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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.

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.

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.

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&ndash, 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.

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.

The most serious aerial disease of garlic is leaf blight caused by Stemphylium spp. Geographical variation in the causal agent of this disease is indicated. Stemphylium vesicarium has been reported in Spain, whereas S. solani is the most prevalent species recorded in China. In this study, Stemphylium isolates were obtained from symptomatic garlic plants sampled from the main Spanish production areas. Sequence data for the ITS1–5.8S–ITS2 region enabled assignation of the isolates to the Pleospora herbarum complex and clearly distinguished the isolates from S. solani. Conidial morphology of the isolates corresponded to that of S. vesicarium and clearly discriminated them from S. alfalfae and S. herbarum on the basis of the size and septation pattern of mature conidia. Conidial morphology as well as conidial length, width and length:width ratio also allowed the Spanish isolates to be distinguished from S. botryosum and S. herbarum. Control of leaf blight of garlic is not well established. Few studies are available regarding the effectiveness of chemical treatments to reduce Stemphylium spp. incidence on garlic. The effectiveness of nine fungicides of different chemical groups to reduce Stemphylium mycelial growth in vitro was tested. Boscalid + pyraclostrobin (group name, succinate dehydrogenase inhibitors + quinone outside inhibitors), iprodione (dicar-boximide), and prochloraz (demethylation inhibitors) were highly effective at reducing mycelial growth in S. vesicarium with EC50 values less than 5 ppm. In general, the effectiveness of the fungicide was enhanced with increasing dosage.

Fusarium proliferatum (Matsushima) Nirenberg is one of the most important Fusarium species considering its worldwide distribution, its pathogenic ability in a wide range of crops, and its mycotoxigenic potential. F. proliferatum conidia are easily carried by rainwater and, in this work, five isolates from rainwater were evaluated as inoculum sources to produce garlic rot and their pathogenic ability were compared with six F. proliferatum isolates from diseased garlic. A phylogenetic analysis was also performed to unravel possible genetic differences among the isolates. Pathogenicity tests were performed in three garlic varieties (purple, white and purple Chinese) and the results revealed that the isolates from rainwater were pathogenic to each of them. However, in all cases, their pathogenic ability was lower than in the case of isolates obtained from rooted garlic. The phylogenetic analysis based on parsimony carried out with the hypervariable region of the intergenic transcribed spacer (IGS) in F. proliferatum showed genetic differences depending on the origin of the isolates. Isolates from rainwater were definitely separated in an independent branch from garlic isolates. This paper shows an interesting approach to the ecological significance of F. proliferatum as a fungal pathogen on garlic. Some degree of relation between genetic variability and pathogenicity was also demonstrated.

Consumer dietary habits have drastically changed in recent decades and functional beverages now have a strong position in the market. The majority of these beverages are produced using simple processes that use raw products, such as cereals, legumes, fruits, and nuts, among others, and these are known to be frequently contaminated with mycotoxins. This review is focused on the occurrence of these toxic compounds in plant-based milks, fruit juices, and herbal teas. The fate of the toxins during processing is discussed to establish the potential risk posed by the consumption of these kind of beverages regarding mycotoxin uptake.

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.

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.

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.

Ochratoxin A (OTA) is the main mycotoxin occurring in wine. This review article is focused on the distribution of this toxin and its producing-fungi in grape berries, as well as on the fate of OTA during winemaking procedures. Due to its toxic properties, OTA levels in wine are regulated in different countries; therefore, it is necessary to apply control and detoxification methods that are also discussed in this revision.

López-Goñi, Ignacio et al., Social networks are already being exploited for searching, storing, and sharing knowledge, demonstrating that they are an efficient vehicle for social learning. Consequently, they could be implemented as a competent tool for formal learning. Twitter is among the 10 most popular online social networks, integrating a community of over 500 million users around the world. Twitter has already been used in several educational programs and evaluated as a positive teaching experience with an outstanding potential in academic and educational environments (1–6). However, there are scarce examples available in the literature about its use in science teaching and communication. In this work, we present and analyze the application of Twitter to create an online space for communication and learning of basic microbiology. With this aim, a team of professionals in the field has imparted, to our knowledge, the first worldwide open access microbiology course via Twitter. Here we assess the results of our experience of using this social network as a tool for teaching, promoting, and communicating scientific knowledge to a wide audience.

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.

The determination of aflatoxin production ability and differentiation of aflatoxigenic strains can be assessed by monitoring the expression of one or several key genes using reverse transcription polymerase chain reaction (RT-PCR). We herein describe the methods for RNA induction, extraction, and quality determination, and the RT-PCR conditions used to evaluate the ability of a given Aspergillus strain to produce aflatoxins.

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.

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.

The determination of aflatoxin production ability and differentiation of aflatoxigenic strains can be assessed by monitoring the expression of one or several key genes using reverse transcription polymerase chain reaction (RT-PCR). We herein describe the methods for RNA induction, extraction, and quality determination, and the RT-PCR conditions used to evaluate the ability of a given Aspergillus strain to produce aflatoxins. info:eu-repo/semantics/publishedVersion

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.

The objective of this study was to gain information about the toxigenic Aspergillus species present in a wide survey of retail samples of paprika and chilli collected in Spain. Detection of these mycotoxigenic species was performed with an optimized protocol for paprika and chilli which includes a set of species-specific PCR assays. Occurrence of toxigenic Aspergillus species was higher in paprika than in chilli samples (83.9% and 64.5%, respectively). Paprika showed also the highest percentage of co-occurrence of two or more different species (43.6%) in comparison with chilli (35.5%). The most common aspergilli were Aspergillus niger aggregate (67.7%), followed by Aspergillus flavus (49.5%). Aspergillus carbonarius, Aspergillus parasiticus and Aspergillus steynii were detected at lower frequency (1.1%). The high co-occurrence of Aspergillus species able to produce ochratoxin A and aflatoxins, particularly in paprika, suggested the need of a more efficient control during processing and storage to reduce fungal contamination, and additional legislation to consider the simultaneous presence of both toxins in these matrices.

Aflatoxins are important mycotoxins that represent a serious risk for human and animal health. These mycotoxins are mainly produced by Aspergillus flavus and Aspergillus parasiticus, two closely related species with different array of aflatoxins. In this work, two specific quantitative PCR (qPCR) assays were developed to detect and quantify both species in wheat flour using primers based on the multicopy ITS2 rDNA target sequence. The species specificity of the assays was tested in a wide range of strains of these species and others colonizing the same commodities. The sensitivity of the assay was estimated in 2.5 pg/reaction in both species. Discrimination capacity for detection and relative quantification of A. flavus and A. parasiticus DNA were analyzed using samples with DNA mixtures containing also other fungal species at different ratios. Both qPCR assays could detect spore concentrations equal or higher than 10(6)spores/g in flour samples without prior incubation. These assays are valuable tools to improve diagnosis at an early stage and in all critical control points of food chain integrated in HACCP strategies.

International audience; Aspergillus parasiticus is considered one of the most important aflatoxin producing species which contaminates foodstuffs and beverages for human consumption. In this work, a specific and highly sensitive PCR protocol was developed to detect A. parasiticus using primers designed on the multicopy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA). The assay proved to be highly specific to A. parasiticus when it was tested in a wide sample of related species and other fungal species commonly found in commodities, and it allowed discrimination from the closely related A. flavus. Accuracy of detection and quantification by conventional PCR were tested with genomic DNA obtained from wheat flour artificially contaminated with spore suspensions of known concentrations. Spore concentrations equal or higher than 106 spore/g could be detected by the assay directly without prior incubation of the samples. The assay described might be incorporated in routine analyses at critical points of the food chain within HAAPC strategies.

Agroproducts contaminated by ochratoxin A (OTA) represent a risk for human and animal health and, therefore, maximum limits have been established by Food Safety Authorities. Reduction of OTA contamination may be accomplished by early detection of OTA-producing fungal species using rapid, specific and sensitive detection and quantification by PCR-based methods. Aspergillus carbonarius is one of the most important OTA-producing species, in particular in grapes and derivatives from Mediterranean regions. In this work, highly efficient quantitative PCR assays using SYBR Green I and TaqMan methods were developed for specific detection of A. carbonarius to be used in grapes. The primers and the TaqMan probe were based on the internal transcribed region 2 multicopy region (internal 2 sequence of the rRNA gene). The specificity and sensitivity of both assays were tested on genomic DNA mixtures of several A. carbonarius strains and other fungal species frequently present in grapes. Both methods were also compared using grapes inoculated with different spore concentrations of A. carbonarius, detecting up to 0.4 pg DNA g(-1) grape berries. The efficiency and sensitivity of both methods were comparable and only the lower cost of SYBR Green might favour its use in routine screenings.

Aspergillus ochraceus and A. westerdijkiae are considered the most important Ochratoxin A (OTA) producing species included in Aspergillus section Circumdati which contaminate foodstuffs and beverages for human consumption. In this work a real-time quantitative PCR protocol was developed to detect both species using SYBR Green and primers designed on the basis of the multicopy ITS1 region of the rDNA. The assay had high efficiency (94%) and showed no inhibition by host or fungal DNA other than the target species. The lower detection limit of the target DNA was 2.5 pg/reaction. Accuracy of detection and quantification by qPCR were tested with genomic DNA obtained from green coffee beans and grapes artificially contaminated with spore suspensions of known concentrations. Spore concentrations equal or higher than 10(6) spore/ml could be detected by the assay directly without prior incubation of the samples and a positive relationship was observed between incubation time and qPCR values. The assay developed would allow rapid, specific, accurate and sensitive detection and quantification of A. ochraceus and A. westerdijkiae to be directly used in a critical point of the food chain, before harvesting green coffee and grape berries, to predict and control fungal growth and OTA production.

Ochratoxin A (OTA) is a highly toxigenic mycotoxin commonly present in a number of diverse agroproducts. Aspergillus Section Circumdati includes some of the most important OTA-producing species: Aspergillus ochraceus, Aspergillus elegans and the recently described Aspergillus steynii and Aspergillus westerdijkiae. In this work, OTA production by several strains of these four species from diverse origins and food matrices was examined. Identification of all the strains was carried out by specific PCR assays. The strains were cultivated in CYA solid medium and OTA was measured by HLPC. This study demonstrated for the first time the high capacity of A. steynii strains to produce OTA at higher levels than A. westerdijkiae,A. ochraceus and A. elegans strains showed low levels or no production at all. In conclusion, the results of this study suggest that the occurrence of A. steynii and A. westerdijkiae might represent the major potential risk for OTA contamination due to their high production and the diversity of commodities that these species may contaminate.

BACKGROUND Fourteen vineyards from two different agro-climatic regions in Spain were sampled in two consecutive years in order to determinate the grape mycobiota and diversity indexes with the final aim to define the potential mycotoxigenic species from both regions and their relationship. RESULTS The most common fungal genera encountered were Aspergillus (30.0%), Alternaria (53.2%), Cladosporium (11.9%) and Penicillium (2.9%). Black aspergilli presence in the hotter region (south) was significantly higher (P < 0.05) than in the northeast in both years. Among black aspergilli, A. tubingensis seemed to be the better adapted species to environmental conditions, while A. carbonarius was the main potentially ochratoxigenic species in both regions and years, owing to the most relevant percentage of ochratoxigenic isolates. Ochratoxin A (OTA)-positive musts were only detected from southern vineyards, although contamination was always lower than 0.1 µg L−1. Finally, none of black aspergilli tested produced fumonisins (FBs) on Czapek yeast extract agar (CYA), while 63% of A. niger tested produced FB2 when inoculated on CYA20S, reaching 100% of isolates from the south. CONCLUSION Climate change scenarios in southern Europe point to an increase in temperature and drought. This could promote particularly adapted species such as A. niger, decreasing OTA risk, but this could lead to an increase in FB2 presence. © 2014 Society of Chemical Industry

Aspergillus steynii and Aspergillus westerdijkiae are important ochratoxin A (OTA)-producing species frequently found in coffee. Although the processing of green coffee beans reduces markedly OTA contamination, levels exceeding the legal limitsmight remain in the final product. Environmental conditions are a crucial factor affecting growth and OTA production in fungal species; therefore, in thiswork,we analysed the effect of different levels of temperature (23, 28, 32, 37, 42 °C) andwater activity (aw) (0.89, 0.91, 0.93, 0.95, 0.97, 0.99) on growth and toxin production by A. steynii and A. westerdijkiae in green coffee-based medium. A. steynii was able to grow and produce OTA in a wider set of conditions than A. westerdijkiae. A new index (OTA risk index) has been described to integrate both fungal growth and OTA production and, according to it, A. steynii would pose a higher risk of OTA contamination in coffee than A. westerdijkiae at all the conditions tested. Neither A. steynii nor A. westerdijkiae were able to grow at the lowest value of aw (0.89) evaluated and OTA production was extremely low at 0.91. Therefore, the application of good practices during storage aiming to maintain low humidity levelsmight be essential to prevent OTA contamination in coffee at this stage. The optimal conditions of both species to grow and produce OTA were established at warm temperatures (28–32 °C) and high aw levels. Therefore, these species could be considered well-adapted in predicted climate change scenarios resulting in a potential high risk source of OTA contamination for this product.

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 cophysiological factors on their growth and OTA production profiles. In this work, the effect of temperature (20, 24 and 28ºC) and water activity (aw) (0.928, 0.964 and 0.995) on rowth, 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.

Sweet sorghum (Sorghum bicolor L.) is considered one of the most promising crops for bioethanol production in many countries and is a focus of bioenergy research worldwide. In July 2011, plants of the sweet sorghum cv. Suchro 506 in Oropesa (Toledo, Spain, 40.048577°N, 5.360298°W) (European Datum 1950 UTM zone 30 N) were observed with severe wilting. Upon examination, the lower internodes were found to be straw colored. When the plant was split, the internal pith was reddish, soft, and disintegrating. Small pieces of symptomatic stems and roots were surface disinfected in sodium hypochlorite (0.5% wt/vol) for 2 min and air dried. The sections were then placed on either PDA (potato dextrose agar) medium or Komada agar and incubated for 5 days at 25°C. Isolations from diseased stem and root tissue consistently yielded Fusarium verticillioides (Sacc.) Nirenberg (3). The small, hyaline, mostly single-celled, oval to club-shaped microconidia of F. verticillioides were produced in long catenate chains arising from monophialides. PCR amplification of the ITS1-5.8S-ITS2 was performed using the primers and protocols described elsewhere (4) and the fragments obtained were subsequently sequenced in both directions. Sequences were deposited in the EMBL Sequence Database (Accession Nos. HE652878, HE652879, HE652880, and HE652881). Four of the recovered F. verticilliodes isolates were tested in pathogenicity assays. One-week-old cultures of each isolate were homogenized in 400 ml of sterile water and 200 ml were used to inoculate water-growth-chamber-grown plants in 500-ml pots. Two pots each with three plants of cv. Suchro 506 were inoculated for each isolate. Water with sterile PDA was used as a control. All plants were kept at 20 to 25°C under a photoperiod of 14 h at 12,000 lux. After 21 days, above- and belowground parts were dried for 24 h at 60°C. Total length and dry weight of both sections were obtained. Inoculated plants produced root rot symptoms characteristic of F. verticillioides with dark red discolorations of the cortex of seedling roots (1), whereas the plants watered with water containing only PDA did not produce symptoms. Inoculated plants also had a decrease in dry weight for above- and belowground sections (P = 0.05) compared with the control with 43 and 47% reductions, respectively. The length of aerial parts was approximately 5% less in inoculated plants compared with control plants. F. verticillioides was reisolated from all inoculated plants. Sorghum stalk and root rot caused by F. verticillioides has been reported in different countries including India (2) and the United States (3). To our knowledge, this is the first report of F. verticillioides causing stalk and root rot of sorghum in Spain. An increase of production of this crop is expected to meet targets of the renewable energy share in Spain and any disease compromising yield may be a threat to this endeavour. References: (1) R. A. Frederiksen and G. N. Odvody. Compendium of Sorghum Diseases. The American Phytopathological Society. St. Paul, MN, 2000. (2) N. N. Khune et al. Indian Phytopathol. 37:316, 1984. (3) J. F. Leslie and B. A. Summerell. The Fusarium Laboratory Manual. Blackwell Publishing, Ames, IA, 2006. (4) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, New York, 1990.

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.

Ochratoxin A (OTA) is one of the most important mycotoxins because of its high toxicity to both humans and animals and its occurrence in a number of basic foods and agro-products. Some of the main OTA-producing species belong to Aspergillus section Circumdati, whose taxonomy has been lately revised with the description of new species. The high morphological similarity of these species (Aspergillus ochraceus, Aspergillus steynii and Aspergillus westerdijkiae) makes difficult discrimination among them and with respect to the other species included in the same section unable to produce OTA. In this work, PCR assays specific for the OTA-producing species, A. ochraceus, A. steynii and A. westerdijkiae, were developed on the basis of the multicopy ITS regions of the rDNA. The assays were tested in a wide sample of isolates from diverse origins and culture collections, confirming their specificity, and revealing that the current identification of strains from section Circumdati might need a revision.