Trichothecene Mycotoxins Research Articles

NRF2/PGC-1α-mediated mitochondrial biogenesis contributes to T-2 toxin-induced toxicity in human neuroblastoma SH-SY5Y cells

The T-2 toxin is a highly toxic trichothecene mycotoxin that would cause serious toxicity in humans and animals. Recent studies suggest that the central nervous system (CNS) is susceptible to T-2 toxin, which can easily cross the blood-brain barrier, accumulate in brain tissues, and cause neurotoxicity. The growing evidence indicates that oxidative damage and mitochondrial dysfunction play a critical role in T-2 toxin-induced neurotoxicity, but the mechanisms are still poorly understood. Our present study showed that T-2 toxin decreased cell viability and increased lactate dehydrogenase leakage in human neuroblastoma SH-SY5Y cells in a concentration- and time-dependent manner. T-2 toxin elicited prominent oxidative stress and mitochondrial dysfunction, as evidenced by the promotion of cellular reactive oxygen species generation, disruption of the mitochondrial membrane potential, depletion of glutathione and reduction of the cellular ATP content. T-2 toxin impaired mitochondrial biogenesis, including decreased mitochondrial DNA copy number and affected the nuclear factor erythroid 2 related factor 2 (NRF2) / peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC-1α) pathway by upregulating NRF2 mRNA and protein expression while inhibiting the expression of PGC-1α, nuclear respiratory factor (NRF1) and mitochondrial transcription factor A (TFAM). NRF2 knockdown was found to significantly exacerbate T-2 toxin-induced cytotoxicity, oxidative stress, and mitochondrial dysfunction, as well as aggravate mitochondrial biogenesis impairment. NRF2 knockdown compromised T-2 toxin-induced upregulation of NRF2, but augmented the inhibition of PGC-1α, NRF1, and TFAM by T-2 toxin. Taken together, these findings suggest that T-2 toxin-induced oxidative stress and mitochondrial dysfunction in SH-SY5Y cells, at least in part by, NRF2/PGC-1α pathway-mediated mitochondrial biogenesis.

Exposure assessment of urinary deoxynivalenol in pregnant women in Wuhan, China

Deoxynivalenol (DON) is a ubiquitous trichothecene mycotoxin in food. DON and its modified forms can cross the placental barrier and influence the foetus’ health. Limited information is available on exposure of dietary DON intake of pregnant women in China recently. We estimated the exposure of dietary DON intake of pregnant women in a large sample size (n = 2534) from China. The total DON (tDON) and free DON (fDON) biomarkers in urine were detected by liquid chromatography–tandem mass spectrometry. The mean concentration of tDON was 34.8 ng/mg creatinine, and the fDON was 15.9 ng/mg creatinine. The estimated exposure of dietary DON intake was 0.96–1.91 μg/kg bw/day, and the difference in exposure of DON between the first and second trimesters was statistically insignificant. Approximately 26%–60% of individuals exceeded the provisional maximum tolerable daily intake (PMTDI). The age, pre-pregnancy BMI, and the sampling seasons may be the risk factor for the occurrence of exceeding PMTDI. This survey is the first exposure biomarker for DON in a large sample size from pregnant women in China. We found that the dietary DON exposure in pregnant women was relatively high and particular concern should be paid to the DON exposure during pregnancy.

T-2 toxin and its cardiotoxicity: New insights on the molecular mechanisms and therapeutic implications

T-2 toxin is one of the most toxic and common trichothecene mycotoxins, and can cause various cardiovascular diseases. In this review, we summarized the current knowledge-base and challenges as it relates to T-2 toxin related cardiotoxicity. The molecular mechanisms and potential treatment approaches were also discussed. Pathologically, T-2 toxin-induced cardiac toxicity is characterized by cell injury and death in cardiomyocyte, increased capillary permeability, necrosis of cardiomyocyte, hemorrhage, and the infiltration of inflammatory cells in the heart. T-2 toxin exposure can cause cardiac fibrosis and finally lead to cardiac dysfunction. Mechanistically, T-2 toxin exposure-induced cardiac damage involves the production of ROS, mitochondrial dysfunction, peroxisome proliferator-activated receptor-gamma (PPAR-γ) signaling pathway, endoplasmic reticulum (ER stress), transforming growth factor beta 1 (TGF-β1)/smad family member 2/3 (Smad2/3) signaling pathway, and autophagy and inflammatory responses. Antioxidant supplementation (e.g., catalase, vitamin C, and selenium), induction of autophagy (e.g., rapamycin), blockade of inflammatory signaling (e.g., methylprednisolone) or treatment with PPAR-γ agonists (e.g., pioglitazone) may provide protective effects against these detrimental cardiac effects caused by T-2 toxin. We believe that our review provides new insights in understanding T-2 toxin exposure-induced cardiotoxicity and fuels effective prevention and treatment strategies against this important food-borne toxin-induced health problems.

Скрининг штаммов грибов Trichoderma pers. для защиты растений от Albifimbria verrucaria, ранее известного как Myrothecium verrucaria

The phytopathogen Albifimbria verrucaria (Myrothecium) produces macrocyclic trichothecene mycotoxins. When developed on animal feed, it can cause poisoning. The research aims to select strains of Trichoderma fungi that are promising for plant protection against the pathogen Alb. verrucaria. The antagonistic activity of 21 Trichoderma cultures against Alb. verrucaria was studied using the double-culture method. On day 10, three isolates of Trichoderma sp. 3K, 7T and 12T inhibited the pathogen by 67-71%. Furthermore, in seven variants of the double cultures, inhibition of Trichoderma fungi by the pathogen with the formation of sterile zones with a radius of 2-10 mm was observed. By day 15 of double-culture incubation, pathogen accretion was noted on some Trichoderma cultures, including 3K and 12T. As a result of the microscopy of crop connection zones and growth zones, three isolates, namely 1K, 4T and 13N, were noted as promising for plant protection against Alb. verrucaria.

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.

Nivalenol affects Cyclin B1 level and activates SAC for cell cycle progression in mouse oocyte meiosis.

ObjectivesNivalenol (NIV) is a secondary metabolite of type B trichothecene mycotoxin produced by Fusarium genera, which is widely found in contaminated food and crops such as corn, wheat and peanuts. NIV is reported to have hepatotoxicity, immunotoxicity, genotoxicity, and reproductive toxicity. Previous studies indicate that NIV disturbs mammalian oocyte maturation. Here, we reported that delayed cell cycle progression might be the reason for oocyte maturation defect caused by NIV exposure.Methods and ResultsWe set up a NIV exposure model and showed that NIV did not affect G2/M transition for meiosis resumption, but disrupted the polar body extrusion of oocytes. Further analysis revealed that oocytes were arrested at metaphase I, which might be due to the lower expression of Cyclin B1 after NIV exposure. After cold treatment, the microtubules were disassembled in the NIV‐exposed oocytes, indicating that NIV disrupted microtubule stability. Moreover, NIV affected the attachment between kinetochore and microtubules, which further induced the activation of MAD2/BUBR1 at the kinetochores, suggesting that spindle assemble checkpoint (SAC) was continuously activated during oocyte meiotic maturation.ConclusionsTaken together, our study demonstrated that exposure to NIV affected Cyclin B1 expression and activated microtubule stability‐dependent SAC to ultimately disturb cell cycle progression in mouse oocyte meiosis.

Draft Genome Sequences of Prototrophic and Biotin-Auxotrophic Fusarium langsethiae Strains Isolated from an Oat Grain in the Northern Region of Russia

ABSTRACTFusarium langsethiae is a suspected plant-pathogenic fungus causing cereal contamination with trichothecene mycotoxins. Here, we report the genome sequences of two F. langsethiae strains, MFG217701 (a prototroph) and MFG217702 (a biotin auxotroph), isolated from a grain of oat harvested in Russia.

Computational Strategy for Minimizing Mycotoxins in Cereal Crops: Assessment of the Biological Activity of Compounds Resulting from Virtual Screening.

Cereal crops are frequently affected by toxigenic Fusarium species, among which the most common and worrying in Europe are Fusarium graminearum and Fusarium culmorum. These species are the causal agents of grain contamination with type B trichothecene (TCTB) mycotoxins. To help reduce the use of synthetic fungicides while guaranteeing low mycotoxin levels, there is an urgent need to develop new, efficient and environmentally-friendly plant protection solutions. Previously, F. graminearum proteins that could serve as putative targets to block the fungal spread and toxin production were identified and a virtual screening undertaken. Here, two selected compounds, M1 and M2, predicted, respectively, as the top compounds acting on the trichodiene synthase, a key enzyme of TCTB biosynthesis, and the 24-sterol-C-methyltransferase, a protein involved in ergosterol biosynthesis, were submitted for biological tests. Corroborating in silico predictions, M1 was shown to significantly inhibit TCTB yield by a panel of strains. Results were less obvious with M2 that induced only a slight reduction in fungal biomass. To go further, seven M1 analogs were assessed, which allowed evidencing of the physicochemical properties crucial for the anti-mycotoxin activity. Altogether, our results provide the first evidence of the promising potential of computational approaches to discover new anti-mycotoxin solutions

Study of the toxic effect of trichothecene mycotoxin produced by <i>Fusarium sporotrichioides</i> in an experiment on pigs

Aim . To assess the consequences of long‐term consumption by pigs of feed experimentally contaminated with T‐2 toxin in an amount close to the most commonly found in feed. Material and Methods . The experiments were carried out on pigs, which were fed food contaminated with T‐2 toxin in the amount of 200 mkg/kg for 45 days. During the experiment, the clinical condition of animals, feed and water consumption, the dynamics of live weight, analysis of blood parameters, the state of internal organs and tissues and statistical processing of the data obtained by conventional methods were undertaken. Results . Long‐term consumption of feed contaminated with mycotoxin T‐2 by pigs did not cause a pronounced clinical picture of toxicosis. However, the treated animals had diarrhea and lag in growth and development. According to the results of the blood test, it was concluded that the toxin suppressed the hematopoiesis process, inhibited protein synthesis, reduced resistance and microphagocytic function of the body, metabolic disorders and the functioning of most organs and systems. Pathological conditions and degenerative changes of cells and tissues of internal organs were recorded and mediated by the initiation of oxidative stress, inflammatory processes and cytotoxic effect. Conclusions . The consumption of feed contaminated with T‐2 toxin in a dose of 200 mkg /kg by pigs was accompanied by disturbances of metabolic processes, functions of organs and systems, manifested by a decrease in growth intensity, changes in the blood picture, activity of enzyme systems, induction of oxidative stress, immunosuppressive and inflammatory reactions and pathomorphological disorders. Thus, the oral intake of T‐2 toxin, even in small concentrations, was found to have a complex negative effect on the body.

Fusarium Mycotoxins in Maize Field Soils: Method Validation and Implications for Sampling Strategy.

While mycotoxins are generally regarded as food contamination issues, there is growing interest in mycotoxins as environmental pollutants. The main sources of trichothecene and zearalenone mycotoxins in the environment are mainly attributed to Fusarium infested fields, where mycotoxins can wash off in infested plants or harvest residues. Subsequently, mycotoxins inevitably enter the soil. In this context, investigations into the effects, fate, and transport are still needed. However, there is a lack of analytical methods used to determine Fusarium toxins in soil matrices. We aimed to validate an analytical method capable of determining the toxins nivalenol (NIV), deoxynivalenol (DON), 15-acetyl-deoxynivalenol (15-AcDON), and zearalenone (ZEN), at environmentally relevant concentrations, in five contrasting agricultural soils. Soils were spiked at three levels (3, 9 and 15 ng g−1), extracted by solid-liquid extraction assisted with ultrasonication, using a generic solvent composition of acetonitrile:water 84:16 (v:v) and measured by LC–HRMS. Method validation was successful for NIV, DON, and 15-AcDON with mean recoveries > 93% and RSDr < 10%. ZEN failed the validation criteria. The validated method was applied to eight conventionally managed maize field soils during harvest season, to provide a first insight into DON, NIV, and 15-AcDON levels. Mycotoxins were present in two out of eight sampled maize fields. Soil mycotoxin concentrations ranged from 0.53 to 19.4 ng g−1 and 0.8 to 2.2 ng g−1 for DON and NIV, respectively. Additionally, we found indication that “hot-spot” concentrations were restricted to small scales (<5 cm) with implications for field scale soil monitoring strategies.

Simultaneous determination of trichothecene mycotoxins in cereals by LC-MS/MS.

This study was designed to determine the residual trichothecene mycotoxins in cereal samples. The optimal solvent for extraction was 84% (v/v) aqueous acetonitrile with 1% (v/v) formic acid. The best performing clean-up method was dispersive-solid phase with a mixture octadecyl silica and primary-secondary amine. The recoveries for the studied mycotoxins ranged from 83.3 to 92.8%. The methodology was successfully applied for monitoring 100 cereal samples obtained from a Korean market. The bean sample were found to be co-contamination with deoxynivalenol and HT-2 toxin. Deoxynivalenol possessed the highest detection freauency (4/100) and amount (727.38µg/kg) among the trichothecene mycotoxins. The hazard index was less than 1.0 for all the observed mycotoxins in all cereal samples except one white rice sample (1.2681). This results indicated that periodic risk assessments of trichothecene mycotoxin through cereal intake are necessary for the health and safety.

Production of type-B trichothecenes by Fusarium meridionale, F. graminearum, and F. austroamericanum in wheat plants and rice medium.

Food security goes beyond food being available; the food needs to be free of contaminants. Trichothecenes mycotoxins, produced by Fusarium fungus, are. among the most frequently found contaminants of wheat. In this study, we evaluated the production of trichothecenes Deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), 15-acetyldeoxynivalenol (15-AcDON), and nivalenol (NIV) by Fusarium meridionale, F. austroamericanum, and F. graminearum grown in wheat plants and rice medium. Fusarim meridionale was efficient only in the production of NIV (production range (pr) from 1340 to 2864µgkg-1 in wheat plant), and F. austroamericanum in the production of 3-AcDON (pr from 50 to 192µgkg-1 in wheat plant, and from 986 to 7045µgkg-1 in rice medium) and DON (pr from 4076 to 13,701µgkg-1 in wheat plant, and from 184 to 43,395µgkg-1 in rice medium). Already, F. graminearum was efficient in the production of 3-AcDON only in rice medium (pr from 81 to 2342µgkg-1), 15-AcDON in wheat plant (pr from 80 to 295µgkg-1) and in rice medium (pr from 436 to 8597µgkg-1), and DON also in wheat plant (pr from 7746 to 12,046µgkg-1) and in rice medium (pr from 695 to 49,624µgkg-1). The specificity of F. meridionale in the production of NIV but not the production of DON could generate a food security problem in regions where this species occurs and the amounts of NIV in grains and derivatives are not regulated in the food chain, as in Brazil.

Transcriptome analysis reveals infection strategies employed by Fusarium graminearum as a root pathogen

The fungal pathogen Fusarium graminearum (Fg) infects both heads and roots of cereal crops causing several economically important diseases such as head blight, seedling blight, crown rot and root rot. Trichothecene mycotoxins such as deoxynivalenol (DON), a well-known virulence factor, produced by Fg during disease development is also an important health concern. Although how Fg infects above-ground tissues is relatively well studied, very little is known about molecular processes employed by the pathogen during below-ground infection. Also unknown is the role of DON during root infection. In the present study, we analyzed the transcriptome of Fg during root infection of the model cereal Brachypodium distachyon (Bd). We also compared our Fg transcriptome data obtained during Bd root infection with those reported during wheat head infection. These analyses suggested that both shared and unique infection strategies were employed by the pathogen during colonization of different host tissues. Several metabolite biosynthesis genes induced in Fg during root infection could be linked to phytohormone production, implying that the pathogen likely interferes with root specific defenses. In addition, to understand the role of DON in Fg root infection, we analyzed the transcriptome of the DON deficient Tri5 mutant. These analyses showed that the absence of DON had a significant effect on fungal transcriptional responses. Although DON was produced in infected roots, this mycotoxin did not act as a Fg virulence factor during root infection. Our results reveal new mechanistic insights into the below-ground strategies employed by Fg that may benefit the development of new genetic tools to combat this important cereal pathogen.

T-2 Toxin Induces Ferroptosis by Increasing Lipid Reactive Oxygen Species (ROS) and Downregulating Solute Carrier Family 7 Member 11 (SLC7A11).

T-2 toxin is a trichothecene mycotoxin commonly found in animal feed and agricultural products. Evidence indicates that T-2 toxin induces apoptosis and autophagy. This study investigated the role of ferroptosis in T-2 toxin cytotoxicity. RAS-selective lethal compound 3 (RSL3) and Erastin were applied to initiate ferroptosis. RSL3- and Erastin-initiated cell death were enhanced by T-2 toxin. Treatment with the ferroptosis inhibitor ferrostatin-1 markedly restored the sensitizing effect of T-2 toxin to RSL3- or Erastin-initiated apoptosis, suggesting that ferroptosis plays a vital role in T-2 toxin-induced cytotoxicity. Mechanistically, T-2 toxin promoted ferroptosis by inducing lipid reactive oxygen species (ROS), as N-acetyl-l-cysteine significantly blocked T-2 toxin-induced ferroptosis. Moreover, T-2 toxin decreased the expression of solute carrier family 7 member 11 (SLC7A11) and failed to further enhance ferroptosis in SLC7A11-deficient cells. SLC7A11 overexpression significantly rescued the enhanced ferroptosis caused by T-2 toxin. T-2 toxin induces ferroptosis by downregulating SLC7A11 expression. Ferroptosis mediates T-2 toxin-induced cytotoxicity by increasing ROS and downregulating SLC7A11 expression.

GPTransformer: A Transformer-Based Deep Learning Method for Predicting Fusarium Related Traits in Barley.

Fusarium head blight (FHB) incited by Fusarium graminearum Schwabe is a devastating disease of barley and other cereal crops worldwide. Fusarium head blight is associated with trichothecene mycotoxins such as deoxynivalenol (DON), which contaminates grains, making them unfit for malting or animal feed industries. While genetically resistant cultivars offer the best economic and environmentally responsible means to mitigate disease, parent lines with adequate resistance are limited in barley. Resistance breeding based upon quantitative genetic gains has been slow to date, due to intensive labor requirements of disease nurseries. The production of a high-throughput genome-wide molecular marker assembly for barley permits use in development of genomic prediction models for traits of economic importance to this crop. A diverse panel consisting of 400 two-row spring barley lines was assembled to focus on Canadian barley breeding programs. The panel was evaluated for FHB and DON content in three environments and over 2 years. Moreover, it was genotyped using an Illumina Infinium High-Throughput Screening (HTS) iSelect custom beadchip array of single nucleotide polymorphic molecular markers (50 K SNP), where over 23 K molecular markers were polymorphic. Genomic prediction has been demonstrated to successfully reduce FHB and DON content in cereals using various statistical models. Herein, we have studied an alternative method based on machine learning and compare it with a statistical approach. The bi-allelic SNPs represented pairs of alleles and were encoded in two ways: as categorical (–1, 0, 1) or using Hardy-Weinberg probability frequencies. This was followed by selecting essential genomic markers for phenotype prediction. Subsequently, a Transformer-based deep learning algorithm was applied to predict FHB and DON. Apart from the Transformer method, a Residual Fully Connected Neural Network (RFCNN) was also applied. Pearson correlation coefficients were calculated to compare true vs. predicted outputs. Models which included all markers generally showed marginal improvement in prediction. Hardy-Weinberg encoding generally improved correlation for FHB (6.9%) and DON (9.6%) for the Transformer network. This study suggests the potential of the Transformer based method as an alternative to the popular BLUP model for genomic prediction of complex traits such as FHB or DON, having performed equally or better than existing machine learning and statistical methods.

Multi-mycotoxins and Fungal Contaminants of Wheat from Anambra State, Nigeria

This study was carried out to determine the fungal and mycotoxins contamination of 36 Wheat (Triticum aestivium) samples purchased randomly from the seller of the agricultural produce in local markets of Anambra State, Nigeria. Results from the studies showed that two hundred and three fungal isolates consisting of 18 species of moulds and 5 species of yeasts contaminated the wheat samples at varying degrees. For moulds, Aspergillus species contaminated the samples mostly with (28) isolates followed by Penicillum species (19) isolates while Verticillium species and Cladosporium species had equal least contaminations with (3) isolates each. Among the yeast species, Candida rugosahad the highest number of contamination with (37) isolates followed by Cryptococcus laurentii (31) isolates while Candida stellatoides (9) isolates had the least contamination. Twenty-four fungal metabolites were also recovered. The concentration of trichothecene mycotoxin Deoxynivalenol (2067µg/kg), a protein synthesis and cell proliferation inhibitor in animals exceeded the maximum acceptable limits for human consumption. It can be deduced therefore that wheat circulating in Anambra State, Nigeria are variously contaminated with different xerophilic moulds and mycotoxins which can exert adverse health problems to consumers.&#x0D; Keyword: Wheat samples, fungal contaminants, multi-mycotoxins and market zones

A Novel Deoxynivalenol-Activated Wheat Arl6ip4 Gene Encodes an Antifungal Peptide with Deoxynivalenol Affinity and Protects Plants against Fusarium Pathogens and Mycotoxins.

Deoxynivalenol (DON) is one of the most widespread trichothecene mycotoxins in contaminated cereal products. DON plays a vital role in the pathogenesis of Fusarium graminearum, but the molecular mechanisms of DON underlying Fusarium–wheat interactions are not yet well understood. In this study, a novel wheat ADP-ribosylation factor-like protein 6-interacting protein 4 gene, TaArl6ip4, was identified from DON-treated wheat suspension cells by suppression subtractive hybridization (SSH). The qRT-PCR result suggested that TaArl6ip4 expression is specifically activated by DON in both the Fusarium intermediate susceptible wheat cultivar Zhengmai9023 and the Fusarium resistant cultivar Sumai3. The transient expression results of the TaARL6IP4::GFP fusion protein indicate that TaArl6ip4 encodes a plasma membrane and nucleus-localized protein. Multiple sequence alignment using microscale thermophoresis showed that TaARL6IP4 comprises a conserved DON binding motif, 67HXXXG71, and exhibits DON affinity with a dissociation constant (KD) of 91 ± 2.6 µM. Moreover, TaARL6IP4 exhibited antifungal activity with IC50 values of 22 ± 1.5 µM and 25 ± 2.6 µM against Fusarium graminearum and Alternaria alternata, respectively. Furthermore, TaArl6ip4 interacted with the plasma membrane of Fusarium graminearum spores, resulting in membrane disruption and the leakage of cytoplasmic materials. The heterologous over-expression of TaArl6ip4 conferred greater DON tolerance and Fusarium resistance in Arabidopsis. Finally, we describe a novel DON-induced wheat gene, TaArl6ip4, exhibiting antifungal function and DON affinity that may play a key role in Fusarium–wheat interactions.

First Report of Maize Stalk Rot Caused by Fusarium kyushuense in China.

During 2017 to 2019, a field survey for maize stalk rot was conducted in 21 counties (districts) across the Guangxi province of China. This disease caused yield losses ranging from 20% to 30%. Maize plants with stalk rot were collected during the late milk stage and pieces of diseased pith tissue were cultured as previously described (Shan et al. 2017). Fungal colonies and mycelia with morphological characteristics of Fusarium species were subcultured onto fresh potato dextrose agar (PDA) and carnation leaf agar (CLA) plates. Based on morphological characteristics and molecular detection by amplification of Fusarium genus-specific primers (Duan et al. 2016), 39 Fusarium isolates were identified. Among them, five isolates from Du'an, Pingguo, Debao, and Daxin had abundant, pale orange to yellow aerial mycelium with deep red pigments when grown on PDA (Fig. 1A; 1B). The average growth rate was 8.0 to 12.0 mm per day at 25°C in the dark. The fungi produced two types of spores on CLA. Microconidia were ovoid to clavate, generally 0- to 3-septate, and 4.6 to 9.4 μm in length (n = 30) (Fig. 1D); Macroconidia were slightly curved with an acute apical cell, mostly 3- to 4- septate, and 19.4 to 38.2 μm in length (n = 30) (Fig. 1C). No chlamydospores were observed. These five isolates were initially identified as Fusarium kyushuense based on morphological features. PCR was performed to amplify three phylogenetic genes (TEF1-α, RPB1, and RPB2) (O'Donnell et al. 1998) and species specific primers kyuR1F/kyuR1R (5-TTTTCCTCACCAAGGAGCAGATCATG-3/5-TCCAATGGACTGGGCAGCCAAAACACC-3), kyuR2F/kyuR2R (5-CAGATATACATTTGCCTCGACAC-3/5-TACTTGAGCACGGAGCTTG-3) were used to confirm species identity. The obtained sequences were deposited in GenBank under the accession numbers MT997084, MT997080, MT997081 (TEF1-α); MT550012, MT997085, MT997086 (RPB1); MT550009, MT997089, and MT997090 (RPB2), respectively. Using BLAST, sequences of TEF1-α, RPB1, and RPB2 of the isolates were 99.33% (MH582297.1) to 100% (MG282364.1) similar to those of F. kyushuense strains (Supplementary Table 1). Based on phylogenetic analysis with maximum likelihood methods using tools of the website of CIPRES (http://www.phylo.org), isolates GX27, GX167, and GX204 clustered with F. kyushuense with 100% bootstrap support (Fig. 2). The pathogenicity of the three isolates was tested using young seedlings and adult plants as previously described with modification (Ye et al. 2013; Zhang et al. 2016). The primary roots of three-leaf-old seedlings were inoculated by immersing the roots into a 1 × 106 macroconidia solution, incubating for 6 h at 25°C, and transferring to normal growth conditions (26°C, 16 h light/22°C, 8 h dark). The second or third internode above the soil surface of flowering stage plants grown in a greenhouse was bored with a Bosch electric drill to make a hole (ca. 8 mm in diameter) and inoculated with 0.5 mL of mycelia plug then sealed with petrolatum. The inoculum was created by homogenizing five plates of flourish hyphal mats (approximately 125 mL) with kitchen blender and adjusting to a final volume of 200 mL with sterilized ddH2O. No symptoms were observed in the seedlings or adult plants that were mock-inoculated with PDA plugs. Three days post-inoculation (dpi), roots of the infected seedling turned dark-brown and shrunk and the leaves wilted (Fig. 1E). Typical stalk rot symptoms observed in the inoculated plants were premature wilting of entire plant and hollow and weak stalks, leading to lodging; the longitudinal section of the internodes exhibited obvious dark brown necrosis and reddish discoloration at 14 dpi and 30 dpi, respectively (Fig. 1F). Fusarium kyushuense was re-isolated from the inoculated stalk lesions but not from the control. This is the first record of stalk rot caused by F. kyushuense on maize plants in China. However, F. kyushuense is known to cause maize ear rot in China (Wang et al. 2014) and can produce type A and type B trichothecene mycotoxins in kernels (Aoki and O'Donnell 1998). The occurrence of maize stalk rot and ear rot caused by F. kyushuense should be monitored in China due to the potential risk for crop loss and mycotoxin contamination.

Evaluating the effects of cellulolytic enzymes and Lactobacillus bulgaricus on mycotoxins production and the quality of maize silage

Fungal spoilage and mycotoxin contamination are two of the greatest hazards of silage. The present work was carried out to evaluate the impact of Lactobacillus bulgaricus and cellulolytic enzymes on the maize silage (MS) quality. Fungal analysis of different MS samples showed different mycotoxigenic fungi. The highest frequency (62.8%) was associated with Fusarium spp. Four species with different relative densities were found: F. graminearum (71.1%), F. culmorum (15.2%), F. proliferatum (11.2%), and F. oxysporum (2.50 %). High-performance liquid chromatography analysis showed the presence of trichothecene, nivalenol, zearalenone, and fumonisins mycotoxins in MS inoculated by F. graminearum. The inhibition % of trichothecene, nivalenol, and zearalenone synthesis was 50.2%, 47.5%, and 23.5%, respectively, in MS inoculated by Lactobacillus bulgaricus after a 30 d incubation period. Trichoderma harzianum succeeded in producing cellulolytic enzymes, i.e., carboxymethyl cellulase, manganase peroxidase, and laccase, with a maximum production of 350 µg/mL, 5.47 µg/mL, and 16.0 µg/mL, respectively, after 21 d using MS as the substrate. Treatment by the extracted cellulolytic enzyme with L. bulgaricus enhanced unfavorable conditions for MS fungal contamination, i.e., the production of lactic acid, a lowered pH, and increased L. bulgaricus colony-forming units, compared to the addition of enzyme extract or L. bulgaricus alone.

Effects of Double-Stranded RNAs Targeting Fusarium graminearum TRI6 on Fusarium Head Blight and Mycotoxins.

Fusarium graminearum is the causal agent of Fusarium head blight (FHB), which reduces crop yield and contaminates grains with poisonous trichothecene mycotoxins, including deoxynivalenol (DON). DON functions as an important virulence factor that promotes FHB spread in wheat; therefore, reducing DON production will decrease yield losses to FHB and increase food safety. Recent progress in the topical application of double-stranded RNA (dsRNA) to reduce F. graminearum infection has provided encouraging results. In this study, we designed and synthesized dsRNA targeting the transcription factor TRI6 (TRI6-dsRNA), which is a key regulator of DON biosynthesis. The expression of F. graminearum TRI6 was significantly lower in detached wheat heads treated with TRI6-dsRNA solution compared with the controls. Furthermore, TRI6-dsRNA treatments reduced disease and DON accumulation in inoculated detached wheat heads. Therefore, topical applications of TRI6-dsRNA on wheat heads of intact plants were assessed for their ability to reduce FHB and DON under growth chamber and greenhouse conditions. When wheat heads were treated with TRI6-dsRNA solution in growth chamber conditions, TRI6-dsRNA treatments failed to prevent FHB spread. However, when wheat heads were treated with TRI6-dsRNA solution under greenhouse conditions, FHB and DON were significantly reduced, and infection was restricted to the inoculated floret. In addition, addition of TRI6-dsRNA to toxin induction liquid media had no effect on F. graminearum 15-ADON production. Our study demonstrates that the efficacy of dsRNA applications is strongly dependent on application methods and environmental conditions.