Naturally Occurring Fusarium Species and Mycotoxins in Oat Grains from Manitoba, Canada.
ABSTRACT: Fusarium head blight (FHB) can lead to dramatic yield losses and mycotoxin contamination in small grain cereals in Canada. To assess the extent and severity of FHB in oat, samples collected from 168 commercial oat fields in the province of Manitoba, Canada, during 2016-2018 were analyzed for the occurrence of Fusarium head blight and associated mycotoxins. Through morphological and molecular analysis, F. poae was found to be the predominant Fusarium species affecting oat, followed by F. graminearum, F. sporotrichioides, F. avenaceum, and F. culmorum. Deoxynivalenol (DON) and nivalenol (NIV), type B trichothecenes, were the two most abundant Fusarium mycotoxins detected in oat. Beauvericin (BEA) was also frequently detected, though at lower concentrations. Close clustering of F. poae and NIV/BEA, F. graminearum and DON, and F. sporotrichioides and HT2/T2 (type A trichothecenes) was detected in the principal component analysis. Sampling location and crop rotation significantly impacted the concentrations of Fusarium mycotoxins in oat. A phylogenetic analysis of 95 F. poae strains from Manitoba was conducted using the concatenated nucleotide sequences of Tef-1α, Tri1, and Tri8 genes. The results indicated that all F. poae strains belong to a monophyletic lineage. Four subgroups of F. poae strains were identified; however, no correlations were observed between the grouping of F. poae strains and sample locations/crop rotations.
Project description:<i>Fusarium</i> head blight (FHB) is one of the most important diseases of barley in Manitoba province (western Canada), and other major barley producing regions of the world. Little is known about the <i>Fusarium</i> species and mycotoxin spectra associated with FHB of barley in Manitoba. Hence, barley grain samples were collected from 149 commercial fields from 2017 to 2019, along with information on respective cropping history, and analyzed with respect to <i>Fusarium</i> species spectra, abundance, chemotype composition, and mycotoxin profiles. <i>Fusarium poae</i> was the predominant <i>Fusarium</i> species associated with FHB of barley in Manitoba, followed by <i>F. graminearum</i>, and <i>F. sporotrichioides</i>; <i>F. equiseti</i> and <i>F. avenaceum</i> were also detected but at low levels. <i>F. poae</i> strains with the nivalenol (NIV) chemotype and <i>F. graminearum</i> strains with 3-acetyl deoxynivalenol (3-ADON) and 15-acetyl deoxynivalenol (15-ADON) chemotypes were commonly detected in the barley grain samples. Nivalenol (597.7, 219.1, and 412.4 µg kg<sup>-1</sup>) and deoxynivalenol (DON) (264.7, 56.7, and 65.3 µg kg<sup>-1</sup>) were the two most prevalent mycotoxins contaminating Manitoba barley in 2017, 2018 and 2019, respectively. A substantially higher DON content was detected in grain samples from barley fields with cereals as a preceding crop compared to canola and flax. Furthermore, <i>F. poae</i> proved less sensitive to four triazole fungicides (metconazole, prothioconazole+tebuconazole, tebuconazole, and prothioconazole) than <i>F. graminearum</i>. Findings from this research will assist barley producers with improved understanding of FHB threat levels and optimizing practices for the best management of FHB in barley.
Project description:The current study investigated the fungal diversity in freshly harvested oat samples from the two largest production regions in Brazil, Paraná (PR) and Rio Grande do Sul (RS), focusing primarily on the <i>Fusarium</i> genus and the presence of type B trichothecenes. The majority of the isolates belonged to the <i>Fusarium sambucinum</i> species complex, and were identified as <i>F. graminearum</i> sensu stricto (s.s.), <i>F. meridionale</i>, and <i>F. poae</i>. In the RS region, <i>F. poae</i> was the most frequent fungus, while <i>F. graminearum</i> s.s. was the most frequent in the PR region. The <i>F. graminearum</i> s.s. isolates were 15-ADON genotype, while <i>F. meridionale</i> and <i>F. poae</i> were NIV genotype. Mycotoxin analysis revealed that 92% and 100% of the samples from PR and RS were contaminated with type B trichothecenes, respectively. Oat grains from PR were predominantly contaminated with DON, whereas NIV was predominant in oats from RS. Twenty-four percent of the samples were contaminated with DON at levels higher than Brazilian regulations. Co-contamination of DON, its derivatives, and NIV was observed in 84% and 57.7% of the samples from PR and RS, respectively. The results provide new information on <i>Fusarium</i> contamination in Brazilian oats, highlighting the importance of further studies on mycotoxins.
Project description:Fusarium Head Blight (FHB) is one of the major diseases affecting small-grain cereals, worldwide spread and responsible for severe yield and quality losses annually. Diagnostic tools, able to track Fusarium species even in the early stages of infection, can contribute to mycotoxins' risk control. Among DNA-based technologies for Fusarium detection, qPCR (single and multiplex assays) is currently the most applied method. However, pathogen diagnostics is now enforced by digital PCR (dPCR), a breakthrough technology that provides ultrasensitive and absolute nucleic acid quantification. In our work, a panel of chip digital PCR assays was developed to quantify Fusarium graminearum, F.culmorum, F. sporotrichioides, F. poae and F. avenaceum. The primers/probes combinations were evaluated on pure fungal samples with cdPCR technique, in comparison with the qPCR approach. Moreover, the cdPCR assays were applied to quantify Fusarium in durum wheat and oat samples, naturally contaminated or spiked with fungal DNA. For a better evaluation of infection level in plants, duplex assays were developed, able to co-amplify both plant and fungal DNA. To the best of our knowledge, this is the first study directed to the application of digital PCR to Fusarium diagnosis in plants.
Project description:Oat is susceptible to several Fusarium species that cause contamination with different trichothecene mycotoxins. The molecular mechanisms behind Fusarium resistance in oat have yet to be elucidated. In the present work, we identified and characterised two oat UDP-glucosyltransferases orthologous to barley HvUGT13248. Overexpression of the latter in wheat had been shown previously to increase resistance to deoxynivalenol (DON) and nivalenol (NIV) and to decrease disease the severity of both Fusarium head blight and Fusarium crown rot. Both oat genes are highly inducible by the application of DON and during infection with <i>Fusarium graminearum</i>. Heterologous expression of these genes in a toxin-sensitive strain of <i>Saccharomyces cerevisiae</i> conferred high levels of resistance to DON, NIV and HT-2 toxins, but not C4-acetylated trichothecenes (T-2, diacetoxyscirpenol). Recombinant enzymes AsUGT1 and AsUGT2 expressed in <i>Escherichia coli</i> rapidly lost activity upon purification, but the treatment of whole cells with the toxin clearly demonstrated the ability to convert DON into DON-3-O-glucoside. The two UGTs could therefore play an important role in counteracting the Fusarium virulence factor DON in oat.
Project description:Recent increases of Fusarium head blight (FHB) disease caused by infections with F. poae (FP) and F. langsethiae (FL) have been observed in oats. These pathogens are producers of nivalenol (NIV) and T-2/HT-2 toxin (T-2/HT-2), respectively, which are now considered major issues for cereal food and feed safety. To date, the impact of FP and FL on oat grains has not yet been identified, and little is known about oat resistance elements against these pathogens. In the present study, the impact of FL and FP on oat grains was assessed under different environmental conditions in field experiments with artificial inoculations. The severity of FP and FL infection on grains were compared across three field sites, and the resistance against NIV and T-2/HT2 accumulation was assessed for seven oat genotypes. Grain weight, ?-glucan content, and protein content were compared between infected and non-infected grains. Analyses of grain infection showed that FL was able to cause infection on the grain only in the field site with the highest relative humidity, whereas FP infected grains in all field sites. The FP infection of grains resulted in NIV contamination (between 30-500 ?g/kg). The concentration of NIV in grains was not conditioned by environmental conditions. FL provoked an average contamination of grains with T-2/HT-2 (between 15-132 ?g/kg). None of the genotypes was able to fully avoid toxin accumulation. The general resistance of oat grains against toxin accumulation was weak, and resistance against NIV accumulation was strongly impacted by the interaction between the genotype and the environment. Only the genotype with hull-less grains showed partial resistance to both NIV and T-2/HT-2 contamination. FP and FL infections could change the ?-glucan content in grains, depending on the genotypes and environmental conditions. FP and FL did not have a significant impact on the thousand kernel weight (TKW) and protein content. Hence, resistance against toxin accumulation remains the only indicator of FHB resistance in oat. Our results highlight the need for new oat genotypes with enhanced resistance against both NIV and T-2/HT-2 to ensure food and feed safety.
Project description:In this study, twenty of the most common <i>Fusarium</i> species were molecularly characterized and inoculated on potato dextrose agar (PDA), rice and maize medium, where thirty three targeted mycotoxins, which might be the secondary metabolites of the identified fungal species, were detected by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Statistical analysis was performed with principal component analysis (PCA) to characterize the mycotoxin profiles for the twenty fungi, suggesting that these fungi species could be discriminated and divided into three groups as follows. Group I, the fusaric acid producers, were defined into two subgroups, namely subgroup I as producers of fusaric acid and fumonisins, comprising of <i>F. proliferatum</i>, <i>F. verticillioides</i>, <i>F. fujikuroi</i> and <i>F. solani</i>, and subgroup II considered to only produce fusaric acid, including <i>F. temperatum</i>, <i>F. subglutinans</i>, <i>F. musae</i>, <i>F. tricinctum</i>, <i>F. oxysporum</i>, <i>F. equiseti</i>, <i>F. sacchari</i>, <i>F. concentricum</i>, <i>F. andiyazi</i>. Group II, as type A trichothecenes producers, included <i>F. langsethiae</i>, <i>F. sporotrichioides</i>, <i>F. polyphialidicum</i>, while Group III were found to mainly produce type B trichothecenes, comprising of <i>F. culmorum</i>, <i>F. poae</i>, <i>F. meridionale</i> and <i>F. graminearum</i>. A comprehensive picture, which presents the mycotoxin-producing patterns by the selected fungal species in various matrices, is obtained for the first time, and thus from an application point of view, provides key information to explore mycotoxigenic potentials of <i>Fusarium</i> species and forecast the <i>Fusarium</i> infestation/mycotoxins contamination.
Project description:<i>Fusarium</i> species produce important mycotoxins, such as deoxynivalenol (DON), nivalenol (NIV) and T-2/HT-2-toxins in cereals. The highest DON and T-2/HT-2 toxin levels in northern Europe have been found in oats. About 12%-24% of Finnish oat samples in 2012 contained >1.75 mg·kg<sup>-1</sup> of DON, which belongs to type B trichothecenes. <i>Fusarium graminearum</i> is the most important DON producer in northern Europe and Asia and it has been displacing the closely related <i>F. culmorum</i> in northern Europe. The 3ADON chemotype of <i>F. graminearum</i> is dominant in most northern areas, while the 15ADON chemotype of <i>F. graminearum</i> is predominating in Central and southern Europe. We suggest that the northern population of <i>F. graminearum</i> may be more specialized to oats than the southern population. Only low levels of <i>F. culmorum</i> DNA were found in a few oat samples and no correlation was found between <i>F. culmorum</i> DNA and DON levels. DNA levels of <i>F. graminearum</i> were in all cases in agreement with DON levels in 2011 and 2012, when DON was measured by gas chromatography-mass spectrometry (GC-MS). When the <i>RIDA</i><sup>®</sup><i>QUICK</i> SCAN kit results (DON) were compared to DNA levels of <i>F. g</i><i>raminearum</i>, the variation was much higher. The homogenization of the oats flour by grinding oats with 1 mm sieve seems to be connected to this variation. There was a significant correlation between the combined T-2 and HT-2 and the combined DNA levels of <i>F. langsethiae</i> and <i>F. sporotrichioides</i> in Finland in 2010-2012.
Project description:Two of the most common species of toxin-producing Fusarium contaminating small cereal grains are Fusarium graminearum and F. poae; with both elaborating diverse toxins, especially deoxynivalenol (DON) and nivalenol (NIV), respectively. The objective of our work during the 2012-2014 growing seasons was to screen crops for the most commonly isolated Fusarium species and to quantify DON and NIV toxins in natural malting-barley samples from different producing areas of Argentina. We identified 1180 Fusarium isolates in the 119 samples analyzed, with 51.2% being F. graminearum, 26.2% F. poae and 22.6% other species. We found high concentrations of mycotoxins, at maximum values of 12 ?g/g of DON and 7.71 ?g/g of NIV. Of the samples, 23% exhibited DON at an average of 2.36 ?g/g, with 44% exceeding the maximum limits (average of 5.24 ?g/g); 29% contained NIV at an average of 2.36 ?g/g; 7% contained both DON and NIV; and 55% were without DON or NIV. Finally, we report the mycotoxin contamination of the grain samples produced by F. graminearum and F. poae, those being the most frequent Fusarium species present. We identified the main Fusarium species affecting natural malting-barley grains in Argentina and documented the presence of many samples with elevated concentrations of DON and NIV. To our knowledge, the investigation reported here was the first to quantify the contamination by Fusarium and its toxins in natural samples of malting barley in Argentina.
Project description:Within the European Union (EU), edible insects need to be approved as "Novel Food" according to Regulation (EU) 2015/2283 and must comply with the requirements of European food law with regard to microbiological and chemical food safety. Substrates used for feeding insects are susceptible to the growth of Fusarium spp. and consequently to contamination with trichothecene mycotoxins. Therefore, the current study aimed to investigate the influence of T-2 and HT-2 toxins on the larval life cycle of yellow mealworm (Tenebrio molitor (L.)) and to study the transfer of T-2, HT-2, T-2 triol and T-2 tetraol in the larvae. In a 4-week feeding study, T. molitor larvae were kept either on naturally (oat flakes moulded with Fusarium sporotrichioides) or artificially contaminated oat flakes, each at two levels (approximately 100 and 250 ?g/kg total T-2 and HT-2). Weight gain and survival rates were monitored, and mycotoxins in the feeding substrates, larvae and residues were determined using LC-MS/MS. Larval development varied between the diets and was 44% higher for larvae fed artificially contaminated diets. However, the artificially contaminated diets had a 16% lower survival rate. No trichothecenes were detected in the surviving larvae after harvest, but T-2 and HT-2 were found both in the dead larvae and in the residues of naturally and artificially contaminated diets.
Project description:<i>Fusarium</i>&nbsp;<i>culmorum</i> is a major pathogen of grain crops. Infected plants accumulate deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3-ADON), or nivalenol (NIV), which are mycotoxins of the trichothecene B group. These toxins are also produced by <i>F.</i>&nbsp;<i>graminearum</i> species complex. New trichothecenes structurally similar to trichothecenes B but lacking the carbonyl group on C-8, designated NX toxins, were recently discovered in atypical isolates of <i>F.</i>&nbsp;<i>graminearum</i> from North America. Only these isolates and a few strains of a yet to be characterized <i>Fusarium</i> species from South Africa are known to produce NX-2 and other NX toxins. Here, we report that among 20 <i>F. culmorum</i> strains isolated from maize, wheat, and oat in Europe and Asia over a period of 70 years, 18 strains produced NX-2 simultaneously with 3-ADON and DON or NIV. Rice cultures of strains producing 3-ADON accumulated NX-2 in amounts corresponding to 2-8% of 3-ADON (1.2-36 mg/kg). A strain producing NIV accumulated NX-2 and NIV at comparable amounts (13.6 and 10.3 mg/kg, respectively). In <i>F. graminearum</i>, producers of NX-2 possess a special variant of cytochrome P450 monooxygenase encoded by <i>TRI1</i> that is unable to oxidize C-8. In <i>F.</i>&nbsp;<i>culmorum</i>, producers and nonproducers of NX-2 possess identical <i>TRI1</i>; the reason for the production of NX-2 is unknown. Our results indicate that the production of NX-2 simultaneously with trichothecenes B is a common feature of <i>F.</i>&nbsp;<i>culmorum</i>.