PanTools v3: functional annotation, classification and phylogenomics.

SUMMARY: The ever-increasing number of sequenced genomes necessitates the development of pangenomic approaches for comparative genomics. Introduced in 2016, PanTools is a platform that allows pangenome construction, homology grouping and pangenomic read mapping. The use of graph database technology makes PanTools versatile, applicable from small viral genomes like SARS-CoV-2 up to large plant or animal genomes like tomato or human. Here, we present our third major update to PanTools that enables the integration of functional annotations and provides both gene-level analyses and phylogenetics. AVAILABILITY AND IMPLEMENTATION: PanTools is implemented in Java 8 and released under the GNU GPLv3 license. Software and documentation are available at https://git.wur.nl/bioinformatics/pantools. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The Pectobacterium pangenome, with a focus on Pectobacterium brasiliense, shows a robust core and extensive exchange of genes from a shared gene pool.

BACKGROUND: Bacterial plant pathogens of the Pectobacterium genus are responsible for a wide spectrum of diseases in plants, including important crops such as potato, tomato, lettuce, and banana. Investigation of the genetic diversity underlying virulence and host specificity can be performed at genome level by using a comprehensive comparative approach called pangenomics. A pangenomic approach, using newly developed functionalities in PanTools, was applied to analyze the complex phylogeny of the Pectobacterium genus. We specifically used the pangenome to investigate genetic differences between virulent and avirulent strains of P. brasiliense, a potato blackleg causing species dominantly present in Western Europe. RESULTS: Here we generated a multilevel pangenome for Pectobacterium, comprising 197 strains across 19 species, including type strains, with a focus on P. brasiliense. The extensive phylogenetic analysis of the Pectobacterium genus showed robust distinct clades, with most detail provided by 452,388 parsimony-informative single-nucleotide polymorphisms identified in single-copy orthologs. The average Pectobacterium genome consists of 47% core genes, 1% unique genes, and 52% accessory genes. Using the pangenome, we zoomed in on differences between virulent and avirulent P. brasiliense strains and identified 86 genes associated to virulent strains. We found that the organization of genes is highly structured and linked with gene conservation, function, and transcriptional orientation. CONCLUSION: The pangenome analysis demonstrates that evolution in Pectobacteria is a highly dynamic process, including gene acquisitions partly in clusters, genome rearrangements, and loss of genes. Pectobacterium species are typically not characterized by a set of species-specific genes, but instead present themselves using new gene combinations from the shared gene pool. A multilevel pangenomic approach, fusing DNA, protein, biological function, taxonomic group, and phenotypes, facilitates studies in a flexible taxonomic context.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

Contamination and Translocation of Deoxynivalenol and Its Derivatives Associated with Fusarium Crown Rot of Wheat in Northern China.

Fusarium crown rot (FCR) is one of the most important wheat diseases in northern China. The main causal agent of FCR, Fusarium pseudograminearum, can produce mycotoxins such as type B trichothecenes. Therefore, FCR could be an additional source of mycotoxin contamination during wheat production. Field inoculation experiments demonstrated that FCR disease severity strongly impacts the distribution pattern of trichothecenes in different wheat tissues. Mycotoxins were mainly observed in lower internodes, and a low amount was detected in the upper parts above the fourth internode. However, high levels of trichothecene accumulation were detected in the upper segments of wheat plants under field conditions, which would threaten the feed production. The variation of mycotoxin content among sampling sites indicated that besides disease severity, other factors like climate, irrigation, and fungicide application may influence the mycotoxin accumulation in wheat. A comprehensive survey of deoxynivalenol (DON) and its derivatives in wheat heads with FCR symptoms in natural fields was conducted at 80 sites in seven provinces in northern China. Much higher levels of mycotoxin were observed compared with inoculation experiments. The mycotoxin content varied greatly among sampling sites, but no significant differences were observed if compared at province level, which indicated the variation is mainly caused by local conditions. Trace amounts of mycotoxin appeared to be translocated to grains, which revealed that FCR infection in natural fields poses a relatively small threat to contamination of grains but a larger one to plant parts that may be used as animal feed. To our knowledge, this is the first report of trichothecene accumulation in wheat stems and heads, as well as grains after FCR infection in natural field conditions. These investigations provide novel insights into food and feed safety risk caused by FCR in northern China.

The Synchytrium endobioticum AvrSen1 Triggers a Hypersensitive Response in Sen1 Potatoes While Natural Variants Evade Detection.

Synchytrium endobioticum is an obligate biotrophic fungus of division Chytridiomycota. It causes potato wart disease, has a worldwide quarantine status and is included on the Health and Human Services and United States Department of Agriculture Select Agent list. S. endobioticum isolates are grouped in pathotypes based on their ability to evade host resistance in a set of differential potato varieties. Thus far, 39 pathotypes are reported. A single dominant gene (Sen1) governs pathotype 1 (D1) resistance and we anticipated that the underlying molecular model would involve a pathogen effector (AvrSen1) that is recognized by the host. The S. endobioticum-specific secretome of 14 isolates representing six different pathotypes was screened for effectors specifically present in pathotype 1 (D1) isolates but absent in others. We identified a single AvrSen1 candidate. Expression of this candidate in potato Sen1 plants showed a specific hypersensitive response (HR), which cosegregated with the Sen1 resistance in potato populations. No HR was obtained with truncated genes found in pathotypes that evaded recognition by Sen1. These findings established that our candidate gene was indeed Avrsen1. The S. endobioticum AvrSen1 is a single-copy gene and encodes a 376-amino-acid protein without predicted function or functional domains, and is the first effector gene identified in Chytridiomycota, an extremely diverse yet underrepresented basal lineage of fungi.

The linear mitochondrial genome of the quarantine chytrid Synchytrium endobioticum; insights into the evolution and recent history of an obligate biotrophic plant pathogen.

BACKGROUND: Chytridiomycota species (chytrids) belong to a basal lineage in the fungal kingdom. Inhabiting terrestrial and aquatic environments, most are free-living saprophytes but several species cause important diseases: e.g. Batrachochytrium dendrobatidis, responsible for worldwide amphibian decline; and Synchytrium endobioticum, causing potato wart disease. S. endobioticum has an obligate biotrophic lifestyle and isolates can be further characterized as pathotypes based on their virulence on a differential set of potato cultivars. Quarantine measures have been implemented globally to control the disease and prevent its spread. We used a comparative approach using chytrid mitogenomes to determine taxonomical relationships and to gain insights into the evolution and recent history of introductions of this plant pathogen. RESULTS: We assembled and annotated the complete mitochondrial genome of 30 S. endobioticum isolates and generated mitochondrial genomes for five additional chytrid species. The mitochondrial genome of S. endobioticum is linear with terminal inverted repeats which was validated by tailing and PCR amplifying the telomeric ends. Surprisingly, no conservation in organisation and orientation of mitochondrial genes was observed among the Chytridiomycota except for S. endobioticum and its sister species Synchytrium microbalum. However, the mitochondrial genome of S. microbalum is circular and comprises only a third of the 72.9 Kbp found for S. endobioticum suggesting recent linearization and expansion. Four mitochondrial lineages were identified in the S. endobioticum mitochondrial genomes. Several pathotypes occur in different lineages, suggesting that these have emerged independently. In addition, variations for polymorphic sites in the mitochondrial genome of individual isolates were observed demonstrating that S. endobioticum isolates represent a community of different genotypes. Such communities were shown to be complex and stable over time, but we also demonstrate that the use of semi-resistant potato cultivars triggers a rapid shift in the mitochondrial haplotype associated with increased virulence. CONCLUSIONS: Mitochondrial genomic variation shows that S. endobioticum has been introduced into Europe multiple times, that several pathotypes emerged multiple times, and that isolates represent communities of different genotypes. Our study represents the most comprehensive dataset of chytrid mitogenomes, which provides new insights into the extraordinary dynamics and evolution of mitochondrial genomes involving linearization, expansion and reshuffling.

Mitochondrial genomes reveal recombination in the presumed asexual Fusarium oxysporum species complex.

BACKGROUND: The Fusarium oxysporum species complex (FOSC) contains several phylogenetic lineages. Phylogenetic studies identified two to three major clades within the FOSC. The mitochondrial sequences are highly informative phylogenetic markers, but have been mostly neglected due to technical difficulties. RESULTS: A total of 61 complete mitogenomes of FOSC strains were de novo assembled and annotated. Length variations and intron patterns support the separation of three phylogenetic species. The variable region of the mitogenome that is typical for the genus Fusarium shows two new variants in the FOSC. The variant typical for Fusarium is found in members of all three clades, while variant 2 is found in clades 2 and 3 and variant 3 only in clade 2. The extended set of loci analyzed using a new implementation of the genealogical concordance species recognition method support the identification of three phylogenetic species within the FOSC. Comparative analysis of the mitogenomes in the FOSC revealed ongoing mitochondrial recombination within, but not between phylogenetic species. CONCLUSIONS: The recombination indicates the presence of a parasexual cycle in F. oxysporum. The obstacles hindering the usage of the mitogenomes are resolved by using next generation sequencing and selective genome assemblers, such as GRAbB. Complete mitogenome sequences offer a stable basis and reference point for phylogenetic and population genetic studies.

GRAbB: Selective Assembly of Genomic Regions, a New Niche for Genomic Research.

GRAbB (Genomic Region Assembly by Baiting) is a new program that is dedicated to assemble specific genomic regions from NGS data. This approach is especially useful when dealing with multi copy regions, such as mitochondrial genome and the rDNA repeat region, parts of the genome that are often neglected or poorly assembled, although they contain interesting information from phylogenetic or epidemiologic perspectives, but also single copy regions can be assembled. The program is capable of targeting multiple regions within a single run. Furthermore, GRAbB can be used to extract specific loci from NGS data, based on homology, like sequences that are used for barcoding. To make the assembly specific, a known part of the region, such as the sequence of a PCR amplicon or a homologous sequence from a related species must be specified. By assembling only the region of interest, the assembly process is computationally much less demanding and may lead to assemblies of better quality. In this study the different applications and functionalities of the program are demonstrated such as: exhaustive assembly (rDNA region and mitochondrial genome), extracting homologous regions or genes (IGS, RPB1, RPB2 and TEF1a), as well as extracting multiple regions within a single run. The program is also compared with MITObim, which is meant for the exhaustive assembly of a single target based on a similar query sequence. GRAbB is shown to be more efficient than MITObim in terms of speed, memory and disk usage. The other functionalities (handling multiple targets simultaneously and extracting homologous regions) of the new program are not matched by other programs. The program is available with explanatory documentation at https://github.com/b-brankovics/grabb. GRAbB has been tested on Ubuntu (12.04 and 14.04), Fedora (23), CentOS (7.1.1503) and Mac OS X (10.7). Furthermore, GRAbB is available as a docker repository: brankovics/grabb (https://hub.docker.com/r/brankovics/grabb/).

An Efficient Triplex TaqMan Quantitative PCR to Detect a Blackleg-Causing Lineage of Pectobacterium brasiliense in Potato Based on a Pangenome Analysis.

P. brasiliense is an important bacterial pathogen causing blackleg (BL) in potatoes. Nevertheless, P. brasiliense is often detected in seed lots that do not develop any of the typical blackleg symptoms in the potato crop when planted. Field bioassays identified that P. brasiliense strains can be categorized into two distinct classes, some able to cause blackleg symptoms and some unable to do it. A comparative pangenomic approach was performed on 116 P. brasiliense strains, of which 15 were characterized as BL-causing strains and 25 as non-causative. In a genetically homogeneous clade comprising all BL-causing P. brasiliense strains, two genes only present in the BL-causing strains were identified, one encoding a predicted lysozyme inhibitor Lprl (LZI) and one encoding a putative Toll/interleukin-1 receptor (TIR) domain-containing protein. TaqMan assays for the specific detection of BL-causing P. brasiliense were developed and integrated with the previously developed generic P. brasiliense assay into a triplex TaqMan assay. This simultaneous detection makes the scoring more efficient as only a single tube is needed, and it is more robust as BL-causing strains of P. brasiliense should be positive for all three assays. Individual P. brasiliense strains were found to be either positive for all three assays or only for the P. brasiliense assay. In potato samples, the mixed presence of BL-causing and not BL-causing P. brasiliense strains was observed as shown by the difference in Ct value of the TaqMan assays. However, upon extension of the number of strains, it became clear that in recent years additional BL-causing lineages of P. brasiliense were detected for which additional assays must be developed.

Detecting Introgression Between Members of the Fusarium fujikuroi and F. oxysporum Species Complexes by Comparative Mitogenomics.

The Fusarium fujikuroi species complex (FFSC) and F. oxysporum species complex (FOSC) are two related groups of plant pathogens causing a wide diversity of diseases in agricultural crops world wide. The aims of this study are (1) to clarify the phylogeny of the FFSC, (2) to identify potential deviation from tree-like evolution, (3) to explore the value of using mitogenomes for these kinds of analyses, and (4) to better understand mitogenome evolution. In total, we have sequenced 24 species from the FFSC and a representative set of recently analyzed FOSC strains was chosen, while F. redolens was used as outgroup for the two species complexes. A species tree was constructed based on the concatenated alignment of seven nuclear genes and the mitogenome, which was contrasted to individual gene trees to identify potential conflicts. These comparisons indicated conflicts especially within the previously described African clade of the FFSC. Furthermore, the analysis of the mitogenomes revealed the presence of a variant of the large variable (LV) region in FFSC which was previously only reported for FOSC. The distribution of this variant and the results of sequence comparisons indicate horizontal genetic transfer between members of the two species complexes, most probably through introgression. In addition, a duplication of atp9 was found inside an intron of cob, which suggests that even highly conserved mitochondrial genes can have paralogs. Paralogization in turn may lead to inaccurate single gene phylogenies. In conclusion, mitochondrial genomes provide a robust basis for phylogeny. Comparative phylogenetic analysis indicated that gene flow among and between members of FFSC and FOSC has played an important role in the evolutionary history of these two groups. Since mitogenomes show greater levels of conservation and synteny than nuclear regions, they are more likely to be compatible for recombination than nuclear regions. Therefore, mitogenomes can be used as indicators to detect interspecies gene flow.

Population Genomic Analysis Reveals a Highly Conserved Mitochondrial Genome in Fusarium asiaticum.

Fusarium asiaticum is one of the pivotal members of the Fusarium graminearum species complex (FGSC) causing Fusarium head blight (FHB) on wheat, barley and rice in large parts of Asia. Besides resulting in yield losses, FHB also causes the accumulation of mycotoxins such as nivalenol (NIV) and deoxynivalenol (DON). The aim of this study was to conduct population studies on F. asiaticum from Southern China through mitochondrial genome analyses. All strains were isolated from wheat or rice from several geographic areas in seven provinces in Southern China. Based on geographic location and host, 210 isolates were selected for next generation sequencing, and their mitogenomes were assembled by GRAbB and annotated to explore the mitochondrial genome variability of F. asiaticum. The F. asiaticum mitogenome proves extremely conserved and variation is mainly caused by absence/presence of introns harboring homing endonuclease genes. These variations could be utilized to develop molecular markers for track and trace of migrations within and between populations. This study illustrates how mitochondrial introns can be used as markers for population genetic analysis. SNP analysis demonstrate the occurrence of mitochondrial recombination in F. asiaticum as was previously found for F. oxysporum and implied for F. graminearum. Furthermore, varying degrees of genetic diversity and recombination showed a high association with different geographic regions as well as with cropping systems. The mitogenome of F. graminearum showed a much higher SNP diversity while the interspecies intron variation showed no evidence of gene flow between the two closely related and sexual compatible species.

Diversity of Mobile Genetic Elements in the Mitogenomes of Closely Related Fusarium culmorum and F. graminearum sensu stricto Strains and Its Implication for Diagnostic Purposes.

Much of the mitogenome variation observed in fungal lineages seems driven by mobile genetic elements (MGEs), which have invaded their genomes throughout evolution. The variation in the distribution and nucleotide diversity of these elements appears to be the main distinction between different fungal taxa, making them promising candidates for diagnostic purposes. Fungi of the genus Fusarium display a high variation in MGE content, from MGE-poor (Fusarium oxysporum and Fusarium fujikuroi species complex) to MGE-rich mitogenomes found in the important cereal pathogens F. culmorum and F. graminearum sensu stricto. In this study, we investigated the MGE variation in these latter two species by mitogenome analysis of geographically diverse strains. In addition, a smaller set of F. cerealis and F. pseudograminearum strains was included for comparison. Forty-seven introns harboring from 0 to 3 endonucleases (HEGs) were identified in the standard set of mitochondrial protein-coding genes. Most of them belonged to the group I intron family and harbored either LAGLIDADG or GIY-YIG HEGs. Among a total of 53 HEGs, 27 were shared by all fungal strains. Most of the optional HEGs were irregularly distributed among fungal strains/species indicating ancestral mosaicism in MGEs. However, among optional MGEs, one exhibited species-specific conservation in F. culmorum. While in F. graminearum s.s. MGE patterns in cox3 and in the intergenic spacer between cox2 and nad4L may facilitate the identification of this species. Thus, our results demonstrate distinctive traits of mitogenomes for diagnostic purposes of Fusaria.

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.

FgPex3, a Peroxisome Biogenesis Factor, Is Involved in Regulating Vegetative Growth, Conidiation, Sexual Development, and Virulence in Fusarium graminearum.

Peroxisomes are involved in a wide range of important cellular functions. Here, the role of the peroxisomal membrane protein PEX3 in the plant-pathogen and mycotoxin producer Fusarium graminearum was studied using knock-out and complemented strains. To fluorescently label peroxisomes' punctate structures, GFP and RFP fusions with the PTS1 and PTS2 localization signal were transformed into the wild type PH-1 and ΔFgPex3 knock-out strains. The GFP and RFP transformants in the ΔFgPex3 background showed a diffuse fluorescence pattern across the cytoplasm suggesting the absence of mature peroxisomes. The ΔFgPex3 strain showed a minor, non-significant reduction in growth on various sugar carbon sources. In contrast, deletion of FgPex3 affected fatty acid ß-oxidation in F. graminearum and significantly reduced the utilization of fatty acids. Furthermore, the ΔFgPex3 mutant was sensitive to osmotic stressors as well as to cell wall-damaging agents. Reactive oxygen species (ROS) levels in the mutant had increased significantly, which may be linked to the reduced longevity of cultured strains. The mutant also showed reduced production of conidiospores, while sexual reproduction was completely impaired. The pathogenicity of ΔFgPex3, especially during the process of systemic infection, was strongly reduced on both tomato and on wheat, while to production of deoxynivalenol (DON), an important factor for virulence, appeared to be unaffected.

First steps towards mitochondrial pan-genomics: detailed analysis of Fusarium graminearum mitogenomes.

There is a gradual shift from representing a species' genome by a single reference genome sequence to a pan-genome representation. Pan-genomes are the abstract representations of the genomes of all the strains that are present in the population or species. In this study, we employed a pan-genomic approach to analyze the intraspecific mitochondrial genome diversity of Fusarium graminearum. We present an improved reference mitochondrial genome for F. graminearum with an intron-exon annotation that was verified using RNA-seq data. Each of the 24 studied isolates had a distinct mitochondrial sequence. Length variation in the F. graminearum mitogenome was found to be largely due to variation of intron regions (99.98%). The "intronless" mitogenome length was found to be quite stable and could be informative when comparing species. The coding regions showed high conservation, while the variability of intergenic regions was highest. However, the most important variable parts are the intron regions, because they contain approximately half of the variable sites, make up more than half of the mitogenome, and show presence/absence variation. Furthermore, our analyses show that the mitogenome of F. graminearum is recombining, as was previously shown in F. oxysporum, indicating that mitogenome recombination is a common phenomenon in Fusarium. The majority of mitochondrial introns in F. graminearum belongs to group I introns, which are associated with homing endonuclease genes (HEGs). Mitochondrial introns containing HE genes may spread within populations through homing, where the endonuclease recognizes and cleaves the recognition site in the target gene. After cleavage of the "host" gene, it is replaced by the gene copy containing the intron with HEG. We propose to use introns unique to a population for tracking the spread of the given population, because introns can spread through vertical inheritance, recombination as well as via horizontal transfer. We demonstrate how pooled sequencing of strains can be used for mining mitogenome data. The usage of pooled sequencing offers a scalable solution for population analysis and for species level comparisons studies. This study may serve as a basis for future mitochondrial genome variability studies and representations.

Genomic Understanding of an Infectious Brain Disease from the Desert.

Rhinocladiella mackenziei accounts for the majority of fungal brain infections in the Middle East, and is restricted to the arid climate zone between Saudi Arabia and Pakistan. Neurotropic dissemination caused by this fungus has been reported in immunocompromised, but also immunocompetent individuals. If untreated, the infection is fatal. Outside of humans, the environmental niche of R. mackenziei is unknown, and the fungus has been only cultured from brain biopsies. In this paper, we describe the whole-genome resequencing of two R. mackenziei strains from patients in Saudi Arabia and Qatar. We assessed intraspecies variation and genetic signatures to uncover the genomic basis of the pathogenesis, and potential niche adaptations. We found that the duplicated genes (paralogs) are more susceptible to accumulating significant mutations. Comparative genomics with other filamentous ascomycetes revealed a diverse arsenal of genes likely engaged in pathogenicity, such as the degradation of aromatic compounds and iron acquisition. In addition, intracellular accumulation of trehalose and choline suggests possible adaptations to the conditions of an arid climate region. Specifically, protein family contractions were found, including short-chain dehydrogenase/reductase SDR, the cytochrome P450 (CYP) (E-class), and the G-protein ß WD-40 repeat. Gene composition and metabolic potential indicate extremotolerance and hydrocarbon assimilation, suggesting a possible environmental habitat of oil-polluted desert soil.

Recent Asian origin of chytrid fungi causing global amphibian declines.

Globalized infectious diseases are causing species declines worldwide, but their source often remains elusive. We used whole-genome sequencing to solve the spatiotemporal origins of the most devastating panzootic to date, caused by the fungus Batrachochytrium dendrobatidis, a proximate driver of global amphibian declines. We traced the source of B. dendrobatidis to the Korean peninsula, where one lineage, BdASIA-1, exhibits the genetic hallmarks of an ancestral population that seeded the panzootic. We date the emergence of this pathogen to the early 20th century, coinciding with the global expansion of commercial trade in amphibians, and we show that intercontinental transmission is ongoing. Our findings point to East Asia as a geographic hotspot for B. dendrobatidis biodiversity and the original source of these lineages that now parasitize amphibians worldwide.

A mobile pathogenicity chromosome in Fusarium oxysporum for infection of multiple cucurbit species.

The genome of Fusarium oxysporum (Fo) consists of a set of eleven 'core' chromosomes, shared by most strains and responsible for housekeeping, and one or several accessory chromosomes. We sequenced a strain of Fo f.sp. radicis-cucumerinum (Forc) using PacBio SMRT sequencing. All but one of the core chromosomes were assembled into single contigs, and a chromosome that shows all the hallmarks of a pathogenicity chromosome comprised two contigs. A central part of this chromosome contains all identified candidate effector genes, including homologs of SIX6, SIX9, SIX11 and SIX 13. We show that SIX6 contributes to virulence of Forc. Through horizontal chromosome transfer (HCT) to a non-pathogenic strain, we also show that the accessory chromosome containing the SIX gene homologs is indeed a pathogenicity chromosome for cucurbit infection. Conversely, complete loss of virulence was observed in Forc016 strains that lost this chromosome. We conclude that also a non-wilt-inducing Fo pathogen relies on effector proteins for successful infection and that the Forc pathogenicity chromosome contains all the information necessary for causing root rot of cucurbits. Three out of nine HCT strains investigated have undergone large-scale chromosome alterations, reflecting the remarkable plasticity of Fo genomes.

ToxGen: an improved reference database for the identification of type B-trichothecene genotypes in Fusarium.

Type B trichothecenes, which pose a serious hazard to consumer health, occur worldwide in grains. These mycotoxins are produced mainly by three different trichothecene genotypes/chemotypes: 3ADON (3-acetyldeoxynivalenol), 15ADON (15-acetyldeoxynivalenol) and NIV (nivalenol), named after these three major mycotoxin compounds. Correct identification of these genotypes is elementary for all studies relating to population surveys, fungal ecology and mycotoxicology. Trichothecene producers exhibit enormous strain-dependent chemical diversity, which may result in variation in levels of the genotype's determining toxin and in the production of low to high amounts of atypical compounds. New high-throughput DNA-sequencing technologies promise to boost the diagnostics of mycotoxin genotypes. However, this requires a reference database containing a satisfactory taxonomic sampling of sequences showing high correlation to actually produced chemotypes. We believe that one of the most pressing current challenges of such a database is the linking of molecular identification with chemical diversity of the strains, as well as other metadata. In this study, we use the Tri12 gene involved in mycotoxin biosynthesis for identification of Tri genotypes through sequence comparison. Tri12 sequences from a range of geographically diverse fungal strains comprising 22 Fusarium species were stored in the ToxGen database, which covers descriptive and up-to-date annotations such as indication on Tri genotype and chemotype of the strains, chemical diversity, information on trichothecene-inducing host, substrate or media, geographical locality, and most recent taxonomic affiliations. The present initiative bridges the gap between the demands of comprehensive studies on trichothecene producers and the existing nucleotide sequence databases, which lack toxicological and other auxiliary data. We invite researchers working in the fields of fungal taxonomy, epidemiology and mycotoxicology to join the freely available annotation effort.

The complete mitogenome of Fusarium culmorum.

The structure of the Fusarium culmorum mitogenome is similar to that of closely related Fusarium spp.: it has a total length of 103,844 bp, the base composition of the genome is the following: A (35.4%), T (32.9%), C (14.6%), and G (17.1%). The mitogenome contains 13 protein-coding genes, 2 ribosomal RNA (rRNA), and 28 transfer RNA (tRNA) genes, all coded on the same strand of DNA. The gene order is identical to that of the other Fusarium and Hypocreales mitogenomes. Maximum likelihood and Bayesian analysis based on the concatenated amino acid dataset of mitochondrial protein-coding genes confirmed close genetic relationship of F. culmorum to the other type B trichothecene producers F. graminearum and F. gerlachii.