Containment of Fusarium culmorum and Its Mycotoxins in Various Biological Systems by Antagonistic Trichoderma and Clonostachys Strains.

Prevention of fungal diseases caused by Fusarium species, including F. culmorum, and thus the accumulation of mycotoxins in wheat ears, is a constant challenge focused on the development of new, effective crop management solutions. One of the currently most ecologically attractive approaches is biological control using natural antagonistic microorganisms. With this in mind, the antagonistic potential of thirty-three Clonostachys and Trichoderma strains was assessed in this work. Screening tests were carried out in in vitro cultures, and the observed potential of selected Trichoderma and Clonostachys strains was verified in field and semi-field experiments with two forms of wheat: winter cv. Legenda and spring cv. Bombona. Three strains, namely C. rosea AN291, T. atroviride AN240 and T. viride AN430 were reported to be most effective in inhibiting the growth of F. culmorum KF846 and the synthesis of DON, 3AcDON and ZEN under both laboratory and semi-controlled field conditions. Observations of the contact zones of the tested fungi in dual cultures exposed their mycoparasitic abilities against KF846. In addition, studies on liquid cultures have demonstrated the ability of these strains to eliminate F. culmorum toxins. Meanwhile, the strains of T. atroviride AN35 and T. cremeum AN392 used as soil inoculants in the field experiment showed a different effect on the content of toxins in ears (grains and chaffs), while improved wheat yield parameters, mainly grain health in both wheat cultivars. It is concluded that the selected Trichoderma and Clonostachys strains have a high potential to reduce the adverse effects of F. culmorum ear infection; therefore, they can be further considered in the context of potential biocontrol factors and as wheat crop improvers.

Prevention of Aflatoxin Occurrence Using Nuts-Edible Coating of Ginger Oil Nanoemulsions and Investigate the Molecular Docking Strategy.

The modern utilization of essential oils such as ginger oil (GO) as an anti-aflatoxin represents a potential target for food preservation and safety; however, the mechanism of action is still unclear. Nanoemulsions, through an edible coating, can enhance the oil's bioactivity, increase its hydrophilicity, and extend the final product's shelf-life. In the present study, two edible films for the GO nanoemulsion were prepared by ultrasonication using carboxymethyl cellulose (FB1-GO) and sodium alginate (FB2-GO). The droplet size of FB2-GO was finer (126.54 nm) compared to FB1-GO (289.77 nm). Meanwhile, both had high stability proved by z-potential; +31.54 mV (FB1-GO) and +46.25 mV (FB2-GO) with low PDI values (<0.4). Using gas chromatography-mass spectrometry, the hydrodistilled GO showed 25 compounds, representing 99.17% of the total oil, with α-zingiberene (29.8%), geranial (10.87%), ß-bisabolene (8.19%), and ar-curcumene (5.96%) as the predominant. A dramatic increase in α-zingiberene, α-bisabolene and ar-curcumene was due to the homogenization conditions in both FB1-GO and FB2-GO compared to the GO. The FB1-GO exhibited superior antibacterial activity against the examined strains of bacterial pathogens, while FB2-GO was more effective as an antifungal agent on the tested Aspergillus fungi strains. In a simulated liquid media, FB2-GO inhibited the total growth of fungi by 84.87−92.51% and showed the highest reduction in the aflatoxin amount produced. The in silico study presented that, among the GO volatile constituents, sesquiterpenes had the highest binding free energies against the enzymes responsible for aflatoxin production compared to monoterpenes. α-Bisabolene showed the highest affinity toward polyketide synthase (−7.5 Kcal/mol), while ar-curcumene was the most potent against cytochrome P450 monooxygenase (−8.3 Kcal/mol). The above findings clarify the reasons for aflatoxin reduction in simulated media during incubation with FB1-GO and FB2-GO.

A Comprehensive Study of Lupin Seed Oils and the Roasting Effect on Their Chemical and Biological Activity.

The present investigation aimed to study the impact of roasting on the chemical composition and biological activities of sweet and bitter lupin seed oils. Lupin oils were extracted using petroleum ether (40-60) with ultrasonic assisted method. Lupin Fatty acids, phytosterols, carotenoids, and total phenolic contents were determined. In addition, antioxidant, antimicrobial, and antifungal activities were evaluated. The results showed a ratio between 7.50% to 9.28% of oil content in lupin seed. Unroasted (bitter and sweet) lupin oil contained a high level of oleic acid ω9 (42.65 and 50.87%), followed by linoleic acid ω6 (37.3 and 34.48%) and linolenic acid ω3 (3.35 and 6.58%), respectively. Concerning phytosterols, unroasted (bitter and sweet lupin) seed oil reflected high values (442.59 and 406.18 mg/100 g oil, respectively). Bitter lupin oil contains a high amount of phenolics, although a lower antioxidant potency compared to sweet lupin oil. This phenomenon could be connected with the synergistic effect between phenolics and carotenoids higher in sweet lupin oil. The results reflected a more efficiently bitter lupin oil against anti-toxigenic fungi than sweet lupin oil. The roasting process recorded enhances the antimicrobial activity of bitter and sweet lupin seed oil, which is linked to the increment in bioactive components during the roasting process. These results concluded that lupin oil deems a novel functional ingredient and a valuable dietary fat source. Moreover, lupin oil seemed to have antifungal properties, which recommended its utilization as a carrier for active-antifungal compounds in food products.

Bioactive Molecules of Mandarin Seed Oils Diminish Mycotoxin and the Existence of Fungi.

Mandarin is a favorite fruit of the citrus family. Mandarin seeds are considered a source of nontraditional oil obtained from byproduct materials. This investigation aimed to assess the biomolecules of mandarin seeds and evaluated their antimycotic and antimycotoxigenic impact on fungi. Moreover, it evaluated the protective role of mandarin oil against aflatoxin toxicity in cell lines. The two types of extracted oil (fixed and volatile) were ecofriendly. The fatty acid composition, tocopherol, sterols, and carotenoids were determined in the fixed oil, whereas volatiles and phenolics were estimated in the essential oil. A mixture of the two oils was prepared and evaluated for its antimicrobial impact. The reduction effect of this mixture was also investigated to reduce mycotoxin secretion using a simulated experiment. The protective effect of the oil was evaluated using healthy strains of cell lines. Fixed oil was distinguished by the omega fatty acid content (76.24%), lutein was the major carotenoid (504.3 mg/100 g) and it had a high ß-sitosterol content (294.6 mg/100 g). Essential oil contained limonene (66.05%), α-pinene (6.82%), ß-pinene (4.32%), and γ-terpinene (12.31%) in significant amounts, while gallic acid and catechol were recorded as the dominant phenolics. Evaluation of the oil mix for antimicrobial potency reflected a considerable impact against pathogenic bacteria and toxigenic fungi. By its application to the fungal media, this oil mix possessed a capacity for reducing mycotoxin secretion. The oil mix was also shown to have a low cytotoxic effect against healthy strains of cell lines and had potency in reducing the mortality impact of aflatoxin B1 applied to cell lines. These results recommend further study to involve this oil in food safety applications.

Expression Patterns of miR398, miR167, and miR159 in the Interaction between Bread Wheat (Triticum aestivum L.) and Pathogenic Fusarium culmorum and Beneficial Trichoderma Fungi.

Bread wheat (Triticum aestivum L.) is an agronomically significant cereal cultivated worldwide. Wheat breeding is limited by numerous abiotic and biotic stresses. One of the most deleterious factors is biotic stress provoked by the Fusarium culmorum fungus. This pathogen is a causative agent of Fusarium root rot and Fusarium head blight. Beneficial fungi Trichoderma atroviride and T. cremeum are strong antagonists of mycotoxigenic Fusarium spp. These fungi promote plant growth and enhance their tolerance of negative environmental conditions. The aim of the study was to determine and compare the spatial (in above- and underground organs) and temporal (early: 6 and 22 hpi; and late: 5 and 7 dpi reactions) expression profiles of three mature miRNAs (miR398, miR167, and miR159) in wheat plants inoculated with two strains of F. culmorum (KF846 and EW49). Moreover, the spatial expression patterns in wheat response between plants inoculated with beneficial T. atroviride (AN35) and T. cremeum (AN392) were assessed. Understanding the sophisticated role of miRNAs in wheat-fungal interactions may initiate a discussion concerning the use of this knowledge to protect wheat plants from the harmful effects of fungal pathogens. With the use of droplet digital PCR (ddPCR), the absolute quantification of the selected miRNAs in the tested material was carried out. The differential accumulation of miR398, miR167, and miR159 in the studied groups was observed. The abundance of all analyzed miRNAs in the roots demonstrated an increase in the early and reduction in late wheat response to F. culmorum inoculation, suggesting the role of these particles in the initial wheat reaction to the studied fungal pathogen. The diverse expression patterns of the studied miRNAs between Trichoderma-inoculated or F. culmorum-inoculated plants and control wheat, as well as between Trichoderma-inoculated and F. culmorum-inoculated plants, were noticed, indicating the need for further analysis.

The Presence of Mycotoxins in Human Amniotic Fluid.

Mycotoxin exposure assessments through biomonitoring studies, based on the analysis of amniotic fluid, provides useful information about potential exposure of mothers and fetuses to ubiquitous toxic metabolites that are routinely found in food and the environment. In this study, amniotic fluid samples (n = 86) were collected via abdominal amniocentesis at 15-22 weeks of gestation from pregnant women with a high risk of chromosomal anomalies or genetic fetal defects detected during 1st trimester prenatal screening. These samples were analyzed for the presence of the most typical Aspergillus, Penicillium and Fusarium mycotoxins, with a focus on aflatoxins, ochratoxins and trichothecenes, using the LC-FLD/DAD method. The results showed that the toxin was present in over 75% of all the tested samples and in 73% of amniotic fluid samples from fetuses with genetic defects. The most frequently identified toxins were nivalenol (33.7%) ranging from

Sarocladium and Lecanicillium Associated with Maize Seeds and Their Potential to Form Selected Secondary Metabolites.

The occurrence and diversity of Lecanicillium and Sarocladium in maize seeds and their role in this cereal are poorly understood. Therefore, the present study aimed to investigate Sarocladium and Lecanicillium communities found in endosphere of maize seeds collected from fields in Poland and their potential to form selected bioactive substances. The sequencing of the internally transcribed spacer regions 1 (ITS 1) and 2 (ITS2) and the large-subunit (LSU, 28S) of the rRNA gene cluster resulted in the identification of 17 Sarocladium zeae strains, three Sarocladium strictum and five Lecanicillium lecanii isolates. The assay on solid substrate showed that S. zeae and S. strictum can synthesize bassianolide, vertilecanin A, vertilecanin A methyl ester, 2-decenedioic acid and 10-hydroxy-8-decenoic acid. This is also the first study revealing the ability of these two species to produce beauvericin and enniatin B1, respectively. Moreover, for the first time in the present investigation, pyrrocidine A and/or B have been annotated as metabolites of S. strictum and L. lecanii. The production of toxic, insecticidal and antibacterial compounds in cultures of S. strictum, S. zeae and L. lecanii suggests the requirement to revise the approach to study the biological role of fungi inhabiting maize seeds.

Occurrence of Mycotoxigenic Fusarium Species and Competitive Fungi on Preharvest Maize Ear Rot in Poland.

Maize has become one of the most important crops for food and feed production-both as a silage and crop residue worldwide. The present study aimed to identify the co-occurrence of Fusarium subglutinans, Fusarium verticillioides, Trichoderma atroviride, Sarocladium zeae, and Lecanicillium lecanii on maize ear rot. Further, the accumulation of mycotoxins as secondary metabolites of Fusarium spp. in maize ear samples was also analyzed. Maize ear samples were collected between 2014 and 2017 from two main maize growing areas in Poland (Greater Poland and Silesia region). A significant difference was found in the frequency of two main Fusarium spp. that infect maize ears, namely F. subglutinans and F. verticillioides. In addition to Fusarium spp. T. atroviride, S. zeae, and L. lecanii were also identified. T. atroviride species was found in 14% of maize samples examined between 2014 and 2017, particularly with a high percentage of Trichoderma spp. recorded in 2014, i.e., in 31% of samples. However, mycotoxin content (beauvericin and fumonisins) varied, depending on both the location and year of sampling. The interaction of fungi and insects inhabiting maize ear and kernel is very complex and not yet elucidated. Therefore, further research is required in this area.

Suppressive Effect of Trichoderma spp. on toxigenic Fusarium species.

The aim of the present study was to examine the abilities of twenty-four isolates belonging to ten different Trichoderma species (i.e., Trichoderma atroviride, Trichoderma citrinoviride, Trichoderma cremeum, Trichoderma hamatum, Trichoderma harzianum, Trichoderma koningiopsis, Trichoderma longibrachiatum, Trichoderma longipile, Trichoderma viride and Trichoderma viridescens) to inhibit the mycelial growth and mycotoxin production by five Fusarium strains (i.e., Fusarium avenaceum, Fusarium cerealis, Fusarium culmorum, Fusarium graminearum and Fusarium temperatum). Dual-culture bioassay on potato dextrose agar (PDA) medium clearly documented that all of the Trichoderma strains used in the study were capable of influencing the mycelial growth of at least four of all five Fusarium species on the fourth day after co-inoculation, when there was the first apparent physical contact between antagonist and pathogen. The qualitative evaluation of the interaction between the colonies after 14 days of co-culturing on PDA medium showed that ten Trichoderma strains completely overgrew and sporulated on the colony at least one of the tested Fusarium species. Whereas, the microscopic assay provided evidence that only T. atroviride AN240 and T. viride AN255 formed dense coils around the hyphae of the pathogen from where penetration took place. Of all screened Trichoderma strains, T. atroviride AN240 was also found to be the most efficient (69-100% toxin reduction) suppressors of mycotoxins (deoxynivalenol, 3-acetyl-deoxynivalenol, 15-acetyl-deoxynivalenol, nivalenol, zearalenone, beauvericin, moniliformin) production by all five Fusarium species on solid substrates. This research suggests that T. atroviride AN240 can be a promising candidate for the biological control of toxigenic Fusarium species.

The role of wastewater treatment in reducing pollution of surface waters with zearalenone.

Zearalenone (ZEA) is a mycotoxin produced by some Fusarium species in food and feed. The toxicity of ZEA and its metabolites is related to the chemical structure of the mycotoxin, which is similar to naturally occurring oestrogens. Currently, there is increasing awareness of the presence of fungi and their toxic metabolites in the aquatic environment. One of the sources of these compounds are the effluents from wastewater treatment plants. The average annual efficiency of zearalenone reduction in the Leczyca plant in our three-year study was in the range from 51.35 to 69.70 %. The threeway analysis of variance (year, month, and kind of wastewater) shows that the main effects of all factors and all interactions between them were significant for zearalenone and dissolved organic carbon content. Our findings suggest that wastewater is not the main source of surface water pollution with zearalenone. Future research should investigate the means to reduce ZEA and its migration from the fields through prevention strategies such as breeding for crops, plant debris management (crop rotation, tillage), and/or chemical and biological control.

Zearalenone lactonohydrolase activity in Hypocreales and its evolutionary relationships within the epoxide hydrolase subset of a/b-hydrolases.

BACKGROUND: Zearalenone is a mycotoxin produced by several species of Fusarium genus, most notably Fusarium graminearum and Fusarium culmorum. This resorcylic acid lactone is one of the most important toxins causing serious animal and human diseases. For over two decades it has been known that the mycoparasitic fungus Clonostachys rosea (synonym: Gliocladium roseum, teleomorph: Bionectria ochroleuca) can detoxify zearalenone, however no such attributes have been described within the Trichoderma genus. RESULTS: We screened for the presence of zearalenone lactonohydrolase homologs in isolates of Clonostachys and Trichoderma genera. We report first finding of expressed zearalenone lactonohydrolase in Trichoderma aggressivum. For three isolates (T. aggressivum, C. rosea and Clonostachys catenulatum isolates), we were able to reconstruct full coding sequence and verify the biotransformation ability potential. Additionally, we assessed progression of the detoxification process (in terms of transcript accumulation and mycotoxin decomposition in vitro).In silico, search for origins of zearalenone lactonohydrolase activity in model fungal and bacterial genomes has shown that zearalenone lactonohydrolase homologs form a monophyletic fungal clade among the a/b hydrolase superfamily representatives. We corroborated the finding of functional enzyme homologs by investigating the functional sites (active site pocket with postulated, noncanonical Ser-Glu-His catalytic triad) conserved in both multiple sequence alignment and in homology-based structural models. CONCLUSIONS: Our research shows the first finding of a functional zearalenone lactonohydrolase in mycoparasitic Trichoderma aggressivum (an activity earlier characterised in the Clonostachys rosea strains). The supporting evidence for presence and activity of functional enzyme homologs is based on the chemical analyses, gene expression patterns, homology models showing conservation of key structural features and marked reduction of zearalenone content in cultured samples (containing both medium and mycelium). Our findings also show divergent strategies of zearalenone biotransformation ability (rapid induced expression and detoxification vs. gradual detoxification) present in several members of Hypocreales order (Trichoderma and Clonostachys genera). The potential for lactonhydrolase activity directed towards zearalenone and/or similar compounds is likely ancient, with homologs present in several divergent filamentous fungi among both Sordariomycetes (Bionectria sp., Trichoderma sp., Apiospora montagnei) and Leotiomycetes (Marssonina brunnea f. sp. 'multigermtubi').

Polymorphism of mycotoxin biosynthetic genes among Fusarium equiseti isolates from Italy and Poland.

Fusarium equiseti (Corda) Saccardo is a soil saprophyte and a weak pathogen, associated with several diseases of fruit and other crops in subtropical and tropical areas, but also in countries with temperate climate. A wide range of secondary metabolites has been identified among natural F. equiseti populations, with zearalenone (ZEA), fusarochromanone and fusarenon-X being the most common. In present study, the genetic diversity of strains from two populations (from Italy and Poland) was evaluated by analysing the translation elongation factor 1α (tef-1α) sequences, two polyketide synthases from the ZEA biosynthetic pathway (PKS13 and PKS4) and the TRI5 gene from the trichothecene biosynthetic pathway. ZEA was produced in rice cultures by 20 of the 27 tested isolates in concentrations ranging from 1.34 ng/g to 34,000 ng/g). The ability to produce enniatins and trichothecenes was evaluated in all strains by identifying esyn1, TRI13 and TRI4 genes. The presence of PKS4 and PKS13 genes was confirmed by polymerase chain reaction (PCR) in only some ZEA-producing isolates. Similarly, the TRI5 gene was found in 14 of the 27 isolates tested. This is likely to have been caused by the divergence of those genes between F. equiseti and F. graminearum (the latter species was used for the primers design) and can be exploited in phylogenetic studies. The analysis of the mycotoxin biosynthetic gene sequences can be used to differentiate the studied genotypes even more precisely than the analysis of the non-coding regions (like tef-1α).

Zearalenone contamination of the aquatic environment as a result of its presence in crops.

The aim of this study was to establish a relation between zearalenone contamination of crops in the Polish province of Wielkopolska and its occurrence in aquatic ecosystems close by the crop fields. Water samples were collected from water bodies such as drainage ditches, wells, or watercourses located in four agricultural areas. Moreover, control water samples were collected from the Bogdanka river, which was located outside the agricultural areas and near an urban area. Cereal samples were collected in the harvest season from each agricultural area close to tested water bodies. Zearalenone (ZEA) was found in all water and cereal samples. The highest concentrations were recorded in the postharvest season (September to October) and the lowest in the winter and spring. Mean ZEA concentrations in water ranged between 1.0 ng L(-1) and 80.6 ng L(-1), and in cereals from 3.72 ng g(-1) to 28.97 ng g(-1). Our results confirm that mycotoxins are transported to aquatic systems by rain water through soil.

Occurrence of estrogenic mycotoxin - Zearalenone in aqueous environmental samples with various NOM content.

Zearalenone is a mycotoxin produced by some Fusarium species in food and feed. Toxicity of ZEA and its metabolites is related to the chemical structure of the mycotoxins, similar to naturally occurring estrogens. Currently, there is increasing awareness of the significance of fungi and their toxic metabolites in water. Thus it was considered essential to determine the concentration of estrogenic toxin in surface waters, groundwater and wastewater in Poland. The application of ZearalaTest immunoaffinity columns for zearalenone determination by HPLC method in water samples characterized by different natural organic matter (NOM) content gave the recovery rate ranging from 74 to 86%. Obtained results showed that the natural organic micropollutants of water with low molecular masses have an effect on zearalenone recovery. Zearalenone was present in water samples in the range from 0 to 43.7 ngL(-1).