Trichothecene-producing Species Research Articles

Fusarium chaquense, sp. nov, a novel type A trichothecene–producing species from native grasses in a wetland ecosystem in Argentina

ABSTRACT The Chaco wetland is among the most biologically diverse regions in Argentina. In collections of fungi from asymptomatic native grasses (Poaceae) from the wetlands, we identified isolates of Fusarium that were morphologically similar to F. armeniacum, but distinct from it by their production of abundant microconidia. All the isolates had identical, or nearly identical, partial sequences of TEF1 and RPB2. But they were distinct from reference sequences from F. armeniacum and Fusarium species closely related to it. Phylogenetic analysis of 34 full-length housekeeping gene sequences retrieved from whole genome sequences of three Chaco wetland isolates, 29 genes resolved the isolates as an exclusive clade within the F. sambucinum species complex. Based on results of the morphological and phylogenetic analysis, we concluded that the Chaco wetland isolates are a distinct and novel species, herein described as Fusarium chaquense, sp. nov., which is closely related to F. armeniacum. F. chaquense in culture can produce the trichothecenes T-2 and HT-2 toxin, neosolaniol, diacetoxyscirpenol, and monoacetoxyscirpenol, as well as beauvericin and the pigment aurofusarin. Genome sequence analysis also revealed the presence of three previously described loci required for trichothecene biosynthesis. This research represents the first study of Fusarium in a natural ecosystem in Argentina.

Efflux pumps as an additional source of resistance to trichothecenes in Fusarium proliferatum and Fusarium oxysporum isolates

Role of efflux-mediated toxin resistance to trichothecenes is known in trichothecene-producing species. However, the role of trichothecene efflux pump homologues in non-producing fusaria such as F. oxysporum and F. proliferatum was not investigated in detail. Analysis of the homologues of trichothecene efflux pump from multiple fungal species allowed us to uncover and catalogue functional gene copies of conserved structure. Putative Tri12 candidates in Fusarium oxysporum and F. proliferatum were characterised via expression profiling in response to different trigger compounds, providing supporting evidence for role of Tri12 homologues in the resistance to trichothecenes. Our analysis of Tri12 phylogeny also suggests that efflux-mediated trichothecene resistance is likely to predate the divergence of Trichoderma and Fusarium species. On the regulatory level, we posit that the increased tolerance of trichothecenes by F. oxysporum is possibly related to the decoupling of Tri12 homologue expression from pH, due to the deletion of PACC/RIM101 transcription factor binding site in its promoter region.

Influence of inoculum and climatic factors on the severity of Fusarium head blight in German spring and winter barley

ABSTRACTFusarium head blight (FHB) of small cereals is a disease of global importance with regard to economic losses and mycotoxin contamination harmful to human and animal health. In Germany, FHB is predominantly associated with wheat and F. graminearum is recognised as the major causal agent of the disease, but little is known about FHB of barley. Monitoring of the natural occurrence of FHB on Bavarian barley revealed differences for individual Fusarium spp. in incidence and severity of grain infection between years and between spring and winter barley. Parallel measurement of fungal DNA content in grain and mycotoxin content suggested the importance of F. graminearum in winter barley and of F. langsethiae in spring barley for FHB. The infection success of these two species was associated with certain weather conditions and barley flowering time. Inoculation experiments in the field revealed different effects of five Fusarium spp. on symptom formation, grain yield and mycotoxin production. A significant association between fungal infection of grain and mycotoxin content was observed following natural or artificial infection with the type B trichothecene producer F. culmorum, but not with the type A trichothecene-producing species F. langsethiae and F. sporotrichioides. Trichothecene type A toxin contamination also occurred in the absence of significant damage to grain and did not necessarily promote fungal colonisation.

Variation in Type A Trichothecene Production and Trichothecene Biosynthetic Genes in Fusarium goolgardi from Natural Ecosystems of Australia

Fusarium goolgardi, isolated from the grass tree Xanthorrhoea glauca in natural ecosystems of Australia, is closely related to fusaria that produce a subgroup of trichothecene (type A) mycotoxins that lack a carbonyl group at carbon atom 8 (C-8). Mass spectrometric analysis revealed that F. goolgardi isolates produce type A trichothecenes, but exhibited one of two chemotypes. Some isolates (50%) produced multiple type A trichothecenes, including 4,15-diacetoxyscirpenol (DAS), neosolaniol (NEO), 8-acetylneosolaniol (Ac-NEO) and T-2 toxin (DAS-NEO-T2 chemotype). Other isolates (50%) produced only DAS (DAS chemotype). In the phylogenies inferred from DNA sequences of genes encoding the RNA polymerase II largest (RPB1) and second largest (RPB2) subunits as well as the trichothecene biosynthetic genes (TRI), F. goolgardi isolates were resolved as a monophyletic clade, distinct from other type A trichothecene-producing species. However, the relationships of F. goolgardi to the other species varied depending on whether phylogenies were inferred from RPB1 and RPB2, the 12-gene TRI cluster, the two-gene TRI1-TRI16 locus, or the single-gene TRI101 locus. Phylogenies based on different TRI loci resolved isolates with different chemotypes into distinct clades, even though only the TRI1-TRI16 locus is responsible for structural variation at C-8. Sequence analysis indicated that TRI1 and TRI16 are functional in F. goolgardi isolates with the DAS-NEO-T2 chemotype, but non-functional in isolates with DAS chemotype due to the presence of premature stop codons caused by a point mutation.

Real-time PCR detection of Fusarium species in Swedish oats and correlation to T-2 and HT-2 toxin content

Contamination of Fusarium moulds and subsequent production of mycotoxins is a common problem in cereals grown in Northern Europe. Oats is the third most important cereal crop in Sweden and is commonly contaminated by Fusarium. To date there are no published reports on the distribution of trichothecene-producing Fusaria in Swedish oats and correlation to T-2 and HT-2, the two most dominant toxins. Identification of Fusarium species by traditional methods requires specific skill and experience and quantification of specific species is not possible. Using modern molecular methods such as real-time PCR both identification and quantification of specific Fusarium species are possible directly from the food or feed samples. In this study, specific methods for Fusarium langsethiae, Fusarium sporotrichioides, Fusarium graminearum, Fusarium culmorum and Fusarium poae as well as for the total amount of DNA from trichothecene-producing species were evaluated with respect to precision, robustness and efficiency and then used in the analysis of oat samples. Sixty-two oat samples were taken from field trial harvests from 2006-2008 in the central and southern parts of Sweden and analysed for the presence of Fusarium species by using species-specific real-time PCR-based methods with TaqMan or SYBR Green biochemistry. The T-2 and HT-2 toxin content was analysed by gas chromatography with electron capture detection of the toxins' pentafluoropropyl derivatives. The results showed that F. langsethiae and F. poae were the most dominant Fusarium species among those tested in Swedish oats during this period. The DNA level of F. langsethiae correlated well with the level of T-2 and HT-2 toxin (r=0.7), indicating that this species was the most important producer of T-2 and HT-2 toxins in Swedish oats during the sampling period (2006 to 2008). It can also be concluded that real-time PCR is a powerful tool when studying Fusarium species distribution in oats.

A molecular based strategy for rapid diagnosis of toxigenic Fusarium species associated to cereal grains from Argentina

Fusarium species are worldwide causal agents of ear rot in cereals. Their toxigenic potential is a health risk for both humans and animals. In Argentina, most identification of these fungi has been based on morphological and cross-fertility criteria which are time consuming and require considerable expertise in Fusarium taxonomy and physiology. DNA based approaches have been reported as rapid, sensitive and specific alternatives to identify the main fumonisin and trichothecene-producing Fusarium species. In this work, we used PCR assays and the partial sequence of TEF1-α gene (Translation Elongation Factor-1α) to identify the fumonisin and trichothecene-producing species in Fusarium isolates from diverse regions of Argentina. The relative efficiency and reliability of those methods to improve mycotoxin risk prediction in this country were also assessed. Species-specific PCR assays were targeted toward multicopy IGS (Intergenic Spacer of rDNA units) and on the toxin biosynthetic genes FUM1 (fumonisins) and TRI13 and TRI7 genes (trichothecenes). PCR assays based on FUM1 gene and IGS sequences allowed detection and discrimination of the fumonisin producers Fusarium proliferatum and Fusarium verticillioides. Molecular identification of nonfumonisin producers from Gibberella fujikuroi species complex was possible after determination of TEF1-α gene sequences, which indicated the presence of Fusarium subglutinans, Fusarium andiyazi and Fusarium thapsinum. TEF-1α gene sequences also allowed discrimination of the different species of the Fusarium graminearum complex ( F. graminearum sensu lato) as F. graminearum sensu stricto, Fusarium meridionale and Fusarium boothii. The last two species belonged to NIV chemotype and were detected for the first time in the subtropical region of Argentina while F. graminearum sensu stricto was DON producer only, which was also confirmed by specific PCR assays based on TRI137/ TRI7 genes. Our results indicated that the PCR assays evaluated in this work are reliable diagnostic tools to detect the main toxigenic Fusarium species associated to cereal grains in Argentina. An extensive epidemiological survey based on the approach presented in this work is currently in progress to know the mycotoxigenic hazard of Fusarium species in cereal grains from the subtropical region of Argentina.

PCR-based strategy to detect contamination with mycotoxigenic Fusarium species in maize

Contamination of cereals with mycotoxigenic species of Fusarium is an important source of trichothecenes, fumonisins and other mycotoxins which cause serious diseases in human and animals. In addition, these species are phytopathogenic and produce severe losses in cereal yield. Methods for early detection of these Fusarium species are crucial to prevent toxins entering the food chain and are a useful tool in disease management practices. We have developed an integrated protocol for diagnosis of mycotoxigenic Fusarium contamination in maize which can also be used for other cereals. The protocol consisted in an easy and rapid DNA extraction from maize samples (grain and germ), and subsequent group-specific polymerase chain reaction (PCR) assays for genus Fusarium, Gibberella fujikuroi complex, and trichothecene-producing species of Fusarium, that orientate the search of the critical species. We have additionally developed a PCR assay for the identification of F. proliferatum. The primers were designed on the basis of IGS sequence (Intergenic Spacer of rDNA), a multi-copy region in the genome that permits to enhance the sensitivity of the assay in comparison with PCR assays based on single-copy sequences. The suitability of the protocol and the relative efficacy of single and multi-copy sequence-based PCR assays have been tested in a wide range of fumonisin-contaminated maize samples.

The use of tri5 gene sequences for PCR detection and taxonomy of trichothecene-producing species in the Fusarium section Sporotrichiella

Purified DNA from isolates of Fusarium poae, Fusarium sporotrichioides, Fusarium kyushuense and Fusarium langsethiae was used as a template to amplify a 658-bp fragment from the trichodiene synthase ( tri5) gene of these fungi with the gene-specific PCR primer pair Tox5-1/Tox5-2. Fragments obtained were isolated and sequenced. DNA sequence alignments revealed high similarity between the sequences derived from F. sporotrichioides and F. langsethiae (98.7%) and less similarity between the latter species and F. poae (90.9%). Phylogenetic analysis of the aligned sequences using the tri5 sequence of Fusarium pseudograminearum as an outgroup revealed clear separation between one group consisting of F. poae and F. kyushuense and another consisting of F. sporotrichioides and F. langsethiae. The two latter species could not be distinguished phylogenetically on the basis of their tri5 sequences. Taxon-specific reverse primers were designed from the aligned sequences and combined with the tri5 gene-specific forward primer Tox5-1. The new reverse primers enabled specific amplification of a fragment of approximately 400 bp from DNA isolated from F. sporotrichioides, F. poae, F. langsethiae and F. kyushuense, respectively. All primers were tested for cross-reactivity with DNA from 26 fungal species potentially capable of producing trichothecenes. Only the primer designed for F. langsethiae cross-reacted with F. sporotrichioides. PCR assays were applied in analysis of artificially and naturally infected samples of barley and oats. On artificially infected barley, species were selectively detected by the corresponding primers. In naturally infected oats, F. langsethiae was identified by the combination of two PCR assays designed for detection of F. sporotrichioides and F. langsethiae, respectively .

Multiplex Polymerase Chain Reaction Assay for the Differential Detection of Trichothecene- and Fumonisin-Producing Species of Fusarium in Cornmeal

The genus Fusarium comprises a diverse group of fungi including several species that produce mycotoxins in food commodities. In this study, a multiplex polymerase chain reaction (PCR) assay was developed for the group-specific detection of fumonisin-producing and trichothecene-producing species of Fusarium. Primers for genus-level recognition of Fusarium spp. were designed from the internal transcribed spacer regions (ITS1 and ITS2) of rDNA. Primers for group-specific detection were designed from the TRI6 gene involved in trichothecene biosynthesis and the FUM5 gene involved in fumonisin biosynthesis. Primer specificity was determined by testing for cross-reactivity against purified genomic DNA from 43 fungal species representing 14 genera, including 9 Aspergillus spp., 9 Fusarium spp., and 10 Penicillium spp. With purified genomic DNA as a template, genus-specific recognition was observed at 10 pg per reaction; group-specific recognition occurred at 100 pg of template per reaction for the trichothecene producer Fusarium graminearum and at 1 ng of template per reaction for the fumonisin producer Fusarium verticillioides. For the application of the PCR assay, a protocol was developed to isolate fungal DNA from cornmeal. The detection of F. graminearum and its differentiation from F. verticillioides were accomplished prior to visible fungal growth at <105 CFU/g of cornmeal. This level of detection is comparable to those of other methods such as enzyme-linked immunosorbent assay, and the assay described here can be used in the food industry's effort to monitor quality and safety.

Molecular Phylogenetic, Morphological, and Mycotoxin Data Support Reidentification of the Quorn Mycoprotein Fungus asFusarium venenatum

Molecular phylogenetic, morphological, and mycotoxin data were obtained in order to investigate the relationships and identity of the Quorn mycoprotein fungus withinFusariumand to examine Quorn strains and commercial Quorn food products for trichothecene mycotoxins. Phylogenetic analyses of aligned DNA sequences obtained via the polymerase chain reaction from the nuclear 28S ribosomal DNA, nuclear ribosomal internal transcribed spacer region, and β-tubulin gene exons and introns indicate that the Quorn fungus isFusarium venenatum,rather thanF. graminearumas previously reported. All of the Quorn strains examined were morphologically degenerate aconidial colonial mutants except for NRRL 25139, which produced chlamydospores in recurved terminal chains together with mostly 5-septate sporodochial conidia on doliform monophialides diagnostic ofF. venenatum.Bootstrap and decay analyses provide strong support for a monophyletic lineage containingF. venenatumand several other type A trichothecene-producing species, while reference strains ofF. graminearumwere nested in a separate clade of species that produce type B trichothecenes and/or zearalenone. Analysis of mycotoxins from rice cultures inoculated with Quorn strain NRRL 25416 revealed that four type A trichothecenes are produced, but at low levels relative to strain NRRL 22198 ofF. venenatum.No trichothecene mycotoxins, however, were detected from the analysis of three commercial Quorn products marketed for human consumption in England.