Accumulation Of Trichothecenes Research Articles

A Role in 15-Deacetylcalonectrin Acetylation in the Non-Enzymatic Cyclization of an Earlier Bicyclic Intermediate in Fusarium Trichothecene Biosynthesis.

The trichothecene biosynthesis in Fusarium begins with the cyclization of farnesyl pyrophosphate to trichodiene, followed by subsequent oxygenation to isotrichotriol. This initial bicyclic intermediate is further cyclized to isotrichodermol (ITDmol), a tricyclic precursor with a toxic trichothecene skeleton. Although the first cyclization and subsequent oxygenation are catalyzed by enzymes encoded by Tri5 and Tri4, the second cyclization occurs non-enzymatically. Following ITDmol formation, the enzymes encoded by Tri101, Tri11, Tri3, and Tri1 catalyze 3-O-acetylation, 15-hydroxylation, 15-O-acetylation, and A-ring oxygenation, respectively. In this study, we extensively analyzed the metabolites of the corresponding pathway-blocked mutants of Fusarium graminearum. The disruption of these Tri genes, except Tri3, led to the accumulation of tricyclic trichothecenes as the main products: ITDmol due to Tri101 disruption; a mixture of isotrichodermin (ITD), 7-hydroxyisotrichodermin (7-HIT), and 8-hydroxyisotrichodermin (8-HIT) due to Tri11 disruption; and a mixture of calonectrin and 3-deacetylcalonectrin due to Tri1 disruption. However, the ΔFgtri3 mutant accumulated substantial amounts of bicyclic metabolites, isotrichotriol and trichotriol, in addition to tricyclic 15-deacetylcalonectrin (15-deCAL). The ΔFgtri5ΔFgtri3 double gene disruptant transformed ITD into 7-HIT, 8-HIT, and 15-deCAL. The deletion of FgTri3 and overexpression of Tri6 and Tri10 trichothecene regulatory genes did not result in the accumulation of 15-deCAL in the transgenic strain. Thus, the absence of Tri3p and/or the presence of a small amount of 15-deCAL adversely affected the non-enzymatic second cyclization and C-15 hydroxylation steps.

Performance of Winter Wheat Cultivars Grown Organically and Conventionally with Focus on Fusarium Head Blight and Fusarium Trichothecene Toxins.

Growing acreage and changing consumer preferences cause increasing interest in the cereal products originating from organic farming. Lack of results of objective test, however, does not allow drawing conclusions about the effects of cultivation in the organic system and comparison to currently preferred conventional system. Field experiment was conducted in organic and conventional fields. Thirty modern cultivars of winter wheat were sown. They were characterized for disease infection including Fusarium head blight, seed sowing value, the amount of DNA of the six species of Fusarium fungi as well as concentration of ergosterol and trichothecenes in grain. The intensity Fusarium head blight was at a similar level in both systems. However, Fusarium colonization of kernels expressed as ergosterol level or DNA concentration was higher for the organic system. It did not reflect in an increased accumulation of trichothecenes in grain, which was similar in both systems, but sowing value of organically produced seeds was lower. Significant differences between analyzed cropping systems and experimental variants were found. The selection of the individual cultivars for organic growing in terms of resistance to diseases and contamination of grain with Fusarium toxins was possible. Effects of organic growing differ significantly from the conventional and grain obtained such way can be recommended to consumers. There are indications for use of particular cultivars bred for conventional agriculture in the case of organic farming, and the growing organic decreases plant stress resulting from intense fertilization and chemical plant protection.

Expression of the Fusarium graminearum terpenome and involvement of the endoplasmic reticulum-derived toxisome

The sesquiterpenoid deoxynivalenol (DON) is an important trichothecene mycotoxin produced by the cereal pathogen Fusarium graminearum. DON is synthesized in specialized subcellular structures called toxisomes. The first step in DON synthesis is catalyzed by the sesquiterpene synthase (STS), Tri5 (trichodiene synthase), resulting in the cyclization of farnesyl diphosphate (FPP) to produce the sesquiterpene trichodiene. Tri5 is one of eight putative STSs in the F. graminearum genome. To better understand the F. graminearum terpenome, the volatile and soluble fractions of fungal cultures were sampled. Stringent regulation of sesquiterpene accumulation was observed. When grown in trichothecene induction medium, the fungus produces trichothecenes as well as several volatile non-trichothecene related sesquiterpenes, whereas no volatile terpenes were detected when grown in non-inducing medium. Surprisingly, a Δtri5 deletion strain grown in inducing conditions not only ceased accumulation of trichothecenes, but also failed to produce the non-trichothecene related sesquiterpenes. To test whether Tri5 from F. graminearum may be a promiscuous STS directly producing all observed sesquiterpenes, Tri5 was cloned and expressed in E. coli and shown to produce primarily trichodiene in addition to minor, related cyclization products. Therefore, while Tri5 expression in F. graminearum is necessary for non-trichothecene sesquiterpene biosynthesis, direct catalysis by Tri5 does not explain the sesquiterpene deficient phenotype observed in the Δtri5 strain. To test whether Tri5 protein, separate from its enzymatic activity, may be required for non-trichothecene synthesis, the Tri5 locus was replaced with an enzymatically inactive, but structurally unaffected tri5N225D S229T allele. This allele restores non-trichothecene synthesis but not trichothecene synthesis. The tri5N225D S229T allele also restores toxisome structure which is lacking in the Δtri5 deletion strain. Our results indicate that the Tri5 protein, but not its enzymatic activity, is also required for the synthesis of non-trichothecene related sesquiterpenes and the formation of toxisomes. Toxisomes thus not only may be important for DON synthesis, but also for the synthesis of other sesquiterpene mycotoxins such as culmorin by F. graminearum.

Resistance-Related l-Pyroglutamic Acid Affects the Biosynthesis of Trichothecenes and Phenylpropanoids by F. graminearum Sensu Stricto.

Fungicide application remains amongst the most widely used methods of fungal control in agroecosystems. However, the extensive use of fungicides poses hazards to human health and the natural environment and does not always ensure the effective decrease of mycotoxins in food and feed. Nowadays, the rising threat from mycotoxin contamination of staple foods has stimulated efforts in developing alternative strategies to control plant pathogenic fungi. A substantial effort is focused on the identification of plant-derived compounds inhibiting mycotoxin production by plant pathogenic fungi. l-Pyroglutamic acid has recently been suggested as playing a role in the response of barley to toxigenic Fusaria. Considering the above, we studied the response of various strains of F. graminearum sensu stricto to different levels of l-pyroglutamic acid on solid YES (yeast extract sucrose) media. l-Pyroglutamic acid decreased the accumulation of trichothecenes in all examined strains. Gene expression studies addressing Tri genes (Tri4, Tri5, and Tri10), which induce the biosynthesis of trichothecenes, revealed the production of mycotoxins by l-pyroglutamic acid to be inhibited at the transcriptional level. Besides inhibitory effects on mycotoxin production, l-pyroglutamic acid exhibited variable and concentration-related effects on phenylpropanoid production by fungi. Accumulation of most of the fungal-derived phenolic acids decreased in the presence of 100 and 400 µg/g of l-pyroglutamic acid. However, a higher dose (800 µg/g) of l-pyroglutamic acid increased the accumulation of trans-cinnamic acid in the media. The accumulation of fungal-derived naringenin increased in the presence of l-pyroglutamic acid. Contrasting results were obtained for quercetin, apigenin, luteolin, and kaempferol, the accumulation of which decreased in the samples treated with 100 and 400 µg/g of l-pyroglutamic acid, whereas the highest l-pyroglutamic acid concentration (800 µg/g) seemed to induce their biosynthesis. The results obtained in this study provide new insights for breeders involved in studies on resistance against Fusaria.

Functional characterization of a soluble NADPH-cytochrome P450 reductase from Fusarium graminearum

Fusarium head blight is a devastating disease in wheat caused by some fungal pathogens of the Fusarium genus mainly F. graminearum, due to accumulation of toxic trichothecenes. Most of the trichothecene biosynthetic pathway has been mapped, although some proteins of the pathway remain uncharacterized, including an NADPH-cytochrome P450 reductase. We subcloned a F. graminearum cytochrome P450 reductase that might be involved in the trichothecene biosynthesis. It was expressed heterologously in E. coli as N-terminal truncated form with an octahistidine tag for purification. The construct yielded a soluble apoprotein and its incubation with flavins yielded the corresponding monomeric holoprotein. It was characterized for activity in the pH range 5.5–9.5, using thiazolyl blue tetrazolium bromide (MTT) or cytochrome c as substrates. Binding of the small molecule MTT was weaker than for cytochrome c, however, the rate of MTT reduction was faster. Contrary to other studies of cytochrome reductase proteins, MTT reduction proceeded in a cooperative manner in our studies. Optimum kinetic activity was found at pH 7.5–8.5 for bothMTT and cytochrome c. This is the first paper presenting characterization of a cytochrome P450 reductase from F. graminearum which most likely is involved in mycotoxin biosynthesis or some primary metabolic pathway such as sterol biosynthesis in F. graminearum.

Correlation of Deoxynivalenol Accumulation in Fusarium-Infected Winter and Spring Wheat Cultivars with Secondary Metabolites at Different Growth Stages.

Fusarium infection in wheat causes Fusarium head blight, resulting in yield losses and contamination of grains with trichothecenes. Some plant secondary metabolites inhibit accumulation of trichothecenes. Eighteen Fusarium infected wheat cultivars were harvested at five time points and analyzed for the trichothecene deoxynivalenol (DON) and 38 wheat secondary metabolites (benzoxazinoids, phenolic acids, carotenoids, and flavonoids). Multivariate analysis showed that harvest time strongly impacted the content of secondary metabolites, more distinctly for winter wheat than spring wheat. The benzoxazinoid 2-β-glucopyranoside-2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA-glc), α-tocopherol, and the flavonoids homoorientin and orientin were identified as potential inhibitors of DON accumulation. Several phenolic acids, lutein and β-carotene also affected DON accumulation, but the effect varied for the two wheat types. The results could form a basis for choosing wheat cultivars using metabolite profiling as a marker for selecting wheat cultivars with improved resistance against Fusarium head blight and accumulation of trichothecene toxins in wheat heads.

2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) inhibits trichothecene production by Fusarium graminearum through suppression of Tri6 expression

Fusarium head blight (FHB) is a devastating disease of wheat (Triticum aestivum L.) caused by a mycotoxigenic fungus Fusarium graminearum resulting in significantly decreased yields and accumulation of toxic trichothecenes in grains. We tested 7 major secondary metabolites from wheat for their effect on trichothecene production in liquid cultures of F. graminearum producing trichothecene 15-acetyldeoxynivalenol (15-ADON). 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) benzoxazinoid completely abolished toxin production without any apparent effect on fungal growth. DIMBOA strongly affected the expression of Tri6, encoding a major transcriptional regulator of several genes of the trichothecene biosynthesis pathway. DIMBOA also repressed expression of Tri5, encoding trichodiene synthase, the first enzyme in the trichothecene biosynthesis pathway. Thus, DIMBOA could play an important role against the accumulation of trichothecenes in wheat grain. Breeding or engineering of wheat with increased levels of benzoxazinoids could provide varieties with increased resistance against trichothecene contamination of grain and lower susceptibility to FHB.

Sublethal concentrations of azoles induce tri transcript levels and trichothecene production in Fusarium graminearum

The effect of sublethal concentrations (below the recommended field doses) of propiconazole and tebuconazole on the amount of tri transcripts and accumulation of trichothecenes by three Fusarium graminearum isolates of 3ADON, 15ADON, and NIV chemotypes was examined on yeast extract sucrose agar (YES) medium. RT-qPCR analyses showed higher tri4, tri5, and tri11 transcript levels in cultures of all three F. graminearum isolates supplemented with sublethal concentrations of azoles as compared to those in nontreated control, although the fold changes in the amount of tri transcripts differed according to the type of azole used. Mycotoxin analysis revealed higher increase in trichothecene accumulation in most of the tebuconazole-treated samples of all chemotypes tested. A huge increase in all trichothecene compounds was revealed in samples of all F. graminearum isolates treated with 5mgL(-1) of tebuconazole. An inducing effect of azoles on trichothecene accumulation in the grain was confirmed in an in planta experiment; however, the results obtained were inconsistent. A higher amount of trichothecenes and fungal DNA was quantitated in two grain samples treated with sublethal propiconazole concentrations. In contrast, no significant increase in trichothecene levels was revealed in grain samples treated with sublethal concentrations of tebuconazole.

Trichothecene Mycotoxins Associated with Potato Dry Rot Caused by Fusarium graminearum

Fusarium graminearum, a known producer of trichothecene mycotoxins in cereal hosts, has been recently documented as a cause of dry rot of potato tubers in the United States. Due to the uncertainty of trichothecene production in these tubers, a study was conducted to determine the accumulation and diffusion of trichothecenes in potato tubers affected with dry rot caused by F. graminearum. Potato tubers of cv. Russet Burbank were inoculated with 14 F. graminearum isolates from potato, sugar beet, and wheat and incubated at 10 to 12 degrees C for 5 weeks to determine accumulation of trichothecenes in potato tubers during storage. Twelve of the isolates were classified as deoxynivalenol (DON) genotype and two isolates were as nivalenol (NIV) genotype. Trichothecenes were detected only in rotted tissue. DON was detected in all F. graminearum DON genotype isolates up to 39.68 microg/ml in rotted potato tissue. Similarly, both NIV genotype isolates accumulated NIV in rotted potato tissue up to 18.28 microg/ml. Interestingly, isolates classified as genotype DON accumulated both DON and NIV in the dry rot lesion. Potato tubers were then inoculated with two isolates of F. graminearum chemotype DON and incubated up to 7 weeks at 10 to 12 degrees C and assayed for DON diffusion. F. graminearum was recovered from >53% of the isolations from inoculated tubers at 3 cm distal to the rotted tissue after 7 weeks of incubation but DON was not detected in the surrounding tissue. Based in this data, the accumulation of trichothecenes in the asymptomatic tissue surrounding dry rot lesions caused by F. graminearum is minimal in cv. Russet Burbank potato tubers stored for 7 weeks at customary processing storage temperatures.

Macrocyclic trichothecene production and sporulation by a biological control strain of Myrothecium verrucaria is regulated by cultural conditions

Myrothecium verrucaria is a pathogen of several invasive weed species, including kudzu, and is currently being evaluated for use as a bioherbicide. However, the fungus also produces macrocyclic trichothecene mycotoxins. The safety of this biological control agent during production and handling would be improved if an inoculum could be produced without concomitant accumulation of macrocyclic trichothecenes. Sporulation and trichothecene production by M. verrucaria was evaluated on standard potato dextrose agar (PDA) and a series of complex and defined media. Sporulation on PDA and on agar media with nitrogen as ammonium nitrate or potassium nitrate was more than ten-fold greater then sporulation on the medium with ammonium sulphate as the nitrogen source. Accumulation of macrocyclic trichothecenes was strongly affected by the media composition, with higher levels often associated with higher carbon content in the media. Overall, incubation in continuous darkness resulted in higher macrocyclic trichothecene concentrations. Results support the hypothesis that accumulation of macrocyclic trichothecenes by this fungus can be altered by manipulating carbon and nitrogen sources. Furthermore, the biosynthesis of these mycotoxins may be independent of sporulation, demonstrating that the bioherbicide can be readily produced on solid substrates while simultaneously yielding conidia that are less threatening to worker safety. A more detailed implementation of the concepts demonstrated in this study will facilitate the safe and economical production of this bioherbicide.

Natural mechanisms for cereal resistance to the accumulation of Fusarium trichothecenes

This review describes the naturally occurring mechanisms in cereals that lead to a reduction of Fusarium trichothecene mycotoxin accumulation in grains. A reduction in mycotoxin contamination in grains could also limit fungal infection, as trichothecenes have been reported to act as virulence factors. The mechanisms explaining the low toxin accumulation trait, generally referred to as type V resistance to Fusarium, can be subdivided into two classes. Class 1 includes mechanisms by which the plants chemically transform the trichothecenes, leading to their degradation or detoxification. Among the detoxification strategies, glycosylation of trichothecenes is a natural process already reported in wheat. According to the structure and the toxicity of trichothecenes, two other detoxification processes, acetylation and de-epoxidation, can be expressed, at least in transgenic plants. Class 2 comprises mechanisms that lead to reduced mycotoxin accumulation by inhibition of their biosynthesis through the action of plant endogenous compounds. These include both grain constitutive compounds and compounds induced in response to pathogen infection. There are already many compounds with antioxidant properties, like phenolic compounds, peptides or carotenoids, and with prooxidant properties, like hydrogen peroxide or linoleic acid-derived hydroperoxides, that have been described as ‘modulators’ of mycotoxin biosynthesis. This review addresses for the first time different studies reporting specific in vitro effects of such compounds on the biosynthesis of Fusarium mycotoxins. A better understanding of the natural processes limiting accumulation of trichothecenes in the plant will open the way to the development of novel breeding varieties with reduced ‘mycotoxin risk’.

Accumulation of deoxynivalenol and its 15-acetylated form is significantly modulated by oxidative stress in liquid cultures of Fusarium graminearum

Liquid cultures of Fusarium graminearum were supplemented with H2O2 or other oxidative compounds. The accumulation kinetics of the resulting trichothecenes were monitored. At non-lethal concentrations, the H2O2 treatments modulated toxin accumulation, dependent on the method of supplementation. When H2O2 was added at the same time as the inoculation, higher levels of toxins accumulated 30 days later. Conversely, adding H2O2 2 or 7 days after inoculation had little effect. When H2O2 was added daily over the course of the culture, the accumulation of trichothecenes was rapidly and strongly enhanced. The fungus may adapt to oxidative stress when the first exposure to H2O2 occurs at the beginning of the culture course. The highest toxin levels were measured when the H2O2 was added daily. The importance of the first hours of culture was confirmed: pre-treating conidia with H2O2 does not affect their germination kinetics but leads to a reduction in the yield of trichothecenes 40 days later. The H2O2 regulation of this trichothecene accumulation may be specific, as paraquat, another pro-oxidant compound, inhibits their production. Since H2O2 is a major component of the oxidative burst occurring in pathogen/host interactions, these data support the theory that trichothecenes may act as virulence factors.