Biocontrol of Mycotoxigenic Fungi Using Bacteria Isolated from Ecological Vineyard Soils.

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.

Searching for the Fusarium spp. Which Are Responsible for Trichothecene Contamination in Oats Using Metataxonomy to Compare the Distribution of Toxigenic Species in Fields from Spain and the UK.

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.

Role of Sesamia nonagrioides and Ostrinia nubilalis as Vectors of Fusarium spp. and Contribution of Corn Borer-Resistant Bt Maize to Mycotoxin Reduction.

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.

Diversity of Mycobiota in Spanish Grape Berries and Selection of Hanseniaspora uvarum U1 to Prevent Mycotoxin Contamination.

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.

The Genomic Regions That Contain Ochratoxin A Biosynthetic Genes Widely Differ in Aspergillus Section Circumdati Species.

Aspergillus section Circumdati includes 27 species, some of which are considered ochratoxin A (OTA) producers. However, there is considerable controversy about their potential OTA synthesis ability. In this work, the complete genomes of 13 species of Aspergillus section Circumdati were analyzed in order to study the cluster of OTA biosynthetic genes and the region was compared to those previously reported in A. steynii and A. westerdijkiae. The results obtained reveal that the genomes of some species in this section, including A. affinis, A. cretensis, A. elegans, A. muricatus, A. pulvericola, A. roseoglobulosus, and A. subramanianii, contain a potentially functional OTA biosynthetic cluster. Therefore, they might be able to synthesize the toxin. On the contrary, A. melleus, A. ochraceus, A. ostianus, A. persii, A. sclerotiorum, A. sesamicola, and A. westlandensis contain a truncated version of the cluster that lacks many of the genes involved in OTA biosynthesis, which might be related to their inability to produce OTA. The gain/loss pattern is different in all species, which suggests that the genetic evolution of this region might be due to independent events.

Assessment of the Effect of Satureja montana and Origanum virens Essential Oils on Aspergillus flavus Growth and Aflatoxin Production at Different Water Activities.

Aflatoxin contamination of foodstuffs poses a serious risk to food security, and it is essential to search for new control methods to prevent these toxins entering the food chain. Several essential oils are able to reduce the growth and mycotoxin biosynthesis of toxigenic species, although their efficiency is strongly influenced by the environmental conditions. In this work, the effectiveness of Satureja montana and Origanum virens essential oils to control Aspergillus flavus growth was evaluated under three water activity levels (0.94, 0.96 and 0.98 aw) using a Bioscreen C, a rapid in vitro spectrophotometric technique. The aflatoxin concentrations at all conditions tested were determined by HPLC-FLD. Aspergillus flavus growth was delayed by both essential oil treatments. However, only S. montana essential oil was able to significantly affect aflatoxin production, although the inhibition percentages widely differed among water activities. The most significant reduction was observed at 0.96 aw, which is coincident with the conditions in which A. flavus reached the highest levels of aflatoxin production. On the contrary, the treatment with S. montana essential oil was not effective in significantly reducing aflatoxin production at 0.94 aw. Therefore, it is important to study the interaction of the new control compounds with environmental factors before their application in food matrices, and in vitro ecophysiological studies are a good option since they provide accurate and rapid results.

A Comprehensive Study on the Occurrence of Mycotoxins and Their Producing Fungi during the Maize Production Cycle in Spain.

Mycotoxin contamination is one of the main problems affecting corn production, due to its significant risk to human and animal health. The Fusarium and Aspergillus species are the main producers of mycotoxins in maize, infecting both pre-harvest and during storage. In this work, we evaluated the presence of mycotoxins and their producing species along maize production cycles in three different stages (anthesis, harvest, and storage) during three consecutive seasons (2016-2018). Fungal occurrences were studied using species-specific PCR protocols, whereas mycotoxin levels were determined by LC-MS/MS. Fumonisin-producing Fusarium species (F. verticillioides and F. proliferatum), as well as the aflatoxin producer Aspergillus flavus, were the most predominant species at all stages; although, during some seasons, the presence of F. graminearum and A. niger aggregate species were also identified. Contrastingly, fumonisins were the only mycotoxins detected and levels were always under legal regulations. The results presented here demonstrate that even when fungal contamination occurs at the early stages of the maize production cycle, the application of good agricultural and storage practices might be crucial to ensure mycotoxin-free grains.

Genetic regulation of aflatoxin, ochratoxin A, trichothecene, and fumonisin biosynthesis: A review.

Mycotoxins are a significant food safety concern. Aflatoxins, trichothecenes, fumonisins, and ochratoxin A are considered the most important mycotoxins due to their frequent occurrence in food products and their well-known toxicity. The regulation of mycotoxin biosynthesis occurs mainly at transcriptional level, and specific regulators have been described in every biosynthetic cluster. Secondary metabolite production, including mycotoxin synthesis, is also regulated by general regulator pathways affected by light, osmotic stress and oxidative stress, among others. This review is focused on this genetic regulation of mycotoxin biosynthesis by specific genes and global regulators.

A Novel Niosome-Encapsulated Essential Oil Formulation to Prevent Aspergillus flavus Growth and Aflatoxin Contamination of Maize Grains During Storage.

Aflatoxin (AF) contamination of maize is a major concern for food safety. The use of chemical fungicides is controversial, and it is necessary to develop new effective methods to control Aspergillus flavus growth and, therefore, to avoid the presence of AFs in grains. In this work, we tested in vitro the effect of six essential oils (EOs) extracted from aromatic plants. We selected those from Satureja montana and Origanum virens because they show high levels of antifungal and antitoxigenic activity at low concentrations against A. flavus. EOs are highly volatile compounds and we have developed a new niosome-based encapsulation method to extend their shelf life and activity. These new formulations have been successfully applied to reduce fungal growth and AF accumulation in maize grains in a small-scale test, as well as placing the maize into polypropylene woven bags to simulate common storage conditions. In this latter case, the antifungal properties lasted up to 75 days after the first application.

Significance of Aspergillus niger aggregate species as contaminants of food products in Spain regarding their occurrence and their ability to produce mycotoxins.

The Aspergillus niger aggregate contains 15 morphologically indistinguishable species which presence is related to ochratoxin A (OTA) and fumonisin B2 (FB2) contamination of foodstuffs. The taxonomy of this group was recently reevaluated and there is a need of new studies regarding the risk that these species might pose to food security. 258 isolates of A. niger aggregate obtained from a variety of products from Spain were classified by molecular methods being A. tubingensis the most frequently occurring (67.5%) followed by A. welwitschiae (19.4%) and A. niger (11.7%). Their potential ability to produce mycotoxins was evaluated by PCR protocols which allow a rapid detection of OTA and FB2 biosynthetic genes in their genomes. OTA production is not widespread in A. niger aggregate since only 17% of A. niger and 6% of A. welwitschiae isolates presented the complete biosynthetic cluster whereas the lack of the cluster was confirmed in all A. tubingensis isolates. On the other hand, A. niger and A. welwitschiae seem to be important FB2 producers with 97% and 29% of the isolates, respectively, presenting the complete cluster. The genes involved in OTA and FB2 were overexpressed in producing isolates and their expression was related to mycotoxin synthesis.

Characterization of a novel cysteine-rich antifungal protein from Fusarium graminearum with activity against maize fungal pathogens.

Filamentous fungi are an invaluable source for biocontrol strategies and for production and development of different antifungal polypeptides. Within this context, cysteine-rich antifungal AFP-like peptides stand out among many different antimicrobial compounds given their production easiness, stability, versatility, and efficacy. AFP from Aspergillus giganteus represents the hallmark of this still increasing family of antifungal polypeptides. Close in silico analyses of the Fusarium graminearum genome revealed the presence of an AFP-like peptide, here designated as FgAFP. This new peptide was cloned, produced in the yeast Pichia pastoris, and characterized. The results obtained showed its strong and specific antifungal activity against several well-recognized maize pathogens, but inefficacy against F. oxysporum, which has not been described as a natural biological competitor of other fungal pathogens assayed. All results together suggest that this small peptide is an important factor for the fungal interplays involved in maize infection and reveals unforeseen potential biotechnological applications for FgAFP in maize production and storage.

Educating in antimicrobial resistance awareness: adaptation of the Small World Initiative program to service-learning.

The Small World Initiative (SWI) and Tiny Earth are a consolidated and successful education programs rooted in the USA that tackle the antibiotic crisis by a crowdsourcing strategy. Based on active learning, it challenges young students to discover novel bioactive-producing microorganisms from environmental soil samples. Besides its pedagogical efficiency to impart microbiology content in academic curricula, SWI promotes vocations in research and development in Experimental Sciences and, at the same time, disseminates the antibiotic awareness guidelines of the World Health Organization. We have adapted the SWI program to the Spanish academic environment by a pioneering hierarchic strategy based on service-learning that involves two education levels (higher education and high school) with different degrees of responsibility. Throughout the academic year, 23 SWI teams, each consisting of 3-7 undergraduate students led by one faculty member, coordinated off-campus programs in 22 local high schools, involving 597 high school students as researchers. Post-survey-based evaluation of the program reveals a satisfactory achievement of goals: acquiring scientific abilities and general or personal competences by university students, as well as promoting academic decisions to inspire vocations for science- and technology-oriented degrees in younger students, and successfully communicating scientific culture in antimicrobial resistance to a young stratum of society.

Description of an orthologous cluster of ochratoxin A biosynthetic genes in Aspergillus and Penicillium species. A comparative analysis.

Ochratoxin A (OTA) is one of the most important mycotoxins due to its toxic properties and worldwide distribution which is produced by several Aspergillus and Penicillium species. The knowledge of OTA biosynthetic genes and understanding of the mechanisms involved in their regulation are essential. In this work, we obtained a clear picture of biosynthetic genes organization in the main OTA-producing Aspergillus and Penicillium species (A. steynii, A. westerdijkiae, A. niger, A. carbonarius and P. nordicum) using complete genome sequences obtained in this work or previously available on databases. The results revealed a region containing five ORFs which predicted five proteins: halogenase, bZIP transcription factor, cytochrome P450 monooxygenase, non-ribosomal peptide synthetase and polyketide synthase in all the five species. Genetic synteny was conserved in both Penicillium and Aspergillus species although genomic location seemed to be different since the clusters presented different flanking regions (except for A. steynii and A. westerdijkiae); these observations support the hypothesis of the orthology of this genomic region and that it might have been acquired by horizontal transfer. New real-time RT-PCR assays for quantification of the expression of these OTA biosynthetic genes were developed. In all species, the five genes were consistently expressed in OTA-producing strains in permissive conditions. These protocols might favour futures studies on the regulation of biosynthetic genes in order to develop new efficient control methods to avoid OTA entering the food chain.

Targeting Conserved Genes in Fusarium Species.

Fumonisins are important mycotoxins contaminating foods and feeds which are mainly produced by F. verticillioides and F. proliferatum. Additionally, both are pathogens of maize and other cereals. We describe two highly sensitive, rapid, and species-specific PCR protocols which enable detection and discrimination of these closely related species in cereal flour or grain samples. The specific primer pairs of these assays were based on the intergenic spacer region of the multicopy rDNA unit which highly improves the sensitivity of the PCR assay in comparison with single-copy target regions.

Species-specific optical genosensors for the detection of mycotoxigenic Fusarium fungi in food samples.

Plant-pathogenic Fusarium species, Fusarium verticillioides and Fusarium proliferatum, are the major producers of fumonisins which are one of the most common mycotoxins found in maize. Herein, we report the development of specific and sensitive genosensors for detecting these two closely related Fusarium species in food samples. The sensors are based on species-specific capture and detection probes, which bind to the intergenic spacer region of rDNA (IGS). Oligonucleotide functionalized magnetic microbeads are used to capture the target DNA which is then detected using biotinylated detection probes and a streptavidin-coupled label. The developed genosensors had detection limits of 1.8 pM and 3.0 pM for F. proliferatum and F. verticillioides, respectively, using synthetic DNA targets. Furthermore, the biosensors were used to analyze natural fungal contamination of commercial maize samples. After amplification of the genomic DNA the sensors detected the presence of the fungi, in accordance with previous results obtained with PCR. No cross-reactivity between F. verticillioides and F. proliferatum, or other fungi species tested, was observed. The developed biosensors can provide a valuable tool to evaluate the potential for mycotoxin contamination in conditions where detection of mycotoxins directly is challenging.

Clustered array of ochratoxin A biosynthetic genes in Aspergillus steynii and their expression patterns in permissive conditions.

Aspergillus steynii is probably the most relevant species of section Circumdati producing ochratoxin A (OTA). This mycotoxin contaminates a wide number of commodities and it is highly toxic for humans and animals. Little is known on the biosynthetic genes and their regulation in Aspergillus species. In this work, we identified and analysed three contiguous genes in A. steynii using 5'-RACE and genome walking approaches which predicted a cytochrome P450 monooxygenase (p450ste), a non-ribosomal peptide synthetase (nrpsste) and a polyketide synthase (pksste). These three genes were contiguous within a 20742 bp long genomic DNA fragment. Their corresponding cDNA were sequenced and their expression was analysed in three A. steynii strains using real time RT-PCR specific assays in permissive conditions in in vitro cultures. OTA was also analysed in these cultures. Comparative analyses of predicted genomic, cDNA and amino acid sequences were performed with sequences of similar gene functions. All the results obtained in these analyses were consistent and point out the involvement of these three genes in OTA biosynthesis by A. steynii and showed a co-ordinated expression pattern. This is the first time that a clustered organization OTA biosynthetic genes has been reported in Aspergillus genus. The results also suggested that this situation might be common in Aspergillus OTA-producing species and distinct to the one described for Penicillium species.

Aspergillus steynii and Aspergillus westerdijkiae as potential risk of OTA contamination in food products in warm climates.

Aspergillus steynii and Aspergillus westerdijkiae are the main ochratoxin A (OTA) producing species of Aspergillus section Circumdati. Due to its recent description, few data are available about the influence of ecophysiological factors on their growth and OTA production profiles. In this work, the effect of temperature (20, 24 and 28 °C) and water activity (aw) (0.928, 0.964 and 0.995) on growth, sporulation and OTA production by these fungi was examined in CYA and media prepared from paprika, green coffee, anise, grapes, maize and barley. Growth was positively affected by the highest temperature and aw values indicating that both species might be expected in warm climates or storage conditions. However, optimal growth conditions showed differences depending on the medium. OTA production was markedly affected by substrate and showed qualitative and quantitative differences. Both species, especially A. steynii, represent a great potential risk of OTA contamination due to their high production in a variety of conditions and substrates, in particular in barley and paprika-based media. Additionally, neither growth nor sporulation did result good indicators of OTA production by A. steynii or A. westerdijkiae; therefore, specific and highly-sensitive detection methods become essential tools for control strategies to reduce OTA risk by these species.

Structural variation and dynamics of the nuclear ribosomal intergenic spacer region in key members of the Gibberella fujikuroi species complex.

The intergenic spacer (IGS) region of the ribosomal DNA was cloned and sequenced in eight species within the Gibberella fujikuroi species complex with anamorphs in the genus Fusarium, a group that includes the most relevant toxigenic species. DNA sequence analyses revealed two categories of repeated elements: long repeats and short repeats of 125 and 8 bp, respectively. Long repeats were present in two copies and were conserved in all the species analyzed, whereas different numbers of short repeat elements were observed, leading to species-specific IGS sequences with different length. In Fusarium subglutinans and Fusarium nygamai, these differences seemed to be the result of duplication and deletion events. Here, we propose a model based on unequal crossing over that can explain these processes. The partial IGS sequence of 22 Fusarium proliferatum isolates was also obtained to study variation at the intraspecific level. The results revealed no differences in terms of number or pattern of repeated elements and detected frequent gene conversion events. These results suggest that the homogenization observed at the intraspecific level might not be achieved primarily by unequal crossing-over events but rather by processes associated with recombination such as gene conversion events.

Mechanisms involved in reduction of ochratoxin A produced by Aspergillus westerdijkiae using Debaryomyces hansenii CYC 1244.

Aspergillus westerdijkiae is one of the most relevant ochratoxin A (OTA) producing species within the Section Circumdati contaminating a number of agroproducts. The yeast Debaryomyces hansenii CYC 1244 was previously reported to be able to reduce growth and extracellular OTA produced by A. westerdijkiae. In this work, we examined several mechanisms possibly involved in this OTA reduction in in vitro experiments. OTA biosynthesis was evaluated by quantitation of expression levels of pks (polyketide synthase) and p450-B03 (cytochrome p450 monooxygenase) genes using newly developed and specific real time RT-PCR protocols. Both genes showed significant lower levels in presence of D. hansenii CYC 1244 suggesting an effect on regulation of OTA biosynthesis at transcriptional level. High levels of removal of extracellular OTA were observed by adsorption to yeast cell walls, particularly at low pH (98% at pH 3). On the contrary, no evidences were obtained of absorption of OTA into yeast cells or the production of constitutively expressed enzymes that degrade OTA by D. hansenii CYC 1244. These results described the potential of this yeast strain as a safe and efficient biocontrol agent to decrease OTA in A. westerdijkiae and two important mechanisms involved which may permit its application at different points of the food chain.

Aflatoxins and ochratoxin A in stored barley grain in Spain and impact of PCR-based strategies to assess the occurrence of aflatoxigenic and ochratoxigenic Aspergillus spp.

Contamination of barley by moulds and mycotoxins results in quality and nutritional losses and represents a significant hazard to the food chain. The presence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) and ochratoxin A (OTA) in stored barley in Spain has been studied. Species-specific PCR assays were used for detection of Aspergillus flavus, A. parasiticus, A. ochraceus, A. steynii, A. westerdijkiae, A. carbonarius and A. niger aggregate in mycotoxin-positive barley samples at different incubation times (0, 1 and 2 days). Classical enumeration techniques (CFU/g) in different culture media for evaluation of Aspergillus in sections Flavi, Circumdati and Nigri were also used. One hundred and five barley kernel samples were collected in Spanish grain stores from 2008 to 2010, and analyzed using a previously optimized method involving accelerated solvent extraction, cleanup by immunoaffinity column, liquid chromatographic separation, post-column derivatization with iodine and fluorescence detection. Twenty-nine samples were contaminated with at least one of the studied mycotoxins. AFB1, AFB2, AFG1, AFG2, and OTA were detected in 12.4%, 2.9%, 4.8%, 2.9%, and 20% of the samples, respectively. Aflatoxins and OTA co-occurred in 4.8% of the samples. Maximum mycotoxin levels (ng/g) were 0.61 (AFB1), 0.06 (AFB2), 0.26 (AFG1), 0.05 (AFG2), and 2.0 (OTA). The results of PCR assays indicated the presence of all the studied species, except A. westerdijkiae. The PCR assays showed high levels of natural contamination of barley with the studied species of Aspergillus which do not correspond to the expected number of CFU/g in the cultures. These results suggest that a high number of non-viable spores or hyphae may exist in the samples. This is the first study carried out on the levels of aflatoxins and OTA in barley grain in Spain. Likewise, this is the first report on the presence of aflatoxigenic and ochratoxigenic Aspergillus spp. in barley grain naturally contaminated with those mycotoxins using a species-specific PCR approach.