Trichothecene Production Research Articles

Characterization of the acivicin effects on trichothecene production by Fusarium graminearum species complex.

Characterization of the acivicin effects on trichothecene production by Fusarium graminearum species complex.

Population genetic analysis and trichothecene profiling of Fusarium graminearum from wheat in Uruguay.

Fusarium graminearum sensu stricto (F. graminearum s.s.) is the major causal agent of Fusarium head blight of wheat worldwide, and contaminates grains with trichothecene mycotoxins that cause serious threats to food safety and animal health. An important aspect of managing this pathogen and reducing mycotoxin contamination of wheat is knowledge regarding its population genetics. Therefore, isolates of F. graminearum s.s. from the major wheat-growing region of Uruguay were analyzed by amplified fragment length polymorphism assays, PCR genotyping, and chemical analysis of trichothecene production. Of the 102 isolates identified as having the 15-ADON genotype via PCR genotyping, all were DON producers, but only 41 strains were also 15-ADON producers, as determined by chemical analysis. The populations were genotypically diverse but genetically similar, with significant genetic exchange occurring between them. Analysis of molecular variance indicated that most of the genetic variability resulted from differences between isolates within populations. Multilocus linkage disequilibrium analysis suggested that the isolates had a panmictic population genetic structure and that there is significant recombination occurs in F. graminearum s.s. In conclusion, tour findings provide the first detailed description of the genetic structure and trichothecene production of populations of F. graminearum s.s. from Uruguay, and expands our understanding of the agroecology of F. graminearum and of the correlation between genotypes and trichothecene chemotypes.

Phylogenetic analyses of the Fusarium graminearum species complex isolated from soybean in Argentina and Brazil

Soybean is one of the most economically important crops in Argentina and Brazil. However, there is limited information on the biodiversity of the FGSC from soybean as compared to other crops of large-scale growing such as wheat and maize. A phylogenetic recognition of the Fusarium graminearum species complex (FGSC) isolated from soybean in Argentina and Brazil was performed in order to identify species responsible for trichothecene production. Sequences of genes encoding for the partial translation elongation factor, the 3-O-acetyltransferase and a putative reductase were analysed by the Maximum Parsimony method. Although the present study has focused on a limited number of isolates, this is the first report that provides evidence of the presence of at least four species within the FGSC associated with soybean in Argentina: F. graminearum sensu stricto, F. cortaderiae, F. meridionale and F. boothii. In addition, F. graminearum sensu stricto was detected for the first time among Brazilian isolates from soybean.

Pathogenicity and mycotoxin chemotypes of Iranian Fusarium culmorum isolates on durum wheat, and comparisons with Italian and Syrian isolates

Fusarium isolates obtained from Iran and Italy were identified by morphological characters and confirmed by using species-specific PCR assays. The genetic chemotyping for each strain, as a preliminary assessment for trichothecene production, was defined using PCR. Subsequently, artificial infection on durum wheat (cv. Normanno) was carried out in the greenhouse to study the pathogenicity and aggressiveness of Iranian, Italian and Syrian F. culmorum strains, the causal agent of crown and root rot of wheat. All F. culmorum strains from Iran and Italy belonged to the 3-acetyl-deoxynivalenol (3Ac-DON) chemotype, while F. culmorum strains from Syria, previously characterized, belonged to the 3Ac-DON and nivalenol (NIV) chemotypes. All of the strains were pathogenic and caused typical Fusarium crown rot (FCR) symptoms. Italian and Iranian strains showed similar mean aggressiveness levels (19.1 and 18.7% respectively), while the mean aggressiveness level for Syrian strains was 11.8%. There were statistically significant differences among the strains. Two Italian strains, F383 and F1126, had the greatest level of aggressiveness while FC9 (Italian) and F961 (Syrian) were the least aggressive strains. No significant differences relating to agro-ecological origin were detected among Iranian, Italian and Syrian strains. This is the first genetic chemotyping characterization and comparison of F. culmorum strains, isolated from different agro-ecological countries, Iran, Syria (Middle East) and Italy (Europe), which has estimated their potential for producing mycotoxins, and the aggressiveness levels of F. culmorum for development of FCR. These results support the increasing concerns about the risk of FCR in many wheat producing countries, particularly in Iran and Syria.

Precocene II, a Trichothecene Production Inhibitor, Binds to Voltage-Dependent Anion Channel and Increases the Superoxide Level in Mitochondria of Fusarium graminearum.

Precocene II, a constituent of essential oils, shows antijuvenile hormone activity in insects and inhibits trichothecene production in fungi. We investigated the molecular mechanism by which precocene II inhibits trichothecene production in Fusarium graminearum, the main causal agent of Fusarium head blight and trichothecene contamination in grains. Voltage-dependent anion channel (VDAC), a mitochondrial outer membrane protein, was identified as the precocene II-binding protein by an affinity magnetic bead method. Precocene II increased the superoxide level in mitochondria as well as the amount of oxidized mitochondrial proteins. Ascorbic acid, glutathione, and α-tocopherol promoted trichothecene production by the fungus. These antioxidants compensated for the inhibitory activity of precocene II on trichothecene production. These results suggest that the binding of precocene II to VDAC may cause high superoxide levels in mitochondria, which leads to stopping of trichothecene production.

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.

The complete mitogenome of Fusarium culmorum

The structure of the Fusarium culmorum mitogenome is similar to that of closely related Fusarium spp.: it has a total length of 103,844 bp, the base composition of the genome is the following: A (35.4%), T (32.9%), C (14.6%), and G (17.1%). The mitogenome contains 13 protein-coding genes, 2 ribosomal RNA (rRNA), and 28 transfer RNA (tRNA) genes, all coded on the same strand of DNA. The gene order is identical to that of the other Fusarium and Hypocreales mitogenomes. Maximum likelihood and Bayesian analysis based on the concatenated amino acid dataset of mitochondrial protein-coding genes confirmed close genetic relationship of F. culmorum to the other type B trichothecene producers F. graminearum and F. gerlachii.

Molecular identification and determination of the trichothecene chemotypes of fungi causing crown and root in different growth stages of wheat in north of Iran

In order to determine the crown and root agents and their mycotoxins produced in different growth stages of wheat including seedling, tillering and heading, sampling was done in north of Iran, during 2011–2012. From 160 isolates of Fusarium, eight species were obtained including F. graminearum, F. culmorum, F. equiseti, F. nygamai, F. semitectum, F. solani, F. acuminatum and F. oxysporum. Sampling at different growth stages showed that F. graminearum was the predominant causal agent of crown and root at the heading stage, whereas other species of Fusarium were mostly observed at the seedling and tillering stages. Moreover, identification of pathogenic species was confirmed using species-specific primers pairs. In F. graminearum isolates, presence of Tri13 gene, responsible for nivalenol (NIV) and deoxynivalenol (DON) mycotoxins biosynthesis, was detected using specific PCR primers. Finally, the ability of trichothecene production of five F. graminearum isolates was confirmed with high-performance liquid chromatography.

Fusarium graminearum Isolates from Wheat and Maize in New York Show Similar Range of Aggressiveness and Toxigenicity in Cross-Species Pathogenicity Tests.

This study aimed to assess whether pathogenic Fusarium graminearum isolates from wheat and maize were more aggressive on their host of origin and whether aggressiveness was influenced further by B-trichothecene chemotype. Fifteen isolates were selected from a contemporary collection of isolates surveyed in New York in 2011 to 2012 to represent diversity of host of origin and chemotype. Three pathogenicity assays were used to evaluate and compare these isolates. Fusarium head blight (FHB) severity and trichothecene production in wheat, and maize seedling blight were evaluated in greenhouse inoculation experiments, and Gibberella ear rot (GER) severity and trichothecene production were evaluated in maize ears inoculated in the field. Our results showed among F. graminearum isolates a wide variation in aggressiveness and mycotoxin production toward wheat and maize and these isolates could not be structured by their host of origin or by chemotype. Moreover, aggressiveness rank order changed according to the host/organ evaluated. This indicates that relative susceptibility at the seedling stage may not predict susceptibility of ears. Significant correlations were observed of total trichothecenes (deoxynivalenol [DON] and its acetylated derivatives) produced with FHB and GER severity on wheat and maize, respectively. One isolate did not produce DON or ADON in wheat or maize kernels, yet was aggressive on both hosts. Nine of the fifteen isolates produced small amounts of zearalenone (ZON) in maize kernels, but not in wheat kernels, and ZON level was not correlated with GER severity. F. graminearum isolates from New York showed wide variation in aggressiveness and mycotoxin production toward susceptible wheat and maize. Neither host of origin nor trichothecene chemotype appeared to structure the populations we sampled.

Fusarium head blight and trichothecene production in wheat by Fusarium graminearum and F. meridionale applied alone or in mixture at post-flowering

A greenhouse study was conducted to expand our knowledge of the disease and mycotoxin production by Fusarium graminearum (Fgra) and F. meridionale (Fmer) inoculated at post-flowering, either alone or in equal mixture, into three cultivars varying in their reaction to Fusarium head blight (FHB) in the field. In the first experiment, the entire spike was spray-inoculated and the proportion of diseased spikes (or incidence, INC) was monitored up to 15 days after inoculation (dai). In the second experiment, the inoculum was dispensed into the central-spikelet and the proportion of disease spikelets (or severity, SEV) was evaluated at 15 dai. The inoculum composition and the inoculum x cultivar interaction on both experiments were not significant. However, BRS 194, a standard for susceptibility, had significantly higher INC and SEV than BRS Parrudo and BRS 179, the latter a standard for moderate resistance. Fmer was less damaging to grain yield than Fgra and grain yield of BRS Parrudo was the least affected among the cultivars. Trichothecenes (DON + NIV) were detected in both experiments, irrespectively of the cultivar, at levels up to 2,000 and 15,000 μg/kg, in the spray-inoculation and central-spikelet inoculation experiment, respectively. Fgra alone produced only DON and Fgra+Fmer produced mainly DON and small amount of NIV. Fmer produced mainly NIV, but also trace amounts of DON. Our results suggest that post-flowering infections may contribute with trichothecenes in mature grain, especially DON. Nevertheless, NIV should be considered in analytical surveys due to its high toxicity.

The prevalence of selected genes involved in the biosynthesis of trichothecenes assessed with the specific PCR tests in Fusarium spp. isolated from cereals in southern Poland

The analysis was conducted using 50 isolates of fungi of the genus Fusarium belonging to the species classified as major trichothecene mycotoxin producers: F. graminearum, F. culmorum, F. sporotrichioides, and F. poae. The tested fungi were isolated from ears of cereal crops in southern Poland during the two growing seasons (2011 and 2012). The aim of this study was to evaluate the prevalence of genes involved in the biosynthesis of trichothecene mycotoxins using the specific PCR tests. Molecular analyses indicated that the genes responsible for the production of trichothecenes (Tri3, Tri5, Tri7, Tri13) were abundant in the examined genetic material. The tested fungal isolates were characterized by a large diversity in terms of the number and composition of the possessed Tri genes. On the other hand, 14 of 50 isolates were found not to carry any of Tri genes.

Involvement of FgMad2 and FgBub1 in regulating fungal development and carbendazim resistance in Fusarium graminearum

Resistance to carbendazim of Fusarium graminearum is conferred by point mutation in the β2‐tubulin gene that plays an important role in spindle assembly. The spindle assembly checkpoint is a cellular surveillance system that is critical for maintaining genomic stability. Predicted protein Mad2‐ and Bub1‐encoding genes in F. graminearum (FgMad2 and FgBub1) were isolated and characterized. There was no difference in FgMad2 and FgBub1 expression levels between carbendazim‐sensitive and ‐resistant strains; however, after carbendazim treatment FgMad2 expression increased while FgBub1 expression stayed the same. Both the FgMad2 and FgBub1 deletion mutants became more sensitive to carbendazim. The FgMad2 deletion mutants grew more slowly, produced fewer conidia and both hyphae and conidia were malformed. Conversely, deletion of FgBub1 had no effect on fungal development other than a reduction in conidia production. FgMad2 deletion mutants exhibited a severe decrease in perithecia production and pathogenicity along with a down‐regulation of trichothecene production, whereas FgBub1 deletion mutants exhibited only a slight reduction in perithecia production and was accompanied by a twofold increase in trichothecene production. Overall, the results indicate that both FgMad2 and FgBub1 are involved in carbendazim resistance and trichothecene biosynthesis, and FgMad2 plays an important role in fungal development in F. graminearum.

Detection, Identification and Quantification of Fusarium graminearum and Fusarium culmorum in Wheat Kernels by PCR Techniques

This study was carried out on 172 samples of winter wheat. The samples consisted of various cultivars that had been randomly collected from farmers’ fields in different areas of Bavaria, South Germany. The objectives of this study were detecting the presence of Tri-5 gene producing fungus that generates trichothecene mycotoxins, especially Deoxynivalenol (DON), by using conventional qualitative PCR; determining the correlation between the presence of Tri-5 gene and DON content; evaluating the Fusarium graminearum and Fusarium culmorum infection by Real-Time PCR and estimating the correlation between DON content and the severity of F. graminearum and F. culmorum contamination. This study showed that 86% of all infected samples had a Tri-5 gene and amplified a single 544bp fragment associated with a detectable amount of DON (ranged from 10 to 2990 μg kg-1). This study demonstrated that F. graminearum is the predominant species associated with Fusarium head blight (FHB) and was considered as the predominant trichothecene producer that associated with FHB since there was a highly significant correlation (R2=0.7) between DON and F. graminearum DNA content, compared to a weak correlation (R2=0.03) between DON and DNA content of F. culmorum infected wheat kernels.

Occurrence of Stachybotrys chartarum chemotype S in dried culinary herbs.

Stachybotrys (S.) chartarum is an omnipresent cellulolytic mould which produces secondary metabolites, such as the highly toxic macrocyclic trichothecenes. While it is known to occur in animal feed like hay and straw as well as in water-damaged indoor environments, there is little knowledge about the occurrence of S. chartarum and its secondary metabolites in food. The objective of the present study was to examine selected dried culinary herbs for the presence of S. chartarum chemotype S, to assess the potential risk of a contamination of foods with macrocyclic trichothecenes. In total, 50 Stachybotrys isolates from different types of culinary herbs (n=100) such as marjoram (Origanum majorana Linné (L.)), oregano (Origanum vulgare L.), thyme (Thymus vulgaris L.), and savory (Satureja hortensis L.) were examined by MTT-cell culture test (effect-based bioassay), ELISA, and by liquid chromatography tandem mass spectrometry (LC-MS/MS). Selected toxic and non-toxic isolates (n=15) were genetically characterized by PCR and sequencing. Five isolates (10%) were highly toxic in the MTT-cell culture test, and the production of macrocyclic trichothecenes was proven by ELISA and LC-MS/MS. These five isolates were genetically confirmed as S. chartarum chemotype S. To the best of our knowledge, this is the first report about a contamination of dried culinary herbs with toxigenic S. chartarum.

Metabolomics deciphers the host resistance mechanisms in wheat cultivar Sumai-3, against trichothecene producing and non-producing isolates of Fusarium graminearum

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum, reduces grain yield and contaminates grains with trichothecene mycotoxins. Host resistance to FHB is quantitatively inherited and more than 100 QTLs have been mapped, but the host resistance mechanisms are poorly understood. Non-targeted metabolic profiling was applied to elucidate the host resistance mechanisms to FHB spread through rachis of wheat cultivar Sumai-3 against both trichothecene producing and non-producing isolates of Fusarium graminearum. The accumulation of deoxynivalenol (DON) in Sumai-3 was low, however the resistance to spread was not due to its detoxification into DON-3-O-glucoside (D3G), as the proportion of total DON converted to D3G in the resistant was not significantly different from that in the susceptible cultivar Roblin. Instead, the resistance was considered to be due to the accumulation of resistance related (RR) metabolites belonging to the phenylpropanoid pathway that reduced pathogen advancement through increased host cell wall thickening and also reduced pathogen growth due to antifungal and/or antioxidant properties which, in turn, reduced subsequent trichothecene biosynthesis. The RR phenylpropanoids accumulated in Sumai-3 were mainly the preformed syringyl rich monolignols and their glucosides, which are precursors of lignin biosynthesis, as well as antimicrobial flavonoids. The resistant cultivar Sumai-3 inoculated with trichothecene producing F. graminearum not only accumulated less RR metabolites but also the abundance of many RR metabolites was lesser than in the trichothecene non-producing F. graminearum. This implies repression of host resistance mechanisms by trichothecenes/DON, which is a protein biosynthesis inhibitor. Enhancement of resistance in wheat against FHB can be exploited through stacking of candidate phenylpropanoid pathway genes.

Fusarium species, chemotype characterisation and trichothecene contamination of durum and soft wheat in an area of central Italy.

Fusarium head blight (FHB) of wheat is an important disease causing yield losses and mycotoxin contamination. The aim of the work was to detect and characterise trichothecene producing Fusarium species in durum and soft wheat cultivated in an area of central Italy in 2009 and 2010 and to determine trichothecene contamination by LC-MS/MS in the grain. F. graminearum s. str. was the most frequent species. In 2009, the occurrence of F. avenaceum and F. poae was higher than in 2010. Among F. graminearum strains, the 15-acetyl deoxynivalenol (15-ADON) chemotype could be found more frequently, followed by nivalenol (NIV) and 3-ADON chemotypes, while all F. culmorum isolates belonged to the 3-ADON chemotype. All F. poae strains were NIV chemotypes. In vitro trichothecene production confirmed molecular characterisation. Durum wheat was characterised by a higher average DON contamination with respect to soft wheat, NIV was always detected at appreciable levels while type-A trichothecenes were mostly found in durum wheat samples in 2009 with 6% of samples exceeding the contamination level recently recommended by the European Union. Climatic conditions were confirmed to be predominant factors influencing mycotoxigenic species composition and mycotoxin contaminations. However, NIV contamination was found to occur irrespective of climatic conditions, suggesting that it may often represent an under-estimated risk to be further investigated.

Effects of acivicin on growth, mycotoxin production and virulence of phytopathogenic fungi.

Acivicin is an inhibitor of γ-glutamyl transpeptidase and glutamine amidotransferase. When grown on a synthetic minimal agar medium, acivicin strongly inhibited the growth of Magnaporthe oryzae and Alternaria brassicicola, and to a lesser extent, Botrytis cinerea. However, only partial or marginal growth inhibition was observed with regard to Fusarium sporotrichioides and Fusarium graminearum. The growth retardation caused by acivicin was significantly alleviated by cultivating the fungus on a nutrient-rich medium. The inhibition of M. oryzae growth caused by 1 μmol l(-1) of acivicin on minimal agar medium was subdued by the addition of specific single amino acids, including His, a branched-chain amino acid (Leu, Ile or Val), an aromatic amino acid (Trp, Tyr or Phe), Met or Gln, at a concentration of 0·4 mmol l(-1). Trichothecene production by F. graminearum in trichothecene-inducing liquid medium was reduced significantly in the presence of acivicin despite its inability to inhibit growth in the trichothecene-inducing liquid medium. Foliar application of conidia in the presence of acivicin reduced the severity of rice blast disease caused by M. oryzae. These results suggest the usefulness of this modified amino acid natural product to mitigate agricultural problems caused by some phytopathogenic fungi. Significance and impact of the study: Fusarium head blight or scab disease and rice blast, caused by Fusarium graminearum and Magnaporthe oryzae, respectively, are major diseases of cereal crops that cause a significant loss of yield and deterioration in the quality of the grain. The present study investigated the effects of acivicin, a glutamine amino acid analog, on the physiology of various phytopathogenic fungi. Application of acivicin to a fungal culture and conidial suspension reduced mycotoxin production by the wheat scab fungus and the severity of rice blast, respectively. These results suggest the possibility that acivicin may serve as a lead compound to develop agricultural chemicals for the control of some plant diseases.

MYT3, a Myb-like transcription factor, affects fungal development and pathogenicity of Fusarium graminearum.

We previously characterized members of the Myb protein family, MYT1 and MYT2, in Fusarium graminearum. MYT1 and MYT2 are involved in female fertility and perithecium size, respectively. To expand knowledge of Myb proteins in F. graminearum, in this study, we characterized the functions of the MYT3 gene, which encodes a putative Myb-like transcription factor containing two Myb DNA-binding domains and is conserved in the subphylum Pezizomycotina of Ascomycota. MYT3 proteins were localized in nuclei during most developmental stages, suggesting the role of MYT3 as a transcriptional regulator. Deletion of MYT3 resulted in impairment of conidiation, germination, and vegetative growth compared to the wild type, whereas complementation of MYT3 restored the wild-type phenotype. Additionally, the Δmyt3 strain grew poorly on nitrogen-limited media; however, the mutant grew robustly on minimal media supplemented with ammonium. Moreover, expression level of nitrate reductase gene in the Δmyt3 strain was decreased in comparison to the wild type and complemented strain. On flowering wheat heads, the Δmyt3 strain exhibited reduced pathogenicity, which corresponded with significant reductions in trichothecene production and transcript levels of trichothecene biosynthetic genes. When the mutant was selfed, mated as a female, or mated as a male for sexual development, perithecia were not observed on the cultures, indicating that the Δmyt3 strain lost both male and female fertility. Taken together, these results demonstrate that MYT3 is required for pathogenesis and sexual development in F. graminearum, and will provide a robust foundation to establish the regulatory networks for all Myb-like proteins in F. graminearum.

Plant lignans inhibit growth and trichothecene biosynthesis in Fusarium graminearum

Lignans are a group of diphenolic compounds with anticancer and antioxidant properties which are present in various grains, although their effect on toxigenic fungi has been poorly examined to date. In this study, the impact of the plant lignans pinoresinol and secoisolariciresinol on growth and trichothecene biosynthesis by five Fusarium graminearum strains of different chemotypes was examined in vitro. Both tested lignans exhibited radial growth inhibition against the fungal strains. RT-qPCR analyses of tri4, tri5 and tri11 genes encoding the first steps of the trichothecene biosynthesis pathway revealed a decrease in tri mRNA levels in lignan-treated fungal cultures. Correspondingly, decreased accumulation of toxins in lignan-treated cultures was confirmed by GC-MS analysis. This is the first study to demonstrate the inhibitory effect of both pinoresinol and secoisolariciresinol on growth and trichothecene biosynthesis in F.graminearum. Knowledge of the regulation of trichothecene production in Fusarium graminearum by environmental cues is key to the design of novel strategies to reduce mycotoxin levels in grains. Here, we show that the lignans pinoresinol and secoisolariciresinol, which occur in wheat grains, inhibit radial growth and decrease trichothecene levels in five F.graminearum strains. RT-qPCR analysis reveals that the reduction in trichothecene level in lignan-treated fungal cultures is associated with decreased mRNA transcript levels for the tri4, tri5 and tri11 genes that are involved in the trichothecene biosynthesis pathway.

Toxigenic Capacity and Trichothecene Production by Fusarium graminearum Isolates from Argentina and Their Relationship with Aggressiveness and Fungal Expansion in the Wheat Spike

At least 20 epidemics of Fusarium head blight (FHB) of wheat have been registered in the last 50 years in Argentina, with variable intensity. Damage induced by the disease is further aggravated by the presence of mycotoxins in affected grains that may cause health problems to humans and animals. The trichothecene chemotype was analyzed for 112 isolates of Fusarium graminearum from Argentina by polymerase chain reaction and two field trials were conducted to study the aggressiveness of a subsample of 14 representative isolates and to analyze deoxynivalenol (DON) production in planta and in vitro. All isolates belonged to the 15-acetyl-DON chemotype. Significant differences were observed in both the symptom severity induced in wheat spikes and the in vivo DON production, and a close correlation was found between these two variables. However, in vitro toxigenic potential was not correlated with the capacity of F. graminearum isolates to produce DON under natural conditions. The progress of infection in the rachis of inoculated wheat spikes was analyzed and the pathogen presence verified in both symptomatic and symptomless spikes. Even isolates with a limited capacity to induce symptoms were able to colonize the vascular tissue and to produce considerable amounts of DON in planta.