Belén Patiño

Ochratoxin A (OTA) accumulation by Penicillium nordicum is enhanced at high salt concentrations. The genes involved in OTA biosynthesis include a polyketide synthase (PKS), although other PKSs outside the cluster could influence OTA production. This study aimed to evaluate a potential new PKS involved in OTA biosynthesis and the metabolites related to this mycotoxin. Optimised protocols were designed for the gene expression analysis of five genes that encoded the previously reported PKS to be overexpressed in dry-cured meat products (n2, n3, n4, n5 and n6). Their expression was tested in two P. nordicum strains (Pn15 and Pn856) grown in CYA medium with different salt concentrations. Fungal mycelia were used for gene expression analyses and metabolomics. Three genes (n3, n4 and n6) seem to be related to OTA biosynthesis in Pn856. However, n2 might be involved in OTA degradation. The lack of correlations between the genes studied and OTA in Pn15 could be due to the low OTA levels quantified. The metabolites coniferyl alcohol and 6-hydroxy-8-methoxy-3-methylisochroman were positively correlated to OTA, and 3-methylbutanoic acid and cephalosporolide E might be used as indicators to predict OTA. These results show the complex regulation and influence of PKS proteins on OTA levels.

Cereal safety is a critical public health concern, as crops are frequently compromised by mycotoxin-producing fungi, particularly those from the genus Fusarium. Although metabarcoding is a powerful tool for characterizing these fungal communities, the universal internal transcribed spacer 2 (ITS2) marker lacks the resolution required to distinguish closely related species with distinct toxigenic profiles. The objective of this work was to accurately characterize the mycobiota and specific Fusarium community structure in Spanish cereal field samples. We propose a high-resolution metabarcoding workflow within the QIIME 2 environment to optimize Fusarium identification using the translation elongation factor 1-α (TEF1) gene. We developed a TEF1 Naive Bayes classifier trained on a curated database derived from FUSARIUM-ID v.3.0, expanded with eukaryotic sequences to prevent false-positive assignments. This approach was validated through direct comparison with ITS2 in the same samples. As expected, ITS2 significantly underestimates Fusarium diversity, whereas TEF1 enables precise species-level resolution within this genus. General mycobiota analysis performed using ITS2 revealed that fungal communities are shaped primarily by geographical location rather than host cereal species. Crucially, our approach confirmed the persistence of key toxigenic species, such as F. langsethiae and F. graminearum, and revealed broader diversity through the consistent detection of species often overlooked by traditional methods, including F. equiseti, F. acuminatum, and F. culmorum. We conclude that our metabarcoding framework reveals a high Fusarium diversity in Spanish cereal grains, and this knowledge is essential for designing targeted strategies to predict and mitigate mycotoxin contamination in these crops. Graphical Abstract

The presence of mycotoxins in food poses a significant risk to food safety, and it is essential to develop effective and safe detoxification strategies. In this study, we demonstrate the strong ability of Pichia fermentans KCB21_L2, a yeast isolated from kefir, to eliminate aflatoxin B₁, fumonisin B₁ and ocratoxin A. Viable cells removed aflatoxin B₁ and fumonisin B₁ more efficiently than heat-inactivated cells, particularly at pH values of 5.5 and 7.0, suggesting the involvement of an active removal process. Subsequently, we evaluated the capacity of P. fermentans KCB21_L2 to remove mycotoxins at high concentrations and investigated the underlying molecular and cellular responses. The yeast effectively eliminated high levels of all three mycotoxins. Transcriptional analysis revealed the activation of metabolic pathways related to amino acid catabolism and fatty acid metabolism, likely reflecting an adaptive stress response. However, no significant upregulation of specific genes related to mycotoxin-degrading enzymes was observed. In conclusion, the reduction process may involve multiple factors, including stress response pathways, possible production of organic acids, adsorption of mycotoxins into the cell wall, and constitutively expressed enzymes capable of degrading mycotoxins. In general, these findings highlight the multifactorial nature of yeast-mediated mycotoxin removal and establish P. fermentans KCB21_L2 as a promising candidate for safe biological decontamination in food systems.

Grapes are frequently contaminated by Aspergillus section Nigri fungi and ochratoxin A (OTA), with A. niger also capable of producing substantial fumonisin B₂ (FB₂) levels. Emerging evidence suggests that aflatoxigenic fungi may eventually replace ochratoxigenic fungi in certain regions due to better adaptation to changes in climatic conditions. However, research on the toxigenic fungal community and mycotoxins in grapes from organic vineyards remains limited. Research on Spanish conventional grapes is also deficient, with most of the available literature being outdated. The present study investigates the diversity of toxigenic fungi and the presence of mycotoxins in organically cultivated grape berries in Spain, which are renowned for their significant oenological tradition. This study employed species-specific PCR protocols for fungal characterization and optimized methods for the analysis of OTA, FB₂, and aflatoxin B₁ (AFB₁) by UPLC–ESI–MS/MS. The most prevalent species present were Aspergillus flavus, A. niger, A. parasiticus, A. steynii, A. carbonarius, and A. westerdijkiae (67.1%, 43.5%, 20.0%, 14.1%, 14.1%, and 11.8% of the samples, respectively). OTA was detected only in 16 samples (19%), averaging 0.48 ng/g and peaking at 0.7 ng/g, which were lower than previously reported for conventional grapes. There was no FB₂ or AFB₁ detected. This study is pioneering in its exploration of the occurrence of toxigenic mycobiota, beyond Nigri fungi, and subsequent potential for other serious mycotoxins to contaminate Spain’s organic grapes.

Aspergillus flavus is a xerophilic fungus whose geographical distribution is expected to change due to the current climate change scenario. Grapes are one of the most important crops worldwide, and it is essential to evaluate the risk posed by their contamination with potential mycotoxigenic species. Recently, a few reports have described A. flavus as an emerging contaminant in vineyards, which has led to a discussion on the need to legislate aflatoxin contents in grapes. Using a specific PCR assay, the occurrence of A. flavus was demonstrated in 43 out of 61 grape samples collected from Spanish vineyards. Considering the high incidence observed, the risk of the grapes becoming contaminated with aflatoxin was subsequently evaluated. Aspergillus flavus isolates from grapes can grow in grape-based media under a variety of environmental conditions, but they were unable to produce either aflatoxin B₁ (AFB₁) or aflatoxin B₂ (AFB₂) even though their ability to produce these toxins was confirmed in a permissive medium (CYA). These results confirm that climate change is affecting the distribution of mycotoxigenic fungi, thereby increasing the occurrence of A. flavus in vineyards, although the risk of the grapes becoming contaminated with aflatoxin needs to be reconsidered.

The presence of mycotoxin-producing Aspergillus species in vineyards is a problem for food safety and the economy. In addition, rising temperatures due to climate change are modifying microbial communities, causing the replacement of some fungal species and the rise of mycotoxins such as aflatoxins. The use of microorganisms as biological control agents (BCAs) is one of the most promising strategies to prevent fungal growth and toxin production. In this study, 513 microorganisms were isolated from organic vineyard soils in different regions of Spain. The 480 bacteria and 33 yeasts isolated were sequentially screened to select those with the most suitable characteristics to be used as BCAs. After identifying 16 isolates meeting all requirements, six bacterial isolates were selected to test their potential to control three relevant toxigenic grape fungi in vitro: A. carbonarius, A. niger and A. flavus. Isolates of Arthrobacter sp., Rhodococcus sp. and Bacillus mycoides showed an excellent ability to reduce the growth and mycotoxin concentration of the above-mentioned fungi and represent potential candidates for further study regarding their possible industrial application as a BCA.

The contamination of oats with Fusarium toxins poses a high risk for food safety. Among them, trichothecenes are the most frequently reported in European oats, especially in northern countries. The environmental conditions related to the climate change scenario might favour a distribution shift in Fusarium species and the presence of these toxins in Southern European countries. In this paper, we present an ambitious work to determine the species responsible for trichothecene contamination in Spanish oats and to compare the results in the United Kingdom (UK) using a metataxonomic approach applied to both oat grains and soil samples collected from both countries. Regarding T-2 and HT-2 toxin producers, F. langsethiae was detected in 38% and 25% of the oat samples from the UK and Spain, respectively, and to the best of our knowledge, this is the first report of the detection of this fungus in oats from Spain. The relevant type B trichothecene producer, F. poae, was the most frequently detected Fusarium species in oats from both origins. Other important trichothecene producers, such as the Fusarium tricinctum species complex or Fusarium cerealis, were also frequently detected in oat fields. Many Fusarium toxins, including T-2 and HT-2 toxins, deoxynivalenol, or nivalenol, were detected in oat samples. The results obtained in this work revealed a clear change in the distribution of trichothecene producers and the necessity to establish the potential of these species to colonize oats and their ability to produce mycotoxins.

Maize expressing Cry1Ab insecticidal toxin (Bt maize) is an effective method to control Sesamia nonagrioides and Ostrinia nubilalis, the most damaging corn borers of southern Europe. In this area, maize is prone to Fusarium infections, which can produce mycotoxins that pose a serious risk to human and animal health, causing significant economic losses in the agrifood industry. To investigate the influence of corn borer damage on the presence of Fusarium species and their mycotoxins, Bt maize ears and insect-damaged ears of non-Bt maize were collected from commercial fields in three Bt maize growing areas in Spain, and differences in contamination were assessed. Additionally, larvae of both borer species were collected to evaluate their role as vectors of these molds. Non-Bt maize ears showed significantly higher presence of F. verticillioides, F. proliferatum, and F. subglutinans than Bt maize ears. For the first time, Fusarium species have been isolated from larvae of the two species. The most frequently found mycotoxins in ears were fumonisins, with non-Bt ears being significantly more contaminated than those of Bt maize. High levels of fumonisins were shown to correlate with the occurrence of corn borers in the ear and the presence of F. verticillioides and F. proliferatum.

The occurrence of mycotoxins on grapes poses a high risk for food safety; thus, it is necessary to implement effective prevention methods. In this work, a metagenomic approach revealed the presence of important mycotoxigenic fungi in grape berries, including Aspergillus flavus, Aspergillus niger aggregate species, or Aspergillus section Circumdati. However, A. carbonarius was not detected in any sample. One of the samples was not contaminated by any mycotoxigenic species, and, therefore, it was selected for the isolation of potential biocontrol agents. In this context, Hanseniaspora uvarum U1 was selected for biocontrol in vitro assays. The results showed that this yeast is able to reduce the growth rate of the main ochratoxigenic and aflatoxigenic Aspergillus spp. occurring on grapes. Moreover, H. uvarum U1 seems to be an effective detoxifying agent for aflatoxin B₁ and ochratoxin A, probably mediated by the mechanisms of adsorption to the cell wall and other active mechanisms. Therefore, H. uvarum U1 should be considered in an integrated approach to preventing AFB₁ and OTA in grapes due to its potential as a biocontrol and detoxifying agent.

Colonization of crops by toxigenic fungi causes important economic losses, which are expected to increase with global change. The presence of mycotoxins in crops affects food security by reducing the edible yield and nutritional values and making cereal consumption unsafe. An efficient method to reduce mycotoxin content is to avoid the appearance of fungi or toxigenesis. This has been traditionally achieved with antifungal chemicals that negatively affect soil and ecosystem health. Current research aims for methods that less harmful for the environment, such as natural proteins or biocontrol. We tested crop-associated fungi against two possible growth-inhibiting proteins: Fusarium graminearum Antifungal Protein (Fg-AFP) and Latrodectin-II (Ltd-II). The first is produced by Fusarium graminearum to compete with other fungi, and the latter is found in Latrodectus hesperus venom and has just recently been correctly purified. In a first assay, we exposed four Aspergillus strains cultured on PDA (potato dextrose agar) against on-surface cellulose discs with 10 µL of different Fg-AFP and Ltd-II concentrations (7 µg/µL, 3.5 µg/µL, 1.4 µg/µL, and 0.7 µg/µL). All tests were carried out in triplicate. Results show that Fg-AFP inhibited development of three of the strains, while Ltd-II did not but may have affected secondary metabolism due to a variation in spore production and pigmentation (no further analysis regarding this event was fulfilled). We subsequently selected Aspergillus niger and A. flavus strains based on their importance in maize crops and sensitivity to the proteins, for a second assay, in which we evenly extended 30 µL of the proteins (1.5 µg/µL) separately on plaques containing PDA. The growth surface was represented over time, the specific growth rate corresponded with the curve slope, and latent period was calculated by obtaining the equation of the line from the slope and the intersection point. An initial diameter of 4 mm was assumed for 2 µL of spore suspension (10⁵ spores/mL) inoculated on the center of the plaque. All tests were carried out in triplicate. Results show that Fg-AFP reduced A. niger and A. flavus growth by 46.3% and 24.3%, respectively, extending fungal latent period by 68.4% and 52.6%, respectively. Ltd-II had no effect on A. flavus, but increased A. niger growth by 18.4%, prolonging the latent period by 67.5%. Toxin production was assessed by thin-layer chromatography under UV light after following a toxin extraction protocol with chloroform and toluene:acetonitrile for quantifying ochratoxin A, and chloroform and methanol for aflatoxin B1 production. Regarding toxigenesis results, ochratoxin A production by A. niger did not vary when it was exposed to the proteins, but aflatoxin B1 synthesis by A. flavus increased with both treatments. Though these proteins have growth-limiting potential, they must be evaluated under wider concentration ranges to assess their effect on toxigenesis and usage as an alternative to harmful chemicals.