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This study identified secondary metabolites produced by Alternaria alternata , Colletotrichum gloeosporioides , and Penicillium digitatum in fruits of two blood orange cultivars before harvest. Analysis was performed by UHPLC-Q-TOF-MS. Three types of fruits were selected, asymptomatic, symptomatic showing necrotic lesions caused by hail, and mummified. Extracts from peel and juice were analyzed separately. Penicillium digitatum was the prevalent species recovered from mummified and hail-injured fruits. Among 47 secondary metabolites identified, 16, 18, and 13 were of A. alternata , C. gloeosporioides , and P. digitatum , respectively. Consistently with isolations, indicating the presence of these fungi also in asymptomatic fruits, the metabolic profiles of the peel of hail-injured and asymptomatic fruits did not differ substantially. Major differences were found in the profiles of juice from hail-injured and mummified fruits, such as a significant higher presence of 5,4-dihydroxy-3,7,8-trimethoxy-6C-methylflavone and Atrovenetin, particularly in the juice of mummified fruits of the Tarocco Lempso cultivar. Moreover, the mycotoxins patulin and Rubratoxin B were detected exclusively in mummified fruits. Patulin was detected in both the juice and peel, with a higher relative abundance in the juice, while Rubratoxin B was detected only in the juice. These findings provide basic information for evaluating and preventing the risk of contamination by mycotoxins in the citrus fresh fruit supply chain and juice industry.

Corn ( Zea mays ) is a worldwide crop subjected to infection by toxigenic fungi such as Fusarium verticillioides during the pre-harvest stage. Fusarium contamination can lead to the synthesis of highly toxic mycotoxins, such as Fumonisin B 1 (FB 1 ) and Fumonisin B 2 (FB 2 ), which compromises human and animal health. The work aimed to study the antifungal properties of fermented yellow and oriental mustard extracts using nine lactic acid bacteria (LAB) in vitro. Moreover, a chemical characterization of the main phenolic compounds and organic acids were carried out in the extracts. The results highlighted that the yellow mustard, fermented by Lactiplantibacillus plantarum strains, avoided the growth of Fusarium spp. in vitro, showing Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) values, ranging from 7.8 to 15.6 g/L and 15.6 to 31.3 g/L, respectively. Then, the lyophilized yellow mustard fermented extract by L. plantarum TR71 was applied through spray-on corn ears contaminated with F. verticillioides to study the antimycotoxigenic activity. After 14 days of incubation, the control contained 14.71 mg/kg of FB 1 , while the treatment reduced the content to 1.09 mg/kg (92.6% reduction). Moreover, no FB 2 was observed in the treated samples. The chemical characterization showed that lactic acid, 3-phenyllactic acid, and benzoic acid were the antifungal metabolites quantified in higher concentrations in the yellow mustard fermented extract with L. plantarum TR71. The results obtained confirmed the potential application of fermented mustard extracts as a solution to reduce the incidence of mycotoxins in corn ears.

The present study describes the manufacture of an antifungal device composed of oriental mustard flour and hydroxyethyl-cellulose (H-OMF) and evaluates its efficacity in inhibiting Aspergillus flavus growth and aflatoxin B1 (AFB 1 ) production in almonds. Additionally, it compares the H-OMF with allyl isothiocyanate (AITC) and a freeze-dried extract of yellow mustard flour (YMF-E); such substances were previously described as antifungal. Minimum inhibitory concentration (MIC), Minimum fungicidal concentration (MFC), the H-OMF in vitro antifungal activity, and the residual fungal population, as well as the production of AFB 1 in almonds were determined. AITC and YMF-E showed significant antifungal activity in vitro. Additionally, the in vitro activity of H-OMF avoided mycelial growth by applying 30 mg/L. Almonds treated with AITC (5.07, 10.13, and 20.26 mg/L) and H-OMF (2000 and 4000 mg/L) showed a reduction in the population of A. flavus and the production of AFB 1 to values below the limit of detection. YMF-E showed effectiveness by in vitro methodologies (MIC and MFC) but did not show efficacy when applied in almonds. Our findings indicated that the hydroxyethyl-cellulose-based device containing oriental mustard flour might be utilised as a fumigant to increase the safety of almonds and could be extended to other cereals or dry fruits.

The presence of mycotoxins in cereals and cereal products remains a significant issue. The use of natural ingredients such as pumpkin and whey, which contain bioactive compounds, could be a strategy to reduce the use of conventional chemical preservatives. The aim of the present work was to study the bioaccessibility of aflatoxin B 1 (AFB1) and ochratoxin (OTA) in bread, as well as to evaluate the effect of milk whey (with and without lactic acid bacteria fermentation) and pumpkin on reducing mycotoxins bioaccessibility. Different bread typologies were prepared and subjected to an in vitro digestion model. Gastric and intestinal extracts were analyzed by HPLC-MS/qTOF and mycotoxins bioaccessibility was calculated. All the tested ingredients but one significantly reduced mycotoxin intestinal bioaccessibility. Pumpkin powder demonstrated to be the most effective ingredient showing significant reductions of AFB1 and OTA bioaccessibility up to 74% and 34%, respectively. Whey, fermented whey, and the combination of pumpkin-fermented whey showed intestinal bioaccessibility reductions between 57-68% for AFB1, and between 11-20% for OTA. These results pointed to pumpkin and milk whey as potential bioactive ingredients that may have promising applications in the bakery industry.

Food bio-preservatives are requested as substituents of chemical pesticides in food. The aim of this study was to carry out a screening of twenty biocontrol agents (BCAs) for their potential fungicidal activity in vitro. Twenty BCAs were tested against ten pathogenic fungi. Some of the cell-free supernatants (CFS) tested showed in vitro antifungal activity versus pathogenic fungi. The highest fungicidal activity was observed in the fermented CFS of Paenibacillus chibensis CECT 375, Bacillus amyloliquefaciens CECT 493, and Pantoea agglomerans CECT 850, which showed a minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values of 125 and 250 g/L, respectively. The compounds responsible for the antifungal activity, such as organic and phenolic acids, were determined. Lactic acid, acetic acid, benzoic acid, and phenyllactic acid among others can be related to antifungal activity. HPLC-MS/MS analysis showed a reduction of ochratoxin A (OTA) and aflatoxin B 1 (AFB 1 ) up to 26% ( Paenibacillus alvei CECT 2) and 55% ( Paenibacillus polymyxa CECT 155), respectively. The present study prompts that metabolism products of BCAs are propitious for the bioconservation of food, due to their ability to reduce the proliferation of mycotoxigenic fungi and mycotoxins production.

The tomato is one of the most consumed agri-food products in Lebanon. Several fungal pathogens, including Alternaria species, can infect tomato plants during the whole growing cycle. Alternaria infections cause severe production and economic losses in field and during storage. In addition, Alternaria species represent a serious toxicological risk since they are able to produce a wide range of mycotoxins, associated with different toxic activities on human and animal health. Several Alternaria species were detected on tomatoes, among which the most important are A. solani , A. alternata , and A. arborescens . A set of 49 Alternaria strains isolated from leaves and stems of diseased tomato plants were characterised by using a polyphasic approach. All strains were included in the recently defined phylogenetic Alternaria section and grouped in three well-separated sub-clades, namely A. alternata (24 out of 49), A. arborescens (12 out of 49), and A. mali morpho-species (12 out of 49). One strain showed high genetic similarity with an A. limoniasperae reference strain. Chemical analyses showed that most of the Alternaria strains, cultured on rice, were able to produce alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT) and tenuazonic acid (TA), with values up to 5634, 16,006, 5156, and 4507 mg kg -1 , respectively. In addition, 66% of the strains were able to co-produce simultaneously the four mycotoxins investigated. The pathogenicity test carried out on 10 Alternaria strains, representative of phylogenetic sub-clades, revealed that they were all pathogenic on tomato fruits. No significant difference among strains was observed, although A. alternata and A. arborescens strains were slightly more aggressive than A. mali morpho-species strains. This paper reports new insights on mycotoxin profiles, genetic variability, and pathogenicity of Alternaria species on tomatoes.

Fungal spoilage is one of the main reasons of economic losses in the food industry, especially in the wine sector. Consequently, the search for safer and new preservation techniques has gained importance in recent years. The objective of this study was to investigate the antifungal and anti-mycotoxigenic activity from 28 microorganisms (MO) isolated from red grape. The antifungal activity of a cell free supernatant of fermented medium by the isolated MO (CFS) was tested with the agar diffusion method and the minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) assay. Additionally, different antifungal compounds from the CFS were identified and quantified (organic acids, phenolic compounds, and volatile organic compounds). Finally, the most active CFS were tested as red grape bio-preservative agents. Results evidenced that CFS fermented by the strain UTA 6 had the highest antifungal activity, above all isolates, and produced a wide pool of antifungal compounds. The use of UTA 6 CFS as bio-preservative agent showed a reduction of 0.4 and 0.6 log 10 spores per gram of fruit in grapes contaminated by A. flavus and B. cinerea , respectively. Moreover, UTA 6 CFS treatment reduced the occurrence of aflatoxin B 1 and fumonisin (B 2 , B 3 , and B 4 ) production in grapes contaminated by 28-100%.

Black point is a fungal disease of wheat, mainly associated with mycotoxigenic Alternaria species. Affected wheat kernels are characterized by dark brown discolouration of the embryo region and reduction of grain quality. Potential risk is the possible accumulation of Alternaria mycotoxins, alternariol (AOH), alternariol-monomethyl ether (AME), tenuazonic acid (TA), and altenuene (ALT), provided by haemato-toxic, genotoxic, and mutagenic activities. One hundred and twenty durum wheat samples belonging to 30 different genotypes grown in Bologna and Modena areas, in Italy, showing black point symptoms, were analyzed for Alternaria species and their mycotoxin contamination. Alternariol was selected as an indicator of the capability of the Alternaria species to produce mycotoxin in vivo in field conditions. The data showed that Alternaria species occurred in 118 out of 120 wheat kernels samples, with the incidence of infected kernels ranging between 1% and 26%. Moreover, AOH was detected by using a HPLC with a diode array detector (LC-DAD) in 98 out of 120 samples with values ranging between 24 and 262 µg Kg -1 . Ninety-two Alternaria representative strains, previously identified morphologically, were identified at species/section level using gene sequencing, and therefore were analyzed for their mycotoxin profiles. Eighty-four strains, phylogenetically grouped in the Alternaria section, produced AOH, AME, and TA with values up to 8064, 14,341, and 3683 µg g -1 , respectively, analyzed by using a LC-DAD. On the other hand, eight Alternaria strains, included in Infectoriae Section, showed a very low or no capability to produce mycotoxins.

Fungal spoilage is an important issue for the food industry, leading to food sensory defects, food waste, economic losses and public health concern through the production of mycotoxins. Concomitantly, the search for safer natural products has gained importance since consumers began to look for less processed and chemically treated foods. In this context, the aim of this study was to evaluate the antifungal and antimycotoxigenic effect of seven strains of Lactobacillus plantarum . Lactic acid bacteria (LAB) were grown on Man Rogosa Sharpe (MRS) broth at 37 ºC in anaerobic conditions. After that, the cell-free supernatant (CFS) were recovered to determine its antifungal activity by halo diffusion agar test. In addition, minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) was determined for each L. plantarum CFS by 96-well microplates method. Additionally, CFS was used as a natural biocontrol agent on corn kernels and corn ears contaminated with Aspergillus flavus and Fusarium verticillioides, respectively. The L. plantarum CECT 749 CFS showed the highest antifungal effect against all essayed strains. Moreover, the employment of this CFS in food reduced the mycotoxin production at a percentage ranging from 73.7 to 99.7%. These results suggest that the L. plantarum CECT 749 CFS could be promising for the biocontrol of corn., Competing Interests: The authors declare no conflicts of interest.

The aims of this study were to evaluate the antifungal activity of the bioactive compound allyl isothiocyanate (AITC) against Aspergillus flavus (8111 ISPA) aflatoxins (AFs) producer and Penicillium verrucosum (D-01847 VTT) ochratoxin A (OTA) producer on corn, barley, and wheat. The experiments were carried out initially in a simulated silo system for laboratory scale composed of glass jars (1 L). Barley and wheat were contaminated with P. verrucosum and corn with A. flavus . The cereals were treated with a hydroxyethylcellulose gel disk to which 500 µL/L of AITC were added; the silo system was closed and incubated for 30 days at 21 °C. After that, simulated silos of 100 L capacity were used. Barley, wheat, and corn were contaminated under the same conditions as the previous trial and treated with disks with 5 mL of AITC, closed and incubated for 90 days at 21 °C. In both cases, the control test did not receive any antifungal treatment. The growth of the inoculated fungi and the reduction in the formation of AFs and OTA were determined. In the lab scale silo system, complete inhibition of fungal growth at 30 days has been observed. In corn, the reduction of aflatoxin B1 (AFB₁) was 98.5%. In the 100 L plastic drums, a significant reduction in the growth of A. flavus was observed, as well as the OTA formation in wheat (99.5%) and barley (92.0%).

Wheat, the main source of carbohydrates worldwide, can be attacked by a wide number of phytopathogenic fungi, included Alternaria species. Alternaria species commonly occur on wheat worldwide and produce several mycotoxins such as tenuazonic acid (TA), alternariol (AOH), alternariol-monomethyl ether (AME), and altenuene (ALT), provided of haemato-toxic, genotoxic, and mutagenic activities. The contamination by Alternaria species of wheat kernels, collected in Tuscany, Italy, from 2013 to 2016, was evaluated. Alternaria contamination was detected in 93 out of 100 field samples, with values ranging between 1 and 73% (mean of 18%). Selected strains were genetically characterized by multi-locus gene sequencing approach through combined sequences of allergen alt1a, glyceraldeyde-3-phosphate dehydrogenase, and translation elongation factor 1α genes. Two well defined groups were generated; namely sections Alternaria and Infectoriae . Representative strains were analyzed for mycotoxin production. A different mycotoxin profile between the sections was shown. Of the 54 strains analyzed for mycotoxins, all strains included in Section Alternaria produced AOH and AME, 40 strains (99%) produced TA, and 26 strains (63%) produced ALT. On the other hand, only a very low capability to produce both AOH and AME was recorded among the Section Infectoriae strains. These data show that a potential mycotoxin risk related to the consumption of Alternaria contaminated wheat is high.

MycoKey, an EU-funded Horizon 2020 project, includes a series of "Roundtable Discussions" to gather information on trending research areas in the field of mycotoxicology. This paper includes summaries of the Roundtable Discussions on Chemical Detection and Monitoring of mycotoxins and on the role of genetics and biodiversity in mycotoxin production. Discussions were managed by using the nominal group discussion technique, which generates numerous ideas and provides a ranking for those identified as the most important. Four questions were posed for each research area, as well as two questions that were common to both discussions. Test kits, usually antibody based, were one major focus of the discussions at the Chemical Detection and Monitoring roundtable because of their many favorable features, e.g., cost, speed and ease of use. The second area of focus for this roundtable was multi-mycotoxin detection protocols and the challenges still to be met to enable these protocols to become methods of choice for regulated mycotoxins. For the genetic and biodiversity group, both the depth and the breadth of trending research areas were notable. For some areas, e.g., microbiome studies, the suggested research questions were primarily of a descriptive nature. In other areas, multiple experimental approaches, e.g., transcriptomics, proteomics, RNAi and gene deletions, are needed to understand the regulation of toxin production and mechanisms underlying successful biological controls. Answers to the research questions will provide starting points for developing acceptable prevention and remediation processes. Forging a partnership between scientists and appropriately-placed communications experts was recognized by both groups as an essential step to communicating risks, while retaining overall confidence in the safety of the food supply and the integrity of the food production chain., Competing Interests: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript or in the decision to publish the results.