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.

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.

Food authentication and traceability is one of the major concern to the food industry, strictly correlatedto food fraud and food adulteration. Rice (Oryza sativa L.) is one of the most important crops, supplyingfood for over half of the world's population. Authenticity of rice products has become a key issue in thefood industry addressed to protect the interests of quality conscious consumers, stakeholders, andimporting countries. DNA markers offer a powerful tool to address the validation of food authenticityand traceability of primary products. Progress in NGS technology has provided opportunities to detectlarge number of DNA polymorphisms, even in the closely related cultivars. In this study, a whole-genomesequencing of Italian rice cultivar Carnaroli has been carried out from genetically pure certified seed.The sequencing yielded about 22.5 million reads. After quality trimming 21.5 million reads were mappedonto the reference sequence of Oryza sativa ssp. japonica cv. Nipponbare (IRGSP-1.0), providing about90% coverage of the rice genome and an average coverage of 15.12x. Preliminary results, found 450,414candidate DNA polymophisms between cultivar Nipponbare and Carnaroli. These were classified into383,080 SNPs (85%) and 67,334 InDels (15%) by polymorphism types, 150,688 homozygous (85%) and299,726 (15%) heterozygous by zygosity type, 371,801 intergenic (82.5%) and 78613 (17.6%) by genomiclocation. The distribution of DNA polymorphisms was found to be uneven across and within the ricechromosomes. In particular, chromosome 8 and 10 showed the highest density of DNA polymorphisms(14.7% and 14.5%, respectively). This study represents the first report of whole genome sequencing ofItalian rice cultivar Carnaroli and will contribute to develop targeted and un-targeted method for riceauthentication and traceabilityThis work has been supported by the European project FOODINTEGRITY (FP7-KBBE-2013-single-stage, No 613688).

During 2009-2013, 302 single-spore isolates of Botrytis cinerea were collected from vineyards located in the most important site of table grape production in Sicily, recognized by the European Community as Protected Geographical Indication (PGI) 'Mazzarrone grape'. In preliminary studies, all isolates were tested invitro for their sensitivity to six fungicides belonging to the following groups: benzimidazoles, dicarboximides, anilinopyrimidines, succinate dehydrogenase inhibitors, hydroxyanilides and phenylpyrroles. In these tests, 45.7% of the isolates were found to be resistant to at least one fungicide. Specific resistance to pyrimethanil was found in 30.8% of the isolates, whereas 13.9, 10.3 and 7.6% of the isolates exhibited resistance to carbendazim, iprodione and boscalid, respectively. No isolates resistant to fenhexamid and fludioxonil were detected within our dataset of B.cinerea isolates. However, 30 B.cinerea isolates possessed multiple resistance to two or more fungicides. In detail, 8 isolates were simultaneously resistant to four fungicides, whereas 5 and 17 isolates were resistant to three and two fungicides, respectively. For boscalid, 11/23 of isolates showing invitro resistance possessed a mutation at the SdhB gene, whereas all isolates resistant to carbendazim and iprodione possessed mutations at ?-tubulin and BcOS1 histidine kinase genes, respectively. Accordingly, these fungicides failed to control gray mould infections caused by resistant or reduced sensitivity isolates on grape berries and grapevine leaves whereas the sensitive isolates were effectively managed by all fungicides applied at label rates. This study represents the first report of B.cinerea field isolates resistant and/or with simultaneous resistance to several botryticides from table grape vineyards in Sicily. Therefore, current strategies for fungicide resistance management of B.cinerea could be negatively affected in future.

Food authentication and traceability is a complex problem, strictly correlated to fraud andadulteration detections that dramatically affect the consumer protection. Analysis of protein,metabolite and DNA represents robust tools for food authentication. In particular, DNA-basedmethods are more reliable, thanks to the stability of DNA under production and processing techniquesapplied along the food-chain. Therefore, DNA markers offer a powerful tool to address the validationof food authenticity and traceability of primary products. Single nucleotide polymorphism (SNP)markers have become the most used markers in genetic characterization studies as well as intranslational genomic even in plants. SNP are, in fact, the most abundant forms of genetic variationamong individuals of a species. In particular, SNP analysis by next generation sequencing (NGS)(e.g.genotyping by sequencing (GBS) and double-digest restriction site-associated DNA sequencing(ddRAD-Seq) or by high resolution melting analysis (HRM), e.g. single-base variants and smallinsertions or deletions, have rapidly become popular due to their flexibility and relatively low cost.The ddRAD-Seq technology has the advantage over GBS of high accuracy read mapping by paired-endsequencing of identical loci. Progress in NGS technology has led to the availability of several plantgenomes. This situation makes it possible to simulate ddRAD-Seqin silico, allowing prediction of thenumbers, sizes, and genome positions of digested fragments. However, few reports have evaluatedthe in silico predictions by comparative experiments using several combinations of restriction enzymesand multiple samples with different SNP density. HRM analysis has several advantages over traditionalmethods for gene scanning and genotyping, making it faster, less laborious and more suitable for highsample throughput. In this study, two approaches are proposed for the authentication of the Italianrice cultivars Carnaroli and Roma: in silico and empirical ddRAD-Seq analysis and HRM analysistargeting an A/C SNP in exon 6, responsible for the Wxin allele. The ddRAD-Seq approach consisted ofa workflow, as follows:(i) in silico prediction of optimum restriction enzymes from the reference ricegenome,(ii) verification of the prediction by ddRAD-Seq data of Carnaroli and Roma genomes (iii)establishment of a computational data processing pipeline for high confidence SNP calling, and (iv)validation of SNP accuracy. In silico prediction prior to sequencing analysis will contribute tooptimization of the experimental conditions for ddRAD-Seq and could help to accelerate the detectionof DNA markers useful for the authentication of rice cultivars Carnaroli and Roma. Preliminary resultsof HRM analysis show potential for rice cultivar differentiation since Carnaroli was distinguished fromRoma, among others (Carnise/Karnak, Gladio, Sant'Andrea and others) with high level of confidence(>98%). Acknowledgments: This work has

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.

Fungal starter, such as Penicillium nalgiovense, are commonly used to inoculate sausages before seasoning process. However, P. nordicum, a well-known ochratoxin A (OTA) producer frequently isolated from seasoning rooms, could colonize the casing surface during the early stage of production. The relationship between OTA accumulation and simultaneous inoculation of P. nalgiovense and P. nordicum at different rates was evaluated. After 14 days of seasoning, the persistence of P. nordicum was assessed by LAMP assay revealing its capability to colonize and grow on salami surface at all the contamination rates. At the end of seasoning, OTA was accumulated both in mycelium and dry-cured meat when P. nordicum contamination rate ranged from 25% to 100% of inoculum, while no OTA was detected in dry-cured meat at 2.5% and 0.25%. Results demonstrated that contamination of fungal starter by P. nordicum could represent a serious concern during salami production and therefore represents an important critical point to be monitored.

RNA interference (RNAi) is a powerful approach for elucidating gene functions in a variety of organisms, including phytopathogenic fungi. In such fungi, RNAi has been induced by expressing hairpin RNAs delivered through plasmids, sequences integrated in fungal or plant genomes, or by RNAi generated in planta by a plant virus infection. All these approaches have some drawbacks ranging from instability of hairpin constructs in fungal cells to difficulties in preparing and handling transgenic plants to silence homologous sequences in fungi grown on these plants. Here we show that RNAi can be expressed in the phytopathogenic fungus Colletotrichum acutatum (strain C71) by virus-induced gene silencing (VIGS) without a plant intermediate, but by using the direct infection of a recombinant virus vector based on the plant virus, tobacco mosaic virus (TMV). We provide evidence that a wild-type isolate of TMV is able to enter C71 cells grown in liquid medium, replicate, and persist therein. With a similar approach, a recombinant TMV vector carrying a gene for the ectopic expression of the green fluorescent protein (GFP) induced the stable silencing of the GFP in the C. acutatum transformant line 10 expressing GFP derived from C71. The TMV-based vector also enabled C. acutatum to transiently express exogenous GFP up to six subcultures and for at least 2 mo after infection, without the need to develop transformation technology. With these characteristics, we anticipate this approach will find wider application as a tool in functional genomics of filamentous fungi.

Knowledge on the genetic basis underlying interactions between beneficial bacteria and woody plants is still very limited, and totally absent in the case of olive. We aimed to elucidate genetic responses taking place during the colonization of olive roots by the native endophyte Pseudomonas fluorescens PICF7, an effective biocontrol agent against Verticillium wilt of olive. Roots of olive plants grown under non-gnotobiotic conditions were collected at different time points after PICF7 inoculation. A Suppression Subtractive Hybridization cDNA library enriched in induced genes was generated. Quantitative real time PCR (qRT-PCR) analysis validated the induction of selected olive genes. Computational analysis of 445 olive ESTs showed that plant defence and response to different stresses represented nearly 45% of genes induced in PICF7-colonized olive roots. Moreover, quantitative real-time PCR (qRT-PCR) analysis confirmed induction of lipoxygenase, phenylpropanoid, terpenoids and plant hormones biosynthesis transcripts. Different classes of transcription factors (i.e., bHLH, WRKYs, GRAS1) were also induced. This work highlights for the first time the ability of an endophytic Pseudomonas spp. strain to mount a wide array of defence responses in an economically-relevant woody crop such as olive, helping to explain its biocontrol activity.

In the framework of a phytoremediation project in the Apulia region (Italy) a field experiment was carried out in multi-metal contaminated soils. The accumulation and distribution of metals in different plant parts of durum wheat and barley were studied. Further, the application of Bacillus licheniformis strain BLMBI to soil was evaluated as a means to enhance metal accumulation in plants. The translocation and the bioconcentration factors indicated that wheat and barley do not act as metal accumulators in the field conditions tested, thus phytoextraction by these species would not be recommended as a soil remediation alternative. Application of B. licheniformis improved the accumulation of all metals in roots of wheat and barley, and increased Cd, Cr, and Pb contents in the shoots of barley. Low health risk for humans and animals was evaluated to exist if straw and grain from both cereal crops grown in these contaminated sites are consumed.

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.

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.

IntroductionEnergy crisis and environmental pollution have led to an increasing interest in renewable energies. Biogas production from plant material, agricultural residual products and food wastes represents one of the most economically attractive alternative technology for biofuel production. In this regards, anaerobic digestion has been widely applied to produce methane for biofuel. Complex consortia of microorganisms are responsible for biomass degradation and biogas production involving several stages such as substrate hydrolysis, acidogenesis, acetogenesis and methanogenesis. In this sense, the next-generation high-throughput sequencing provides a powerful tool for dissecting microbial community structure and methane-producing pathways in anaerobic digestion. Here, a taxonomic and functional metagenomic analysis of microbial community residing in an industrial-scale biogas fermenter has been carried out at different steps of biogas production.MethodsSample were collected from an industrial-scale mesophilic plant, daily fed with maize silage, consisting of a three steps production taking place in a bioreactor, post-reactor and a storage tank. Total DNA was extracted from samples belonging to each stage of biogas production. Metagenomic analysis were carried out by 16S and shotgun sequencing approach. The 16S datasets were generated by sequencing the bacterial and archaeal V4 hypervariable region. Reads from 16S sequencing were aligned against SILVA ribosomal RNA sequence database by using MALT (1), while shotgun reads were aligned against NCBI-nr sequence database by using DIAMOND (2). Taxonomic binning and functional annotation were performed with MEGAN 6 software (3). ResultsOver 14.5 million high quality reads (about 3.4 gigabases) were generated on the Ion Torrent S5 Sequencing System. About 2.4 and 3 million reads were assigned for 16S and shotgun approach, respectively. Although the average number of assigned taxa for 16S analysis was considerably lower than shotgun analysis, the overall taxa distribution resulting from both sequencing strategies was conserved. In detail, metagenomic analysis revealed that the superkingdom of Bacteria was dominant (~93%) along the production steps, whereas Archaea were less represented (~4%). Within Bacteria the most abundant phyla were Firmicutes, mostly represented by Clostridia, followed by Bacteroidetes, Synergistetes and Proteobacteria. Within the superkingdom of Archaea, only microorganisms belonging to the phylum of Euryarchaeota were detected. Within Euryarchaeota the dominant genera were Methanosarcina and Methanoculleus, notably to be key microorganisms involved in methanogenesis. Data showed that during biogas production steps the abundance of Methanosarcina genus decreased from bioreactor to storage tank, with a simultaneous increase of Methanoculleus genus. Functional analysis of assigned reads also supported a shift from acetotrophic methanogens to hydrogenotrophic methanogens.

Biogas production represents one of the most economically attractive alternative technology for biofuel production from renewable resources. Generally, biogas plants are fed with agricultural residual products and food wastes, but the rising up of agricultural products contaminated by mycotoxins, such as maize silage not suitable for animal feeding, has pointed the question on the possibility to use this agricultural productfor biogas production. In this regards, a preliminary metagenomic analysis of microbial community residing in a mesophilic industrial-scale biogas fermenter, daily fed with contaminated maize silage, has been carried out to characterize the evolution of microbial community under the operating conditions and the mycotoxin content. Sample were collected from a biogas plant consisting of a three steps production taking place in a bioreactor, post-reactor and a storage tank. Total DNA was extracted from samples belonging to each steps of biogas production. Metagenomic analysis was carried out by analyzing the V4 variable region of bacterial and archaeal 16S rRNA gene. Mycotoxin content was analyzed in maize silage feeding the biogas plant and in the digestate from bioreactor, post-reactor and storage tank by immunoaffinity column clean-up (Myco6in1+®) and detected with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Over 3million high quality reads (about 1Gb) were generated on the Ion Torrent S5 Sequencing System. About 2.4 million reads were assigned for 16S analysis. In detail, metagenomic analysis revealed that Bacteria superkingdom was dominant (~96%) along the production steps, whereas Archaea were less represented (~4%). Within Bacteria the most abundant phylum was Firmicutes, mostly represented by Clostridia, followed by Bacteroidetes and Synergistetes. Within the superkingdom of Archaea, only microorganisms belonging to the phylum of Euryarchaeota were detected. Within Euryarchaeota the dominant genera were Methanosarcina and Methanoculleus. Chemical analysis on maize silage feeding the plants showed an initial mycotoxin contamination by DON (410 µg/kg), FB1 (3570 µg/kg), FB2 (810 µg/kg) and T-2 toxin (20 µg/kg), while AfB1, HT-2 Toxin, NIV, OTA and ZEA were not detected. After the first step of biogas production, a complete reduction of DON and T-2 content was achieved. These preliminary results suggest a possible absorption/degradation of mycotoxins in bioreactor tank and therefore further studies are needed to better elucidate the possible involvement of specific microbial taxa capable of mycotoxins reduction and the enzymatic pathways potentially involved in mycotoxin degradation.

Energy crisis and environmental pollution have led to an increasing interest in renewable energies. Biogas production from plant material, agricultural residual products and food wastes represents one of the most economically attractive alternative technology for biofuel production. Complex consortia of microorganisms are responsible for biomass degradation and biogas production involving several stages. Next-generation high-throughput sequencing provides a powerful tool for dissecting microbial community structure and methane-producing pathways in anaerobic digestion. A taxonomic and functional metagenomic analysis of microbial community residing in an industrial-scale biogas fermenter has been carried out at different steps of biogas production. Sample were collected from an industrial-scale mesophilic plant consisting of a three steps production taking place in a bioreactor, post-reactor and a storage tank. Total DNA was extracted from samples belonging to each steps of biogas production. Metagenomic analysis was carried out by using 16S and shotgun sequencing approach. The 16S datasets were generated by sequencing the bacterial and archaeal V4 hypervariable region. Reads from 16S sequencing were aligned against SILVA ribosomal RNA sequence database by using MALT(1), while shotgun reads were aligned against NCBI-nr protein database by using DIAMOND(2). Taxonomic binning and functional annotation were performed with MEGAN 6 software(3). About 2.9 and 11.5 million high quality reads were generated on the Ion Torrent S5 Sequencing System for 16S and shotgun approach, respectively. Metagenomic analysis revealed that the overall taxa distribution resulting from both sequencing strategies was conserved. In details, the superkingdom of Bacteria was dominant (~93%) along the production steps, whereas Archaea were less represented (~4%). Within the superkingdom of Archaea, only microorganisms belonging to the phylum of Euryarchaeota were detected. Within the key microorganisms involved in methanogenesis, data showed that during biogas production steps the abundance of Methanosarcina genus decreased from bioreactor to storage tank, with a simultaneous increase of Methanoculleus genus. Considering the key methanogenesis pathways, functional analysis supported a shift from acetotrophic methanogens to hydrogenotrophic methanogens. Results showed that the combination of both 16S and shotgun sequencing approach successfully addressed the taxonomical and functional analysis of microbial community, revealing new insights in microbial and functional dynamics during biogas production steps.

È stata messa a punto con successo una nuova metodologia di studio qui schematizzata:- Effettuare foto digitali di uno o più campioni di salsicce e salami, in maniera standardizzata, riprendendole dall'alto adagiate su un piano a fronte di due righelli tra loro perpendicolari, in giorni successivi durante la stagionatura in una cella industriale- Selezionare una opportuna serie di immagini di uno stesso campione nel tempo (ad es. in base alla vista del salame per intero o parziale)-Trattare ogni immagine attraverso un opportuno software di elaborazione per prepararla alla successiva analisi- Riconoscere nell'immagine e misurare attraverso uno specifico software la superficie del budello ricoperto dalle muffe- Costruire una serie temporale di dati (ad es. percentuale di superficie totale ricoperta dalle muffe, distribuzione superficiale delle muffe, ecc.) dalle misure effettuate- Confrontare e correlare i dati derivanti da analisi d'immagine con le determinazioni microbiologiche su campioni "di controllo"All'atto di questo lavoro, la metodologia non distingue se la distribuzione delle muffe è più/meno uniforme sul budello, né se è casuale per posizione e dimensione delle colonie; ulteriore lavoro di sviluppo è in corso. La metodologia messa a punto ed i risultati ottenuti sui lotti oggetto d'indagine promettono di essere molto interessanti, specialmente nell'ottica che essa possa essere applicata per controllare da remoto l'andamento della colonizzazione fungina in celle industriali di stagionatura

Fermented and cured meat products are unique and often represented as element of culinary heritage and gastronomic identity. Together with meat enzymes and bacteria, molds are very important part of microbial community involved the ripening of some dry fermented meat products. They contribute to the development of the typical sausage flavor, prevent lipid oxidations and counteract undesirable microorganisms. Various genera of fungi could colonize salami, but Penicillium species are predominant, and above all P. nalgiovense, P. chrysogenum and P. salami, a new recently described species. On the other hand, depending on its peculiar composition, the surface could be colonized by undesirable molds like P. nordicum, an important and consistent producer of the potent nephrotoxic ochratoxin A (OTA). Addressing the safety of seasoning of meat products we developed different molecular approaches to monitor the presence of P. nordicum and its related OTA contamination risk. A sensitive and easy to use Loop-mediated isothermal amplification (LAMP) assay, for P. nordicum detection on salami surface, was set up targeting otapksPN gene, a key gene in the biosynthesis of OTA in P. nordicum. Positive reactions were detected directly in-tube by color transition of hydroxynaphthol blue from violet to sky blue. The assay was proved to be specific for P. nordicum and able to detect down to 100 fg of target DNA. In addition, gene expression of otapksPN gene and OTA production of P. nordicum were monitored throughout the seasoning process, up to 30 days in a small-scale experiment. The expression of otapksPN gene was early detected after 4 days of seasoning and increased significantly after 7 days, reaching the maximum expression level after 10 days. Consistently with gene expression data, OTA was detected from the 4th day and its content increased significantly from the 7th day, reaching the maximum level after 10 days. Finally, the LAMP assay was tested to detect the persistence of P. nordicum during the seasoning process of sausages co-inoculated with P. nalgiovense at different contamination rates. After 14 days of seasoning, LAMP assay was able to detect the presence of P. nordicum down to 2.5% contamination interspersed with 97.5 % of P. nalgiovense. The analysis of toxin content at the end of seasoning, revealed that OTA was accumulated both in mycelium and dry-cured meat when P. nordicum contamination rate ranged from 25% to 100% of inoculum, while OTA was not detected in dry-cured meat at 2.5% and 0.25%. These results evidenced that 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 to prevent and control OTA risk in dry-cured meat production.

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.

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.

This paper reports the development and the results of a procedure aimed at measuring, modelling and validating the growth of surface molds in industrial salami ripening by relying on techniques of image analysis. The sausages under investigation were inoculated with fungal starters and ripened in a test carried out at SSICA (Parma) under closely monitored and controlled conditions in a pilot-scale ripening chamber based on the "Air flow from bottom upward" technology. The work has been carried out within the R&D PON01_01409 "Safemeat" project. Among the various investigations, digital images were purposely acquired in a standardized way throughout the experimental test of sausage ripening. The graphical procedures here discussed involve a bit of image pre-processing, a digital image analysis work and some data post-processing. A pre-processing software i ntroduces a black background around each photographed sausage. The open-source ImageJ software is used for recognizing and measuring the gut area covered by molds as a whole, distinguishing each individual mold colony, measuring its surface area and counting the overall number of colonies. Further data post- processing provides results in terms of percent surface covered by molds, number of mold colonies per unit gut surface and size distribution of colonies as a function of their individual area. Microbiological analyses confirmed that the fungal population established on the salami casing immediately after the surface inoculum was exactly corresponding to the mold starters. The developed methodology and the encouraging results obtained so far promise to be a rather simple and cheap way to control the onset and progress of the fungal colonization in industrial ripening chambers.

Traditional sausages are often considered of superior quality to sausages inoculated with commercial starter cultures and this is partially due to the action of the typical house microflora. Penicillium nalgiovense is the species commonly used as starter culture for dry-cured meat production. Recently a new species, Penicillium salamii, was described as typical colonizer during salami seasoning. In order to understand its contribution to the seasoning process, two different experiments on curing of fresh pork sausages were conducted using P. salamii ITEM 15302 in comparison with P. nalgiovense ITEM 15292 at small and industrial scale, and the dry-cured sausages were subjected to sensory analyses. Additionally, proteolytic and lipolytic in vitro assays were performed on both strains. P. salamii ITEM 15302 proved to be a fast growing mould on dry-cured sausage casings, well adapted to the seasoning process, with high lipolytic and proteolytic enzymatic activity that confers typical sensory characteristics to meat products. Therefore, P. salamii ITEM 15302 was shown to be a good candidate as new starter for meat industry.

Fermented meat products, praised for their culinary heritage and identity, represent crossroads of innovation and tradition, quality and healthiness. Mold growth of some Penicillium species is highly desired in some dry-cured meat products, due to their contribution to flavour, anti-oxidative effects and protective role against detrimental microorganisms. Penicillium salamii has been recently described as a promising candidate for starter formulations for meat industry. Otherwise, undesirable species, like Penicillium nordicum, could colonize and contaminate cured meat with ochratoxin A or other related mycotoxins. To this aim, LAMP and other DNA-based assays represent useful tools for early detection of toxigenic fungi and therefore for effective mycotoxins risk managing.

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.

The availability of rapid diagnostic methods for monitoring ochratoxigenic species during the seasoning processes for dry-cured meats is crucial and constitutes a key stage in order to prevent the risk of ochratoxin A (OTA) contamination. A rapid, easy-to-perform and noninvasive method using an electronic nose (e-nose) based on metal oxide semiconductors (MOS) was developed to discriminate dry-cured meat samples in two classes based on the fungal contamination: class P (samples contaminated by OTA-producing Penicillium strains) and class NP (samples contaminated by OTA non-producing Penicillium strains). Two OTA-producing strains of P. nordicum and two OTA non-producing strains of P. nalgiovense and P. salamii, were tested. The feasibility of this approach was initially evaluated by e-nose analysis of 480 samples of both Yeast Extract Sucrose (YES) and meat-based agar media inoculated with the tested Penicillium strains and incubated up to 14 days. The high recognition percentages (higher than 82%) obtained by Discriminant Function Analysis (DFA), either in calibration and cross-validation (leave-more-out approach), for both YES and meat-based samples demonstrated the validity of the used approach. The e-nose method was subsequently developed and validated for the analysis of dry-cured meat samples. A total of 240 e-nose analyses were carried out using inoculated sausages, seasoned by a laboratory-scale process and sampled at 5, 7, 10 and 14 days. DFA provided calibration models that permitted discrimination of dry-cured meat samples after only 5 days of seasoning with mean recognition percentages in calibration and cross-validation of 98 and 88%, respectively. A further validation of the developed enose method was performed using 60 dry-cured meat samples produced by an industrialscale seasoning process showing a total recognition percentage of 73%. The pattern of volatile compounds of dry-cured meat samples was identified and characterized by a developed HS-SPME/GC-MS method. Seven volatile compounds (2-methyl-1-butanol, octane, 1R-?-pinene, D-limonene, undecane, tetradecanal, 9-(Z)-octadecenoic acid methyl ester) allowed discrimination between dry-cured meat samples of classes P and NP. These results demonstrate that MOS-based electronic nose can be a useful tool for a rapid screening in preventing OTA contamination in the cured meat supply chain.

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.

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.

Two organic amendments (OMW-M1 and OMW-M2), based on olive mill wastes (OMWs) subjected to advanced processes of aerobic static storage or composting, were tested for their suppressive activity against Verticillium dahliae , the causal agent of olive Verticillium wilt. OMW-M1 and OMW-M2 drastically inhibited the pathogen growth in vitro and then were further tested in suppressive pot experiments. The amendments, mixed at 15 % (v/v) with a nursery standard plant-growth matrix, were tested alone or in combination with two biocontrol bacteria (Bacillus amyloliquefaciens and Burkholderia cepacia ) selected from suppressive soils. All mixtures were artificially contaminated with V. dahliae microsclerotia (MS), the density of which was periodically monitored by either a semi-selective medium or a specific real-time PCR technique. In plant-less pot assays conducted in a growth chamber, OMW-M1 was the most effective amendment, reducing V. dahliae MS density by 100 % after 90 days with respect to the untreated control. In nursery experiments with pot-growing olive plants, OMW-M1, particularly when combined with the biocontrol bacteria, confirmed its strong suppressive activity reducing up to 100 % the density of V. dahliae MS in the rhizosphere behaving even better than a commercial biofungicide (Trichoderma asperellum TV1) used as a control. The best combined treatment also reduced plant mortality and increased root and shoot extension. We conclude that organic amendments from stabilized olive mill by-products showed positive agronomic and phytosanitary properties on pot-growing olive plants and, particularly when enriched with biocontrol agents, they are potentially suitable for use in sustainable agriculture.

The selection of appropriate plant species is critical in the successful application of phytoremediation techniques. The present study is an attempt to assess the capability of three brassicaceae, Brassica alba (L) Rabenh, Brassica carinata A. Braun and Brassica nigra (L) Koch, for the phytoextraction of Cr, Cu, Pb and Zn from an unpolluted and polluted silty loamy soil added with either Bacillus licheniformis BLMB1 or compost or both. Experiments were conducted in a greenhouse in pots filled with the soils. In all experiments metals were shown to accumulate in shoots and roots of plants grown on polluted soils, and both compost and B. licheniformis BLMB1 strain were able to enhance the accumulation of metals, especially Cr. In particular, Cr accumulation in B. alba resulted higher than the Cr threshold for hyperaccumulator plants (1000 mg kg(-1)). This result provides a new plant resource that may have a potential use for phytoextraction of Cr from contaminated soil. However, because of the low bioconcentration factors (< 1) for all studied metals, these species cannot be regarded as suitable for the phytoextraction of excessive Cr, Cu, Pb and Zn from polluted soils. Thus, these species may be used with success only for low metal polluted soils. (C) 2011 Elsevier B.V. All rights reserved.

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.