Production Of Trichothecene Mycotoxins Research Articles

A Combined1H Nuclear Magnetic Resonance and Electrospray Ionization–Mass Spectrometry Analysis to Understand the Basal Metabolism of Plant-PathogenicFusariumspp.

Many ascomycete Fusarium spp. are plant pathogens that cause disease on both cereal and noncereal hosts. Infection of wheat ears by Fusarium graminearum and F. culmorum typically results in bleaching and a subsequent reduction in grain yield. Also, a large proportion of the harvested grain can be spoiled when the colonizing Fusarium mycelia produce trichothecene mycotoxins, such as deoxynivalenol (DON). In this study, we have explored the intracellular polar metabolome of Fusarium spp. in both toxin-producing and nonproducing conditions in vitro. Four Fusarium spp., including nine well-characterized wild-type field isolates now used routinely in laboratory experimentation, were explored. A metabolic "triple-fingerprint" was recorded using (1)H nuclear magnetic resonance and direct-injection electrospray ionization-mass spectroscopy in both positive- and negative-ionization modes. These combined metabolomic analyses revealed that this technique is sufficient to resolve different wild-type isolates and different growth conditions. Principal components analysis was able to resolve the four species explored-F. graminearum, F. culmorum, F. pseudograminearum, and F. venenatum-as well as individual isolate differences from the same species. The external nutritional environment was found to have a far greater influence on the metabolome than the genotype of the organism. Conserved responses to DON-inducing medium were evident and included increased abundance of key compatible solutes, such as glycerol and mannitol. In addition, the concentration of γ-aminobutyric acid was elevated, indicating that the cellular nitrogen status may be affected by growth on DON-inducing medium.

Comparisons of water activity and temperature impacts on growth of Fusarium langsethiae strains from northern Europe on oat-based media

This study has examined the effect of water activity (a w, 0.995–0.90) and temperature (10–37 °C) on the lag phases prior to growth, growth rates and used models to develop two dimensional profiles for optimum and marginal conditions for two strains of Fusarium langsethiae from four northern European countries (UK, Norway, Sweden, and Finland) on an oat-based medium. Results showed that the optimum a w for growth was at 0.98–0.995 and 25 °C. The limit for growth of the strains was at 0.92–0.93 a w with minima of 10 °C. No growth occurred at 37 °C. The lag phases prior to growth were lowest under optimum conditions and extended to > 10 days at marginal conditions. Statistical analyses of intra and inter-strain differences in terms of both lag phases prior to growth and growth rates were not statistically significant. However, a w and temperature were statistically significant factors. Two dimensional profiles for strains from each country of origin were built to identify optimum and marginal conditions for F. langsethiae for the first time. These environmental profiles will be beneficial for improving the ecological knowledge of this species which is able to produce trichothecene mycotoxins in a range of temperate cereals.

Synthetic Medium for Production of Trichothecene Mycotoxins by Fusarium Species

Synthetic Medium for Production of Trichothecene Mycotoxins by Fusarium Species

Early activation of wheat polyamine biosynthesis during Fusarium head blight implicates putrescine as an inducer of trichothecene mycotoxin production

BackgroundThe fungal pathogen Fusarium graminearum causes Fusarium Head Blight (FHB) disease on wheat which can lead to trichothecene mycotoxin (e.g. deoxynivalenol, DON) contamination of grain, harmful to mammalian health. DON is produced at low levels under standard culture conditions when compared to plant infection but specific polyamines (e.g. putrescine and agmatine) and amino acids (e.g. arginine and ornithine) are potent inducers of DON by F. graminearum in axenic culture. Currently, host factors that promote mycotoxin synthesis during FHB are unknown, but plant derived polyamines could contribute to DON induction in infected heads. However, the temporal and spatial accumulation of polyamines and amino acids in relation to that of DON has not been studied.ResultsFollowing inoculation of susceptible wheat heads by F. graminearum, DON accumulation was detected at two days after inoculation. The accumulation of putrescine was detected as early as one day following inoculation while arginine and cadaverine were also produced at three and four days post-inoculation. Transcripts of ornithine decarboxylase (ODC) and arginine decarboxylase (ADC), two key biosynthetic enzymes for putrescine biosynthesis, were also strongly induced in heads at two days after inoculation. These results indicated that elicitation of the polyamine biosynthetic pathway is an early response to FHB. Transcripts for genes encoding enzymes acting upstream in the polyamine biosynthetic pathway as well as those of ODC and ADC, and putrescine levels were also induced in the rachis, a flower organ supporting DON production and an important route for pathogen colonisation during FHB. A survey of 24 wheat genotypes with varying responses to FHB showed putrescine induction is a general response to inoculation and no correlation was observed between the accumulation of putrescine and infection or DON accumulation.ConclusionsThe activation of the polyamine biosynthetic pathway and putrescine in infected heads prior to detectable DON accumulation is consistent with a model where the pathogen exploits the generic host stress response of polyamine synthesis as a cue for production of trichothecene mycotoxins during FHB disease. However, it is likely that this mechanism is complicated by other factors contributing to resistance and susceptibility in diverse wheat genetic backgrounds.

First report of Fusarium poae on tomato in Argentina

During the spring of 2003, blighted flowers were observed on numerous plants (ca. 20%) of a tomato (Solanum lycopersicum formerly Lycopersicon esculentum) crop in two glasshouses (approx. 300–400 m2 each) in San Pedro, Buenos Aires, Argentina. Pedicels of flowers turned chlorotic and later became necrotic. Subsequently, the calyx and the petals turned brown, and the flowers shrivelled and generally abscised. Small pieces of diseased tissue were surface sterilized with 0·5% NaOCl, plated on 2% potato dextrose agar (PDA) at pH 6, and incubated at 22 to 24°C. Dense, whitish mycelium developed within 72–96 h. Microconidia were abundant, globose to piriform, 0–1 septate, 4–10 × 4·5–7 µm, and formed on unbranched and branched monophialides. Cultures produced a fruity aroma similar to amyl acetate. Spores from 14-day-old colonies that developed on PDA were removed with 4 mL of sterile water. The pathogenicity of the fungus was tested by spraying five healthy inflorescences of tomato with a 5-mL suspension (2 × 105 conidia mL−1 of sterile distilled water). Another five healthy inflorescences were sprayed with sterile distilled water. The plants were placed in a growth chamber with a 12-h photoperiod at 22 ± 2°C and covered with polyethylene bags that were removed after 3 days when plants were moved to a glasshouse. While control flowers were healthy, all inoculated flowers showed symptoms similar to those observed previously. A fungus was consistently isolated from diseased tissue in the inoculated plants and identified as Fusarium poae on the basis of fungal morphology (Nelson et al., 1983) and production of an amplicon of the appropriate size with primers 5′-CAAGCAAACAGGCTCTTCACC-3′-forward and 5′-TGTTCCACCTCAGTGACAGGTT-3′-reverse (Parry & Nicholson, 1996). This is the first report of this fungus on tomato in Argentina. Fusarium poae is one of the main causal agents of fusarium head blight of wheat. The increasing occurrence of this fungus in Argentinean cereals and the finding of F. poae infecting tomato could be of importance due to the close proximity of the two crops in some areas, as this may represent an alternative host. In addition, because of the potential for F. poae to produce trichothecene mycotoxins such as nivalenol, this is of significance as it may pose toxicological risks to consumers if tomato fruit become infected. This research was supported by FONCYT-SECYT PICT 2006-N°327 and an external stay CONICET fellow (N°3351/2007) of S.A.S.

Enzyme‐Linked Immunosorbent Assay Detection of Trichothecenes Produced by the Bioherbicide Myrothecium verrucaria in Cell Cultures, Extracts, and Plant Tissues

A rapid technique for trichothecene detection was needed in screening tests of the potential bioherbicide Myrothecium verrucaria (MV), in order to select strains, mutants, or formulations that were void of or that possessed low amounts of these undesirable mycotoxin compounds. Commercially available enzyme‐linked immunosorbent assay (ELISA) plates for trichothecene detection, possessing cross‐reactivity with several trichothecene mycotoxins (e.g., verrucarin A, and J, roridin A, L‐2, E, and H), were tested for their ability to detect trichothecenes produced by a strain of Myrothecium verrucaria (MV) in cell cultures, in plant tissues (hemp sesbania and kudzu) treated with purified roridin A, or ethyl acetate fractions of MV cultures. Evaluations of ELISA assays showed linear responses for standards of verrucarin A and roridin A over a concentration range of 0.2 to 20 ppb. Ethyl acetate or aqueous extractions were used to obtain samples from MV cultures and plant tissues for testing. Trichothecenes were detected in conidia and mycelia of MV, and in agar upon which wild‐type MV was grown, indicating secretion into the growth media. Two MV sectors (morphological variants of wild type) also tested positive for trichothecenes. Purified roridin A and concentrated extracts containing trichothecenes from MV spore cultures exhibited phytotoxicity (growth inhibition or necrosis) when applied to excised shoots of hemp sesbania seedlings and intact kudzu leaf tissues. Evidence of some translocation of trichothecenes from the application point in kudzu was found, but translocation to the upper shoot portion of hemp sesbania was not detected at the lowest limit of detection in this assay (0.14 ppb). This assay is also being employed to identify induced mutants and/or other naturally occurring sectors deficient in trichothecene mycotoxin production. Results indicated that ELISA is a sensitive and rapid assay method to quantify trichothecenes produced by this bioherbicidal fungus and in certain plant tissues treated with trichothecenes.

The Fusarium mycotoxin deoxynivalenol elicits hydrogen peroxide production, programmed cell death and defence responses in wheat

Fusarium species infect cereal crops worldwide and cause the important diseases Fusarium head blight and crown rot in wheat. Fusarium pathogens reduce yield and some species also produce trichothecene mycotoxins, such as deoxynivalenol (DON), during infection. These toxins play roles in pathogenesis on wheat and have serious health effects if present in grain consumed by humans or animals. In the present study, the response of wheat tissue to DON has been investigated. Infusion of wheat leaves with DON induced hydrogen peroxide production within 6 h followed by cell death within 24 h that was accompanied by DNA laddering, a hallmark of programmed cell death. In addition, real-time PCR analysis revealed that DON treatment rapidly induced transcription of a number of defence genes in a concentration-dependent manner. Co-treatment with DON and the antioxidant ascorbic acid reduced these responses, suggesting their induction may be at least partially mediated by reactive oxygen species (ROS), commonly known to be signalling molecules in plants. Wheat defence genes were more highly expressed in wheat stems inoculated with a DON-producing fungal strain than those inoculated with a DON-non-producing mutant, but only at a late stage of infection. Taken together, the results are consistent with a model in which DON production during infection of wheat induces ROS, which on the one hand may stimulate programmed host cell death assisting necrotrophic fungal growth, whereas, on the other hand, the ROS may contribute to the induction of antimicrobial host defences.

Effect of temperature and duration of wetness during initial infection periods on disease development, fungal biomass and mycotoxin concentrations on wheat inoculated with single, or combinations of, Fusarium species

Experiments were conducted under controlled environment conditions to study the relationship between environmental conditions, development of fusarium head blight (FHB) and mycotoxin production. A single isolate from each of four Fusarium species (F. avenaceum, F. culmorum, F. graminearum and F. poae) was used to inoculate wheat ears separately. Combinations of two or three isolates were also used to inoculate ears simultaneously. Inoculated ears were subjected to various combinations of duration of wetness (6–48 h) and temperature (10–30°C). For all inoculations, both incidence of spikelets with FHB symptoms and concentration of mycotoxins generally increased with increasing length of wetness period and temperature. There were significant positive correlations among disease incidence, fungal biomass (quantified as total amount of fungal DNA) and mycotoxins. Mycotoxin production was also greatly enhanced by high temperatures (≥ 20°C) during initial infection periods. In single‐isolate inoculations, F. poae was the least aggressive. There was no evidence to support synergetic interactions between fungal isolates in causing visual symptoms; rather the results suggest, in most cases, the presence of competitive interactions. Furthermore, the competition led to large reductions in fungal biomass compared to single‐isolate inoculations, often > 90% reduction for the weaker isolate(s). In contrast, mycotoxin productivity increased dramatically in the co‐inoculations, by as much as 1000 times, suggesting that competition resulted in greater production of trichothecene mycotoxins. The F. graminearum isolate was most competitive and isolates of the other three species were similar in their competitiveness.

Microbial Diversity in Milled Rice as Revealed by Riosomal Intergenic Spacer Analysis

Microbial diversity in milled rice was examined using culture-independent methods. Environmental DNA samples were extracted from commercial products of milled rice, and the intergenic spacer regions between the small and large subunit ribosomal RNA genes were amplified by ribosomal intergenic spacer analysis (RISA). The results indicated the presence of microbial communities in milled rice and subsequent sequencing of RISA amplicons has identified 17 unique microbial sequences. These include several sequences highly similar to known plant-associated microbes, including Pseudomonas fluorescens, Xanthomonas sacchari, and Pseudozyma antarctica, as well as to several uncultured bacteria. In addition, one sequence shows significant similarity to the sequence of Fusarium graminearum, a potent pathogenic fungus in foodstuffs that produces trichothecene mycotoxins. The potential presence of a pathogenic Fusarium fungus in milled rice was also suggested by a diagnostic PCR procedure that detects the genes involved in the production of trichothecene mycotoxins.

Influence of environmental factors on the biosynthesis of type B trichothecenes by isolates of Fusarium spp. from Spanish crops

Various species of Fusarium can produce trichothecene mycotoxins that contaminate food commodities and can represent a risk for human and animal health. In this paper, a full factorial design was applied to study the influence of incubation temperature, water activity ( a w) and type of isolate on the production of deoxynivalenol (DON), nivalenol (NIV) and 3-acetyldeoxynivalenol (3-AcDON) in corn kernel cultures by three isolates of Fusarium graminearum and three isolates of Fusarium culmorum from crops grown in Spain. The tested temperatures were 15, 20, 28 and 32 °C. The a w-values were 0.960, 0.970 and 0.980. Moisture of cultures (within the studied range) did not affect significantly production of trichothecenes; however, the temperature affected significantly mycotoxin production and the optimal values were 28, 20 and 15 °C for DON, NIV and 3-AcDON, respectively. Four additional isolates of F. graminearum and two additional isolates of F. culmorum were examined for production of these mycotoxins at the optimal temperatures. Of the seven isolates of F. graminearum, four produced DON (0.88–3.97 μg/g), seven produced NIV (1.53–124 μg/g), and three produced 3-AcDON (0.65–10.6 μg/g). Of the five isolates of F. culmorum, four produced DON (1.20–4.93 μg/g), four produced NIV (6.94–701 μg/g), and four produced 3-AcDON (0.83–7.70 μg/g). Practically all isolates seem to belong to the NIV-chemotype. This is the first study done with regard to interaction between strain and ecological variables on type B trichothecene production by isolates of these two species from crops grown in Spain.

Tri16 is required for esterification of position C-8 during trichothecene mycotoxin production by Fusarium sporotrichioides.

We previously characterized Tri1, a gene required for hydroxylation of the C-8 position during trichothecene mycotoxin biosynthesis in Fusarium sporotrichioides NRRL 3299. Sequence analysis of the region surrounding Tri1 revealed a gene, named Tri16, which could encode an acyltransferase. Unlike the wild-type parent strain NRRL 3299, which accumulates primarily T-2 toxin along with low levels of diacetoxyscirpenol (DAS) and neosolaniol (NEO) and trace amounts of 8-propionyl-neosolaniol (P-NEO) and 8-isobutyryl-neosolaniol (B-NEO), mutants containing a disruption of Tri16 were blocked in the production of the three C-8 esterified compounds T-2 toxin, P-NEO, and B-NEO and accumulated the C-8-hydroxylated compound NEO along with secondary levels of DAS. These data indicate that Tri16 encodes an acyltransferase that catalyzes the formation of ester side groups at C-8 during trichothecene biosynthesis. We also report the presence of a Tri16 ortholog in Gibberella pulicaris R-6380 that is likely linked to a presumably inactive ortholog for Tri1.

Development of PCR assays to Tri7 and Tri13 trichothecene biosynthetic genes, and characterisation of chemotypes of Fusarium graminearum, Fusarium culmorum and Fusarium cerealis

Fusarium graminearum, Fusarium culmorum and Fusarium cerealis are major causal agents of Fusarium Head Blight (scab) which is a disease of global significance in all cereal growing areas. These fungi produce trichothecene mycotoxins, principally nivalenol (NIV) and deoxynivalenol (DON). Genes Tri13 and Tri7 from the trichothecene biosynthetic gene cluster convert DON to NIV ( Tri13) and NIV to 4-acetyl-NIV ( Tri7). We have developed positive–negative PCR assays based on these two genes, which accurately indicate a DON or NIV chemotype in F. graminearum, F. culmorum and F. cerealis. These assays are useful in assessing the risk of trichothecene contamination, and can be informative in epidemiological studies. All NIV chemotype isolates studied have functional copies of both Tri13 and Tri7, and all DON-producing isolates have both genes disrupted or deleted. We have identified several mutations in these genes, which are conserved across F. graminearum lineage, RAPD and SCAR groupings and between the three species. There appears to be evidence of inter-species hybridisation within the trichothecene biosynthetic gene cluster.

Identification of new genes positively regulated by Tri10 and a regulatory network for trichothecene mycotoxin production.

Tri10, a regulatory gene in trichothecene mycotoxin-producing Fusarium species, is required for trichothecene biosynthesis and the coordinated expression of four trichothecene pathway-specific genes (Tri4, Tri5, Tri6, and Tri101) and the isoprenoid biosynthetic gene for farnesyl pyrophosphate synthetase (FPPS). We showed that six more trichothecene genes (Tri3, Tri7, Tri8, Tri9, Tri11, and Tri12) are regulated by Tri10. We also constructed a cDNA library from a strain of Fusarium sporotrichioides that overexpresses Tri10 ( upward arrow Tri10) and used cDNA derived from the upward arrow Tri10 strain and a non-Tri10-expressing strain (DeltaTri10) to differentially screen macroarrays prepared from the cDNA library. This screen identified 15 additional Tri10-regulated transcripts. Four of these transcripts represent Tri1, Tri13, and Tri14 and a gene designated Tri15. Three other sequences are putative orthologs of genes for isoprenoid biosynthesis, the primary metabolic pathway preceding trichothecene biosynthesis. The remaining eight sequences have been designated Ibt (influenced by Tri10) genes. Of the 26 transcripts now known to be positively regulated by Tri10, 22 are positively coregulated by Tri6, a gene that encodes a previously characterized trichothecene pathway-specific transcription factor. These 22 Tri10- and Tri6-coregulated sequences include all of the known Tri genes (except for Tri10), the FPPS gene, and the other three putative isoprenoid biosynthetic genes. Tri6 also regulates a transcript that is not regulated by Tri10. Thus, Tri10 and Tri6 regulate overlapping sets of genes that include a common group of multiple genes for both primary and secondary metabolism.

Polymorphism of trichothecene biosynthesis genes in deoxynivalenol-and nivalenol-producing Fusarium graminearum isolates

Diversity in trichothecene mycotoxin production by 167 isolates of Fusarium graminearum was examined by chemical and molecular methods. Isolates from barley, corn, and wheat grown in Korea produced either deoxynivalenol (DON) or nivalenol (NIV), whereas isolates from corn grown in the United States produced DON only. Southern blotting of MseI-digested genomic DNA's from these isolates was performed using a 0.6-kb fragment of Tri5, a key enzyme for trichothecene production, as a probe. This technique revealed a single-band polymorphism between these isolates, with 1.8- and 2.2-kb bands arising from DON and NIV producers, respectively. The same set of isolates was subjected to previously developed PCR assays using primers derived from Tri7 or Tri13. These assays also revealed a single-band polymorphism between NIV- and DON-producing chemotypes. The polymorphisms at Tri5, Tri7, or Tri13 in all of the US isolates were consistent with their chemotypes as identified by GC-MS. However, for seven Korean isolates, chemical and molecular analyses yielded seemingly inconsistent results. This issue was resolved by Southern blot analysis with the Tri5 probe using two other restriction enzymes and sequence comparison of a 3.8-kb region spanning Tri5. In addition, one of these exceptional isolates was found to carry both DON and NIV chemotype-specific regions, possibly resulting from recombination between the two chemotypes.

Isolation of the β-tubulin gene and characterization of thiabendazole resistance in Gibberella pulicaris

Gibberella pulicaris (anamorph: Fusarium sambucinum), is a common potato pathogen that produces trichothecene mycotoxins and reduces plant yield and tuber quality. Field isolates have been recovered from western Canada that are resistant to thiabendazole, a benzimidazole fungicide registered in many countries to control dry rot in storage. To characterize this resistance, homokaryons were derived from single ascospores that were produced in vitro from crosses of thiabendazole-resistant G. pulicaris field isolates with sensitive isolates of an opposite mating type. The β-tubulin gene was amplified from these homokaryons using degenerate oligonucleotides synthesized to conserved sequences of the β-tubulin gene derived from related fungi. An open reading frame containing three putative introns and a deduced amino acid sequence of 446 amino acids exhibited a high level of homology to the β-tubulin gene of other fungi. Although nucleotide and amino acid differences were observed in the β-tubulin gene, none were linked to the thiabendazole resistance. Linkage analysis confirmed that the isolated β-tubulin gene is single copy and was not linked to thiabendazole resistance in G. pulicaris.

Double-stranded RNA mycovirus from Fusarium graminearum.

Double-stranded RNA (dsRNA) viruses in some fungi are associated with hypovirulence and have been used or proposed as biological control agents. We isolated 7.5-kb dsRNAs from 13 of 286 field strains of Fusarium graminearum isolated from maize in Korea. One of these strains, DK21, was examined in more detail. This strain had pronounced morphological changes, including reduction in mycelial growth, increased pigmentation, reduced virulence towards wheat, and decreased (60-fold) production of trichothecene mycotoxins. The presence or absence of the 7.5-kb dsRNA was correlated with the changes in pathogenicity and morphology. The dsRNA could be transferred to virus-free strains by hyphal fusion, and the recipient strain acquired the virus-associated phenotype of the donor strain. The dsRNA was transmitted to approximately 50% of the conidia, and only colonies resulting from conidia carrying the mycovirus had the virus-associated phenotype. Partial nucleotide sequences of the purified dsRNA identify an RNA-dependent RNA polymerase sequence and an ATP-dependent helicase that are closely related to those of Cryphonectria hypovirus and Barley yellow mosaic virus. Collectively, these results suggest that this dsRNA isolated from F. graminearum encodes traits for hypovirulence.

Analysis of double-stranded RNA and virus-like particles in trichothecene-producing strains ofFusarium graminearum

Double-stranded RNAs (dsRNAs) have been found in two isolates of the plant pathogenic fungus Fusarium graminearum which produce trichothecene mycotoxins. The isolates 8.2 and 19.2 had dsRNAs in the size of about 2.0 kb and 6.0 kb, respectively, which were associated with capsid proteins and persisted within the cytoplasm of the infected host cells as encapsidated virus-like particles (VLPs). The dsRNAs contained in the VLP pellets were the same size as the dsRNA isolated in total nucleic acid preparations. In the VLP pellets the isolate 19.2 had a second dsRNA with the size of about 1.6 kb. After mycovirus purification one icosahedral particle of about 28 nm in diameter from the isolate 8.2 and two icosahedral particles of about 28 nm and 38 to 40 nm in diameter from the isolate 19.2 could be identified with electron microscopy. SDS-PAGE analysis of the VLPs from the isolate 8.2 revealed one major protein component of approximately 65 kDa, while the isolate 19.2 had two major protein bands at about 94 kDa and 105 kDa. Both isolates were studied for potential trichothecene production. Tox5 PCR showed a 658 bp fragment in each isolate. In addition, both strains were able to produce the trichothecenes deoxynivalenol (DON), the derivatives acetyl-DON (3-A-DON, 15-A-DON) and nivalenol (NIV) in vitro.

Fluconazole efficacy in tineas and pityriasis versicolor

Recent publications have argued that there are potentially serious consequences for researchers in recognising distinct genera in the terminal fusarioid clade of the family Nectriaceae. Thus, an alternate hypothesis, namely a very broad concept of the genus Fusarium was proposed. In doing so, however, a significant body of data that supports distinct genera in Nectriaceae based on morphology, biology, and phylogeny is disregarded. A DNA phylogeny based on 19 orthologous protein-coding genes was presented to support a very broad concept of Fusarium at the F1 node in Nectriaceae. Here, we demonstrate that re-analyses of this dataset show that all 19 genes support the F3 node that represents Fusarium sensu stricto as defined by F. sambucinum (sexual morph synonym Gibberella pulicaris). The backbone of the phylogeny is resolved by the concatenated alignment, but only six of the 19 genes fully support the F1 node, representing the broad circumscription of Fusarium. Furthermore, a re-analysis of the concatenated dataset revealed alternate topologies in different phylogenetic algorithms, highlighting the deep divergence and unresolved placement of various Nectriaceae lineages proposed as members of Fusarium. Species of Fusarium s. str. are characterised by Gibberella sexual morphs, asexual morphs with thin- or thick-walled macroconidia that have variously shaped apical and basal cells, and trichothecene mycotoxin production, which separates them from other fusarioid genera. Here we show that the Wollenweber concept of Fusarium presently accounts for 20 segregate genera with clear-cut synapomorphic traits, and that fusarioid macroconidia represent a character that has been gained or lost multiple times throughout Nectriaceae. Thus, the very broad circumscription of Fusarium is blurry and without apparent synapomorphies, and does not include all genera with fusarium-like macroconidia, which are spread throughout Nectriaceae (e.g., Cosmosporella, Macroconia, Microcera). In this study four new genera are introduced, along with 18 new species and 16 new combinations. These names convey information about relationships, morphology, and ecological preference that would otherwise be lost in a broader definition of Fusarium. To assist users to correctly identify fusarioid genera and species, we introduce a new online identification database, Fusarioid-ID, accessible at www.fusarium.org. The database comprises partial sequences from multiple genes commonly used to identify fusarioid taxa (act1, CaM, his3, rpb1, rpb2, tef1, tub2, ITS, and LSU). In this paper, we also present a nomenclator of names that have been introduced in Fusarium up to January 2021 as well as their current status, types, and diagnostic DNA barcode data. In this study, researchers from 46 countries, representing taxonomists, plant pathologists, medical mycologists, quarantine officials, regulatory agencies, and students, strongly support the application and use of a more precisely delimited Fusarium (= Gibberella) concept to accommodate taxa from the robust monophyletic node F3 on the basis of a well-defined and unique combination of morphological and biochemical features. This F3 node includes, among others, species of the F. fujikuroi, F. incarnatum-equiseti, F. oxysporum, and F. sambucinum species complexes, but not species of Bisifusarium [F. dimerum species complex (SC)], Cyanonectria (F. buxicola SC), Geejayessia (F. staphyleae SC), Neocosmospora (F. solani SC) or Rectifusarium (F. ventricosum SC). The present study represents the first step to generating a new online monograph of Fusarium and allied fusarioid genera (www.fusarium.org).

Production of trichothecene mycotoxins on water damaged gypsum boards in Danish buildings

Water damaged gypsum boards heavily infested with Stachybotrys chartarum were found in a school and in a domestic residence in Copenhagen. Samples were extracted with dichloromethane and cleaned up on Sep-Pak ® C 18 modules. The trichothecenes were detected as the heptafluorobutyrlated derivatives by gas chromatography ion trap mass spectrometry with negative ion chemical ionisation. Extracts of samples from both locations yielded verrucarol after hydrolysis, strongly indicating the presence of toxic macrocyclic trichothecenes, probably satratoxin H and G. In addition the sample from the domestic residence yielded trichodermol.

Myrothecium: A new indoor contaminant?

In the natural environmentMyrothecium species occur as soil or leaf surface saprobes or as weak plant pathogens. In addition, some species ofMyrothecium are known to produce trichothecene mycotoxins. During a previous aerobiological investigation at two Las Vegas elementary schools,Myrothecium conidia were found to be the second most abundant spore type identified indoors from Burkard personal spore trap samples. The present study was undertaken to re-examine the schools to locate the source ofMyrothecium spores and to examine the ability ofMyrothecium to grow on indoor substrates. There were no obvious signs of contamination in the classrooms; however,Myrothecium spores occurred on about 30% of the Burkard samples. Two colonies ofMyrothecium were identified from subcultures of the Andersen samples, and three colonies were identified from carpet dust samples. Culture studies showed that a strain ofMyrothecium cinctum was able to grow on various culture media as well as on various indoor substrates including paper, cardboard, wallpaper, ceiling tiles, dry wall, carpets and cotton rug. Although there was no attempt to estimate any human health risks, these investigations are believed to be the first to document abundantMyrothecium spores from indoor air samples.