Nivalenol Research Articles

Relationship Between Mycotoxin Production and Gene Expression in Fusarium graminearum Species Complex Strains Under Various Environmental Conditions.

The Fusarium graminearum species complex (FGSC) can produce various mycotoxins and is a major concern for food quantity and quality worldwide. In this study, we determined the effects of water activity (aw), temperature, incubation time and their interactions on mycotoxin accumulation and the expression levels of biosynthetic genes in FGSC strains from maize samples in China. The highest deoxynivalenol (DON), 3-acetyldeoxynivalenol(3ADON) and 15-acetyldeoxynivalenol (15ADON) levels of the F. boothii and F. graminearum strains were observed at 0.98 aw/30°C or 0.99 aw/25°C. F. asiaticum and F. meridionale reached maximum nivalenol (NIV) and 4-acetylnivalenol (4ANIV) contents at 0.99 aw and 30°C. With the extension of the incubation time, the concentrations of DON and NIV gradually increased, while those of their derivatives decreased. F. boothii, F. meridionale and one F. asiaticum strain had the highest zearalenone (ZEN) values at 0.95 aw and 25°C, while the optimum conditions for the other F. asiaticum strain and F. graminearum were 0.99 aw and 30°C. Four genes associated with trichothecene and zearalenone synthesis were significantly induced under higher water stress in the early stage of production. The results indicated independence of mycotoxin production and gene expression, as maximum amounts of these toxic metabolites were observed at higher aw in most cases. This study provides useful information for the monitoring and prevention of such toxins entering the maize production chain.

Effect of Crop Rotation on Fusarium Mycotoxins and Fusarium Species in Cereals in Sichuan Province (China).

The present study was performed to evaluate the effect of crop rotation on Fusarium mycotoxins and species in cereals in Sichuan Province. A total of 311 cereal samples were randomly collected and analyzed from 2018 to 2019 in Sichuan Province. The results of mycotoxin analysis showed that the major trichothecene mycotoxins in Sichuan Province were nivalenol (NIV) and deoxynivalenol (DON), and the mean concentration of total trichothecenes (including NIV, fusarenone X [4ANIV], DON, 3-acetyldeoxynivalenol [3ADON], and 15-acetyldeoxynivalenol [15ADON]) in wheat was significantly higher than that in maize and rice. The concentration of total trichothecenes in the succeeding crops was significantly higher than that in the previous crops. In addition, wheat grown after maize had reduced incidence and concentration of trichothecene mycotoxins compared with that grown after rice, and ratooning rice grown after rice had increased incidence and concentration of trichothecene mycotoxins. Our data indicated that Fusarium asiaticum with the NIV chemotype was predominant in wheat and rice samples, while the number of the NIV chemotypes of F. asiaticum and Fusarium meridionale and the 15ADON chemotype of Fusarium graminearum in maize were almost the same. Although the composition of Fusarium species was affected by crop rotations, there were no differences when comparing the same crop rotation except for the maize-wheat rotation. Moreover, the same species and chemotype of Fusarium strains originated from different crops in various rotations, but there were no significant differences in pathogenicity in wheat and rice. These results contribute to the knowledge of the effect of crop rotation on Fusarium mycotoxins and species affecting cereals in Sichuan Province, which may lead to improved strategies for control of Fusarium mycotoxins and fungal disease in China.

Inter-laboratory study on simultaneous quantification of ten trichothecenes in feed.

An inter-laboratory study was performed in eight laboratories to evaluate the simultaneous quantification method for HT-2 toxin (HT-2), T-2 toxin (T-2), diacetoxyscirpenol (DAS), neosolaniol (NES), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), deoxynivalenol (DON), deoxynivalenol-3-glucoside (D3G), nivalenol (NIV), and fusarenon-X (FUS-X) in feed. The mycotoxins in the samples were extracted with hydrous acetonitrile, purified using a multifunctional column (InertSep® VRA-3) and a phospholipid removal column (Hybrid SPE®-Phospholipid), and then quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with atmospheric pressure chemical ionisation mode. The mean recovery, repeatability, reproducibility, and Horwitz ratio from the inter-laboratory validation study were 99.8-109%, 3.1-9.8%, 4.3-9.8%, and 0.19-0.45, respectively, for type A trichothecenes (HT-2, T-2, DAS, and NES). Those values for type B trichothecenes (3-AcDON, 15-AcDON, DON, NIV, and FUS-X) were 89.9-116%, 3.4-9.1%, 5.6-14%, and 0.25-0.70, and the values for modified mycotoxin (D3G) were 78.2-96.7%, 3.5-6.4%, and 13-22%, respectively.

Substance P and Glucagon-like Peptide-17-36 Amide Mediate Anorexic Responses to Trichothecene Deoxynivalenol and Its Congeners.

Type B trichothecenes commonly contaminate cereal grains and include five structurally related congeners: deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), fusarenon X (FX), and nivalenol (NIV). These toxins are known to have negative effects on human and animal health, particularly affecting food intake. However, the pathophysiological basis for anorexic effect is not fully clarified. The purpose of this study is to explore the potential roles of the brain-gut peptides substance P (SP) and glucagon-like peptide-17-36 amide (GLP-1) in anorexic responses induced by type B trichothecenes following both intraperitoneal (IP) and oral administration. SP and GLP-1 were elevated at 1 or 2 h and returned to basal levels at 6 h following exposure to DON and both ADONs. FX induced the production of both brain gut peptides with initial time at 1 or 2 h and duration > 6 h. Similar to FX, exposing IP to NIV caused elevations of SP and GLP-1 at 1 h and lasted more than 6 h, whereas oral exposure to NIV only increased both brain gut peptides at 2 h. The neurokinin-1 receptor (NK-1R) antagonist Emend® dose-dependently attenuated both SP- and DON-induced anorexic responses. Pretreatment with the GLP-1 receptor (GLP-1R) antagonist Exending9-39 induced a dose-dependent attenuation of both GLP-1- and DON-induced anorexic responses. To summarize, the results suggest that both SP and GLP-1 play important roles in anorexia induction by type B trichothecenes.

Trichoderma versus Fusarium—Inhibition of Pathogen Growth and Mycotoxin Biosynthesis

This study evaluated the ability of selected strains of Trichoderma viride, T. viridescens, and T. atroviride to inhibit mycelium growth and the biosynthesis of mycotoxins deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEN), α-(α-ZOL) and β-zearalenol (β-ZOL) by selected strains of Fusarium culmorum and F. cerealis. For this purpose, an in vitro experiment was carried out on solid substrates (PDA and rice). After 5 days of co-culture, it was found that all Trichoderma strains used in the experiment significantly inhibited the growth of Fusarium mycelium. Qualitative assessment of pathogen-antagonist interactions showed that Trichoderma colonized 75% to 100% of the medium surface (depending on the species and strain of the antagonist and the pathogen) and was also able to grow over the mycelium of the pathogen and sporulate. The rate of inhibition of Fusarium mycelium growth by Trichoderma ranged from approximately 24% to 66%. When Fusarium and Trichoderma were co-cultured on rice, Trichoderma strains were found to inhibit DON biosynthesis by about 73% to 98%, NIV by about 87% to 100%, and ZEN by about 12% to 100%, depending on the pathogen and antagonist strain. A glycosylated form of DON was detected in the co-culture of F. culmorum and Trichoderma, whereas it was absent in cultures of the pathogen alone, thus suggesting that Trichoderma is able to glycosylate DON. The results also suggest that a strain of T. viride is able to convert ZEN into its hydroxylated derivative, β-ZOL.

A novel toxic effect of foodborne trichothecenes: The exacerbation of genotoxicity

Trichothecenes (TCT) are very common mycotoxins. While the effects of DON, the most prevalent TCT, have been extensively studied, less is known about the effect of other trichothecenes. DON has ribotoxic, pro-inflammatory, and cytotoxic potential and induces multiple toxic effects in humans and animals. Although DON is not genotoxic by itself, it has recently been shown that this toxin exacerbates the genotoxicity induced by model or bacterial genotoxins. Here, we show that five TCT, namely T-2 toxin (T-2), diacetoxyscirpenol (DAS), nivalenol (NIV), fusarenon-X (FX), and the newly discovered NX toxin, also exacerbate the DNA damage inflicted by various genotoxins. The exacerbation was dose dependent and observed with phleomycin, a model genotoxin, captan, a pesticide with genotoxic potential, and colibactin, a bacterial genotoxin produced by the intestinal microbiota. For this newly described effect, the trichothecenes ranked in the following order: T-2>DAS > FX > NIV ≥ DON ≥ NX. The genotoxic exacerbating effect of TCT correlated with their ribotoxic potential, as measured by the inhibition of protein synthesis. In conclusion, our data demonstrate that TCT, which are not genotoxic by themselves, exacerbate DNA damage induced by various genotoxins. Therefore, foodborne TCT could enhance the carcinogenic potential of genotoxins present in the diet or produced by intestinal bacteria.

Preparation of Monoclonal Antibodies Specifically Reacting with the Trichothecene Mycotoxins Nivalenol and 15-Acetylnivalenol via the Introduction of a Linker Molecule into Its C-15 Position.

Nivalenol (NIV) is a trichothecene mycotoxin that is more toxic than deoxynivalenol. It accumulates in grains due to infection with Fusarium species, which are the causative agents of scab or Fusarium head blight. An immunoassay, which is a rapid and easy analytical method, is necessary for monitoring NIV in grains. However, a specific antibody against NIV has not been prepared previously. To establish an immunoassay, we prepared NIV, introduced a linker, and generated antibodies against it. NIV was prepared from a culture of Fusarium kyushuense obtained from pressed barley through chromatographic procedures with synthetic adsorbents and silica gel. NIV was reacted with glutaric anhydride, and the reaction was stopped before mono-hemiglutaryl-NIV was changed to di-hemiglutaryl-NIV. 15-O-Hemiglutaryl-NIV was isolated via preparative HPLC and bound to keyhole limpet hemocyanin (KLH) using the active ester method. Two different monoclonal antibodies were prepared by immunizing mice with the NIV-KLH conjugate. The 50% inhibitory concentration values were 36 and 37 ng/mL. These antibodies also showed high reactivity in a direct competitive enzyme-linked immunosorbent assay and specifically reacted with NIV and 15-acetyl-NIV but not with deoxynivalenol and 4-acetyl-NIV.

Temporal and spatial dynamics of Fusarium spp. and mycotoxins in Swedish cereals during 16 years

We analysed the dynamics of Fusarium spp. and mycotoxin contamination in Swedish cereals during 2004–2018. More than 1400 cereal samples from field trials were included, collected in a monitoring programme run by the Swedish Board of Agriculture. Five Fusarium mycotoxins were quantified with LC-MS/MS and fungal DNA from four species was quantified using quantitative real-time PCR. Correlation analyses revealed that deoxynivalenol (DON) and zearalenone (ZEN) were mainly associated with Fusarium graminearum, but stronger correlations with F. culmorum was seen some years. Nivalenol (NIV) was associated with F. poae and the HT-2 and T-2 toxins with F. langsethiae. Clear differences in mycotoxin contamination between different cereal crops and geographical regions were identified. The highest levels of DON and ZEN were found in spring wheat in Western Sweden. For NIV, HT-2 and T-2 toxins, the levels were highest in spring oats and spring barley. Regional differences were not detected for NIV, while HT-2 and T-2 toxins were associated with the northernmost region. We found that delayed harvest was strongly associated with increased levels of DON and ZEN in several crops. However, harvest date did not influence the levels of NIV or HT-2 and T-2 toxins. Our results suggest similar distribution patterns of DON and ZEN, in contrast to NIV and HT-2 and T-2 toxins, probably mirroring the differences in the ecology of the toxin-producing Fusarium species. Timely harvest is important to reduce the risk of DON and ZEN contamination, especially for fields with other risk factors.

Multiple Mycotoxin Contamination in Medicinal Plants Frequently Sold in the Free State Province, South Africa Detected Using UPLC-ESI-MS/MS.

Medicinal plants are important in the South African traditional healthcare system, the growth in the consumption has led to increase in trade through muthi shops and street vendors. Medicinal plants are prone to contamination with fungi and their mycotoxins. The study investigated multiple mycotoxin contamination using Ultra High Pressure Liquid Chromatography–Tandem Mass Spectrometry (UPLC-ESI-MS/MS) for the simultaneous detection of Aflatoxin B1 (AFB1), Deoxynivalenol (DON), Fumonisins (FB1, FB2, FB3), Nivalenol (NIV), Ochratoxin A (OTA) and Zearalenone (ZEN) in frequently sold medicinal plants. Medicinal plant samples (n = 34) were purchased and analyzed for the presence of eight mycotoxins. DON and NIV were not detected in all samples analyzed. Ten out of thirty-four samples tested positive for mycotoxins —AFB1 (10.0%); OTA (10.0%); FB1 (30.0%); FB2 (50.0%); FB3 (20.0%); and ZEN (30.0%). Mean concentration levels ranged from AFB1 (15 µg/kg), OTA (4 µg/kg), FB1 (7–12 µg/kg), FB2 (1–18 µg/kg), FB3 (1–15 µg/kg) and ZEN (7–183 µg/kg). Multiple mycotoxin contamination was observed in 30% of the positive samples with fumonisins. The concentration of AFB1 reported in this study is above the permissible limit for AFB1 (5 µg/kg). Fumonisin concentration did not exceed the limits set for raw maize grain (4000 µg/kg of FB1 and FB2). ZEN and OTA are not regulated in South Africa. The findings indicate the prevalence of mycotoxin contamination in frequently traded medicinal plants that poses a health risk to consumers. There is therefore a need for routine monitoring of multiple mycotoxin contamination, human exposure assessments using biomarker analysis and establishment of regulations and standards.

Toxigenicity of F. graminearum Residing on Host Plants Alternative to Wheat as Influenced by Environmental Conditions.

Fusarium graminearum is an important pathogen that causes Fusarium head blight (FHB) in several cereal crops worldwide. The potential of this pathogen to contaminate cereals with trichothecene mycotoxins presents a health risk for both humans and animals. This study aimed to evaluate the potential of different trichothecene genotypes of F. graminearum isolated from an alternative host plant to produce mycotoxins under different spring wheat grain incubation conditions. Fourteen F. graminearum strains were isolated from seven alternative host plants and identified as 3-acetyl-deoxynivalenol (3-ADON) and 15-acetyl-deoxynivalenol (15-ADON) genotypes. These strains were cultivated on spring wheat grains at 25 °C and 29 °C for 5 weeks. The mycotoxins produced were analysed with a high-performance liquid chromatograph (HPLC) coupled to a Thermo Scientific TSQ Quantiva MS/MS detector. The obtained results showed that the F. graminearum strains from alternative host plants could produce nivalenol (NIV), deoxynivalenol (DON), fusarenon-X (FUS-X), 3-ADON, deoxynivalenol-3-ß-d-glucoside (D3G), 15-ADON, and zearalenone (ZEA). F. graminearum strains produced DON and ZEA under both temperatures, with the mean concentrations varying from 363 to 112,379 µg kg−1 and from 1452 to 44,816 µg kg−1, respectively. Our results indicated the possible role of dicotyledonous plants, including weeds, as a reservoir of inoculum sources of F. graminearum-induced Fusarium head blight, associated with the risk of mycotoxin contamination in spring wheat.

Nivalenol Mycotoxin Concerns in Foods: An Overview on Occurrence, Impact on Human and Animal Health and Its Detection and Management Strategies.

Mycotoxins are secondary metabolites produced by fungi that infect a wide range of foods worldwide. Nivalenol (NIV), a type B trichothecene produced by numerous Fusarium species, has the ability to infect a variety of foods both in the field and during post-harvest handling and management. NIV is frequently found in cereal and cereal-based goods, and its strong cytotoxicity poses major concerns for both human and animal health. To address these issues, this review briefly overviews the sources, occurrence, chemistry and biosynthesis of NIV. Additionally, a brief overview of several sophisticated detection and management techniques is included, along with the implications of processing and environmental factors on the formation of NIV. This review’s main goal is to offer trustworthy and current information on NIV as a mycotoxin concern in foods, with potential mitigation measures to assure food safety and security.

Implications of Crop Rotation and Fungicide on Fusarium and Mycotoxin Spectra in Manitoba Barley, 2017-2019.

Fusarium 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 Fusarium 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 Fusarium species spectra, abundance, chemotype composition, and mycotoxin profiles. Fusarium poae was the predominant Fusarium species associated with FHB of barley in Manitoba, followed by F. graminearum, and F. sporotrichioides; F. equiseti and F. avenaceum were also detected but at low levels. F. poae strains with the nivalenol (NIV) chemotype and F. graminearum 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−1) and deoxynivalenol (DON) (264.7, 56.7, and 65.3 µg kg−1) 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, F. poae proved less sensitive to four triazole fungicides (metconazole, prothioconazole+tebuconazole, tebuconazole, and prothioconazole) than F. graminearum. 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.

Fusarium culmorum Produces NX-2 Toxin Simultaneously with Deoxynivalenol and 3-Acetyl-Deoxynivalenol or Nivalenol

Fusarium culmorum 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 F. graminearum 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 F. graminearum from North America. Only these isolates and a few strains of a yet to be characterized Fusarium species from South Africa are known to produce NX-2 and other NX toxins. Here, we report that among 20 F. culmorum 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 F. graminearum, producers of NX-2 possess a special variant of cytochrome P450 monooxygenase encoded by TRI1 that is unable to oxidize C-8. In F. culmorum, producers and nonproducers of NX-2 possess identical TRI1; 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 F. culmorum.

Survey of the Presence of Nivalenol and Deoxynivalenol in Wheat Flour Factories of Khuzestan Province, Iran

Background: Wheat is frequently contaminated by Deoxynivalenol (DON) and Nivalenol (NIV), which are type B trichothecenes produced by Fusarium fungi. Most problems related to these contaminants involve prolonged feed intake at low levels of contamination. This study investigated the occurrence of NIV and Deoxynivalenol (DON) in wheat flour in flour factories in Khuzestan Providence, Iran. Methods: In total, 104 samples were collected in this study. An acetonitrile/water mixture (84:16, v/v) was used to extract the samples. The extracts were filtered and purified using a Whatman No. 4 paper and MycoSepTM 227 column. Then, they were evaporated to dryness at 40˚C under a nitrogen stream. After dissolving the dry residue in the mobile phase, containing a mixture of methanol, acetonitrile, and water (5:5:90, v/v/v), the contents of NIV and DON in the samples were measured in High-Performance Liquid Chromatography (HPLC) system with a column C18 (150 mm×4.6 mm ID, 5 μm) and a UV detector (220 nm). Results: The results showed that among 104 wheat flour samples, 28 (26.8%) and 54 (51.9%) samples were contaminated with NIV and DON, respectively. The mean and maximum concentrations were 118.75 and 2278 ng/g for NIV, and 593 and 67.88 ng/g for DON, respectively. Conclusion: Based on the findings, DON and NIV had significantly lower concentrations than the maximum tolerated level (1 μg/g), established by the Institute of Standards and Industrial Research of Iran. Therefore, there were no health risks for consumers at the studied contamination levels.

Identification and Functional Characterisation of Two Oat UDP-Glucosyltransferases Involved in Deoxynivalenol Detoxification.

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 Fusarium graminearum. Heterologous expression of these genes in a toxin-sensitive strain of Saccharomyces cerevisiae 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 Escherichia coli 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.

Infection timing affects Fusarium poae colonization of bread wheat spikes and mycotoxin accumulation in the grain.

Fusarium poae is one of the most common Fusarium head blight (FHB) causal agents in wheat. This species can biosynthesize a wide range of mycotoxins, in particular nivalenol (NIV). In FHB epidemiology, infection timing is important for disease occurrence, kernel development, symptom appearance and mycotoxin accumulation in grain. The present study explored, both in a controlled environment and in a 2-year field plot experiment in Central Italy, the influence of five infection timings (from beginning of flowering to medium milk growth stage) on F. poae colonization and mycotoxin accumulation in bread wheat spikes (spring cv. A416 and winter cv. Ambrogio). Both climate chamber and field experiments showed that early infection timings (from beginning of flowering to full flowering) especially favoured F. poae colonization and accumulation of its mycotoxins (particularly NIV) in grain. By contrast, later infection timings (watery ripe and medium milk) reduced F. poae development and mycotoxin levels. The time window of host susceptibility in the field was shorter than that observed under controlled conditions. Symptom expression in kernels also differed among infection timings. In general, F. poae biomass was higher in the chaff than in the grain. These results enhance knowledge of a common member of the FHB complex worldwide, and could be useful in forecasting the risk of F. poae infection and mycotoxin contamination. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Development and evaluation of a novel visual and rapid detection assay for toxigenic Fusarium graminearum in maize based on recombinase polymerase amplification and lateral flow analysis

Maize ear rot caused by Fusarium graminearum is one of the most severe maize diseases in global maize-growing regions. It reduces maize yield in the field and is also responsible for mycotoxin contamination of grains during the postharvest period. F. graminearum is one of the major deoxynivalenol (DON), nivalenol (NIV), and zearalenone (ZEN) producers. The ingestion of these mycotoxins represents a risk for human and animal health. Hence, early detection and identification of F. graminearum are crucial to controlling these mycotoxins along the food or feed supply chains. In this study, the recombinase polymerase amplification with lateral flow dipstick (RPA-LFD) assay targeting the gaoA gene that codes for galactose oxidase was developed. The reaction conditions were optimized to make the method rapid, sensitive, and cost-effective. The developed RPA-LFD assay could detect the presence of 20 fg of the target genomic DNA per reaction within 25 min at 40 °C. Moreover, 52 field samples were tested using the developed RPA-LFD assay and compared with conventional PCR-based methods. The positive rate between RPA-LFD and the conventional PCR-based method was 100%. In conclusion, the developed method provides a novel alternative for the rapid, sensitive, and specific detection and identification of F. graminearum. It is not only workable for bulk maize samples without using sophisticated lab equipment but is also potentially useful for other agriculturally important toxigenic fungi.

Trichothecene Genotype Profiling of Wheat Fusarium graminearum Species Complex in Paraguay

Paraguay is a non-traditional wheat-producing country in one of the warmest regions in South America. Fusarium Head Blight (FHB) is a critical disease affecting this crop, caused by the Fusarium graminearum species complex (FGSC). A variety of these species produce trichothecenes, including deoxynivalenol (DON) and its acetylated forms (3-ADON and 15-ADON) or nivalenol (NIV). This study characterized the phylogenetic relationships, and chemotype diversity of 28 strains within FGSC collected from wheat fields across different country regions. Phylogenetic analysis based on the sequence of elongation factor-1α gene (EF-1α) from 28 strains revealed the presence of four species in the FGSC: F. graminearum sensu stricto, F. asiaticum, F. meridionale and F. cortaderiae. Ten strains selected for further analysis revealed that all F. graminearum strains were 15-ADON chemotype, while the two strains of F. meridionale and one strain of F. asiaticum were NIV chemotype. Thus, the 15-ADON chemotype of F. graminearum sensu stricto was predominant within the Fusarium strains isolated in the country. This work is the first report of phylogenetic relationships and chemotype diversity among Fusarium strains which will help understand the population diversity of this pathogen in Paraguay.

The FaFlbA mutant of Fusarium asiaticum is significantly increased in nivalenol production.

Cereals contaminated with type B trichothecene nivalenol (NIV) and its acetylated derivative 4-acetyl-nivalenol (4-AcNIV) are a global mycotoxicological problem threatening the health of humans and livestock. Toxicological studies, quantitative determinations and screening for biodegrading micro-organisms require massive amounts of pure toxins. However, the low yield from fungal cultures and high prices of NIV and 4-AcNIV limit research progress in these areas. This work aimed to select Fusarium asiaticum mutant strains with enhanced production of NIV and 4-AcNIV. A total of 62 NIV-producing F. asiaticum strains were isolated and compared regarding their ability to produce NIV. Strain RR108 had the highest yield of NIV among 62 field isolates surveyed and was then genetically modified for higher production. Targeted deletion of the FaFlbA gene, encoding a regulator of G protein signalling protein, resulted in a significant increase in NIV and 4-AcNIV production in the FaFlbA deletion mutant ΔFaFlbA. The expression of three TRI genes involved in the trichothecene biosynthetic pathway was upregulated in ΔFaFlbA. ΔFaFlbA produced the highest amount of NIV and 4-AcNIV when cultured in brown long-grain rice for 21days, and the yields were 2.07 and 2.84gkg-1 , respectively. The mutant showed reduced fitness, including reduced conidiation, loss of perithecial development and decreased virulence on wheat heads, which makes it biologically safe for large-scale preparation and purification of NIV and 4-AcNIV. The F. asiaticum mutant strain ΔFaFlbA presented improved production of NIV and 4-AcNIV with reduced fitness and virulence in plants. Targeted deletion of the FaFlbA gene resulted in increased NIV and 4-AcNIV production. Our results provide a practical approach using genetic modification for large-scale mycotoxin production.

Dynamics of Deoxynivalenol and Nivalenol Glucosylation in Wheat Cultivars Infected with Fusarium culmorum in Field Conditions─A 3 Year Study (2018-2020).

Fusarium head blight (FHB) caused by pathogenic species of Fusarium fungi is one of the most important diseases of cereal plants and a factor contributing to losses in plant production. The growth of FHB-associated species is often accompanied by biosynthesis of secondary metabolites─mycotoxins, which serve as a virulence factor. The aim of the study was to evaluate the ratios between deoxynivalenol (DON) and nivalenol (NIV) and their derivatives in the ears of six cultivars of winter wheat with varying resistance to FHB, taking into account a range of factors (weather conditions, location, cultivar, and year) after inoculation with Fusarium culmorum, during a 3 year field experiment, 2018-2020. The presence of toxins in the ears was measured within 21 days of inoculation. The toxins were found in the ears as soon as on the third day from the start of the experiment, whereas relative humidity higher than 80% was a decisive factor for FHB incidence. All wheat cultivars showed the ability to biotransform DON and NIV present in the ears to glucosides, that is, deoxynivalenol-3-glucoside (DON-3G) and nivalenol-3-glucoside (NIV-3G). The levels of these metabolites showed significant correlation with the levels of their basic analogues. In most cases, higher levels of DON and NIV in wheat ears and higher levels of their metabolites were observed, but the relative levels of DON-3G/DON and NIV-3G/NIV at relatively high levels of toxins were lower compared to the ear samples with relatively low toxin levels. The presented results are the first studies, which systematically correlate a variety of wheat cultivars with their extent to glucosylate trichothecenes.