Production Of Trichothecene Mycotoxins Research Articles

Genome Sequence Resource of the Head Blight Pathogens Fusarium asiaticum and F. graminearum Isolated from Cereal Crops and Gramineous Weeds in Korea and China

Head blight is a serious fungal disease that results in yield loss and mycotoxin contamination in cereal crops worldwide. Head blight is primarily caused by members of the Fusarium graminearum species complex (FGSC), which consists of at least 16 species, including Fusarium asiaticum and Fusarium graminearum. FGSC members produce trichothecene mycotoxins and can be divided into four groups (chemotypes) depending on the type of trichothecenes produced: NIV (production of nivalenol), 3ADON (production of 3-acetyldeoxynivalenol), 15ADON (production of 15-acetyldeoxynivalenol), and NX-2 (production of 3α-acetoxy, 7α, 15-dihydroxy-12, and 13-epoxytrichothe-9-ene). Whereas F. graminearum mainly causes head blight in wheat-producing areas, F. asiaticum is associated primarily with rice head blight in eastern Asia. To date, the NCBI database archives 116 assembled genome sequences of F. graminearum, but only four accessions correspond to F. asiaticum. Accessions for three F. graminearum strains (PH-1 = NRRL 31084, CS3005, and FG-12) are chromosome assemblies (i.e., include contigs/scaffolds consisting of full-length or nearly full-length chromosomes), but none of the F. asiaticum accessions is a chromosome assembly. This announcement reports the assembled genome sequences of nine F. asiaticum strains and seven F. graminearum strains isolated from rice, wheat, barley, and other gramineous plants in Korea and China. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

The Fusarium graminearum Transporters Abc1 and Abc6 Are Important for Xenobiotic Resistance, Trichothecene Accumulation, and Virulence to Wheat.

The plant pathogenic fungus Fusarium graminearum is the causal agent of Fusarium head blight (FHB) disease on small-grain cereals. F. graminearum produces trichothecene mycotoxins such as deoxynivalenol (DON) that are required for full virulence. DON must be exported outside the cell to cause FHB disease, a process that may require the involvement of membrane-bound transporters. In this study, we show that the deletion of membrane-bound transporters results in reduced DON accumulation as well as reduced FHB symptoms on wheat. Deletion of the ATP-binding cassette (ABC) transporter gene Abc1 results in the greatest reduction in DON accumulation and virulence. Deletion of another ABC transporter gene, Abc6, also reduces FHB symptoms to a lesser degree. Combining deletions fails to reduce DON accumulation or virulence in an additive fashion, even when a ∆abc1 deletion is included. Heterologous expression of F. graminearum transporters in a DON-sensitive strain of yeast confirms Abc1 as a major DON resistance mechanism; furthermore, it suggests that Abc1 is directly participating in DON transport rather than facilitating DON transport though other means. Yeast expression further indicates that multiple transporters, including Abc1, play an important role in resistance to the wheat phytoalexin 2-benzoxazolinone (BOA) and other xenobiotics. Thus, Abc1 may contribute to virulence on wheat both by facilitating export of DON and by providing resistance to the wheat phytoalexin BOA. This research provides useful information that may aid in designing novel management techniques of FHB or other destructive plant diseases.

Identification of Candidate Genes Associated with Trichothecene Biosynthesis in Fusarium graminearum Species Complex Combined with Transcriptomic and Proteomic Analysis.

The Fusarium graminearum species complex is the main causal agent of wheat head blight worldwide. Trichothecenes produced by the pathogen in infected grains have important food safety implications. Previously reported studies on trichothecene production have all focused on the conditions conducive to mycotoxin production, while the molecular mechanisms of trichothecene biosynthesis in Fusarium strains under normal or non-inducing conditions are still unclear. Here, a global analysis of the fungal gene expression of three strains using the Affymetrix Fusarium GeneChip under non-inducing conditions is reported. Differentially expressed genes were identified among strains with different trichothecene-production ability, and some novel genes associated with trichothecene biosynthesis were found by bioinformatics analysis. To verify the transcriptome results, proteomic analyses of the three strains were conducted under the same culture conditions. In total, 69 unique fungal proteins were identified in 77 protein spots. Combined with transcriptome and proteome analysis, 27 novel genes were predicted to be associated with trichothecene mycotoxin production. A protein, encoded by FGSG_01403, was found to be associated with trichothecene production via proteome analysis. Gene knock-out mutations of FGSG_01403 resulted in mutants with increased production of trichothecenes. Future functional analysis of the candidate genes identified in this study may reveal new insights into the negative regulation of trichothecene production in the Fusarium graminearum species complex.

Investigation of the inflammatory and oxidative stress-inducing effects of deoxynivalenol and T-2 toxin exposure in non-tumorigenic human intestinal cell model

Fungi in the Fusarium genus produce trichothecene mycotoxins including deoxynivalenol (DON) and T-2 toxin which may elicit their damaging effects on the gastrointestinal tract following the consumption of contaminated cereal-based foods. The aim of our study was to evaluate the effects of these commonly occurring fusarotoxins alone and in combination using the human, non-cancerous intestinal epithelial cell line HIEC-6. Based on our experimental data, 24 h after treatment with fusarotoxins, hydrogen peroxide levels, intracellular oxidative stress and the amounts of inflammatory interleukins IL-6 and IL-8 significantly increased. Cell membrane localization of the tight junction protein claudin-1 decreased, whereas distribution of occludin remained unchanged. Taken together, the HIEC-6 cell line appears to be a suitable experimental model for monitoring the combined effects of mycotoxins at the cellular level including changes in the redox states of cells.

Fusarium: more than a node or a foot-shaped basal cell.

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).

Impact of nitrogen metabolism-associated culture pH changes on regulation of Fusarium trichothecene biosynthesis: revision of roles of polyamine agmatine and transcription factor AreA.

Fusarium graminearum produces trichothecene mycotoxins in infected grains and axenic liquid culture. A proposed regulatory model of trichothecene biosynthesis was examined in relation to nitrogen utilization. First, we showed that an important factor for the stimulation of trichothecene biosynthesis was not the occurrence of agmatine as a specific inducer molecule, but rather continuous acidification of the liquid culture medium arising from agmatine catabolism. When the pH of the L-Gln synthetic medium was frequently adjusted to the pH of the agmatine culture, trichothecene productivity of the L-Gln culture was equal to that of the agmatine culture. For efficient trichothecene biosynthesis, the culture pH should be lowered at an appropriate time point during the early growth stage. Second, we re-evaluated the role of the nitrogen regulatory GATA transcription factor AreA in trichothecene biosynthesis. Since Tri6 encodes a transcription factor indispensable for trichothecene biosynthesis, all fifteen AreA-binding consensus sequences in the Tri6 promoter were mutated. The mutant could catabolize L-Phe as the sole nitrogen source; furthermore, the pH profile of the synthetic L-Phe medium (initial pH 4.2) was the same as that of the wild-type (WT) strain. Under such conditions, the promoter mutant exhibited approximately 72% of the trichothecene productivity compared to the WT strain. Thus, F. graminearum AreA (FgAreAp) is dispensable for the functioning of the Tri6 promoter, but it contributes to the increased production of mycotoxin under mildly acidic conditions to some extent. Further investigations on the culture pH revealed that extremely low pH bypasses the function of FgAreAp.

Clove oil-in-water nanoemulsion: Mitigates growth of Fusarium graminearum and trichothecene mycotoxin production during the malting of Fusarium infected barley

Fusarium mycotoxin contamination in malting barley is of great concerns in malting industry. Our recent study found that clove oil nanoemulsions can act as highly efficient antifungal agents in vitro. Therefore, we explored the efficacy of clove oil nanoemulsions on Fusarium growth and mycotoxin during malting process. The impact of emulsifier types (Tween 80, BSA and quillaja saponins) on the formation of clove oil nanoemulsion, the mitigation effects on mycotoxin levels and fungal biomass, and the clove oil flavor residues on malts were measured. We observed that 1.5 mg clove oil/g nanoemulsion showed a negligible influence on germinative energy of barley, while still efficiently eliminated the DON levels and toxicogenic fungal biomass as quantified by Tri5 DNA content. Tween 80-stablized clove oil nanoemulsion displayed higher mycotoxin inhibitory activity and less flavor impact on the final malt. The results indicated the potential application of essential oil nanoemulsion during the malting process.

Selection of Fusarium Trichothecene Toxin Genes for Molecular Detection Depends on TRI Gene Cluster Organization and Gene Function.

Food security is a global concern. Fusarium are among the most economically important fungal pathogens because they are ubiquitous, disease management remains a challenge, they produce mycotoxins that affect food and feed safety, and trichothecene mycotoxin production can increase the pathogenicity of some Fusarium species depending on the host species. Although trichothecenes may differ in structure by their patterns of hydroxylation or acetylation, these small changes have a significant impact on toxicity and the biological activity of these compounds. Therefore, detecting and identifying which chemotype is present in a given population are important to predicting the specific toxins that may be produced and, therefore, to evaluating the risk of exposure. Due to the challenges of inducing trichothecene production by Fusarium isolates in vitro for subsequent chemical analysis, PCR assays using gene-specific primers, either singly or in combination, designed against specific genes of the trichothecene gene cluster of multiple species of Fusarium have been developed. The establishment of TRI genotypes that potentially correspond to a specific chemotype requires examination of an information and knowledge pipeline whose critical aspects in sequential order are: (i) understanding the TRI gene cluster organization which differs according to Fusarium species under study; (ii) knowledge of the re-arrangements to the core TRI gene cluster over evolutionary time, which also differs according to Fusarium species; (iii) the functions of the TRI genes in the biosynthesis of trichothecene analogs; and (iv) based on (i)–(iii), selection of appropriate target TRI gene(s) for primer design in PCR amplification for the Fusarium species under study. This review, therefore, explains this pipeline and its connection to utilizing TRI genotypes as a possible proxy to chemotype designation.

L-Threonine and its analogue added to autoclaved solid medium suppress trichothecene production by Fusarium graminearum

Fusarium graminearum produces trichothecene mycotoxins under certain nutritional conditions. When L-Thr and its analogue L-allo-threonine were added to brown rice flour solid medium before inoculation, trichothecene production after 4 days of incubation was suppressed. A time-course analysis of gene expression demonstrated that L-Thr suppressed transcription of Tri6, a trichothecene master regulator gene, and a terpene cyclase Tri5 gene. Regulation of trichothecene biosynthesis by altering major primary metabolic processes may open up the possibility to develop safe chemicals for the reduction of mycotoxin contamination might be developed.

Transgenic Wheat Expressing a Barley UDP-Glucosyltransferase Detoxifies Deoxynivalenol and Provides High Levels of Resistance to Fusarium graminearum.

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is a devastating disease of wheat that results in economic losses worldwide. During infection, F. graminearum produces trichothecene mycotoxins, including deoxynivalenol (DON), that increase fungal virulence and reduce grain quality. Transgenic wheat expressing a barley UDP-glucosyltransferase (HvUGT13248) were developed and evaluated for FHB resistance, DON accumulation, and the ability to metabolize DON to the less toxic DON-3-O-glucoside (D3G). Point-inoculation tests in the greenhouse showed that transgenic wheat carrying HvUGT13248 exhibited significantly higher resistance to disease spread in the spike (type II resistance) compared with nontransformed controls. Two transgenic events displayed complete suppression of disease spread in the spikes. Expression of HvUGT13248 in transgenic wheat rapidly and efficiently conjugated DON to D3G, suggesting that the enzymatic rate of DON detoxification translates to type II resistance. Under field conditions, FHB severity was variable; nonetheless, transgenic events showed significantly less-severe disease phenotypes compared with the nontransformed controls. In addition, a seedling assay demonstrated that the transformed plants had a higher tolerance to DON-inhibited root growth than nontransformed plants. These results demonstrate the utility of detoxifying DON as a FHB control strategy in wheat.

Leucine metabolism regulates TRI6 expression and affects deoxynivalenol production and virulence in Fusarium graminearum.

TRI6 is a positive regulator of the trichothecene gene cluster and the production of trichothecene mycotoxins [deoxynivalenol (DON)] and acetylated forms such as 15-Acetyl-DON) in the cereal pathogen Fusarium graminearum. As a global transcriptional regulator, TRI6 expression is modulated by nitrogen-limiting conditions, sources of nitrogen and carbon, pH and light. However, the mechanism by which these diverse environmental factors affect TRI6 expression remains underexplored. In our effort to understand how nutrients affect TRI6 regulation, comparative digital expression profiling was performed with a wild-type F. graminearum and a Δtri6 mutant strain, grown in nutrient-rich conditions. Analysis showed that TRI6 negatively regulates genes of the branched-chain amino acid (BCAA) metabolic pathway. Feeding studies with deletion mutants of MCC, encoding methylcrotonyl-CoA-carboxylase, one of the key enzymes of leucine metabolism, showed that addition of leucine specifically down-regulated TRI6 expression and reduced 15-ADON accumulation. Constitutive expression of TRI6 in the Δmcc mutant strain restored 15-ADON production. A combination of cellophane breach assays and pathogenicity experiments on wheat demonstrated that disrupting the leucine metabolic pathway significantly reduced disease. These findings suggest a complex interaction between one of the primary metabolic pathways with a global regulator of mycotoxin biosynthesis and virulence in F. graminearum.

Structure elucidation and biological activity of two new trichothecenes from an endophyte, Myrothecium roridum.

Worldwide, many different grains are infected by various fungi that may produce trichothecene mycotoxins. Fungi that produce trichothecenes, as well as the trichothecenes themselves, are potential problems for public health. On the other hand, trichothecenes possess multiple biological activities. Reduced toxicity may result in their applications in the pharmaceutical field. Two new trichothecenes along with seven known trichothecenes were isolated from an endophyte of the herb plant Ajuga decumbens. Their structures were deduced from 1D and 2D NMR data. The results of MTT assays revealed that new trichothecene 2',3'-epoxymyrothecine A, 1, and myrothecine A, 3, exhibited much lower toxicity compared to other trichothecenes. New trichothecene 2',3'-epoxymyrothecine A, 1, could induce phosphorylation of JNK (c-Jun N-terminal protein kinase) protein and the PARP (poly ADP-ribose polymerase) cleavage, and eventually induce apoptosis in cancer cells. These results point out the possibility for application of trichothecenes as chemotherapeutic agent.

Impact of selected antagonistic fungi on Fusarium species – toxigenic cereal pathogens

<em>Fusarium</em>-ear blight is a destructive disease in various cereal-growing regions and leads to significant yield and quality losses for farmers and to contamination of cereal grains with mycotoxins, mainly deoxynivalenol and derivatives, zearalenone and moniliformin. <em>Fusarium</em> pathogens grow well and produce significant inoculum on crop resiudues. Reduction of mycotoxins production and pathogen sporulation may be influenced by saprophytic fungi, exhibiting antagonistic effect. Dual culture bioassays were used to examine the impact of 92 isolates (belonging to 29 fungal species) against three toxigenic species, i.e. <em>Fusarium avenaceum</em> (Corda) Saccardo, <em>F. culmorum</em> (W.G.Smith) Saccardo and <em>F. graminearum</em> Schwabe. Both <em>F.culmorum</em> and <em>F. graminearum</em> isolates produce trichothecene mycotoxins and mycohormone zearalenone and are considered to be the most important cereal pathogens worldwide. Infection with those pathogens leads to accumulation of mycotoxins: deoxynivalenol (DON) and zearalenone (ZEA) in grains. <em>Fusarium avenaceum</em> isolates are producers of moniliformin (MON) and enniatins. Isolates of <em>Trichoderma</em> sp. were found to be the most effective ones to control the growth of examined <em>Fusarium</em> species. The response of <em>Fusarium</em> isolates to antagonistic activity of <em>Trichoderma</em> isolates varied and also the isolates of <em>Trichoderma</em> differed in their antagonistic activity against <em>Fusarium</em> isolates. The production of MON by two isolates of F. avenaceum in dual culture on rice was reduced by 95% to 100% by <em>T. atroviride</em> isolate AN 35. The same antagonist reduced the amount of moniliformin from 100 μg/g to 6.5 μg/g when inoculated to rice culture contaminated with MON, which suggests the possible decomposition of this mycotoxin.

First Report of Fusarium sporotrichioides Causing Foliar Spots on Forage Corn in Chile.

In southern Chile, forage corn (Zea mays L.) is grown for feeding animals in milk diaries and livestock production. In December 2010, corn plants with small circular spots on leaves were collected from three fields located in Río Negro (Los Lagos region). Symptoms began as small, circular white to brown spots of 5 to 10 mm on different parts of the leaf and necrotic tissue with irregular brown to burgundy margins on the border and tip of the leaf. Estimated visual severity was ~5 to 40% for each leaf from field samples. Twelve small blocks of tissue were taken from the edge of necrotic spots from infected leaves, surface disinfected (2 min in 95% ethanol, 2 min in 0.5% NaOCl, followed by three rinses with sterile distilled water), and then placed on PDA and incubated for 7 days at 24 ± 1°C. Seventy five percent of the sampled tissues developed fungal colonies and a 4-mm3 block of agar that contained the advancing hyphal edge of each colony was transferred to PDA and carnation leaf agar and incubated for 10 days at 24 ± 1°C. Colonies were fast growing with pink-white and dense mycelia; with a carmine red color on the undersurface of the plate and orange sporodochia; polyphialides abundant; microconidia abundant, oval or pear-shaped or spindle-shaped, thin walled, hyaline, often with a papilla at the base, and 5.5 to 12.2 × 2.0 to 3.2 μm. Macroconidia were sickle-shaped, 3 to 5 septate, moderately curved to straight, hyaline, thick walled, and 20.5 to 42.9 × 3.5 to 5.0 μm. Morphology of colonies and conidia matched the description of Fusarium sporotrichioides Sherb. (3). Identity of the fungus was confirmed by molecular characterization of the ITS and 18SrRNA regions (universal primers ITS4/5 and NS1/2, respectively) and the β-tubulin gene (primers Bt1a/Bt1b) of three isolates. BLAST searches of the obtained sequences had between 99 to 100% homology with several isolates of F. sporotrichioides from GenBank (Accession Nos. KC866343 to KC866351). Pathogenicity tests were conducted by dispensing 10 μl of a prepared spore suspension (107 spores/ml) on corn leaves (16 leaves). Negative controls were corn leaves inoculated with sterile distilled water. Inoculated corn leaves were kept at 25 ± 1°C in glass bell jars and monitored for the onset of symptoms for 10 days. The test was conducted twice. Additionally, 20 corn plants of four hybrid lines were inoculated with ~5 ml of a spore suspension (104 macroconidia/ml) 2 months after seeding under field conditions in Valdivia, Los Ríos region, Chile. Seventy five days after sowing, similar lesions to those initially observed on field infected leaves were observed on inoculated leaves but not on water controls. Under field conditions, an extended damage on borders of basal leaves and spots on stems and cobs was observed. The pathogen was reisolated from infected tissues, thereby fulfilling Koch's postulates. F. sporotrichioides is a frequent pathogen in corn silage (1) and cereal crops (3,4), and produces trichothecene mycotoxins that cause toxicosis in animals (2,3). To our knowledge, this is the first report of F. sporotrichioides causing foliar spot on forage corn in Chile and this disease could represent a serious risk of mycotoxin contamination in this crop.

Multiplex Real‐Time PCR Method for Detection and Quantification of Mycotoxigenic Fungi Belonging to Three Different Genera

Cereal crop plants are colonized by many fungal species such as Aspergillus ochraceus and Penicillium verrucosum, which produce ochratoxins, and Fusarium graminearum, which produces trichothecene mycotoxins. A multiplex real-time PCR method using TaqMan probes was developed to simultaneously detect and quantify these mycotoxigenic Fusarium, Penicillium and Aspergillus species in cereal grains. Primers and probes used in this method were designed targeting the trichothecene synthase (Tri5) gene in trichothecene-producing Fusarium, rRNA gene in Penicillium verrucosum, and polyketide synthase gene (Pks) in Aspergillus ochraceus. The method was highly specific in detecting fungal species containing these genes and was sensitive, detecting up to 3 pg of genomic DNA. These PCR products were detectable over five orders of magnitude (3 pg to 30 ng of genomic DNA). The method was validated by evaluating sixteen barley culture samples for the presence of deoxynivalenol (DON) and ochratoxin A (OTA) producing fungi. Among the barley culture samples tested, 9 were positive for Fusarium spp, 5 tested positive for Penicillium spp, and 2 tested positive for Aspergillus spp. Results were confirmed by traditional microbiological methods. These results indicate that DON- and OTA-producing fungi can be detected and quantified in a single reaction tube using this multiplex real-time PCR method. This method would be helpful in detecting and quantifying the mycotoxin producing fungi such as Fusarium, Aspergillus, and Penicillium in cereal grains and cereal-based foods.

Chlorogenic Acid and Maize Ear Rot Resistance: A Dynamic Study InvestigatingFusarium graminearumDevelopment, Deoxynivalenol Production, and Phenolic Acid Accumulation

Fusarium graminearum is the causal agent of Gibberella ear rot and produces trichothecene mycotoxins. Basic questions remain unanswered regarding the kernel stages associated with trichothecene biosynthesis and the kernel metabolites potentially involved in the regulation of trichothecene production in planta. In a two-year field study, F. graminearum growth, trichothecene accumulation, and phenolic acid composition were monitored in developing maize kernels of a susceptible and a moderately resistant variety using quantitative polymerase chain reaction and liquid chromatography coupled with photodiode array or mass spectrometry detection. Infection started as early as the blister stage and proceeded slowly until the dough stage. Then, a peak of trichothecene accumulation occurred and infection progressed exponentially until the final harvest time. Both F. graminearum growth and trichothecene production were drastically reduced in the moderately resistant variety. We found that chlorogenic acid is more abundant in the moderately resistant variety, with levels spiking in the earliest kernel stages induced by Fusarium infection. This is the first report that precisely describes the kernel stage associated with the initiation of trichothecene production and provides in planta evidence that chlorogenic acid may play a role in maize resistance to Gibberella ear rot and trichothecene accumulation.

Evaluation of Potential Reference Genes for Quantitative RT-PCR Analysis in Fusarium graminearum under Different Culture Conditions

Department of Medical Biotehcnology, Soonchunhyang University, Asan 336-745, Korea(Received on October 25, 2011; Accepted on November 2, 2011)The filamentous fungus Fusarium graminearum is animportant cereal pathogen. Although quantitative real-time PCR (qRT-PCR) is commonly used to analyze theexpression of important fungal genes, no detailedvalidation of reference genes for the normalization ofqRT-PCR data has been performed in this fungus.Here, we evaluated 15 candidate genes as references,including those previously described as housekeepinggenes and those selected from the whole transcriptomesequencing data. By a combination of three statisticalalgorithms (BestKeeper, geNorm, and NormFinder),the variation in the expression of these genes wasassessed under different culture conditions that favoredmycelial growth, sexual development, and trichothecenemycotoxin production. When favoring mycelial growth,GzFLO and GzUBH expression were most stable incomplete medium. Both EF1A and GzRPS16 expressionwere relatively stable under all conditions on carrotagar, including mycelial growth and the subsequentperithecial induction stage. These two genes were alsomost stable during trichothecene production. For thecombined data set, GzUBH and EF1A were selected asthe most stable. Thus, these genes are suitable referencegenes for accurate normalization of qRT-PCR data forgene expression analyses of F. graminearum and otherrelated fungi. Keywords : Fusarium graminearum, gene expression,quantitative real-time PCR, reference genesQuantitative real-time PCR (qRT-PCR) is the most sensi-tive and specific method for quantifying mRNA expressionlevels of individual target genes of interest. qRT-PCRexpression analysis has several advantages over otherconventional methods, but requires data normalization withappropriate reference gene(s) whose expression should beunaffected throughout the study conditions (Anderson etal., 2004; Bustin et al., 2004; Gutierrez et al., 2008). Severalhousekeeping genes involved in basic cellular functions,such as ribosomal genes, and those encoding actin, beta-tubulin (BTUB), translation elongation factor (EF1), andglyceraldehyde-3-phosphate dehydrogenase (GAPDH) havebeen used as reference genes for qRT-PCR in humans andother model organisms. However, several studies havedemonstrated that some of these traditional housekeepinggenes are unsuitable references because their expressionlevels vary under certain conditions (Czechowski et al.,2005; Dheda et al., 2004; Lee et al., 2002; Suzuki et al.,2000). Therefore, it is necessary to evaluate candidatereference genes for qRT-PCR expression analysis underspecific experimental conditions in the organism to bestudied. The filamentous fungus Fusarium graminearum (telo-morph: Gibberella zeae) is an important cereal pathogen,and produces mycotoxins that are harmful to humans andanimals (Desjardins and Proctor, 2007; McMullen et al.,1997). Complete genome sequencing of F. graminearum(Cuomo et al., 2007) has allowed for genome-wide expre-ssion profiling using DNA microarrays under variousdevelopmental and physiological conditions, such as my-celial growth (Guldener et al., 2006) sexual development(Hallen et al., 2007), germination (Seong et al., 2008) andpathogenesis (Guldener et al., 2006; Stephens et al., 2008).Although the microarray data in these studies were notvalidated by qRT-PCR, it has become the most commonmethod to obtain specific expression patterns of genesidentified from gene expression profiles (Chen et al., 2011;Liu et al., 2011; Pandolfi et al., 2010) or functional studies(Lee et al., 2009; Lysoe et al., 2009). However, theexpression stabilities of the housekeeping genes used asreferences for qRT-PCR analysis in even these studies havenot been properly validated. The objective of this study was to identify F. gramine-arum reference genes suitable for normalization of qRT-PCR data under different conditions such as mycelialgrowth, sexual development, and mycotoxin production.We selected 15 candidate references, including the commonhousekeeping genes used in previous studies (Chen et al.,2011; Lee et al., 2009; Liu et al., 2011; Lysoe et al., 2009;Pandolfi et al., 2010) and a new set of genes selected from

Trichothecenes: from simple to complex mycotoxins.

As the world’s population grows, access to a safe food supply will continue to be a global priority. In recent years, the world has experienced an increase in mycotoxin contamination of grains due to climatic and agronomic changes that encourage fungal growth during cultivation. A number of the molds that are plant pathogens produce trichothecene mycotoxins, which are known to cause serious human and animal toxicoses. This review covers the types of trichothecenes, their complexity, and proposed biosynthetic pathways of trichothecenes.

The genetic basis for 3-ADON and 15-ADON trichothecene chemotypes in Fusarium

Certain Fusarium species cause head blight of wheat and other small grains worldwide and produce trichothecene mycotoxins. These mycotoxins can induce toxicoses in animals and humans and can contribute to the ability of some fusaria to cause plant disease. Production of the trichothecene 3-acetyldeoxynivalenol (3-ADON) versus 15-acetyldeoxynivalenol (15-ADON) is an important phenotypic difference within and among some Fusarium species. However, until now, the genetic basis for this difference in chemotype has not been identified. Here, we identified consistent DNA sequence differences in the coding region of the trichothecene biosynthetic gene TRI8 in 3-ADON and 15-ADON strains. Functional analyses of the TRI8 enzyme (Tri8) in F. graminearum, the predominant cause of wheat head blight in North America and Europe, revealed that Tri8 from 3-ADON strains catalyzes deacetylation of the trichothecene biosynthetic intermediate 3,15-diacetyldeoxynivalenol at carbon 15 to yield 3-ADON, whereas Tri8 from 15-ADON strains catalyzes deacetylation of 3,15-diacetyldeoxynivalenol at carbon 3 to yield 15-ADON. Fusarium strains that produce the trichothecene nivalenol have a Tri8 that functions like that in 15-ADON strains. TRI3, which encodes a trichothecene carbon 15 acetyltransferase, was found to be functional in all three chemotypes. Together, our data indicate that differential activity of Tri8 determines the 3-ADON and 15-ADON chemotypes in Fusarium.

Brachypodium distachyon: a new pathosystem to study Fusarium head blight and other Fusarium diseases of wheat

BackgroundFusarium species cause Fusarium head blight (FHB) and other important diseases of cereals. The causal agents produce trichothecene mycotoxins such as deoxynivalenol (DON). The dicotyledonous model species Arabidopsis thaliana has been used to study Fusarium-host interactions but it is not ideal for model-to-crop translation. Brachypodium distachyon (Bd) has been proposed as a new monocotyledonous model species for functional genomic studies in grass species. This study aims to assess the interaction between the most prevalent FHB-causing Fusarium species and Bd in order to develop and exploit Bd as a genetic model for FHB and other Fusarium diseases of wheat.ResultsThe ability of Fusarium graminearum and Fusarium culmorum to infect a range of Bd tissues was examined in various bioassays which showed that both species can infect all Bd tissues examined, including intact foliar tissues. DON accumulated in infected spike tissues at levels similar to those of infected wheat spikes. Histological studies revealed details of infection, colonisation and host response and indicate that hair cells are important sites of infection. Susceptibility to Fusarium and DON was assessed in two Bd ecotypes and revealed variation in resistance between ecotypes.ConclusionsBd exhibits characteristics of susceptibility highly similar to those of wheat, including susceptibility to spread of disease in the spikelets. Bd is the first reported plant species to allow successful infection on intact foliar tissues by FHB-causing Fusarium species. DON appears to function as a virulence factor in Bd as it does in wheat. Bd is proposed as a valuable model for undertaking studies of Fusarium head blight and other Fusarium diseases of wheat.