Skip to main content
NCBI home page
ACS AuthorChoice logoLink to ACS AuthorChoice
. 2024 Feb 20;72(8):3949–3957. doi: 10.1021/acs.jafc.3c08437

Debunking the Myth of Fusarium poae T-2/HT-2 Toxin Production

Thomas E Witte †,‡, Carmen Hicks †, Anne Hermans †, Sam Shields †, David P Overy †,*
PMCID: PMC10905990  PMID: 38375818

Abstract

graphic file with name jf3c08437_0004.jpg

Fusarium poae is commonly detected in field surveys of Fusarium head blight (FHB) of cereal crops and can produce a range of trichothecene mycotoxins. Although experimentally validated reports of F. poae strains producing T-2/HT-2 trichothecenes are rare, F. poae is frequently generalized in the literature as a producer of T-2/HT-2 toxins due to a single study from 2004 in which T-2/HT-2 toxins were detected at low levels from six out of forty-nine F. poae strains examined. To validate/substantiate the observations reported from the 2004 study, the producing strains were acquired and phylogenetically confirmed to be correctly assigned as F. poae; however, no evidence of T-2/HT-2 toxin production was observed from axenic cultures. Moreover, no evidence for a TRI16 ortholog, encoding a key acyltransferase shown to be necessary for T-2 toxin production in other Fusarium species, was observed in any of the de novo assembled genomes of the F. poae strains. Our findings corroborate multiple field-based and in vitro studies on FHB-associated Fusarium populations which also do not support the production of T-2/HT-2 toxins with F. poae and therefore conclude that F. poae should not be generalized as a T-2/HT-2 toxin producing species of Fusarium.

Keywords: Fusarium poae, T-2 toxin, trichothecenes, mycotoxins, Fusarium head blight, metabolomics

Introduction

Fusarium poae is frequently isolated from Fusarium head blight (FHB)-damaged wheat, barley, and oat samples and is considered to be globally distributed.1,2 Often described as a “weak pathogen” in comparison to F. graminearum, F. poae infection during plant challenge experiments typically exhibits low levels of disease symptoms.3−8 Nevertheless, F. poae is a species of concern to cereal growers primarily because of its frequent detection in cereal samples and its ability to produce both type A and B trichothecene mycotoxins. F. poae trichothecene profiles from in vitro experiments are typically characterized by the presence of the type A trichothecenes diacetoxyscirpenol, monoacetoxyscirpenol, neosolaniol, and scirpentriol and the type B trichothecenes fusarenon-X and nivalenol (Table 1). Interestingly, in rare instances, subsets of F. poae isolates have been reported to produce the highly toxic type A trichothecenes T-2 and HT-2 toxins,9,10 which are of utmost concern to cereal producers because T-2 and HT-2 toxins are acutely toxic to consumers11,12 and are strictly regulated in feed.13,14 Given the gravity of the association of F. poae and T-2/HT-2 toxin production, and the rarity of observed production, it is imperative to critically evaluate previous claims of T-2/HT-2 production by F. poae strains.

Table 1. Summary of F. poae Trichothecene Profiling Resultsa.

    no. of strains producing detected trichothecenes
research study F. poae strains T-2/HT-2 DAS MAS FX NIV SCR NEO
Thrane et al., 2004 49 6 46 45 38 41 33 7
Vogelgsang et al., 2008a 3 0 3 3 <3 3 – <3
Kokkonen et al., 2010 3 0 0 – – – – 0
Somma et al., 2010 81 0 59 – 59 69 – 62
Stenglein et al., 2014 5 0 – – – 5 – –
Vanheule et al., 2017 61 0 59 – 16 12 – 54
O’Donnell et al., 2018 3 0 3 – – – – –
Witte et al., 2021 38 0 36 36 36 33 35 36
Kahla et al., 2023 2 0 1 – – – – 0
Totals 245 6 207 84 149 163 68 159
Open in a new tab
a

Abbreviations: DAS, 3,15-diacetoxyscirpenol; MAS, 15-monoacetoxyscirpenol; FX, fusarenon-X; NIV, nivalenol; SCR, scirpentriol; NEO, neosolaniol; −, not tested.

Trichothecenes are a diverse group of nonvolatile sesquiterpenoid natural products that cause deleterious effects on plant and animal cells through impairment of ribosomal peptide synthesis and mitochondrial function. Composed of highly functionalized 12,13-epoxytrichothecene scaffolds, trichothecenes are broadly classified as types A and B based on scaffold substitution profiles (detected in Fusarium spp.), or as types C and D by the presence of longer side chains which may be macrocyclized (not associated with Fusarium spp.). Trichothecene biosynthesis follows a multistep pathway involving enzymes encoded by TRI genes that are mostly clustered at a single genomic locus; however, three enzymes involved in tailoring trichothecene scaffolds are encoded by genes residing on two additional loci in many Fusarium species,15 including the TRI101 locus and a two-gene TRI1-TRI16 cluster which plays an essential role in the production of T-2/HT-2 toxins. Proposed biosynthetic routes for Fusarium trichothecenes have been extensively reviewed (for example, see ref (16)); with particular relevance to our current study, a critical biosynthetic step in the production of T-2/HT-2 toxins in Fusarium spp. involves the action of the TRI16 acyltransferase to acylate a hydroxyl at the C8 position of the trichothecene scaffold.

When comparing literature summarizing trichothecene production in F. poae, we noticed that the association of T-2/HT-2 toxin production with F. poae can be traced almost entirely to a single, influential study of secondary metabolites produced by F. poae, F. sporotrichioides, and F. langsethiae by Thrane et al.9 In that study, Thrane et al. focused on clarifying the mycotoxin profile of F. langsethiae, which at that time was only recently described, in contrast to F. sporotrichioides and F. poae, two other commonly detected species of head-blight associated Fusaria. Thrane et al.9 identified T-2 toxin associated trichothecenes (including T-2 toxin, HT-2 toxin and T-2 tetraol) in extracts of all F. langsethiae and F. sporotrichioides strains, as well as from a few of the F. poae strains examined (Table 1). The production of T-2/HT-2 toxins from F. poae was considered rare by Thrane et al.,9 detected in only 6/49 strains profiled, and was investigated by three independent laboratories, either via GC–MS/MS analysis of pentafluoropropionoinyl esterified derivatives (2 laboratories), or by GC electron capture detection of heptafluorbutyryl and trimethylsilyl derivatives (1 laboratory), in comparison to commercially available standards of T-2, HT-2, and T-2 tetraol toxins. Although the strains were grown on various media conditions by the different contributing laboratories, the published results neither specify which of the laboratories positively identified the presence of the toxins, on which media conditions the toxins were produced, nor is there reported biological replication employed in any experiments to confirm initial observations. Furthermore, beyond the labeling of “trace” amounts, no MS mass feature signal intensities were published, although the rarity of the F. poae-associated production of T-2 toxin is noted and described as “low amounts” by Thrane et al.9 Thrane et al.9 justifiably concluded that F. langsethiae and F. sporotrichioides are the primary culprits for T-2/HT-2 toxin production in the FHB disease complex.

What other historical evidence is there supporting the production of the T-2/HT-2 toxin by F. poae? In the time since the publication of Thrane et al.,9 subsequent mycotoxin profiling of reliably identified F. poae strains by multiple researchers has consistently reported the absence of T-2/HT-2 production from this species.1,17−24 To date, from literature accounts, 245 different strains of F. poae have been profiled for trichothecene content, with only the six strains identified by Thrane et al.9 being reported as T-2/HT-2 producers (Table 1). Although a few reports tentatively link F. poae-inoculated plants or in vitro media conditions with T-2 toxin detection, those reports describe unconvincingly “trace” T-2 or HT-2 signals observed at or below the limit of detection and were not reported on a per-strain basis,25,26 or the reports described field-sampling experiments seeking to correlate the quantitation of T-2/HT-2 in bulk homogenized field samples with that of the presence of Fusarium species’ DNA amplified from the bulk samples.10,27 Although this latter type of correlation analysis is a useful method for generating broad hypotheses linking toxins to groups of species within a complex such as FHB, the analysis of bulk homogenized field samples prohibits conclusive links between toxins and fungal species due to the frequent, in-field co-occurrence of F. poae with other known T-2 toxins producers, such as F. langsethiae or F. sporotrichioides. In contrast to these few studies, the majority of correlation analyses from cereal grain sampling surveys have consistently demonstrated that T-2/HT-2 detection is not positively correlated with the presence of F. poae DNA.28−37

The chemical profiling work published by Thrane et al.9 has been cited over 250 times in the past two decades, and of these, at least 43 citations attribute T-2 toxin production to the concept of F. poae as a species (based solely on observations from 6/49 strains reported in the Thrane et al. study). Additionally, many review articles and manuscript discussions simply refer to F. poae as a known T-2 toxin producer without providing any supporting evidence, even though in the study by Thrane et al.9 the reported occurrence of observed T-2/HT-2 toxin production in F. poae was limited to only 12% of the strains examined, or 2% of all F. poae strains profiled and reported in the literature to date (Table 1). The consistent absence of T-2/HT-2 production in strains of F. poae from numerous pathogenicity surveys and chemical screening has led multiple research groups to speculate that the reported T-2/HT-2-producing strains characterized by Thrane et al.9 were likely misidentified F. langsethiae isolates.23,26,38−40 This speculation is reasonable since F. langsethiae, previously referred to in the literature as “powdery poae” due to the macro- and microphenotypic similarity between the two species, is a known producer of T-2/HT-2 toxins. Indeed, any historical study of F. poae should be interpreted with caution, given that morphology alone cannot reliably distinguish this species from F. langsethiae, which was not formally described until 2004.41,42 Nevertheless, the F. poae strains from Thrane et al.9 were taxonomically supported by phylogenetics and by the simultaneous detection of other secondary metabolites such as beauvericin, a cyclic depsipeptide produced primarily by F. poae and not F. langsethiae, in reportedly T-2/HT-2 producing F. poae strain extracts. This level of support therefore weakens the “misidentification” speculation.

Given the importance of T-2 toxin contamination to agronomic trade and consumer health, we decided to further investigate the T-2 toxin-producing strains identified by Thrane et al.9 using a combination of targeted metabolomics and whole-genome sequencing. The two selected approaches were employed to confirm the chemical detection of T-2/HT-2 toxins (and relevant analogs) from strain extracts on a trichothecene-permissive medium, as well as the detection of TRI16 orthologs within the genomes of the various strains that would support the possibility of T-2/HT-2 toxin production, even in the absence of detected chemical signals from the extracts.

Materials and Methods

Strain Selection

Five F. poae strains identified by Thrane et al.9 as T-2 and/or HT-2 producers (IBT 9924, IBT 9928, IBT 9976, IBT 9988, and IBT 400006) and a sixth strain reported to produce T-2 tetraol (IBT 9973) were requested from the IBT (Institut for Bioteknologi) strain collection at the Denmark Technical University Bioengineering department. All strains were successfully revived and maintained on Spezieller Nahrstoffarmer agar (SNA) plates, except for IBT 9976, which had lost viability in storage. The SNA medium formulation used consisted of 1 g of KH2PO4, 1 g of KNO3, 0.5 g of MgSO4·7H2O, 0.5 g of KCl, 0.2 g of glucose, 0.2 g of sucrose, and 20 g of agar per 1 L of distilled water.

Three additional strains were added to the analysis: F. poae strain Fp157 is a Canadian isolate whose genome and chemical phenotype was previously published,23F. langsethiae strain Fl201059 has been the subject of previous genetic and chemical profiling,43 and F. sporotrichioides strain Fsp184 is available at the Canadian Collection of Fungal Cultures (under accession DAOMC 238877).

Metabolomics Protocol: Fermentation, Extraction, and UPLC–HRMS Analysis

The strain culturing, extract production, and data analysis protocols used in this study closely followed those published in Witte et al.,22 with the exception that only Yeast-Extract-Sucrose (YES) medium, a trichothecene-production eliciting medium, was used as a growth medium for the strains. The YES media formulation consists of 20 g of yeast extract, 150 g of sucrose, and 500 mg of MgSO4·7H2O per 1 L of distilled water. In brief, mycelium plugs were inoculated into slant tubes containing 15 mL of liquid YES medium in four replicates. After 14 days of growth at 25 °C in the dark, the mycelia were separated from the broth, and both broth and mycelium were frozen as separate samples. Samples were thawed, extracted in 15 mL of ethyl acetate, dried, and then resuspended in MS-grade methanol to a concentration of 500 μg/mL. The resuspended extracts were then analyzed on a Thermo Ultimate 3000 ultrahigh pressure liquid chromatograph coupled to a Thermo LTQ Orbitrap XL high resolution mass spectrometer (UPLC–HRMS). Reverse-phase chromatography was performed using a Phenomenex C18 Kinetex column (50 mm × 2.1 mm ID, 1.7 μm) running a gradient of water and acetonitrile exactly as published in Witte et al.,22 and mass spectrometry was performed in positive mode only.

Metabolomics Data Analysis

The resulting high-resolution mass spectrometry RAW files were preprocessed using MZmine v2.59 or else directly examined in Thermo Qual Browser by plotting extracted ion chromatographs for each mass/charge ratio (m/z) and comparing them to standards processed under the same UPLC–HRMS methods. Within MZmine2, mass features were characterized by comparison to known retention times and exact m/z of mycotoxin standards, using the “Targeted feature detection” module with an intensity tolerance of 10%, a noise level of 3.0 E4, a m/z tolerance of 5.0 ppm, and a retention time tolerance of 0.08 min. Parameters were chosen based on careful examination of raw spectra including from methanol blanks processed every tenth sample, and uninoculated media extractions (YES media broth, extracted as described above) as controls. Mass feature annotation was enabled by comparison to commercial standards for T-2 toxin, HT-2 toxin, 3,15-diacetoxyscirpenol, neosolaniol, fusarenon-X, 15-monoacetoxyscirpenol, apicidin, aurofusarin, and beauvericin. If no standards were available, mass features were compared to features annotated from F. poae strain Fp157, including W-493 A/W-493 B, fusarin C, fusarin A, fusarin PM, and diacetoxynivalenol.23 Peak area values for the mass feature putatively annotated as fusarin C were selected to represent the “fusarin-associated” annotation. Other mass features matching the exact mass (<5 ppm) of T-2 toxin analogs, including 4-propanoyl T-2 toxin, 3-hydroxy T-2 toxin, and acetyl T-2 toxin, were tentatively annotated based on exact m/z matches, as no commercial standards were available for these features.

Genomic DNA Purification and Sequencing

Genomic DNA (gDNA) for Illumina genome sequencing was generated by inoculating mycelium from F. poae isolates grown on SNA media into 250 mL Erlenmeyer flasks containing 50 mL first-stage media.44 The cultures were incubated at 25 °C and shaken at 180 rpm for 4 days. The mycelia were isolated by vacuum filtration, washed using sterile water, frozen in liquid nitrogen, and ground using a mortar and pestle until a fine powder was produced. Genomic DNA was extracted using a cetyltrimethylammonium bromide (CTAB) protocol with minor modifications including a scale up for a larger DNA yield, and a sodium acetate DNA precipitation in place of ammonium acetate.45 Isolated DNA was subjected to an RNase treatment post extraction and cleaned using the Genomic DNA Clean and Concentrator (Zymo Research, Irvine, CA).

The gDNA pellet was reconstituted in 200 μL of 10 mM Tris pH 8.0, and the gDNA concentration determined using a FLUOstar OPTIMA fluorometer (BMG LABTECH) and a PicoGreen dsDNA Quantitation Kit (Molecular Probes Inc.). The reconstituted gDNA was mechanically sheared to ∼300 bp fragments with a Covaris LE220 instrument and used as a template to construct PCR free Libraries with the NxSeq AmpFREE Low DNA Library kit (Lucigen) and TruSeq CD dual indices (Illumina) according to the Lucigen’s Library protocol. Indexed libraries were pooled, and sequencing was carried on a NextSeq500/550 (Illumina) using 2 × 150 bp NextSeq High Output Reagent Kit (Illumina) according to the manufacturer’s recommendations in order to obtain paired-end reads.

Genome Assembly

Raw reads were trimmed of adapters, poor quality sites, and trailing G′s using fastp v0.23.2.46 Scaffold assembly was performed using SPAdes v3.10.1.47 The resulting assemblies were assessed for quality using Quast v5.0.2,48 and completeness was predicted via assessment of Hypocreales-associated benchmarked universal single copy ortholog (BUSCO) presence (4,494 orthologs from database hypocreales_odb10) using BUSCO v5.2.2,49 with gene models predicted using Augustus with F. graminearum preset training parameters.50

Phylogenetic Analysis

All genomes not sequenced and assembled in this study were downloaded from the Genbank repository of nucleotide sequences. A total of 4,053 BUSCO genes (Hypocreales_odb10 database) were detected as “single copy” and “complete” in all genomes using BUSCO v5.2.2,49 then aligned using MAFFT v7.47051 and trimmed with automated parameter detection using trimal v1.2.52 IQTREE v2.0 was used to infer phylogenetic relationships, using the Maximum Likelihood method with best model automatically determined per gene sequence using ModelFinder53 as part of the IQ-TREE v2.0.6 pipeline.54 Partition modeling was used, allowing genes to evolve under independent models.55 Bootstraps were calculated using both sh-LRT (n = 1000) and ultrafast values (n = 1000) and the tree was drawn to scale, with substitutions per site used to calculate branch lengths.56

Gene Model Prediction and TRI16 Homologue Search

Gene models were predicted from repeat-masked, de novo assembled genomes using FUNANNOTATE v1.8.14.57 RepeatModeler v2.0.158 was used to generate de novo libraries of repeated sequences for each genome, using the RepBase 2018 library of transposable elements (TE’s) to annotate TE’s, and RepeatMasker v4.2.1-p1 was used to softmask the assemblies in preparation for gene annotation. The FUNANNOTATE predict pipeline then used Evidence Modeler59 to weigh predicted models generated using BUSCO-supplied models to train Augustus v3.5.0 (with conserved gene models predicted from the sordariomycetes database),49,50 and GeneMark-ES.60F. venenatum TRI16 (FVRRES_00063) nucleotide and amino acid sequences (XP_025587271.1) were used as queries to search for related genes in the de novo assemblies and predicted proteomes via BLASTn, tBLASTx, and BLASTp using default parameters within Geneious v 2022.2.2. Although F. venenatum has not yet been associated with T-2/HT-2 toxin production,40 it is more closely related to F. poae than other TRI16-containing species (queries using the F. sporotrichioides TRI16 sequence gave similar results).

Results and Discussion

Taxonomic Confirmation of IBT Strains as Fusarium poae

Five strains of F. poae were reported to produce either T-2 toxin or HT-2 toxin, and a sixth strain was reported to produce T-2 tetraol.9 Of these strains, five were successfully revived (IBT 9924, IBT 9928, IBT 9973, IBT 9988, and IBT 40006), and a sixth (IBT 9976) was reported to have lost viability in storage. The five viable strains were whole-genome sequenced using Illumina Nextseq short-read sequencing to confirm their taxonomic relationship to previously genome sequenced F. poae isolates. All assembled genomes were assessed as high quality, at over 99.7% complete by analysis of Hypocreales benchmarked universal single-copy orthologs (BUSCOs), with an average length of approximately 38.6 Mb (Table 2). To infer evolutionary relationships, a phylogenetic cladogram was constructed using 4,053 BUSCOs modeled from the F. poae strains and a selection of relevant Fusarium species from the Sambucinum species complex, representing the Sambucinum, Sporotrichioides, Graminearum, Longipes, and Brachygibbosum clades, with three strains from the F. incarnatum-equiseti species complex included as an outgroup. The modeled BUSCOs incorporate 7,055,724 base pairs or approximately 20% of the total averaged F. poae assembly lengths and represent putative housekeeping or “core” gene coding sequences. Our analysis confirms all strains sequenced in this study were correctly assigned by Thrane et al.9 to a contemporary understanding of the F. poae taxon (Figure 1) and thereby refutes speculation that these were misidentified F. langsethiae (“powdery poae”) strains. Most are mating type 1–2, which is considered the rarer of the two mating types in profiled populations.1,23

Table 2. Fusarium poae Genome Statistics.

Strain ID IBT 9924 IBT 9928 IBT 9988 IBT 40006 IBT 9973
no. contigs (≥0 bp) 1729 1610 1701 1862 1725
no. contigs (≥1000 bp) 1180 1119 1206 1244 1179
Total length (≥0 bp) 38,587,486 38,642,078 38,591,118 38,557,541 38,447,436
GC (%) 47.33 47.28 47.43 47.58 47.14
N50 169,178 189,771 180,810 177,076 195,830
L50 68 57 63 66 60
no. N’s per 100 kbp 8.84 8.36 6.27 11.31 7.18
Estimated sequencing coverage (X) 133.6 169.6 132.9 121.9 131.5
BUSCO completeness (%) 99.8 99.7 99.7 99.7 99.7
Mating type 1–2 1–2 1–1 1–2 1–2
Genome accession JASDAK010000000 JASDAL010000000 JASDAM010000000 JASDAN0100000000 JASDAO0100000000
SRA accession SAMN34361090 SAMN34361091 SAMN34361092 SAMN34361093 SAMN34361094
Open in a new tab

Figure 1.

Figure 1

Open in a new tab

Maximum likelihood tree of representative isolates from clades within the Fusarium sambucinum species complex, built from nucleotide sequence alignments of 4,053 single copy orthologs (BUSCOs) from whole genome assemblies, including five strains sequenced in this study (red text). Representatives of the F. incarnatum-equiseti species complex were included as an outgroup. All IBT strains sequenced in this study cluster with F. poae strains, supporting their taxonomic assignment as F. poae. Numbers at nodes are bootstrap support values (SH-aLRT, n = 1000). Tree computed using IQTREE2.

Targeted Metabolomics Analysis Does Not Support T-2 Toxin Production

Organic extracts from axenically cultured strains were profiled for trichothecene content as well as all other secondary metabolites associated with F. poae, namely beauvericin, aurofusarin, fusarins, and W-493 A/B,1,9,23 using UPLC–HRMS. In addition to the five strains profiled from Thrane et al.,9 YES culture extracts from F. poae strain Fp157, F. langsethiae strain Fl201059, and F. sporotrichioides strain Fsp184 were also included in the targeted metabolomics analysis, allowing for confirmation that the culturing methods used were suitable for elicitation of T-2/HT-2 toxins from known producers (F. langsethiae and F. sporotrichioides). Our analysis indicated mass features associated with T-2/HT-2 toxins (including T-2 toxin and analogs HT-2 toxin, 4-propanoyl-T-2 toxin, 3′-hydroxy-T-2 toxin, and acetyl-T-2 toxin) were present in the F. sporotrichioides and F. langsethiae extracts but absent from all F. poae extracts (Figure 2). Moreover, the trichothecene profiles of most F. poae strains were consistent with known F. poae chemical phenotypes: specifically, diacetoxyscirpenol was detected as the primary trichothecene from all strains except IBT 9973 and IBT 9928, which had poor growth in YES media and did not produce any trichothecenes above the level of detection. Neosolaniol and fusarenon-X were detected from three strains, and 15-monoacetoxyscirpenol was detected from two strains. Neither nivalenol nor T-2 tetraol was detected in any of the strain extracts. This pattern is consistent with Vanheule et al.’s description of a “hierarchy” of F. poae trichothecene detection from in vitro culturing,1 wherein diacetoxyscirpenol is detected in greatest abundance from all TRI-producing strains, followed by increasingly infrequent detections of neosolaniol, fusarenon-X, and last nivalenol (if nivalenol is detected at all). The profiles are also consistent with Witte et al.’s profiling of Eastern Canadian F. poae strains.23 Other nontrichothecene F. poae-associated secondary metabolite mass features were also characterized, including beauvericin (5/5 strains), aurofusarin (4/5 strains), W-493B (4/5 strains), W-493 A (2/5 strains), and fusarin-associated metabolites (4/5 strains). Taken together, all chemical phenotypes of the strains profiled in Thrane et al.9 are consistent with known F. poae chemical phenotypes, even in the cases where trichothecenes were not detected, suggesting all five IBT strains are all nonexceptional F. poae strains and are not T-2 toxin producers under the conditions tested.

Figure 2.

Figure 2

Open in a new tab

Targeted secondary metabolite profiles from UPLC-HRMS analysis of extracts from six F. poae strains, one F. langsethiae strain (Fl201059), and one F. sporotrichioides strain (Fsp184), cultured axenically on YES medium. Abbreviations: DAS, 3,15-diacetoxyscirpenol; 15-MAS, 15-monoacetoxyscirpenol; and DAN, diacetylnivalenol.

An Intact TRI16 Gene Is Absent in All IBT F. poae Genomes

Recognizing that strain degeneration can occur due to prolonged periods of cryostorage (20+ years in this case) and might therefore lead to irreproducibility of the results observed by Thrane et al.,9 we searched the genomes of the IBT F. poae strains for the presence of homologues of the acyltransferase-encoding gene TRI16, expression of which is required for T-2/HT-2 metabolite production61 (Figure 3A). Whole-genome assemblies were generated for all of the IBT strains to account for the possibility of potential TRI16 homologue sequence divergence (Table 2). This approach was particularly relevant for F. poae since recent genomic and metabolomic studies of F. poae isolates have identified small, strain-specific and transcriptionally active chromosomes associated with the evolution of novel chemical phenotypes, termed “supernumerary” or “accessory” chromosomes,23,62,63 on which a potential TRI16 homologue could reside and would account for unique trichothecene production in a subpopulation of F. poae. Each of the genomes were therefore assembled de novo rather than aligning to a reference assembly, and our TRI16-homologue search was broadened to include divergent genes with predicted acyltransferase function.

Figure 3.

Figure 3

Open in a new tab

(A) Diagram of the final biosynthetic steps of the production of T-2 toxin by F. sporotrichioides, adapted from McCormick et al.16 Proposed biosynthetic steps involved in the production of neosolaniol and fusarenon-X, also detected from F. poae extracts, include alternative degrees of TRI1-mediated oxidation of carbons 7 and 8, and are not shown here. (B) Comparison of the two-gene TRI1–TRI16 biosynthetic gene cluster neighborhood in relevant Fusarium species, including all F. poae strains profiled in this study, F. poae strains 2516 and Fp157, F. venenatum strain A3/5, and strains from T-2 toxin producing species F. langsethiae and F. sporotrichioides. Hollow triangles indicate pseudogenized remnants of TRI16 in F. poae genomes and one other pseudogene in the neighborhood of TRI1 in IBT 40006. The F. poae IBT 40006 genome was also fragmented at a region of low GC content immediately adjacent to the TRI1 gene and TRI16 remnant (relevant contigs were concatenated for the synteny analysis).

The results of our genomic profiling were consistent with previous analyses exploring the evolution of trichothecene biosynthesis in Fusarium:15F. poae isolates likely all evolved from a common ancestral lineage in which the TRI16 gene appears alongside TRI1 in a two-gene cluster. However, in all F. poae strains sequenced so far, including the five published in this study, Belgian isolate 2516, and Canadian isolate Fp157, TRI16 has been pseudogenized via truncation, while TRI1 appears intact (Figure 3B). This pattern was also confirmed in 58 additional unpublished F. poae genomes from Canadian isolates (data not shown). The truncated TRI16 pseudogene sequence appears as an approximately 445 base-pair fragment of the 3′-terminus of the gene, whereas the functional TRI16 gene is approximately 1,450 base pairs long in closely related species F. sporotrichioides, F. langsethiae, and F. venenatum. Apart from the truncated TRI16 pseudogene, no other genes with notable homology (minimum 30% amino acid identity over 70% of the query sequence, see Supporting Information Tables S1 and S2) to F. sporotrichioides or F. venenatum TRI16 was detected anywhere in the assembled F. poae genomes/proteomes. BLASTp searches querying the amino acid sequence of F. venenatum TRI16 against the predicted proteomes of the F. poae strains matched only very distantly related orthologs which are present in all F. poae genomes sequenced to date and are not associated with trichothecene C8 acylation. The nearest detected relative of F. venenatum TRI16 in F. poae is TRI101 (20.4% nt identity over 98% of the query sequence), which is consistent with previously established TRI16/TRI101 evolutionary relationships.61 Taken together, the results strongly indicate that accessory genome-associated TRI16 orthologues are absent in this population. Other putative acyltransferases encoded for in genomes examined were both unrelated to TRI16 and were present in all F. poae genomes published to date (as well as the 58 unpublished genomes we have generated thus far), suggesting that their presences are not specific to the reportedly T-2/HT-2 toxin producing strains from Thrane et al.9 Therefore, it is highly unlikely that the distantly related acyltransferase homologues convergently evolved to encode for enzymes that will acylate the 3-acetylneosolaniol C8 hydroxyl with isovalerate to produce T-2/HT-2 toxins in the strains profiled by Thrane et al.9

Evidence Is Lacking for the Production of T-2 or HT-2 Toxins by F. poae

Precedence in the scientific literature for T-2/HT-2 toxin production as attributed to F. poae is associated with a select few F. poae strains, production of which was reported as rare and of low abundance by Thrane et al.9 Taken as a whole, the genetic and metabolomic evidence presented in our current study indicates that the F. poae strains profiled by Thrane et al.9 are not able to produce T-2 toxin or related analogs. We have confirmed the strains were not misidentified, as supported by whole genome-based phylogenetic analyses and chemical phenotypes, which were consistent with recent work profiling European and Canadian F. poae populations. It is therefore difficult to explain the initial reports of T-2/HT-2 production from said strains. The possibility that these strains once had the capacity to make T-2 associated toxins, due to the presence of a TRI16 homologue (possibly in an accessory region or plasmid), is unlikely since all five strains would have had to have simultaneously lost this ability while in storage. The mycotoxin profiles detected in this study are consistent with the presence of a trichothecene biosynthetic pathway that progresses no further than neosolaniol production. Given that neosolaniol is the last biosynthetic precursor to T-2/HT-2 toxins and involves activation of TRI1, the genetic neighbor of TRI16 needed for T-2/HT-2 toxin production, we can reasonably conclude that all homologous biosynthetic gene clusters associated with T-2/HT-2 toxin production in F. sporotrichioides are activated in the trichothecene-producing F. poae strains included in this study, and the resulting lack of T-2/HT-2 toxin production indicates the strains are in fact not capable of T-2/HT-2 production.

Without a more detailed accounting of the work of Thrane et al.,9 including the diagnostic mass feature signal intensities observed, experimental replication/validation, media conditions tested, and specificity of which laboratories reported positive detection of the T-2/HT-2 toxins for the F. poae strains, it remains difficult to speculate on why these strains were associated with low abundance T-2/HT-2 toxin production. Nevertheless, given the widespread occurrence of F. poae in FHB surveys, the extreme toxicity of T-2 associated mycotoxins, and the lack of metabolomic and genomic evidence for T-2/HT-2 toxin production from any F. poae strain profiled to date, we believe it is important at this time to decouple the association of the species F. poae with the production of the T-2/HT-2 toxin. We reiterate that there has been no convincing proof that any F. poae strain produces T-2/HT-2 toxins, and moving forward, we consider it incumbent on future F. poae researchers to conclusively prove a strains’ capacity to produce T-2/HT-2 toxin if they intend to generalize the F. poae species as such a producer. Additionally, we note that our assertions do not diminish the need to further study F. poae, as it is a proven producer of trichothecene mycotoxins, which include diacetoxyscirpenol, nivalenol, and fusarenon-X, each of which are demonstrably toxic to consumers and should be of concern to cereal producers, particularly of oats.

Acknowledgments

The authors would like to acknowledge Dr. Ulf Thrane, who provided comments on the manuscript prior to submission.

Data Availability Statement

Assembled genomes and raw reads were published at the NCBI Genbank/SRA under bioproject ID PRJNA961673. All assembly versions included in this study are the first versions.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.3c08437.

  • tblastx results of TRI16 search; blastp results of TRI16 search (PDF)

Author Contributions

TW and DO conceived the study; TW performed metabolomic and phylogenetic data analyses, genome assembly and annotation, and wrote the first draft of the manuscript; CH and TW performed DNA extractions; AH performed strain culturing and metabolomic sample preparation; SS performed UPLC-HRMS sample analysis; DO acquisition of funding, project administration, and supervision of TW, CH, AH, and SS; TW and DO edited the manuscript with input from all authors.

This research was funded by the AAFC research grant J-002071 (The population structure of Fusarium pathogens of small grain cereals, their distribution and relationship to mycotoxins). TW is also supported by an NSERC PGS-D scholarship.

The authors declare no competing financial interest.

Supplementary Material

jf3c08437_si_001.pdf (85.1KB, pdf)

References

  1. Vanheule A.; De Boevre M.; Moretti A.; Scauflaire J.; Munaut F.; De Saeger S.; Bekaert B.; Haesaert G.; Waalwijk C.; Van Der Lee T.; et al. Genetic Divergence and Chemotype Diversity in the Fusarium Head Blight Pathogen Fusarium Poae. Toxins 2017, 9 (9), 1–19. 10.3390/toxins9090255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Yli-Mattila T.; Mach R. L.; Alekhina I. A.; Bulat S. A.; Koskinen S.; Kullnig-Gradinger C. M.; Kubicek C. P.; Klemsdal S. S. Phylogenetic Relationship of Fusarium Langsethiae to Fusarium Poae and Fusarium Sporotrichioides as Inferred by IGS, ITS, β-Tubulin Sequences and UP-PCR Hybridization Analysis. Int. J. Food Microbiol. 2004, 95 (3), 267–285. 10.1016/j.ijfoodmicro.2003.12.006. [DOI] [PubMed] [Google Scholar]
  3. Brennan J. M.; Fagan B.; van Maanen A.; Cooke B. M.; Doohan F. M. Studies on in Vitro Growth and Pathogenicity of European Fusarium Fungi. European Journal of Plant Pathology 2003, 109 (6), 577–587. 10.1023/A:1024712415326. [DOI] [Google Scholar]
  4. Browne R. A.; Cooke B. M. Resistance of Wheat to Fusarium Spp. in an in Vitro Seed Germination Assay and Preliminary Investigations into the Relationship with Fusarium Head Blight Resistance. Euphytica 2005, 141 (1), 23–32. 10.1007/s10681-005-4820-0. [DOI] [Google Scholar]
  5. Imathiu S. M.; Hare M. C.; Ray R. V.; Back M.; Edwards S. G. Evaluation of Pathogenicity and Aggressiveness of F. Langsethiae on Oat and Wheat Seedlings Relative to Known Seedling Blight Pathogens. European Journal of Plant Pathology 2010, 126 (2), 203–216. 10.1007/s10658-009-9533-0. [DOI] [Google Scholar]
  6. Martin C.; Schöneberg T.; Vogelgsang S.; Mendes Ferreira C. S.; Morisoli R.; Bertossa M.; Bucheli T. D.; Mauch-Mani B.; Mascher F. Responses of Oat Grains to Fusarium Poae and F. Langsethiae Infections and Mycotoxin Contaminations. Toxins 2018, 10 (1), 1–18. 10.3390/toxins10010047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Tan J.; Ameye M.; Landschoot S.; De Zutter N.; De Saeger S.; De Boevre M.; Abdallah M. F.; Van der Lee T.; Waalwijk C.; Audenaert K. At the Scene of the Crime: New Insights into the Role of Weakly Pathogenic Members of the Fusarium Head Blight Disease Complex. Molecular Plant Pathology 2020, 21 (12), 1559–1572. 10.1111/mpp.12996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Xue A. G.; Ho K. M.; Butler G.; Vigier B. J.; Babcock C. Pathogenicity of Fusarium Species Causing Head Blight in Barley. phyto 2006, 87 (2), 55–61. 10.7202/013973ar. [DOI] [Google Scholar]
  9. Thrane U.; Adler A.; Clasen P. E.; Galvano F.; Langseth W.; Lew H.; Logrieco A.; Nielsen K. F.; Ritieni A. Diversity in Metabolite Production by Fusarium Langsethiae, Fusarium Poae, and Fusarium Sporotrichioides. Int. J. Food Microbiol. 2004, 95 (3), 257–266. 10.1016/j.ijfoodmicro.2003.12.005. [DOI] [PubMed] [Google Scholar]
  10. Vogelgsang S.; Sulyok M.; Hecker A.; Jenny E.; Krska R.; Schuhmacher R.; Forrer H. R. Toxigenicity and Pathogenicity of Fusarium Poae and Fusarium Avenaceum on Wheat. European Journal of Plant Pathology 2008, 122 (2), 265–276. 10.1007/s10658-008-9279-0. [DOI] [Google Scholar]
  11. World Health Organization . Evaluation of Certain Contaminants in Food: Ninety-Third Report of the Joint FAO/WHO Expert Committee on Food Additives; WHO, 2023. https://www.who.int/publications-detail-redirect/9789240068452 (accessed 01-13-2024).
  12. Rocha O.; Ansari K.; Doohan F. M. Effects of Trichothecene Mycotoxins on Eukaryotic Cells: A Review. Food Additives and Contaminants 2005, 22 (4), 369–378. 10.1080/02652030500058403. [DOI] [PubMed] [Google Scholar]
  13. van Egmond H. P.; Jonker M. A.. Worldwide Regulations for Mycotoxins in Food and Feed in 2003; Food and Agriculture Organization of the United Nations, 2004. [Google Scholar]
  14. Draft amendment to EU regulation. Draft of COMMISSION REGULATION (EU) Amending Regulation (EU) 2023/915 as Regards Maximum Levels of T-2 and HT-2 Toxins in Food. CMTD(2023)28: (I) NOVEL FOOD AND TOXICOLOGICAL SAFETY OF THE FOOD CHAIN/ 22 September- In-person meeting; European Commission, 2023. https://ec.europa.eu/transparency/comitology-register/screen/documents/092050/1/consult?lang=en (accessed 01-13-2024).
  15. Proctor R. H.; McCormick S. P.; Alexander N. J.; Desjardins A. E. Evidence That a Secondary Metabolic Biosynthetic Gene Cluster Has Grown by Gene Relocation during Evolution of the Filamentous Fungus Fusarium. Mol. Microbiol. 2009, 74 (5), 1128–1142. 10.1111/j.1365-2958.2009.06927.x. [DOI] [PubMed] [Google Scholar]
  16. McCormick S. P.; Stanley A. M.; Stover N. A.; Alexander N. J. Trichothecenes: From Simple to Complex Mycotoxins. Toxins 2011, 3 (7), 802–814. 10.3390/toxins3070802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kahla A.; Verheecke-Vaessen C.; Delpino-Deelias M.; Gutierrez-Pozo M.; Medina A.; Magan N.; Doohan F. Acclimatisation of Fusarium Langsethiae, F. Poae and F. Sporotrichioides to Elevated CO2: Impact on Fungal Growth and Mycotoxin Production on Oat-Based Media. Int. J. Food Microbiol. 2023, 394, 110176. 10.1016/j.ijfoodmicro.2023.110176. [DOI] [PubMed] [Google Scholar]
  18. Kokkonen M.; Ojala L.; Parikka P.; Jestoi M. Mycotoxin Production of Selected Fusarium Species at Different Culture Conditions. Int. J. Food Microbiol. 2010, 143 (1–2), 17–25. 10.1016/j.ijfoodmicro.2010.07.015. [DOI] [PubMed] [Google Scholar]
  19. Nazari L.; Pattori E.; Somma S.; Manstretta V.; Waalwijk C.; Moretti A.; Meca G.; Rossi V. Infection Incidence, Kernel Colonisation, and Mycotoxin Accumulation in Durum Wheat Inoculated with Fusarium Sporotrichioides, F. Langsethiae or F. Poae at Different Growth Stages. European Journal of Plant Pathology 2019, 153 (3), 715–729. 10.1007/s10658-018-1558-9. [DOI] [Google Scholar]
  20. Somma S.; Alvarez C.; Ricci V.; Ferracane L.; Ritieni A.; Logrieco A.; Moretti A. Trichothecene and Beauvericin Mycotoxin Production and Genetic Variability in Fusarium Poae Isolated from Wheat Kernels from Northern Italy. Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment 2010, 27 (5), 729–737. 10.1080/19440040903571788. [DOI] [PubMed] [Google Scholar]
  21. Stenglein S. A.; Dinolfo M. I.; Barros G.; Bongiorno F.; Chulze S. N.; Moreno M. V. Fusarium Poae Pathogenicity and Mycotoxin Accumulation on Selected Wheat and Barley Genotypes at a Single Location in Argentina. Plant Disease 2014, 98 (12), 1733–1738. 10.1094/PDIS-02-14-0182-RE. [DOI] [PubMed] [Google Scholar]
  22. Vogelgsang S.; Sulyok M.; Bänziger I.; Krska R.; Schuhmacher R.; Forrer H.-R. Effect of Fungal Strain and Cereal Substrate on in Vitro Mycotoxin Production by Fusarium Poae and Fusarium Avenaceum. Food Additives & Contaminants: Part A 2008, 25 (6), 745–757. 10.1080/02652030701768461. [DOI] [PubMed] [Google Scholar]
  23. Witte T. E.; Harris L. J.; Nguyen H. D. T.; Hermans A.; Johnston A.; Sproule A.; Dettman J. R.; Boddy C. N.; Overy D. P. Apicidin Biosynthesis Is Linked to Accessory Chromosomes in Fusarium Poae Isolates. BMC Genomics 2021, 22 (1), 1–18. 10.1186/s12864-021-07617-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. O’Donnell K.; McCormick S. P.; Busman M.; Proctor R. H.; Ward T. J.; Doehring G.; Geiser D. M.; Alberts J. F.; Rheeder J. P.; Marasas; et al. 1984 “Toxigenic Fusarium Species: Identity and Mycotoxicology” Revisited. Mycologia 2018, 110 (6), 1058–1080. 10.1080/00275514.2018.1519773. [DOI] [PubMed] [Google Scholar]
  25. Jestoi M. N.; Paavanen-Huhtala S.; Parikka P.; Yli-Mattila T. In Vitro and in Vivo Mycotoxin Production of Fusarium Species Isolated from Finnish Grains. Archives of Phytopathology and Plant Protection 2008, 41 (8), 545–558. 10.1080/03235400600881547. [DOI] [Google Scholar]
  26. Yli-Mattila T.; Ward T. J.; O’Donnell K.; Proctor R. H.; Burkin A. A.; Kononenko G. P.; Gavrilova O. P.; Aoki T.; McCormick S. P.; Gagkaeva; et al. Fusarium Sibiricum Sp. Nov, a Novel Type A Trichothecene-Producing Fusarium from Northern Asia Closely Related to F. Sporotrichioides and F. Langsethiae. Int. J. Food Microbiol. 2011, 147 (1), 58–68. 10.1016/j.ijfoodmicro.2011.03.007. [DOI] [PubMed] [Google Scholar]
  27. Halstensen A. S.; Nordby K.-C.; Klemsdal S. S.; Elen O.; Clasen P.-E.; Eduard W. Toxigenic Fusarium Spp. as Determinants of Trichothecene Mycotoxins in Settled Grain Dust. Journal of Occupational and Environmental Hygiene 2006, 3 (12), 651–659. 10.1080/15459620600987431. [DOI] [PubMed] [Google Scholar]
  28. Bernhoft A.; Clasen P.-E.; Kristoffersen A. B.; Torp M. Less Fusarium Infestation and Mycotoxin Contamination in Organic than in Conventional Cereals. Food Additives & Contaminants: Part A 2010, 27 (6), 842–852. 10.1080/19440041003645761. [DOI] [PubMed] [Google Scholar]
  29. Edwards S. G.; Imathiu S. M.; Ray R. V.; Back M.; Hare M. C. Molecular Studies to Identify the Fusarium Species Responsible for HT-2 and T-2 Mycotoxins in UK Oats. Int. J. Food Microbiol. 2012, 156 (2), 168–175. 10.1016/j.ijfoodmicro.2012.03.020. [DOI] [PubMed] [Google Scholar]
  30. Fredlund E.; Gidlund A.; Sulyok M.; Börjesson T.; Krska R.; Olsen M.; Lindblad M. Deoxynivalenol and Other Selected Fusarium Toxins in Swedish Oats — Occurrence and Correlation to Specific Fusarium Species. Int. J. Food Microbiol. 2013, 167 (2), 276–283. 10.1016/j.ijfoodmicro.2013.06.026. [DOI] [PubMed] [Google Scholar]
  31. Hofgaard I. s.; Aamot H. u.; Torp T.; Jestoi M.; Lattanzio V. m. t.; Klemsdal S. s.; Waalwijk C.; Van der Lee T.; Brodal G. Associations between Fusarium Species and Mycotoxins in Oats and Spring Wheat from Farmers’ Fields in Norway over a Six-Year Period. World Mycotoxin Journal 2016, 9 (3), 365–378. 10.3920/WMJ2015.2003. [DOI] [Google Scholar]
  32. Hofgaard I. S.; Brodal G.; Almvik M.; Lillemo M.; Russenes A. L.; Edwards S. G.; Aamot H. U. Different Resistance to DON versus HT2 + T2 Producers in Nordic Oat Varieties. Toxins 2022, 14 (5), 313. 10.3390/toxins14050313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Karlsson I.; Mellqvist E.; Persson P. Temporal and Spatial Dynamics of Fusarium Spp. and Mycotoxins in Swedish Cereals during 16 Years. Mycotoxin Res. 2023, 39 (1), 3–18. 10.1007/s12550-022-00469-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Schöneberg T.; Martin C.; Wettstein F. E.; Bucheli T. D.; Mascher F.; Bertossa M.; Musa T.; Keller B.; Vogelgsang S. Fusarium and Mycotoxin Spectra in Swiss Barley Are Affected by Various Cropping Techniques. Food Additives & Contaminants: Part A 2016, 33 (10), 1608–1619. 10.1080/19440049.2016.1219071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Vanheule A.; Audenaert K.; De Boevre M.; Landschoot S.; Bekaert B.; Munaut F.; Eeckhout M.; Höfte M.; De Saeger S.; Haesaert G. The Compositional Mosaic of Fusarium Species and Their Mycotoxins in Unprocessed Cereals, Food and Feed Products in Belgium. Int. J. Food Microbiol. 2014, 181, 28–36. 10.1016/j.ijfoodmicro.2014.04.012. [DOI] [PubMed] [Google Scholar]
  36. Vogelgsang S.; Beyer M.; Pasquali M.; Jenny E.; Musa T.; Bucheli T. D.; Wettstein F. E.; Forrer H. R. An Eight-Year Survey of Wheat Shows Distinctive Effects of Cropping Factors on Different Fusarium Species and Associated Mycotoxins. European Journal of Agronomy 2019, 105, 62–77. 10.1016/j.eja.2019.01.002. [DOI] [Google Scholar]
  37. Yli-Mattila T.; Paavanen-Huhtala S.; Jestoi M.; Parikka P.; Hietaniemi V.; Gagkaeva T.; Sarlin T.; Haikara A.; Laaksonen S.; Rizzo A. Real-Time PCR Detection and Quantification of Fusarium Poae, F. Graminearum, F. Sporotrichioides and F. Langsethiae in Cereal Grains in Finland and Russia. Archives of Phytopathology and Plant Protection 2008, 41 (4), 243–260. 10.1080/03235400600680659. [DOI] [Google Scholar]
  38. Imathiu S. M.; Ray R. V.; Back M.; Hare M.; Edwards S. G. In Vitro Growth Characteristics of Fusarium Langsethiae Isolates Recovered from Oats and Wheat Grain in the UK. Acta Phytopathologica et Entomologica Hungarica 2016, 51 (2), 159–169. 10.1556/038.51.2016.2.1. [DOI] [Google Scholar]
  39. Imathiu S. M.; Edwards S. G.; Ray R. V.; Back M. A. Fusarium Langsethiae - a Ht-2 and t-2 Toxins Producer That Needs More Attention. Journal of Phytopathology 2013, 161 (1), 1–10. 10.1111/jph.12036. [DOI] [Google Scholar]
  40. Laraba I.; McCormick S. P.; Vaughan M. M.; Geiser D. M.; O’Donnell K. Phylogenetic Diversity, Trichothecene Potential, and Pathogenicity within Fusarium Sambucinum Species Complex. PLoS One 2021, 16 (1), e0245037 10.1371/journal.pone.0245037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Torp M.; Langseth W. Production of T-2 Toxin by a Fusarium Resembling Fusarium Poae. Mycopathologia 1999, 147 (2), 89–96. 10.1023/A:1007060108935. [DOI] [PubMed] [Google Scholar]
  42. Torp M.; Nirenberg H. I. Fusarium Langsethiae Sp. Nov. on Cereals in Europe. Int. J. Food Microbiol. 2004, 95 (3), 247–256. 10.1016/j.ijfoodmicro.2003.12.014. [DOI] [PubMed] [Google Scholar]
  43. Lysøe E.; Frandsen R. J. N.; Divon H. H.; Terzi V.; Orrù L.; Lamontanara A.; Kolseth A. K.; Nielsen K. F.; Thrane U. Draft Genome Sequence and Chemical Profiling of Fusarium Langsethiae, an Emerging Producer of Type A Trichothecenes. Int. J. Food Microbiol. 2016, 221, 29–36. 10.1016/j.ijfoodmicro.2016.01.008. [DOI] [PubMed] [Google Scholar]
  44. Miller J. D.; Blackwell B. A. Biosynthesis of 3-Acetyldeoxynivalenol and Other Metabolites by Fusarium Culmorum HLX 1503 in a Stirred Jar Fermentor. Canadian Journal of Botany 1986, 64 (1), 1–5. 10.1139/b86-001. [DOI] [Google Scholar]
  45. Dettman J. R.; Eggertson Q. Phylogenomic Analyses of Alternaria Section Alternaria: A High-Resolution, Genome-Wide Study of Lineage Sorting and Gene Tree Discordance. Mycologia 2021, 113 (6), 1218–1232. 10.1080/00275514.2021.1950456. [DOI] [PubMed] [Google Scholar]
  46. Chen S.; Zhou Y.; Chen Y.; Gu J. Fastp: An Ultra-Fast All-in-One FASTQ Preprocessor. Bioinformatics 2018, 34 (17), i884–i890. 10.1093/bioinformatics/bty560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Bankevich A.; Nurk S.; Antipov D.; Gurevich A. A.; Dvorkin M.; Kulikov A. S.; Lesin V. M.; Nikolenko S. I.; Pham S.; Prjibelski A. D.; et al. SPAdes: A New Genome Assembly Algorithm and Its Applications to Single-Cell Sequencing. Journal of Computational Biology 2012, 19 (5), 455–477. 10.1089/cmb.2012.0021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Gurevich A.; Saveliev V.; Vyahhi N.; Tesler G. QUAST: Quality Assessment Tool for Genome Assemblies. Bioinformatics 2013, 29 (8), 1072–1075. 10.1093/bioinformatics/btt086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Seppey M.; Manni M.; Zdobnov E. M.. BUSCO: Assessing Genome Assembly and Annotation Completeness. In Methods Mol. Biol.; Humana Press Inc., 2019; Vol. 1962, pp 227–245. 10.1007/978-1-4939-9173-0_14. [DOI] [PubMed] [Google Scholar]
  50. Stanke M.; Diekhans M.; Baertsch R.; Haussler D. Using Native and Syntenically Mapped cDNA Alignments to Improve de Novo Gene Finding. Bioinformatics 2008, 24 (5), 637–644. 10.1093/bioinformatics/btn013. [DOI] [PubMed] [Google Scholar]
  51. Katoh K.; Standley D. M. MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Mol. Biol. Evol. 2013, 30 (4), 772–780. 10.1093/molbev/mst010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Capella-Gutiérrez S.; Silla-Martínez J. M.; Gabaldón T. trimAl: A Tool for Automated Alignment Trimming in Large-Scale Phylogenetic Analyses. Bioinformatics 2009, 25 (15), 1972–1973. 10.1093/bioinformatics/btp348. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Kalyaanamoorthy S.; Minh B. Q.; Wong T. K. F.; Von Haeseler A.; Jermiin L. S. ModelFinder: Fast Model Selection for Accurate Phylogenetic Estimates. Nat. Methods 2017, 14 (6), 587–589. 10.1038/nmeth.4285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Minh B. Q.; Schmidt H. A.; Chernomor O.; Schrempf D.; Woodhams M. D.; Von Haeseler A.; Lanfear R.; Teeling E. IQ-Tree 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Mol. Biol. Evol. 2020, 37 (5), 1530–1534. 10.1093/molbev/msaa015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Chernomor O.; Von Haeseler A.; Minh B. Q. Terrace Aware Data Structure for Phylogenomic Inference from Supermatrices. Systematic Biology 2016, 65 (6), 997–1008. 10.1093/sysbio/syw037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Hoang D. T.; Chernomor O.; von Haeseler A.; Minh B. Q.; Vinh L. S. UFboot2: Improving the Ultrafast Bootstrap Approximation. Mol. Biol. Evol. 2018, 35 (2), 518–522. 10.1093/molbev/msx281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Palmer J. M.; Stajich J. . Funannotate v1.8.1: Eukaryotic Genome Annotation 2020. 10.5281/ZENODO.4054262. [DOI] [Google Scholar]
  58. Flynn J. M.; Hubley R.; Goubert C.; Rosen J.; Clark A. G.; Feschotte C.; Smit A. F. RepeatModeler2 for Automated Genomic Discovery of Transposable Element Families. Proc. Natl. Acad. Sci. U. S. A. 2020, 117 (17), 9451–9457. 10.1073/pnas.1921046117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Haas B. J.; Salzberg S. L.; Zhu W.; Pertea M.; Allen J. E.; Orvis J.; White O.; Buell C. R.; Wortman J. R. Automated Eukaryotic Gene Structure Annotation Using EVidenceModeler and the Program to Assemble Spliced Alignments. Genome Biology 2008, 9 (1), R7. 10.1186/gb-2008-9-1-r7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Ter-Hovhannisyan V.; Lomsadze A.; Chernoff Y. O.; Borodovsky M. Gene Prediction in Novel Fungal Genomes Using an Ab Initio Algorithm with Unsupervised Training. Genome Res. 2008, 18 (12), 1979–1990. 10.1101/gr.081612.108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Peplow A. W.; Meek I. B.; Wiles M. C.; Phillips T. D.; Beremand M. N. Tri16 Is Required for Esterification of Position C-8 during Trichothecene Mycotoxin Production by Fusarium Sporotrichioides. Appl. Environ. Microbiol. 2003, 69 (10), 5935–5940. 10.1128/AEM.69.10.5935-5940.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Hoogendoorn K.; Barra L.; Waalwijk C.; Dickschat J. S.; van der Lee T. A. J.; Medema M. H. Evolution and Diversity of Biosynthetic Gene Clusters in Fusarium. Frontiers in Microbiology 2018, 9 (1158), 1–12. 10.3389/fmicb.2018.01158. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Vanheule A.; Audenaert K.; Warris S.; van de Geest H.; Schijlen E.; Höfte M.; De Saeger S.; Haesaert G.; Waalwijk C.; van der Lee T. Living Apart Together: Crosstalk between the Core and Supernumerary Genomes in a Fungal Plant Pathogen. BMC Genomics 2016, 17 (1), 670. 10.1186/s12864-016-2941-6. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

jf3c08437_si_001.pdf (85.1KB, pdf)

Data Availability Statement

Assembled genomes and raw reads were published at the NCBI Genbank/SRA under bioproject ID PRJNA961673. All assembly versions included in this study are the first versions.

Articles from Journal of Agricultural and Food Chemistry are provided here courtesy of American Chemical Society

RESOURCES