A result of the European Project MycoKey is the development and the production at pilot scale of innovative feed additives to decontaminate mycotoxins contaminated feeds and to prevent toxic effects in animals. Lesaffre International (France), specialized in fermentation, developing, producing and marketing special active ingredients to control mycotoxins, collaborated with the Institute of Sciences of Food Production (CNR-ISPA) to develop this yeast-based additive. The new feed additive adsorbed in vitro a large spectrum of mycotoxins with high capacity and affinity, and reduced mycotoxin absorption in animals. The efficacy of the additive in sequestering mycotoxins was confirmed by short-term toxicokinetic studies with lab animals (rats) and piglets, using the biomarker approach. Scaling-up at industrial level for commercial production of this feed additive is ongoing. It can be concluded that dietary supplementation of mycotoxin-contaminated feeds with the new additive can reduce the effects of exposure to a cocktail of mycotoxins, preventing the carry-over of toxic metabolites in animal products.

An alternative and straightforward indirect sampling method for mycotoxin analysis of grain lots has been evaluated against current sampling praxis and sampling according to Commission Regulation (EC) 401/2006. The approach, based on the Eurofins rapidust® system, foresees sampling of dust, which can easily be withdrawn from grain lots by suction during (un)loading of shipments or within grain processing e.g. before grain cleaning after storage. Without time consuming grinding or sample preparation steps, the dust can rapidly be analysed, followed by recalculation of the grain mycotoxin contamination via data conversion by data models.
The trials on wheat and maize performed at industrial level within the MycoKey project demonstrated that the dust sampling was suitable for controls during (un)loading of big bags, trucks, and train waggons, as well as within in-process control of grains after silo storage. In addition, the variability within the dust samples was much smaller as the variability within the results after sampling according to Commission Regulation (EC) 401/2006.
Protocols and standard operation procedures have been developed for the described control situations as well as analytical methods via LC-MS/MS and rapid strip test to measure the main mycotoxins in wheat and maize dust.
The Eurofins rapidust® dust sampling procedure is patented (EP 2614352A2; CA 2810948) and marketed by Eurofins, a subcontractor of the MycoKey project.

The mycotoxin producing fungus Fusarium graminearum causes the devastating cereal disease Fusarium head blight. The infection contaminates the harvested grain with deoxynivalenol (DON) and zearalenone (ZEN). Remaining host crop residues in the field represent the primary inoculum of F. graminearum for the subsequent cereal crop.
Throughout two field seasons, we examined the potential of the promising biological control agent Clonostachys rosea against F. graminearum on infected host crop residues to reduce the primary inoculum and thereby reduce mycotoxin contamination in subsequently grown wheat.
To simulate a maize‐wheat rotation under no‐tillage, we prepared maize stalks inoculated with F. graminearum. The infected stalks were treated with formulations of C. rosea and subsequently exposed to field conditions over winter and spring between winter wheat rows.
To monitor and quantify the disease during wheat growing, fungal spores were trapped, disease symptoms were rated and, after harvest, the biomass and the fungal colonies were measured. 
Mycotoxin analyses revealed significant reductions by C. rosea compared with an untreated control. DON and ZEN contents were reduced between 64-93% and 78-98%, respectively. 
To render this method applicable in practice, we currently evaluate in an on‐farm setting the efficacy of C. rosea directly applied during the mulching of maize crop residues.

Bentonite in the form of dioctahedral smectite is an additive authorized in the EU as a substance for the reduction of the contamination of feed by aflatoxins (EU Regulation 1060/2013). Bentonite adsorbs preferably aflatoxins, with little adsorption efficacy towards other mycotoxins. To overcome this limitation, within the EU Project MycoKey, Laviosa Chimica Mineraria (Italy) and CNR-ISPA (Italy) developed a bio-organoclay acting as a multi-toxin adsorbent. The additive was obtained by functionalization of a Na-smectite with an organic, non-toxic modifier. At low dosages, the bio-organoclay sequestered in vitro more than 95% of aflatoxin B1, fumonisin B1, ochratoxin A, and zearalenone, in a large range of pH values, and with high capacity and affinity. The efficacy of the bio-organoclay in reducing the systemic exposure to these mycotoxins was further assessed in rats and piglets, using the biomarker for exposure approach. Scaling-up at industrial level for commercial production of the additive and patent protection of the product/process will be addressed. Dietary supplementation of mycotoxin-contaminated feeds with the new additive is a practical and feasible approach to reduce the effects of exposure to a cocktail of mycotoxins and to prevent the carry-over of toxic metabolites in animal products.

Maize is frequently affected by the fungal pathogen Fusarium verticillioides, the causal agent of Fusarium ear rot (FER). This fungus is of concern to stakeholders as it affects crop yield and quality, contaminating maize grains with mycotoxins, mainly fumonisins. The most sustainable strategy to prevent pre-harvest contamination by F. verticillioides is to develop maize hybrids resistant to FER, as well as to its associated mycotoxins.
A set of 46 F1 hybrids, originated by crossing different breeding groups at Università Cattolica del Sacro Cuore, Piacenza, was tested for FER resistance and their agronomic performances. 
In 2017, all hybrids were planted and artificially inoculated with a toxigenic strain of F. verticillioides at two locations, and the best performing 17 out of the 46 hybrids were also tested in 2018. Ear rot was present in all hybrids in 2017 and 2018, with percentages of FER ranging from 6.5 to 49.5%. Seven hybrids (PC8, PC15, PC9, PC11, PC14, PC34 and PC17) presented the lowest levels of disease considering the overall locations and growing seasons, and three of these (PC8, PC11 and PC14) were also amongst the least mycotoxin contaminated hybrids in 2017. 

The inbred lines used in hybrid production will provide additional sources of resistance suitable in breeding programs targeting FER and fumonisins.

Assay system for a real time biosensor platform called MiSens for detection of aflatoxin B1 and ochratoxin A in dried fig and raisin is developed. This system includes a portable device and assay kits which can be easily used by end users. The system will allow end users to analyze their crops in their facilities without sending samples to the external laboratories. Beside the analyzing system, on-site extraction of mycotoxins from the foodstuff in the field is one of the bigest challenge, and to overcome this challenge, a prototype of mobile, easy-to-use extraction instrument is developed. This on-site mycotoxin extraction prototype can be easily transferred to the field by car and it allows to do extractions of mycotoxins from the foodstuffs easily by using standard procedures. The extraction system has a software which can be easily prorammable to run different kind of extraction procedures and it also uses closed tanks and tubing system for solvents to avoid harmful effect of them. So, these on-site mycotoxin extraction and analysis systems, developed within MycoKey Project, will allow end-users to carry out their own mycotoxin analysis without additional sample tranfer effort and external analysis budget.

Legislation is in force almost worldwide to fix the maximum amount of mycotoxin allowed in maize and wheat destined to human consumption; in addition, aflatoxin is regulated and recommendation were defined for fusaria toxins also for animal feeding.
Weather conditions are the main driving variable for fungal growth and their metabolism, but a strong mitigation action can be obtained optimising the cropping system.
Many efforts were devoted by scientist to understand the impact of any actions in the cropping system on mycotoxin contamination at harvest. All this information was merged and discussed with experts to finalise guidelines. They were developed in a smart format for the end users.
For each management strategy, risk and critical conditions were described and actions suggested, scoring for each strategy is score the impact on the target mycotoxin, ranging between low and very high.

Mycotoxins such as DON, aflatoxin, and fumonisin are toxins produced by fungi that can accumulate in grains and have adverse health effects on people and livestock. The MycoKey app is developed to aid mycotoxin risk mitigation by growers, grain collectors, governmental planners and policy makers. The MycoKey app predicts the amounts of the most important mycotoxins in winter wheat (DON) and grain maize (Aflatoxin B1 and Fumonisin) based on local weather data and predictive models. It provides direct links to the scientific articles describing the models in detail. Both access to the platform and the MycoKey app is free of charge and user data are private. Enlist at https://akkerweb.eu/en-gb/ and download the free MycoKey app. Growers can calculate the predicted amount of mycotoxins in their crops with local weather information and relevant agronomic measures such as ploughing or the level of plant resistance based on a list of cultivars. Grain collectors, governmental planners and policy makers have access to public databases including satellite data, and can calculate mycotoxin risks based on weather data and land use (if publicly available). Recalculation allows integration of management strategies in the risk model and calculations of “what if” scenarios.

Mycotoxin contamination in the maize supply chain is a major threat both for human and animal health. The grain cleaning process is the most effective post-harvest mitigation strategy to reduce high levels of mycotoxins due to the removal of mold-infected grains and grain fractions with high mycotoxin content. The efficacy of industrial-scale cleaning solutions in reducing aflatoxins and the major Fusarium toxins (i.e. deoxynivalenol, zearalenone and fumonisins), in naturally contaminated maize has been demonstrated in the MycoKey project. The investigated industrial scale cleaning solutions included: mechanical size separation of coarse, small and broken kernels; removal of dust/fine particles through an aspiration channel; separation of kernels based on gravity and optical sorting of spatial and spectral kernel defects. Overall results showed that an accurate maize cleaning by means of appropriate cleaning equipment could allow us to convert biomass/feed quality grains to feed/food quality grains with significant economic gains for the farmers and other stakeholders. This outcome could be particularly useful in critical crop seasons in which the levels of mycotoxins in cereals intended for human or animal consumption might exceed the maximum permitted levels.

A new multiplex rapid test, the 3-Mycosensor, is now available to detect the major Fusarium mycotoxins (deoxynivalenol, zearalenone and fumonisins) in cereals. The 3-Mycosensor is a semi-quantitative immunochromatographic screening test composed by two main components:
(i) a microwell containing freeze-dried antibodies labeled with gold particles. 
(ii) a dipstick made up of a set of membranes with three specific capture lines and one control line. 
The assay protocol requires mycotoxin extraction with a mixture of methanol/water followed by extract migration onto the strip. Then the use of an optical reader allows mycotoxin quantification in cereal samples in ranges of of 500 to 3000 µg/kg, 50 to 750 µg/kg and 500 to 6000 µg/kg respectively for deoxynivalenol, zearalenone and fumonisins. The resulting immunoassay protocol is rapid (total analysis time 15 min for 4 mycotoxins), inexpensive, and easy-to-use. Assay robustness and reliability have been demonstrated through single-laboratory validation, obtaining fit-for-purposes analytical performances (precision, cut off, false suspect rates)
The kit will be commercially available soon and will be distributed by Unisensor (https://unisensor.be/), partner of the MycoKey project. The 3-Mycosensor is the first ever developed rapid screening test allowing the simultaneous quantification of deoxynivalenol, zearalenone and fumonisins in cereals. Food and feed business operators will be able to demonstrate easily and cost-effectively the safety of their own products, thus strengthening their economic and export position in agriculture.

An important key action to reduce mycotoxins in the field is a correct inventarisation of the residing toxigenic mycoflora. Therefore, fast, reliable LAMP tests (loop-mediated isothermal amplification) were developed that can be used in the field, in mills, during storage to assess the residing mycoflora. The tests have been optimized for the most important toxigenic fungi occurring in maize and wheat. This information is gathered in a risk map that gives a global overview of the pathogen population

An important key action to prevent and/or reduce mycotoxins in maize and wheat is the correct prediction of the occurrence and concentration of mycotoxins in the field. Therefore, prediction models have been established and/or optimised to be implemented in practice. This implementation is facilitated through the IT dashboard. A correct prediction of DON, will allow us to tailor intervention strategies (e.g. biocontrol, new fungicides) and remediation strategies (e.g. mycotoxin binders) depending on the occurring mycotoxins. Contamination risk predictions will support a correct harvest time and postharvest grain management, including remediation strategies. Risk maps for the areas were predictive models are applied can be drawn, both using past, present and future meteorological data to make stakeholders aware regarding current and future risks, in climate change scenarios.

The mycotoxin producing fungus Fusarium graminearum causes the devastating cereal disease Fusarium head blight. The infection contaminates the harvested grain with deoxynivalenol (DON) and zearalenone (ZEN). Remaining host crop residues in the field represent the primary inoculum of F. graminearum for the subsequent cereal crop.
Throughout two field seasons, we examined the efficacy of potential antifungal mulch layers on infected crop residues to reduce the primary inoculum and thereby reduce mycotoxin contamination in subsequently grown wheat. To simulate a maize‐wheat rotation under no‐tillage, we prepared maize stalks inoculated with F. graminearum. For mulch layers, a novel cut-and-carry biofumigation was employed. White mustard, Indian mustard or berseem clover were grown in separate fields, harvested in autumn, chopped, and applied directly onto the inoculated maize residues. To monitor and quantify the disease during wheat growing, fungal spores were trapped, disease symptoms were rated and, after harvest, the biomass, the fungal colonies and mycotoxins were measured. 
Mulch layers of white mustard, Indian mustard or clover suppressed Fusarium infection in both years and decreased DON in grain by up 50, 58 and 56% and ZEN by up to 76, 71 and 87%, respectively. Moreover, mulch layer treatments improved grain yield up to 15% compared with the control without mulch layers. 
Within the context of sustainable crop protection, cereal growers could benefit from this prevention strategy by decreasing the risk of mycotoxin contamination and thus improving grain yield and quality.

Deoxynivalenol (DON), T-2 toxin (T-2) and HT-2 toxin (HT-2), due to their incidence and toxicity, are the Fusarium toxins of major concern for cereals and cereal-based products. Moreover, natural occurrence of modified forms of DON and of T-2 and HT-2 have been recently included in the EFSA risk assessment programme. Analytical methods for the simultaneous detection of these mycotoxins and their modified forms are therefore highly demanded since it could meet future requirements of European or international regulations. Two fluorescence polarization immunoassays (FPIAs), of which one for the simultaneous determination, expressed as sum, of DON and modified forms (3-acetyl-DON, 15-acetyl-DON, and DON-3-glucoside) and one for the determination of T-2, HT-2 and modified forms (T-2 glucoside and HT-2 glucoside) in wheat have been developed and validated in the MycoKey project. The FPIAs showed analytical performances, in terms of accuracy and precision that fulfilled the criteria for acceptability established by the European Union. The assays have been also validated according to the harmonized guidelines for the validation of screening methods (Regulation EU, No 519/2014), showing satisfactory analytical performances. The FPIAs are rapid (quantitative response in less than 15 min), cheap, easy-to-use, portable and can be automated for high-throughput screening. For these reasons, they are mainly addressed to non-skilled personnel of laboratories focused on monitoring programs and quality control studies/services.

Aspergillus flavus is the main responsible fungus for aflatoxin contamination in maize. Since 2013, Europe is included in the geographic areas with higher contamination risk and Romania was listed among the countries with severe aflatoxin contents. Research to find out useful preventive actions focused on the biocontrol with atoxigenic strains of the same fungus. This approach is established in USA and in Africa, and available in Italy. The atoxigenic strains used are all different, depending on the geographic area, due to the need of applying native, highly competitive strains. Therefore, grain collection and strain isolation were conducted in Romanian maize. 
A set of around 200 A. flavus strains was collected and characterised using molecular tools, sharing the population between toxigenic and atoxigenic strains. Seven atoxigenic strains were selected and included in competition tests with a toxigenic strain to check their ability to reduce aflatoxin contamination. They all performed well, but one of them was selected as the best candidate for field treatments.
Using the same methodology as in Italy, a two-year field application was performed in Romania. Briefly, sorghum kernels were coated with a suspension of the strain and distributed in fields with maize at the growth stage of 5 true leaves. Due to the overall low aflatoxin contamination, the efficacy could not be determined.
Even if not yet confirmed in Romania, biocontrol is worldwide reported as the only tool with highly satisfactory results. Aflatoxin contamination reduction of >80%, frequently >90%, were reported. Maize grain contaminated with >20mig/kg, the legal limit for maize destined to feed in Europe, almost disappeared, and the limit of 5mig/kg, for human and dairy animal consumption, was commonly complied.

The mycotoxin producing fungus Fusarium graminearum causes the devastating cereal disease Fusarium head blight (FHB). The infection contaminates the harvested grain mainly with deoxynivalenol (DON). Remaining host crop residues in the field represent the primary inoculum of F. graminearum for the subsequent cereal crop.
Throughout two field seasons, we examined the potential of various cover crop species to reduce mycotoxin contamination in subsequently grown wheat. 
To ensure a sufficient level of FHB infection in the field, ten maize plants (first internode above the crown roots) per plot were inoculated at the beginning of grain development with spore suspensions of three F. graminearum isolates. After silage maize harvest and mulching of the residues, white mustard, Indian mustard or winter pea were sown. A herbicide treatment as well as ploughed plots served as controls. To monitor and quantify the disease in subsequent wheat, disease symptoms were rated and after harvest, the fungal incidence and mycotoxins were measured. 
The use of cover crops decreased DON content in grain and increased gross margin of wheat by up to 75 and 34%, respectively, compared with the herbicide treatment without a cover crop. Moreover, growing winter pea decreased the DON content as efficient as the plough treatment.
Within the context of diversifying cropping systems, utilisation of cover cropping can improve wheat safety by reducing mycotoxin levels in grain. We suggest to further explore the best performing system, i.e. winter pea-based, under on-farm conditions with variable disease pressure. Additional ecosystem services should be monitored as well, including the presence of beneficial organisms, weed reduction and soil quality parameters.