated with growth rate in Fusarium circinatum.

Fusarium is a diverse assemblage that includes a large number of species of considerable medical and agricultural importance. Not surprisingly, whole genome sequences for many Fusarium species have been published or are in the process of being determined, the availability of which is invaluable for deciphering the genetic basis of key phenotypic traits. Here we investigated the distribution, genic composition, and evolutionary history of a locus potentially determining growth rate in the pitch canker pathogen F. circinatum. We found that the genomic region underlying this locus is highly conserved amongst F. circinatum and its close relatives, except for the presence of a 12 000 base pair insertion in all of the examined isolates of F. circinatum. This insertion encodes for five genes and our phylogenetic analyses revealed that each was most likely acquired through horizontal gene transfer from polyphyletic origins. Our data further showed that this region is located in a region low in G+C content and enriched for repetitive sequences and transposable elements, which is situated near the telomere of Chromosome 3 of F. circinatum. As have been shown for other fungi, these findings thus suggest that the emergence of the unique 12 000 bp region in F. circinatum is linked to the dynamic evolutionary processes associated with subtelomeres that, in turn, have been implicated in the ecological adaptation of fungal pathogens.

Nine draft genome sequences of Claviceps purpurea s.lat., including C. arundinis, C. humidiphila, and C. cf. spartinae, pseudomolecules for the pitch canker pathogen Fusarium circinatum, draft genome of Davidsoniella eucalypti, Grosmannia galeiformis, Quambalaria eucalypti, and Teratosphaeria destructans.

This genome announcement includes draft genomes from Claviceps purpurea s.lat., including C. arundinis, C. humidiphila and C. cf. spartinae. The draft genomes of Davidsoniella eucalypti, Quambalaria eucalypti and Teratosphaeria destructans, all three important eucalyptus pathogens, are presented. The insect associate Grosmannia galeiformis is also described. The pine pathogen genome of Fusarium circinatum has been assembled into pseudomolecules, based on additional sequence data and by harnessing the known synteny within the Fusarium fujikuroi species complex. This new assembly of the F. circinatum genome provides 12 pseudomolecules that correspond to the haploid chromosome number of F. circinatum. These are comparable to other chromosomal assemblies within the FFSC and will enable more robust genomic comparisons within this species complex.

Architecture and Distribution of Introns in Core Genes of Four Fusarium Species.

Removal of introns from transcribed RNA represents a crucial step during the production of mRNA in eukaryotes. Available whole-genome sequences and expressed sequence tags (ESTs) have increased our knowledge of this process and revealed various commonalities among eukaryotes. However, certain aspects of intron structure and diversity are taxon-specific, which can complicate the accuracy of in silico gene prediction methods. Using core genes, we evaluated the distribution and architecture of Fusarium circinatum spliceosomal introns, and linked these characteristics to the accuracy of the predicted gene models of the genome of this fungus. We also evaluated intron distribution and architecture in F. verticillioides, F. oxysporum, and F. graminearum, and made comparisons with F. circinatum Results indicated that F. circinatum and the three other Fusarium species have canonical 5' and 3' splice sites, but with subtle differences that are apparently not shared with those of other fungal genera. The polypyrimidine tract of Fusarium introns was also found to be highly divergent among species and genes. Furthermore, the conserved adenosine nucleoside required during the first step of splicing is contained within unique branch site motifs in certain Fusarium introns. Data generated here show that introns of F. circinatum, as well as F. verticillioides, F. oxysporum, and F. graminearum, are characterized by a number of unique features such as the CTHAH and ACCAT motifs of the branch site. Incorporation of such information into genome annotation software will undoubtedly improve the accuracy of gene prediction methods used for Fusarium species and related fungi.

IMA Genome-F 4: Draft genome sequences of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygamai, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata.

The genomes of Chrysoporthe austroafricana, Diplodia scrobiculata, Fusarium nygami, Leptographium lundbergii, Limonomyces culmigenus, Stagonosporopsis tanaceti, and Thielaviopsis punctulata are presented in this genome announcement. These seven genomes are from endophytes, plant pathogens and economically important fungal species. The genome sizes range from 26.6 Mb in the case of Leptographium lundbergii to 44 Mb for Chrysoporthe austroafricana. The availability of these genome data will provide opportunities to resolve longstanding questions regarding the taxonomy of species in these genera, and may contribute to our understanding of the lifestyles through comparative studies with closely related organisms.

IMA Genome-F 5: Draft genome sequences of Ceratocystis eucalypticola, Chrysoporthe cubensis, C. deuterocubensis, Davidsoniella virescens, Fusarium temperatum,Graphilbum fragrans, Penicillium nordicum, and Thielaviopsis musarum.

The genomes of Ceratocystis eucalypticola, Chrysoporthe cubensis, Chrysoporthe deuterocubensis, Davidsoniella virescens, Fusarium temperatum, Graphilbum fragrans, Penicillium nordicum and Thielaviopsis musarum are presented in this genome announcement. These seven genomes are from plant pathogens and otherwise economically important fungal species. The genome sizes range from 28 Mb in the case of T. musarum to 45 Mb for Fusarium temperatum. These genomes include the first reports of genomes for the genera Davidsoniella, Graphilbum and Thielaviopsis. The availability of these genome data will provide opportunities to resolve longstanding questions regarding the taxonomy of species in these genera. In addition these genome sequences through comparative studies with closely related organisms will increase our understanding of how these pathogens cause disease.

Genome-wide macrosynteny among Fusarium species in the Gibberella fujikuroi complex revealed by amplified fragment length polymorphisms.

The Gibberella fujikuroi complex includes many Fusarium species that cause significant losses in yield and quality of agricultural and forestry crops. Due to their economic importance, whole-genome sequence information has rapidly become available for species including Fusarium circinatum, Fusarium fujikuroi and Fusarium verticillioides, each of which represent one of the three main clades known in this complex. However, no previous studies have explored the genomic commonalities and differences among these fungi. In this study, a previously completed genetic linkage map for an interspecific cross between Fusarium temperatum and F. circinatum, together with genomic sequence data, was utilized to consider the level of synteny between the three Fusarium genomes. Regions that are homologous amongst the Fusarium genomes examined were identified using in silico and pyrosequenced amplified fragment length polymorphism (AFLP) fragment analyses. Homology was determined using BLAST analysis of the sequences, with 777 homologous regions aligned to F. fujikuroi and F. verticillioides. This also made it possible to assign the linkage groups from the interspecific cross to their corresponding chromosomes in F. verticillioides and F. fujikuroi, as well as to assign two previously unmapped supercontigs of F. verticillioides to probable chromosomal locations. We further found evidence of a reciprocal translocation between the distal ends of chromosome 8 and 11, which apparently originated before the divergence of F. circinatum and F. temperatum. Overall, a remarkable level of macrosynteny was observed among the three Fusarium genomes, when comparing AFLP fragments. This study not only demonstrates how in silico AFLPs can aid in the integration of a genetic linkage map to the physical genome, but it also highlights the benefits of using this tool to study genomic synteny and architecture.

IMA Genome-F 3: Draft genomes of Amanita jacksonii, Ceratocystis albifundus, Fusarium circinatum, Huntiella omanensis, Leptographium procerum, Rutstroemia sydowiana, and Sclerotinia echinophila.

The genomes of fungi provide an important resource to resolve issues pertaining to their taxonomy, biology, and evolution. The genomes of Amanita jacksonii, Ceratocystis albifundus, a Fusarium circinatum variant, Huntiella omanensis, Leptographium procerum, Sclerotinia echinophila, and Rutstroemia sydowiana are presented in this genome announcement. These seven genomes are from a number of fungal pathogens and economically important species. The genome sizes range from 27 Mb in the case of Ceratocystis albifundus to 51.9 Mb for Rutstroemia sydowiana. The latter also encodes for a predicted 17 350 genes, more than double that of Ceratocystis albifundus. These genomes will add to the growing body of knowledge of these fungi and provide a value resource to researchers studying these fungi.