Trichothecene Biosynthetic Genes Research Articles

TRI14 Is Critical for Fusarium graminearum Infection and Spread in Wheat

Trichothecenes are sesquiterpenoid toxins produced by diverse ascomycetes, including Fusarium. The trichothecene analog deoxynivalenol (DON) produced by the Fusarium head blight (FHB) pathogen Fusarium graminearum is a virulence factor on wheat and a major food and feed safety concern. In Fusarium, the trichothecene biosynthetic gene (TRI) cluster consists of 7–14 genes. Most TRI cluster genes are conserved and their specific roles in trichothecene biosynthesis have been determined. An exception is TRI14, which is not required for DON synthesis in vitro but is required for spread of F. graminearum in wheat heads. In the current study, gene expression analyses revealed that TRI14 was highly induced in infected wheat heads. We demonstrated that TRI14 was not only required for F. graminearum spread but also important for initial infection in wheat. Although a prior study did not detect DON in infected seeds, our analyses showed significantly less DON and fungal biomass in TRI14-mutant (designated ∆tri14)-inoculated heads than wild-type-inoculated heads. Gene expression comparison showed that the level of expression of TRI genes was similar in the wheat tissues infected with ∆tri14 or the wild type, indicating the reduced toxin levels caused by ∆tri14 may be due to less fungal growth. ∆tri14 also caused less lesion and grew less in wheat coleoptiles than the wild type. The growth of ∆tri14 in carboxymethylcellulose medium was more sensitive to hydrogen peroxide than the wild type. The data suggest that TRI14 plays a critical role in F. graminearum growth, and potentially protects the fungus from plant defense compounds.

A Novel Trichothecene Toxin Phenotype Associated with Horizontal Gene Transfer and a Change in Gene Function in Fusarium.

Fusarium trichothecenes are among the mycotoxins of most concern to food and feed safety. Production of these mycotoxins and presence of the trichothecene biosynthetic gene (TRI) cluster have been confirmed in only two multispecies lineages of Fusarium: the Fusarium incarnatum-equiseti (Incarnatum) and F. sambucinum (Sambucinum) species complexes. Here, we identified and characterized a TRI cluster in a species that has not been formally described and is represented by Fusarium sp. NRRL 66739. This fungus is reported to be a member of a third Fusarium lineage: the F. buharicum species complex. Cultures of NRRL 66739 accumulated only two trichothecenes, 7-hydroxyisotrichodermin and 7-hydroxyisotrichodermol. Although these are not novel trichothecenes, the production profile of NRRL 66739 is novel, because in previous reports 7-hydroxyisotrichodermin and 7-hydroxyisotrichodermol were components of mixtures of 6-8 trichothecenes produced by several Fusarium species in Sambucinum. Heterologous expression analysis indicated that the TRI13 gene in NRRL 66739 confers trichothecene 7-hydroxylation. This contrasts the trichothecene 4-hydroxylation function of TRI13 in other Fusarium species. Phylogenetic analyses suggest that NRRL 66739 acquired the TRI cluster via horizontal gene transfer from a close relative of Incarnatum and Sambucinum. These findings provide insights into evolutionary processes that have shaped the distribution of trichothecene production among Fusarium species and the structural diversity of the toxins.

Deoxynivalenol Biosynthesis in Fusarium pseudograminearum Significantly Repressed by a Megabirnavirus.

Deoxynivalenol (DON) is a mycotoxin widely detected in cereal products contaminated by Fusarium. Fusarium pseudograminearum megabirnavirus 1 (FpgMBV1) is a double-stranded RNA virus infecting Fusarium pseudograminearum. In this study, it was revealed that the amount of DON in F. pseudograminearum was significantly suppressed by FpgMBV1 through a high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) assay. A total of 2564 differentially expressed genes were identified by comparative transcriptomic analysis between the FpgMBV1-containing F. pseudograminearum strain FC136-2A and the virus-free strain FC136-2A-V-. Among them, 1585 genes were up-regulated and 979 genes were down-regulated. Particularly, the expression of 12 genes (FpTRI1, FpTRI3, FpTRI4, FpTRI5, FpTRI6, FpTRI8, FpTRI10, FpTRI11, FpTRI12, FpTRI14, FpTRI15, and FpTRI101) in the trichothecene biosynthetic (TRI) gene cluster was significantly down-regulated. Specific metabolic and transport processes and pathways including amino acid and lipid metabolism, ergosterol metabolic and biosynthetic processes, carbohydrate metabolism, and biosynthesis were regulated. These results suggest an unrevealing mechanism underlying the repression of DON and TRI gene expression by the mycovirus FpgMBV1, which would provide new methods in the detoxification of DON and reducing the yield loss in wheat.

Effective inhibition of fungal growth, deoxynivalenol biosynthesis and pathogenicity in cereal pathogen Fusarium spp. by cold atmospheric plasma

Fusarium spp. is one of the most destructive plant pathogenic fungi worldwide, not only causing many devastating crop diseases with huge economic losses, but also threatening human and livestock health by producing mycotoxins. Recently, cold atmospheric plasma (CAP) has gained much interest as a promising approach to control fungi and mycotoxins. However, the effects of CAP on fungal toxigenicity and pathogenicity were still rarely reported. Herein, this study investigated the influence of CAP on fungal growth, deoxynivalenol (DON) biosynthesis, and pathogenicity of Fusarium spp. both in vitro and in vivo. The in-vitro results show that CAP generated various reactive oxygen and nitrogen species (RONS) in water, effectively inactivating four major Fusarium strains (2 to 6 log10 reduction) through destructing cell membrane, accumulating intracellular ROS, and depolarizing mitochondrial membrane. Meanwhile, CAP inhibited DON biosynthesis in F. graminearum via reducing acetyl-CoA production, toxisomes formation, and key trichothecene biosynthetic gene (TRI) expression. Further in-vivo results verify that CAP can inactivate fungal spores and reduce DON production on wheat grains, as well as reduce the pathogenicity of Fusarium graminearum in wheat coleoptiles and florets. Overall, these findings provide comprehensive insights into the antifungal and detoxification mechanism of CAP against F. graminearum, which promotes the application of CAP in controlling pathogenic fungus growth and mycotoxin production in grains and crops.

Distribution, Function, and Evolution of a Gene Essential for Trichothecene Toxin Biosynthesis in Trichoderma.

Trichothecenes are terpenoid toxins produced by species in 10 fungal genera, including species of Trichoderma. The trichothecene biosynthetic gene (tri) cluster typically includes the tri5 gene, which encodes a terpene synthase that catalyzes formation of trichodiene, the parent compound of all trichothecenes. The two Trichoderma species, Trichoderma arundinaceum and T. brevicompactum, that have been examined are unique in that tri5 is located outside the tri cluster in a genomic region that does not include other known tri genes. In the current study, analysis of 35 species representing a wide range of the phylogenetic diversity of Trichoderma revealed that 22 species had tri5, but only 13 species had both tri5 and the tri cluster. tri5 was not located in the cluster in any species. Using complementation analysis of a T. arundinaceum tri5 deletion mutant, we demonstrated that some tri5 homologs from species that lack a tri cluster are functional, but others are not. Phylogenetic analyses suggest that Trichoderma tri5 was under positive selection following its divergence from homologs in other fungi but before Trichoderma species began diverging from one another. We propose two models to explain these diverse observations. One model proposes that the location of tri5 outside the tri cluster resulted from loss of tri5 from the cluster in an ancestral species followed by reacquisition via horizontal transfer. The other model proposes that in species that have a functional tri5 but lack the tri cluster, trichodiene production provides a competitive advantage.

Enantioselective Effect of Flutriafol on Growth, Deoxynivalenol Production, and TRI Gene Transcript Levels in Fusarium graminearum.

In recent years, deoxynivalenol (DON) has frequently been detected in wheat grains and their products. The enantioselective impact of flutriafol on the growth and DON biosynthesis of Fusarium graminearum was investigated in relation to water activity (αw, 0.97 and 0.99) and temperature (20, 25, and 30 °C) on the wheat-based medium. R-(-)-flutriafol exhibited higher bioactivity than S-(+)-flutriafol and Rac-flutriafol under the above conditions. Flutriafol enantiomers reduced or stimulated DON biosynthesis depending on αw. DON levels were negligible after 14 or 7 days of incubation times under 0.97 and 0.99 aw, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) analyses showed that the expression levels of trichothecene biosynthetic (TRI) genes of F. graminearum under 0.97 aw were significantly higher than those under 0.99 aw. In addition, R-(-)-flutriafol can induce more TRI gene expression than S-(+)-flutriafol. Taken together, this study indicated that aw and temperature play important roles in regulating DON biosynthesis in F. graminearum with flutriafol enantiomers.

Activation of biosynthetic gene clusters by the global transcriptional regulator TRI6 in Fusarium graminearum

In F. graminearum, the transcription factor TRI6 positively regulates the trichothecene biosynthetic gene cluster (BGC) leading to the production of the secondary metabolite 15-acetyl deoxynivalenol. Secondary metabolites are not essential for survival, instead, they enable the pathogen to successfully infect its host. F. graminearum has the potential to produce a diverse array of secondary metabolites (SMs). However, given high functional specificity and energetic cost, most of these clusters remain silent, unless the organism is subjected to an environment conducive to SM production. Alternatively, secondary metabolite gene clusters (SMCs) can be activated by genetically manipulating their activators or repressors. In this study, a combination of transcriptomic and metabolomics analyses with a deletion and overexpressor mutants of TRI6 was used to establish the role of TRI6 in the regulation of several BGCs in F. graminearum. Evidence for direct and indirect regulation of BGCs by TRI6 was obtained by chromatin immunoprecipitation and yeast two-hybrid experiments. The results showed that the trichothecene genes are under direct control, while the gramillin gene cluster is indirectly controlled by TRI6 through its interaction with the pathway-specific transcription factor GRA2.

Genetic bases for variation in structure and biological activity of trichothecene toxins produced by diverse fungi.

Trichothecenes are sesquiterpene toxins produced by diverse but relatively few fungal species in at least three classes of Ascomycetes: Dothideomycetes, Eurotiomycetes, and Sordariomycetes. Approximately 200 structurally distinct trichothecene analogs have been described, but a given fungal species typically produces only a small subset of analogs. All trichothecenes share a core structure consisting of a four-ring nucleus known as 12,13-epoxytrichothec-9-ene. This structure can be substituted at various positions with hydroxyl, acyl, or keto groups to give rise to the diversity of trichothecene structures that has been described. Over the last 30years, the genetic and biochemical pathways required for trichothecene biosynthesis in several species of the fungi Fusarium and Trichoderma have been elucidated. In addition, phylogenetic and functional analyses of trichothecene biosynthetic (TRI) genes from fungi in multiple genera have provided insights into how acquisition, loss, and changes in functions of TRI genes have given rise to the diversity of trichothecene structures. These analyses also suggest both divergence and convergence of TRI gene function during the evolutionary history of trichothecene biosynthesis. What has driven trichothecene structural diversification remains an unanswered question. However, insight into the role of trichothecenes in plant pathogenesis of Fusarium species and intoplant glucosyltransferases that detoxify the toxins by glycosylating them point to a possible driver. Because the glucosyltransferases can have substrate specificity, changes in trichothecene structures produced by a fungus could allow it to evade detoxification by the plant enzymes. Thus, it is possible that advantages conferred by evading detoxification have contributed to trichothecene structural diversification. KEY POINTS : • TRI genes have evolved by diverse processes: loss, acquisition and changes in function. • Some TRI genes have acquired the same function by convergent evolution. • Someother TRI genes have evolved divergently to have different functions. • Some TRI genes were acquired or resulted from diversification in function of other genes. • Substrate specificity of plant glucosyltransferases could drive trichothecene diversity.

Gain and loss of a transcription factor that regulates late trichothecene biosynthetic pathway genes in Fusarium

Trichothecenes are among the mycotoxins of most concern to food and feed safety and are produced by species in two lineages of Fusarium: the F. incarnatum-equiseti (FIESC) and F. sambucinum (FSAMSC) species complexes. Previous functional analyses of the trichothecene biosynthetic gene (TRI) cluster in members of FSAMSC indicate that the transcription factor gene TRI6 activates expression of other TRI cluster genes. In addition, previous sequence analyses indicate that the FIESC TRI cluster includes TRI6 and another uncharacterized transcription factor gene (hereafter TRI21) that was not reported in FSAMSC. Here, gene deletion analysisindicated that in FIESC TRI6 functions in a manner similar to FSAMSC, whereas TRI21 activated expression of some genes that function late in the trichothecene biosynthetic pathway but not early-pathway genes. Consistent with this finding, TRI21 was required for formation of diacetoxyscripenol, a late-trichothecene-pathway product, but not for isotrichodermin, an early-pathway product. Although intact homologs of TRI21 were not detected in FSAMSC or other trichothecene-producing fungal genera, TRI21 fragments were detected in some FSAMSC species. This suggests that the gene was acquired by Fusarium after divergence from other trichothecene-producing fungi, was subsequently lost in FSAMSC, but was retained in FIESC. Together, our results indicate fundamental differences in regulation of trichothecene biosynthesis in FIESC and FSAMSC.

Synergistic Phytotoxic Effects of Culmorin and Trichothecene Mycotoxins.

Species of the fungus Fusarium cause Fusarium head blight (FHB) of cereal crops and contaminate grain with sesquiterpenoid mycotoxins, including culmorin (CUL) and trichothecenes. While the phytotoxicity of trichothecenes, such as deoxynivalenol (DON), and their role in virulence are well characterized, less is known about the phytotoxicity of CUL and its role in the development of FHB. Herein, we evaluated the phytotoxic effects of purified CUL and CUL-trichothecene mixtures using Chlamydomonas reinhardtii growth and Triticum aestivum (wheat) root elongation assays. By itself, CUL did not affect growth in either system. However, mixtures of CUL with DON, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, or NX-3, but not with nivalenol, inhibited growth in a synergistic manner. Synergistic phytotoxic effects of CUL and DON were also observed on multiple plant varieties and species. The severity of wheat FHB caused by 15 isolates of Fusarium graminearum was negatively correlated with the CUL/DON ratio, but positively correlated with the sum of both CUL and DON. Additionally, during the first week of infection, CUL biosynthetic genes were more highly expressed than the TRI5 trichothecene biosynthetic gene. Furthermore, genomic analysis of Fusarium species revealed that CUL and trichothecene biosynthetic genes consistently co-occur among species closely related to F. graminearum.

A cytochrome P450 monooxygenase gene required for biosynthesis of the trichothecene toxin harzianum A in Trichoderma.

Trichothecenes are sesquiterpene toxins produced by diverse fungi, including some species of Trichoderma that are potential plant disease biocontrol agents. Trichoderma arundinaceum produces the trichothecene harzianum A (HA), which consists of the core trichothecene structure (12,13-epoxytrichothec-9-ene, EPT) with a linear polyketide-derived substituent (octa-2,4,6-trienedioyl) esterified to an oxygen at carbon atom 4. The genes required for biosynthesis of EPT and the eight-carbon polyketide precursor of the octa-2,4,6-trienedioyl substituent, as well as for esterification of the substituent to EPT have been described. However, genes required for conversion of the polyketide (octa-2,4,6-trienoic acid) to octa-2,4,6-trienedioyl-CoA, the immediate precursor of the substituent, have not been described. Here, we identified 91 cytochrome P450 monooxygenase genes in the genome sequence of T. arundinaceum, and provided evidence from gene deletion, complementation, cross-culture feeding, and chemical analyses that one of them (tri23) is required for conversion of octa-2,4,6-trienoic acid to octa-2,4,6-trienedioyl-CoA. The gene was detected in other HA-producing Trichoderma species, but not in species of other fungal genera that produce trichothecenes with an octa-2,4,6-trienoic acid-derived substituent. These findings indicate that tri23 is a trichothecene biosynthetic gene unique to Trichoderma species, which in turn suggests that modification of octa-2,4,6-trienoic acid during trichothecene biosynthesis has evolved independently in some fungi.

Requirement of Two Acyltransferases for 4- O-Acylation during Biosynthesis of Harzianum A, an Antifungal Trichothecene Produced by Trichoderma arundinaceum.

Trichothecenes are sesquiterpenoid toxins produced by multiple fungi, including plant pathogens, entomopathogens, and saprotrophs. Most of these fungi have the acyltransferase-encoding gene tri18. Even though its function has not been determined, tri18 is predicted to be involved in trichothecene biosynthesis because of its pattern of expression and its location near other trichothecene biosynthetic genes. Here, molecular genetic, precursor feeding, and analytical chemistry experiments indicate that in the saprotroph Trichoderma arundinaceum the tri18-encoded acyltransferase (TRI18) and a previously characterized acyltransferase (TRI3) are required for conversion of the trichothecene biosynthetic intermediate trichodermol to harzianum A, an antifungal trichothecene analog with an octa-2,4,6-trienedioyl acyl group. On the basis of the results, we propose that TRI3 catalyzes trichothecene 4- O-acetylation, and subsequently, TRI18 catalyzes replacement of the resulting acetyl group with octa-2,4,6-trienedioyl to form harzianum A. Thus, the findings provide evidence for a previously unrecognized two-step acylation process during trichothecene biosynthesis in T. arundinaceum and possibly other fungi.

Role of Trichoderma arundinaceum tri10 in regulation of terpene biosynthetic genes and in control of metabolic flux

Production of trichothecene toxins occurs in phylogenetically diverse fungi with different lifestyles. In these fungi, most homologs of the trichothecene biosynthetic gene cluster include the transcription factor genes tri6 and tri10. Analyses of phytopathogenic species of Fusarium indicate that the TRI6 and TRI10 proteins positively regulate genes required for synthesis of trichothecenes as well as farnesyl diphosphate (FPP), a precursor of the trichothecene and other terpenoids (e.g., ergosterol). However, the apparent absence of tri6 and tri10 in some trichothecene-producing fungi, and the presence of multiple paralogs of the genes in others suggest considerable variability in genetic regulation of trichothecene biosynthesis. To begin to investigate this variability, we functionally characterized tri10 in the saprotrophic fungus Trichoderma arundinaceum. We found that TRI10 is required for wild-type expression of tri genes and trichothecene production during the first 12 h of growth of T. arundinaceum. Comparison of the effect of tri10 deletion in T. arundinaceum and Fusarium species has provided evidence for similarities in the genetic regulation of trichothecene biosynthesis in these two fungi with different lifestyles. In contrast to trichothecenes, tri10 deletion increased production of ergosterol and the polyketide-derived metabolites aspinolides, which is more likely caused by an increase in the intracellular pool of FPP resulting from loss of trichothecene production. Furthermore, although it is unclear how TRI10 affects polyketide production, one possibility is that it does so by rechanneling terpene precursors.

Effect of deletion of a trichothecene toxin regulatory gene on the secondary metabolism transcriptome of the saprotrophic fungus Trichoderma arundinaceum

Trichothecenes are terpenoid toxins produced by multiple fungal species with diverse lifestyles. In these fungi, the trichothecene biosynthetic gene (tri) cluster includes a gene encoding a Cys2His2 Zn-finger protein (TRI6). Analyses of plant pathogenic Fusarium species indicate that tri6 regulates tri gene expression. Here, we analyzed TRI6 function in the saprotrophic fungus Trichoderma arundinaceum, which produces the antimicrobial trichothecene harzianum A (HA). Deletion of the TRI6-encoding gene, tri6, blocked HA production and reduced expression of tri genes, and mevalonate biosynthetic genes required for synthesis of farnesyl diphosphate (FPP), the primary metabolite that feeds into trichothecene biosynthesis. In contrast, tri6 deletion did not affect expression of ergosterol biosynthetic genes required for synthesis of ergosterol from FPP, but did increase ergosterol production, perhaps because increased levels of FPP were available for ergosterol synthesis in the absence of trichothecene production. RNA-seq analyses indicated that genes in 10 of 49 secondary metabolite (SM) biosynthetic gene clusters in T. arundinaceum exhibited increased expression and five exhibited reduced expression in a tri6 deletion mutant (Δtri6). Despite the metabolic and transcriptional changes, Δtri6 mutants were not reduced in their ability to inhibit growth of fungal plant pathogens. Our results indicate that T. arundinaceum TRI6 regulates expression of both tri and mevalonate pathway genes. It remains to be determined whether the effects of tri6 deletion on expression of other SM clusters resulted because TRI6 can bind to promoter regions of cluster genes or because trichothecene production affects other SM pathways.

A highly efficient genetic system for the identification of a harzianum B biosynthetic gene cluster in Trichoderma hypoxylon.

Trichoderma hypoxylon is a fungicolous species which produces rich secondary metabolites. However, no genetic transformation method is available for further studies. Here, we developed a marker-less transformation system based on the complementation of an uridine/uracil biosynthetic gene by protoplast transformation. An uridine/uracil auxotrophic mutant of Δthpyr4 was obtained by using a positive screening protocol with 5'-fluoroorotic acid as a selective reagent. To improve the homologous integration rates, the orthologues of ku70 and lig4 which play critical roles in non-homologous end-joining recombination were disrupted. The resulting thlig4 mutant showed remarkable transformation rates of 89 %, while no change was found in the thku70 deletion mutant compared with the WT strain. This suggests that thlig4 play a key role in the non-homologous recombination in this strain. Using this system, the biosynthetic gene cluster of trichothecene (tri) harzianum B was identified by deletion of the thtri5 in T. hypoxylon. Comparative genome analysis revealed that the trichothecene biosynthetic gene cluster in T. hypoxylon shared similar organizations with T. arundinaceum and T. brevicompactum, even though their encoded products are different in structures. Taken together, the highly efficient genetic system provides a convenient tool for studying the biosynthetic diversity and mining the novel natural product from the fungi.

Evolution of structural diversity of trichothecenes, a family of toxins produced by plant pathogenic and entomopathogenic fungi

Trichothecenes are a family of terpenoid toxins produced by multiple genera of fungi, including plant and insect pathogens. Some trichothecenes produced by the fungus Fusarium are among the mycotoxins of greatest concern to food and feed safety because of their toxicity and frequent occurrence in cereal crops, and trichothecene production contributes to pathogenesis of some Fusarium species on plants. Collectively, fungi produce over 150 trichothecene analogs: i.e., molecules that share the same core structure but differ in patterns of substituents attached to the core structure. Here, we carried out genomic, phylogenetic, gene-function, and analytical chemistry studies of strains from nine fungal genera to identify genetic variation responsible for trichothecene structural diversity and to gain insight into evolutionary processes that have contributed to the variation. The results indicate that structural diversity has resulted from gain, loss, and functional changes of trichothecene biosynthetic (TRI) genes. The results also indicate that the presence of some substituents has arisen independently in different fungi by gain of different genes with the same function. Variation in TRI gene duplication and number of TRI loci was also observed among the fungi examined, but there was no evidence that such genetic differences have contributed to trichothecene structural variation. We also inferred ancestral states of the TRI cluster and trichothecene biosynthetic pathway, and proposed scenarios for changes in trichothecene structures during divergence of TRI cluster homologs. Together, our findings provide insight into evolutionary processes responsible for structural diversification of toxins produced by pathogenic fungi.

Screening of Deoxynivalenol Producing Strains and Elucidation of Possible Toxigenic Molecular Mechanism.

In this study, seven strains of Fusarium graminearum were isolated from wheat, of which six were identified to produce deoxynivalenol and the production of deoxynivalenol was assessed. F. graminearum strain Fg1 was noted to produce 1.0 μg/g deoxynivalenol during the incubation period in the Czapek yeast broth, while none was detected in F. graminearum strain Fg2. Hence, the differences in proteomes and transcriptomes of Fg1 and Fg2 were compared to analyze the mechanism underlying deoxynivalenol production. Among the 66 significantly differentially expressed proteins in Fg1, 39 and 27 were more or less abundant expressed. Functional analysis suggested that the enzymes involved in the methylerythritol 4-phosphate and mevalonate pathways, which provide a substrate for biosynthesis of farnesyl pyrophosphate, a precursor of DON, were activated in Fg1. The transcriptomics data demonstrated that the expression level of a majority of genes, including trichothecene biosynthetic genes, protein kinases, and transcription factors, involved in trichothecene biosynthesis was higher in Fg1 than in Fg2. The results also revealed differential expression profiles of deoxynivalenol biosynthesis genes in strains Fg1 and Fg2, which emphasized their deoxynivalenol producing ability and the underlying mechanism.

Down-regulatory effect of Thymus vulgaris L. on growth and Tri4 gene expression in Fusarium oxysporum strains.

The aims of this study were to evaluate the efficacy of Thymus vulgaris (T.vulgaris) essential oil on the fungal growth and Tri4 gene expression in Fusarium oxysporum (F.oxysporum) strains. The oil was obtained by water-distillation using a Clevenger-type system. The chemical composition of the essential oil was obtained by gas chromatography- mass spectroscopy (GC-MS) and by retention indices. The antifungal activity was evaluated by broth microdilution assay. A quantitative real time RT-PCR (qRT-PCR) assay was also developed specific for F.oxysporum on the basis of trichothecene biosynthetic gene, Tri4, which allowed discrimination from F.oxysporum. Results showed thymol (32.67%) and p-cymene (16.68%) as the main components of T.vulgaris. Minimum inhibitory concentration (MIC) values varied from 5 to 20μg/ml with T.vulgaris (mean: 10.50μg/ml), while minimum fungicidal concentration (MFC) values ranged from 8 to 30μg/ml with mean value of 16.20μg/ml qRT-PCR results revealed a downregulation from 4.04 to 6.27 fold of Tri4 gene expression of the fungi exposed to T.vulgaris essential oil. The results suggest that T.vulgaris oil can be considered potential alternative natural fungicide to the synthetic chemicals that are currently used to prevent and control seed-borne diseases.

A polyphasic approach for characterization of a collection of cereal isolates of the Fusarium incarnatum-equiseti species complex

DNA-based phylogenetic analyses have resolved the fungal genus Fusarium into multiple species complexes. The F. incarnatum-equiseti species complex (FIESC) includes fusaria associated with several diseases of agriculturally important crops, including cereals. Although members of FIESC are considered to be only moderately aggressive, they are able to produce a diversity of mycotoxins, including trichothecenes, which can accumulate to harmful levels in cereals. High levels of cryptic speciation have been detected within the FIESC. As a result, it is often necessary to use approaches other than morphological characterization to distinguish species. In the current study, we used a polyphasic approach to characterize a collection of 69 FIESC isolates recovered from cereals in Europe, Turkey, and North America. In a species phylogeny inferred from nucleotide sequences from four housekeeping genes, 65 of the isolates were resolved within the Equiseti clade of the FIESC, and four isolates were resolved within the Incarnatum clade. Seven isolates were resolved as a genealogically exclusive lineage, designated here as FIESC 31. Phylogenies based on nucleotide sequences of trichothecene biosynthetic genes and MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) were largely concordant with phylogeny inferred from the housekeeping gene. Finally, Liquid Chromatography (Time-Of-Flight) Mass Spectrometry [LC-(TOF-)MS(/MS)] revealed variability in mycotoxin production profiles among the different phylogenetic species investigated in this study.

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.