Introduction. Fungi (yeasts and moulds) are recognized as one of the main contaminants of dairy products including yogurt and sour milk. These microorganisms can also cause spoilage in a wide range of processed, preserved and refrigerated food products. During the past years, several molecular methods based on immunological and genotypic techniques have been developed for revealing the presence of undesirable microorganisms, including fungi, in different food matrices. However, no commercial kit are already available to detect viable yeasts and moulds in dairy products. Materials and methods. Five antibodies against yeasts and molds were selected from commercially available antibodies and used to produce functionalized magnetic beads to be used to capture and separate microrganisms associated to dairy products. Four yeast type species (Debaryomyces hansenii, Kluyveromyces marxianus, Geotrichum candidum and Pichia anomala) and four mold species (Alternaria alternata, Aspergillus niger, Penicillium italicum and Rhizopus stolonifer) were used. Milk, yogurt and soft cheese were tested as matrices. A RT-PCR protocol was developed for detection of yeast and molds mRNA extracted from contaminated foods. Results. A new method for yeast and molds enrichment from different food dairy products (milk, yogurt and soft cheese) based on the use of antibody coated magnetic beads was developed. A new RT-PCR assay based on a nested amplification was optimized for the detection of yeast and molds in artificially contaminated dairy products.Discussion. The correlation between the amplification signal and the microbial count will allow to use this method for viable contaminants quantification in dairy products. This method can avoid the labor expensive food matrices treatments, often cause of loss of sensitivity. This approach will be transferred also in other food matrices. This procedure can be implemented by the use of automated enrichment systems already available for pathogen microorganisms.

Aflatoxins contamination by Aspergillus flavus is a matter of great concern for oil rich crops among which hazelnuts represent economically important agricultural commodities of Mediterranean countries, mainly used as mixed nuts or as ingredients in the bakery and confectionery industries. Since the biosynthetic pathway of aflatoxin biosynthesis has been elucidated in detail, expression analysis of the genes along the pathway can provide a thorough insight into the molecular mechanisms of toxin production and regulation. In the present work, we carried out a transcriptional analysis of the main genes belonging to aflatoxin biosynthetic cluster of A. flavus, namely the two regulatory genes aflR and aflS and the five structural genes aflD, aflM, aflO, aflP, and aflQ The analysis was carried out at different stages of fungal growth on two different media: hazelnut agar medium and YES medium. The transcripts of all the genes paralleled the synthesis of aflatoxin and both were detected starting around 36 h in YES medium, and 72 h in hazelnut agar medium. Significantly, the amount of anatoxin produced was about one order lower in hazelnut agar compared to YES medium. The expression of two genes encoding a lipase and a metalloprotease. potentially involved in lipid and protein catabolism, was also monitored during fungal growth. Noteworthy, the expression of the metalloprotease gene appeared to be specific for the hazelnut medium, whereas the lipase gene was expressed in both media. Finally, we verified the expression profiles of three genes encoding fatty acid dioxygenases/diol synthases involved in the biosynthesis of fungal oxylipins, namely ppoA, ppoB, ppoC Recent findings have pointed out the importance of fungal oxylipins in fungal growth/mycotoxin production and our results indicated that all the three ppo genes are expressed during A. flavus growth on hazelnut medium. In particular. ppoB appeared to be specifically expressed in this medium. This study reports for the first time on the expression profiles of genes belonging to the biosynthetic cluster and genes potentially involved in the regulation of fungal secondary metabolism during A. flavus colonisation of hazelnuts. (C) 2009 Elsevier B.V. All rights reserved.

Aspergillus carbonarius is the main responsible fungus of ochratoxin A (OTA) contamination of grapes and derived products. Recently, the biosynthetic mechanism of this mycotoxin has been mainly elucidated by experiments of knocking out of the key biosynthetic genes. The mutant strains of A. carbonarius, in which the AcOTAnrps gene had been disrupted, was unable to produce OTA but retained its ability to degrade OTA into OT? when it was grown in presence of exogenous OTA. Microbial degradation of OTA is due to the enzymatic cleavage of the amide bond between L-?-phenylalanine and OT? by proteolytic proteins. Then, an in silico screening has been made on the available genome sequence of A. carbonarius ITEM 5010 to identify genes encoding proteases and to investigate their involvement in the OTA degrading activity of A. carbonarius. Preliminary transcriptomic analysis allowed selecting eight protease encoding genes that were expressed at increased level during OTA production. From the analysis of functional domains of the deduced protein sequences, four identified genes encode for aspartic proteases, three of them encode for serine proteases and one for a metalloprotease. Wild type and three mutant strains of A. carbonarius ITEM 5010 (?AcOTAnrps, ?AcOTApks, ?AcOTAhal) previously obtained and resulted to be unable to produce OTA, have been incubated in presence of OTA under different conditions and time of growth. Expression levels during growth and activation rate of the selected protease genes are under investigation in order to establish their involvement in the degradation activity of A. carbonarius strains.

Aflatoxins and the producing fungi Aspergillus section Flavi are widely known as the most serious and dangerous mycotoxin issue in agricultural products. In Europe, before the outbreak of aflatoxins on maize (2003-2004) due to new climatic conditions, their contamination was confined to imported foods. Little information is available on molecular biodiversity and population structure of Aspergillus section Flavi in Europe. Preliminary reports evidenced the massive presence of Aspergillus flavus L-morphotype as the predominant species in maize field, no evidence of the highly toxigenic S-morphotype and of other aflatoxigenic species are reported. The risk of a shift in traditional occurrence areas for aflatoxins is expected in the world and in particular in South East of Europe due to the increasing average temperatures. Biological control of aflatoxin risk in the field by atoxigenic strains of A. flavus starts to be widely used in Africa and USA. Studies are necessary on the variation of aflatoxin production in populations of A. flavus to characterize stable atoxigenic A. flavus strains. The aim of present article is to give an overview on biodiversity and genetic variation of Aspergillus section Flavi in Europe in relation to the management of aflatoxins risk in the field.

Ochratoxin A (OTA) is a potent pentaketide nephrotoxin diffusely distributed in food and feed products (grains, legumes, coffee, dried fruits, meat derived products, beer and wine); it is also carcinogenic, neurotoxic, teratogenic and immunotoxic. This mycotoxin is produced by species belonging to the genus Aspergillus and Penicillium. OTA is the primary mycotoxin risk in wine and dried vine fruits, its maximum level is regulated by law. Several studies focused on Aspergillus section Nigri, due to their role as causative agents of black rot of grapes, and subsequently as cause of ochratoxin A contamination. In particular, Aspergillus carbonarius has been identified as the major cause of contamination in grape berries. This contamination is strongly related to climatic conditions, geographical regions, grape varieties, damage by insects, growing season; in particular great variations may occur from one year to another. So, climate represents an important key-factor in the agro-ecosystem, influencing fungal colonization and ochratoxin A production in grapes. Climate change is expected to have a profound effect on our landscape worldwide, and also to have an important impact on sustainable food production system. Recent studies have reported how the climate change may affect mycotoxins production in the fields and the relevant risk on economically important crops. In this regard, the interacting effect of water stress (aw 0.99-0.93) and different day/night climate conditions simulating nowadays (18-31 and 15-28°C) and climate change scenarios (18-34 and 20-37 °C) in high OTA risk area of Southern Italy during the ripening season, were studied. Mycelial growth rate, OTA production and molecular expression of key genes (PKS, NRPS, Hal, p450, bZIP) of OTA biosynthetic cluster by A. carbonarius ITEM 5010 were measured. Our results showed that, in water stress conditions (0.93 aw), no OTA production was observed and, except at 20-37°C, the growth rate was slower compared to 0.99 aw. A significantly higher amount of OTA was observed at 0.99 aw and 18-34°C climate change scenario. Gene expression, monitored by quantitative real time RT-PCR, gave evidence of the high expression levels of OTA biosynthetic genes in this condition, in particular NRPS and Hal genes were strongly expressed. These preliminary and new results on A. carbonarius in a climate change scenario suggest that a possible slight increase of temperature may lead to higher OTA contamination and to a possible expansion of the risk area in the Mediterranean basin.

The fungal genus Aspergillus is of critical importance to humankind. Species include those with industrialapplications, important pathogens of humans, animals and crops, a source of potent carcinogenic contaminants offood, and an important genetic model. The genome sequences of eight aspergilli have already been explored toinvestigate 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 thesein detail to sister and more distant genera. Comparative studies of key aspects of fungal biology, including primary andsecondary metabolism, stress response, biomass degradation, and signal transduction, revealed both conservation anddiversity among the species. Observed genomic differences were validated with experimental studies. This revealedseveral highlights, such as the potential for sex in asexual species, organic acid production genes being a key feature ofblack aspergilli, alternative approaches for degrading plant biomass, and indications for the genetic basis of stressresponse. A genome-wide phylogenetic analysis demonstrated in detail the relationship of the newly genomesequenced species with other aspergilli.Conclusions: Many aspects of biological differences between fungal species cannot be explained by current knowledgeobtained from genome sequences. The comparative genomics and experimental study, presented here, allows for the firsttime a genus-wide view of the biological diversity of the aspergilli and in many, but not all, cases linked genomedifferences to phenotype. Insights gained could be exploited for biotechnological and medical applications of fungi.

The need of powerful diagnostic tools for rapid, simple, and cost-effective detection of food-borne fungi has become very important in the area of food safety. Currently, several isothermal nucleic acid amplification methods have been developed as an alternative to PCR-based analyses. Loop-mediated isothermal amplification (LAMP) is one of these innovative methods; it does not require either gel electrophoresis to separate and visualize the products or expensive laboratory equipments and it has already been applied for detection of pathogenic organisms. In the current study, we developed a LAMP assay for the specific detection of Penicillium nordicum, the major causative agent of ochratoxin A contamination in protein-rich food, especially dry-cured meat products. The assay was based on targeting otapksPN gene, a key gene in the biosynthesis of ochratoxin A (OTA) in P. nordicum. Amplification of DNA during the reaction was detected directly in-tube by color transition of hydroxynaphthol blue from violet to sky blue, visible to the naked eye, avoiding further post amplification analyses. Only DNAs isolated from several P. nordicum strains led to positive results and no amplification was observed from non-target OTA and non OTA-producing strains. The assay was able to detect down to 100 fg of purified targeted genomic DNA or 102 conidia/reaction within 60 min. The LAMP assay for detection and identification of P. nordicum was combined with a rapid DNA extraction method set up on serially diluted conidia, providing an alternative rapid, specific and sensitive DNA-based method suitable for application directly "on-site", notably in key steps of dry-cured meat production.

Almonds are among the commodities at risk of aflatoxin contamination by Aspergillus flavus.Temperature and water activity are the two key determinants in pre and post-harvest environmentsinfluencing both the rate of fungal spoilage and aflatoxin production. Varying the combination ofthese parameters can completely inhibit or fully activate the biosynthesis of aflatoxin, so it isfundamental to know which combinations can control or be conducive to aflatoxin contamination.Little information is available about the influence of these parameters on aflatoxin production onalmonds. The objective of this study was to determine the influence of different combinations oftemperature (20°C, 28°C, and 37°C) and water activity (0.90, 0.93, 0.96, 0.99 aw) on growth,aflatoxin B1 (AFB1) production and expression of the two regulatory genes, aflR and aflS, and twostructural genes, aflD and aflO, of the aflatoxin biosynthetic cluster in A. flavus grown on analmond medium solidified with agar. Maximum accumulation of fungal biomass and AFB1production was obtained at 28°C and 0.96 aw; no fungal growth and AFB1 production wereobserved at 20°C at the driest tested conditions (0.90 and 0.93 aw). At 20° and 37°C AFB1production was 70-90% lower or completely suppressed, depending on aw. Reverse transcriptasequantitative PCR showed that the two regulatory genes (aflR and aflS) were highly expressed atmaximum (28°C) and minimum (20°C and 37°C) AFB1 production. Conversely the two structuralgenes (aflD and aflO) were highly expressed only at maximum AFB1 production (28°C and 0.96-0.99 aw). It seems that temperature acts as a key factor influencing aflatoxin production which isstrictly correlated to the induction of expression of structural biosynthesis genes (aflD and aflO),but not to that of aflatoxin regulatory genes (aflR and aflS), whose functional products are mostlikely subordinated to other regulatory processes acting at post translational level.The results of this study are useful to select conditions that could be used in the almond processingchain to suppress aflatoxin production in this important product

Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium species and is a common contaminant of a wide variety of food commodities, with Aspergillus carbonarius being the main producer of OTA contamination in grapes and wine. The molecular structure of OTA comprises a dihydroisocoumarin ring linked to phenylalanine and, as shown in different producing fungal species, a polyketide synthase (PKS) is a component of the OTA biosynthetic pathway. Similar to observations in other filamentous ascomycetes, the genome sequence of A. carbonarius contains a large number of genes predicted to encode PKSs. In this work a pks gene identified within the putative OTA cluster of A. carbonarius, designated as AcOTApks, was inactivated and the resulting mutant strain was unable to produce OTA, confirming the role of AcOTApks in this biosynthetic pathway. AcOTApks protein is characteristic of the highly reduced (HR)-PKS family, and also contains a putative methyltransferase domain likely responsible for the addition of the methyl group to the OTA polyketide structure. AcOTApks is different from the ACpks protein that we previously described in A. carbonarius, which showed an expression profile compatible with OTA production. We performed phylogenetic analyses of the beta-ketosynthase and acyl-transferase domains of the OTA PKSs that had been identified and characterized in different OTA producing fungal species. The phylogenetic results were similar for both domains analyzed and showed that OTA PKS of A. carbonarius, Aspergillus niger and Aspergillus ochraceus clustered in a monophyletic group with 100% bootstrap support suggesting a common origin, while the other OTA PKSs analyzed were phylogenetically distant. A quantitative RT-PCR assay monitored AcOTApks expression during fungal growth and concomitant production of OTA by A. carbonarius in synthetic grape medium. A clear correlation between the expression profile of AcOTApks and kinetics of OTA production was observed, with AcOTApks reaching its maximum level of transcription before OTA accumulation in mycelium reached its highest level, confirming the fact that gene transcription always precedes phenotypic production. (C) 2014 Elsevier B.V. All rights reserved.

Aspergillus carbonarius is the main responsible fungus of ochratoxin A (OTA) contamination of grapes and derived products. Todate, the biosynthetic mechanism of this mycotoxin has been partially elucidated. Availability of genome sequence of A. carbonariushas allowed the identification of a putative gene cluster involved in OTA biosynthesis. This region hosts the previouslycharacterized AcOTAnrps and AcOTApks genes encoding two key enzymes of the biosynthetic pathway. At about 4,400 nucleotidesdownstream of these loci, a gene encoding a putative flavin dependent-halogenase came out from the annotation data. Itsproximity to OTA biosynthetic genes and its sequence analysis have suggested a role in the biosynthesis of OTA, directed to theintroduction of the chlorine atom in the C-5 position of the final molecular structure of this mycotoxin. The deduced proteinsequence of the halogenase gene, we designated AcOTAhal, shows a high similarity to a halogenase that is located in the OTAcluster of A. niger. The deletion of the halogenase gene completely eliminated the production of ochratoxin A in A. carbonariusand determined a significant increase of ochratoxin B, as confirmed by mass spectrometry analysis. Moreover, its expressionprofile was similar to the two biosynthetic genes previously identified, AcOTApks and AcOTAnrps, indicating a strong correlationof the AcOTAhal gene with the kinetics of OTA accumulation in A. carbonarius. Therefore, experimental evidence confirmedthat the chlorination step which converts OTB in OTA represents the final stage of the biosynthetic pathway, supportingour earlier hypothesis on the order of enzymatic steps of OTA biosynthesis in A. carbonarius.

Obiettivo del progetto SIX è sperimentare l'efficacia di un prodotto sanitario a base di argento colloidale e di una sostanza GRAS (Generally Regarded As Safe) di origine naturale nel controllo di Xylella fastidiosa in piante di olivo. I principi attivi vengono somministrati mediante trattamenti combinati di endoterapia di nuova concezione (metodo BITE) e spray fogliare. L'efficacia dei trattamenti verrà valutata mediante ispezione visiva e determinazioni molecolari per consentire la diagnosi e quantificazione dell'agente patogeno anche in piante asintomatiche. La durata del progetto pilota è di 18 mesi

Competition is one of the potential mechanisms of the antagonistic action of Trichoderma harzianumagainst Fusarium oxysporum. The competitive capability of the T. harzianum isolate ITEM 908 (Th908)vs. an isolate of F. oxysporum f. sp. lycopersici was improved via enhancement of the tolerance togrowth-inhibitory metabolites produced by F. oxysporum. HPTLC and HPLC analyses led to the identificationof fusaric acid (FA) as the major metabolite in culture filtrate of the phytopathogenic F. oxysporumstrain ITEM 2797 (Fo2797). FA, a phytotoxin which has also been reported to be released in soil, totallyinhibited the growth of Th908 on PDA containing 120 lg of FA per gram in 3-day trials. Through UV-Cirradiation and subsequent selection of mutants able to grow on PDA supplemented with culture extractsof Fo2797 or FA, one stable tolerant mutant (Th908-5) with unaltered physiological features and rhizospherecompetence was isolated. The biocontrol capability of the UV-mutant Th908-5 was compared tothat of the wild-type strain Th908 on tomato plants grown in a substrate heavily infested with Fo2797 intwo separate trials. The reduction of the disease by Th908-5 was highly (P < 0.01) to extremely (P < 0.001)significant, while only marginally significant (P < 0.05) and inconsistent biocontrol was achieved byTh908. In addition, in non-inoculated vermiculite, Th908-5 increased the emergence and growth oftomato plants compared to the control. Th908 and Th908-5 were investigated for the expression of fivegenes (MDR ProB, MDR BrefA, MDR Protein2, Hydro II, ThPTR2) encoding proteins putatively associatedwith T. harzianum biocontrol function and involved in the mechanisms of multidrug resistance (MDR)or competition for space and nutrients. When the mutant strain was exposed to FA, the expression ofthe gene MDR ProB, encoding a protein associated with MDR was suppressed, suggesting a role for thegene in response to FA. Since UV-mutants are not regarded as genetically modified organisms (GMO)and their circulation and use is not subjected to restrictions that apply to strains derived by genetic transformation,the improved strain Th908-5 could be readily available for application in the field.

Olive quick decline syndrome (OQDS) caused by X. fastidiosa is currently causing severe damages to the production and reducing the life span of the plants in the Salento peninsula of Apulia (Italy). No effective means of control of X. fastidiosa is currently available. The objective of this study was to evaluate in vitro antimicrobial activities against X. fastidiosa (strain Salento-1) of different classes of compounds having diverse origins, i.e. traditional antibiotics, plant-derived natural products, and microbial metabolites. A preliminary bioassay, performed by the agar disc diffusion method, revealed that 17 of the 31 antibiotics tested did not affect bacterial growth at a dose of 5 ?g. Olive mill wastewaters (OMWs), which are known to possess a broad range of antimicrobial activity, are able to inhibit X. fastidiosa in vitro. Most interestingly when we analysed different OMWs derived micro, ultra and nano-filtered fractions as well as some of the single phenolic compounds that they contain, we found that the OMWs micro-filtered fraction is the most effective against the bacterium but only few phenolics are active in their pure form. Also some fungal extracts and bacteria toxins showed noteworthy inhibitory effect to strain Salento-1 growth. The possible use of some of these products for curative/preventive treating OQDS-affected or at-risk olive plants will be discussed.

Olive quick decline syndrome (OQDS) is causing 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 a OQDS affected plant and classified as ST53. A bioassay based on agar disc diffusion method, revealed that 17 out of the 32 tested antibiotics did not affect bacterial growth at a dose of 5 g disk-1. When we assayed micro-, ultra- and nano-filtered fractions of olive mill wastewaters (OMWs) we found that the micro-filtered fraction resulted 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.

Fusarium head blight (FHB) is a world-wide occurring disease of wheat and other grain crops that causes yearly considerable losses in terms of yield and quality of grains. The severity of the disease is aggravated by intensive crop management and some cultural practices, such as monocropping and conservation tillage. Moreover a further increase of FHB is expected in temperate areas as a result of the global climate change. The infection of wheat heads is primarily caused by spores of Fusarium graminearum (teleomorph: Gibberella zeae) that infect the spikes at flowering and impair formation of the embryos and accumulation of starch in the endosperm of the developing kernels. Besides being small, shrunk and whitened, the infected kernels may also contain mycotoxins produced by F. graminearum (mainly deoxynivalenol and zearalenone), which enter the food and feed chains and pose safety concerns for human and animal health. The main source of inoculum for flowers infection are the ascospores, which are formed inside perithecia, the flask-shaped fruiting bodies of the fungus that are developed by the overwintering mycelium on the infected plant debris of previous susceptible crops. Since chemical control is difficult and raises environmental and safety concerns, prevention of perithecia formation and ascospore release appears a feasible means for FHB control. We investigated the capability of seven Trichoderma spp. strains to inhibit perithecia formation in dual culture tests. One isolate of F. graminearum was challenged with the antagonistic Trichoderma spp. strains on carrot-agar medium; after 7 days of co-culture the mycelium was peeled off the plates and production of perithecia was induced by fertilization of cultures. After 7 more days of incubation at 25 °C, the number of perithecia formed was assessed in the plate sectors that were pre-colonized by either Trichoderma or Fusarium. In the Trichoderma pre-colonized sectors, perithecia formation was inhibited by 80 to 100%. In the Fusarium pre-colonized sectors, perithecia formation was totally inhibited by 3 out of 7 tested Trichoderma isolates, while the other 4 isolates showed not significant perithecia inhibition. Further investigations on the mechanism of perithecia inhibition showed that the Trichoderma strains released unidentified metabolites that were able to reduce the number of perithecia formed. The reduction of number of perithecia formed by F. graminearum colonies exposed to Trichoderma cell-free metabolites ranged from 27% to 91%, depending on the Trichoderma strain. To explore the effect of Trichoderma metabolites on the regulatory mechanisms of perithecia formation, we carried out a preliminary study of genes involved in the perithecia developmental process. This study allowed to identify a first group of genes associated with different stages of the perithecia formation, whose expression rate in response to Trichoderma metabolites is under investigation.

Hydroxycinnamic acids (HCAs), phenolic components of wine, are known to have antimicrobial properties. Aspergillus carbonarius is one of the most important ochratoxin A (OTA) producing fungi in wine. Strategies for the control and prevention of A. carbonarius contamination are important for the maintenance of wine safety. This study sought to determine the potential of HCAs, such as caffeic, p-coumaric and ferulic acids, as antifungal natural compounds for the control of A. carbonarius growth and OTA production. The HCAs were tested at the increasing concentrations of 0.30, 0.65 and 1.10 mg/ml in minimal medium (MM) and grape juice. Germination of conidia was not affected in neither of the two media in presence of HCAs. At all the concentrations tested, OTA biosynthesis in MM was reduced and the dose effect was more evident for p-coumaric and ferulic acids; in grape juice the reduction trend was confirmed, and ferulic acid showed the highest inhibitory effect. Moreover, the expression level of genes encoding a polyketide synthase (AcOTApks) and a nonribosomal peptide synthetase (AcOTAnrps) involved in OTA biosynthesis, was evaluated by real-time PCR in A. carbonarius grown in presence of 0.65 mg/ml of HCAs. From gene expression analysis only the AcOTApks gene showed a marked reduction of transcription level in presence of p-coumaric and ferulic acids. On the contrary, caffeic acid seemed to not influence the expression levels of the genes analysed in this study, suggesting a different mechanism of action on the regulation of OTA biosynthesis.

The edible jellyfish world fishery, mostly based in Southeast Asia, generates a global catch annually exceeding 750,000 tons, with increasing demands expanding outside Asian markets. Known for its nutritional and medical value in the Chinese pharmacopeia, jellyfish seem to have characteristics of healthy food. Some Mediterranean jellyfish species (phylum Cnidaria) can represent a low-cost raw material for innovative medical, nutraceutical or cosmeceutical products. Jellyfish are rich in protein, mainly collagen, and are recently designated as novel foods in Europe. Several compounds isolated from jellyfish, including proteins, can exert high antioxidant and other biological activities. GoJelly is a new H2020 funded project addressing the potential exploitation of jellyfish biomass and derived compounds for multiple purposes. Here we report on the protein characterization of Rhizostoma pulmo, an edible scyphozoan jellyfish regularly found with large populations along Italian sandy coastlines. We show that hydrolysed proteins, including hydrolysed jellyfish collagen, have strong antioxidant activity, particularly low molecular weight fractions (less than 10 kDa). Moreover, the cytotoxicity of different fractions of jellyfish hydrolysed protein on cancer cells (MCF7) and human keratinocytes (HEKa) was investigated, as well as their protective effect on UV-irradiated HEKa. In combination with food safety assessments and improvements in jellyfish processing technology, scientific evidence demonstrating edible jellyfish as functional food may promote the future expansion of jellyfish fishery worldwide.

Jellyfish have been considered as food for more than thousand years in Asia, where a multi-phase processing consisting on mixtures of salt and alum are used as traditional procedure. Jellyfish could also become an attractive choice for western food market for many reasons. The need for fishery diversification and for new food resources, the increasing market demand for innovative food products or ingredients, and finally the great availability of a resource whose use would be economically profitable and environmentally sustainable, make jellyfish a good candidate for future exploitation. However, in the attempt to introduce jellyfish as a new food product in Western market, several issues should be considered. Within the EU Project "GoJelly", we are continuing studies on "western style" jellyfish food. Protocols and treatments currently used for fish and sea-foods preparation are under consideration as a starting point to develop ad hoc new handling and storage procedures from fishing to on-boat pre-treatment and new processing methods will be developed to optimize taste, texture, flavor and shelf-life of final products acceptable for European consumers. Moreover, these new practices will be addressed also to maintain or enhance nutritional traits of formulated jellyfish products. In addition, study on the identification of strategies to reduce spoilage and to individuate possible food-borne microbial pathogens have been started up. The setting up of diagnostic and analytical assays to define consumers risk assessment and to monitor safety threat during jellyfish processing steps for preparation of food for human consumption is also in progress.

DNA-based approaches were used to characterize a strain (Salento-1) of Xylella fastidiosa obtained from an olive plant suffering from the syndrome of quick decline in Apulia (South Italy). Salento-1 was indistinguishable from strain CoDiRO previously isolated from olive in Apulia and assigned to X. fastidiosa subsp. pauca. Based on our results and comparative analysis with reported data, the subspecies pauca, multiplex, and fastidiosa may invade olive throughout the world (California, Italy, Argentina and Brazil). The strain Salento-1 has been deposited in the National Collection of Plant Pathogenic Bacteria (NCPPB), England, and in the Belgian Coordinated Collections of Microorganisms (BCCM), Belgium.

An Aspergillus population (67 strains), isolated from maize in 2003, during the first outbreak of aflatoxin contamination documented in Northern Italy, was characterised according to gene sequencing data. All strains were identified as A. flavus by sequencing of ?-tubulin and calmodulin gene fragments. Furthermore, the strains were analysed for the presence of seven aflatoxin biosynthesis genes in relation to their capability to produce aflatoxin B1, targeting the regulatory genes aflR and aflS, and the structural genes aflD, aflM, aflO, aflP, and aflQ. The strains were placed into four groups based on their patterns of amplification products: group I (40 strains) characterised by presence of all seven amplicons; groups II (two strains) and III (nine strains), showing four (AflM, aflP, aflO, and aflQ) and three (aflO, aflP, aflQ) amplicons, respectively; and group IV (16 strains) characterised by total absence of PCR products. Only group I contained strains able to produce aflatoxin B1 (37 out of 40), whereas the strains belonging to the other groups and lacking three, four or all seven PCR products were non-producers. The results obtained in this study pointed out that A. flavus was the only species responsible for aflatoxin contamination in Northern Italy in 2003, and that the aflatoxin gene cluster variability existing in populations can be useful for understanding the toxicological risk as well as the selection of biocontrol agents.

Fungi have an important role in the production of dry-cured meat products, especially during the seasoning period, when the salami surface, both industrially and handmade, is quickly colonized by a composite mycobiota. This mycobiota could have beneficial or undesirable effects on the products depending on its peculiar composition. Various genera of fungi could colonize salami (i.e. Aspergillus¸ Cladosporium, Eurotium, Penicillium), but Penicillium species are predominant, being P. nalgiovense, P. olsonii, P. brevicompactum, P. chrysogenum and a new recently described species P. salamii, the main occurring. As part of the Ministerial project "SAFE-MEAT", aiming to increase food safety and quality of pork-based products, new interesting results to prevent and control ochratoxin A (OTA) risk, and improvements of the quality of salami production have been achieved. In comparison with P. nalgiovense, P. salamii has been proved to be a fast growing mould on dry-cured sausages casing, well adapted to the seasoning process, with higher lipolytic and proteolytic enzymatic activities that could contribute to confer typical sensory characteristics to meat products. Thus, P. salamii resulted a promising candidate for new fungal starter formulations for meat industry. However, salami could be also colonized by P. nordicum, an important and consistent producer of the potent nephrotoxin OTA, widely reported as undesirable contaminant of dry-cured meat products. To this purpose, a high sensitive and easy to use LAMP assay, has been developed for P. nordicum detection on salami surface co-inoculated with P. nalgiovense and P. nordicum at different rates. Moreover, monitoring gene expression of a key gene of OTA biosynthesis in P. nordicum and toxin accumulation in meat during the seasoning process revealed that expression profile was consistent with OTA accumulation. Gene expression was observed since the 4th day after inoculation and progressively increased up to the 10th day when OTA reached the maximum level. Indeed, contamination of dry-cured meat products by P. nordicum could represent a serious concern for salami production and therefore molecular tools, such as LAMP and gene expression assay, should be considered for new HACCP plans in order toprevent and control OTA risk in dry-cured meat production.

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine, and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been elucidated in Penicillium species. In Aspergillus species, only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase (NRPS) in OTA-producing A. carbonarius ITEM 5010 has eliminated the ability of this fungus to produce OTA. This is the first report on the involvement of an nrps gene product in OTA biosynthetic pathway in an Aspergillus species. The absence of OTA and ochratoxin ?, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin ?, the dechloro analog of ochratoxin ?, were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius and that ochratoxin ? is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.

In the nineties fungal secondary metabolites (SMs), such as antibiotics and mycotoxins, started to be genetically characterized. Then, the clustered arrangement of genes involved in the biosynthesis of a single SM was studied. In the pre-genomic era, gene cluster discovery in fungi was complex and time-consuming, involving cumbersome traditional molecular methods. Genomics has revolutionized the research on SM biosynthesis pathways, allowing the bypass of such approaches. The breakthrough of next-generation sequencing (NGS) technologies and the advent of Bioinformatics have opened a new era in the study of biological systems. NGS technologies contributed significantly to the increasing availability of fungal genomes and bioinformatic analysis lead to the identification of SM clusters of known metabolites and to the prediction of novel cryptic clusters for still unknown microbial metabolites. However, most of the clusters identified by genome analysis are still to be deeply examined to completely understand the pathway steps and the regulatory network behind the metabolite biosynthesis. Here, we present the example of how the genomic approach has led to the identification of biosynthetic genes and their role in ochratoxin A (OTA) production by Aspergillus carbonarius. From the genome sequencing and the subsequent prediction of OTA cluster, we demonstrated by gene knock-out approach the key role of three genes (AcOTApks, AcOTAnrps and AcOTAhal) in the OTA biosynthesis. Single gene knock-out mutant allowed us to elucidate the order of the enzymatic steps in the biosynthesis pathway. Other predicted genes in the cluster, such as a p450 monooxygenase and a transcription factor gene, need to be investigated for the full knowledge of the structural and regulatory mechanisms of toxin production. Furthermore, transcriptomic analyses are in progress to study and clarify at a deeper level the complex genetic picture of the fungus during OTA biosynthesis.

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.

Polyketide synthase (PKSs) and nonribosomal peptide synthetase (NRPSs) arelarge multimodular enzymes involved in biosynthesis of polyketide and peptide toxinsproduced by fungi. Furthermore, hybrid enzymes, in which a reducing PKS region is fusedto a single NRPS module, are also responsible of the synthesis of peptide-polyketidemetabolites in fungi. The genes encoding for PKSs and NRPSs have been exposed tocomplex evolutionary mechanisms, which have determined the great number and diversityof metabolites. In this study, we considered the most important polyketide and peptidemycotoxins and, for the first time, a phylogenetic analysis of both PKSs and NRPSsinvolved in their biosynthesis was assessed using two domains for each enzyme:?-ketosynthase (KS) and acyl-transferase (AT) for PKSs; adenylation (A) andcondensation (C) for NRPSs. The analysis of both KS and AT domains confirmed thedifferentiation of the three classes of highly, partially and non-reducing PKSs. HybridPKS-NRPSs involved in mycotoxins biosynthesis grouped together in the phylogenetictrees of all the domains analyzed. For most mycotoxins, the corresponding biosyntheticenzymes from distinct fungal species grouped together, except for PKS and NRPSinvolved in ochratoxin A biosynthesis, for which an unlike process of evolution could behypothesized in different species.

Members of the economically important ascomycete genus Trichoderma are ubiquitously distributed around the world. The mycoparasitic lifestyle and plant defence-inducing interactions of Trichoderma spp. make them ideal biocontrol agents. Of the Trichoderma enzymes that produce secondary metabolites, some of which likely play important roles in biocontrol processes, polyketide synthase (PKSs) have garnered less attention than non-ribosomal peptide synthetases such as those that produce peptaibols. We have taken a phylogenomic approach to study the PKS repertoire encoded in the genomes of Trichoderma reesei, Trichoderma atroviride and Trichoderma virens. Our analysis lays a foundation for future research related to PKSs within the genus Trichoderma and in other filamentous fungi.

Recently, studies on the molecular aspects of ochratoxin A biosynthesis have significantly advanced. Differently from other mycotoxins, the genes and the enzymatic stages involved in biosynthesis pathway of ochratoxin A have remained long unknown. New ecological data have led to the definition of new producing species, responsible of ochratoxin A contamination in several food and feed. In parallel, genomics, transcriptomics and proteomics studies have provided new information to better define the molecular key steps of the mycotoxin biosynthesis. Further studies are still needed to completely clarify the regulatory mechanisms underlying the activation of the production of ochratoxin A. Previous findings on fungal secondary metabolism biosynthesis and the availability of new data from different omics approaches will permit to fill the gap of knowledge in the near future.

Ochratoxin A (OTA) undergoes to enzymatic biodegradation by proteolytic enzymes able to hydrolyze its amide bond with consequent formation of ochratoxin ? (OT?) and L-?-phenylalanine. This mechanism can be regarded as a detoxification method since OT? and L-?-phenylalanine are considered as less and non-toxic, respectively. Different microorganisms belonging to bacterial, yeast and fungal species have been reported to degrade OTA. Several enzymes may be involved in microbiological degradation of OTA, such as carboxypeptidase A, lipase, and acid proteases. Also Aspergillus carbonarius, one of the most important fungal producer of OTA and the major responsible of OTA contamination of grapes, wine and by-products, turned out to be able to degrade OTA. In the attempt to identify the enzyme able to degrade OTA in this microorganism, a protease encoding gene, located in the genomic region recognized as OTA cluster, has driven our attention. In particular, this gene, namely Acap1 of A. carbonarius strain ITEM 5010, encodes for an aspartic protease and is located downstream of the core genes involved in OTA biosynthesis. Acap1gene was isolated and cloned for its characterization. The gene is 1367 bp long and the in silico analyses of the deduced protein sequence of 421 aa revealed that the AcAP1 protein shows the functional typical structure of aspartic protease enzymes. Aspartyl proteases are a highly specific family of proteases that tend to cleave dipeptide bond and they are optimally active at acidic pH. Heterologous recombinant production of the AcAP1 protein has been carried out in order to verify the involvement of AcAP1 in the ability of A. carbonarius in OTA degradation and to analyze its structural and functional properties for a potential biotechnological use of the enzyme. Acap1 gene was cloned in two expression vectors (p426 and pYES), carrying a constitutive and an inducible promoter, respectively, in fusion with a sequence encoding for a His-tag at the 3'-terminus. Three different strains of Saccharomyces cerevisiae, carrying diverse genotypes, have been transformed. Data concerning the protein expression by yeast, evaluation of the protease activity, and purification of the recombinant protein will be produced.

Penicillium nordicum, an important and consistent producer of ochratoxin A (OTA), is a widely distributed contaminant of NaCl and protein rich food. It is usually found on dry-cured meat products and is considered the main responsible of 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 30 days. Gene expression and OTA production were monitored throughout the seasoning process after 4, 5, 6, 7, 10, 14, and 30 days. The expression of otapksPN gene was already detected after 4 days and increased significantly after 7 days of seasoning, reaching the maximum expression level after 10 days (1.69·104 copies/100 mg). Consistently with gene expression monitoring, OTA was detected since the 4th dayand its content increased significantly from the 7th day, reaching the maximum level after 10 days. In the late stages of seasoning process, OTA did not increase further and the number of gene copies was progressively reduced after 14 and 30 days.

Ochratoxin A (OTA) is a potent pentaketide nephrotoxin diffusely distributed in food and feed products (grains, legumes, coffee, dried fruits, meats, beer and wine); it is also carcinogenic, neurotoxic, teratogenic and immunotoxic. This mycotoxin is produced by species of genus Aspergillus and Penicillium. OTA is the primarily mycotoxin risk in wine and dried vine fruits. Several studies focused on Aspergillus section Nigri, due to their role as causative agents of black rot of grapes, and subsequently as cause of ochratoxin A contamination. Nine different black Aspergillus species have been identified on grapes with different secondary metabolites profiles. These species are often difficult to be identified by means of classical methods. The polyphasic approach used in our studies led to characterization of three new non toxigenic species occurring on grapes: A. brasiliensis, A. ibericus and A. uvarum. However, the main source of OTA contamination in grapes is A. carbonarius, followed by A. niger and A. welwitschiae. This contamination is strongly related to climatic conditions, geographical regions (South Mediterranean climate is highly conducive), grape varieties, damage by insects, growing season (high susceptibility from early veraison to harvest, with a peak at ripening), and great variations may occur from one year to another. Differently from other mycotoxins, the genes and the enzymatic stages involved in OTA biosynthesis pathway have remained unknown for long. In last years, genomics, transcriptomics and proteomics studies have provided new information to better define the molecular key steps of OTA biosynthesis. Genome sequencing of A. carbonarius led us to predict OTA cluster and to elucidate the key role of three genes (AcOTApks, AcOTAnrps and AcOTAhal) and the order of the enzymatic steps of the biosynthesis pathway. Other predicted genes in the cluster have been identified and analysed, such as a p450 monooxygenase and a transcription factor gene, likely involved in the structural and regulatory mechanisms of OTA production. Furthermore, transcriptomic analyses are in progress to study and clarify the complex genetic picture of the fungus during OTA biosynthesis at a deeper level. Interestingly, recent studies on climate change effects evidenced the influence of raising temperature and CO2 levels on OTA production increase. Managing OTA contamination to reduce risks in grapes implies several strategies, such as implementations of good agricultural practices and risk maps, in association with the use of insecticides and fungicides when favourable climatic conditions occur. In addition, corrective actions can be adopted in wineries.

The increasing availability of fungal genomes and bioinformatic tools have led to the identification of clusters of known metabolites and to the prediction of novel cryptic clusters for still unknown metabolites. However, most of the clusters identified by genome analysis are still to be deeply examined to completely understand the pathway steps and the regulatory network behind the metabolite biosynthesis (1). The genome sequencing of Aspergillus carbonarius has advanced the knowledge of the molecular mechanism of biosynthesis of ochratoxin A (OTA), one of the most important mycotoxin contaminating several commodities. Differently from other mycotoxins, the elucidation of the genetic background of OTA biosynthesis has remained uncompleted for a long time. Aspergillus carbonarius is the major responsible of OTA contamination of wine and other grape products in the Mediterranean area, constituting a great health risk and cause of important economic losses (2). The analysis of A. carbonarius genome has revealed the presence of a great number of PKSs and NRPSs, enzymes having an essential role in the synthesis of fungal secondary metabolites. Subsequently, the identification of the PKS putatively involved in the biosynthesis of OTA has led to an extensive study of the adjacent genomic region, in the attempt to identify other genes involved and to define the OTA biosynthesis cluster. The roles of three key genes -AcOTApks, AcOTAnrps and AcOTAhal - have been demonstrated by gene knock-out approach and the order of the fundamental enzymatic steps in the biosynthesis pathway of OTA has been clarified. These studies demonstrated that the enzymatic step involving the addition of phenilalanine to the polyketide ring takes place before the chlorination step. Moreover, it was demonstrated that OT? is not a precursor of OTA but rather a product of OTA hydrolysis (3, 4). Other predicted genes in the cluster need to be further investigated to fully clarify the structural and regulatory mechanisms of toxin production, among which the genes coding a p450 monooxygenase, a transcription factor, a transporter protein and an aspartyl protease. Transcriptomic analyses are in progress to study and clarify at a deeper level the complex genetic picture of the fungus during OTA biosynthesis.References1. Brakhage A.A., 2013. Nature Reviews Microbiology 11.1: 21-32.2. Perrone G. et al., 2008. Aspergillus in the genomic era, Academic Publishers, Wageningen, 2008, 179-212.3. Gallo A. et al., 2012. Appl. Environ. Microbiol., 78 (23), 8208-8218.4. Ferrara M. et al., 2016. Appl. Environ. Microbiol., 82 (18), 5631-5641.

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 (rum) 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.

Xylella fastidiosa is a gram-negative, xylem-limited, bacterium which is responsible, in Italy, for the Olive Quick Decline Syndrome (OQDS). The disease is caused by the subspecies pauca and emerged a few years ago in the Apulia province of Lecce, in the Salento peninsula, on Olea europaea plants. X. fastidiosa can infect different plant species and is well known in California as the causal agent of Pierce's disease on grape. Infections of susceptible hosts with X. fastidiosa are known to result in xylem vessel occlusions, water movement impairment, and accordingly to induce the typical desiccation symptoms. In the present study, we investigated xylem vessel occlusions in healthy and naturally infected O. europaea plants grown in open field by analyzing three olive cultivars widespread in the region that show different degree of susceptibility to the disease: the susceptible cultivars "Ogliarola salentina" and "Cellina di Nardò", and the tolerant cultivar "Leccino". Our results show that occlusions were caused by tyloses and gums/pectin gels, and not by bacterial cell aggregates. Our data also indicate that occlusions are not responsible for the symptomatology of the OQDS and, as observed in Leccino plants, they are not a marker of tolerance/resistance to the disease.