Tri5 Gene Research Articles

Trehalose-6-phosphate phosphatase inhibitor: N-(phenylthio) phthalimide, which can inhibit the DON biosynthesis of Fusarium graminearum

Fusarium head blight(FHB)caused by Fusarium graminearum species complex (FGSC) is one of the most important diseases around the world. Deoxynivalenol (DON) is a type of mycotoxin produced by FGSC when infecting cereal crops. It is a serious threat to the health of both humans and livestock. Trehalose-6-phosphate phosphatase (TPP), a conserved metabolic enzyme found in many plants and pathogens, catalyzes the formation of trehalose. N-(phenylthio) phthalimide (NPP) has been reported to inhibit the normal growth of nematodes by inhibiting the activity of TPP, but this inhibitor of nematodes has not previously been tested against F. graminearum. In this study, we found that TPP in F. graminearum (FgTPP) had similar secondary structures and conserved cysteine (Cys356) to nematodes by means of bioinformatics. At the same time, the sensitivity of F. graminearum strains to NPP was determined. NPP exhibited a better inhibitory effect on conidia germination than mycelial growth. In addition, the effects of NPP on DON biosynthesis and trehalose biosynthesis pathway in PH-1 were also determined. We found that NPP decreased DON production, trehalose content, glucose content and TPP enzyme activity but increased trehalose-6-phosphate content and trehalose-6-phosphate synthase (TPS) enzyme activity. Moreover, the expression of TRI1, TRI4, TRI5, TRI6, and TPP genes were downregulated, on the contrary, the TPS gene was upregulated. Finally, in order to further determine the control ability of NPP on DON production in the field, we conducted a series of field experiments, and found that NPP could effectively reduce the DON content in wheat grain and had a general control effect on FHB. In conclusion, the research in this study will provide important theoretical basis for controlling FHB caused by F. graminearum and reducing DON production in the field.

Antifungal Activity of Quinofumelin against Fusarium graminearum and Its Inhibitory Effect on DON Biosynthesis.

Fusarium graminearum, causal agent of Fusarium head blight (FHB), causes a huge economic loss. No information is available on the activity of quinofumelin, a novel quinoline fungicide, against F. graminearum or other phytopathogens. In this study, we used mycelial growth and spore germination inhibition methods to determine the inhibitory effect of quinofumelin against F. graminearum in vitro. The results indicated that quinofumelin excellently inhibited mycelial growth and spore germination of F. graminearum, with the average EC50 values of 0.019 ± 0.007 μg/mL and 0.087 ± 0.024 μg/mL, respectively. In addition, we found that quinofumelin could significantly decrease deoxynivalenol (DON) production and inhibit the expression of DON-related gene TRI5 in F. graminearum. Furthermore, we found that quinofumelin could disrupt the formation of Fusarium toxisome, a structure for producing DON. Western blot analysis demonstrated that the translation level of TRI1, a marker gene for Fusarium toxisome, was suppressed by quinofumelin. The protective and curative assays indicated that quinofumelin had an excellent control efficiency against F. graminearum on wheat coleoptiles. Taken together, quinofumelin exhibits not only an excellent antifungal activity on mycelial growth and spore germination, but also could inhibit DON biosynthesis in F. graminearum. The findings provide a novel candidate for controlling FHB caused by F. graminearum.

The Applicability of Species- and Trichothecene-Specific Primers in Monitoring the Fusarium graminearum Species Complex and Its Impact on the Surveillance of Fusarium Head Blight in Winter Wheat in Serbia

Monitoring changes in the prevalence of Fusarium species and toxin production is an important tool for the integrated control of Fusarium head blight (FHB). However, methods for the high-throughput screening of Fusarium populations have been developed using isolates with limited geographic origins. In this study, we used species- and trichothecene-specific primers to monitor the F. graminearum species complex (FGSC) originating from Serbia. We also tested the applicability of the primers to the surveillance of FHB. We analyzed two hundred and ten isolates collected from thirty two locations and five winter wheat varieties over a three-year period. Using multiple correspondence analysis (MCA), we investigated associations between Fusarium-damaged kernels (FDK) and location, variety, members of the FGSC, and their predisposition for mycotoxin production. The results revealed that the species-specific primers were not specific for 11% of the F. graminearum population. The primer sets were 98.5%, 95.2%, and 92.4% effective in the multilocus genotyping of Tri7, Tri3, and Tri5 genes, respectively. We found that individual wheat varieties were associated with isolates that could not be characterized using species- and trichothecene-specific primers. Alternaria spp. had a significant influence (p < 0.001) on grain infection with F. graminearum, indicating the necessity to further investigate its impact on the pathogenesis of the F. graminearum clade.

Antifungal activity of myriocin against Fusarium graminearum and its inhibitory effect on deoxynivalenol production in wheat grains

Fusarium graminearum, a devastating pathogenic fungus of wheat (Triticum aestivum L.), causes fusarium head blight (FHB) and produces mycotoxins such as deoxynivalenol (DON). This study aimed to explore the potency of myriocin to control postharvest infection of F. graminearum in wheat grains. Myriocin showed excellent antifungal activity against F. graminearum, as evidenced through inhibition on spore germination and mycelial growth. The mechanism of action was investigated by analyzing changes of cell membrane permeability and serine palmitoyl transferase (SPT) activity. Results indicated that myriocin inhibited F. graminearum by disrupting permeability of membrane and result in release of cellular contents. Myriocin suppressed the SPT activity and molecular docking also proved the sC interaction of myriocin-SPT. Significant morphological alterations were observed in SEM imaging, including holes on the hyphae, contraction and disruption of mycelia. In addition, myriocin also showed inhibitory effect on toxigenic activity of F. graminearum in wheat grains by reducing more than 99.0% of deoxynivalenol (DON) and its derivative 15-acetyldeoxynivalenol (15-ADON) synthesis. Quantitative real-time PCR analysis indicated that TRI5 gene expression was downregulated by myriocin and results in the reduction of toxicity. Overall, myriocin exerted effective fungicidal activity to control F. graminearum contamination in wheat grains and provided a lead compound to develop more alternatives for pathogen control.

Bacillus velezensis RC 218 as a biocontrol agent against Fusarium graminearum: effect on penetration, growth and TRI5 expression in wheat spikes

Fusarium head blight (FHB) is one of the main fungal diseases affecting bread (Triticum aestivum L.) and durum wheat (Triticum durum L.) crops worldwide, resulting not only in important economic losses due to reduced grain yield and quality, but also in grain safety due to mycotoxins contamination, mainly deoxynivalenol (DON). TRI5 gene catalyzes the first step in DON biosynthesis and its expression is used as an indicator for trichothecene synthesis induction. In Argentina, FHB is caused by Fusarium graminearum sensu stricto when humid weather and warm temperature conditions predominate around flowering. Commonly used strategies to manage FHB are not highly efficient if individually applied and under this scenario. Biological control appears as a viable approach than can be included in an integrated FHB management. Previous studies carried out by our research group proved the biocontrol efficacy of Bacillus velezensis RC 218 in reducing FHB in bread and durum wheat, under greenhouse and field conditions. In this study, we analyzed F. graminearum sensu stricto penetration, growth and TRI5 gene expression when B. velezensis RC 218 was applied on wheat spikes. Spike tissue sections were obtained and observed under different microscopic techniques. We observed that the biocontrol agent (BCA) was able to reduce F. graminearum infection and trichothecene accumulation in bread wheat susceptible cultivar BioInta1005. Data on tissue analysis suggest B. velezensis RC 218 induced cell wall thickening in the host tissues immediately surrounding the F. graminearum infection site, preventing cell plasmolysis and collapse.

Naturally Occurring Phenols Modulate Vegetative Growth and Deoxynivalenol Biosynthesis in Fusarium graminearum.

To assess the in vitro activity of five naturally occurring phenolic compounds (ferulic acid, apocynin, magnolol, honokiol, and thymol) on mycelial growth and type B trichothecene mycotoxin accumulation by Fusarium graminearum, three complementary approaches were adopted. First, a high-throughput photometric continuous reading array allowed a parallel quantification of F. graminearum hyphal growth and reporter TRI5 gene expression directly on solid medium. Second, RT-qPCR confirmed the regulation of TRI5 expression by the tested compounds. Third, liquid chromatography–tandem mass spectrometry analysis allowed quantification of deoxynivalenol (DON) and its acetylated forms released upon treatment with the phenolic compounds. Altogether, the results confirmed the activity of thymol and an equimolar mixture of thymol–magnolol at 0.5 mM, respectively, in inhibiting DON production without affecting vegetative growth. The medium pH buffering capacity after 72–96 h of incubation is proposed as a further element to highlight compounds displaying trichothecene inhibitory capacity with no significant fungicidal effect.

Commercially available natural inhibitors of trichothecene production in Fusarium graminearum: A strategy to manage Fusarium head blight of wheat

Fusarium Head Blight (FHB) is one of the most important diseases affecting wheat production. The disease causes yield and quality losses and hinders commercialization because of frequent grain contamination with trichothecenes, sesquiterpene mycotoxins that are harmful to human and animals and act as pathogenicity factors for FHB. Although not always effective, application of fungicides is the most common method for FHB management; thus, there is an urgent need to develop new efficient and sustainable tools. We analyzed the inhibitory activity of a group of commercially available naturally occurring compounds on the production of trichothecenes and on the severity of FHB on field-grown wheat spikes inoculated with Fusarium graminearum. The gene TRI5 codifies trichodiene synthase, the enzyme that catalyzes the synthesis of trichodiene, a volatile intermediate to trichothecenes. Our first aim was to obtain a trichothecene non-producing mutant of the pathogen by gene disruption of this gene. Seventeen putative ΔTRI5 mutants produced in the laboratory were characterized for pathogenicity and trichothecene production. The target gene was correctly and efficiently replaced in more than 75% of the transformants obtained, indicating a high efficacy of the approach used for gene disruption. Selected commercially available natural inhibitors -eugenol, apocynin, caffeic acid and propyl gallate-, were used to test their effect on trichodiene production in vitro and in field-grown wheat spikes inoculated with F. graminearum, using a ΔTRI5 mutant as non-trichodiene production control. Fungal volatile organic compounds production was measured by gas chromatography coupled to mass spectrometry. Caffeic acid, apocynin and eugenol produced a significant reduction of trichodiene production in vitro. In field assays, sesquiterpene inhibition was also significant at 2 days post inoculation (dpi), showing that effective inhibition of the trichothecene precursor can be achieved through the action of commercially available naturally occurring compounds. However, at later stages (>7 dpi) the production of trichodiene was similar to that of the F. graminearum-inoculated control. At 21 dpi, FHB severity did not differ between the treatments with inhibitors and the inoculated control. Hence, further experiments are required to identify the most appropriate inhibitors concentrations and formulations to improve their bioavailability and to achieve long lasting protection against F. graminearum infections.

Quinone outside inhibitors affect DON biosynthesis, mitochondrial structure and toxisome formation in Fusarium graminearum

Quinone outside inhibitors (QoIs) are currently extensively used agricultural fungicides. However, the application of QoIs in controlling Fusarium graminearum was rarely reported. No information is available on pharmacological characteristics of QoIs against F. graminearum, as well as their effects on DON biosynthesis. Here, we found that six QoIs exhibited an excellent fungicidal activity against F. graminearum based on mycelial growth and spore germination. ATP production assay further confirmed that QoIs decreased ATP production via inhibiting mitochondrial respiration, which contributes their fungicidal activity. Unfortunately, QoIs can stimulate DON production and up-regulate the expression of Tri5 and Tri6 genes. Additionally, acetyl-CoA, the basic precursor of DON biosynthesis, significantly increased as affected by QoIs, furtherly indicating that QoIs indeed enhance DON biosynthesis. We also found that QoIs can accelerate the formation of toxisomes and enhance the fluorescence signals of Tri-GFP labeled toxisomes, which may be due to the effect of QoIs on toxisome-related endoplasmic reticulum-remodeling. In addition, QoIs could disrupt the homeostasis of mitochondrial dynamics, resulting in the fragmented mitochondria. Finally, the simulated inoculation assay with wheat grains further verified that QoIs can stimulate DON production relative to wheat grain weight, especially relative to mycelial biomass.

Tri5 gene expression analysis during postharvest storage of wheat grain from field plots treated with a triazole and a strobilurin fungicide

Fusarium head blight (FHB) and the associated mycotoxin deoxynivalenol (DON) negatively impact the wheat industry worldwide. In North America, FHB is mainly caused by Fusarium graminearum sensu stricto. The purpose of this study was to evaluate, under storage conditions, the expression of the DON biosynthetic gene trichodiene synthase (Tri5) of F. graminearum in grain of hard red winter wheat cultivars ‘Overley’ (FHB-susceptible) and ‘Overland’ (moderately resistant to FHB) from field plots treated or untreated with the triazole fungicide Prosaro and the strobilurin fungicide Headline. Infected grain was stored and periodically sampled to determine gene expression by qRT-PCR analysis. The F. graminearum housekeeping gene GAPDH was consistently detected, indicative of metabolically active fungi, and Tri5 detection was significantly higher in ‘Overley’ compared with ‘Overland’. Tri5 gene expression and DON concentrations showed little to no correlation; consequently, Tri5 expression levels did not accurately predict DON concentrations. The strobilurin did not significantly reduce Tri5 gene expression compared with untreated wheat. In the triazole treatment, a significant reduction in the relative expression of Tri5 was detected after 120 days, as well as a downregulation of Tri5 from 60 to 120 days of storage in ‘Overley’. In grain from strobilurin-treated plots of both cultivars, the expression of Tri5 increased from 0 to 30 days after tempering. Genetic expression of Tri5 that is necessary for the production of DON can increase during storage of high-moisture grain. Fusarium fungi can persist in wheat kernels for several months postharvest and may actively produce toxin during this period.

Synthesis of Trichodermin Derivatives and Their Antimicrobial and Cytotoxic Activities.

Trichothecene mycotoxins are recognized as highly bioactive compounds that can be used in the design of new useful bioactive molecules. In Trichoderma brevicompactum, the first specific step in trichothecene biosynthesis is carried out by a terpene cyclase, trichodiene synthase, that catalyzes the conversion of farnesyl diphosphate to trichodiene and is encoded by the tri5 gene. Overexpression of tri5 resulted in increased levels of trichodermin, a trichothecene-type toxin, which is a valuable tool in preparing new molecules with a trichothecene skeleton. In this work, we developed the hemisynthesis of trichodermin and trichodermol derivatives in order to evaluate their antimicrobial and cytotoxic activities and to study the chemo-modulation of their bioactivity. Some derivatives with a short chain at the C-4 position displayed selective antimicrobial activity against Candida albicans and they showed MIC values similar to those displayed by trichodermin. It is important to highlight the cytotoxic selectivity observed for compounds 9, 13, and 15, which presented average IC50 values of 2 μg/mL and were cytotoxic against tumorigenic cell line MCF-7 (breast carcinoma) and not against Fa2N4 (non-tumoral immortalized human hepatocytes).

Diversity of trichothecene genotypes of Fusarium graminearum sensu stricto from winter wheat in Serbia

Data related to genetic diversity of F. graminearum chemotypes in Serbia are limited, thus the main objective of this study was to analyze the divergence of genes associated with the trichothecene cluster. F. graminearum was shown to be the dominant Fusarium species in Serbia occurring with frequency of 65.6% in 2016/2017 growing season. Isolates of F. graminearum dominantly belonged to SCAR type 1 (104/105) producing PCR product of 420 bp, but one isolate was proven to be SCAR type 6 producing PCR product of 400 bp. This is the first report of the presence of SCAR type 6 in Southeastern Europe. Results from this study reveal that F. graminearum populations were shown to be DON-DON chemotype with respect to Tri7 and Tri13 genes. All but one of these isolates were 15-AcDON chemotypes based on Tri3 gene. One isolate carried a deletion of Tri7 and was found to be 3-AcDON by using Tri303F/R primer pair. This is the first record on the presence of a 3-AcDON isolate with the Tri7 deletion in Serbia. The Tri7F/Tri7DON primer pair was not effective if isolates carried more than four 11-bp repeats in the Tri7 gene. Primer pair GzTri7f1/r1 was proven to be more effective in comparison with Tri7F/Tri7DON. Genetic variation ranged from 57 to 80% with no significant association of Tri7 alleles with localities. Results of this study have brought new insights into the distribution and population structure of F. graminearum in Serbia.

Impact of Five Succinate Dehydrogenase Inhibitors on DON Biosynthesis of Fusarium asiaticum, Causing Fusarium Head Blight in Wheat.

Deoxynivalenol (DON) is a class of mycotoxin produced in cereal crops infected with Fusarium graminearum species complex (FGSC). In China, FGSC mainly includes Fusarium asiaticum and F. graminearum. DON belongs to the trichothecenes and poses a serious threat to the safety and health of humans and animals. Succinate dehydrogenase inhibitors (SDHIs) are a class of fungicides that act on succinate dehydrogenase and inhibit the respiration of pathogenic fungi. In this study, the fungicidal activities of five SDHIs, including fluopyram, flutolanil, boscalid, benzovindiflupyr, and fluxapyroxad, against FGSC were determined based on mycelial growth and spore germination inhibition methods. The five SDHIs exhibited better inhibitory activities in spore germination than mycelial growth. Fluopyram exhibited a higher inhibitory effect in mycelial growth and spore germination in comparison to the other four SDHIs. In addition, the biological characteristics of F. asiaticum as affected by the five SDHIs were determined. We found that these five SDHIs decreased DON, pyruvic acid and acetyl-CoA production, isocitrate dehydrogenase mitochondrial (ICDHm) and SDH activities, and NADH and ATP content of F. asiaticum but increased the citric acid content. In addition, TRI5 gene expression was inhibited, and the formation of toxisomes was disrupted by the five SDHIs, further confirming that SDHIs can decrease DON biosynthesis of F. asiaticum. Thus, we concluded that SDHIs may decrease DON biosynthesis of F. asiaticum by inhibiting glycolysis and the tricarboxylic acid (TCA) cycle. Overall, the findings from the study will provide important references for managing Fusarium head blight (FHB) caused by FGSC and reducing DON contamination in F. asiaticum-infected wheat grains.

Interacting climate change environmental factors effects on Fusarium langsethiae growth, expression of Tri genes and T-2/HT-2 mycotoxin production on oat-based media and in stored oats

This study examined the effect of climate change (CC) abiotic factors of temperature (20, 25, 30 °C), water activity (aw; 0.995, 0.98) and CO2 exposure (400, 1000 ppm) may have on (a) growth, (b) gene expression of biosynthetic toxin genes (Tri5, Tri6, Tri16), and (c) T-2/HT-2 toxins and associated metabolites by Fusarium langsethiae on oat-based media and in stored oats. Lag phases and growth were optimum at 25 °C with freely available water. This was significantly reduced at 30 °C, at 0.98 aw and 1000 ppm CO2 exposure. In oat-based media and stored oats, Tri5 gene expression was reduced in all conditions except 30 °C, 0.98 aw, elevated CO2 where there was a significant (5.3-fold) increase. The Tri6 and Tri16 genes were upregulated, especially in elevated CO2 conditions. Toxin production was higher at 25 °C than 30 °C. In stored oats, at 0.98 aw, elevated CO2 led to a significant increase (73-fold) increase in T2/HT-2 toxin, especially at 30 °C. Nine T-2 and HT-2 related metabolites were detected, including a new dehydro T-2 toxin (which correlated with T-2 production) and the conjugate, HT-2 toxin, glucuronide. This shows that CC factors may have a significant impact on growth and mycotoxin production by F. langsethiae.

SiRNA quelling of tri4 and tri5 genes related to deoxynivalenol synthesis in Fusarium graminearum and Fusarium culmorum

siRNA quelling of tri4 and tri5 genes related to deoxynivalenol synthesis in Fusarium graminearum and Fusarium culmorum

Prevalence of Fusarium fungi and their toxins in marketed feed

This study aimed to explore the incidence of Fusarium fungi and their mycotoxins in marketed feed in Qatar. Samples of mixed-grain-cereal, maize, wheat, millet, barley, oat and soybean were tested for the presence of zearalenone (ZEN), fumonisins, T2/HT2 and deoxynevalenol (DON) mycotoxins. PCR-based identification of the Fusarium isolates was followed by investigation into genes involved in trichothecenes (tri gene cluster), fumonisins (fum gene cluster) and ZEN (pks gene) biosynthesis. In-vitro mycotoxin production potential of the isolates was compared with their molecular profiles. Mixed-grain cereals showed highest contamination with Fusarium, followed by maize and wheat. The incidence of ZEN was highest in mixed grain samples (87.5%) followed by maize (50%), millet (42.85%) and wheat (40%). In case of DON; 62.5%, 30%, 40%, 14.28%, 50% and 66.67% of the tested mixed cereal grain, maize, wheat, millet, barley and soybean samples were positive. Based on PCR results, F. verticillioides showed highest frequency distribution (34%), followed by F. graminearum (16%), F. oxysporum (15%), F. proliferatum (13%), F. culmorum (8%), F. solani (7%), F. subglutinans (4%) and F. avenaceum (3%) in the feed cereals. All the isolates of F. verticillioides presented both fum1 and fum13 genes, and produced fumonisin mycotoxins in-vitro. All the isolates of F. graminearum and F. culmorum had the tri5 and tri6 genes and resulted in DON accumulation in rice media. In addition to DON, all the F. culmorum isolates produced ZEN in artificially contaminated media, in line with their molecular profile of having tri5, tri6 and pks13 genes. The findings of present study confirmed the occurrence of toxigenic Fusarium and their mycotoxins in animal feed, all below the maximum limit set by EU. Further, the in-vitro mycotoxin production potential of the isolates was in agreement with their tri, fum and pks genetic profiles.

Investigation of Camphor Effects on Fusarium graminearum and F. culmorum at Different Molecular Levels.

Fusarium graminearum and F. culmorum are phytopathogens, which cause destructive diseases in cereals. Epidemics of these phytopathogens are caused by mycotoxin contamination and the reduction of crop quality. In this study, the alteration due to in vitro camphor treatment on F. culmorum 9F and F. graminearum H11 isolates was investigated in terms of epigenetic, cellular, and transcription levels. Camphor with different concentrations (0.2, 0.4, 0.8, 1, 2, and 4 µg/µL) was applied to potato dextrose agar (PDA) growth media. The minimum inhibitory concentration (MIC) and the half maximal inhibitory concentration (IC50) were calculated as 2 and 1 µg/µL, respectively. hog1, mst20, CAT, POD, mgv1, stuA, and tri5 genes, which are related to various cellular processes and pathogenesis, were examined by qPCR assay. qPCR analysis showed that camphor treatment leads to the downregulation of tri5 expression but the upregulation of the remaining genes. Apoptosis and oxidative stress were confirmed via acridine orange/ethidium bromide (AO/EB) and dichlorofluorescin diacetate (DCF-DA) staining, respectively. Moreover, coupled restriction enzyme digestion-random amplification (CRED-RA) assay, used for DNA methylation analysis, was carried out to evaluate epigenetic alterations. The decrease in genomic template stability (GTS) values, which resulted due to the alterations in random amplified polymorphic DNA (RAPD) profiles caused by camphor treatment, were detected as 97.60% in F. culmorum 9F and 66.27% in F. graminearum H-11. The outer and inner methylated cytosine profiles are determined by CRED-RA assay as type I–IV epigenetic alterations. The outcomes indicated that camphor could lead to alterations at several molecular levels of F. graminearum and F. culmorum.

Inhibition of Fusarium trichothecene biosynthesis by yeast extract components extractable with ethyl acetate

While Fusarium graminearum readily produces trichothecenes in complex media containing sucrose as the carbon source (YS_60), the amount of the mycotoxin is quite limited when other sugars, such as glucose and fructose, are used. We found that autoclaving of media containing fructose and yeast extract (YF_60) results in the formation of inhibitors of trichothecene biosynthesis by F. graminearum JCM 9873, a strain that produces 15-acetyldeoxynivalenol (15-ADON) in liquid culture. Removal of the solvent fraction from the autoclaved media after ethyl acetate extraction attenuated the inhibitory activity against trichothecene production. In addition, extraction of the non-autoclaved complex media with ethyl acetate, followed by removal of the solvent fraction, similarly resulted in increased accumulation of the mycotoxin. Although the increase in trichothecene production differed considerably among fungal strains and yeast extract products, F. graminearum species complex generally responded to the medium treatments in the same way. These results suggest that some hydrophobic substances that arise during the drying and heating of yeast extract negatively affected trichothecene production in liquid culture. Modes of actions of inhibitory substances were partially characterized using strain JCM 9873, with focus on the transcriptional and functional analyses of Tri6, a key regulator gene in trichothecene biosynthesis. The presence of the ethyl acetate-extractable substances in autoclaved YF_60 media decreased the relative transcription level of Tri6, as well as that of a trichodiene synthase gene Tri5. Thus, the substances exerted their inhibitory action through suppression of Tri6 expression. By using a yeast extract lot that completely prevented trichothecene production by the wild-type strain in autoclaved YS_60 medium, we prepared YF_60 media and cultured a constitutive Tri6 overexpressor strain described by Maeda et al. (2018). Despite the high transcription level of Tri6, the presence of the ethyl acetate extractable-substances suppressed 15-ADON production. These results suggested that both Tri6p-independent initial activation of Tri6 expression and subsequent Tri6p-dependent activation of Tri expression were affected by the hydrophobic substances in the yeast extract products.

Thiophanate methyl susceptibility and alterations in tri5, Mgv1 and StuA expression among Fusarium graminearum and F. culmorum isolates

Fusarium graminearum and F. culmorum are phytopathogenic species that cause head blight and crown rot diseases in cereals worldwide. These two phytopathogenic fungi show great variation in terms of phenotypic, biochemical and genetic characteristics. In this study, linear growth rate, thiophanate methyl (TPM) resistance, survival and toxin production- associated gene expressions, and beta-tubulin nucleotide sequence knowledge of F. graminearum from Turkey and Iran and F. culmorum isolates from Turkey were investigated. The mean linear growth rate values of isolates ranged from 7.58 to 15.16 mm/day. In TPM resistance analysis, each isolate was characterized as highly sensitive to TPM, with IC50 values between 2 and 4 μg/ml TPM. TPM-subjected isolates showed decreased tri5 gene expression and increased Mgv and StuA expression. Amino acid conservations at codons 198 and 200 in the beta tubulin gene were also investigated in order to confirm TPM resistance and reveal genetic diversity in Fusarium species. Each isolate showed conserved amino acids at 198 and 200 codons, resulting in sensitive genotypes. The bootstrap support value for genetic similarity was more than 70%. Findings obtained from this study showed that Fusarium isolates that cause head blight and crown rot in Turkey have a low level of variation in terms of phenotype and genetic characteristics. Moreover, this is the first report to show that TPM led to a potential decrease in toxin production-associated genes and increased potential of sexual/asexual reproduction genes. Results could provide improved and comprehensive data for the struggle against Fusarium pathogens.

Relationship between environmental conditions,TRI5 gene expression and deoxynivalenol production in storedLentinula edodes infected withFusarium graminearum

This study made the first attempt to relate the production of deoxynivalenol (DON) to the expression ofTRI5 gene inFusarium graminearum as a function of interacting environmental factors (water activity (aw) (0.95-0.98), temperature (20-30 °C) and incubation time (7 day-28 day)), so as to investigate its production mechanisms inLentinula edodes. Changes in temperature, water activity and incubation time could significantly (P<0.01) affect DON production andTRI5 gene expression. The highest DON concentration (793.5±27.4 μg/kg) andTRI5 gene expression (2−ΔΔCt=38.8±4.8) were observed when the cultures were incubated at 20 °C and 0.98 aw for 21 days. Multi-regression analysis was performed and nonlinear models based on polynomial equations were established to uncover the individual effects of temperature, water activity and incubation time as well as their interactions on DON production andTRI5 gene expression. The established model was further used to develop contour maps to predict the DON production andTRI5 gene expression in relation to storage conditions inL. edodes. Highly significant positive correlation between DON production and foldTRI5 gene expression (R=0.5534,P<0.0001) was observed in this commodity. The production mechanisms of DON inL. edodes revealed in the present study will be beneficial for developing more effective targeted control strategies.

Real time PCR: a good tool to estimate mycotoxin contamination in pig diets

Cereals and soybean are the main components of pig diets. Unfortunately, feed materials are often contaminated with fungi and their metabolites, which pose a potential threat to human and animal health. Therefore, this study was undertaken to evaluate the effectiveness of cultural methods and quantitative PCR for detecting fungi and their metabolites in pig diets, and to determine which plant components are responsible for mycotoxin contamination of feed. The presence of mycotoxin-producing fungi of the generaFusarium,Penicillium andAspergillus and their metabolites was determined in pig diets with different inclusion levels of various cereals and transgenic soybean meal. Six farm-made complete diets containing locally produced feed materials and imported soybean meal were investigated. The presence of the following fungi in pig diets was determined by microscopic observations of fungal cultures and by qPCR: trichothecene-producingFusarium spp. (Tri5 gene),Penicillium verrucosum (rRNA) andAspergillus ochraceus (PKS gene). The concentrations of mycotoxins (ochratoxin A (OTA) and zearalenone (ZEA)), trichothecenes (deoxynivalenol (DON), 3-acetyl-deoxynivalenol and T-2 toxin (T-2)) were analysed by HPLC. The results of the qPCR analysis demonstrated that the presence of DNA of mycotoxin-producing fungi and mycotoxins in pig diets was correlated with the inclusion levels of transgenic soybean meal and various cereals. The above correlation was validated by an analysis of Spearman’s rank correlation between the content of transgenic soybean meal and various cereals vs mycotoxin concentrations and the amount of DNA of toxin-producing fungi in pig diets. A significant positive correlation was found between: the percentage content of soybeans vs the concentrations of DON (R=0.93), trichothecenes (R=0.76) and T-2 (R=0.64), the percentage content of barley vs the concentrations of DON (R=0.50) and T-2 (R=0.49), the percentage content of triticale vs OTA levels (R=0.47), the percentage content of oats vs ZEA levels (0.50). A correlation was also noted between the percentage content of soybeans and the amount of DNA of trichothecene-producingFusarium spp. (R=0.96). The results of this study indicate that pig diets are significantly contaminated with toxin-producing fungi and their metabolites, and that the quantification of DNA of mycotoxin-producing fungi is a reliable indicator of mycotoxin contamination of feed. Our findings can contribute to reducing the costs of analyses that should be routinely performed to minimise the entry of mycotoxins into the food chain.