Fusarium graminearum Chemotype-Spring Wheat Genotype Interaction Effects in Type I and II Resistance Response Assays.

Fusarium head blight (FHB), caused by several Fusarium spp., is a worldwide problem that severely impacts cereal grain yield and poses major risks to human and animal health due to production of the mycotoxin deoxynivalenol (DON) and its acetylated forms, 3-acetyl-DON (3-ADON) and 15-acetyl-DON (15-ADON). Recent studies suggest an inconsistent effect of F. graminearum chemotypes and resistance of wheat (Triticum aestivum) genotypes. To gain insight into the interaction effects of F. graminearum chemotypes and spring wheat genotypes on FHB resistance response, 10 spring wheat genotypes with varying levels of FHB resistance were inoculated with 10 F. graminearum isolates, consisting of 5 3-ADON- and 5 15-ADON-producing isolates and evaluated in type I (spray inoculation) and type II (point inoculation) resistance assays. Wheat genotypes carrying the resistance allele of the Fhb1 quantitative trait locus on chromosome 3BS had lower disease in type II evaluations, regardless of F. graminearum isolate or chemotype. Isolates of F. graminearum were also significantly different for disease aggressiveness. In addition, the 3-ADON-producing isolates were 18% more aggressive than the 15-ADON isolates in type I resistance assays. No difference in aggressiveness of the two chemotypes was observed, when tested in type II resistance assays. There was no interaction effect between F. graminearum chemotypes and spring wheat genotypes, suggesting that screening of germplasm for resistance can be performed with limited number of aggressive isolates.

Diversity of Fusarium head blight populations and trichothecene toxin types reveals regional differences in pathogen composition and temporal dynamics.

Analyses of genetic diversity, trichothecene genotype composition, and population structure were conducted using 4086 Fusarium graminearum isolates collected from wheat in eight Canadian provinces over a three year period between 2005 and 2007. The results revealed substantial regional differences in Fusarium head blight pathogen composition and temporal population dynamics. The 3ADON trichothecene type consistently predominated in Maritime provinces (91%) over the sampled years, and increased significantly (P<0.05) between 2005 and 2007 in western Canada, accounting for 66% of the isolates in Manitoba by the end of the sampling period. In contrast, 3ADON frequency was lower (22%, P<0.001) in the eastern Canadian provinces of Ontario and Québec and did not change significantly between 2005 and 2007, resulting in two distinct longitudinal clines in 3ADON frequency across Canada. Overall, genetic structure was correlated with toxin type, as the endemic population (NA1) was dominated by 15ADON isolates (86%), whereas a second population (NA2) consisted largely of 3ADON isolates (88%). However, the percentage of isolates with trichothecene genotypes that were not predictive of their genetic population assignment (recombinant genotypes) increased from 10% in 2005 to 17% in 2007, indicating that trichothecene type became an increasingly unreliable marker of population identity over time. In addition, there were substantial regional differences in the composition of recombinant genotypes. In western and maritime provinces, NA2 isolates with 15ADON genotypes were significantly more common than NA1 isolates with 3ADON genotypes (P<0.001), and the reverse was true in the eastern provinces of Québec and Ontario. Temporal trends in recombinant genotype composition also varied regionally, as the percentage of 15ADON isolates with NA2 genetic backgrounds increased approximately three fold in western and Maritime provinces, while the opposite trends were observed in Québec and Ontario. The results indicate that F. graminearum population dynamics in Canada have been influenced by a complex adaptive landscape comprising different regional selective pressures, and do not reflect a simple model of dispersal and integration following the introduction of a novel pathogen population. In addition, we identified F. graminearum strains that produce the recently discovered A-trichothecene mycotoxin (NX-2) for the first time in Canada, representing a significant expansion of the known range of NX-2 producing strains in North America.

New insights into mycotoxin mixtures: the toxicity of low doses of Type B trichothecenes on intestinal epithelial cells is synergistic.

Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. DON is often present with other type B trichothecenes such as 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), nivalenol (NIV) and fusarenon-X (FX). Although the cytotoxicity of individual mycotoxins has been widely studied, data on the toxicity of mycotoxin mixtures are limited. The aim of this study was to assess interactions caused by co-exposure to Type B trichothecenes on intestinal epithelial cells. Proliferating Caco-2 cells were exposed to increasing doses of Type B trichothecenes, alone or in binary or ternary mixtures. The MTT test and neutral red uptake, respectively linked to mitochondrial and lysosomal functions, were used to measure intestinal epithelial cytotoxicity. The five tested mycotoxins had a dose-dependent effect on proliferating enterocytes and could be classified in increasing order of toxicity: 3-ADON<15-ADON≈DON

Population structure and genetic diversity of Fusarium graminearum from southwestern Russia and the Russian Far East as compared with northern Europe and North America.

Genetic variation at variable number tandem repeat (VNTR) markers was used to assess population structure and diversity among 296 Fusarium graminearum isolates from northern Europe (Finland, northwestern Russia, and Norway), southern Europe (southwestern and western Russia), and Asia (Siberia and the Russian Far East). We identified at least two highly differentiated and geographically structured genetic populations (E1 and E2) in Eurasia (ΦPT = 0.35). Isolates from northern Europe were almost exclusively from the E1 population (95.6%) and had the 3ADON (3-acetyldeoxynivalenol) trichothecene genotype (97.3%). In contrast, all isolates from southern Europe were from the E2 population and 94.4% had the 15ADON (15-acetyldeoxynivalenol) genotype. The E2 population also predominated in the Asian sampling locations (92.7%) where 3ADON and 15ADON genotypes occurred at nearly equal frequencies. Southern European isolates were more closely related to those from Asia (ΦPT = 0.06) than to geographically closer populations from northern Europe (ΦPT ≥ 0.31). Northern European populations also harbored substantially less genetic diversity (Ne ≤ 2.1) than populations in southern Europe or Asia (Ne ≥ 3.4), indicative of a selective sweep or recent introduction and subsequent range expansion in northern Europe. Bayesian analyses incorporating previously described genetic populations from North America (NA1 and NA2) surprisingly identified NA2 and E2 as a single genetic population, consistent with hypotheses of a recent Eurasian origin for NA2. Additionally, more than 10% of the isolates from Asia and southern Europe were assigned to the NA1 population, indicating recent introductions of NA1 into parts of Eurasia. Collectively, these results demonstrate that there are at least three genetic populations of F. graminearum in the Northern Hemisphere and indicate that population-level diversity in Eurasia and North America has been shaped by recent transcontinental introductions.

The effect of technological processes on contamination with B-class trichothecenes and quality of spring wheat products from grain harvested at different times.

The study aimed to investigate the effect of technological processes on deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyl-deoxynivalenol (15-ADON) concentrations and quality of spring wheat products from grain harvested at different times. In this study, 408 samples were analysed for DON, 3-ADON and 15-ADON contamination by the HPLC method with UV detection. Delays in harvesting due to cool and rainy weather conditions resulted in increased DON, 3-ADON and 15-ADON levels. The highest DON concentrations were determined in bran. Higher DON concentrations in the bran indicate the protective function of the grain hull. On the other hand, the highest levels of minerals have been found in bran and whole grain flours, highlighting the importance of consuming these milling fractions as a mineral source with sustained health benefits. Our results showed that DON is stable at 170 °C, and high DON levels in whole-meal flour and white flour could not be converted or decomposed during baking. The levels of 3-ADON and 15-ADON in whole-meal flour bread and white flour bread were significantly reduced but not completely removed. The levels of DON and its derivatives 3-ADON and 15-ADON were significantly reduced in starch and gluten produced from contaminated whole meal flour; however, the washing process did not completely eliminate these toxic compounds. The concentrations of mycotoxins in starch and gluten remained relatively high. Negative correlation was found in highly contaminated samples between DON and bread baking properties. Also, inverse relationship was found between high mycotoxin concentrations and mineral element content in white flour.

Construction and characterization of a cDNA library from wheat infected with Fusarium graminearum Fg 2.

Total RNA from wheat spikes infected with F. graminearum Fg2 was extracted and the mRNA was purified. Switching Mechanism at 5' end of the RNA Transcript (SMART) technique and CDS Ill/3' primer were used for first-strand cDNA synthesis using reverse transcriptase by RT-PCR. Primer extension polymerase chain reaction was used to construct the double-strand cDNA that was digested by proteinase K, then by Sfi I and fractionated. cDNAs longer than 0.5 kb were collected and ligated to λTriplEx2 vector followed λ phage packaging reaction and library amplification. The qualities of both unamplified and amplified cDNA libraries were strictly checked by conventional titer determination. One hundred and sixty five plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. A high quality cDNA library from wheat spikes that have been infected by F. graminearum was successfully constructed.

Population Genetic Structure and Chemotype Diversity of Fusarium graminearum Populations from Wheat in Canada and North Eastern United States.

Fusarium head blight (FHB) is a major disease in wheat causing severe economic losses globally by reducing yield and contaminating grain with mycotoxins. In Canada, Fusarium graminearum is the principal etiological agent of FHB in wheat, producing mainly the trichothecene mycotoxin, deoxynivalenol (DON) and its acetyl derivatives (15-acetyl deoxynivalenol (15ADON) and 3-acetyl deoxynivalenol (3ADON)). Understanding the population biology of F. graminearum such as the genetic variability, as well as mycotoxin chemotype diversity among isolates is important in developing sustainable disease management tools. In this study, 570 F. graminearum isolates collected from commercial wheat crops in five geographic regions in three provinces in Canada in 2018 and 2019 were analyzed for population diversity and structure using 10 variable number of tandem repeats (VNTR) markers. A subset of isolates collected from the north-eastern United States was also included for comparative analysis. About 75% of the isolates collected in the Canadian provinces of Saskatchewan and Manitoba were 3ADON indicating a 6-fold increase in Saskatchewan and a 2.5-fold increase in Manitoba within the past 15 years. All isolates from Ontario and those collected from the United States were 15ADON and isolates had a similar population structure. There was high gene diversity (H = 0.803-0.893) in the F. graminearum populations in all regions. Gene flow was high between Saskatchewan and Manitoba (Nm = 4.971-21.750), indicating no genetic differentiation between these regions. In contrast, less gene flow was observed among the western provinces and Ontario (Nm = 3.829-9.756) and USA isolates ((Nm = 2.803-6.150). However, Bayesian clustering model analyses of trichothecene chemotype subpopulations divided the populations into two clusters, which was correlated with trichothecene types. Additionally, population cluster analysis revealed there was more admixture of isolates among isolates of the 3ADON chemotypes than among the 15ADON chemotype, an observation that could play a role in the increased virulence of F. graminearum. Understanding the population genetic structure and mycotoxin chemotype variations of the pathogen will assist in developing FHB resistant wheat cultivars and in mycotoxin risk assessment in Canada.

Simultaneous quantitation of 3ADON and 15ADON chemotypes of DON-producing Fusarium species in Chinese wheat based on duplex droplet digital PCR assay.

Pathogens within Fusarium species are the primary agents of Fusarium head blight (FHB) of wheat, which bring about yield reduction and deoxynivalenol (DON) contamination and are of great concern worldwide. DON-producing Fusarium species can be classified into 3-acetyldeoxynivalenol (3ADON) and 15-acetyldeoxynivalenol (15ADON) chemotypes according to the trichothecene metabolites they produce. The detection of these two chemotypes of pathogens is paramount to the successful implementation of disease management strategies and pathogen-related DON forecasting models. In this study, a duplex droplet digital PCR (duplex ddPCR) assay was developed that allowed for the simultaneous quantitation of 3ADON and 15ADON chemotypes of DON-producing Fusarium species. The assay specificity was tested against 30 isolates of target Fusarium species and several non-target Fusarium species that are frequently isolated from wheat in China. Analyzing 90 wheat samples collected from the North China plain and Yangtze River plain demonstrated that the duplex ddPCR assay coupled with magnetic bead-based DNA extraction was competent for investigating composition of 3ADON and 15ADON chemotypes in Chinese wheat. This assay will be useful for monitoring the epidemic and geographic distribution of 3ADON and 15ADON chemotypes of FHB pathogens, which will help with the disease control and DON management.

Molecular Phylogenetic Relationships, Trichothecene Chemotype Diversity and Aggressiveness of Strains in a Global Collection of Fusarium graminearum Species.

Fusarium head blight (FHB), caused principally by the species belonging to the Fusarium graminearum species complex (FGSC), is an important disease in wheat, barley, and other small grain crops worldwide. Grain infected with species in the FGSC may be contaminated with trichothecene mycotoxins such as deoxynivalenol (DON) and nivalenol (NIV). In this study, we characterized the phylogenetic relationships, chemotype diversity, phenotypic characters, and aggressiveness of 150 strains in FGSC collected from eight different countries. Phylogenetic analysis based on portions of translation elongation factor 1-α (EF-1α) gene from 150 strains revealed six species in the FGSC, F. graminearum s.s, F. asiaticum, F. meridionale, F. cortaderiae, F. boothii, and F. austroamericanum. In this collection, 50% of the strains were 15-acetyldeoxynivalenol (15-ADON), 35% were 3-acetyldeoxynivalenol (3-ADON) and 15% were NIV. Evaluation of strains on moderately resistant (MR) wheat cultivar Carberry indicated that there is no significant difference among the species for FHB disease severity (DS), fusarium damaged kernel percentage (FDK%) and DON production. However, significant differences were observed among the chemotypes. Results showed significantly higher FHB DS, FDK%, DON production, growth rates, and macroconidia production for the 3-ADON strains than the 15-ADON and NIV strains. In addition, significant differences for FHB response variables were observed among the strains from different countries. Our results demonstrate that type and amount of trichothecene produced by a strain play a key role in determining the level of aggressiveness of that particular strain, regardless of the species.

Micronucleus induction and cell cycle alterations produced by deoxynivalenol and its acetylated derivatives in individual and combined exposure on HepG2 cells.

Mycotoxins are produced by a number of fungal genera spp as e.g. Aspergillus, Penicillium, Alternaria, Fusarium and Claviceps. 3-Acetyl-Deoxynivalenol (3-A-DON) and 15-Acetyl-Deoxynivalenol (15-ADON) which are produced by Fusarium, chemically belong to trichothecenes and occur in significant amounts as modified forms of deoxynivalenol (DON) in various cereal crops and processed grains. This study aims to determine the cytotoxicity, cell cycle and genotoxicity of the mycotoxins DON, 3-A-DON and 15-A-DON on HepG2 cells. Cytotoxic concentration range studied was from 100 to 3.1 µM for DON and 12.5 to 0.04 µM for 3-A-DON and 15-A-DON by the Neutral Red (NR) assay, over 24, 48 and 72 h. Potential of toxicity of 3-ADON in HepG2 cells was the highest at all times assayed. Cell cycle arrest in G0/G1 and G2/M phase was detected for all mycotoxins either in individually or in combined treatment in HepG2 cells. Genotoxicity was performed through micronuclei (MN) induction (TG 487) revealing significant effects for 3-ADON mycotoxin and in several combinations. It was evidenced that cell cycle alterations detected were associated to MN induction at all doses assayed, reaching the highest induction in tertiary combinations and in the binary combination 3-ADON + 15-ADON.

Binary and tertiary combination of alternariol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol on HepG2 cells: Toxic effects and evaluation of degradation products.

Fungi producers of mycotoxins are able to synthesize more than one toxin. Alternariol (AOH) is one of the mycotoxins produced by several Alternaria species, the most common one being Alternaria alternata. The toxins 3-Acetyl-deoxynivalenol (3-ADON) and 15-Acetyl-deoxynivalenol (15-ADON) are acetylated forms of deoxynivalenol (DON) produced by Fusarium graminearum. In the present work it is determined and evaluated the toxic effects of binary and tertiary combination treatment of HepG2 cells with AOH, 3-ADON and 15-ADON, by using the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide), to subsequently apply the isobologram method and elucidate if the mixtures of these mycotoxins produced synergism, antagonism or additive effect; and lastly, to analyze mycotoxins conversion into metabolites produced and released by HepG2 cells after applying the treatment conditions by liquid chromatography tandem mass spectrometry (LC-MS/MS) equipment and extracted from culture media. HepG2 cells were treated at different concentrations over 24, 48 and 72h. IC50 values detected at all times assayed, ranged from 0.8 to >25µM in binary combinations; while in tertiary it ranged from 7.5 to 12µM. Synergistic, antagonism or additive effect detected in the mixtures of these mycotoxins was different depending on low or high concentration. Among all four mycotoxins combinations assayed, 15-ADON+3-ADON presented the highest toxic potential. At all assayed times, recoveries values oscillated depending on the time and combination studied.

Comparative in vitro cytotoxicity of modified deoxynivalenol on porcine intestinal epithelial cells.

The gastrointestinal tract is the first target after ingestion of the mycotoxin deoxynivalenol (DON) via feed and food. Deoxynivalenol is known to affect the proliferation and viability of animal and human intestinal epithelial cells. In addition to DON, feed and food is often co-contaminated with modified forms of DON, such as 3-acetyldeoxynivalenol (3ADON), 15-acetyl-deoxynivalenol (15ADON) and deoxynivalenol-3-ß-D-glucoside (DON3G). The goal of this study was to determine the in vitro intrinsic cytotoxicity of these modified forms towards differentiated and proliferative porcine intestinal epithelial cells by means of flow cytometry. Cell death was assessed by dual staining with Annexin-V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), which allows the discrimination of viable (FITC-/PI-), apoptotic (FITC+/PI-) and necrotic cells (FITC+/PI+). Based on the data from the presented pilot in vitro study, it is concluded that cytotoxicity for proliferative cells can be ranked as follows: DON3G â‰ª 3ADON < DON≈15ADON.

Oral Bioavailability, Hydrolysis, and Comparative Toxicokinetics of 3-Acetyldeoxynivalenol and 15-Acetyldeoxynivalenol in Broiler Chickens and Pigs.

The goal of this study was to determine the absolute oral bioavailability, (presystemic) hydrolysis and toxicokinetic characteristics of deoxynivalenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol in broiler chickens and pigs. Crossover animal trials were performed with intravenous and oral administration of deoxynivalenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol to broilers and pigs. Plasma concentrations were analyzed by using liquid chromatography-tandem mass spectrometry, and data were processed via a tailor-made compartmental toxicokinetic analysis. The results in broiler chickens showed that the absorbed fraction after oral deoxynivalenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol administration was 10.6, 18.2, and 42.2%, respectively. This fraction was completely hydrolyzed presystemically for 3-acetyldeoxynivalenol to deoxynivalenol and to a lesser extent (75.4%) for 15-acetyldeoxynivalenol. In pigs, the absorbed fractions were 100% for deoxynivalenol, 3-acetyldeoxynivalenol, and 15-acetyldeoxynivalenol, and both 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol were completely hydrolyzed presystemically. The disposition properties of 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol demonstrate their toxicological relevance and consequently the possible need to establish a tolerable daily intake.

Development and validation of an LC-MS/MS method for the toxicokinetic study of deoxynivalenol and its acetylated derivatives in chicken and pig plasma.

This study aims to develop an LC-MS/MS method allowing the determination of 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, deoxynivalenol and its main in vivo metabolite, deepoxy-deoxynivalenol, in broiler chickens and pigs. These species have a high exposure to these toxins, given their mainly cereal based diet. Several sample cleanup strategies were tested and further optimized by means of fractional factorial designs. A simple and straightforward sample preparation method was developed consisting out of a deproteinisation step with acetonitrile, followed by evaporation of the supernatant and reconstitution in water. The method was single laboratory validated according to European guidelines and found to be applicable for the intended purpose, with a linear response up to 200ngml(-1) and limits of quantification of 0.1-2ngml(-1). As a proof of concept, biological samples from a broiler chicken that received either deoxynivalenol, 3- or 15-acetyl-deoxynivalenol were analyzed. Preliminary results indicate nearly complete hydrolysis of 3-acetyl-deoxynivalenol to deoxynivalenol; and to a lesser extent of 15-acetyl-deoxynivalenol to deoxynivalenol. No deepoxy-deoxynivalenol was detected in any of the plasma samples. The method will be applied to study full toxicokinetic properties of deoxynivalenol, 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol in broiler chickens and pigs.

Effect of cultivars, Fusarium strains and storage temperature on trichothecenes production in inoculated potato tubers.

Four trichothecenes (Fus-X, 3ADON, DAS and T-2) were detected in potato tubers inoculated with Fusarium spp. by UPLC-MS/MS. The influence of cultivars, Fusarium strains and storage temperature on trichothecenes production was evaluated. The concentration of trichothecenecs was much higher in susceptible cultivar (Longshu No. 3) than in resistant one (Longshu No. 6). The susceptible cultivar infected with Fusarium sulphureum had the maximum concentration of Fus-X, 3ADON and DAS. Among the three Fusarium strains, Fusarium solani had the strongest ability to produce T-2 in both susceptible and resistant cultivars. Room temperature storage was more likely to accumulate trichothecenes than low temperature storage. Meanwhile, the trichothecenes were found not only in the lesion but also in the adjacent asymptomatic tissue. Trichothecenes concentration showed a strong trend of decline with increase in distance from the infection point.