Novel Fermentation Strategies of Strawberry Tree Arbutus unedo Fruits to Obtain High Nutritional Value Products.

The strawberry tree (Arbustus unedo) is a medicinal plant and an important source of biocompounds, potentially useful for pharmaceutical and chemical applications to prevent or treat several human diseases. The strawberry tree fruits have usually been used to produce traditional products such as jams and jellies and to obtain fermented alcoholic drinks, representing the most valuable derivative products. Other fermented products are potentially interesting for their nutritional value; however, the fermentation process needs to be controlled and standardized to obtain high-quality products/ingredients. In this work, we investigated two different fermentative procedures, using strawberry tree whole fruit and fruit paste as matrices inoculated with a selected starter strain of Saccharomyces cerevisiae LI 180-7. The physical, chemical, microbiological and nutritional properties of fermented products were evaluated, as well as their antioxidant activity. The new obtained fermented products are enriched in organic acids (acetic acid varied from 39.58 and 57.21 mg/g DW and lactic acid from 85.33 to 114.1 mg/g DW) and have better nutritional traits showing a higher amount of total polyphenols (phenolic acids, flavonoids and anthocyanins) that ranged from 1852 mg GAE/100 g DW to 2682 mg GAE/100 g DW. Also, the amount of isoprenoid increased ranging from 155.5 µg/g DW to 164.61 µg/g DW. In this regard, the most promising strategy seemed to be the fermentation of the fruit paste preparation; while the extract of fermented whole fruits showed the most powerful antioxidant activity. Finally, a preliminary attempt to produce a food prototype enriched in fermented strawberry tree fruits suggested the whole fruit fermented sample as the most promising from a preliminary sensory analysis.

Cadmium treatment induces endoplasmic reticulum stress and unfolded protein response in Arabidopsisthaliana.

We report about the response of Arabidopsis thaliana to chronic and temporary Cd2+ stress, and the Cd2+ induced activation of ER stress and unfolded protein response (UPR). Cd2+-induced UPR proceeds mainly through the bZIP60 arm, which in turn activates relevant ER stress marker genes such as BiP3, CNX, PDI5 and ERdj3B in a concentration- (chronic stress) or time- (temporary stress) dependent manner. A more severe Cd-stress triggers programmed cell death (PCD) through the activation of the NAC089 transcription factor. Toxic effects of Cd2+ exposure are reduced in the Atbzip28/bzip60 double mutant in terms of primary root length and fresh shoot weight, likely due to reduced UPR and PCD activation. We also hypothesised that the enhanced Cd2+ tolerance of the Atbzip28/bzip60 double mutant is due to an increase in brassinosteroids signaling, since the amount of the brassinosteroid insensitive1 receptor (BRI1) protein decreases under Cd2+ stress only in Wt plants. These data highlight the complexity of the UPR pathway, since the ER stress response is strictly related to the type of the treatment applied and the multifaceted connections of ER signaling. The reduced sensing of Cd2+ stress in plants with UPR defects can be used as a novel strategy for phytoremediation.

Impact of environmental factors on ochratoxin A: From natural occurrence to control strategy.

Ochratoxin A (OTA) contamination and the associated issues of food security, food safety and economic loss are widespread throughout the world. The occurrence of OTA depends on ochratoxigenic fungi, foodstuffs and their environment. In this review, natural occurrence and control strategy of OTA, with a focus on the impact of environmental factors, are summarized. First, this manuscript introduces potentially contaminated foodstuffs, including the emerging ones which are not regulated in international legislation. Secondly, it gives an update of native producers based on foodstuffs and OTA biosynthesis. Thirdly, complicated environmental regulation is disassembled into individual factors in order to clarify their regulatory effect and mechanism. Finally, to emphasize control OTA at all stages of foodstuffs from farm to table, strategies used at crop planting, harvest, storage and processing stages are discussed.

Potential of Trichoderma spp. for Biocontrol of Aflatoxin-Producing Aspergillus flavus.

The inhibitory action of 20 antagonistic Trichoderma isolates against the aflatoxigenic isolate A. flavus ITEM 9 (Af-9) and their efficacy in reducing aflatoxin formation in vitro were examined. Production of metabolites with inhibitory effect by the Trichoderma isolates was also investigated. Antagonistic effect against Af-9 was assessed by inhibition of radial growth of the colonies and by fungal interactions in dual confrontation tests. A total of 8 out of 20 isolates resulted in a significant growth inhibition of 3-day-old cultures of Af-9, ranging from 13% to 65%. A total of 14 isolates reduced significantly the aflatoxin B1 (AfB1) content of 15-day-old Af-9 cultures; 4 were ineffective, and 2 increased AfB1. Reduction of AfB1 content was up to 84.9% and 71.1% in 7- and 15-day-old cultures, respectively. Since the inhibition of Af-9 growth by metabolites of Trichoderma was not necessarily associated with inhibition of AfB1 production and vice versa, we investigated the mechanism of reduction of AfB1 content at the molecular level by examining two strains: one (T60) that reduced both growth and mycotoxin content; and the other (T44) that reduced mycotoxin content but not Af-9 growth. The expression analyses for the two regulatory genes aflR and aflS, and the structural genes aflA, aflD, aflO and aflQ of the aflatoxin biosynthesis cluster indicated that neither strain was able to downregulate the aflatoxin synthesis, leading to the conclusion that the AfB1 content reduction by these Trichoderma strains was based on other mechanisms, such as enzyme degradation or complexation. Although further studies are envisaged to identify the metabolites involved in the biocontrol of A. flavus and prevention of aflatoxin accumulation, as well as for assessment of the efficacy under controlled and field conditions, Trichoderma spp. qualify as promising agents and possible alternative options to other biocontrol agents already in use.

Evidence of the Involvement of a Cyclase Gene in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius.

Ochratoxin A (OTA) is a well-known mycotoxin with wide distribution in food and feed. Fungal genome sequencing has great utility for identifying secondary metabolites gene clusters for known and novel compounds. A comparative analysis of the OTA-biosynthetic cluster in A. steynii, A. westerdijkiae, A. niger, A. carbonarius, and P. nordicum has revealed a high synteny in OTA cluster organization in five structural genes (otaA, otaB, ota, otaR1, and otaD). Moreover, a recent detailed comparative genome analysis of Aspergilli OTA producers led to the identification of a cyclase gene, otaY, located in the OTA cluster between the otaA and otaB genes, encoding for a predicted protein with high similarity to SnoaLs domain. These proteins have been shown to catalyze ring closure steps in the biosynthesis of polyketide antibiotics produced in Streptomyces. In the present study, we demonstrated an upregulation of the cyclase gene in A. carbonarius under OTA permissive conditions, consistent with the expression trends of the other OTA cluster genes and their role in OTA biosynthesis by complete gene deletion. Our results pointed out the involvement of a cyclase gene in OTA biosynthetic pathway for the first time. They represent a step forward in the understanding of the molecular basis of OTA biosynthesis in A. carbonarius.

Current Approaches for Advancement in Understanding the Molecular Mechanisms of Mycotoxin Biosynthesis.

Filamentous fungi are able to synthesise a remarkable range of secondary metabolites, which play various key roles in the interaction between fungi and the rest of the biosphere, determining their ecological fitness. Many of them can have a beneficial activity to be exploited, as well as negative impact on human and animal health, as in the case of mycotoxins contaminating large quantities of food, feed, and agricultural products worldwide and posing serious health and economic risks. The elucidation of the molecular aspects of mycotoxin biosynthesis has been greatly sped up over the past decade due to the advent of next-generation sequencing technologies, which greatly reduced the cost of genome sequencing and related omic analyses. Here, we briefly highlight the recent progress in the use and integration of omic approaches for the study of mycotoxins biosynthesis. Particular attention has been paid to genomics and transcriptomic approaches for the identification and characterisation of biosynthetic gene clusters of mycotoxins and the understanding of the regulatory pathways activated in response to physiological and environmental factors leading to their production. The latest innovations in genome-editing technology have also provided a more powerful tool for the complete explanation of regulatory and biosynthesis pathways. Finally, we address the crucial issue of the interpretation of the combined omics data on the biology of the mycotoxigenic fungi. They are rapidly expanding and require the development of resources for more efficient integration, as well as the completeness and the availability of intertwined data for the research community.

Susceptibility to Xylella fastidiosa and functional xylem anatomy in Olea europaea: revisiting a tale of plant-pathogen interaction.

Xylella fastidiosa is a xylem-limited bacterium causing the Olive Quick Decline Syndrome, which is currently devastating the agricultural landscape of Southern Italy. The bacterium is injected into the xylem vessels of leaf petioles after the penetration of the insect vector's stylet. From here, it is supposed to colonize the xylem vasculature moving against water flow inside conductive vessels. Widespread vessel clogging following the bacterial infection and causing the failure of water transport seemed not to fully supported by the recent empirical xylem anatomical observations in infected olive trees. We tested the hypothesis that the higher susceptibility to the X. fastidiosa's infection in Cellina di Nardò compared with Leccino is associated to the higher vulnerability to air embolism of its larger vessels. Such hypothesis is motivated by the recognized ability of X. fastidiosa in degrading pit membranes and also because air embolism would possibly provide microenvironmental conditions more favourable to its more efficient aerobic metabolism. We revised the relevant literature on bacterium growth and xylem physiology, and carried out empirical field, mid-summer measurements of xylem anatomy and native embolism in olive cultivars with high (Cellina di Nardò) and low susceptibility (Leccino) to the infection by X. fastidiosa. Both cultivars had similar shoot mass traits and vessel length (~80 cm), but the highly susceptible one had larger vessels and a lower number of vessels supplying a given leaf mass. Native air embolism reduced mean xylem hydraulic conductance by ~58 % (Cellina di Nardò) and ~38 % (Leccino). The higher air-embolism vulnerability of the larger vessels in Cellina di Nardò possibly facilitates the X. fastidiosa's infection compared to Leccino. Some important characteristics of the vector-pathogen-plant interactions still require deep investigations acknowledging both the pathogen metabolic pathways and the biophysical principles of xylem hydraulics.

Interacting climate change factors (CO2 and temperature cycles) effects on growth, secondary metabolite gene expression and phenotypic ochratoxin A production by Aspergillus carbonarius strains on a grape-based matrix.

Little is known on the impact that climate change (CC) may have on Aspergillus carbonarius and Ochratoxin A (OTA) contamination of grapes, especially in the Mediterranean region where in CC scenarios temperature are expected to increase by +2-5 °C and CO2 from 400 to 800/1200 ppm. This study examined the effect of (i) current and increased temperature in the alternating 11.5 h dark/12.5 h light cycle (15-28 °C vs 18-34 °C), representative of the North Apulia area, South Italy and (ii) existing and predicted CO2 concentrations (400 vs 1000 ppm), on growth, expression of biosynthetic genes (AcOTApks, AcOTAnrps, AcOTAhal, AcOTAp450, AcOTAbZIP) and regulatory genes of Velvet complex (laeA/veA/velB, "velvet complex") involved in OTA biosynthesis and OTA phenotypic production by three strains of A. carbonarius. The experiments made on a grape-based matrix showed that elevated CO2 resulted in a general stimulation of growth and OTA production. These results were also supported by the up-regulation of both structural and regulatory genes involved in the OTA biosynthesis. Our work has shown for the first time that elevated CO2 concentration in the Mediterranean region may result in an increased risk of OTA contamination in the wine production chain.

Comparative Genomic Analysis of Ochratoxin A Biosynthetic Cluster in Producing Fungi: New Evidence of a Cyclase Gene Involvement.

The widespread use of Next-Generation Sequencing has opened a new era in the study of biological systems by significantly increasing the catalog of fungal genomes sequences and identifying gene clusters for known secondary metabolites as well as novel cryptic ones. However, most of these clusters still need to be examined in detail to completely understand the pathway steps and the regulation of the biosynthesis of metabolites. Genome sequencing approach led to the identification of the biosynthetic genes cluster of ochratoxin A (OTA) in a number of producing fungal species. Ochratoxin A is a potent pentaketide nephrotoxin produced by Aspergillus and Penicillium species and found as widely contaminant in food, beverages and feed. The increasing availability of several new genome sequences of OTA producer species in JGI Mycocosm and/or GenBank databanks led us to analyze and update the gene cluster structure in 19 Aspergillus and 2 Penicillium OTA producing species, resulting in a well conserved organization of OTA core genes among the species. Furthermore, our comparative genome analyses evidenced the presence of an additional gene, previously undescribed, located between the polyketide and non-ribosomal synthase genes in the cluster of all the species analyzed. The presence of a SnoaL cyclase domain in the sequence of this gene supports its putative role in the polyketide cyclization reaction during the initial steps of the OTA biosynthesis pathway. The phylogenetic analysis showed a clustering of OTA SnoaL domains in accordance with the phylogeny of OTA producing species at species and section levels. The characterization of this new OTA gene, its putative role and its expression evidence in three important representative producing species, are reported here for the first time.

Effects of temperature and water activity change on ecophysiology of ochratoxigenic Aspergillus carbonarius in field-simulating conditions.

Ochratoxin A (OTA) is the primary mycotoxin threat in wine and dried vine fruits. Its presence in grape and wine is strongly related to climatic conditions and the expected climate change could represent a risk of increasing fungal colonization and OTA contamination in grapes. In this regard, the interacting effect of i) different conditions of water availability (0.93 and 0.99aw) and ii) different 10 h/14 h dark/light alternating temperature conditions simulating a nowadays (18/31 °C) and climate change scenario (20/37 °C) in high OTA risk areas of Apulia region, were studied. Lag phases prior to growth, mycelial growth rate, the expression of biosynthesis, transcription factors and regulatory genes of OTA cluster and OTA production were analysed in Aspergillus carbonarius ITEM 5010 under the combined effect of different climatic factors. At 18/31 °C and under water stress conditions (0.93 aw) the growth rate was slower than at 0.99 aw; on the contrary, at 20/37 °C a higher growth rate was observed at 0.93 aw. An over-expression of OTA genes and genes belonging to the global regulator Velvet complex was observed at 18/31 °C and 0.99 aw, with the specific OTA pathway transcription factor bZIP showing the highest expression level. The up-regulated transcription profile of the genes positively correlated with OTA production higher at 18/31 °C than at 20/37 °C and 0.99 aw; while no OTA production was detected at 0.93 aw at each of the temperature conditions tested. These findings provide preliminary evidence that the possible increase of the temperature, likely to happen in some areas of the Apulia region, may results in a reduction of both A. carbonarius spoilage and OTA production in grapes.

Identification of Safety and Quality Parameters for Preparation of Jellyfish Based Novel Food Products.

Edible jellyfish are mainly consumed and marketed in Southeastern Countries, generally produced by a multi-phase drying process, using mixtures of salt and alum. Recently, jellyfish have become very attractive also for Western food markets. They are novel food in Europe and no recognized handling/processing steps have been set up yet. Moreover, no specific food safety and quality parameters are available. In this study, we identified a set of safety and quality parameters for jellyfish, based on standards and process hygiene criteria used in Europe for other products. These assays were tested on three different jellyfish preparations that can be used as raw materials for subsequent food processing. All jellyfish samples revealed the absence of pathogens (Salmonella spp. and Listeria monocytogenes), Enterobacteriaceae and Pseudomonas spp., even if a limited presence of Staphylococci was observed. No biogenic amine histamine was detected and negligible levels of total volatile basic nitrogen (TVB-N) were revealed. Total bacterium, yeast and mold counts were negligible or undetectable by conventional accredited methods, and conversely the results were higher when optimized saline conditions were used. This study, for the first time, established a set of quality and safety parameters necessary for first-operations and subsequent processing of jellyfish as novel food. Highlights: Jellyfish can represent a novel food in Europe. Identification of safety and quality parameters for jellyfish food products. Saline conditions are essential for improving safety and quality assessment of jellyfish as food.

Oxylipin dynamics in Medicago truncatula in response to salt and wounding stresses.

Multiple stresses are becoming common challenges in modern agriculture due to environmental changes. A large set of phytochemicals collectively known as oxylipins play a key role in responses to several stresses. Understanding the fine-tuned plant responses to multiple and simultaneous stresses could open new perspectives for developing more tolerant varieties. We carried out the molecular and biochemical profiling of genes, proteins and active compounds involved in oxylipin metabolism in response to single/combined salt and wounding stresses on Medicago truncatula. Two new members belonging to the CYP74 gene family were identified. Gene expression profiling of each of the six CYP74 members indicated a tissue- and time-specific expression pattern for each member in response to single/combined salt and wounding stresses. Notably, hormonal profiling pointed to an attenuated systemic response upon combined salt and leaf wounding stresses. Combined, these results confirm the important role of jasmonates in legume adaptation to abiotic stresses and point to the existence of a complex molecular cross-talk among signals generated by multiple stresses.

EFR-Mediated Innate Immune Response in Arabidopsis thaliana is a Useful Tool for Identification of Novel ERQC Modulators.

Plants offer a simpler and cheaper alternative to mammalian animal models for the study of endoplasmic reticulum glycoprotein folding quality control (ERQC). In particular, the Arabidopsis thaliana (At) innate immune response to bacterial peptides provides an easy means of assaying ERQC function in vivo. A number of mutants that are useful to study ERQC in planta have been described in the literature, but only for a subset of these mutants the innate immune response to bacterial elicitors has been measured beyond monitoring plant weight and some physio-pathological parameters related to the plant immune response. In order to probe deeper into the role of ERQC in the plant immune response, we monitored expression levels of the Phosphate-induced 1 (PHI-1) and reticulin-oxidase homologue (RET-OX) genes in the At ER α-Glu II rsw3 and the At UGGT uggt1-1 mutant plants, in response to bacterial peptides elf18 and flg22. The elf18 response was impaired in the rsw3 but not completely abrogated in the uggt1-1 mutant plants, raising the possibility that the latter enzyme is partly dispensable for EF-Tu receptor (EFR) signaling. In the rsw3 mutant, seedling growth was impaired only by concomitant application of the At ER α-Glu II NB-DNJ inhibitor at concentrations above 500 nM, compatibly with residual activity in this mutant. The study highlights the need for extending plant innate immune response studies to assays sampling EFR signaling at the molecular level.

In vitro activity of antimicrobial compounds against Xylella fastidiosa, the causal agent of the olive quick decline syndrome in Apulia (Italy).

Olive quick decline syndrome (OQDS) causes severe damages to the olive trees in Salento (Apulia, Italy) and poses a severe threat for the agriculture of Mediterranean countries. DNA-based typing methods have pointed out that OQDS is caused by a single outbreak strain of Xylella fastidiosa subsp. pauca referred to as CoDiRO or ST53. Since no effective control measures are currently available, the objective of this study was to evaluate in vitro antimicrobial activities of different classes of compounds against Salento-1 isolated by an OQDS affected plant and classified as ST53. A bioassay based on agar disk diffusion method revealed that 17 out of the 32 tested antibiotics did not affect bacterial growth at a dose of 5 µg disk-1. When we assayed micro-, ultra- and nano-filtered fractions of olive mill wastewaters, we found that the micro-filtered fraction resulted to be the most effective against the bacterium. Moreover, some phenolics (4-methylcathecol, cathecol, veratric acid, caffeic acid, oleuropein) were active in their pure form. Noteworthy, also some fungal extracts and fungal toxins showed inhibitory effects on bacterial growth. Some of these compounds can be further explored as potential candidate in future applications for curative/preventive treating OQDS-affected or at-risk olive plants.

Comparative genomics reveals high biological diversity and specific adaptations in the industrially and medically important fungal genus Aspergillus.

BACKGROUND: The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrial applications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants of food, and an important genetic model. The genome sequences of eight aspergilli have already been explored to investigate aspects of fungal biology, raising questions about evolution and specialization within this genus. RESULTS: We have generated genome sequences for ten novel, highly diverse Aspergillus species and compared these in detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary and secondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation and diversity among the species. Observed genomic differences were validated with experimental studies. This revealed several highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature of black aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stress response. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genome sequenced species with other aspergilli. CONCLUSIONS: Many aspects of biological differences between fungal species cannot be explained by current knowledge obtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the first time a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genome differences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

Aspergillus Species and Their Associated Mycotoxins.

The genus Aspergillus is among the most abundant and widely distributed organism on earth, and at the moment comprises 339 known species. It is one of the most important economically fungal genus and the biotechnological use of Aspergillus species is related to production of soy sauce, of different hydrolytic enzymes (amylases, lipases) and organic acid (citric acid, gluconic acid), as well as biologically active metabolites such as lovastatin. Although they are not considered to be major cause of plant diseases, Aspergillus species are responsible for several disorders in various plants and plant products, especially as opportunistic storage moulds. The notable consequence of their presence is contamination of foods and feeds by mycotoxins, among which the most important are aflatoxins, ochratoxin A, and, at a less extent, fumonisins. Aflatoxins B1, B2, G1, G2 are the most toxic and carcinogenic mycotoxins, due to their extreme hepatocarcinogenicity; ochratoxin A is a potent nephrotoxin, it is also carcinogenic, teratogenic, and immunotoxic in rats and possibly in humans; fumonisins are hepatotoxic and nephrotoxic with potential carcinogenic effects on rat and mice. In this chapter we summarize the main aspects of morphology, ecology, epidemiology, and toxigenicity of Aspergillus foodborne pathogens which belong to sections Flavi, Circumdati, and Nigri, occurring in several agricultural products and responsible of aflatoxin, ochratoxin A, and fumonisins contamination of food and feed.

Targeting Ochratoxin Biosynthetic Genes.

The pathway of ochratoxin A (OTA) biosynthesis has not yet been completely elucidated. Essentially, two kind of genes have been demonstrated to be involved in the biosynthesis of OTA. One of them is the nrps gene encoding a non-ribosomal peptide synthetase (NRPS) which catalyzes the ligation between the isocoumarin group, constituting the polyketide group of OTA molecule, and the amino acid phenylalanine.Here we describe a conventional PCR method developed for the detection of OTA-producing molds belonging to Penicillium and Aspergillus genera by Luque et al. (Food Control 29:270-278, 2013). This method is based on the OTA nrps gene of Penicillium nordicum. It produces a specific amplicon of 459 bp and its functionality in naturally infected samples was also demonstrated.

Variation in Fumonisin and Ochratoxin Production Associated with Differences in Biosynthetic Gene Content in Aspergillus niger and A. welwitschiae Isolates from Multiple Crop and Geographic Origins.

The fungi Aspergillus niger and A. welwitschiae are morphologically indistinguishable species used for industrial fermentation and for food and beverage production. The fungi also occur widely on food crops. Concerns about their safety have arisen with the discovery that some isolates of both species produce fumonisin (FB) and ochratoxin A (OTA) mycotoxins. Here, we examined FB and OTA production as well as the presence of genes responsible for synthesis of the mycotoxins in a collection of 92 A. niger/A. welwitschiae isolates from multiple crop and geographic origins. The results indicate that (i) isolates of both species differed in ability to produce the mycotoxins; (ii) FB-nonproducing isolates of A. niger had an intact fumonisin biosynthetic gene (fum) cluster; (iii) FB-nonproducing isolates of A. welwitschiae exhibited multiple patterns of fum gene deletion; and (iv) OTA-nonproducing isolates of both species lacked the ochratoxin A biosynthetic gene (ota) cluster. Analysis of genome sequence data revealed a single pattern of ota gene deletion in the two species. Phylogenetic analysis suggest that the simplest explanation for this is that ota cluster deletion occurred in a common ancestor of A. niger and A. welwitschiae, and subsequently both the intact and deleted cluster were retained as alternate alleles during divergence of the ancestor into descendent species. Finally, comparison of results from this and previous studies indicate that a majority of A. niger isolates and a minority of A. welwitschiae isolates can produce FBs, whereas, a minority of isolates of both species produce OTA. The comparison also suggested that the relative abundance of each species and frequency of FB/OTA-producing isolates can vary with crop and/or geographic origin.

Identification of a Halogenase Involved in the Biosynthesis of Ochratoxin A in Aspergillus carbonarius.

UNLABELLED: Aspergillus carbonarius is the main responsible fungus of ochratoxin A (OTA) contamination of grapes and derived products. To date, the biosynthetic mechanism of this mycotoxin has been partially elucidated. Availability of genome sequence of A. carbonarius has allowed the identification of a putative gene cluster involved in OTA biosynthesis. This region hosts the previously characterized AcOTAnrps and AcOTApks genes encoding two key enzymes of the biosynthetic pathway. At about 4,400 nucleotides downstream of these loci, a gene encoding a putative flavin dependent-halogenase came out from the annotation data. Its proximity to OTA biosynthetic genes and its sequence analysis have suggested a role in the biosynthesis of OTA, directed to the introduction of the chlorine atom in the C-5 position of the final molecular structure of this mycotoxin. The deduced protein sequence of the halogenase gene, we designated AcOTAhal, shows a high similarity to a halogenase that is located in the OTA cluster of A. niger The deletion of the halogenase gene completely eliminated the production of ochratoxin A in A. carbonarius and determined a significant increase of ochratoxin B, as confirmed by mass spectrometry analysis. Moreover, its expression profile was similar to the two biosynthetic genes previously identified, AcOTApks and AcOTAnrps, indicating a strong correlation of the AcOTAhal gene with the kinetics of OTA accumulation in A. carbonarius. Therefore, experimental evidence confirmed that the chlorination step which converts OTB in OTA represents the final stage of the biosynthetic pathway, supporting our earlier hypothesis on the order of enzymatic steps of OTA biosynthesis in A. carbonarius IMPORTANCE: Ochratoxin A is a potent mycotoxin classified as a possible carcinogen for humans, and Aspergillus carbonarius is the main agent responsible for OTA accumulation in grapes. We demonstrate here that a flavin-halogenase is implicated in the biosynthesis of OTA in A. carbonarius The encoding gene, AcOTAhal, is contiguous to biosynthetic genes that we have already described (nrps and pks), resulting as part of the biosynthetic cluster. The encoded protein is responsible of the introduction of chlorine atom in the final molecular structure and acts at the last step in the pathway. This study can be considered a continuation of an earlier study wherein we started to clarify the molecular basis of OTA biosynthesis in A. carbonarius, which has not been completely elucidated until now. This research represents an important step forward to a better understanding of the production mechanism, which will contribute to the development of improved control strategies to reduce the risk of OTA contamination in food products.

Study of gene expression and OTA production by Penicillium nordicum during a small-scale seasoning process of salami.

Penicillium nordicum, an important and consistent producer of ochratoxin A (OTA), is a widely distributed contaminant of protein rich food with elevated NaCl. It is usually found on dry-cured meat products and is considered the main species responsible for their contamination by OTA. The aim of this work was to study the gene expression of a polyketide synthase (otapksPN) involved in P. nordicum OTA biosynthesis, and OTA production during a small-scale seasoning process. Fresh pork sausages were surface inoculated with P. nordicum and seasoned for 30days. Gene expression and OTA production were monitored throughout the seasoning process after 4, 5, 6, 7, 10, 14, and 30days. The expression of otapksPN gene was already detected after 4days and increased significantly after 7days of seasoning, reaching the maximum expression level after 10days (1.69×10(4)copies/100mg). Consistently with gene expression monitoring, OTA was detected from the 4th day and its content increased significantly from the 7th day, reaching the maximum level after 10days. In the late stages of the seasoning process, OTA did not increase further and the number of gene copies was progressively reduced after 14 and 30days.